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Biomedical subjects

Y Zhao

Publications and source records attributed to Y Zhao.

At least 469 records · Page 26Linked to original sources
PubMed1999

Finite element analysis of the lift on a slightly deformable and freely rotating and translating cylinder in two-dimensional channel flow.

Motivated by the lateral migration phenomena of fresh and glutaraldehyde-fixed red blood cells in a field flow fractionation (FFF) separation system, we studied the transverse hydrodynamic lift on a slightly flexible cylinder in a two-dimensional channel flow. The finite element method was used to analyze the flow field with the cylinder at different transverse locations in the channel. The shape of the cylinder was determined by the pressure on the surface of the cylinder from the flow field solution and by the internal elastic stress. The cylinder deformation and the flow field were solved simultaneously. The transverse lift exerted on the cylinder was then calculated. The axial and angular speed of the cylinder were iterated such that the drag and torque on the cylinder were nulled to represent a freely translating and rotating state. The results showed that the transverse lift on a deformable cylinder increased greatly and the equilibrium position moved closer to the center of the channel compared to a rigid cylinder. Also, with the same elastic modulus but a higher flow rate, a larger deformation and higher equilibrium location were found. The maximum deformation of the cylinder occurred when the cylinder was closest to the wall where a larger shear rate existed. The numerical results and experimental studies are discussed.

Blood Flow Velocity↗
PubMed1999

Endothelial cell-specific transcriptional targeting from a hybrid long terminal repeat retrovirus vector containing human prepro-endothelin-1 promoter sequences.

For many applications, specificity of gene expression by recombinant retroviral vectors is necessary. We wished to obtain transcriptional targeting in endothelial cells as part of an antivasculature approach to cancer treatment and have achieved specificity by using the promoter for human prepro-endothelin-1. In particular, we have inserted this heterologous promoter within the 3' long terminal repeat (LTR), replacing all viral upstream transcriptional regulatory sequences, to generate a hybrid LTR with precise fusion at the TATA box for initiation of transcription at the viral start site. Reverse transcription and integration resulted in duplication of this hybrid promoter in the 5' LTR of the provirus for transcription of the internal transgene. An important feature of our vectors is the absence of a selectable marker gene or additional promoters to avoid potential complications of silencing or interference and because selection will be inappropriate for clinical application. This vector design showed endothelial cell specificity of beta-galactosidase expression when tested on a panel of human cell lines and primary breast microvascular endothelial cells, matching the specificity of expression of the endogenous promoter. Such simplified vectors exhibiting transcriptional specificity are likely to be useful for the development of a gene therapy approach to targeting tumor vasculature.

3T3 Cells↗
PubMed1999

Mutational analysis of Vpr-induced G2 arrest, nuclear localization, and cell death in fission yeast.

Cell cycle G2 arrest, nuclear localization, and cell death induced by human immunodeficiency virus type 1 Vpr were examined in fission yeast by using a panel of Vpr mutations that have been studied previously in human cells. The effects of the mutations on Vpr functions were highly similar between fission yeast and human cells. Consistent with mammalian cell studies, induction of cell cycle G2 arrest by Vpr was found to be independent of nuclear localization. In addition, G2 arrest was also shown to be independent of cell killing, which only occurred when the mutant Vpr localized to the nucleus. The C-terminal end of Vpr is crucial for G2 arrest, the N-terminal alpha-helix is important for nuclear localization, and a large part of the Vpr protein is responsible for cell killing. It is evident that the overall structure of Vpr is essential for these cellular effects, as N- and C-terminal deletions affected all three cellular functions. Furthermore, two single point mutations (H33R and H71R), both of which reside at the end of each alpha-helix, disrupted all three Vpr functions, indicating that these two mutations may have strong effects on the overall Vpr structure. The similarity of the mutant effects on Vpr function in fission yeast and human cells suggests that fission yeast can be used as a model system to evaluate these Vpr functions in naturally occurring viral isolates.

Cell Death↗
PubMed1999

A sodium-dependent neutral-amino-acid transporter mediates infections of feline and baboon endogenous retroviruses and simian type D retroviruses.

