bluuebunny/crossref_metadata_2025 huggingface dataset:
Two distinct inputs to an avian song nucleus activate different glutamate receptor subtypes on individual neurons.
Authors: Mooney R , Konishi M
Published: May 2006

Although neural circuits mediating various simple behaviors have been delineated, those generating more complex behaviors are less well described. The discrete structure of avian song control nuclei promises that circuits controlling complex behaviors, such as birdsong, can also be understood. To this end, we developed an in vitro brain slice preparation containing the robust nucleus of the archistriatum (RA), a forebrain song control nucleus, and its inputs from two other song nuclei, the caudal nucleus of the ventral hyperstriatum (HVc) and the lateral part of the magnocellular nucleus of the anterior neostriatum (L-MAN). Using intracellular recordings, we examined the pharmacological properties of the synapses made on RA neurons by L-MAN and HVc axons. Electrical stimulation of the L-MAN and the HVc fiber tracts evoked excitatory postsynaptic potentials (EPSPs) from >70% of RA neurons when slices were prepared from male birds of 40-90 days of age, suggesting that many individual RA neurons receive excitatory input from L-MAN and HVc axons. The "L-MAN" EPSPs were blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D-(-)-2-amino-5-phosphonovaleric acid (D-APV) as well as the broad-spectrum glutamate receptor antagonist kynurenic acid but were relatively unaffected by the non-NMDA receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, "HVc" EP-SPs were relatively insensitive to D-APV but almost completely abolished by CNQX. These experiments suggest that L-MAN and HVc axons provide pharmacologically distinct types of excitatory input to many of the same RA neurons.
Electroporation by using bipolar oscillating electric field: an improved method for DNA transfection of NIH 3T3 cells.
Authors: Tekle E , Astumian R D , Chock P B
Published: May 2006

Using the plasmid DNA pSV2-neo (which, when integrated into the cellular genome confers resistance to the antibiotic G418 for selection), we examined and compared the transfection efficiency on NIH 3T3 cells electropermeabilized by applying a sequence of high-frequency unipolar or bipolar square waves or a single square pulse. Results show that a bipolar square wave is, at least, 1.7- and 5.5-fold more efficient than the unipolar square wave and single square pulse, respectively. In the range of electric field strength used for optimum transfection, the survivability of electropermeabilized cells was comparable between the unipolar and bipolar square waves but fell considerably with the single square pulse. Qualitative comparison of cell permeabilization induced by the three types of wave forms and monitored by ethidium bromide uptake revealed that only the bipolar square wave permeabilizes the cell membrane symmetrically at the two hemispheres facing the electrodes. With unipolar square wave or single square pulse, the membrane is permeabilized either on one side or asymmetrically. Taken together, our result suggests that permeabilization of the membrane at multiple sites without affecting cell survivability may account for the improvements in transfection efficiency observed with bipolar oscillating electric fields.
Endothelin as an autocrine factor in the regulation of parathyroid cells.
Authors: Fujii Y , Moreira J E , Orlando C , Maggi M , Aurbach G D , Brandi M L , Sakaguchi K
Published: May 2006

Endothelin, originally purified from porcine aortic endothelial cells, is widely distributed in tissues and is recognized as a product of epithelial cells, glial cells, and neurons in addition to endothelial cells. We found evidence by mRNA content and immunoreactivity that this peptide is synthesized in rat parathyroid epithelial cells (PT-r cells) and bovine parathyroid chief cells. The peptide synthesized by PT-r cells comigrated with synthetic endothelin 1 in reverse-phase HPLC and was diluted out in radioimmunoassay in parallel with the synthetic peptide. Bovine parathyroid endothelial cells (BPE-1 cells) did not express this peptide. Preproendothelin 1 mRNA expression by PT-r cells and endothelin 1 peptide production were regulated by calcium. Shifts in extracellular calcium either from high to low concentrations or vice versa elicited similar evanescent increases in expression of mRNA with a peak at 1 h. Synthesis of the peptide seems to be controlled by mRNA expression, and peptide in the medium appears to be continuously degraded or taken up by cells because its concentration in the medium showed a time course similar to that of mRNA expression. PT-r cells also bear a single class of receptors highly specific for endothelin 1, suggesting an autocrine regulation by endothelin 1 of the parathyroid. The facile regulation of endothelin concentrations in the medium by shifts in extracellular calcium concentration and possible autocrine regulation by endothelin 1 suggest that this peptide may mediate, at least in part, effects of calcium on the parathyroid system.
Human X-box-binding protein 1 is required for the transcription of a subset of human class II major histocompatibility genes and forms a heterodimer with c-fos.
Authors: Ono S J , Liou H C , Davidon R , Strominger J L , Glimcher L H
Published: May 2006

