bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium influx stimulates a second pathway for sustained diacylglycerol production in leukocytes activated by chemoattractants.
Authors: Truett A P , Verghese M W , Dillon S B , Snyderman R
Published: May 2006

Metabolic pathways involved in the activation of polymorphonuclear leukocytes (PMNs) were characterized by using chemoattractants with equivalent chemotactic activity but widely disparate ability to stimulate superoxide production [N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) much greater than leukotriene B4]. Leukotriene B4 stimulated a low level of superoxide production that plateaued at 60 sec, whereas with fMet-Leu-Phe the response continued to increase for 5 min. Both agents produced equivalent initial rises in diacylglycerol (acyl2Gro) (less than or equal to 30 sec); however, only fMet-Leu-Phe induced a second increase of acyl2Gro peaking at ca. 120 sec. Both chemoattractants also caused an equivalent initial (less than or equal to 10 sec) rise in intracellular calcium; however, the elevation induced by fMet-Leu-Phe was more sustained. We sought to determine the biochemical mechanisms underlying these discrepancies. Superoxide production and the second phase of acyl2Gro generation were both inhibited ca. 56% by depleting extracellular calcium or ca. 79% by buffering intracellular calcium. Cytochalasin B greatly enhanced the respiratory burst, acyl2Gro production, and calcium influx, but not inositolphospholipid turnover in PMNs stimulated with chemoattractants. These data indicate that sequential metabolic pathways activate the respiratory burst in PMNs stimulated by chemoattractants. The response is initiated by inositolpolyphospholipid hydrolysis, which results in rapid (less than or equal to 5 sec) calcium mobilization from intracellular stores and acyl2Gro release (peak at ca. 30 sec). To fully activate the respiratory burst, the chemoattractant must also trigger calcium influx, which leads to a sustained cytosolic calcium elevation. This supports a prolonged new phase of acyl2Gro production that is independent of inositolphospholipid hydrolysis and is correlated with superoxide production.
Partial reversion of the transformed phenotype in HRAS-transfected tumorigenic cells by transfer of a human gene.
Authors: Schaefer R , Iyer J , Iten E , Nirkko A C
Published: May 2006

The transformed phenotype of rat FE-8 cells transfected by an activated human HRAS gene was suppressed upon fusion with normal cells. An experimental approach was developed to identify and isolate a human gene capable of suppressing the transforming activity of the HRAS oncogene in FE-8 cells. Genomic DNA from human placenta was introduced into FE-8 cells by cotransfection with the plasmid pY3 conferring hygromycin B resistance. Transfectants were selected in medium containing hygromycin B. HRAS-transformed FE-8 cells showed an increased sensitivity toward ouabain when compared to their normal counterparts. Therefore, the population of transfected hygromycin B-resistant cells was treated with ouabain to eliminate cells with a transformed phenotype. Ouabain selection resulted in a small number of cell clones exhibiting a more normal phenotype. The clones had lost the morphology of transformed cells and required anchorage for growth. The tumorigenicity of transfectants in nude mice was reduced but not completely abolished. FE-8 revertants continued to express the p21 RAS protein. Human repetitive sequences contained in the DNA of a secondary transfectant were used for isolation of the suppressor gene from reverted FE-8 cells. The cloned DNA fragment was transfected into tumorigenic FE-8 cells and conferred a partial reversion of the transformed phenotype.
Hepatitis B synthetic immunogen comprised of nucleocapsid T-cell sites and an envelope B-cell epitope.
Authors: Milich D R , Hughes J L , McLachlan A , Thornton G B , Moriarty A
Published: May 2006

Previous studies located T-cell recognition of the nucleocapsid of the hepatitis B virus (HBcAg) to residues 120-140 in mice bearing the H-2s or H-2b haplotypes. Herein, we demonstrate that B10.S (H-2s) and B10 (H-2b) H-2 congenic strains recognize distinct T-cell sites within the p120-140 (a synthetic peptide corresponding to residues 120-140 of HBcAg) sequence defined by p120-131 and p129-140, respectively. Peptide p120-131 stimulates B10.S HBcAg-primed T cells, and reciprocally p120-131-primed T cells recognize HBcAg. Similarly, the p129-140 sequence is a T-cell recognition site relevant to the native HBcAg in the B10 strain. It is also shown that these 12-residue peptides efficiently prime T-helper cells, which are capable of eliciting antibody production to HBcAg in vivo. These observations prompted us to examine the ability of the HBcAg-specific p120-140 sequence to function as a T-cell carrier moiety as a component of a totally synthetic hepatitis B vaccine. For this purpose a synthetic B-cell epitope from the pre-S(2) region (p133-140) of the viral envelope was chosen because this sequence represents a dominant antibody-binding site of the envelope. Immunization of B10.S and B10 strains with the synthetic composite peptide c120-140-(133-140) elicited anti-peptide antibody production, which was crossreactive with the native viral envelope. Furthermore, c120-140-(133-140) immunization primed p120-131-specific T cells in the B10.S strain and p129-140-specific T cells in the B10 strain, which recognized HBcAg and provided T-helper cell function for anti-envelope antibody production in vivo. These results demonstrate the feasibility of constructing complex synthetic immunogens that represent multiple proteins of a pathogen and are capable of engaging both T and B cells relevant to the native antigens.
Cytomegalovirus replicates efficiently in human kidney mesangial cells.
Authors: Heieren M H , van der Woude F J , Balfour H H
Published: May 2006

