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Biomedical subjects

Y Zhao

Publications and source records attributed to Y Zhao.

At least 883 records · Page 49Linked to original sources
PubMed1995

Experimental study of NADPH-diaphorase positive neurons in nucleus accumbens of rats.

BACKGROUND: In order to explain the mechanism for the sparing of the nucleus accumbens in several pathologic conditions, the accumbens, caudate and putamen were examined in an experimental model. METHODS: The neurons in these regions from twenty adult rats were stained for the enzyme nicotinamide adenine dinucleotide phosphate (NADPH-diaphorase) and quantified. RESULTS: Positive neurons in the nucleus accumbens were more abundant than in the caudate and putamen. CONCLUSIONS: Since these neurons have been shown to be resistant to ischemia and degeneration, it is suggested that the mechanism for the sparing of the nucleus accumbens is related to the presence of a large number of NADPH-diaphorase positive neurons.

Animals↗
PubMed1995

Schizosaccharomyces pombe: a model for molecular studies of eukaryotic genes.

Several features of the fission yeast Schizosaccharomyces pombe make it exceptionally well suited for the study of eukaryotic genes. It is a relatively simple eukaryote that can be readily grown and manipulated in the laboratory, using a variety of highly developed and sophisticated methodologies. Schizosaccharomyces pombe cells share many molecular, genetic, and biochemical features with cells from multicellular organisms, making it a particularly useful model to study the structure, function, and regulation of genes from more complex species. For examples, this yeast divides by binary fission, has many genes that contain introns, is capable of using mammalian gene promoters and polyadenylation signals, and has been used to clone mammalian genes by functional complementation of mutants. We present a summary of the biology of S. pombe, useful features that make it amenable to laboratory studies, and molecular techniques available to manipulate the genome of this organism as well as other eukaryotic genes within the fission yeast cellular environment.

Cloning, Molecular↗
PubMed1995

Mus spretus LINE-1 sequences detected in the Mus musculus inbred strain C57BL/6J using LINE-1 DNA probes.

The inbred mouse strain, C57BL/6J, was derived from mice of the Mus musculus complex. C57BL/6J can be crossed in the laboratory with a closely related mouse species, M. spretus to produce fertile offspring; however there has been no previous evidence of gene flow between M. spretus and M. musculus in nature. Analysis of the repetitive sequence LINE-1, using both direct sequence analysis and genomic Southern blot hybridization to species-specific LINE-1 hybridization probes, demonstrates the presence of LINE-1 elements in C57BL/6J that were derived from the species M. spretus. These spretus-like LINE-1 elements in C57BL/6J reveal a cross to M. spretus somewhere in the history of C57BL/6J. It is unclear if the spretus-like LINE-1 elements are still embedded in flanking DNA derived from M. spretus or if they have transposed to new sites. The number of spretus-like elements detected suggests a maximum of 6.5% of the C57BL/6J genome may be derived from M. spretus.

Animals↗
PubMed1995

Cross-linking of the TCR-CD3 complex with CD4, CD8 or LFA-1 induces an anti-apoptotic signal in thymocytes: the signal is canceled by FK506.

The immunosuppressant FK506 did not block differentiation of double-negative thymocytes into double-positive (DP) cells, but interfered with differentiation of DP cells into mature single-positive cells in a fetal thymus organ culture system, suggesting that FK506 inhibits positive selection. The drug also reduced the number of DP cells recovered after the culture. As positive selection depends on the inhibition of thymocyte apoptosis at its DP stage by signaling through the TCR-CD3 complex and some of the accessory molecules, including CD4, CD8 and LFA-1, we studied the possibility that FK506 enhanced apoptosis by itself or canceled the inhibition of apoptosis. The results indicated that FK506 was hardly toxic or hardly affected anti-CD3-induced DNA fragmentation in isolated thymocytes in vitro. On the other hand, upon cross-linking TCR-CD3 together with CD4, CD8 or LFA-1, FK506 markedly enhanced DNA fragmentation and cytolysis. The drug, however, hardly or only slightly enhanced these responses upon cross-linking TCR-CD3 together with CD2, CD28, Thy-1 or H-2Kd. Cross-linking of TCR-CD3 together with CD4, CD8 or LFA-1 markedly inhibited glucocorticoid-induced death and the inhibition was canceled by FK506. Furthermore, cross-linking of TCR-CD3 together with LFA-1 potentially induces both an apoptosis-inducing signal and an FK506-sensitive anti-apoptotic signal, and that the latter signal may be related to an essential signal for positive selection.

