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The effects of the chlorinated pesticide chlordecone on food intake, body weight, and water intake were examined in adult male rats. Chlordecone treatment produced a dose dependent suppression of food intake. Loss of body weight accompanied the reduced food intake. However, chlordecone did not suppress water intake. Chlordecone treated animals maintained on a liquid diet also demonstrated reduced food intake, suggesting that chlordecone has a specific effect on feeding behavior and not a general effect on ingestive behaviors. The potential contribution of chlordecone-induced tremor to the suppressed food intake and the loss of body weight was considered. When treatment occurred immediately before a 24 hr fast, controls and animals given 75 mg/kg showed no differences in the body weight decline, even though tremor occurred in the pesticide-treated rats. Thus, it is unlikely that tremor alone produced the body weight loss observed in the present experiment. Similarly, animals were capable of initiating eating behavior even though tremor was present. In addition, chlordecone treatment inhibited food intake within 2 hr. Consequently, these results suggest that chlordecone suppresses food intake which in turn produces the decline in body weight. | US |
An active model of ocular anaphylaxis was developed in guinea pigs to evaluate the histopathology of the early (EPR) and late (LPR) phase reaction, focusing on the role of mast cells. Five groups (n = 6) of animals were actively immunized by first injecting into each of the axillary and inguinal lymph node areas, 0.25 ml of an emulsion containing 1 mg dinitrophenyl bovine gamma-globulin (DNP-BCG) with 0.5 ml complete Freund's adjuvant. After two weeks, an intramuscular injection of 0.5 ml of an emulsion containing 1 mg DNP-BGG with 0.5 ml of incomplete adjuvant was administered. One month after the first injection, animals were sacrificed after topical ocular challenge with 10 microliters of 1 mg/ml divalent hapten, di-DNP-lysine, in one eye and phosphate buffered saline (PBS) in the fellow eye as control. Clinical reactions were graded over time and histology evaluated at the endpoint (time 0, 0.5, 3, 9, and 24 h). Results showed that all animals clearly developed both an EPR and an LPR, as either a biphasic, multiphasic or prolonged clinical response. A small percentage of mast cells were degranulated at baseline, whereas, at 0.5 h, 95% of mast cells were degranulated in the eyes treated with specific hapten and 25% in the control eyes treated with PBS. At 3 h, 84% of the mast cells were degranulated. This value rose to 89% at 9 h, and remained unchanged at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS) | US |
We examined 142 biopsy specimens of smokeless tobacco-associated oral mucosal lesions from 133 professional baseball players. Four types of epithelial change were observed in the specimens: hyperparakeratosis, hyperorthokeratosis, pale surface staining, and basal cell hyperplasia. These types of epithelial change were associated with the type of smokeless tobacco used (snuff or chewing tobacco) but not with the duration (years) or amount (hours per day) of use. The thickness of hyperkeratosis in a specimen correlated directly with the amount of smokeless tobacco use. The use of snuff was more frequently associated with development of oral mucosal lesions than was the use of chewing tobacco, and snuff appeared to cause a greater variety and severity of epithelial change than did chewing tobacco. | US |
We have cloned and sequenced the human KIT proto-oncogene, which contains 21 exons and spans more than 34 kb of DNA on chromosome segment 4q12. We also establish physical linkage between the KIT gene and the related PDGFRA gene. The organization of the KIT gene is virtually identical to that of the homologous FMS gene, located on chromosome 5. Together, these data suggest that the KIT and PDGFRA genes on chromosome 4 and the FMS and PDGFRB genes on chromosome 5 arose by duplication of a common ancestral gene, followed by duplication of a chromosome. | US |
Cocaine solution has traditionally been the agent of choice for vasoconstriction and anesthesia when applied topically to the nasal mucosa during nasal operative procedures. Because of the relative scarcity and resulting expense of cocaine, there has arisen an impetus for an alternative intranasal solution for mucosal anesthesia and vasoconstriction. As a logical alternative, we have used a mixed solution of xylometazoline and lidocaine with reasonable results. No clinical studies comparing the efficacy of the two solutions exist, however, and there is presently no such solution commercially available. A double-blind, randomized, placebo-controlled study was undertaken to assess the relative efficacy of the preparations. Both solutions resulted in a marked and roughly equivalent degree of mucosal vasoconstriction (as evidenced by comparable increases in nasal airway cross-sectional area). Subjective pain ratings of mucosal pin-prick decreased a surprisingly small degree after application of both solutions. It appears that xylometazoline/lidocaine solution is comparable to cocaine solution for purposes of vasoconstriction and anesthesia during intranasal operative procedures. | US |
In the present paper, we have characterized the specificity of a series of monoclonal antibodies (MoAbs) against Plasmodium berghei sporozoites, selected for their lack of reactivity with the repeat domain of the circumsporozoite (CS) protein. We found that these MoAbs recognize Pb44, the mature membrane form of the CS protein, but they do not react with Pb54, its precursor. Furthermore, these MoAbs do not react with any of the synthetic peptides representing the linear sequence of the P. berghei CS protein nor do they react with a recombinant CS (rCS) protein. These observations indicate that the epitope recognized by these antibodies is expressed only after the processing of the CS protein has occurred. This 'processing dependent epitope' is present in sporozoites of P. berghei, and also in those of P. falciparum, P. yoelii and P. brasilianum. It is not present in sporozoites of the P. cynomolgi-P. vivax complex and of P. gallinaceum. These anti-sporozoite MoAbs strongly inhibit sporozoite invasion of hepatoma cells, in vitro, however, they displayed no protective effect in an in vivo assay. | US |
The existing bleomycin (BLM)-rodent model of lung fibrosis requires large doses and is often associated with morbidity and high mortality. We have developed an intratracheal multiple-dose BLM-hamster model of lung fibrosis. In this model, 3 consecutive doses of BLM (2.5 U, 2.0 U and 1.5 U/5mL/kg) were instilled intratracheally, one dose per week. The hamsters were killed at 10, 20, 30, 60 and 90 days after the last IT instillation and the lungs were lavaged or perfused with saline. This regimen of BLM administration was devoid of morbidity and caused only 6% overall mortality. Lung prolyl hydroxylase activity at 10 days and hydroxyproline content at 20, 30, 60 and 90 days were significantly higher than noted for the controls. Bronchoalveolar lavage fluid-supernatant protein and the total number of recovered cells of all types were significantly higher than observed for the controls at all times, except at 90 days. Lungs showed a multifocal mixed mononuclear infiltrate at 10 and 20 days and septal fibrosis, which was most severe and organized at 30 days and less severe at 60 and 90 days. The parenchymal lesions were significantly greater than those of the controls at all times, except at 10 days. This model, which required only 6 U BLM/kg, induced a moderate level of lung fibrosis. It has been concluded, therefore, that this model, inasmuch as it is not associated with an overwhelmingly acute inflammation, would be more applicable for screening potential antifibrotic agents than existing models of lung fibrosis. | US |
L-Selectin, previously known as LEC.CAM-1, LECAM-1, LAM1, and as the MEL-14, Leu-8, TQ1, and DREG-56 antigens, is a leukocyte membrane protein that participates in adhesion to endothelium. We studied its expression on eosinophils using flow cytometry and the MAb Dreg-56 and Leu-8. Unstimulated peripheral blood eosinophils from healthy adults expressed about one third the level of L-selectin as neutrophils (mean +/- SD specific fluorescence: 20.9 +/- 3.2 versus 54.5 +/- 8.4, p = 0.0001, n = 18). After stimulation with A23187, L-selectin expression on eosinophils was rapidly lost. This was temporally correlated with increased expression of Mac-1 (CD11b/CD18); the kinetics on eosinophils and neutrophils were similar. Eosinophil expression of L-selectin decreased modestly after stimulation with platelet activating factor, but was minimally affected by N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, or C5a compared with their effects on neutrophils. Eosinophils from cord blood of healthy neonates born at term expressed less L-selectin than adult eosinophils (10.4 +/- 3.8 versus 19.4 +/- 2.7, p = 0.0001, n = 9); the relative reduction was the same as on cord blood neutrophils (36.4 +/- 8.2 versus 55.5 +/- 4.8, p = 0.0001, n = 9). Relative to baseline expression, the responses of neonatal and adult cells to stimulation did not differ. We conclude that neonatal eosinophils have abnormalities in L-selectin expression similar to neonatal neutrophils and suggest that decreased expression of L-selectin and a diminished responsiveness to direct stimulation with chemotactic factors are possible mechanisms that may limit the exudation of eosinophils. | US |
Children with inborn errors of urea synthesis who survive neonatal hyperammonemic coma commonly exhibit cognitive deficits and neurologic abnormalities. Yet, there is evidence that ammonia is not the only neurotoxin. Hyperammonemia appears to induce a number of neurochemical alterations. In rodent models of hyperammonemia, uptake of L-tryptophan into brain is increased. It has been reported that in an experimental rat model of hepatic encephalopathy, in the ammonium acetate-injected rat, and in patients with hepatic failure and inborn errors of ammonia metabolism, quinolinate, a tryptophan metabolite, is increased. Elevations in quinolinate are of particular concern, as quinolinate could excessively activate the N-methyl-D-aspartate subclass of excitatory amino acid receptors, thereby causing selective neuronal necrosis. We sought to identify an animal model that would replicate the increases in quinolinate that have been associated with hyperammonemia in humans. Levels of quinolinate were measured in hyperammonemic urease-infused rats and ammonium acetate-injected rats. In the urease-infused rat, brain tryptophan was doubled, and serotonin and its metabolite 5-hydroxyindoleacetic acid were significantly increased. Yet, despite the increase in tryptophan and evidence for increased metabolism of tryptophan to serotonin, there were no observed increases of quinolinate in brain, cerebrospinal fluid, or plasma. In the ammonium acetate-injected rat, significant increases of 5-hydroxyindoleacetic acid in cerebral cortex were also observed, but quinolinate did not change in cerebrospinal fluid or cerebral cortex. In summary, we were unable to demonstrate an increase of quinolinate in brain or cerebrospinal fluid in these rat models of hyperammonemia. | US |
Circularly permuted group I intron precursor RNAs, containing end-to-end fused exons which interrupt half-intron sequences, were generated and tested for self-splicing activity. An autocatalytic RNA can form when the primary order of essential intron sequence elements, splice sites, and exons are permuted in this manner. Covalent attachment of guanosine to the 5' half-intron product, and accurate exon ligation indicated that the mechanism and specificity of splicing were not altered. However, because the exons were fused and the order of the splice sites reversed, splicing released the fused-exon as a circle. With this arrangement of splice sites, circular exon production was a prediction of the group I splicing mechanism. Circular RNAs have properties that would make them attractive for certain studies of RNA structure and function. Reversal of splice site sequences in a context that allows splicing, such as those generated by circularly permuted group I introns, could be used to generate short defined sequences of circular RNA in vitro and perhaps in vivo. | US |
Skeletal muscle has been used for biomechanical assist in experimental and clinical studies. Central to the success of these procedures is the generation of sufficient muscle force for the lifetime of the subject. Burst (tetanic) stimulation results in summation of individual twitches and generates higher power output. However, the superiority of paraneural versus intramuscular as well as proximal versus middle and distal intramuscular stimulations remains unclear. Electrophysiological mapping and mechanical performance of seven canine latissimus dorsi muscles were analyzed. The mechanism of higher tension generation produced by: (1) increased temporal summation; (2) greater motor units activated; or (3) result of both were determined. The parameters primarily dependent on the number of activated motor units are significantly greater following paraneural and proximal intramuscular stimulations. The parameters mainly related to temporal summation are not different between various electrode configurations. For intramuscular stimulation, it is the location of interelectrode field rather than the location of the cathode per se that determines the mechanical performance of the skeletal muscle. Furthermore, tension development of skeletal muscle is primary nerve activation rather than direct muscle stimulation. The higher tension generation that resulted from different electrode configurations is produced by activating a higher number of muscle fibers through the neuromuscular junctions. | US |
Coexistence of immunoreactivity for calcitonin gene-related peptide (CGRP) and galanin (GAL) was examined in varicose nerve endings in female rat pelvic paracervical ganglia (PG) and in perikarya of lumbosacral dorsal root ganglia (DRG). Varicose peptide-containing nerves were closely adjacent to somata of neurons in the PG, certain somata being virtually surrounded by immunoreactive varicosities. Some nerve endings were immunoreactive for either CGRP or GAL; in others, immunoreactivity for CGRP and GAL coexisted. Likewise, many perikarya in DRG were CGRP immunoreactive, fewer were GAL immunoreactive, and in some immunoreactivity for CGRP and GAL coexisted. The results suggest there are subpopulations of neuropeptide-containing sensory nerve endings in the PG; some contain CGRP, some contain GAL, and in some CGRP and GAL coexist. These substances contained in sensory nerve endings could have important roles in pelvic ganglionic functions. | US |
Of the various types of potent bombesin(Bn)/gastrin releasing peptide receptor antagonists that have been discovered, the desMet14-methyl ester peptides are devoid of residual agonist activity and are among the most potent in terms of in vitro receptor blockade and also in terms of their prolonged inhibition of bombesin-stimulated amylase and protein release in the rat. We have now examined the in vitro and in vivo properties of a new series of methyl ester analogues, [D-Phe6]Bn(6-13)OMe, [D-Phe6,D-Ala11]Bn(6-13)OMe, N alpha-propionyl-[D-Ala24]GRP(20-26)OMe, and [D-pentafluoro-Phe6,D-Ala11]Bn(6-13)OMe, which have an additional D-amino acid substituent and some highly lipophilic moieties at the N-terminus. All analogues were able to potently antagonize the ability of Bn to stimulate amylase release from rat acinar cells, with IC50 values of 2.4, 2.5, 0.6, and 1.3 nM, respectively. The four peptides were found to have binding affinities for these cells comparable to Bn itself, with K(i)s of 10.3, 2.8, 5.5, and 3.6 nM, respectively, but all had little or no affinity for neuromedin B receptors on murine C6 cells. Single bolus IV injections of these peptides were found to potently inhibit amylase and protein release caused by IV infusion of bombesin into the rat. Generally the peptides containing the D-Ala substituent were longer acting than [D-Phe6]Bn(6-13)OMe, so that [D-Phe6,D-Ala11]Bn(6-13)OMe and N alpha-propionyl-[D-Ala24]GRP(20-26)OMe displayed significant inhibitory effects for up to 1.5 h after administration.(ABSTRACT TRUNCATED AT 250 WORDS) | US |