Human cytomegalovirus (HCMV) is a major renal pathogen in congenitally infected infants and renal allograft recipients. We postulated that a specific renal cell type was involved in HCMV infection and reactivation. Human fetal kidney cortex cell cultures were assayed for their ability to support HCMV infection. Infectious center assays indicated that the low level of viral replication observed by virus yield assay occurred from a fraction of the cells in the mixed cultures. Virus-specific immunofluorescence and in situ hybridization documented the presence of HCMV-specific protein and nucleic acid, respectively, in a morphologically distinct cell type. These cells were purified, were identified as kidney mesangial cells, and were observed to support efficient HCMV replication. Our research identifies mesangial cells as a renal cell type that supports HCMV replication and provides evidence to implicate these cells in the pathogenesis of HCMV-induced renal disease.
Spatially localized 1H NMR spectra of metabolites in the human brain.
Authors: Hanstock C C , Rothman D L , Prichard J W , Jue T , Shulman R G
Published: May 2006

Using a surface coil, we have obtained 1H NMR spectra from metabolites in the human brain. Localization was achieved by combining depth pulses with image-selected in vivo spectroscopy magnetic field gradient methods. 1H spectra in which total creatine (3.03 ppm) has a signal/noise ratio of 95:1 were obtained in 4 min from 14 ml of brain. A resonance at 2.02 ppm consisting predominantly of N-acetylaspartate was measured relative to the creatine peak in gray and white matter, and the ratio was lower in the white matter. The spin-spin relaxation times of N-acetylaspartate and creatine were measured in white and gray matter and while creatine relaxation times were the same in both, the N-acetylaspartate relaxation time was longer in white matter. Lactate was detected in the normoxic brain and the average of three measurements was approximately equal to 0.5 mM from comparison with the creatine plus phosphocreatine peak, which was assumed to be 10.5 mM.
Human and rat mast cell high-affinity immunoglobulin E receptors: characterization of putative alpha-chain gene products.
Authors: Shimizu A , Tepler I , Benfey P N , Berenstein E H , Siraganian R P , Leder P
Published: May 2006

We have cloned and determined the entire nucleotide sequence of cDNAs corresponding to the putative alpha subunits of the human and rat mast cell high-affinity IgE receptors. Both human and rat cDNAs encode an NH2-terminal signal peptide, two immunoglobulin-like extracellular domains (encoded by discrete exons), a hydrophobic transmembrane region, and a positively charged cytoplasmic tail. The human and rat alpha subunits share an overall homology with one another and the immunoglobulin gene family, suggesting that they arose from a common ancestral gene and continue to share structural homology with their ligands. In addition, the rat gene is transcribed into at least three distinct forms, each of which yields a somewhat different coding sequence.
Allospecific cytotoxic T lymphocytes recognize an H-2 peptide in the context of a murine major histocompatibility complex class I molecule.
Authors: Song E S , Linsk R , Olson C A , McMillan M , Goodenow R S
Published: May 2006

We have isolated cytotoxic T lymphocytes (CTL) preferentially reactive with the alpha 1 external domain of the H-2Ld antigen by selecting for T cells capable of recognizing a variant major histocompatibility complex (MHC) class I antigen sharing alpha 1 sequences with H-2Ld. Using these CTL, we demonstrate that a synthetic alpha 1 peptide corresponding to one of the helices derived from the H-2Ld molecule can be presented by a class I restriction element to reconstitute a CTL determinant borne by intact H-2Ld. Moreover, several other H-2L-reactive CTL generated independently were also able to recognize H-2Ld either as an intact alloantigen or as a peptide in conjunction with appropriate class I restriction elements. These data demonstrate that an H-2 peptide can reconstitute a CTL target structure and suggest that some alloreactive T cells in fact might be directed against allogeneic class I peptides in the context of a class I framework.
Epinephrine enhances Ca2+ current-regulated Ca2+ release and Ca2+ reuptake in rat ventricular myocytes.
Authors: Callewaert G , Cleemann L , Morad M
Published: May 2006