Animals↗
PubMed1995

Expression and selective cellular localization of granulocyte-macrophage colony-stimulating factor (GM-CSF) and GM-CSF alpha and beta receptor messenger ribonucleic acid and protein in human ovarian tissue.

Reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemical observations revealed that human ovarian tissue expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as GM-CSF alpha and beta receptor (R) mRNA and protein. The RT-PCR products revealed the predicted 286-, 546-, and 380-bp fragments for GM-CSF, GM-CSF alpha R, and GM-CSF beta R, respectively, which were further verified by restriction enzyme digestion analysis. In situ hybridization revealed that the theca interna of the large follicles and luteal cells are the exclusive site of GM-CSF mRNA expression. Furthermore, immunohistochemical studies using specific monoclonal antibodies indicated that the theca interna of the large follicles are the exclusive site of GM-CSF protein, whereas theca externa and to a lesser extent the granulosa cells are the major sites of GM-CSF alpha R and beta R proteins. Atretic follicles and follicular cysts showed very low or no detectable levels of GM-CSF and GM-CSF alpha R and beta R. In the luteal tissue, both the small and large luteal cells of early luteal (Days 14-19) and midluteal (Days 20-25) phase expressed GM-CSF mRNA and protein as well as GM-CSF alpha R and beta R proteins, and their immunostaining intensity was similar to that seen with theca cells. Luteal cells from late luteal phase (Days 26-28), corpus albicans, and ectopic pregnancy expressed a low level of GM-CSF, GM-CSF alpha R, and GM-CSF beta R mRNA and protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1995

Actions of 1,4-dihydropyridines in isolated mesenteric vascular beds.

Recent studies indicate that the vasorelaxation induced by nitrendipine may be mediated partially by increased release of nitric oxide (NO). To study this effect in more detail and to examine the vasodilating effect of other 1,4-dihydropyridines (1,4-DHP) with regard to a possible involvement of NO, we investigated the effects of nitrendipine, nifedipine, nisoldipine, and nimodipine on isolated mesenteric vascular bed an the influence of L-NG-nitroarginine (L-NNA) on 1,4-DHP-induced vasorelaxation. Perfusion with these 1,4-DHP resulted in a concentration-dependent increase in global flow and vascular diameter in all vessel branches. Nifedipine exhibited a more pronounced effect on G4 vessels, whereas the actions of the other 1,4-DHP on the investigated vascular tree were more homogeneous. The dilating and flow increasing effects of nitrendipine and nifedipine could be significantly antagonized by treatment with LNNA. The vasodilating effects of nisoldipine and nimodipine could also be antagonized with L-NNA. We conclude that NO release plays an additional role in the relaxation of small resistance vessels by 1,4-DHP.

Animals↗
PubMed1995

Three antigenic regions in p17 of human immunodeficiency virus type 1 (HIV-1) revealed by mouse monoclonal antibodies and human antibodies in HIV-1 carrier sera.

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (KA = 1.9 x 10(5)-1.4 x 10(8) M-1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 x 10(5)-1.8 x 10(7) M-1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.

Amino Acid Sequence↗
PubMed1995

Regulatory function of the equine herpesvirus 1 ICP27 gene product.

The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in transient transfection assays. These assays demonstrated that the EHV-1 UL3 gene product is a regulatory protein that can independently trans activate the EHV-1 IE promoter; however, this effect can be inhibited by the repressive function of the IE gene product on the IE promoter (R. H. Smith, G. B. Caughman, and D. J. O'Callaghan, J. Virol. 66:936-945, 1992). In the presence of the IE gene product, the UL3 gene product significantly augments gene expression directed by the promoters of three EHV-1 early genes (thymidine kinase; IR4, which is the homolog of HSV-1 ICP22; and UL3 [ICP27]) and the promoter of the EHV-1 late gene IR5, which is the homolog of HSV-1 US10. Sequences located at nucleotides -123 to +20 of the UL3 promoter harbor a TATA box, SP1 binding site, CAAT box, and octamer binding site and, when linked to the CAT reporter gene, are trans activated to maximal levels by the pSVIE construct in transient expression assays. Results from CAT assays also suggest that the first 11 amino acids of the UL3 protein are not essential for the regulatory function of the UL3 gene product.