Hypothalamic galanin gene expression was investigated during reproductive maturation in peripubertal rats. Rat galanin-like immunoreactivity (rGAL-LI) increased in the median eminence and anterior pituitary during the extended first estrous and diestrous phases relative to anestrous phase female or male rats. In the neurointermediate lobe, rGAL-LI was elevated in first diestrous phase compared to anestrous phase females or males. In a second study, hypothalamic tissue was divided into quadrants for analysis of rat galanin (rGAL) mRNA by Northern blot hybridization. Two days after the injection of pregnant mare's serum gonadotropin (PMSG), rGAL mRNA increased approximately twofold in the paraventricular area and preoptic area quadrants. No effects of PMSG on galanin gene expression were found in medial basal or supraoptic hypothalamic quadrants. Because PMSG acts through the stimulation of ovarian estrogen secretion, these studies conclude that galanin gene expression in dorsal hypothalamic nuclei is under the stimulatory influence of estrogen and suggest that galanin may be a mediator of central ovarian steroid feedback. | US |
Tumor necrosis factor beta (TNF-beta) (lymphotoxin) may play an important role in the immune response and pathologic inflammatory diseases. Insulitis is an important early step in the development of insulin-dependent diabetes mellitus. To understand better the role of TNF-beta in the regulation of inflammation and type 1 diabetes, we produced transgenic mice in which the murine TNF-beta gene was regulated by the rat insulin II promoter. The transgene was expressed in the pancreas, kidney, and skin of transgenic mice. The expression of TNF-beta in the pancreas of transgenic mice resulted in a leukocytic inflammatory infiltrate consisting primarily of B220+ IgM+ B cells and CD4+ and CD8+ T cells. The insulitis is reminiscent of the early stages of diabetes, though the mice did not progress to diabetes. | US |
Antisense DNA inhibition of gene expression was explored as an approach toward elucidating mechanisms regulating development of preimplantation mammalian embryos. Specifically, a role for the c-myc protooncogene was examined. Detection of c-myc mRNA and immunoreactive nuclear c-myc protein in preimplantation mouse embryos at the eight-cell/morula and blastocyst stages suggested that this DNA-binding protein could be important during early embryo-genesis. The effects of c-myc oligodeoxyribonucleotides (oligos) on the in vitro development of two-cell mouse embryos were examined. Embryos cultured in medium containing an unmodified (phosphodiester) antisense c-myc oligo complementary to the translation initiation codon and spanning the first seven codons exhibited a dose-dependent arrest at the eight-cell/morula stage. At lower concentrations (7.5 microM) this inhibitory effect was specific to the antisense oligo and did not occur with the sense-strand complement or with duplexes of the antisense and sense oligos. However, at 4-fold higher concentrations of DNA (30 microM), all unmodified c-myc oligos were embryotoxic, causing embryos to arrest at the two-cell to four-cell stages. In contrast, almost all (98%) two-cell embryos cultured with a modified (chimeric phosphorothioate/phosphodiester) antisense c-myc oligo (7.5 microM) exhibited developmental arrest at the eight-cell/morula stage, whereas no developmental arrest occurred following incubation with high concentrations of the modified sense complement (30 microM). Culture of freshly recovered eight-cell embryos with antisense c-myc led to the absence of c-myc protein but no change in epidermal growth factor receptor in those embryos that developed a blastocoel. These effects on c-myc were specific for the antisense oligo. These results suggest that c-myc function becomes particularly critical for preimplantation mouse embryos at the eight-cell/morula stage of development and establish that antisense DNA can be successfully applied as an approach toward elucidating the roles of specific genes in preimplantation mammalian embryo development. | US |
The results of anterograde and retrograde axonal transport experiments in the rat indicate that the dorsal premammillary nucleus (PMd) gives rise to a branched pathway ending in the anterior thalamic group and brainstem, like the medial and lateral mammillary nuclei. However, unlike these nuclei, the ascending PMd projection courses through and to the anterior hypothalamic nucleus, and the descending PMd projection ends in the periaqueductal gray, superior colliculus, and adjacent parts of the reticular formation. Also unlike the traditional mammillary nuclei, the PMd does not receive a direct input from the columns of the fornix; instead, it receives a bilateral input from the anterior hypothalamic nucleus, which in turn receives inputs from areas related to the prefrontal cortex, amygdala, and hippocampus. The results provide interesting perspectives on the organization of medial hypothalamic circuits underlying the goal-oriented behaviors associated with hunger, thirst, and reproduction. | US |
A previously unknown collagen cDNA clone, PF19, was isolated from a human placenta library. The 2.1-kilobase insert has a complete open reading frame of 709 amino acids that includes 12 amino acids of the NH2-terminal domain, a principally collagenous region of 577 residues, and 120 residues of the noncollagenous COOH terminus. The collagenous part of the sequence encoded by PF19 is characterized by 13 interruptions ranging in size from 2 to 45 amino acids. Within four interruptions are consensus sequences for attachment of serine-linked glycosaminoglycans and asparagine-linked oligosaccharides suggesting that this collagen may be extensively glycosylated. A synthetic decapeptide representing a sequence at the beginning of the COOH-terminal noncollagenous domain was used to prepare an antibody in rabbits. This antiserum detected a 125-kDa bacterial collagenase-sensitive protein in Western blots of HeLa cell lysate. Consistent with the size of the collagen chain, Northern blot hybridization revealed a major transcript of 5.3 kilobases and two minor ones of 4.7 and 4.4 kilobases that are present in cultured human fibroblasts but absent from umbilical vein endothelial cells. We propose that the previously unidentified polypeptide described in this report be designated the alpha 1 chain of type XV collagen. | US |
Apical nonselective cation channels with an average single-channel conductance of 34 +/- 2.3 pS were found in M-1 mouse cortical collecting duct cells. Channel activity is increased by depolarization and abolished by cytoplasmic calcium removal. Cytoplasmic application of 0.1 mM cGMP decreases channel open probability by 27%. cDNAs corresponding to approximately 40% of the coding region of the photoreceptor channel were isolated by the polymerase chain reaction from M-1 cells and a rat kidney cDNA library. The rat kidney-derived sequence differs by a single base, and the M-1-cell-derived sequence differs by only two bases, from the photoreceptor sequence. A second clone from M-1 cells differs by 20 out of 426 bases from the photoreceptor sequence. In all three clones, the deduced amino acid sequence is identical to that of the rat photoreceptor channel. Northern blot analysis of poly(A)+ RNA from M-1 cells reveals the presence of a 3.2-kilobase band hybridizing with a retinal cGMP-gated cation channel probe. The results suggest the expression in M-1 cells of more than one gene coding for nonselective cation channels or channel subunits, one of which is identical to the cGMP-gated cation channel gene of rod photoreceptors. | US |
Protein-tyrosine kinase and protein-tyrosine phosphatase (PTPase) activities are essential for T-cell antigen receptor-mediated signaling. To assess the functional consequences of alteration of the levels of tyrosine phosphorylation in normal human T cells, the effects of vanadate and hydrogen peroxide were studied. In combination, these agents induced tyrosine phosphorylation of cellular substrates, elevated cytosolic free calcium, and induced interleukin 2 receptor (IL-2R) alpha chain expression but not IL-2 secretion. However, anti-CD28 antibody in combination with vanadate and hydrogen peroxide induced IL-2 secretion, consistent with the requirement for a costimulatory signal in the induction of this gene. The effects of vanadate and hydrogen peroxide were enhanced in the absence of the T-cell PTPase, CD45. Thus, acute pharmacologic manipulation of the level of tyrosine phosphorylation in normal T cells correlates with partial, but not full, activation of these cells; in concert with a costimulatory signal provided by perturbation of the CD28 molecule, the complete program of activation is initiated. These agents should prove useful in dissecting signaling pathways involved in the regulation of genes critical to the immune response. | US |
Autonomic neurons help to regulate immune responses, and there are reciprocal interactions between the nervous and immune systems. This study seeks to define some of the molecular mechanisms that may underlie such interactions. Immunoblot analysis indicated that cultured sympathetic neurons synthesize and release the cytokine interleukin 1 beta (IL-1 beta). In addition, RNA blot analysis of cultured sympathetic neurons demonstrated that the neurons contain mRNA encoding IL-1 beta. It was previously shown that explant cultures of sympathetic ganglia and dissociated cocultures of neurons with ganglionic nonneuronal cells synthesize substance P, whereas in situ levels of substance P and its mRNA are low. An antagonist at the interleukin 1 receptor markedly depressed this increase in substance P in cultures, suggesting that endogenous IL-1 beta mediates the synthetic response, at least in part. Because pure neuronal cultures do not contain substance P and neurons synthesize and release IL-1 beta, the actions of the cytokine require the presence of ganglion nonneuronal cells. These observations suggest a role for autonomic neurons in influencing immune responses by synthesizing and secreting at least two known immunoregulators, the cytokine IL-1 beta and the neuropeptide substance P. | US |
The preferential inactivation of the paternal X chromosome in extraembryonic cells during early mouse development is an example of parental imprinting, but it has not been studied at the transcriptional level because standard methods of measuring RNA levels do not allow detection of allele-specific RNAs in individual early embryos. We sought to determine whether the paternal allele of the X chromosome-linked gene for 3-phosphoglycerate kinase 1 (Pgk-1), which is located very near the center of X chromosome inactivation, is transcribed prior to differentiation of extraembryonic lineages. Previous reports indicated that in heterozygous embryos there is a delay in the appearance of the phosphoglycerate kinase 1 allozyme encoded by the paternal X chromosome until 2 days after the appearance of the corresponding maternal allozyme. We report results obtained by use of a reverse transcription/PCR-based method which allows the quantitative measurement of allele-specific RNA. The assay is sensitive enough for the quantitative analysis in single embryos of allele-specific transcripts differing by only one nucleotide. We have used this assay to analyze mouse embryos heterozygous at the Pgk-1 and Hprt [hypoxanthine (guanine) phosphoribosyltransferase] loci, and we find that individual 8-cell and blastocyst embryos express both Hprt and Pgk-1 paternal transcripts, as do pooled 2- to 4-cell embryos. These results are discussed in view of the apparent temporal delay in paternal expression of the Pgk-1 gene at the enzyme level. | US |
Active, transepithelial, Ca2+ reabsorption in kidney occurs primarily in the distal convoluted tubule. Recent evidence suggests that entry of Ca2+ at the apical membrane through channels bearing resemblance to those of the voltage-dependent L type may be the rate-determining step in Ca2+ reabsorption. To determine the molecular identity of the pore-forming subunit of voltage-dependent Ca2+ channel(s) in the kidney, a homology-based PCR cloning strategy was employed. Nondegenerate primers, based on conserved regions of the published cDNA sequences of voltage-dependent Ca2+ channel alpha 1 subunits, were used to amplify cDNA from rat kidney, and the products were subcloned and sequenced. A family of molecular species was identified, representing alternatively spliced transcripts of four known genes encoding these channel subunits. Northern blot analysis indicated that the expression of each of the genes exhibits a distinct spatial distribution within the kidney. One gene, CaCh4, is expressed primarily in the cortex, and by microdissected-tubule PCR was found predominantly in the distal convoluted tubule, consistent with a role in transepithelial Ca2+ reabsorption at this site. | US |
Caveolae are flask-shaped plasma membrane invaginations abundant in endothelium and muscle but may be present in all cells. They contain a filamentous coat material thought to be important in their structure and function. Recent studies have demonstrated that a 22-kDa protein (caveolin) phosphorylated on tyrosine in Rous sarcoma virus-transformed chicken fibroblasts is a component of the caveolae coat on the inner aspect of the membrane. We now report the deduced protein sequence of chicken caveolin derived from cDNA PCR products and genomic DNA clones. Caveolin is a unique protein of 178 amino acids and displays little sequence similarity to other proteins in the GenBank data base. Hydrophobicity predictions indicate an unusual 40-amino acid hydrophobic region near the C terminus that may be used to anchor the protein to the membrane. When chicken caveolin was expressed in mouse 3T3 cells and detected by immunofluorescence microscopy, the typical caveolae pattern was observed. This includes brightly fluorescent membrane patches in many cases concentrated at the margin of cells and in arrays. Caveolae may be distinct from other membrane domains due at least in part to caveolin. | US |