The voltage dependence of the intracellular Ca2+ transients was measured in single rat ventricular myocytes with the fluorescent Ca2+ indicator dye fura-2. The whole-cell voltage clamp technique was used to measure the membrane current, and 0.9 mM fura-2 was loaded into the cell by including it in the dialyzing solution of the patch electrode. A mechanical light chopper operating at 1200 Hz was used to obtain simultaneous measurements of the intracellular Ca2+ activity with fluorescence excitation on either side of the isosbestic point (330 nm and 410 nm). The symmetry of the two optical Ca2+ signals was used as a criterion to guard against artifacts resulting, for instance, from motion. The voltage dependence of peak Ca2+ current and the Ca2+ transient measured 25 ms after depolarizing clamps from a holding potential of -40 mV were bell-shaped and virtually identical. The Ca2+ entry estimated from the integral of the Ca2+ current (0 mV, 25 ms) corresponds to a 5-10 microM increase in the total intracellular Ca2+ concentration, whereas the optical signal indicated a 100 microM increase in total intracellular Ca2+. Repolarization of clamp pulses from highly positive potentials were accompanied by a second Ca2+ transient, the magnitude of which, when summed with that measured during depolarization, was nearly constant. Ryanodine (10 microM) had little or no effect on the peak Ca2+ current but reduced the magnitude of the early Ca2+ transients by 70-90%. Epinephrine (1 microM) increased the Ca2+ current and the Ca2+ transients, accelerated the rate of decline of the Ca2+ transients at potentials between -30 and +70 mV, and reduced the intracellular [Ca2+] below baseline at potentials positive to +80 or negative to -40 mV, where clamp pulses did not elicit any Ca2+ release. Elevation of intracellular cAMP mimicked the relaxant effect of epinephrine at depolarizing potentials, whereas elevation of extracellular [Ca2+] did not. These results suggest that most of the activator Ca2+ in rat ventricular cells is released from the sarcoplasmic reticulum as a graded response to sarcolemmal Ca2+ influx. Consistent with a graded Ca2+-induced Ca2+ release we find that epinephrine increases the internal Ca2+ release by increasing the Ca2+ current. Epinephrine may also increase the Ca2+ content of the sarcoplasmic reticulum that may, in turn, increase the Ca2+-induced Ca2+ release. The relaxant effect of epinephrine appears to be caused by enhanced rate of Ca2+ resequestration and is mediated by adenylate cyclase system.
Purification of a yeast protein that binds to origins of DNA replication and a transcriptional silencer.
Authors: Diffley J F , Stillman B
Published: May 2006

Two factors that interact specifically with the chromosomal replicator, autonomously replicating sequence 1 (ARS1), were identified in whole-cell yeast extracts. One of these proteins, ARS binding factor I (ABF-I), was purified to homogeneity as a polypeptide of 135 kDa. ABF-I binds within a region previously shown to be essential for the function of ARS1 when yeast are grown under certain nutritional conditions, and deletion analysis presented here suggests that the ARS1 domain containing the ABF-I-binding site, domain B, is a complex element composed of at least two subdomains. ABF-I also binds within an ARS at the HMR silent mating locus. This ARS functions as a cis-acting transcriptional silencer, and the ABF-I-binding site within this ARS is important for both ARS and silencer function.
Active suppression of major histocompatibility complex class II gene expression during differentiation from B cells to plasma cells.
Authors: Latron F , Jotterand-Bellomo M , Maffei A , Scarpellino L , Bernard M , Strominger J L , Accolla R S
Published: May 2006

Constitutive expression of major histocompatibility complex class II genes is acquired very early in B-cell ontogeny and is maintained up to the B-cell blast stage. Terminal differentiation in plasma cells is, however, accompanied by a loss of class II gene expression. In B cells this gene system is under the control of several loci encoding transacting factors with activator function, one of which, the aIr-1 gene product, operates across species barriers. In this report human class II gene expression is shown to be extinguished in somatic cell hybrids between the human class II-positive B-cell line Raji and the mouse class II-negative plasmacytoma cell line P3-U1. Since all murine chromosomes are retained in these hybrids and no preferential segregation of a specific human chromosome is observed, the results are compatible with the presence of suppressor factors of mouse origin, operating across species barriers and inhibiting class II gene expression. Suppression seems to act at the level of transcription or accumulation of class II-specific mRNA, since no human, and very few murine, class II transcripts are detectable in the hybrids.
Immunogenic (tum-) variants of mouse tumor P815: cloning of the gene of tum- antigen P91A and identification of the tum- mutation.
Authors: De Plaen E , Lurquin C , Van Pel A , Mariamé B , Szikora J P , Wölfel T , Sibille C , Chomez P , Boon T
Published: May 2006

Mutagen treatment of mouse P815 tumor cells produces tum- variants that are rejected by syngeneic mice because these variants express new surface antigens. These "tum- antigens" are recognized by cytolytic T lymphocytes but induce no detectable antibody response. Transfection of P815 cell line P1.HTR with DNA of tum- variant P91 yielded transfectants expressing tum- antigen P91A. They were detected by their ability to stimulate proliferation of cytolytic T lymphocytes [Wölfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L. & Boon, T. (1987) Immunogenetics 26, 178-187]. A cosmid library of a cell line expressing antigen P91A was transfected into P1.HTR. Transfectants expressing the antigen were obtained. By packaging directly the DNA of a transfectant with lambda phage extracts, we obtained a small cosmid population containing as major component a cosmid that transferred the expression of P91A. The assay of various restriction fragments of this cosmid led to the isolation of an 800-base-pair fragment containing the P91A sequence required for transfection. Comparison with a homologous cDNA showed that this fragment contained only one of the several exons of the P91A gene. The normal and the tum- forms of the gene differ by one nucleotide located in this 137-base-pair exon. The essential role of this mutation, which produces an amino acid change, was confirmed by site-directed mutagenesis. No significant sequence similarity was found between the 800-base-pair fragment and any recorded gene.
Enhanced osteoblast proliferation and collagen gene expression by estradiol.
Authors: Ernst M , Schmid C , Froesch E R
Published: May 2006