The type D simian retroviruses cause immunosuppression in macaques and have been reported as a presumptive opportunistic infection in a patient with AIDS. Previous evidence based on viral interference has strongly suggested that the type D simian viruses share a common but unknown cell surface receptor with three type C viruses: feline endogenous virus (RD114), baboon endogenous virus, and avian reticuloendotheliosis virus. Furthermore, the receptor gene for these viruses has been mapped to human chromosome 19q13.1-13.2. We now report the isolation and characterization of a cell surface receptor for this group of retroviruses by using a human T-lymphocyte cDNA library in a retroviral vector. Swiss mouse fibroblasts (NIH 3T3), which are naturally resistant to RD114, were transduced with the retroviral library and then challenged with an RD114-pseudotyped virus containing a dominant selectable gene for puromycin resistance. Puromycin selection yielded 12 cellular clones that were highly susceptible to a beta-galactosidase-encoding lacZ(RD114) pseudotype virus. Using PCR primers specific for vector sequences, we amplified a common 2.9-kb product from 10 positive clones. Expression of the 2.9-kb cDNA in Chinese hamster ovary cells conferred susceptibility to RD114, baboon endogenous virus, and the type D simian retroviruses. The 2.9-kb cDNA predicted a protein of 541 amino acids that had 98% identity with the previously cloned human Na+-dependent neutral-amino-acid transporter Bo. Accordingly, expression of the RD114 receptor in NIH 3T3 cells resulted in enhanced cellular uptake of L-[3H]alanine and L-[3H]glutamine. RNA blot (Northern) analysis suggested that the RD114 receptor is widely expressed in human tissues and cell lines, including hematopoietic cells. The human Bo transporter gene has been previously mapped to 19q13.3, which is closely linked to the gene locus of the RD114 receptor.

3T3 Cells↗
PubMed1999

Receptor-mediated Moloney murine leukemia virus entry can occur independently of the clathrin-coated-pit-mediated endocytic pathway.

To investigate receptor-mediated Moloney murine leukemia virus (MoMuLV) entry, the green fluorescent protein (GFP)-tagged ecotropic receptor designated murine cationic amino acid transporter (MCAT-1) (MCAT-1-GFP) was constructed and expressed in 293 cells (293/MCAT-1-GFP). 293/MCAT-1-GFP cells displayed green fluorescence primarily at the cell membrane and supported wild-type levels of MoMuLV vector binding and transduction. Using immunofluorescence labeling and confocal microscopy, it was demonstrated that the surface envelope protein (SU) gp70 of MoMuLV virions began to appear inside cells 5 min after virus binding and was colocalized with MCAT-1-GFP. However, clathrin was not colocalized with MCAT-1-GFP, suggesting that MoMuLV entry, mediated by MCAT-1, does not involve clathrin. Double immunofluorescence labeling of SU and clathrin in 293 cells expressing untagged receptor (293/MCAT-1) gave the same results, i.e., SU and clathrin did not colocalize. In addition, we examined the transduction ability of MoMuLV vector on HeLa cells overexpressing the dominant-negative GTPase mutant of dynamin (K44A). HeLa cells overexpressing mutant dynamin have a severe block in endocytosis by the clathrin-coated-pit pathway. No significant titer difference was observed when MoMuLV vector was tranduced into HeLa cells overexpressing either wild-type or mutant dynamin, while the transduction ability of vesicular stomatitis virus glycoprotein pseudotyped vector into HeLa cells overexpressing mutant dynamin was decreased significantly. Taken together, these data suggest that MoMuLV entry does not occur through the clathrin-coated-pit-mediated endocytic pathway.

3T3 Cells↗
PubMed1999

Role of Ca(2+) channels in NE-induced increase in [Ca(2+)](i) and tension in fetal and adult cerebral arteries.