Animals↗
PubMed1995

RSK3 encodes a novel pp90rsk isoform with a unique N-terminal sequence: growth factor-stimulated kinase function and nuclear translocation.

A novel pp90rsk Ser/Thr kinase (referred to as RSK3) was cloned from a human cDNA library. The RSK3 cDNA encodes a predicted 733-amino-acid protein with a unique N-terminal region containing a putative nuclear localization signal. RSK3 mRNA was widely expressed (but was predominant in lung and skeletal muscle). By using fluorescence in situ hybridization, the human RSK3 gene was localized to band q27 of chromosome 6. Hemagglutinin epitope-tagged RSK3 was expressed in transiently transfected COS cells. Growth factors, serum, and phorbol ester stimulated autophosphorylation of recombinant RSK3 and its kinase activity toward several protein substrates known to be phosphorylated by RSKs. However, the relative substrate specificity of RSK3 differed from that reported for other isoforms. RSK3 also phosphorylated potential nuclear target proteins including c-Fos and histones. Furthermore, although RSK3 was inactivated by protein phosphatase 2A in vitro, the enzyme was not activated by ERK2/mitogen-activated protein (MAP) kinase. In contrast, the kinase activity of another epitope-tagged RSK isoform (RSK-1) was significantly increased by in vitro incubation with ERK2/MAP kinase. Finally, we used affinity-purified RSK3 antibodies to demonstrate by immunofluorescence that endogenous RSK3 undergoes serum-stimulated nuclear translocation in cultured HeLa cells. These results provide evidence that RSK3 is a third distinct isoform of pp90rsk which translocates to the cell nucleus, phosphorylates potential nuclear targets, and may have a unique upstream activator. RSK3 may therefore subserve a discrete physiologic role(s) that differs from those of the other two known mammalian RSK isoforms.

Amino Acid Sequence↗
PubMed1995

TGF-beta regulates expression of tenascin alternative-splicing isoforms in fetal rat lung.

Two distinct mRNA-splice isoforms of tenascin (TN) are expressed differentially during rat lung development. The unique temporal expression pattern of two TN isoforms suggests the expression of tenascin is strictly developmentally regulated in rat lung tissue. We investigated molecular mechanisms which modulate alternative-splicing expression of TN in lung development. The effect of transforming growth factor-beta 1 (TGF-beta 1) on regulation of expression of TN isoforms was examined by in vitro lung explant culture. Immunoblotting with anti-TN antibody detected two TN polypeptides in rat lung explant culture, the larger [relative molecular weight (M(r)) 230, TN230] polypeptide and the smaller (M(r) 180, TN180). TGF-beta 1 markedly induced the TN180 isoform and caused only a moderate increase of the TN230 isoform. The effects of TGF-beta 1 were shown to be dose dependent over a physiological range of TGF-beta 1 protein concentration. The induction of TN isoform biosynthesis by TGF-beta 1 was detected 12 h after addition of the growth factor, and the effects endured for up to 48 h at a dose of 5 ng/ml. By reverse transcriptase-polymerase chain reaction through amplification of the entire fibronectin type III (FN-III) splicing domain, two distinct TN isoforms were detected in total RNA isolated from gestational day 21 rat lung explant culture treated with TGF-beta 1 and from postnatal day 8 rat lung. The larger isoform contained five FN-III alternative splicing repeats [1,420 base pairs (bp)], but the shorter splicing isoform lacked four FN-III alternative splicing repeats (340 bp).(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative Splicing↗
PubMed1995

Expression of transforming growth factor-beta type II receptor in rat lung is regulated during development.

Transforming growth factor-beta (TGF-beta) is an autocrine/paracrine growth factor that regulates cell proliferation, differentiation, extracellular matrix production and various other cell functions in the lung. TGF-beta exerts its effects on cells by binding to transmembrane heteromeric serine-threonine kinase receptors. The expression and localization of specific TGF-beta receptors in the lung, however, have not yet been investigated. In the present studies, we isolated a 1,762-base pair cDNA containing the full-length coding sequence for TGF-beta type II receptor (T beta RII) from rat fetal lung with the use of polymerase chain reaction methods. The expression of T beta RII during lung development was examined by Northern analysis. A 5.1-kilobase T beta RII mRNA was detected in rat lung tissue. T beta RII mRNA was expressed in rat fetal lung tissue early in development, increased as development proceeded, reached maximal concentration postnatally, and then decreased to the adult level. The localization of T beta RII in fetal and postnatal rat lung tissue was investigated with the use of in situ hybridization performed with an antisense RNA probe. T beta RII gene was expressed in the mesenchymal tissue and in the epithelial lining of the developing airway at day 16 of gestation. The hybridization signal of T beta RII mRNA was also observed in the adventitial layer of small blood vessels. Expression of T beta RII gene in the developing airway epithelium occurred along a proximal-distal gradient. In postnatal lung, T beta RII mRNA was detected mainly in parenchymal tissues and blood vessels. Expression of T beta RII remained high in the interstitium of interalveolar septa.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1995

Tenascin in rat lung development: in situ localization and cellular sources.