Corticosteroids are the preeminent antiinflammatory agents although the molecular mechanisms that impart their efficacy have not been defined. The endothelium plays a critical role in inflammation by directing circulating leukocytes into extravascular tissues by expressing adhesive molecules for leukocytes [e.g., endothelial-leukocyte adhesion molecule 1 (ELAM-1) and intercellular adhesion molecule 1 (ICAM-1)]. We therefore determined whether corticosteroids suppress inflammation by inhibiting endothelial expression of adhesion molecules for neutrophils (polymorphonuclear leukocytes). Preincubation of endothelial cells with endotoxin [lipopolysaccharide (LPS), 1 microgram/ml] led to a 4-fold increase in subsequent adherence of polymorphonuclear leukocytes (P < 0.0001, n = 10) to endothelial cells, an increase that was markedly attenuated when endothelial cells were treated with dexamethasone (IC50 < 1 nM, P < 0.0001, n = 6 or 7) during preincubation with LPS. Moreover, the steroid receptor agonist cortisol (10 microM), but not its inactive metabolite tetrahydrocortisol (10 microM), diminished LPS-induced endothelial cell adhesiveness. Further evidence that the action of dexamethasone was mediated through ligation of corticosteroid receptors [human glucocorticoid receptors (hGRs)] was provided by experiments utilizing the steroid antagonist RU-486. RU-486 (10 microM), which prevents translocation of ligated hGR to the nucleus by inhibiting dissociation of hGR from heat shock protein 90, completely aborted the effect of dexamethasone on adhesiveness of endothelial cells (P < 0.0005, n = 3). Treatment of endothelial cells with LPS (1 microgram/ml) stimulated transcription of ELAM-1, as shown by Northern blot analysis, and expression of membrane-associated ELAM-1 and ICAM-1, as shown by quantitative immunofluorescence (both P < 0.001, n = 9). Dexamethasone markedly inhibited LPS-stimulated accumulation of mRNA for ELAM-1 and expression of ELAM-1 and ICAM-1 (IC50 < 10 nM, both P < 0.001, n = 4-9); inhibition of expression by dexamethasone was reversed by RU-486 (both P < 0.005, n = 4-6). As in the adhesion studies, cortisol but not tetrahydrocortisol inhibited expression of ELAM-1 and ICAM-1 (both P < 0.005, n = 3 or 4). In contrast, sodium salicylate (1 mM) inhibited neither adhesion nor expression of these adhesion molecules. These studies suggest that antagonism by dexamethasone of endotoxin-induced inflammation is a specific instance of the general biological principle that the glucocorticoid receptor is a hormone-dependent regulator of transcription. | US |
Diazepam-binding inhibitor (DBI) is a 9-kDa polypeptide that colocalizes in glial, adrenocortical, and Leydig cells with the mitochondrial DBI receptor (MDR). By binding with high affinity to the MDR, DBI and one of its processing products--DBI-(17-50)--regulate pregnenolone synthesis and have been suggested to participate in the immediate activation of adrenal steroidogenesis by adrenocorticotropic hormone (ACTH). In adrenals of hypophysectomized rats (1 day after surgery), ACTH failed to acutely affect the amount of adrenal DBI and the density of MDR but increased the rate of DBI processing, as determined by the HPLC profile of DBI-(17-50)-like immunoreactivity. The similar latency times for this effect and for ACTH stimulation of adrenal steroidogenesis suggest that the two processes are related. The ACTH-induced increase in both adrenal steroidogenesis and rate of DBI processing were completely inhibited by cycloheximide; this result suggests the requirement for the de novo synthesis of a protein with a short half-life, probably an endopeptidase. This enzyme, under the influence of ACTH, may activate formation of a DBI-processing product that stimulates steroidogenesis via the MDR. In support of this hypothesis is the demonstration that in hypophysectomized rats the MDR antagonist PK 11195 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide completely inhibited the adrenal steroidogenesis stimulated by ACTH and by the high-affinity MDR ligand 4'-chlorodiazepam. | US |
Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl- secretion by airway epithelial cells. In CF, cAMP does not activate Cl- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[beta-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl- currents and restore cAMP-activated Cl- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[gamma-thio]triphosphate and AlF4- reduce Cl- currents and inhibit cAMP from activating Cl- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[beta-thio]diphosphate and in normal cells, cAMP activates a Cl- conductance that has properties similar to CF transmembrane-conductance regulator Cl- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl- secretion in CF. | US |
Human immunodeficiency virus type 1 (HIV-1) infections of humans have a natural history characterized by a variable but usually slow progression to an immunodeficient state. We have described a molecular model of HIV-1 proviral latency in certain cell lines, characterized by extremely low or undetectable levels of unspliced genomic HIV-1-specific RNA but significant levels of multiply spliced HIV-1-specific RNA. We have utilized a quantitative reverse transcriptase-initiated polymerase chain reaction to measure the levels of various HIV-1 RNA species in peripheral blood mononuclear cells. The median level of multiply spliced HIV-1 RNA was dramatically higher than the median level of unspliced viral RNA in asymptomatic individuals. In addition, HIV-1 RNA patterns characterized by at least a 10-fold excess of multiply spliced to unspliced viral RNA were significantly more common in asymptomatic individuals than in patients with the acquired immunodeficiency syndrome. We suggest that asymptomatic clinical HIV-1 infection is characterized by a preponderance of HIV-1-infected peripheral blood cells blocked at an early stage of HIV-1 infection. This viral expression pattern, which we have called blocked early-stage latency, may constitute a reservoir of latently infected cells in certain HIV-1-infected persons. | US |
Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene encoding FGF-1 secrete FGF-1 into their conditioned medium in response to heat shock. The form of FGF-1 released by NIH 3T3 cells in response to increased temperature (42 degrees C, 2 hr) in vitro is not biologically active and does not associate with either heparin or the extracellular NIH 3T3 monolayer matrix. However, it was possible to derive biologically active FGF-1 from the conditioned medium of heat-shocked NIH 3T3 cell transfectants by ammonium sulfate fractionation. The form of FGF-1 exposed by ammonium sulfate fractionation is similar in size to cytosolic FGF-1 and can bind and be eluted from immobilized heparin similarly to the recombinant human FGF-1 polypeptide. Further, the release of FGF-1 by NIH 3T3 cell transfectants in response to heat shock is reduced significantly by both actinomycin D and cycloheximide. These data indicate that increased temperature may upregulate the expression of a factor responsible for the secretion of FGF-1 as a biologically inactive complex that requires an activation step to exhibit the biological activity of the extracellular polypeptide mitogen. | US |
Genomic nomenclature has not kept pace with the levels and depth of analyzing and understanding genomic structure, function, and evolution. We wish to propose a general terminology that might aid the integrated study of evolution and molecular biology. Here we designate as a "nuon" any stretch of nucleic acid sequence that may be identifiable by any criterion. We show how such a general term will facilitate contemplation of the structural and functional contributions of such elements to the genome in its past, current, or future state. We focus in this paper on pseudogenes and dispersed repetitive elements, since their current names reflect the prevalent view that they constitute dispensable genomic noise (trash), rather than a vast repertoire of sequences with the capacity to shape an organism during evolution. This potential to contribute sequences for future use is reflected in the suggested terms "potonuons" or "potogenes." If such a potonuon has been coopted into a variant or novel function, an evolutionary process termed "exaptation," we employ the term "xaptonuon." If a potonuon remains without function (nonaptive nuon), it is a "nonaptation" and we term it "naptonuon." A number of examples for potonuons and xaptonuons are given. | US |
As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Transfer of the recombinant plasmid to three LPS-rough strains of P. aeruginosa resulted in synthesis of IT-2 O antigen, and two of these transconjugant strains also synthesized a second O polysaccharide, presumably representing expression of a repressed, or an incomplete, set of genes for an endogenous O polysaccharide. Rabbits injected with the purified recombinant LPS made antibody specific for P. aeruginosa IT-2 O side chains, as did mice fed the recombinant E. coli strain. Expression of P. aeruginosa O antigens by enteric bacteria makes it possible to study these recombinant strains as oral vaccines to prevent P. aeruginosa infections. | US |
We have isolated a cDNA from a mouse brain library that encodes a protein whose predicted amino acid sequence is 42% identical and 64% similar to that of the amyloid beta protein precursor (APP). This 653-amino acid protein, which we have termed the amyloid precursor-like protein (APLP), appears to be similar to APP in overall structure as well as amino acid sequence. The amino acid homologies are concentrated within three distinct regions of the two proteins where the identities are 47%, 54%, and 56%. The APLP cDNA hybridizes to two messages of approximately 2.4 and 1.6 kilobases that are present in mouse brain and neuroblastoma cells. Polyclonal antibodies raised against a peptide derived from the C terminus of APLP stain the cytoplasm in a pattern reminiscent of Golgi staining. In addition to APP, APLP also displays significant homology to the Drosophila APP-like protein APPL and a rat testes APP-like protein. These data indicate that the APP gene is a member of a strongly conserved gene family. Studies aimed at determining the functions of the proteins encoded by this gene family should provide valuable clues to their potential role in Alzheimer disease neuropathology. | US |
The spatial and temporal relationship between the polymerase and RNase H activities of human immunodeficiency virus type 1 reverse transcriptase has been examined by using a 40-mer RNA template and a series of DNA primers of lengths ranging from 15 to 40 nucleotides, hybridized to the RNA, as substrates. The experiments were executed in the absence and presence of heparin, an efficient trap to sequester any free or dissociated reverse transcriptase, thus facilitating the study of events associated with a single turnover of the enzyme. The results indicate a spatial separation of 18 or 19 nucleotides between the two sites. To examine the effect of concomitant polymerization on the RNase H activity, the substrate was doubly 5' end labeled on the RNA and DNA. This enabled the study of RNase H activity as a function of polymerization in a single experiment, and the results in the absence and presence of heparin indicate a tight temporal coupling between the two activities. | US |
The expression of a molecular cDNA clone (P1 KIN) of the human RNA-dependent P1/eIF-2 alpha protein kinase (PKR) was examined in transfected monkey cells and in cell-free protein-synthesizing systems. Expression of the wild-type (wt) P1 KIN cDNA, which encodes an active protein kinase, was compared with that of the phosphotransfer catalytic domain II Lys-296-->Arg (K296R) mutant cDNA, which does not encode an active kinase. wt and K296R mutant P1 mRNAs prepared by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of P1 ribosome-associated protein with comparable efficiency in the rabbit reticulocyte system. The K296R mutant P1 protein was also efficiently synthesized in vivo in transfected COS monkey cells. However, synthesis of the wt P1 protein was reduced about 30-fold in transfected COS cells as compared with the K296R mutant P1 protein. Cotransfection of wt P1 KIN cDNA with either K296R mutant P1 KIN cDNA or reovirus S4 cDNA greatly reduced the synthesis of K296R mutant P1 protein and reovirus sigma 3 protein, respectively. Although the wt and K296R mutant P1 KIN plasmid expression vectors replicated with comparable efficiencies in COS cells, the steady-state amount of P1 mRNA was about 3-fold less in COS cells transfected with the wt as compared with the K296R mutant P1 KIN cDNA. These results suggest that RNA-dependent P1 protein kinase expression is autoregulated in vivo in transfected mammalian cells primarily at the level of translation by a mechanism that is likely dependent upon catalytically active P1 kinase. | US |
Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative immunosuppressant-binding site is probably distinct from the hsp90-binding site, and we predict that FKBP52 has different structural domains to accommodate these functions. hFKBP52 contains 12 protein kinase phosphorylation-site motifs and a potential calmodulin-binding site, implying that posttranslational phosphorylation could generate multiple isoforms of the protein and that calmodulin and intracellular Ca2+ levels could affect FKBP52 function. FKBP52 transcripts are present in a variety of human tissues and could vary in abundance and/or stability. | US |
The 40 years since the seminal papers of Hodgkin and Huxley appeared have been extraordinarily productive in terms of understanding the molecular basis for electrical activity. The Hodgkin-Huxley proposal that electrical excitability should be understood in terms of voltage-dependent changes in discrete sites has been resoundingly verified. Indeed, the Hodgkin-Huxley framework is remarkable in that its essential elements have remained largely intact as molecular understanding has advanced. This robustness is, at least in part, a result of the fact that Hodgkin and Huxley developed a mathematical model, based on simple physical arguments, that was sufficiently comprehensive to describe the kinetics of the voltage-clamped currents and yet simple enough to be predictive. The predictive features were demonstrated early by the reconstruction of both space-clamped and propagated action potentials on a desk-top calculator (293) and, later, when the sites of Hodgkin and Huxley developed into being well-characterized molecular structures. Voltage- and ligand-dependent ion-selective channels are now the established framework within which cellular electrophysiology is being pursued. Moreover, electrophysiological measurements of membrane and single-channel currents have become essential tools to examine molecular questions pertaining to channel structure and activity. The last 10 years have witnessed spectacular activity, which has resulted from two developments, the giga-seal patch clamp (249) and the elucidation of primary sequences of a number of channel-forming proteins (494), along with the first outlines of their low-resolution three-dimensional structures (651). The stage is now set for 1) applying a variety of convergent techniques to decipher molecular structural details at high resolution, and 2) seeking to understand the complex dynamic functions, gating, and ion selectivity at the molecular level. The early successes are likely to be in understanding the molecular determinants of ion conductance and selectivity, initially in terms of quantitative descriptions of how a sequence modification can alter a channel's permeability characteristics. Channel gating is a far more elusive target because it involves molecular rearrangements, which are poorly understood at any level of description and which may be modified by the channel's environment. The general mechanisms of ion permeation and gating will differ among different classes of ion channels, but a molecular understanding of either phenomenon must eventually be based on an understanding of intermolecular forces, which are invariant among all channel types.(ABSTRACT TRUNCATED AT 400 WORDS) | US |