In vascular smooth muscle, elevation of agonist-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) occurs via both Ca(2+) release from intracellular stores and Ca(2+) influx across the plasma membrane. In the cerebral vasculature of the fetus and adult the relative roles of these mechanisms have not been defined. To test the hypothesis that plasma membrane L-type and receptor-operated Ca(2+) channels play a key role in NE-induced vasoconstriction via alterations in plasma membrane Ca(2+) flux and that this may change with developmental age, we performed the following study. In main branch middle cerebral arteries (MCA) from near-term fetal ( approximately 140 days) and nonpregnant adult sheep, we quantified NE-induced responses of vascular tension and [Ca(2+)](i) (by use of fura 2) under standard conditions in response to several Ca(2+) channel blockers and in response to zero extracellular Ca(2+). In fetal and adult MCA, maximal NE-induced tensions (g) were 0.91 +/- 0.12 (n = 10) and 1.61 +/- 0.13 (n = 12), respectively. The pD(2) values for NE-induced tension were both 6.0 +/- 0.1, whereas the fetal and adult maximum responses (%K(max)) were 107 +/- 16 and 119 +/- 7, respectively. The fetal and adult pD(2) values for NE-induced increase of [Ca(2+)](i) were 6.2 +/- 0.1 and 6.4 +/- 0.1, respectively, whereas maximum [Ca(2+)](i) responses were 81 +/- 9 and 103 +/- 15% of K(max), respectively. After 10(-5) M NE-induced contraction, nifedipine resulted in dose-dependent decrease in vessel tone and [Ca(2+)](i) with pIC(50) values for fetal and adult tensions of 7.3 +/- 0.1 and 6.6 +/- 0.1, respectively (P < 0.01; n = 4 each), whereas pIC(50) for [Ca(2+)](i) responses were 7.2 +/- 0.1 and 6.9 +/- 0.1, respectively. The pIC(50) values for tension for diltiazem and verapamil were somewhat lower but showed a similar relationship. The receptor-operated Ca(2+) channel blocker 2-nitro-4 carboxyphenyl-N,N-diphenyl carbamate showed little effect on NE-induced vessel contractility or [Ca(2+)](i). In the absence of extracellular Ca(2+) for 2 min, 10(-5) M NE resulted in markedly attenuated responses of adult MCA tension and [Ca(2+)](i) to 39 +/- 7 and 73 +/- 8% of control values (n = 4). For fetal MCA, exposure to extracellular Ca(2+) concentration resulted in essentially no contractile or [Ca(2+)](i) response (n = 4). Similar blunting of NE-induced tension and [Ca(2+)](i) was seen in response to 10(-3) M lanthanum ion. These findings provide evidence to suggest that especially in fetal, but also in adult, ovine MCA, Ca(2+) flux via L-type calcium channels plays a key role in NE-induced contraction. In contrast, Ca(2+) flux via receptor-operated Ca(2+) channels is of less importance. This developmental difference in the role of cerebrovascular plasma membrane Ca(2+) channels may be an important association with increased Ca(2+) sensitivity of the fetal vessels.

Age Factors↗
PubMed1999

beta-hexosaminidase-induced activation of p44/42 mitogen-activated protein kinase is dependent on p21Ras and protein kinase C and mediates bovine airway smooth-muscle proliferation.

Late-phase and sustained activation of p44/42(MAPK) has been reported to be a critical factor in cell mitogenesis. We therefore hypothesized that p44/42(MAPK) is involved in mannosyl-rich glycoprotein-induced mitogenesis in bovine airway smooth-muscle cells (ASMC). Treatment of adherent ASMC with beta-hexosaminidase A (Hex A, 50 nM), an endogenous mannosyl-rich glycoprotein, resulted in a late-onset (30-min) activation of p44/42(MAPK) that lasted for 4 h. Activation of p44/42(MAPK) induced by Hex A was inhibited by an 18-mer phosphorothioate-derivatized antisense oligonucleotide (1-5 microM) directed to human p44(MAPK); the mitogen-activated protein kinase kinase (MEK1) inhibitor PD98059 (5 microM); the p42(MAPK) inhibitor Tyrphostin AG-126 (0.2 microM); the farnesyl transferase inhibitors SCH-56582 (10 microM) and FPT III (10 miroM), which inhibit p21Ras activation; and Calphostin C (0.2 microM), an inhibitor of protein kinase C. These agents also inhibited Hex A-induced cell proliferation in bovine ASMC. These data suggest that Hex A activates p44/42(MAPK) in a p21Ras- and PKC-dependent manner and that this activation mediates Hex A- induced mitogenesis in bovine ASMC.

Alkyl and Aryl Transferases↗
PubMed1999

Characterization of novel cathepsin K mutations in the pro and mature polypeptide regions causing pycnodysostosis.