A complementary DNA encoding a member of the leucine-zipper class of proteins (human X-box-binding protein, hXBP-1) that binds to the 3' end of the conserved X box (X2) of the HLA-DRA major histocompatibility complex gene was recently described. Further gel-retardation analysis has demonstrated that hXBP-1 also binds to HLA-DPB X2 but not to other X2 sequences. Transient transfection of a mammalian expression vector with the hXBP-1 cDNA inserted in the antisense orientation represses the surface expression of HLA-DR and HLA-DP in Raji cells. Cotransfection of the antisense hXBP-1 vector with a HLA-DRA/chloramphenicol acetyltransferase (but not a HLA-DQB/chloramphenicol acetyltransferase) reporter plasmid decreases chloramphenicol acetyltransferase activity in Raji cells and in gamma-interferon-treated HeLa cells relative to cells cotransfected with a control antisense vector. Moreover, hXBP-1 is shown to form a stable heterodimer with the product of the c-fos protooncogene. These data suggest that the hXBP-1 c-fos heterodimer is critical for the transcription of a subset of the human class II major histocompatibility complex genes and that the regulatory mechanisms for the different class II genes are distinct.
Autoantibody production in hepatitis B e antigen transgenic mice elicited with a self T-cell peptide and inhibited with nonself peptides.
Authors: Milich D R , McLachlan A , Raney A K , Houghten R , Thornton G B , Maruyama T , Hughes J L , Jones J E
Published: May 2006

Studies in hepatitis B e antigen (HBeAg)-expressing transgenic mice indicate that self tolerance to two T-cell determinants on the same transgenic self molecule can differ markedly. The dominant T-cell site on HBeAg is tolerogenic, whereas a proportion of T cells recognizing a second T-cell site evade tolerance induction, persist in the periphery, and can be activated in vivo by a single injection of a 12-residue T-cell self peptide. The self-reactive T cells mediate in vivo autoantibody production sufficient to neutralize detection of the autoantigen in serum. Furthermore, autoantibody production can be inhibited by nonself peptides that compete with the self peptide for binding to major histocompatibility complex molecules. This model illustrates that T cells specific for an immunogenic T-cell site on a nonsequestered autoantigen can escape tolerance induction and, more importantly, can mediate autoreactivity in vivo. Furthermore, these results suggest that synthetic T-cell sites may be useful as immunotherapeutic agents for the purpose of circumventing nonresponse to HBeAg during persistent hepatitis B virus infection.
The ligand specificities of the insulin receptor and the insulin-like growth factor I receptor reside in different regions of a common binding site.
Authors: Kjeldsen T , Andersen A S , Wiberg F C , Rasmussen J S , Schäffer L , Balschmidt P , Møller K B , Møller N P
Published: May 2006

To identify the region(s) of the insulin receptor and the insulin-like growth factor I (IGF-I) receptor responsible for ligand specificity (high-affinity binding), expression vectors encoding soluble chimeric insulin/IGF-I receptors were prepared. The chimeric receptors were expressed in mammalian cells and partially purified. Binding studies revealed that a construct comprising an IGF-I receptor in which the 68 N-terminal amino acids of the insulin receptor alpha-subunit had replaced the equivalent IGF-I receptor segment displayed a markedly increased affinity for insulin. In contrast, the corresponding IGF-I receptor sequence is not critical for high-affinity IGF-I binding. It is shown that part of the cysteine-rich domain determines IGF-I specificity. We have previously shown that exchanging exons 1, 2, and 3 of the insulin receptor with the corresponding IGF-I receptor sequence results in loss of high affinity for insulin and gain of high affinity for IGF-I. Consequently, it is suggested that the ligand specificities of the two receptors (i.e., the sequences that discriminate between insulin and IGF-I) reside in different regions of a binding site with common features present in both receptors.
Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.
Authors: Molinari A M , Abbondanza C , Armetta I , Medici N , Minucci S , Moncharmont B , Nigro V , Puca G A
Published: May 2006

The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity.
Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation in intact Swiss 3T3 cells.
Authors: Zachary I , Gil J , Lehmann W , Sinnett-Smith J , Rozengurt E
Published: May 2006

The mitogenic neuropeptides bombesin and vasopressin markedly increased tyrosine and serine phosphorylation of multiple substrates in quiescent Swiss 3T3 fibroblasts, including two major bands of Mr 90,000 and 115,000. Tyrosine phosphorylation of these proteins was increased as judged by immunoprecipitation of 32Pi-labeled cells and immunoblotting of unlabeled cells with monoclonal antiphosphotyrosine antibodies, elution with phenyl phosphate, and phospho amino acid analysis. Phosphotyrosyl proteins generated by bombesin and vasopressin did not correspond either by apparent molecular weight or by immunological and biochemical criteria to several known tyrosine kinase substrates, including phospholipase C gamma, the microtubule-associated protein 2 kinase, GTPase-activating protein, or phosphatidylinositol kinase. The effect was rapid (within seconds), concentration dependent, and inhibited by specific receptor antagonists for both bombesin and vasopressin. The endothelin-related peptide, vasoactive intestinal contractor, also elicited a rapid and concentration-dependent tyrosine/serine phosphorylation of a similar set of substrates. These results demonstrate that neuropeptides, acting through receptors linked to GTP-binding proteins, stimulate tyrosine phosphorylation of a common set of substrates in quiescent Swiss 3T3 cells and suggest the existence of an additional signal transduction pathway in neuropeptide-induced mitogenesis.
Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products.
Authors: Hu Y W , Kang C Y
Published: May 2006

Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity.
Unusual isomeric polyunsaturated fatty acids in liver phospholipids of rats fed hydrogenated oil.
Authors: Holman R T , Pusch F , Svingen B , Dutton H J
Published: May 2006

Linoleic acid (18:2 omega 6) and linolenic acid (18:3 omega 3) are precursors of two series of essential fatty acids (EFA) formed by alternate desaturations and elongations. In EFA deficiency (EFAD), oleic acid (18:1 omega 9) and palmitoleic acid (16:1 omega 7) undergo the same reactions to form polyunsaturated fatty acids (PUFA) of other structures. Partially hydrogenated soybean oil (PHSO) contains isomeric 18:1 acids that can be converted to unusual isomers of 18:2 by liver microsomes. To test whether 18:2, 20:3, and 20:4 of unusual structure occur in phospholipids as a consequence of EFAD or ingestion of PHSO, rats were fed corn oil, an EFA-deficient diet, or PHSO to provide isomeric 18:1 acids. At 2.5 months the phospholipids were isolated from livers and converted to methyl esters, and the 18:2, 20:2, 20:3, and 20:4 fractions were isolated. The 18:2 and 20:2 fractions were ozonized, and, by using a computer solution of simultaneous equations, the structures and proportions of each isomer were calculated. The 20:3 and 20:4 fractions were analyzed by ozonolysis and capillary gas chromatography. When corn oil was fed, the major isomer in each group was 9,12-18:2, 11,14-20:2, 8,11,14-20:3, and 5,8,11,14-20:4. Patterns in EFAD- and PHSO-fed groups were more diverse, with large proportions of unusual isomers. Feeding EFA-deficient diet and PHSO induced measurable amounts of unusual PUFA at each step of the cascade, and these PUFA may compete in metabolism of normal PUFA and are substrates for oxidative formation of autacoids of unknown structures and function.
Reduction to homozygosity involving p53 in esophageal cancers demonstrated by the polymerase chain reaction.
Authors: Meltzer S J , Yin J , Huang Y , McDaniel T K , Newkirk C , Iseri O , Vogelstein B , Resau J H
Published: May 2006

Loss of heterozygosity affecting chromosome 17p has been detected at high frequencies in a variety of human tumors, including cancers of the colon, breast, lung, and brain. One presumed target of these losses is p53, a tumor suppressor gene located on 17p. To our knowledge, loss of heterozygosity has not yet been reported at any locus, including p53, in human esophageal cancer. Moreover, current methods of detecting loss of heterozygosity depend on the availability of large amounts of high molecular weight DNA, making the study of small biopsy specimens or paraffin-embedded tissues problematic. We examined 52 primary human esophageal neoplasms for loss of heterozygosity affecting the p53 gene by using the polymerase chain reaction. Loss of one allele was detected in 52% of informative cases and was more common in squamous carcinomas than in adenocarcinomas. Southern blot analysis was used to confirm polymerase chain reaction-derived data. The identification of allelic loss in approximately half of the tumors analyzed supports the hypothesis that inactivation of p53 is involved in the pathogenesis of esophageal cancer.
Effect of surgical menopause and estrogen replacement on cytokine release from human blood mononuclear cells.
Authors: Pacifici R , Brown C , Puscheck E , Friedrich E , Slatopolsky E , Maggio D , McCracken R , Avioli L V
Published: May 2006

To determine whether mononuclear cell secretory products contribute to the changes in bone turnover that characterize the development of postmenopausal osteoporosis, we evaluated the effects of oophorectomy and subsequent estrogen replacement on the spontaneous secretion of interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) and on the phytohemagglutinin A-induced secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) from peripheral blood mononuclear cells. In 15 healthy premenopausal women who underwent oophorectomy, increases in GM-CSF activity were observed as early as 1 week after surgery, whereas elevations in IL-1 and TNF-alpha and in hydroxyproline/creatinine and calcium/creatinine ratios, two urinary indices of bone resorption, were detectable 2 weeks after the surgical procedure. Six of the oophorectomized women received no estrogen therapy after surgery and in these subjects hydroxyproline/creatinine and calcium/creatinine ratios plateaued 6 weeks postoperatively, and all three cytokines reached the highest levels 8 weeks after oophorectomy, when the study ended. In the remaining 9 women, who were started on estrogen replacement therapy 4 weeks after oophorectomy, decreases in the indices of bone resorption paralleled decreases in the secretion of the cytokines, with lower levels detected after 2 weeks of therapy. In the women who did not receive estrogen therapy, circulating osteocalcin, a marker of bone formation, increased beyond preoperative levels 8 weeks after oophorectomy, whereas in the estrogen-treated subjects osteocalcin remained unchanged in the entire study period. In 9 female controls who underwent simple hysterectomy, cytokine release and biochemical indices of bone turnover did not change after surgery. These data indicate that changes in estrogen status in vivo are associated with the secretion of mononuclear cell immune factors in vitro and suggest that alterations in the local production of bone-acting cytokines may underlie changes in bone turnover caused by surgically induced menopause and estrogen replacement.
Polarized intestinal hybrid cell lines derived from primary culture: establishment and characterization.
Authors: Aponte G W , Keddie A , Halldén G , Hess R , Link P
Published: May 2006