Tenascin (TN) is a hexameric extracellular matrix glycoprotein that may play an important role during lung development. TN protein is temporally and spatially restricted during lung organogenesis. The temporo-spatial and cellular expression of TN mRNA in lung remains unclear. Localization of message expression of TN in rat lung tissue was first investigated by using in situ hybridization performed with an antisense RNA probe. TN mRNA was present primarily within the mesenchyme of day 16 gestational age fetal rat lung tissue, whereas immunoreactive TN protein was found along the basement membrane. In postnatal day 3 rat lung tissue, TN mRNA was detected along alveolar septal walls and was concentrated at secondary septal tips. Expression of TN message was consistent with localization of immunoreactive TN protein. Accumulation of TN mRNA in alveolar septal tips suggests that mesenchyme may be the major source of TN mRNA. To investigate the cellular source of TN in rat lung, we studied the expression of TN in cultured rat lung fibroblasts, endothelial cells, and alveolar epithelial cells. Two TN isoforms having molecular mass of 230 and 180 kDa were in conditioned medium and in cellular extracts of lung fibroblasts and endothelial cells. TN was secreted and deposited in the extracellular matrix closely associated with the surface of lung fibroblasts and endothelial cells. Lung alveolar epithelial cells showed undetectable or barely detectable amounts of TN. These studies demonstrated that TN isoforms are expressed not only by lung fibroblasts but also by lung endothelial cells. The unique spatial localization of TN mRNA during lung development and expression of TN by different lung cell types suggested TN may be involved in matrix organization and cell-cell interactions during lung development.

Aging↗
PubMed1995

Characterization of recombinant human apoB-48-containing lipoproteins in rat hepatoma McA-RH7777 cells transfected with apoB-48 cDNA. Overexpression of apoB-48 decreases synthesis of endogenous apoB-100.

We studied the effect of overexpression of apolipoprotein (apo) B-48 on the synthesis and secretion of endogenous apoB-100 in rat hepatoma McA-RH7777 cell lines stably transfected with human apoB-48 cDNA under the control of the cytomegalovirus promoter. Three cell lines that secrete 40 to 60 ng human apoB.mg cell protein-1.h-1 were used. The recombinant human apoB-48 exhibited physicochemical characteristics (buoyant density, 1.06 to 1.21 g/mL; beta-electrophoretic mobility and diameters, 16 to 20 nm) indistinguishable from those of endogenous rat apoB-48. Overexpression of the recombinant human apoB-48 resulted in a 50% decrease in the secretion of endogenous apoB-100 but did not affect the secretion of apoE or apoA-I. Several possible mechanisms for the decreased secretion of apoB-100 were evaluated. First, recruitment of lipids into lipoproteins was shown to be unaffected since no major changes in the physicochemical properties of apoB-100-containing lipoproteins were observed. Second, the intracellular degradation of apoB-100 was not altered as the intracellular retention half-time and secretion efficiency remained unaffected by apoB-48 overexpression. Third, the posttranslational regulatory mechanisms for apoB-100 remained normal, as demonstrated by a twofold increase in apoB-100 secretion after supplementation with oleic acid. Unexpectedly, a 35% to 50% decrease in the steady-state synthesis of endogenous apoB-100 was observed in apoB-48-transfected cells compared with control cells. These data suggested that decreased secretion of apoB-100 was secondary to decreased synthesis. The decreased apoB-100 synthesis was not due to decreased steady-state levels of rat apoB-100 mRNA. These results suggest that overexpression of recombinant human apoB-48 may interfere with posttranscriptional events, possibly at the translation-translocation level, and decrease translational yield of apoB-100. These posttranscriptional events prior to the complete synthesis of the apoB-100 polypeptide can be important in the control of apoB-100 secretion.

Amino Acid Sequence↗