Cathepsin K, a lysosomal cysteine protease critical for bone remodeling by osteoclasts, was recently identified as the deficient enzyme causing pycnodysostosis, an autosomal recessive osteosclerotic skeletal dysplasia. To investigate the nature of molecular lesions causing this disease, mutations in the cathepsin K gene from eight families were determined, identifying seven novel mutations (K52X, G79E, Q190X, Y212C, A277E, A277V, and R312G). Expression of the first pro region missense mutation in a cysteine protease, G79E, in Pichia pastoris resulted in an unstable precursor protein, consistent with misfolding of the proenzyme. Expression of five mature region missense defects revealed that G146R, A277E, A277V, and R312G precursors were unstable, and no mature proteins or protease activity were detected. The Y212C precursor was activated to its mature form in a manner similar to that of the wild-type cathepsin K. The mature Y212C enzyme retained its dipeptide substrate specificity and gelatinolytic activity, but it had markedly decreased activity toward type I collagen and a cathepsin K-specific tripeptide substrate, indicating that it was unable to bind collagen triple helix. These studies demonstrated the molecular heterogeneity of mutations causing pycnodysostosis, indicated that pro region conformation directs proper folding of the proenzyme, and suggested that the cathepsin K active site contains a critical collagen-binding domain.

Cathepsin K↗
PubMed1999

Why is erythropoietin present in human milk? Studies of erythropoietin receptors on enterocytes of human and rat neonates.

Erythropoietin receptors (Epo-R) are expressed on cells in the small bowel of human fetuses, but their function has not been defined. We hypothesized that intestinal Epo-R are present postnatally, and that recombinant erythropoietin (rEpo) would increase enterocyte migration and decrease cytokine-induced apoptosis. We used reverse transcriptase-polymerase chain reaction and immunohistochemistry to evaluate the presence of Epo-R mRNA and protein in rat intestinal epithelial cells (IEC-6), and in postnatal human and rat bowel. The effect of rEpo on rates of cell migration and proliferation were established in IEC-6 cells by using cell counting and incorporation of bromodeoxyuridine. To determine whether rEpo affects response to injury, cells were pretreated with rEpo, then were damaged with 25 or 50 ng/mL tumor necrosis factor-alpha plus 2.5 microg/mL cycloheximide. Cell death was determined by colorimetric bioassay. We found that Epo-R mRNA and protein were expressed by IEC-6 cells and by enterocytes of postnatal rat and human small bowel. Cells that had been exposed to 0.05 or 5.00 U/mL rEpo migrated faster than did the controls (p < 0.05), but no difference was noted in cell proliferation. Treatment of IEC-6 cells with rEpo before or at the time of injury resulted in a lower percentage of cell death, and this effect was neutralized by anti-Epo antibody. We conclude that Epo-R is expressed in enterocytes postnatally in rats and humans. Recombinant Epo increases the rate of migration of IEC-6 cells and decreases cytokine-induced apoptosis. These studies suggest that Epo within human milk has actions on neonate's intestinal function.

Animals↗
PubMed1999

The study of maternal and fetal plasma catecholamines levels during pregnancy and delivery.

BACKGROUND: To study maternal and fetal plasma levels of catecholamines (CA) during pregnancy and delivery, especially changes in CA levels during fetal distress and conditions of different modes of delivery. METHODS: Maternal and fetal plasma NE, E and DA levels were determined by high performance liquid chromatography (HPLC) for 16 non-pregnant women, 19 cases of early pregnancy, 17 cases of mid pregnancy, late pregnancy, spontaneous vaginal delivery and 53 cases of cesarean section. RESULTS: Plasma NE and DA levels decreased gradually with the advance of gestational weeks, and levels of plasma NE were significantly lower than those of non-pregnant women (P < 0.05). The levels of plasma CA in patients who had elective cesarean section were significantly lower than those who had vaginal delivery and emergency cesarean section (P < 0.01). However, CA levels of the cord artery in the vaginal delivery group were significantly higher than those in the cesarean section group (P < 0.01). CONCLUSION: Vaginal delivery is better than cesarean section for the newborn. If cesarean section is necessary, it is best for the newborn after onset of labor.

Adult↗
PubMed1999

Oestrogen and progesterone receptor immunoreactivity and c-fos expression in the ovine cervix.