A cell culture system has been developed that produces stable gastrointestinal (GI) polarized cell lines capable of maintaining hormone secretion. A spontaneously transformed rat mucosal epithelial cell was selected for hypoxanthine/guanine phosphoribosyltransferase deficiency and transfected with a plasmid conferring hygromycin resistance (BRIE 291 cells). Fusion of these cells with dispersed small intestinal epithelia cells resulted in hybrid cell lines that retained characteristic properties of the native GI cell more effectively than the transformed tumorigenic parental cell line. Hybrid hBRIE 380 cells are uniformly cuboidal with microvilli, contain villin, are contact inhibited, are anchorage dependent, require serum supplementation for growth, and are more sensitive to virus infection than the parental BRIE 291 cells. Fusion of BRIE 291 with dispersed pancreatic islet cells has resulted in a variety of pancreatic-hormone-producing cell lines. One of these, hybrid hBRIE 291-i2, forms confluent monolayers capable of synthesizing insulin-like immunoreactivity. These studies demonstrate that functionally polarized GI cells can be generated from primary cultures of nondividing committed epithelial cells by somatic cell hybridization and make feasible the selection and maintenance of specific GI epithelial cell types in confluent monolayer cultures.
Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: a model for adrenal growth regulation.
Authors: Mesiano S , Mellon S H , Gospodarowicz D , Di Blasio A M , Jaffe R B
Published: May 2006

Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in extracts of whole human fetal adrenal glands and primary cultures of human fetal adrenal cells. Northern blot analysis of total RNA from whole human fetal adrenal glands revealed a characteristic 7-kilobase bFGF mRNA, indicating that the fetal adrenal bFGF bioactivity was most likely due to local synthesis. Slot blot and ribonuclease protection analysis showed that bFGF mRNA was present in very low amounts in total RNA from primary cultures of unstimulated human fetal adrenal cells but was increased 2- to 3-fold in cells exposed to 10 nM ACTH-(1-24) or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate for 24 hr. bFGF mRNA was localized to adrenocortical cells and not fibroblasts by in situ hybridization. bFGF mRNA was barely detectable in unstimulated cells, whereas it was markedly increased in cells exposed to either ACTH or 8-bromoadenosine 3',5'-cyclic monophosphate. These data support our hypothesis that the regulation of human fetal adrenal growth by ACTH at midgestation may be mediated by the stimulation of local growth factor production, and we suggest that bFGF may play a major role in this process.
Putative receptor for the plant growth hormone auxin identified and characterized by anti-idiotypic antibodies.
Authors: Prasad P V , Jones A M
Published: May 2006

The molecular mechanism of action of auxins, which are an important class of plant growth regulators, is not known. While they are thought to influence cellular processes such as gene expression and membrane hyperpolarization by binding to specific receptors, no receptor mediating these processes has been unambiguously identified. We describe the identification of a putative auxin receptor by using anti-idiotypic antibodies. We have demonstrated that a 65-kilodalton protein found in a wide range of plants specifically binds auxins. Furthermore, since it is localized to the nucleus, it is a candidate for an auxin receptor that mediates some nuclear process.
Regulation of a fos-lacZ fusion gene: a paradigm for quantitative analysis of stimulus-transcription coupling.
Authors: Schilling K , Luk D , Morgan J I , Curran T
Published: May 2006

Expression of the c-fos protooncogene is induced by a great variety of extracellular stimuli. A fos-lacZ fusion gene has been constructed that recapitulates this regulation. The fos-lacZ gene was introduced into B104 neuroblastoma cells for use in a quantitative assay for stimulus-transcription coupling. Both alpha- and beta-adrenergic agonists, dibutyryl cAMP, and phorbol ester induced beta-galactosidase activity in a dose-dependent manner. Thus, the interactions of receptors with agonists and antagonists, as well as intracellular second messenger-mediated signaling events, can be analyzed quantitatively. This approach represents a prototypic method for investigating stimulus-response coupling based upon gene expression.
Functional complementation of ataxia-telangiectasia group D (AT-D) cells by microcell-mediated chromosome transfer and mapping of the AT-D locus to the region 11q22-23.
Authors: Lambert C , Schultz R A , Smith M , Wagner-McPherson C , McDaniel L D , Donlon T , Stanbridge E J , Friedberg E C
Published: May 2006