Estrogens play a crucial role in the development of postmenopausal osteoporosis. However, the mechanism by which estrogens exert their effects on bone is unknown. To examine possible direct effects of 17 beta-estradiol on bone-forming cells, we used pure rat osteoblast-like cells in vitro as a model. Osteoblast-like cells prepared from calvaria of newborn rats were cultured serum-free in methylcellulose-containing medium for 21 days. Osteoblast-like cells proliferate selectively into clonally derived cell clusters of spherical morphology. 17 beta-Estradiol at concentrations of 0.1 nM and 1 nM enhanced osteoblast-like cell proliferation by 41% and 68% above vehicle-treated controls. The biologically inactive stereoisomer 17 alpha-estradiol (same concentrations) had no effect. Moreover, the antiestrogen tamoxifen abolished the stimulation of osteoblast-like cell proliferation by 17 beta-estradiol. After 21 days of culture, RNA was prepared and analyzed in a dot-hybridization assay for the abundance of pro alpha 1(I) collagen mRNA. Steady-state mRNA levels were increased in cultures treated with 17 beta-estradiol in a dose-dependent manner with maximal stimulation at 1 nM and 10 nM. At the same concentrations, the percentage of synthesized protein (labeled by [3H]proline pulse) that was digestible by collagenase was increased, indicating that 17 beta-estradiol acts at pretranslational levels to enhance synthesis of bone collagen. These data show that the osteoblast is a direct target for 17 beta-estradiol.
Close correlation between restriction fragment length polymorphism of the L-MYC gene and metastasis of human lung cancer to the lymph nodes and other organs.
Authors: Kawashima K , Shikama H , Imoto K , Izawa M , Naruke T , Okabayashi K , Nishimura S
Published: May 2006

Restriction length fragment polymorphism of the L-MYC gene was examined in DNAs from lung cancer tissues and normal tissues of 51 Japanese patients with lung cancer. In individual patients, no difference was seen between the restriction length fragments of the two alleles of L-MYC [6-kilobase (kb) and 10-kb fragments in EcoRI digests] in lung cancer tissues and normal tissues. But a striking correlation was found between the restriction length fragment polymorphism pattern of L-MYC and the extent of metastasis, particularly to the lymph nodes at the time of surgery: Patients with only the L band (10 kb) had few lymph node metastatic lesions, whereas patients with either the S band (6 kb) or the S and L bands almost always had lymph node metastatic lesion. A similar correlation was found between the presence of the S band and metastases to other organs. This correlation was particularly marked in cases of adenocarcinoma. These results indicate a clear genetic influence on metastases and a consequent poor prognosis for certain patients of lung cancer; L-MYC restriction length fragment polymorphism is thus shown to be a useful marker for predicting the metastatic potential of human lung cancer.
Channel protein engineering: synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers.
Authors: Oiki S , Danho W , Montal M
Published: May 2006

A synthetic 22-mer peptide that mimics the sequence of a putative pore segment of the voltage-dependent sodium channel forms transmembrane ionic channels in lipid bilayers. Several features of the authentic sodium channel are exhibited by the synthetic peptide: (i) The single channel conductance of the most frequent event is 20 pS in 0.5 M NaCl. (ii) The single channel open and closed lifetimes are in the ms time range. (iii) The synthetic channel discriminates cations over anions but is nonselective between Na+ and K+. However, the synthetic channel displays no significant voltage dependence. Energetic considerations suggest a bundle of four parallel amphipathic alpha-helices as the most plausible channel structure. The synthetic 22-mer channel-forming peptide allows study of the mechanisms of ion permeation through sodium channels by protein engineering techniques.
On the active site thiol of gamma-glutamylcysteine synthetase: relationships to catalysis, inhibition, and regulation.
Authors: Huang C S , Moore W R , Meister A
Published: May 2006

gamma-Glutamylcysteine synthetase (glutamate-cysteine ligase; EC 6.3.2.2) was isolated from an Escherichia coli strain enriched in the gene for this enzyme by recombinant DNA techniques. The purified enzyme has a specific activity of 1860 units/mg and a molecular weight of 56,000. Comparison of the E. coli enzyme with the well-characterized rat kidney enzyme showed that these enzymes have similar catalytic properties (apparent Km values, substrate specificities, turnover numbers). Both enzymes are feedback-inhibited by glutathione but not by gamma-glutamyl-alpha-aminobutyrylglycine; the data indicate that glutathione binds not only at the glutamate binding site but also at a second site on the enzyme that interacts with the thiol moiety of glutathione but not with a methyl group. Both enzymes are inactivated by buthionine sulfoximine in the presence of ATP, suggesting a common gamma-glutamyl phosphate intermediate. However, unlike the rat kidney enzyme that has an active center thiol, the bacterial enzyme is insensitive to cystamine, gamma-methylene glutamate, and S-sulfo amino acids, indicating that it does not have an active site thiol. Thus, the rat kidney and E. coli enzymes share several catalytic features but differ in active site structure. If the active site thiol of the rat kidney enzyme is involved in catalysis, which seems likely, there would appear to be differences in the mechanisms of action of the two gamma-glutamylcysteine synthetases.
The v-rel oncogene product is complexed to a 40-kDa phosphoprotein in transformed lymphoid cells.
Authors: Tung H Y , Bargmann W J , Lim M Y , Bose H R
Published: May 2006