Immunocytochemistry was used to detect the presence of oestrogen and progesterone receptors in the cervices of prepubertal lambs, seasonally anoestrous ewes, cyclic ewes, and pregnant ewes of known gestational stages, to define the roles of gonadal steroids in cervical function. The presence of the immediate early gene product, c-Fos, a marker for cellular activation, was also investigated using immunocytochemistry and in situ hybridization. Oestrogen receptor immunoreactivity was restricted to the endometrium on days 0-3 of the oestrous cycle (day 0 = oestrus). In immature animals, very few scattered nuclei in the endometrium were immunoreactive. Oestrogen receptor immunoreactivity was not apparent in the endometrium during the remainder of the oestrous cycle or in this region in anoestrous animals. In pregnant ewes, oestrogen receptor immunostaining appeared as relatively few isolated nuclei in the connective tissue stroma. Progesterone receptor immunoreactivity was found in the endometrium at days 0-3 of the oestrous cycle and also in the luminal epithelium, the myometrium and the blood vessels. Progesterone receptor immunoreactivity was also found in these regions, with the exception of the endometrium, at all other stages examined. Immunostaining for c-Fos was present in the endometrium at days 0-3 of the oestrous cycle, and some scattered immunopositive nuclei were present in prepubertal animals. c-Fos immunoreactivity was also found in the myometrium and in blood vessels at all other stages examined. Visualization of c-fos gene expression by in situ hybridization showed that it occurred in the luminal epithelium and blood vessels at oestrus, but was restricted to the blood vessels in all other samples examined.

Anestrus↗
PubMed1999

Strong immunoreactivity of platelet-derived growth factor and its receptor at human and mouse neuromuscular junctions.

Platelet-derived growth factor (PDGF) and its alpha-receptor were localized at human and mouse neuromuscular junctions (NMJs) using specific polyclonal antibodies against each, anti-PDGF-A and anti-PDGF alpha-receptor, respectively. By applying double fluorescence labeling, immunoreactivity for PDGF and its receptor was closely co-localized with acetylcholine receptors, which were identified with alpha-bungarotoxin. PDGF might be involved in the interaction between the presynaptic and postsynaptic components. This is the first demonstration of PDGF and its receptor concentrated at human and mouse NMJs.

Animals↗
PubMed1999

[Rapid detection of non-deletional alpha-thalassemia mutations by PCR-LIS-SSCP].

OBJECTIVE: To establish a rapid and convenient single strand conformation polymorphism (SSCP) analysis method for detecting the point mutation of non-deletional alpha-thalassemia. METHODS: The 543bp DNA fragment spanning the hot spot region mainly responsible for non-deletional alpha-thalassemia was nested amplified using the selective amplification of alpha2 globin gene as a template and was denatured with low ionic strength(LIS) solution followed by SSCP analysis. RESULTS: LIS buffer was more efficient for ssDNA formation than formamide buffer was and the formation of ssDNA was very stable. In addition to a normal electrophoresis pattern, at least three SSCP profiles can be detected by the present method when the DNA samples bearing non-deletional genes of Hb H disease were screened. Confirmed by DNA sequencing analysis, the DNAs represented these profiles have turned out to be the different three mutants, i.e., the alpha&supCS; mutation, the alpha &supQS; mutation and the alpha&supWestmead; mutation, respectively. Only 3 hours were needed to complete the electrophoresis procedure of this method. CONCLUSION: PCR-LIS-SSCP can be used as a tool in rapid screening for the alterations in human alpha-globin gene.

Humans↗
PubMed1999

LDL receptor binds newly synthesized apoE in macrophages. A precursor pool for apoe secretion.