The hereditary human disease ataxia-telangiectasia (AT) is characterized by phenotypic complexity at the cellular level. We show that multiple mutant phenotypes of immortalized AT cells from genetic complementation group D (AT-D) are corrected after the introduction of a single human chromosome from a human-mouse hybrid line by microcell-mediated chromosome transfer. This chromosome is cytogenetically abnormal. It consists primarily of human chromosome 18, but it carries translocated material from the region 11q22-23, where one or more AT genes have been previously mapped by linkage analysis. A cytogenetically normal human chromosome 18 does not complement AT-D cells after microcell-mediated transfer, whereas a normal human chromosome 11 does. We conclude that the AT-D gene is located on chromosome 11q22-23.
Domain of a yeast site-specific recombinase (Flp) that recognizes its target site.
Authors: Chen J W , Evans B R , Yang S H , Teplow D B , Jayaram M
Published: May 2006

Binding of a partial proteolytic digest by V8 enzyme of the yeast site-specific recombinase Flp to its target site gives rise to DNA-protein complexes that are smaller than those produced by the full-sized protein. The smallest of these complexes (occupancy of one peptide monomer per site) contains either one of two polypeptides (32 and 28 kDa) of the V8 digestion mixture. The amino termini of both polypeptides map to Ser-129 of Flp, corresponding to V8 cleavage at Glu-128. The relative mobilities of the complexes formed by the V8 peptides indicate that they lack the sharp substrate bend that is characteristic of Flp-derived complexes. A hybrid protein consisting of the amino-terminal one-third of the R recombinase (from Zygosaccharomyces rouxii) and the carboxyl-terminal two-thirds of Flp recognizes the Flp target site.
Identification of aminopeptidase activity in the secretory granules of mouse mast cells.
Authors: Serafin W E , Guidry U A , Dayton E T , Kamada M M , Stevens R L , Austen K F
Published: May 2006

Sonicates of mouse bone marrow-derived mast cells (BMMC) differentiated in vitro and of mouse serosal mast cells differentiated in vivo contained small but approximately equal amounts of aminopeptidase activity, as determined by cleavage of leucine-beta-naphthylamide and resolution of the reaction products by reverse-phase high-performance liquid chromatography. Aminopeptidase activity was exocytosed from antigen-activated, IgE-sensitized BMMC in proportion to the secretory granule enzyme beta-hexosaminidase, thereby localizing approximately 60% of the total cell-associated aminopeptidase activity to the secretory granules of the mast cells. A prominent secretory granule location for aminopeptidase was confirmed by activity measurement in subcellular fractions of disrupted BMMC. The secretory granule aminopeptidase had a pH optimum of 6.0-8.0 and a Km of 0.36 +/- 0.06 mM (mean +/- SD; n = 3) for leucine-beta-naphthylamide. When various amino acid beta-naphthylamides were used as substrates, the preference of the secretory granule enzyme was Ala greater than Leu greater than Phe much greater than Arg much greater than Asp = Tyr. Most of the aminopeptidase activity that was exocytosed from calcium ionophore-activated BMMC was bound to 35S-labeled proteoglycans in complexes of greater than 1 x 10(7) kDa as defined by exclusion during Sepharose CL-2B gel-filtration chromatography. We postulate that the amino-peptidase in the mast cell protease/proteoglycan complexes allows the removal of N-terminal amino acids from peptides that are generated by the action of mast cell endopeptidases.
Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase.
Authors: Furman P A , Painter G , Wilson J E , Cheng N , Hopkins S
Published: May 2006

Substrate inhibition was observed with the heterodimeric (p66/p51) and the homodimeric (p66/p66, p51/p51) forms of human immunodeficiency virus type 1 reverse transcriptase (RNA-dependent DNA polymerase, EC 2.7.7.49). An apparent Ki value of 195 +/- 37 microM was determined for dTTP using the bacterial cloned and expressed heterodimer. Similar values were obtained with the homodimeric and the virus-encoded enzymes. When poly-(rC).p(dG)10 was used as template-primer, dGTP exhibited substrate inhibition with an apparent Ki value of 189 +/- 32 microM. Substrate inhibition was not observed with dTTP when DNA.DNA template-primers were used. Hill coefficients for substrate binding determined in the presence of saturating concentrations of template-primer were equal to 1.0, suggesting that substrate inhibition of the heterodimer is not the result of an allosteric mechanism involving the p51 subunit. Furthermore, UV crosslinking experiments with [gamma-32P]dTTP showed crosslinking only to the p66 subunit. Substrate inhibition was not as pronounced with other retroviral reverse transcriptases as it was with human immunodeficiency type 1 reverse transcriptase.
Retinoic acid is a negative regulator of AP-1-responsive genes.
Authors: Schüle R , Rangarajan P , Yang N , Kliewer S , Ransone L J , Bolado J , Verma I M , Evans R M
Published: May 2006