The insulin-like growth factors (IGF)-I and IGF-II are peptides with structural homology to insulin and potent mitogenic and anabolic actions in vitro and in vivo. IGF-I levels are growth hormone (GH)-dependent and vary strikingly with age. IGF-I levels are typically low in infancy and childhood, increase dramatically during puberty, and then gradually decline with advancing age. Whether age-associated changes in GH production or sex steroid secretion, or other unknown factors, cause diminished IGF production in the elderly remains to be determined. In the brain, IGF-II appears to be the most prevalent IGF, but a truncated form of IGF-I also has been recognized. IGF actions are mediated by binding to a family of receptors, which includes the insulin receptor, the structurally homologous type I IGF receptor, and the IGF-II/M-6P receptor, all of which are found in the central nervous system. Additionally, the IGFs bind with high affinity to a family of IGF-binding proteins (IGFBPs). Of the six known IGFBPs, IGFBP-2 appears to be the major one in the mammalian brain and is a major component of CSF. Immunoreactive IGFBP-2 has been identified in astrocytes, and its mRNA has been identified in fetal and adult brain and choroid plexus. The IGFBPs transport the IGFs in serum and other body fluids and appear to regulate IGF access to receptors. In vivo regulation of IGFBPs includes tissue-specific proteases, which cleave specific IGFBPs, altering their affinities for IGF peptides.(ABSTRACT TRUNCATED AT 250 WORDS) | US |
A simplified and rapid screening method for detecting radiation-induced neoplastically transformed foci in the HeLa x skin fibroblast human hybrid cell assay system has been developed. The method is based on the recent identification of the tumor-associated antigen in this system as intestinal alkaline phosphatase (IAP), and on the recent commercial development of a stable alkaline phosphatase chromogenic substrate solution, Western blue (WB). Cleavage of the substrate results in the production of a blue insoluble precipitate. It is shown that WB can be used on both viable and paraformaldehyde-fixed cells. Fixation does not noticeably reduce the IAP enzymatic activity. A direct comparison with the current method of immunoperoxidase (IMPO) staining indicates that the WB method is not only easier, but appears to be more sensitive in picking up weakly positive foci with a resulting higher (factor of 2.5) induced transformation frequency for 7 Gy of 137Cs gamma radiation. Whereas the IMPO staining procedure is time-consuming and requires access to large amounts of expensive IAP-specific BD6 monoclonal antibody and peroxidase-labeled secondary antibody, the WB staining procedure is rapid and utilizes an inexpensive and readily available reagent. It should now allow this assay system to enter general use. | US |
The neurotoxic effect of capsaicin has been shown to be selective on a subpopulation of small dorsal root ganglion neurons in newborn animals. The aim of this study was to provide evidence of the long lasting effect of capsaicin and its ultrapotent analog resiniferatoxin (RTX) on sensory peptidergic neurons maintained in organotypic cultures. The effects of the two irritants were examined on neurons that contained substance P (SP) and calcitonin gene-related peptide (CGRP). Exposure of the cultures to 10 microM capsaicin and 100 nM RTX for periods of 2 days or longer resulted in almost complete elimination of SP-immunoreactive (IR) neurites and reduction, but not elimination, of CGRP-IR neurites. In addition, both 10 microM capsaicin and 100 nM RTX significantly reduced the number of SP- and CGRP-IR cell bodies within DRG explants. Capsaicin in 100 microM concentration produced complete elimination of SP-IR fibers and a greater decrease in the number of CGRP-IR fibers, but failed to completely eliminate IR cell bodies. Exposure of the cultures to the irritants in the same concentrations for 90 min did not produce a measurable effect on SP- or CGRP-IR in neurites or cell bodies. It is important to establish that the effect of capsaicin and RTX on cultured neurons was of long duration (longer than 4 days) and is therefore different from depletion of peptides. These findings demonstrate that processes of cultured sensory neurons are much more sensitive to capsaicin and RTX than cell bodies. Furthermore, our results show that SP-IR neuronal elements are more sensitive to capsaicin than CGRP-IR elements. These data suggest that cultured sensory neurons express the functional properties of differentiated sensory neurons in vivo. | US |
The study of the immunobiology of FVIII inhibitors may lead to new therapies for this potentially severe complication of haemophilia A and to new principles for the use of therapeutic proteins. In order to characterize the idiotype-anti-idiotype networks regulating FVIII inhibitors, we developed rabbit anti-idiotypic sera to 7 murine inhibitors and found at least 12 independent FVIII loci to which inhibitors could be raised. Rabbit antisera to the FVIII peptide, Ser1687-Thr1695, characterized one functional site to which about 46% of patients' inhibitor sera reacted. The multiplicity of inhibitor-recognized epitopes in FVIII makes it impractical, at the present time, to develop clinically useful specific anti-idiotypic therapies for FVIII inhibitors. Alternatively, one might induce genomic mutations in recombinant FVIII molecules to decrease immunogenicity of epitopes recognized by T helper cells. Methods to design such altered therapeutic proteins are presented, based on changing the longitudinal hydrophobic strip-of-helix which is in or near many T-cell-presented epitopes. | US |
Ligation of the antigen receptor on B cells induces the rapid phosphorylation of tyrosine on a number of cellular proteins. A monoclonal antibody that recognized a tyrosine-phosphorylated cell surface protein that was present in activated B cells was generated. Amino acid sequence analysis showed that this 140-kilodalton protein was CD22, a B cell-specific cell surface glycoprotein and putative extracellular ligand of the protein tyrosine phosphatase CD45. Tyrosine phosphorylation of CD22 may be important in B cell signal transduction, possibly through regulation of the adhesiveness of activated B cells. | US |
Plasticity of the developing visual system has been regarded as the best model for changes of neuronal connections under the influence of the environment. N-methyl-D-aspartate (NMDA) receptors are crucial for experience-dependent synaptic modifications that occur in the developing visual cortex. NMDA-mediated excitatory postsynaptic currents (EPSCs) in layer IV neurons of the visual cortex lasted longer in young rats than in adult rats, and the duration of the EPSCs became progressively shorter, in parallel with the developmental reduction in synaptic plasticity. This decrease in NMDA receptor-mediated EPSC duration is delayed when the animals are reared in the dark, a condition that prolongs developmental plasticity, and is prevented by treatment with tetrodotoxin, a procedure that inhibits neural activity. Application of L-glutamate to outside-out patches excised from layer IV neurons of young, but not of adult, rats activated prolonged bursts of NMDA channel openings. A modification of the NMDA receptor gating properties may therefore account for the age-dependent decline of visual cortical plasticity. | US |
Bacterial transport proteins mediate passive and active transport of small solutes across membranes. Comparison of amino acid sequences shows strong conservation not only among bacterial transporters, but also between them and many transporters of animal cells; thus the study of bacterial transporters is expected to contribute to our understanding of transporters in more complex cells. During the last few years, structures of three bacterial outer membrane transporters were solved by x-ray crystallography. Much progress has also occurred in the biochemical and molecular genetic studies of transporters in the cytoplasmic membranes of bacteria, and a unifying design among membrane transporters is gradually emerging. Common structural motives and evolutionary origins among transporters with diverse energy-coupling mechanisms suggest that many transporters contain a central module forming a transmembrane channel through which the solute may pass. Energy-coupling mechanisms can be viewed as secondary features added on to these fundamental translocation units. | US |
Fibronectin type III domains are found in many different proteins including cell surface receptors and cell adhesion molecules. The crystal structure of one such domain from the extracellular matrix protein tenascin was determined. The structure was solved by multiwavelength anomalous diffraction (MAD) phasing of the selenomethionyl protein and has been refined to 1.8 angstrom resolution. The folding topology of this domain is identical to that of the extracellular domains of the human growth hormone receptor, the second domain of CD4, and PapD. Although distinct, this topology is similar to that of immunoglobulin constant domains. An Arg-Gly-Asp (RGD) sequence that can function for cell adhesion is found in a tight turn on an exposed loop. | US |
Two DNA strand transfer reactions occur during retroviral reverse transcription. The mechanism of the first, minus strand strong-stop DNA, transfer has been studied in vitro with human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT) and a model template-primer system derived from the HIV-1 genome. The results reveal that HIV-1 RT alone can catalyze DNA strand transfer reactions. Two kinetically distinct ribonuclease (RNase) H activities associated with HIV-1 RT are required for removal of RNA fragments annealed to the nascent DNA strand. Examination of the binding of DNA.RNA duplex and single-stranded RNA to HIV-1 RT during strand transfer supports a model where the enzyme accommodates both the acceptor RNA template and the nascent DNA strand before the transfer event is completed. The polymerase activity incorporated additional bases beyond the 5' end of the RNA template, resulting in a base misincorporation upon DNA strand transfer. Such a process occurring in vivo during retroviral homologous recombination could contribute to the hypermutability of the HIV-1 genome. | US |
The overall sequence similarity between the voltage-activated K+ channels and cyclic nucleotide-gated ion channels from retinal and olfactory neurons suggests that they arose from a common ancestor. On the basis of sequence comparisons, mutations were introduced into the pore of a voltage-activated K+ channel. These mutations confer the essential features of ion conduction in the cyclic nucleotide-gated ion channels; the mutant K+ channels display little selectivity among monovalent cations and are blocked by divalent cations. The property of K+ selectivity is related to the presence of two amino acids that are absent from the pore-forming region of the cyclic nucleotide-gated channels. These data demonstrate that very small differences in the primary structure of an ion channel can account for extreme functional diversity, and they suggest a possible connection between the pore-forming regions of K+, Ca2+, and cyclic nucleotide-gated ion channels. | US |
Studies of a series of short oligonucleotide double and triple helices containing either all RNA, all DNA, or a mixture of the two show strand-dependent variation in their stability and structure. The variation in stability for both groups falls over a range of greater than 10 kilocalories per mole. In forming the triple helix, RNA is favored on both pyrimidine strands, whereas DNA is favored on the purine strand. In general, relatively unstable duplexes form particularly stable triplexes and vice versa. Structural data indicate that the strands in hybrid helices adopt a conformation that is intermediate between molecules containing all DNA and all RNA. Thus, RNA-DNA hybrids were not forced into the conformation of the RNA (A-form). The provocative stability of the triplex with an RNA third strand+DNA duplex points to novel antisense strategies and opens the possibility of an in vivo role of these structures. Overall, the data emphasize the fundamental role of sugars in determining the properties of nucleic acid complexes. | US |
The cystic fibrosis gene product (CFTR) is a complex protein that functions as an adenosine 3,5-monophosphate (cAMP)-stimulated ion channel and possibly as a regulator of intracellular processes. In order to determine whether the CFTR molecule contains a functional aqueous pathway, anion, water, and urea transport were measured in Xenopus oocytes expressing CFTR. Cyclic AMP agonists induced a Cl- conductance of 94 microsiemens and an increase in water permeability of 4 x 10(-4) centimeter per second that was inhibited by a Cl- channel blocker and was dependent on anion composition. CFTR has a calculated single channel water conductance of 9 x 10(-13) cubic centimeter per second, suggesting a pore-like aqueous pathway. Oocytes expressing CFTR also showed cAMP-stimulated transport of urea but not the larger solute sucrose. Thus CFTR contains a cAMP-stimulated aqueous pore that can transport anions, water, and small solutes. The results also provide functional evidence for water movement through an ion channel. | US |
Experimental autoimmune encephalomyelitis (EAE), a demyelinating disease of the central nervous system that can be induced in susceptible strains of mice by immunization with myelin basic protein (MBP) or its immunodominant T cell determinants, serves as a model of human multiple sclerosis. Tolerance to MBP in adult mice was induced by intraperitoneal injection of synthetic peptides of immunodominant determinants of MBP and prevented MBP-induced EAE. Furthermore, tolerance-inducing regimens of peptides administered to mice after the disease had begun (10 days after induction with MBP) blocked the progression and decreased the severity of EAE. Peptide-induced tolerance resulted from the induction of anergy in proliferative, antigen-specific T cells. | US |
Synaptic events at the neuromuscular junction are integer multiples of a quantum, the postsynaptic response to transmitter released from one presynaptic vesicle. At central synapses where quanta are small, it has been suggested they are invariant due to occupation of all postsynaptic receptors, a concept neglecting inherent fluctuations in channel behavior. If this did occur, the quantal release model would not apply there and could not be used to localize sites of synaptic modification. Monte Carlo simulations of quanta include transmitter diffusion and interactions with postsynaptic receptors that are treated probabilistically. These models suggest that when there are few postsynaptic channels available at a synapse, their stochastic behavior produces significant intrinsic variance in response amplitude and kinetics, and saturation does not occur. These results were confirmed by analysis of inhibitory quanta in embryonic and adult Mauthner cells involving a small and large number of channels, respectively. The findings apply to excitatory synapses as well. | US |
Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis. | US |
Serotonin, via 5-HT2 receptors, exerts an excitatory effect on CA1 neurons and may play a role in ischemia-induced excitotoxic damage. To evaluate the role of serotonin in ischemia, both neurochemical and histopathological studies were performed. | US |