The transforming protein encoded by the v-rel oncogene of avian reticuloendotheliosis virus (REV-T) is a very low copy number molecule in the cytosol of transformed cells. Analysis of cytosolic extracts from a REV-T-transformed lymphoid cell line by gel filtration on Sephacryl S-300 indicated that most of the v-rel oncogene product, pp59v-rel, eluted with an apparent molecular mass of 400 kDa. The size of this complex was confirmed by analysis on a fast-protein liquid chromatography gel filtration column. A 40-kDa cellular protein copurified with pp59v-rel on sequential gel filtration on Sephacryl S-200 and immunoaffinity chromatography with a monoclonal antibody directed against pp59v-rel. The 40-kDa cellular protein could also be immunoprecipitated together with pp59v-rel from cell extracts of [35S]methionine-labeled cells, suggesting that pp59v-rel is complexed with the 40-kDa protein in transformed lymphoid cells. Both the 59- and 40-kDa proteins were phosphorylated when the highly purified preparation containing pp59v-rel was incubated with [gamma-32P]ATP and 10 mM MgCl2 in vitro. The identity of the kinase in the highly purified preparation containing pp59v-rel, however, is unknown. Immune complexes recovered from extracts of REV-T-transformed lymphoid cells labeled with [32P]orthophosphate also contained the 59- and 40-kDa phosphoproteins. These observations suggest that pp59v-rel is complexed with a 40-kDa cellular phosphoprotein to form a 400-kDa heteropolymer in the cytoplasm of transformed lymphoid cells.
Nonmyristoylated p60v-src fails to phosphorylate proteins of 115-120 kDa in chicken embryo fibroblasts.
Authors: Linder M E , Burr J G
Published: May 2006

We have used anti-phosphotyrosine antibodies to identify a large number of tyrosine phosphoproteins in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Most of these proteins were found in the 100,000 X g supernatant when cells were separated into soluble and particulate fractions; however, one group of proteins, of 115-120 kDa, was found in the particulate fraction. The phosphotyrosine content of the 115- to 120-kDa proteins was greatly reduced in chicken embryo fibroblasts infected with mutants of RSV (NY314 and SD10) encoding nonmyristoylated forms of the viral src gene product that do not associate with cellular membranes. Since RSV mutants NY314 and SD10 do not transform cells, phosphorylation of this group of 115- to 120-kDa membrane proteins may be related to the process of transformation.
Pathogenic and host range determinants of the feline aplastic anemia retrovirus.
Authors: Riedel N , Hoover E A , Dornsife R E , Mullins J I
Published: May 2006

Feline leukemia virus (FeLV) C-Sarma (or FSC) is a prototype of subgroup C FeLVs, which induce fatal aplastic anemia in outbred specific-pathogen-free (SPF) cats. FeLV C isolates also possess an extended host range in vitro, including an ability, unique among FeLVs, to replicate in guinea pig cells. To identify the viral determinants responsible for the pathogenicity and host range of FSC we constructed a series of proviral DNAs by exchanging gene fragments between FSC and FeLV-61E (or F6A), the latter of which is minimally pathogenic and whose host range in vitro is restricted to feline cells. Transfer of an 886-base-pair (bp) fragment of FSC, encompassing the codons for 73 amino acids at the 3' end of pol (the integrase/endonuclease gene) and the codons for 241 amino acids of the N-terminal portion of env [the extracellular glycoprotein (gp70) gene], into the F6A genome was sufficient to confer onto chimeric viruses the ability to induce fatal aplastic anemia in SPF cats. In contrast, no chimera lacking this sequence induced disease. When assayed in vitro, all chimeric viruses containing the 886-bp fragment of FSC acquired the ability to replicate in heterologous cells, including dog and guinea pig cells. Thus, the pathogenic and the host range determinants of the feline aplastic anemia retrovirus colocalize to a 3' pol-5' env region of the FSC genome and likely reside within a region encoding 241 amino acid residues of the N terminus of the extracellular glycoprotein.
Myocardial ischemia and reperfusion: direct evidence for free radical generation by electron spin resonance spectroscopy.
Authors: Baker J E , Felix C C , Olinger G N , Kalyanaraman B
Published: May 2006

Electron spin resonance spectroscopy has recently been used by others to detect directly radical species in isolated perfused hearts. Sample processing prior to spectroscopy in this study involved pulverization of tissue, which can artifactually generate radical species. We assessed in isolated perfused hearts the influence of tissue pulverization on the identity of radical species detected by spectroscopy and then, using a processing technique less likely to induce artifacts, whether myocardial ischemia and reperfusion generate radical species. Rat and rabbit hearts (n = 8) were perfused aerobically for 10 min and freeze-clamped to -196 degrees C. Frozen tissue was processed at -196 degrees C for spectroscopic analysis by pulverization vs. chopping. Spectra of pulverized tissue consisted of three components: a semiquinone (g = 2.004), a lipid peroxy radical (g [ = 2.04 and g = 2.006), and a carbon-centered radical that is possibly a lipid radical (giso = 2.002 and AHzz approximately equal to 50 G). Chopped tissue consisted of a single component, a semiquinone (g = 2.004). Rat hearts (n = 8 per group) also underwent 10-min global no-flow normothermic ischemia followed by 5-60 sec of either aerobic or anaerobic reperfusion, with frozen tissue chopped prior to spectroscopy. Spectra of ischemic tissue consisted of an iron-sulfur center and a semiquinone. Aerobic reperfusion resulted in a spectrum similar to the control but with increased amplitude that peaked after 10-15 sec of reflow. Anaerobic reperfusion yielded a spectrum identical to that of ischemic tissue. We conclude that pulverization of frozen myocardial tissue arti-factually generates radical species. Using a nonpulverization technique for tissue processing, we found that myocardial ischemia and reperfusion produce radical species but that molecular oxygen is necessary for the burst of radical production during reflow.
Endothelium-derived relaxing factor reduces platelet adhesion to bovine endothelial cells.
Authors: Sneddon J M , Vane J R
Published: May 2006