We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression.
Cytosolic rat brain synapsin I is a diacylglycerol kinase.
Authors: Kahn D W , Besterman J M
Published: May 2006

The phosphorylation of diacylglycerol (DG), a reaction catalyzed by DG kinase, may be critical in the termination of effector-induced signals mediated by protein kinase C. Synapsin I is a principal target of intracellular protein kinases and is thought to be involved in the release of neurotransmitter from axon terminals. We present several lines of evidence which indicate that rat brain synapsin, in addition to this role, may function as a DG kinase. Purified rat brain DG kinase was digested with trypsin, which produced three major fragments whose sequence was identical to three regions in synapsin I. Using a rabbit anti-synapsin polyclonal antiserum, the elution profile of synapsin immunoreactivity coincided exactly with that of DG kinase activity in column fractions from the final step in the DG kinase purification procedure. As is the case with synapsin, the purified enzyme was a strongly basic protein with an isoelectric point greater than 10.0. Finally, incubating the DG kinase with highly purified bacterial collagenase, an enzyme that partially degrades the proline- and glycine-rich synapsin, resulted in the simultaneous loss of DG kinase activity and synapsin immunoreactivity. We conclude that cytosolic rat brain synapsin is capable of functioning as a DG kinase.
The Barr body is a looped X chromosome formed by telomere association.
Authors: Walker C L , Cargile C B , Floy K M , Delannoy M , Migeon B R
Published: May 2006

We examined Barr bodies formed by isodicentric human X chromosomes in cultured human cells and in mouse-human hybrids using confocal microscopy and DNA probes for centromere and subtelomere regions. At interphase, the two ends of these chromosomes are only a micron apart, indicating that these inactive X chromosomes are in a nonlinear configuration. Additional studies of normal X chromosomes reveal the same telomere association for the inactive X but not for the active X chromosome. This nonlinear configuration is maintained during mitosis and in a murine environment.
Phosphatidate-dependent protein phosphorylation.
Authors: Bocckino S B , Wilson P B , Exton J H
Published: May 2006

Phosphatidate-dependent protein phosphorylation was observed in soluble extracts from rat liver, brain, lung, and testis. The phosphorylation was stimulated by free Ca2+ in the range of 360-800 nM. Incubation mixtures containing phosphatidate provided markedly different profiles of protein phosphorylation from those with phosphatidylserine plus 1,2-diolein. Phosphatidate-dependent phosphorylation of a 30-kDa protein in the soluble fraction from heart was also observed. This phosphorylation did not require Ca2+. Soluble fractions from liver, testis, brain, and lung phosphorylated the 30-kDa heart protein in a phosphatidate-dependent Ca(2+)-independent manner. We propose that part of the action of phosphatidate in cells may be mediated by a protein kinase(s).
Direct proliferative actions of stem cell factor on murine bone marrow cells in vitro: effects of combination with colony-stimulating factors.
Authors: Metcalf D , Nicola N A
Published: May 2006

Stem cell factor (SCF), the ligand for the c-kit protooncogene product, was able to stimulate blast cell and granulocytic colony formation by precursors from normal murine bone marrow. The blast cell colonies contained a high content of progenitor cells able to form macrophage and/or granulocyte colonies. Clone transfer studies, the secondary culture of colony cells, and the culture of populations freed of accessory cells all indicated a direct proliferative action of SCF. SCF receptors were present in high numbers on blast cells and in lower numbers on immature granulocytic, monocytic, and eosinophilic cells. Combination of SCF with granulocyte, granulocyte-macrophage, or multipotential colony-stimulating factors, but not macrophage colony-stimulating factor, resulted in enhancement of colony size. Granulocyte colony-stimulating factor enhanced cell proliferation initiated by SCF, but not vice-versa, and resulted in a 10-fold increase in colony cell numbers and a 7-fold increase in progenitor cells in blast colonies. No evidence was obtained that SCF, alone or in combination with granulocyte colony-stimulating factor, could stimulate self-generation by blast colony-forming cells.
Bacterial chemotaxis signaling complexes: formation of a CheA/CheW complex enhances autophosphorylation and affinity for CheY.
Authors: McNally D F , Matsumura P
Published: May 2006