Sulfated glycoprotein 2 (SGP-2) from rat, and similar molecules from cow, dog, human, pig, ram and quail are known by 11 or more acronyms. SGP-2 is associated with the responses of brain and other tissues to injury; it and related molecules are also normally secreted by the adrenal gland, the liver and the testes. The mRNA of this protein is found in increased levels in Alzheimer's disease. In rats, after perforant path or excitotoxin lesions, levels of the protein or mRNA are elevated in astrocytes, and also in neurons. In rats, brain SGP-2 is regulated by gonadal and adrenal steroids. However, these increases after brain lesions may relate to a function that is associated with the human protein, namely that of inhibiting complement-mediated cell lysis. Other activities suggested for SGP-2 are lipid transport and cell-cell interactions, which are consistent with sequence data that predict binding of dinucleotides, heparin and lipids. The emerging neurobiology of SGP-2 encompasses the subjects of cell death, synaptic remodelling, neuroendocrinology and neurodegenerative diseases. | US |
Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles' medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, alpha 1-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was alpha-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. gamma-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed. | US |
A number of observations support molecular mimicry as a possible pathogenetic mechanism in diseases such as acute rheumatic fever, reactive arthritis after enteric infection or associated with Reiter's syndrome, myasthenia gravis, or even in rheumatoid arthritis. Molecular mimicry can be defined as a sharing of epitopes in linear or 3-dimensional presentation on disparate proteins from entirely different sources--for instance, group A streptococcal membranes and human cardiac myosin. How exposure to or infection with organisms sharing molecular similarity with antigens of the human host can evade tolerance and actually induce a self-reacting humoral or cellular immune response is still not clear; however, a large body of evidence has now been accumulated that documents apparent molecular mimicry mechanisms in these disorders. In some diseases, the molecular mimicry appears to involve human target organs and specific components of the infectious organism, whereas in others the host HLA cell surface molecules appear to share antigens with presumed bacterial or viral initiators of disease. | US |
We have previously shown that human piebaldism results from mutations of the KIT gene, which encodes the receptor for the mast/stem cell growth factor and is located in chromosome segment 4q12. Using DNA of a patient with piebaldism, mental retardation, and multiple congenital anomalies associated with a 46,XY,del(4) (q12q21.1) karyotype, we carried out quantitative Southern blot hybridization analyses of the KIT gene and the adjacent PDGFRA (platelet-derived growth factor receptor alpha subunit) genes. The patient was hemizygous for both the KIT and PDGFRA genes, indicating that both of these genes are included within the deleted region. Therefore, deletion of the KIT and PDGFRA genes may account for the piebald phenotype in this patient. | US |
This study sought to determine whether cyclooxygenase products modulate angiotensin II-induced contractions of human placental arteries. | US |
AIDS encephalitis is a common sequela to HIV-1 infection in humans and simian immunodeficiency virus (SIVmac) infection in macaques. Although lentiviral-infected macrophages comprise parenchymal inflammatory infiltrates in affected brain tissue, the mechanisms responsible for leukocyte trafficking to the central nervous system in AIDS are unknown. In this study, we investigated the expression of various endothelial-derived leukocyte adhesion proteins in SIVmac-induced AIDS encephalitis. Encephalitic brains from SIVmac-infected macaques, but not uninflamed brains from other SIVmac-infected animals, were found to express abundant vascular cell adhesion molecule-1 (VCAM-1) protein on the majority of arteriolar, venular, and capillary endothelial cells. Soluble VCAM-1 concentrations in cerebrospinal fluid (CSF) from encephalitic animals were increased approximately 20-fold above those from animals without AIDS encephalitis. Expression of other endothelial-related adhesion molecules, including E-selectin, P-selectin, and intercellular adhesion molecule-1 (ICAM-1), was not uniformly associated with AIDS encephalitis. Thus, the presence of VCAM-1 in both brain and CSF was uniformly associated with SIVmac-induced disease of the central nervous system, and this expression may, at least in part, influence monocyte and lymphocyte recruitment to the central nervous system during the development of AIDS encephalitis. Moreover, measurement of soluble VCAM-1 in CSF may assist in the clinical assessment of animals or people with AIDS. | US |
This study sought to characterize the action of neurokinin B (NKB) and senktide, a selective synthetic agonist for NK3 receptors, on the myenteric plexus of the guinea pig small intestine. Both peptides stimulated a dose-dependent release of [3H]-acetylcholine (ACh). The mean effective dose values were 1 x 10(-9) for NKB and 3 x 10(-11) M for senktide. The action of these two neurokinins was blocked by the removal of Ca2+ and was sensitive to tetrodotoxin. The release of [3H]ACh was antagonized by omega-conotoxin GVIA, implying the involvement of N-type voltage-sensitive calcium channels. Senktide-evoked ACh release was also insensitive to nifedipine or flunarizine but was blocked by diltiazem. Treatment with protein kinase C (PKC) inhibitors (H-7 and polymyxin B) or activators (12-tetradecanoylphorbol 13-acetate and SC-9) failed to alter the NK3 receptor-mediated ACh output. Our data did not support an action mediated via PKC upon the activation of NK3 receptors on myenteric cholinergic neurons. | US |
The objective of this study was to determine whether substance P and calcitonin gene-related peptide (CGRP), at physiologically relevant concentrations, affect leukocyte-endothelial cell adhesion. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated (40 min) with freshly isolated human neutrophils in the presence or absence of substance P or CGRP (10(-11) M). Both substance P and CGRP caused a significant increase (2-fold) in neutrophil adherence to HUVEC. Monoclonal antibodies (MAb) directed against the leukocyte adhesion glycoproteins CD11/CD18 (MAb IB4) and L-selectin (MAb DREG56) did not attenuate substance P-induced adhesion. Antibodies directed against the endothelial cell adhesion molecules E-selectin (MAb CL2) and ICAM-1 (MAb R6.5) were also without effect on substance P-induced neutrophil adhesion. Similar results were obtained when either MAb IB4, DREG56, CL2, or R6.5 was coincubated with CGRP-stimulated neutrophils and endothelial cells. Phorbol 12-myristate 13-acetate-stimulated neutrophil adherence was significantly attenuated by MAb IB4, indicating that CD11/CD18 participates in this adhesion process. The results of this study indicate that 1) the neuropeptides substance P and CGRP promote neutrophil adherence to venular endothelium and 2) the neuropeptide-induced adhesion is not mediated by the adhesion molecules CD11/CD18, L-selectin, E-selectin, or ICAM-1. | US |
We have recently isolated cDNAs encoding a Na(+)-H+ exchanger isoform, referred to as NHE-1, from rabbit kidney and LLC-PK1 cells. To identify the NHE-1 protein and to establish its cellular and subcellular localization in the rabbit kidney, we prepared antibodies to a NHE-1 fusion protein. cDNA encoding the COOH-terminal 41 amino acids of NHE-1 was subcloned into a maltose-binding protein vector and the purified fusion protein (FP347A) used to immunize guinea pigs. To identify the NHE-1 protein, we performed Western blot analysis against membrane fractions prepared from rabbit renal cortex. Anti-FP347A antibody specifically reacted with a polypeptide with an apparent molecular mass of 100-110 kDa that was enriched in basolateral membrane fractions. When indirect immunofluorescence was performed on semithin (0.5 micron) cryosections of paraformaldehyde-lysine-periodate-fixed rabbit kidney, anti-FP347A specifically stained the basolateral plasma membrane of cells of the proximal tubule, thick ascending limb, and distal convoluted tubule. Anti-FP347A similarly stained connecting tubule cells and principal cells. No staining was detected on the apical membrane of any cells of the rabbit nephron. We conclude that NHE-1 is a 100- to 110-kDa protein expressed on the basolateral membrane of multiple nephron segments. | US |
Tumor necrosis factor-alpha (TNF) causes a spontaneously reversible increase in tight junction permeability. TNF was the only cytokine tested that produced this effect. The effect on transepithelial permeability proceeds in four distinct phases: 1) a 60- to 90-min delay from time of application of TNF, 2) a rapid decrease in transepithelial resistance, 3) a recovery of transepithelial resistance to control level within 1 h, and 4) a further increase of transepithelial resistance above control levels. The recovery of transepithelial resistance occurs with or without TNF in the culture medium. Different protein kinase inhibitors affected different phases of this overall process. The tyrosine kinase inhibitor genistein significantly blocked the TNF effect. Neither transcription nor protein synthesis was required for transepithelial permeability to increase, but were required for the recovery. After the tight junctions have opened at 2 h in response to TNF, a second application of TNF will not produce the effect again for at least 12 h. The tight junctions will, however, open in response to phorbol esters during this time frame. Electron microscopy studies using apically applied ruthenium red suggest that TNF action results in < 10% of the junctions having increased permeability at any given time during the resistance decrease. The role of epithelial barrier permeability changes in TNF action in vivo is discussed. | US |
Rat ventricular cardiac muscle has previously been shown to contain exceptionally high levels of preproenkephalin mRNA (ppEnk mRNA). We have recently determined that the level of ppEnk mRNA is developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and in cultured myocytes (J. P. Springhorn and W. C. Claycomb. Biochem. J. 258: 73-77, 1989). We demonstrate in the current study that heart ppEnk mRNA is structurally identical at the 5' end to brain ppEnk mRNA using a ribonuclease protection assay and that heart ppEnk mRNA can be translated in vitro using a rabbit reticulocyte lysate system. In vitro synthesized preproenkephalin peptides were immunoprecipitated with a polyclonal antibody directed to the carboxy-terminal seven amino acids of preproenkephalin. We have also established by radioimmunoassay that enkephalin-containing peptides are secreted from cultured neonatal and adult rat ventricular cardiac muscle cells. This secretion is linear with respect to time and can be stimulated by phorbol 12-myristate 13-acetate (PMA) and adenosine 3',5'-cyclic monophosphate (cAMP). It was determined by column chromatography that cAMP induced neonatal rat ventricular cardiac muscle cells to secrete Met5-enkephalin-Arg6-Phe7, whereas PMA plus 3-isobutyl-1-methylxanthine induced adult rat ventricular cardiac muscle cells to secrete Met5-enkephalin. These studies establish that ventricular heart muscle ppEnk mRNA can be translated and that enkephalin peptides are secreted from ventricular cardiac muscle cells. | US |
We used cell pairs electrically coupled with relatively high intercellular resistance to investigate the involvement of calcium current in the origin of the source current during the conduction process of the action potential (AP). Three interventions were used to reduce the calcium current: a specific calcium channel blocker [nifedipine (NIF)], premature stimulation, and increments in the frequency of stimulation of the cell. The ionic membrane current (Iion) after the peak of the AP of the stimulated cell was positive and small when the cell was uncoupled. However, when the stimulated cell was coupled to a cell model or to another cell, Iion during this period became negative and large to supply the coupling current. A rapid early repolarization of the AP occurred in the stimulated cell because of the removal of charge from the stimulated cell. NIF decreased the magnitude of the net negative Iion during this period and caused a more rapid early repolarization in the stimulated cell. NIF increased the delay between the activations of two coupled cells at a given coupling resistance (Rc) but decreased the longest delay that could be produced without conduction failure for a given cell pair. The highest Rc below which conduction of AP occurred was also decreased by NIF. Premature stimulation and an increase of the stimulation frequency also caused an increase in the extent of the early repolarization and increased the delay between two cell activations at a given Rc. Conduction block occurred with sufficient prematurity or at a sufficiently high frequency of stimulation even though activation of the stimulated cell occurred for each stimulus. The Iion that flows during the early plateau phase of the AP in the stimulated cell became negative and significantly large by coupling two cardiac cells together. This current flow is a major component needed to supply the coupling current through the intercellular resistance. The decrease of calcium current caused a decrease in the magnitude of this net inward ionic current, resulting in an increase of the rate of early repolarization and an increase in the conduction delay between two cells at a given Rc. These results suggest the involvement of calcium current in the conduction process when cells are coupled at relatively high Rc. | US |
The present study examines whether in vivo administration of granulocyte colony-stimulating factor (G-CSF) and the resultant neutrophilia alters basal glucose metabolism or modulates the glucose metabolic response to a subsequent endotoxin [lipopolysaccharide (LPS)] challenge. Rats were injected with human recombinant G-CSF (50 micrograms/kg sc) twice daily for 2 days preceding an injection of LPS. Animals treated with G-CSF showed an eightfold increase in blood polymorphonuclear leukocytes (PMNs) but no detectable changes in hemodynamics or glucose metabolism. In control animals, LPS transiently decreased circulating PMN number, but by 4 h neutrophils had returned to control levels. LPS produced a greater reduction in circulating neutrophils in G-CSF-treated animals, which did not return to pretreatment levels by 4 h. G-CSF also produced marked changes in the glucose metabolic response to LPS. Rates of whole body glucose production and utilization in both control and G-CSF-treated rats were rapidly increased by LPS; however, the increment in glucose flux was 55-100% greater in the latter group. The enhanced rate of hepatic glucose production in this group occurred despite lower plasma levels of lactate and glucagon. The elevated rate of whole body glucose utilization was attributable to the G-CSF-enhanced LPS-induced increase in glucose uptake by the ileum, spleen, liver, and lung. Furthermore, LPS increased glucose uptake by skeletal muscle in G-CSF-treated rats but not in control animals. The enhanced glucose disposal in G-CSF-treated rats was not mediated by increases in plasma glucose or insulin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) | US |
An analysis of Plasmodium falciparum antigen-specific antibodies was performed on 44 serum samples from Guatemala, a region endemic for P. vivax. Most sera showed positive reactivity to P. falciparum asexual stage antigens by indirect immunofluorescent assay, enzyme-linked immunosorbent assay, and immunoprecipitation analysis using biosynthetically labeled parasites. Although several antigens were recognized by these sera, proteins with molecular weights of 195 kD, 140 kD, and in the range of 70-80 kD were strongly recognized by many of the sera studied. No such reactivity was observed for any of the surface antigens in the male and female gametes and zygotes of P. falciparum. These studies suggest that P. vivax and P. falciparum, two major human parasites, may share certain epitopes in several antigens of immunologic significance. | US |