The adhesion of thrombin-stimulated human blood platelets to either the endothelial surface of intact bovine aorta or cultured bovine aortic endothelial cells was studied to determine the role of endothelium-derived relaxing factor in the regulation of platelet adhesion. Endothelial cells and platelets were pretreated with indomethacin to prevent the formation of prostaglandins. The adhesion of thrombin-stimulated platelets to endothelial cells was reduced by superoxide dismutase and bradykinin. The inhibitory effect of both drugs was abolished by hemoglobin and was absent in strips of bovine aorta where the endothelial cells had been removed by scraping. It is suggested that the effects of bradykinin are mediated by the release of endothelium-derived relaxing factor, which is protected from destruction by superoxide dismutase, and that endothelium-derived relaxing factor contributes to the nonadhesive properties of the vascular endothelium.
cDNA cloning and complete primary structure of skeletal muscle phosphorylase kinase (alpha subunit).
Authors: Zander N F , Meyer H E , Hoffmann-Posorske E , Crabb J W , Heilmeyer L M , Kilimann M W
Published: May 2006

We have isolated and sequenced a cDNA encoding the alpha subunit of phosphorylase kinase from rabbit fast-twitch skeletal muscle. The cDNA molecule consists of 388 nucleotides of 5'-nontranslated sequence, the complete coding sequence of 3711 nucleotides, and 342 nucleotides of 3'-nontranslated sequence followed by a poly(dA) tract. It encodes a polypeptide of 1237 amino acids and a deduced molecular mass of 138,422 Da. Nearly half of the deduced amino acid sequence is confirmed by peptide sequencing. Seven positions of endogenously phosphorylated serine residues and autophosphorylation sites, identified by peptide sequencing, could be assigned. They cluster in a segment of only 60 amino acids. RNA blot hybridization analysis demonstrates a predominant RNA species of approximately equal to 4500 nucleotides and a less abundant RNA of 8700 nucleotides.
Do exons code for structural or functional units in proteins?
Authors: Traut T W
Published: May 2006

In considering the origin and evolution of proteins, the possibility that proteins evolved from exons coding for specific structure-function modules is attractive for its economy and simplicity but is not systematically supported by the available data. However, the number of correspondences between exons and units of protein structure-function that have so far been identified appears to be greater than expected by chance alone. The available data also show (i) that exons are fairly limited in size but are large enough to specify structure-function modules in proteins; (ii) that the position of introns for homologous domains in the same gene is reasonably stable, but there is also evidence for mechanisms that alter the position or existence of introns; and (iii) that it is possible that the observed relationship of exons to protein structure represents a degenerate state of an ancestral correspondence between exons and structure-function modules in proteins.
Tracer diffusion of globular proteins in concentrated protein solutions.
Authors: Muramatsu N , Minton A P
Published: May 2006

The diffusion of tracer proteins at low concentration was measured in solutions containing "background" proteins at concentrations of up to 200 g/liter. The fractional reduction of the diffusion coefficient of tracer in the presence of a given weight/volume concentration of background species generally increases with increasing size of tracer species and with decreasing size of background species. The dependence of the diffusion constants of three out of four tracer species upon the concentrations of four background species is accounted for semiquantitatively by a simple hard particle model. Extrapolation of model calculations to higher background concentrations suggests that in solutions containing proteins at concentrations comparable to those found in biological fluid media, the diffusive transport of larger proteins and aggregates may be slower than in dilute solution by several orders of magnitude.
Identification of a GTP-binding protein alpha subunit that lacks an apparent ADP-ribosylation site for pertussis toxin.
Authors: Fong H K , Yoshimoto K K , Eversole-Cire P , Simon M I
Published: May 2006

Recent molecular cloning of cDNA for the alpha subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called Tr and Tc, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, we have identified a G-protein alpha subunit, which we refer to as Gz alpha, by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine Tr alpha-subunit cDNA. The deduced amino acid sequence of Gz alpha is 41-67% identical with those of other known G-protein alpha subunits. However, the 355-residue Gz alpha lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein alpha subunits. We suggest that Gz alpha, which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.
Intercellular adhesion molecule 1 (ICAM-1) has a central role in cell-cell contact-mediated immune mechanisms.
Authors: Boyd A W , Wawryk S O , Burns G F , Fecondo J V
Published: May 2006