We have demonstrated that a complex of the proteins CheA (CheAL and CheAS) and CheW can be isolated and constitutes a functional unit that responds to the signaling state of the chemoreceptors. The autophosphorylation rate of CheAL is much greater when CheAL and CheAS are complexed with CheW. Moreover, the presence of mutant chemoreceptors that cause cells to tumble increases this rate. At wild-type levels of expression, the isolated CheAL/CheAS/CheW complex accounts for about 10% of the total number of CheAL, CheAS, and CheW molecules and exists in a 1:1:1 stoichiometry. This complex is also required for CheAL/CheAS and CheW binding to the phosphorylation substrate, CheY. A separate interaction between CheY and another chemotaxis component, CheZ, was also detected. The CheY-CheZ interaction does not require participation of the CheAL/CheAS/CheW complex.
Detection of a key tertiary interaction in the highly conserved GTPase center of large subunit ribosomal RNA.
Authors: Ryan P C , Draper D E
Published: May 2006

Searches of ribosomal RNA sequences for compensatory base changes preserving Watson-Crick base pairing have led to detailed models of the conserved secondary structures of these RNAs. In principle, tertiary interactions can also be detected by searches for phylogenetically covariant bases. Within a highly conserved region of the large subunit ribosomal RNA termed the "GTPase center," the bases G-1056-U-1082.A-1086 are found in all eubacteria (Escherichia coli numbering), while A-1056.C-1082.G-1086 are found at the homologous positions in eukaryotes; archaebacteria fall into either category with some exceptions. Either sequence can potentially form a similar set of hydrogen bonds connecting the 3 bases. To determine the contribution of these 3 bases to RNA tertiary structure, sequence variants were made in RNA fragments covering the GTPase center. Correct folding of the RNA fragments was assayed by measuring the binding affinities of two different ligands that recognize the RNA tertiary structure: the highly conserved ribosomal protein L11, which is normally associated with the GTPase center RNA, and the peptide antibiotic thiostrepton, which inhibits the GTPase activity of eubacterial and some archaebacterial ribosomes. The results strongly support the existence of a base pair between positions 1082 and 1086: single mutations at either position weaken both L11 and thiostrepton binding by approximately 10-fold or more, while compensatory double mutations bind the ligands nearly as well as the wild-type E. coli sequence. Variants at position 1056 have little effect on either L11 or thiostrepton binding; a 3-base interaction is therefore not supported by these experiments. A base pair between positions 1082 and 1086 strongly constrains the geometry with which three helical segments join in the middle of the GTPase center.
Interleukin 7 receptor engagement stimulates tyrosine phosphorylation, inositol phospholipid turnover, proliferation, and selective differentiation to the CD4 lineage by human fetal thymocytes.
Authors: Uckun F M , Tuel-Ahlgren L , Obuz V , Smith R , Dibirdik I , Hanson M , Langlie M C , Ledbetter J A
Published: May 2006

The purposes of this study were to elucidate the effects of recombinant human interleukin 7 (rhIL-7) on proliferation as well as differentiation of human fetal thymocytes and to analyze the biochemical nature of the IL-7 receptor-linked transmembrane signal. In the absence of costimulants, rhIL-7 stimulated the in vitro proliferation and colony formation of CD4+CD8+ double-positive immature fetal thymocytes. Furthermore, rhIL-7 promoted partial differentiation of immature thymocytes with a selective advantage for the development of CD4+CD8- single-positive thymocytes. Our observations suggest that IL-7 likely has an important regulatory role during the earliest stages of human T-cell ontogeny. Stimulation of fetal thymocytes with rhIL-7 resulted in enhanced tyrosine phosphorylation of three distinct phosphoproteins with molecular masses of 72, 98, 123, and 190 kDa and induced a rapid and biphasic increase in the production of inositol 1,4,5-trisphosphate, which was inhibitable by the tyrosine protein kinase inhibitor genistein. Thus, the transmembrane signal triggered by engagement of the IL-7 receptor is intimately linked to a functional tyrosine protein kinase pathway and stimulates the inositol phospholipid turnover and proliferation, as well as selective differentiation to the CD4 lineage, by human fetal thymocytes.
Expression of biologically active human corticosteroid binding globulin by insect cells: acquisition of function requires glycosylation and transport.
Authors: Ghose-Dastidar J , Ross J B , Green R
Published: May 2006

Human corticosteroid binding globulin (hCBG) is a 50- to 55-kDa serum glycoprotein that binds cortisol and progesterone with high affinity. To map the steroid-binding domain and to investigate the folding pathways of hCBG, we have established an expression system based on infection of insect cells with a recombinant baculovirus encoding hCBG. Infected Spodoptera frugiperda (Sf9) cells secrete immunoreactive hCBG at high levels (16-24 pmol per 10(6) cells per 40 h), and the recombinant protein binds cortisol with an affinity and specificity equivalent to that of human serum-derived hCBG. Thus, this system has the potential to provide large amounts of wild-type and mutant hCBGs for physical-chemical analysis. Cotranslational asparagine-linked glycosylation is essential for acquisition of steroid-binding capability, as shown by the lack of cortisol-binding activity of unglycosylated hCBG secreted in the presence of tunicamycin. Golgi-associated oligosaccharide processing, however, is not required for activity, as demonstrated by the endoglycosidase H susceptibility of the fully active, secreted glycoprotein. Comparison of the steroid-binding properties of intracellular and secreted hCBG with that synthesized in vitro in the rabbit reticulocyte lysate system suggests that this protein undergoes a maturation process during transport through the secretory pathway. This system will be useful for identifying the molecular determinants of biological function in hCBG.
The herpes simplex virus 1 segment inversion site is specifically cleaved by a virus-induced nuclear endonuclease.
Authors: Wohlrab F , Chatterjee S , Wells R D
Published: May 2006