A simple, rapid method of epitope mapping has been developed that avoids the often cumbersome requirement of obtaining amino acid sequence information. The protein antigen is digested with various concentrations of carboxypeptidase into a nearly continuous series of polypeptides of different molecular weights, all containing a common N-terminus. The peptides are separated by polyacrylamide gel electrophoresis and then transferred to nitrocellulose paper. After developing the blot with the antibody to be mapped, a nearly continuous stain is observed extending from the position of the intact antigen to the molecular weight of the smallest N-terminal fragment still containing the antibody's epitope. By noting the molecular weight where the stain terminates, the position of the epitope relative to the N-terminus can be determined. Using this methodology, and taking special precautions to inhibit all endoproteinases in the carboxypeptidase preparation, the previously mapped epitopes of six nonoverlapping antibodies to the erythrocyte anion transporter were confirmed. | US |
We have investigated the complex formation between an immobilized monoclonal antibody and antigens that differ in size about 50-fold. As a model system, we used an iodinated progesterone derivative and a progesterone-horseradish peroxidase conjugate as tracer and a monoclonal antibody as binding protein. The antibody was immobilized by four different methods: physical adsorption, chemical binding, and binding via protein G in the absence or presence of a protective protein (gelatin). These investigations have shown that the performance of competitive immunoassays is determined by a combination of factors: (a) the relative size of the analyte and the tracer, (b) the antibody density on the solid matrix, (c) the method of immobilization of the antibody, and (d) the binding constants between antibody-analyte and antibody-tracer. All of these interactions have to be considered in designing an optimal immunoassay. The smaller antigen can form a 3- to 35-fold higher maximal complex density than the larger antigen. Dose-response curves are less affected by the size of the tracer than by the binding constant with the antibody. A large enzyme tracer with a relatively low binding constant can, therefore, provide a more sensitive assay. On the other hand, the increase in complex density achieved with a smaller tracer yields a higher signal that in turn can provide a better signal-to-noise ratio in highly sensitive competitive solid-phase immunoassays. We have suggested a model for antibody immobilization that accounts for the interdependence of tracer size, complex formation, and antibody density. The methods described can be used to design and optimize immunoassays of predefined performance characteristics. The results are particularly useful for converting radioimmunoassays to enzyme immunoassays. | US |
A highly sensitive analytical procedure was developed to assess the site of glycosylation in a model glycoprotein, bovine fetuin. First, sample cleavage with immobilized trypsin and the peptide map development are accomplished by microcolumn liquid chromatography. Second, the sialic acids content is measured fluorometrically using their precolumn conversion to quinoxaline derivatives. A unique preconcentration system was developed to secure sensitivity of the second measurement. | US |
A solid-phase assay for the activity of CMPNeuAc:Gal beta 1-4GlcNAc-R alpha-2,6-sialyltransferase (2,6ST) has been developed. In the assay an acceptor glycoprotein is immobilized onto microtiter plate wells. The two glycoprotein acceptors used were asialofetuin (ASF), which contains oligosaccharides terminating in the sequence Gal beta 1-4GlcNAc-R, and a neoglycoprotein of bovine serum albumin containing covalently attached Gal beta 1-4GlcNAc-R units. Samples containing the donor CMPNeuAc and the 2,6ST were incubated with the immobilized acceptor to generate the product NeuAc alpha 2-6Gal beta 1-4GlcNAc-R. The product was detected by a biotin-streptavidin system using the biotinylated plant lectin Sambucus nigra agglutinin (SNA), which binds to sialic acid in alpha-2,6, but not in alpha-2,3, linkage. The biotinylated SNA bound to the product was then detected with streptavidin and biotinylated forms of either alkaline phosphatase or the recombinant bioluminescent protein aequorin. The assay was optimized with respect to the commercially available 2,6ST and shown to be dependent on the concentration of acceptor and CMPNeuAc and proportional to the 2,6ST activity in the range of 20 to 400 microU in a 1-h assay. The solid-phase assay also allows for the selective detection of 2,6ST activity in human and fetal bovine serum, where the activity was proportional in the range of 0.1 to 2 microliters of serum. | US |
The clastogenicity of lobeline and possible interactions between lobeline and ethyl alcohol were investigated in a mutagen-sensitivity assay on cultures of human lymphoblastoid cell lines. Lobeline (10(-6) M) and ethyl alcohol (1% and 2%) were added to the cultures alone or in different combinations prior to or simultaneously with bleomycin treatment. The number of chromatid breaks per cell served as the indicator of clastogenicity. Lobeline alone was not clastogenic, but there was a marked increase in genetic damage resulting from a coclastogenic interaction between lobeline and ethyl alcohol. These data emphasize the significance of interactions between compounds that may increase genotoxicity in cells exposed to mutagenic environments. | US |
The most common mechanism of antibiotic resistance in multiply resistant Pseudomonas cepacia is decreased porin-mediated outer membrane permeability. In some gram-negative organisms this form of antibiotic resistance can be induced by growth in the presence of weak acids, such as salicylates, which suppress porin synthesis. To determine the effects of salicylates on outer membrane permeability of P. cepacia, a susceptible laboratory strain, 249-2, was grown in 10 mM sodium salicylate. Antibiotic susceptibility and uptake, as well as outer membrane protein patterns, were compared between strain 249-2 grown with and without salicylates. The MICs of chloramphenicol, trimethoprim, ciprofloxacin, and ceftazidime were compared between organisms grown in standard and salicylate-containing medium and are as follows: chloramphenicol, 12.5 versus 100 micrograms/ml; trimethoprim, 0.78 versus 3.125 micrograms/ml; ciprofloxacin, 0.4 versus 1.56 micrograms/ml; ceftazidime, 3.125 versus 3.125 micrograms/ml. The permeability of beta-lactam antibiotics was calculated from the rate of hydrolysis of the chromogenic cephalosporin, PADAC. There was no significant difference between strains grown in the presence and absence of salicylate. By using high-pressure liquid chromatography quantitation of loss from culture medium, the effect of 10 mM salicylate on the cellular permeability of chloramphenicol was measured in strain 249-2 by introduction of a plasmid which encodes production of chloramphenicol acetyltransferase. After 1 h of incubation, 18.5% +/- 1.54% versus 70.1% +/- 3.52%, and after 2 h, 4.20% +/- 1.65% versus 41.90% +/- 2.16% remained in supernatants from organisms grown in the absence and presence of 10 mM salicylate, respectively. Outer membrane protein pattern analysis demonstrated the absence of a protein of apparent molecular weight of 40,000 when strain 249-2 was grown in the presence of 10 mM salicylate. To determine whether this protein functioned as a porin, reconstituted membrane vesicles were constructed to assess antibiotic permeability. Vesicles constructed with this salicylate-suppressible outer membrane protein (OpcS) were permeable to chloramphenicol but not to penicillin G. These findings suggest that OpcS is a selective, antibiotic-permeable porin which can be suppressed by growth in the presence of salicylate. Further investigation will be required to determine the biochemical effects of salicylate on porin synthesis. | US |
The polymerase chain reaction (PCR) is used widely to recover rRNA genes from naturally occurring communities for analysis of population constituents. We have found that this method can result in differential amplification of different rRNA genes. In particular, rDNAs of extremely thermophilic archaebacteria often cannot be amplified by the usual PCR methods. The addition of 5% (wt/vol) acetamide to a PCR mixture containing both archaebacterial and yeast DNA templates minimized nonspecific annealing of the primers and prevented preferential amplification of the yeast small-subunit rRNA genes. | US |
The serum-type mannose-binding protein (MBP) is a defense molecule that has carbohydrate-dependent bactericidal effects. It shares with mammalian and chicken hepatic lectins similarity in the primary structure of the carbohydrate-recognition domain, as well as the ligand-binding mode: a high affinity (KD approximately nM) is generated by clustering of approximately 30 terminal target sugar residues on a macromolecule, such as bovine serum albumin, although the individual monosaccharides have low affinity (KD 0.1-1 mM). On the other hand, MBP does not manifest any significant affinity enhancement toward small, di- and trivalent ligands, in contrast to the hepatic lectins whose affinity toward divalent ligands of comparable structures increased from 100- to 1000-fold. Such differences may be explained on the basis of different subunit organization between the hepatic lectins and MBP. | US |
Previously, we demonstrated that the Heymann nephritis autoantigen, gp330, can serve as a receptor site for plasminogen. This binding was not significantly inhibited by the lysine analogue epsilon-amino caproic acid (EACA), indicating that plasminogen binding was not just through lysine binding sites as suggested for other plasminogen binding sites. We now report that once plasminogen is bound to gp330, it can be converted to its active form of plasmin by urokinase. This conversion of plasminogen to plasmin proceeds at a faster rate when plasminogen is first prebound to gp330. Although there is a proportional increase in the Vmax of the urokinase-catalyzed reaction with increasing gp330 concentrations, no change in Km was observed. Once activated, plasmin remains bound to gp330 in an active state capable of cleaving the chromogenic tripeptide, S-2251. The binding of plasmin to gp330 did not significantly change its enzymatic activity; however, gp330 did have a stabilizing effect on plasmin activity at 37 degrees C. While bound to gp330, plasmin is protected from inactivation by its natural inhibitor alpha 2-antiplasmin. The binding of plasmin to gp330 as analyzed by ELISA was shown to be time dependent, reversible, saturable, and specific for gp330. Inhibition of binding of both plasminogen and plasmin to gp330 by benzamidine was similar, although EACA inhibited the binding of plasmin to gp330 slightly more than the binding of plasminogen to gp330. These results indicate that the binding of plasminogen to gp330 serves as an effective means of increasing the rate of plasmin production on the glomerular and tubular epithelial cell surface while protecting the active plasmin from natural inhibitors. | US |
Hepatitis C virus (HCV) is the principal cause of nonenteric non-A, non-B hepatitis worldwide. While it has been well documented that people with developmental disabilities are at an increased risk for infections with hepatitis B virus, little is known of the prevalence of HCV infection among this population. | US |
Antibodies to the proliferating cell nuclear antigen allow identification of proliferating cells in fresh tissue specimens using routine immunocytochemical methods. However, the use of such proliferation markers has not been verified for autopsy-derived tissue specimens, in which there is often a significant delay between the time of death and tissue specimen fixation. To assess the reliability of anti-proliferating cell nuclear antigen antibodies to identify proliferating cells in autopsy tissue specimens, an autopsy simulation was performed using fresh monkey and rat tissue specimens. These tissue specimens were kept at room temperature for predetermined numbers of hours before fixation. The proliferation specific staining was most reliable for tissue specimens obtained within 6 hours of death. There was reliable staining of proliferating regions up to 12 hours, although sensitivity was decreased. The only exception was skin, which was able to withstand much longer periods. Quantitative data from monkey spleen white-pulp regions showed 63% of the cells to stain for proliferating cell nuclear antigen when fixed immediately; this decreased to 29% of the cells after 12 hours and only 19% by 18 hours of postmortem simulation. Representative tissue specimens obtained from human autopsy material revealed similar postmortem staining patterns. Rapid procurement and fixation of tissue specimens and the use of control tissue specimens derived from the same autopsy material (eg, lymph node tissue) are recommended. These studies do suggest that anti-proliferating cell nuclear antigen antibodies can be used to identify proliferating cells in human autopsy tissue specimens obtained within approximately 12 hours of death, with some compromise in overall sensitivity. | US |
Circumstantial evidence suggests that gallstone-induced pancreatitis is triggered by obstruction of the pancreatic duct. In this report I will review the results of studies conducted during the last decade that have employed the diet-induced, secretagogue-induced, and duct obstruction-induced models of experimental pancreatitis to investigate the early events that lead to the development of acute pancreatitis. In each of these models, digestive enzyme zymogens and the lysosomal hydrolase cathepsin B were found to become colocalized. These observations have led to the hypothesis that intra-acinar cell activation of digestive enzyme zymogens by lysosomal hydrolases may be an important critical event in the development of acute pancreatitis. Recent morphologic studies evaluating the initial 24 hours after ligation of the opossum pancreatic duct indicate that the earliest lesions in this model of hemorrhagic pancreatitis occur in acinar cells. | US |
The role of protein kinase A (PKA) in the release of amylase from permeabilized pancreatic acini was investigated. Addition of cyclic AMP (cAMP) to permeabilized acini resulted in a potentiation of Ca(2+)-dependent amylase release, shifting the Ca2+ dose/response curve leftwards. As with protein kinase C (PKC) activation, this is due to an increase in the time of active discharge. The effect of cAMP was shown to be blocked by two inhibitors of PKA, H89 and the PKI-(5-24)-peptide. At low concentration, cAMP synergizes from phorbol 12-myristate 13-acetate (PMA), while at optimal concentrations cAMP and PMA are additive. PKA and PKC appear to work via similar, but not identical mechanisms. | US |