The role of intercellular adhesion molecule 1 (ICAM-1) in immune function was probed by using the Wehi-CAM-1 (W-CAM-1) monoclonal antibody. This antibody blocks aggregation of cell lines mediated by the ICAM-1 molecule and is here shown to block homotypic binding of purified populations of activated T and B lymphocytes (blasts) and also aggregation of mixed T- and B-cell blasts. We also demonstrate that W-CAM-1 inhibited T-cell adhesion to normal human endothelial cells, the first step in lymphocyte egress into the tissues. In tests of immune function, W-CAM-1 had a modest inhibitory effect on T- and B-cell activation by potent mitogens and no effect on the response of activated lymphocytes to lymphokines. By contrast, activation induced by cell-cell contact (mixed lymphocyte reaction, T-cell-mediated B-cell activation) was significantly inhibited. Moreover, the antibody was shown to block elements of both effector arms of the immune system (cytotoxic cell function and immunoglobulin production). These findings show that the ICAM-1 molecule is a central component of the mechanism of lymphocyte-endothelial cell adhesion. The studies of lymphoid function demonstrate a pivotal role for this molecule in both the T-cell/T-cell and T-cell/B-cell interactions, which underpin the regulation of the immune response, and in the mechanism of cell-mediated cytotoxicity.
Conversion of encapsulated 5-fluoro-2'-deoxyuridine 5'-monophosphate to the antineoplastic drug 5-fluoro-2'-deoxyuridine in human erythrocytes.
Authors: De Flora A , Zocchi E , Guida L , Polvani C , Benatti U
Published: May 2006

The fluoropyrimidine deoxyribonucleotide 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was encapsulated in human erythrocytes by a procedure based on hypotonic hemolysis and isotonic resealing. Encapsulated FdUMP (up to 9 mumol/ml of packed erythrocytes) did not affect erythrocyte metabolism or morphology. Hemolysates were found to catalyze efficient dephosphorylation of FdUMP to yield nearly stoichiometric amounts of the corresponding deoxyribonucleoside 5-fluoro-2'-deoxyuridine (FdUrd), an antineoplastic drug showing selective cytotoxicity toward liver metastases from colorectal carcinomas. The dephosphorylation reaction had an apparent Km of 7.7 +/- 1.2 mM FdUMP at pH 7.4 and was remarkably slower at pH 8.2. ATP, GTP, and UTP inhibited both the disappearance of FdUMP and the formation of FdUrd in hemolysates. The enzyme responsible for the FdUMP-to-FdUrd conversion was identified with the deoxyribonucleotide-specific isozyme of erythrocyte pyrimidine 5'-nucleotidase (EC 3.1.3.5). Intracellular formation and subsequent release of FdUrd were observed in intact erythrocytes loaded with FdUMP. Inhibition of FdUrd release from these erythrocytes was obtained by raising the pH intracellularly and, alternatively, by coencapsulation of ATP. Autologous FdUMP-loaded erythrocytes might be used as endogenous bioreactors designed for time-programmed and liver-targeted delivery of FdUrd.
Paradoxical relationship between atriopeptin plasma levels and diuresis-natriuresis induced by acute volume expansion.
Authors: Sakata M , Greenwald J E , Needleman P
Published: May 2006

Surgical removal of one or both atrial appendages was employed in rats to reduce the intrinsic stores of atriopeptin (AP). In conscious rats (with intact baroreceptor reflexes), bilateral or unilateral atrial appendectomy suppressed the diuresis and natriuresis produced by acute volume expansion. Surprisingly, volume expansion (with 4% bovine serum albumin in saline at 1.5 ml/kg per min for 15 min) did not result in an increase in plasma AP immunoreactivity (APir) in control or atrial-appendectomized conscious rats. Previous studies demonstrated that acute volume expansion in anesthetized animals caused increased plasma APir. Indeed, we found that volume expansion causes comparable diuresis-natriuresis in conscious and chloral hydrate-anesthetized rats, but only the latter group exhibits an increase in plasma APir. Brattleboro rats, which are deficient in vasopressin, exhibit the same response as Long-Evans controls in that acute volume expansion in conscious animals produces a pronounced diuresis and natriuresis but no APir release, but when these same animals are anesthetized, there is a simultaneous induction of diuresis-natriuresis and APir release by volume expansion. Plasma AP does not increase in conscious rats despite a large volume load, 30-40% of the total blood volume given in 15 min, and the natriuresis-diuresis appears to also be independent of vasopressin. On the other hand, the diuresis induced by acute volume expansion in anesthetized rats seems dependent on the elevated APir, since rats made autoimmune to AP (which are nonresponsive to exogenous AP infusions) exhibit a diuresis in conscious but not anesthetized rats. We therefore conclude that the participation of AP in volume homeostasis is more likely in pathophysiological states and that another mechanism or possibly another atrial factor mediates the diuresis-natriuresis induced by volume expansion in conscious rats.
Poliovirus type 1/type 3 antigenic hybrid virus constructed in vitro elicits type 1 and type 3 neutralizing antibodies in rabbits and monkeys.
Authors: Murray M G , Kuhn R J , Arita M , Kawamura N , Nomoto A , Wimmer E
Published: May 2006

Poliovirus exists as three stable serotypes (PV-1, PV-2, and PV-3). These viruses display three antigenic sites each, designated N-AgI, N-AgII, and N-AgIII. When mice are immunized with poliovirus, N-AgI is the major neutralization antigenic site for PV-3, whereas N-AgII and N-AgIII are immunodominant over N-AgI for PV-1. To study the relationship between structure and antigenicity, a hybrid virus was constructed in which N-AgI of PV-1 was replaced by N-AgI of PV-3. PV-3- and PV-1-specific antisera, including those elicited by PV-3 in primates, neutralized the hybrid virus. Injection of the hybrid virus into rabbits or into primates resulted in the production of antisera that neutralized both PV-1 and PV-3. The data show that sequence replacement at N-AgI of poliovirus is compatible with viral proliferation, an observation useful for the development of multivalent picornavirus vaccines.
Neural and humoral factors separately regulate neuropeptide Y, enkephalin, and chromogranin A and B mRNA levels in rat adrenal medulla.
Authors: Fischer-Colbrie R , Iacangelo A , Eiden L E
Published: May 2006