There appear to be multiple post-translational sites for regulation of macrophage apolipoprotein (apo)E secretion, including the presence of a distinct cell surface pool of apoE. Cell surface proteoglycans have been shown to be involved in forming this pool. The current studies were designed to investigate the role of an additional cell surface site, i.e., the low density lipoprotein (LDL) receptor. Antiserum to the LDL receptor displaced apoE from the macrophage cell surface and into the medium during a 4 degrees C incubation from apoE-expressing J774 cells, from proteoglycan-depleted apoE-expressing J774 cells, and from human monocyte-derived macrophages. Similar results were obtained when purified monoclonal antibody to the LDL receptor was added to human monocyte-derived macrophages. J774 cells transfected to express an LDL receptor binding-defective mutant of apoE did not show a similar response to addition of LDL receptor antibody. Studies were conducted in which cells were pulse labeled for 30 min, followed by various periods of chase at 4 degrees C or 37 degrees C in the presence or absence of LDL receptor antibody. The results of these studies indicated that nascent macrophage-derived apoE binds to the LDL receptor, and that this apoE served as a precursor pool for apoE released into the medium. These studies establish a role for the LDL receptor in forming the cell surface pool of apoE and, along with data regarding the importance of proteoglycans, indicate that cell surface binding sites for nascent macrophage-derived apoE are heterogeneous. The heterogeneity of such sites could have implication for the size and turnover of this cell surface pool.

Animals↗
PubMed1999

[Mutation in the promoter region of 21-hydroxylase gene of patients with congenital adrenal hyperplasia].

OBJECTIVE: To investigate the mutations in the 21-hydroxylase gene promoter region of patients with congenital adrenal hyperplasia (CAH). METHODS: The authors amplified a 1.6 kb fragment of the CYP21 gene which was confirmed by endonuclease digestion. The PCR product was re-amplified with promoter-region-specific primers. The amplified fragments were analyzed using SSCP and endonuclease digestion methods. RESULTS: Among 12 patients, 6 patients presented an abnormal band on SSCP analysis. By endonuclease digestion test, the authors found a patient who presented one mutation in KpnI recognition site located in the CK-2 (-101) binding region and one in Taq I recognition site located -201. These mutations were confirmed by sequencing. CONCLUSION: Novel mutations were found in the promoter of 21-hydroxylase gene in CAH patients and the findings spurred on further expression studies.

Adrenal Hyperplasia, Congenital↗
PubMed1999

[Effects of retrovirus-mediated HSV-tk/GCV on human hepatocellular carcinoma].

OBJECTIVE: The efficacy of the herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) suicide-gene therapy system on human hepatocellular carcinoma was observed in vitro as well as ex vivo. METHODS: The recombinant retroviral vector containing HSV-tk gene was constructed and HCC9204 human hepatocellular carcinoma cell line was infected with the recombinant retrovirus. Following selection the HCC9204/tk cell line which stably expressed tk was cloned. The sensitivity of HCC9204/tk cells to GCV was examined in vitro. Antitumor effects of GCV were also observed after the administration of GCV in nude mice bearing tumor derived from HCC9204/tk cells. RESULTS: The HSV-tk gene expressed stably in HCC9204/tk cells. In vitro the growth inhibition studies showed that HCC9204/tk cells were highly sensitive to GCV (IC50 1.2 mg/L in 72 hour), and the HSV-tk gene-modified HCC9204/tk cells were toxic to nearby unmodified HCC9204 tumor cells that were resistant to GCV. This phenomenon was termed "bystander effect". Ex vivo studies showed similar results that significant tumor inhibition was found in the treatment group than in control group. CONCLUSION: Tumor cells expressing HSV-tk gene can be eradicated by GCV in vitro and ex vivo. The HSV-tk/GCV suicide-gene therapy system mediated retrovirally may provide an effective approach to treatment of human hepatoma.

Animals↗
PubMed1999

[Detection of methylation status of tumor suppressor gene p16 by methylation specific polymerase chain reaction].

OBJECTIVE: To introduce a new method for studies on methylation status of CpG island: methylation specific polymerase chain reaction (MSP) with some modifications. METHODS: Target DNA was modified by sodium bisulfite, converting all unmethylated, but not methylated, cytodines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. The status of 5'CpG island of tumor suppressor gene p16 in maxilliofacial squamous cell carcinoma (MSCC) was analyzed by modified MSP assay. RESULTS: To get single strand DNA, the authors denatured the target DNA by heating instead of alkaline treatment. DNA purification kit was also replaced by dialysis. A cheaper restriction enzyme Taq I was used in place of BstU I to distinguish methylated fragment from unmethylated fragment. The 5'CpG island of p16 gene was methylated in some MSCC tumors. CONCLUSION: MSP is a simple, sensitive, and specific method for detecting the methylation status of any CpG-rich region and the modifications make it more simple to perform.

CpG Islands↗