Mitogen-activated protein (MAP) kinases are regarded as switch kinases in the phosphorylation cascade initiated by various agonists. We have investigated whether endothelins (ET), which are constrictor and mitogenic isopeptides, can increase MAP kinase activity in rat mesangial cells, using bovine myelin basic protein (MBP) as a substrate for an in vitro kinase assay. Treatment of quiescent mesangial cells with ET-1 rapidly stimulated a kinase activity which phosphorylated exogenous MBP. This stimulation was dose-dependent, with threshold responses at 1 nM-ET-1. Epidermal growth factor and thrombin also activated this kinase in mesangial cells. We also examined the ET signal transduction pathways leading to activation of MBP kinase. Pertussis toxin had no effect on ET-stimulated MBP kinase activity. Stimulation of protein kinase C by phorbol ester increased MBP kinase activity, and down-regulation of PKC partially inhibited ET-stimulated MBP kinase as well as phorbol ester-stimulated MBP kinase activity. Interestingly, genestein, an inhibitor of protein tyrosine kinases, partially inhibited MBP kinase stimulated by ET but not by phorbol esters. These results suggest that ET stimulates MBP kinase activity in rat mesangial cells via at least two pathways: one which is protein kinase C-dependent and a second one that involves a protein tyrosine kinase. Finally, by raising rabbit antibodies against the two forms of MAP kinase, p44mapk and p42mapk, we demonstrated that both isoforms are expressed in mesangial cells. Antibody alpha 1 Cp42 specifically immunoprecipitated p42mapk and allowed us to demonstrate that ET stimulates MBP kinase activity in the p42mapk immunocomplex. In conclusion, we have provided evidence that, in rat mesangial cells, MAP kinases are rapidly activated by ET-1, a regulatory process that involves at least protein kinase C activation and also a contribution of a tyrosine kinase not yet characterized. | US |
The poly(A)(+)-binding protein (PABP) is a highly conserved protein that binds to the poly(A)+ tail of mRNAs. PABP has been shown to regulate message stability and translational efficiency, yet the mechanisms remain unknown. To facilitate further dissection of the functions of this protein, we have expressed and purified Xenopus PABP using a baculovirus expression system. At 48 h after infection of insect Spodoptera frugiperda (Sf9) cells with recombinant virus, approx. 3% of cell protein was PABP. Purification of PABP was achieved by affinity chromatography on poly(A)(+)-Sepharose. The purified protein was indistinguishable from Xenopus PABP with respect to its immunoreactivity and electrophoretic mobility. Furthermore, the recombinant PABP was expressed and purified as a functional protein as indicated by its ability to bind to poly(A)(+)-Sepharose and its ability to enhance the translation of adenylated messages in vitro. By comparing protein extracts from various developmental stages of Xenopus embryos with known amounts of purified PABP, we determined the amount of PABP per embryo. This analysis suggested that there is less than one PABP molecule available per PABP-binding site at early stages of development, and only a slight excess of PABP at later stages. | US |
The amounts of NG-methylarginine derivatives in myelin basic protein (MBP) purified from dysmyelinating mutant and different stages of normal myelinating mouse brains have been studied by using h.p.l.c. with a highly sensitive post-column o-phthaldialdehyde derivative-formation method. All three naturally occurring derivatives (NG-monomethylarginine (MeArg), NGN'G-dimethylarginine [Me2(sym)Arg] and NGNG-dimethylarginine [Me2(asym)Arg]) were found in MBP; however, their relative concentrations varied significantly with the age of the animal. The amounts of MeArg and Me2(sym)Arg in MBP increased as a function of the age of the brain, whereas that of Me2(asym)Arg decreased. MBP from early-myelinating mouse brain was shown to contain a high proportion of Me2(asym)Arg, which was hardly detectable in older brain MBP. This derivative, Me2(asym)Arg, was also absent from MBP embedded in the most compact multilamellar myelin, but was present in MBP in the least compact myelin (P3B). Comparing the extent of total methylation in vivo (sum of all three arginine derivatives), MBP extracted from less-compact myelin (P3A and P3B) showed a level approx. 40% higher than that from compact myelin. MBPs isolated from dysmyelinating mutant mouse brains, such as jimpy (jp/y) and quaking (qk/qk), contained a much higher level of Me2(asym)Arg relative to the other two methyl derivatives and also in comparison with those levels in the mother brain MBP. SDS/PAGE analysis of MBPs extracted from the mutant (both jp/y and qk/qk) as well as young normal (6-13 days old) mouse brains indicated the presence of a high-molecular-mass isoform of MBP (about 32 kDa), but this isoform was not found in adult brains. These results therefore indicate that structural integrity of myelin membrane in which MBP is embedded appears to play a pivotal role in determining the extent and the kind of Me2Arg formation in MBP at the post-translational level. | US |
Platelet-derived growth factor (PDGF) AB and BB isoforms were potent mitogens for cultured vascular smooth muscle cells from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). PDGF-AA promotes protein synthesis in a dose-dependent manner in SHR cells, whereas DNA synthesis was stimulated only slightly. However, this isoform did not activate either DNA or protein synthesis in WKY cells. PDGF-AA stimulated tyrosine phosphorylation of its receptor protein and phospholipase C-gamma 1 in SHR cell but not in WKY cells. These results indicate that vascular smooth muscle cell of SHR is uniquely responsive to PDGF-AA, presumably due to abnormality in receptor expression, in its hypertrophic response. | US |
Calcitonin gene-related peptides I and II (CGRP I and II) were found to stimulate cAMP levels by approximately 4-6 fold in human nonpigmented ciliary epithelial cells with half-maximal effective concentrations of 20 x 10(-10) and 3 x 10(-10) M, respectively. Prior exposure of cells to 6 x 10(-7) M phorbol 12-myristate, 13-acetate for 15 min resulted in a 40-50% inhibition of CGRP II-dependent cAMP stimulation. Phorbol didecanoate and dioctanoylglycerol also effectively inhibited, whereas 4 alpha phorbol didecanoate, an ineffective activator of protein kinase C, had no effect. Staurosporine, a protein kinase C inhibitor, blocked the inhibition of cAMP formation by phorbol esters. cAMP stimulation by forskolin or cholera toxin was not inhibited by phorbol esters, suggesting that neither a Gs protein nor adenylyl cyclase is the site of inhibition by protein kinase C. These data therefore suggest that CGRP receptors are required for inhibition of adenylate cyclase by protein kinase C. | US |
The activity of the rat class I alcohol dehydrogenase (ADH) is enriched in certain tissues including the liver, intestine and testis. The tissue-specific expression of the gene encoding ADH in the rat was studied and found to closely correlate with tissue isozymic activity. A factor designated enhancer-site downstream binding protein (EDBP) was recently identified in the rat liver and found to interact with the proximal promoter of the class I ADH gene. The distribution of EDBP in nuclear extracts obtained from various tissues was examined based on its sequence-specific DNA binding property and found to correlate with tissue ADH expression. These findings suggest that EDBP is potentially a positive regulatory factor which is involved in controlling the tissue-specific expression of the ADH gene. | US |
The bradykinin (BK) B2 receptor cDNA was synthesized by rt-PCR and transfected into the Chinese hamster lung fibroblasts, CCL39. The CCL39 do not contain the mRNA for this receptor and do not bind BK. Clones of transfected cells were screened for BK receptor mRNA, binding of BK, and for [Ca2+]i response to BK. The clones showed various levels of receptor mRNA. Scatchard analysis of three clones, B6, B5 and B1, each gave a Kd of approximately 1.0nM while the Bmax for each clone differed at 320, 38.7, and 5.39 fmoles per 10(6) cells respectively. The [Ca2+]i response of the three clones to BK decreased with the receptor number/cell. Thus, levels of mRNA, BK binding and [Ca2+]i response proved proportionally related in the transfected clones. The actions of BK and alpha-thrombin, which has an endogenous receptor in these cells, were assessed in clone B6. BK proved active but also distinct from thrombin. BK at 10nM and thrombin at 2units/ml both effectively increased cytosolic [Ca2+]i. BK at 10nM stimulated PGE2 production three fold over basal, while thrombin only marginally elevated PGE2 levels. Alone, BK stimulated a small increase in 3H-thymidine incorporation into DNA. However, in combination with insulin, BK stimulated DNA synthesis to 76% of thrombin, a potent mitogen in these cells. These results illustrate that the BK-B2 receptor cDNA can be stably transfected into a mammalian cell and can activate transmembrane signalling pathways. | US |
Using FDC-P1 cells stably transfected with a murine erythropoietin receptor cDNA as a model, we recently have shown that erythropoietin (EPO), IL-3 and GM-CSF each induce the rapid phosphorylation of a common cytosolic target, i.e., a M(r) 100,000 phosphoprotein "pp100". Presently, we demonstrate that cytokine-induced phosphorylation of pp100 is primarily at tyrosine residues. This is shown by Western blotting with the anti-phosphotyrosine antibody PY20, and by the resistance of [32P]-pp100 to hydroxide-mediated hydrolysis of phosphates. These data, together with the recent observation by Linnekin et al. that pp100/p97 apparently associates directly with EPO receptors, suggest that pp100 may comprise an immediate common component in the signal transduction pathways of EPO, IL-3, GM-CSF and possibly other type I/II cytokine receptors. Additional analyses suggest that pp100 is distinct from a previously described M(r) 100,000 cytosolic target which is tyrosine phosphorylated in hematopoietic cells upon activation of T-cell receptors. | US |
Protein-tyrosine phosphatases (PTPases) play an essential role in the control of signalling through phosphotyrosine pathways. Since little is known about the regulation of these enzymes, we examined the effect of insulin and phorbol 12-myristate 13-acetate (PMA) treatment of well-differentiated rat hepatoma (Fao) cells on the expression of mRNAs encoding three major PTPase homologs in liver: PTPase1B, an intracellular enzyme with a single conserved PTPase domain, and two tandem-domain, transmembrane PTPases, known as LAR and LRP. Treatment of serum-deprived cells with 100 nM insulin increased the abundance of the 4.3 kb and 1.6 kb mRNAs encoding PTPase1B on Northern analysis by 1.6 and 3.1-fold, respectively (p < or = 0.02). Similarly, exposure to 100 ng/ml PMA increased the 4.3 and 1.6 kb PTPase1B mRNAs by 4.5 and 5.7-fold, respectively (p < or = 0.035). In contrast, treatment with insulin or PMA had no significant effect of the abundance of mRNA encoding either LAR or LRP. PMA appeared to have a transcriptional effect on the PTPase1B gene by a protein kinase C-mediated mechanism. The increase in PTPase1B mRNA expression by insulin and PMA suggests that this PTPase may provide feed-back regulation of signalling through the insulin action pathway as well as a potential link between the action of protein kinase C and the regulation of specific phosphotyrosine residues in cells. | US |
Nuclear localization of fibroblast growth factors (FGF) have been reported by many laboratories. We demonstrate here that FGF-1, the precursor for acidic FGF contains a putative nuclear translocation sequence (NTS) NYKKPKL, which is able to direct the expression of the bacterial beta galactosidase (beta gal) gene to the nucleus of transfected NIH 3T3 cells. However, this NTS is unable to target either FGF-1 itself or a FGF-1-beta gal fusion protein into the nucleus, suggesting that FGF-1 may contain an additional sequence which prevents endogenously expressed FGF-1 from being translocated into the nucleus. Indeed, when FGF-1 was fused to the NTS derived from the yeast histone 2B gene, the chimeric construct also failed to be transported into the nucleus either by itself or as a beta gal fusion protein. Interestingly, when 125I-FGF-1 was used to stimulate quiescent NIH 3T3 cells, a significant amount of internalized 125I-FGF-1 (approximately 10%) was found within the nucleus and the nuclear localization of FGF-1 through the exogenous pathway could be significantly reduced by suramin, an inhibitor of the interaction of FGF-1 with its receptor. These data suggest that while FGF-1 contains a NTS, nuclear translocation requires an exogenous and not an endogenous pathway. | US |
Several recent autopsy reports indicate an increased prevalence of coronary atherosclerosis in ischemic heart disease temporally associated with cocaine abuse. The objective of this study was to conduct a retrospective analysis of sudanophilic lesions in young asymptomatic individuals who abused cocaine. Twenty-six cases (15-34-year-old black males) were examined from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. Sixteen subjects (mean age 25 +/- 1 years) had a positive toxicologic screen for cocaine and/or its major metabolites at autopsy and were confirmed habitual cocaine abusers. The remaining 10 cases (mean age 24 +/- 2 years) were subjects with a negative toxicologic screen at autopsy and no history of illicit drug abuse. Post-mortem blood was collected for lipoprotein analysis and determination of smoking status. The aorta and right coronary arteries were stained with Sudan IV and the degree and extent of sudanophilia was quantitated by image analysis. Multiple linear regression analysis of cocaine, age, smoking status, VLDL+LDL-C/HDL-C ratio and HDL-C as predictor variables of percentage intimal surface involvement, revealed an association between cocaine abuse and the extent of sudanophilia in both the thoracic and abdominal aorta (P = 0.002 and 0.049, respectively). Analysis of risk factors or of cocaine abuse as predictors of sudanophilia did not achieve statistical significance in the right coronary artery. These preliminary results suggest that habitual use of cocaine, through unknown mechanism(s), increases aortic sudanophilia independent of traditional risk factors. | US |
The effects of age and lesion of the cholinergic nucleus basalis magnocellularis (NBm) were assessed behaviorally, morphologically, and biochemically. Groups consisted of rats lesioned 1 month before testing, rats lesioned 13 months before testing, and their respective age-matched controls. Both age and lesion independently induced behavioral deficits in performance on two water maze tasks. The combined effect of these two factors produced behavioral deficits equal to the sum of the individual impairments. NBm lesion produced a 28% decrease in anterior cortical choline acetyltransferase activity and a 20% decrease in synaptophysin immunoreactivity in the neocortex that was stable over a 12-month period. Neither neuritic plaque nor neurofibrillary-tanglelike structures were found in the brains of 18-month-old control rats, nor were they found in NBm-lesioned rats examined 15 months postlesion. There was an age-related decrease in homovanillic acid levels in both control and NBm groups, which suggests a decrease in dopamine turnover. These results show a lack of biochemical and behavioral recovery after NBm lesion and suggest that the effects of age on behavior are independent of NBm-cortical dysfunction. | US |