Nuclear extracts from several tissue culture cell lines (human, primate, and murine) contain an endonuclease that specifically cleaves sequences at the herpes simplex virus 1 (HSV-1) segment inversion site. Mapping studies identified the preferential site of cleavage as a set of tandemly repeated dodecamers, the DR2 repeats. Endonuclease levels vary according to the proliferative state of the cell; little or no activity is detectable in extracts from quiescent cells, whereas high levels are expressed in dividing cells. Also, infection of density-arrested BSC-1 cells with HSV-1 induces a substantial increase (at least 35-fold) in endonucleolytic activity, which is first detectable at about 1 hr after infection at 32 degrees C. The elevated levels of enzyme activity then persist throughout the viral life cycle. In addition to the HSV-1 DR2 repeats, certain other G+C-rich sequences with an asymmetric distribution of purines and pyrimidines on the DNA strands and with appropriate sequences and lengths are substrates for the nuclease. These data indicate that target site recognition by the enzyme is conformation specific rather than sequence specific.
Natriuretic peptide receptors regulate endothelin synthesis and release from parathyroid cells.
Authors: De Feo M L , Bartolini O , Orlando C , Maggi M , Serio M , Pines M , Hurwitz S , Fujii Y , Sakaguchi K , Aurbach G D
Published: May 2006

Cloned rat parathyroid cells (PTr cell line) that produce parathyroid hormone-related peptide plus endothelin 1 and primary cultures of human parathyroid cells were tested for growth and differentiation responses to atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). High- and low-affinity binding sites for ANP were found on PTr cells; BNP appeared to bind to the same receptors with similar affinities. Either ANP or BNP stimulated production of cGMP and caused a 30% decrease in Na(+)-K(+)-Cl- cotransport. Each peptide increased synthesis and secretion of endothelin 1 by PTr cells in a dose-dependent fashion, but cell growth was not affected. Human parathyroid cells (normal and pathological) also responded to ANP or BNP with an increase in cGMP production. The finding of receptors for natriuretic hormones on parathyroid cells with consequent effects on release of endothelin 1 might be of relevance in understanding the clinical association between hyperparathyroidism and hypertension.
Monomeric erythrocyte band 3 protein transports anions.
Authors: Lindenthal S , Schubert D
Published: May 2006

The anion transport system of the human erythrocyte membrane was reconstituted in egg phosphatidylcholine membranes by using either the unmodified transport protein, band 3, or covalently crosslinked band 3 dimers. Unilamellar vesicles of a diameter of 32 +/- 3 nm were then isolated from the sample by passage through a French press and subsequent gel filtration. According to sedimentation equilibrium measurements, around 85% of the vesicles were devoid of protein. The remaining 15% contained either a single band 3 monomer or, when crosslinked band 3 protein was used, a single band 3 dimer. Vesicles containing either single monomers or single dimers showed a rapid, inhibitor-sensitive sulfate efflux, and the turnover numbers of band 3 for the inhibitor-sensitive flux component were identical in both systems. This shows that monomeric band 3 protein is able to transport anions and that dimerization of the protein does not change its transport activity.
Expression of an antisense viral gene in transgenic tobacco confers resistance to the DNA virus tomato golden mosaic virus.
Authors: Day A G , Bejarano E R , Buck K W , Burrell M , Lichtenstein C P
Published: May 2006

Transgenic tobacco plants carrying a genetic cassette including an antisense DNA sequence of the virally encoded AL1 gene of the geminivirus tomato golden mosaic virus (TGMV) were constructed; AL1 encodes a protein absolutely required for TGMV DNA replication. These genetic cassettes also contained, on the same transcription unit, a gene encoding hygromycin resistance, which allowed selection for concomitant expression of the antisense gene. In transgenic lines, RNA transcripts of the predicted size and strand specificity were detected in antisense plants and sense controls. After infection of plants with TGMV, by agroinoculation, the frequency of symptom development was very significantly reduced in a number of antisense lines and correlated, broadly, with the abundance of antisense RNA transcript and with a reduction in viral DNA harvested from infected leaf tissue. We used an in vitro assay to study viral DNA replication in the absence of cell-to-cell spread; no replication was seen in five of the six antisense lines studied, in contrast to controls.