The influence of neurogenic versus humoral factors on mRNA levels of several secretory proteins of rat adrenal medulla was studied in vivo. Increased splanchnic nerve activity was generated (reflexly) with insulin treatment. Twenty-four hours after insulin injection, levels of mRNAs encoding neuropeptides (enkephalin and neuropeptide Y) were increased 6.5-fold, whereas those of mRNAs for the major secretory proteins (chromogranins A and B) were unchanged. Bilateral transection of the splanchnic nerves completely prevented this increase. Hypophysectomy decreased levels of chromogranin A mRNA to 32% of control, suggesting a dependence on hormones of the pituitary-adrenal axis. Treatment of hypophysectomized rats with dexamethasone restored chromogranin A mRNA to basal levels. Chromogranin B mRNA levels were not changed by either insulin treatment or hypophysectomy. These results demonstrate (i) that different classes of secretory proteins present in chromaffin granules are regulated by different mechanisms, (ii) that this regulation occurs at a pretranslational site, and (iii) that the relative concentration of secretory constituents of chromaffin granules may vary. The significance of an altered composition of secretory-granule constituents, which may be important in hypotension or stress, is discussed.
Evolution of the genome and the genetic code: selection at the dinucleotide level by methylation and polyribonucleotide cleavage.
Authors: Beutler E , Gelbart T , Han J H , Koziol J A , Beutler B
Published: May 2006

Noting the scarcity of CpG dinucleotide in total genomic DNA derived from higher organisms and the scarcity of TpA dinucleotide in total genomic DNA derived from most life forms, we examined the distribution of these dinucleotides in sequences derived from functionally distinct types of human DNA, including mitochondrial DNA, intergenic DNA, intron DNA, and DNA destined to be represented in the cytoplasm as mRNA, tRNA, or rRNA. While CpG frequency has fallen to its lowest levels in DNA that is transcriptionally silent, TpA is most stringently excluded in DNA destined to be expressed as mRNA in the cytosol. This observation suggests that the selective pressures leading to the removal of CpG and TpA operate at different levels. With respect to TpA, dinucleotide scarcity may reflect a requirement for mRNA stability and may indicate the action of UpA-selective ribonucleases. We propose that, by reason of its instability, UpA must have been very rare in primordial RNA. Therefore, tRNA with the anticodon for this dinucleotide may have failed to evolve, making UpA the primordial doublet "stop" codon. The modern triplet code has faithfully conserved this arrangement in the two universal stop codons, UAA and UAG.
Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9.
Authors: Lowrey D M , Aebischer T , Olsen K , Lichtenheld M , Rupp F , Hengartner H , Podack E R
Published: May 2006

The nucleotide sequence coding for the cytotoxic T-lymphocyte (CTL) protein perforin 1 (P1) has been determined and the corresponding protein sequence has been derived. Murine CTL cDNA libraries contained in the vector lambda gt11 were screened by using a monospecific antiserum to purified P1. Three recombinant phages were isolated and their cDNA inserts were sequenced. The derived protein sequence contains 554 amino acids and displays, as expected, considerable homology with certain functional domains in the complement components C9, C8 alpha, C8 beta, and C7. The identity of P1 cDNA clones was verified by prokaryotic expression and the reactivities of antisera produced to the expressed proteins. In addition, antisera were produced to two synthetic peptides located in the center and C-terminal portions of P1. All antisera reacted with purified P1. In Northern blot analyses, P1 cDNA probes recognized a 2.9-kilobase mRNA only in CTL. Perforin mRNA was found in all cloned CTL and in all mixed lymphocyte reactions that gave rise to cytotoxic cells. Perforin mRNA was also detected in virus-specific CTL that had been generated in vivo and isolated from liver tissue of mice infected with lymphocytic choriomeningitis virus. The cell-specific expression of perforin is consistent with its postulated role in cytolysis.
Phenylethanolamine N-methyltransferase-containing neurons in the limbic system of the young rat.
Authors: Mezey E
Published: May 2006

Fifteen years ago epinephrine cells were shown to be present in the medulla oblongata of the rat. These cell groups (C1 and C2) were thought to supply the epinephrine innervation in the rest of the central nervous system. In this study I demonstrate the presence of epinephrine-producing neurons in the forebrain of the young rat. Neurons that are immunopositive for phenylethanolamine N-methyltransferase (S-adenosyl-L-methionine:phenylethanolamine N-methyltransferase, EC 2.1.1.29) are present in the central nucleus of the amygdala as well as in the bed nucleus of the stria terminalis. Neurons in the same location are also immunopositive for tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydrobiopterine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2]. The phenylethanolamine N-methyltransferase immunopositivity disappears by day 35, while a small amount of tyrosine hydroxylase-positive cells still can be found in the adult. In situ hybridization reveals tyrosine hydroxylase mRNA in the above nuclei in both young and adult animals. The number of the positive cells decreases in adulthood. RNA blot-hybridization analysis showed the presence of phenylethanolamine N-methyltransferase mRNA in the amygdala and the bed nucleus of the stria terminalis in the young and in the adult rat brain. Neurons that are immunopositive for phenylethanolamine N-methyltransferase are also present in the human amygdala.