The orientation of the valine-1 side chain of gramicidin was determined by solid-state 2H NMR using valine-1-deuterated (d8) gramicidin. The peptide was incorporated into DMPC bilayers that were oriented between glass plates. When the plates were oriented with their normal perpendicular to the magnetic field, four quadrupolar splittings were observed of 106, 68, 9.7, and 2.0 kHz. These resonances were assigned to C alpha D, C beta D, and the deuterons of each of the C gamma D3 methyl groups, respectively. The average orientation of the various C-D bonds was calculated with respect to the helix axis. The angle obtained for the C alpha-D resonance was consistent with a single-stranded beta 6.3-helical model for the backbone but not with double-helical models. The angles of the side chain were then fitted to a model for the right-handed beta 6.3-helix. Rotation of the valine-1 side chain yielded a set of torsion angles that matched the angles as determined from the 2H NMR measurements. The corresponding orientation of the valine-1 side chain (chi 1 = -5 degrees) was found to be quite unusual, but it explains well the importance of a branched side chain at position 1 for channel formation and stability. A van der Waals interaction between valine-1 of one monomer and alanine-5 of the other helps to stabilize the gramicidin dimer. | US |
It is important for the understanding of protein kinase action to differentiate between regulation at the enzyme and at the substrate levels. For example, the inhibitors dinitrophenol-tyrosine and tyrphostins act at the enzyme level to inhibit phosphorylation of all substrates by c-Src and v-Src kinases. In contrast, polylysine acts at the substrate level to stimulate Src-mediated phosphorylation of beta-casein but to inhibit phosphorylation of alpha-casein. Here we demonstrate novel enzyme-specific and substrate-specific modulations of Src kinase activity of potential physiological significance. At the enzyme level, we observed that c-Src kinase preferentially phosphorylates alpha-casein, while the v-Src kinase prefers beta-casein. At the substrate level we observed substrate-specific modulation by physiological factors including sphingosine, sphingosine derivatives and the ganglioside GM3. Galactosyl-sphingosine (psychosine) was more effective in stimulating phosphorylation of beta-casein and poly(E1A1Y1) than sphingosine. Glucosyl- and lactosyl-sphingosine were ineffective. Rat was extensively phosphorylated by c-Src in the presence of polylysine, and to a lesser extent in the sphingosine and galactosyl-sphingosine. These unexpected differences point out another potential mechanism for regulation of c-Src and v-Src kinase activities and may help to explain some of the pleotyptic manifestations of protein tyrosine kinase actions. | US |
Resonance Raman (RR) spectra are reported for the [2Fe-2S] Rieske protein from Thermus thermophilus (TRP) and phthalate dioxygenase from Pseudomonas cepacia (PDO) as a function of pH and excitation wavelength. Depolarization ratio measurements are presented for the RR spectra of spinach ferredoxin (SFD), TRP, and PDO at 74 K. By comparison with previously published RR spectra of SFD, we suggest reasonable assignments for the spectra of TRP and PDO. The spectra of PDO exhibit virtually no pH dependence, while significant changes are observed in TRP spectra upon raising the pH from 7.3 to 10.1. One band near 270 cm-1, which consists of components at 266 cm-1 and 274 cm-1, is attributed to Fe(III)-N(His) stretching motions. We suggest that these two components arise from conformers having a protonated-hydrogen-bonded imidazole (266 cm-1) and deprotonated-hydrogen-bonded imidazolate (274 cm-1) coordinated to the Fe/S cluster and that the relative populations of the two species are pH-dependent; a simple structural model is proposed to account for this behavior in the respiratory-type Rieske proteins. In addition, we have identified RR peaks associated with the bridging and terminal sulfur atoms of the Fe-S-N cluster. The RR excitation profiles of peaks associated with these atoms are indistinguishable from each other in TRP (pH 7.3) and PDO and differ greatly from those of [2Fe-2S] ferrodoxins. The profiles are bimodal with maxima near 490 nm and > approx. 550 nm. By contrast, bands associated with the Fe-N stretch show a somewhat different enhancement profile. Upon reduction, RR peaks assigned to Fe-N vibrations are no longer observed, with the resulting spectrum being remarkably similar to that reported for reduced adrenodoxin. This indicates that only modes associated with Fe-S bonds are observed and supports the idea that the reducing electron resides on the iron atom coordinated to the two histidine residues. Taken as a whole, the data are consistent with an St2FeSb2Fe[N(His)]t2 structure for the Rieske-type cluster. | US |
The objective of this study was to evaluate reference sites for recording the middle- and long-latency scalp potentials elicited by painful and non-painful sural nerve stimulation. Somatosensory evoked potentials (SEPs) were recorded from the scalp, the mastoid, the earlobe, the neck, and the wrist. Each site was referenced to the sterno-vertebral (SV) electrode, which is a balanced non-cephalic reference with essentially no ECG contamination. There was little or no activity recorded between the wrist and SV, and the SV was located within a region extending from the rostral neck to the wrist where the potentials were stable over space. Hence, the SV reference is indifferent for the middle- and long-latency potentials evoked by painful and non-painful sural nerve stimulation. There was, however, significant activity recorded from the earlobe and mastoid, sites which are frequently used as references for the SEP. It is important that investigators using these cephalic references to study the middle- and long-latency peaks of the SEP be aware of this activity as it will distort SEPs recorded from single sites and the SEP scalp topography, distortions which could unnecessarily complicate their interpretation. | US |
In contrast to rat granulosa cells (GC), GC of the pig and cow produce very low levels of transforming growth factor-beta (TGF-beta)-like activity in vitro. Because cultured rat GC predominantly express TGF-beta 2 messenger RNA (mRNA) and secrete high levels of the protein, we hypothesized that TGF-beta 2 mRNA expression by porcine GC might be absent or diminished, thus providing a molecular explanation(s) for their relatively low levels of TGF-beta production. We tested this hypothesis by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay analysis. When analyzed by RT-PCR, porcine GC RNA from 1-3 mm follicles did not yield the expected 489 base pair (bp) TGF-beta 2 product but instead generated a smaller than anticipated 240 bp species; porcine testis RNA generated both the 240 and the anticipated 489 bp products. Sequencing these species indicated that the smaller form was not a novel TGF-beta 2 splice variant, and that the 489-bp product was porcine TGF-beta 2. This is the first reported nucleotide sequence for porcine TGF-beta 2; it is 90% and 91% identical to murine and human TGF-beta 2 sequences, respectively. Further RT-PCR analysis of porcine GC RNA resulted in the identification of bp products representing TGF-beta 1 and TGF-beta 3 mRNA. Enzyme-linked immunosorbent assay analysis of porcine GC conditioned medium confirmed the presence of TGF-beta 1 at very low levels. TGF-beta 2 was undetectable. Comparable analysis of GC from the diethylstilbestrol-treated prepubertal rat demonstrated the presence of TGF-beta 1 and TGF-beta 3 mRNA by RT-PCR and very low levels of the corresponding protein products in conditioned culture medium. Collectively, these results suggest that the inability of porcine GC to express TGF-beta 2 mRNA could explain the very low levels of TGF-beta activity secreted by these cells in vitro. | US |
FSH, which stimulates cAMP in the Sertoli cell, markedly lowers the concentration of insulin-like growth factor-binding protein-3 (IGFBP-3) in Sertoli cell-conditioned medium; in contrast, insulin-like growth factor-I (IGF-I) increases BP-3 expression. In this study, the mechanisms controlling the contrasting effects of cAMP and IGF-I were investigated. The abundance of BP-3 mRNA was dramatically lowered by (Bu)2cAMP, but was unaffected by IGF-I. Analyzed by ligand blot of conditioned medium, coincubation of (Bu)2cAMP and IGF-I largely eliminated the increase observed with IGF-I alone. Based on the following evidence, the effect of IGF-I appeared to be solely related to the capacity of IGF-I to interact directly with BP-3. 1) Insulin at micromolar concentrations failed to increase BP-3 abundance despite documentation by affinity cross-linking that insulin displaced [125I]IGF-I from the IGF-I receptor. 2) A synthetic IGF-I analog, [Leu24,1-62]IGF-I, which has reduced binding affinity for rat IGF-I receptor but displays high affinity for rat Sertoli cell-conditioned medium BPs, increased BP-3 abundance. 3) A synthetic IGF-I analog, B-chain mutant, which has reduced affinity for rat Sertoli cell BPs but displays normal affinity for the rat IGF-I receptor, failed to increase BP-3 abundance. 4) Human recombinant glycosylated [125I]BP-3 when added to cultured Sertoli cells was preserved in the medium when IGF-I or analogs with BP-3 affinity were present. 5) IGF-I, in dose-responsive manner, both retarded the disappearance from the medium of exogenously added human recombinant nonglycosylated BP-3 and decreased the amount of membrane-associated BP-3. These results indicate that whereas cAMP lowers BP-3 abundance in medium, most likely by markedly decreasing synthesis, IGF-I increases BP-3 accumulation by retarding its clearance by the Sertoli cell. | US |
The release of GnRH evoked by dopamine (DA) was studied in the GT1 GnRH neuronal cell lines. Superfusion of GT1-1 cells with DA or the D1-dopaminergic agonist SKF 38393, but not with the D2-dopaminergic agonist bromocriptine, increased 2-fold the amplitude of the spontaneous GnRH pulses. Treatment with DA for 30 min also stimulated GnRH release from static cultures of GT1-7 cells. This effect was mimicked by the selective D1-dopaminergic agonist SKF 38393 and blocked by the D1-dopaminergic antagonist SCH 23390. However, the D2-dopaminergic agonist bromocriptine had no effect, and the stimulation of GnRH release by DA was not blocked by the D2-dopaminergic antagonist spiroperidol. In parallel to the stimulation of GnRH release, DA also rapidly increased (first observed at 120 sec) in a dose-dependent fashion, the intracellular concentration of cAMP in isobutylmethylxanthine-pretreated GT1-7 cells. The pharmacological profile of the increase in cAMP was identical to that for GnRH release. The cAMP responses to DA and norepinephrine were lost after long term treatment with SKF 38393, i.e. heterologous desensitization. GT1 cells also express the mRNA for the dopamine- and cAMP-regulated phospho-protein (mol wt, 32,000; DARPP-32) only seen in cells expressing DA D1-receptors. These results demonstrate a direct stimulatory effect of DA on GnRH release via DA D1-receptors positively coupled to adenylate cyclase in GnRH neuronal cell lines. | US |
cAMP is involved in the regulation of secretory activity in lactotrope, thyrotrope, and gonadotrope cells. The present study examined whether pulsatile or intermittent changes in cAMP are more effective than a continuous stimulation in increasing pituitary hormone gene expression. Pituitaries from adult female rats were dissociated, plated for 48 h (7-8 x 10(6) cells per well) to allow attachment to Matrigel-coated plastic coverslips, then inserted into perifusion chambers (five to eight chambers per group). After 24 h of treatment, the cells were recovered, RNA extracted, and messenger RNAs (mRNAs) determined by dot blot hybridization. Perfused cells were exposed to either hourly pulses of monobutyryl cAMP (Bt cAMP, 0.01, 0.1, or 1 mM; 1 mM butyrate pulses to controls), or continuously to forskolin (10 microM). Bt cAMP pulses increased both PRL and alpha-subunit mRNAs, maximal after the 0.1 mM dose for PRL (51% increase vs. butyrate controls) and after the 1 mM dose for alpha (60% increase). However, forskolin was ineffective in increasing PRL or alpha mRNA concentrations. TSH, LH, and FSH beta-subunit mRNAs were not altered by Bt cAMP pulses or forskolin. To confirm the different effects of pulsatile vs. continuous cAMP on PRL and alpha-subunit mRNAs, the response to pulsatile 8-bromo cAMP (1 mM) or Bt cAMP (0.5 mM) was compared to continuous Bt cAMP (0.5 mM). PRL and alpha-subunit mRNAs were increased by both cAMP analogs given in a pulsatile manner but not by continuous Bt cAMP. PRL and LH secretory responses (determined in perifusate samples after 2 h and 22 h of treatment) revealed that both PRL and LH release was increased by cAMP stimulation, given either in a pulsatile or continuous manner. These results show that PRL and alpha-subunit gene expression were sensitive to changes in cAMP stimulation, whereas that of TSH, LH, and FSH beta were unaltered. Only intermittent cAMP stimuli were effective in increasing PRL and alpha mRNAs. These data suggest that pulsatile fluctuations in intracellular cAMP may be essential for maximal expression of the PRL and alpha genes. Thus, intermittent changes in intracellular second messengers may be a necessary part of the pathway involved in the transduction of signals from the plasma membrane to the nucleus. | US |
The inability to detect insulin-like growth factor binding protein-3 (IGFBP-3) in some circumstances by Western ligand blot analysis has emphasized the need to characterize IGFBPs by both ligand binding and immunological techniques. In this study, we have: 1) characterized and quantified IGFBP-3 in nonpregnancy, pregnancy, and fetal cord serum, follicular, peritoneal, and amniotic fluid, seminal plasma, cerebrospinal fluid (CSF), and urine; 2) established a new IGFBP-3 RIA that detects both intact and fragments of IGFBP-3; 3) identified both intact and fragments of IGFBP-3 by Western immunoblot techniques; and 4) addressed the discordance between Western ligand blot analysis and RIA by assessing fluids for IGFBP proteolytic activity. All fluids examined, except pregnancy serum, CSF, and amniotic fluid, displayed a 44-34-kilodalton (kDa) IGFBP-3 doublet by Western ligand blot analysis. Western immunoblot analysis using specific IGFBP-3 antiserum showed a 44-34-kDa IGFBP-3 doublet and a 28-kDa fragment in nonpregnancy serum, fetal cord serum, follicular fluid, and peritoneal fluid. The immunoreactive 42-38-kDa doublet was faint in urine and seminal plasma. IGFBPs in CSF did not cross-react with IGFBP-3 antiserum. Pregnancy serum and amniotic fluid contained only the 28-kDa fragment when compared against equal volumes of nonpregnancy serum. With the development of an IGFBP-3 RIA, IGFBP-3 could be accurately measured; urine, CSF, and seminal plasma contained the lowest levels of IGFBP-3 at 27 +/- 3 ng/ml (mean +/- SEM), 110 +/- 26 ng/ml, and 209 +/- 56 ng/ml, respectively. In increasing concentration: fetal cord serum contained 753 +/- 101 ng/ml; peritoneal fluid, 1124 +/- 130 ng/ml; follicular fluid, 2356 +/- 211 ng/ml; nonpregnancy serum, 3556 +/- 508 ng/ml; pregnancy serum, 3718 +/- 842 ng/ml; and amniotic fluid, 5150 +/- 688 ng/ml. The measurable concentrations of IGFBP-3 in CSF and the high concentrations measured in pregnancy serum and amniotic fluid conflicted with Western blot analysis. Thus, fluids were assessed for IGFBP proteolytic activity by incubation with a source of IGFBP-3, either nonpregnancy serum or purified IGFBP-3. All fluids displayed some proteolytic activity with either assay. Fluids with little protease activity (nonpregnancy serum, follicular fluid, and urine) showed a close relationship between immunoassayable IGFBP-3 by RIA and IGFBP-3 band intensity by Western ligand blot. Fluids with high proteolytic activity (pregnancy serum, CSF, seminal plasma, peritoneal fluid, and amniotic fluid) gave discrepant IGFBP-3 values between RIA and Western ligand blot.(ABSTRACT TRUNCATED AT 400 WORDS) | US |