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Biomedical subjects

A Zhou

Publications and source records attributed to A Zhou.

At least 19 recordsLinked to original sources
PubMed2006

Role of 2-5A-dependent RNase-L in senescence and longevity.

Senescence is a permanent growth arrest that restricts the lifespan of primary cells in culture, and represents an in vitro model for aging. Senescence functions as a tumor suppressor mechanism that can be induced independent of replicative crisis by diverse stress stimuli. RNase-L mediates antiproliferative activities and functions as a tumor suppressor in prostate cancer, therefore, we examined a role for RNase-L in cellular senescence and aging. Ectopic expression of RNase-L induced a senescent morphology, a decrease in DNA synthesis, an increase in senescence-associated beta-galactosidase activity, and accelerated replicative senescence. In contrast, senescence was retarded in RNase-L-null fibroblasts compared with wild-type fibroblasts. Activation of endogenous RNase-L by 2-5A transfection induced distinct senescent and apoptotic responses in parental and Simian virus 40-transformed WI38 fibroblasts, respectively, demonstrating cell type specific differences in the antiproliferative response to RNase-L activation. Replicative senescence is a model for in vivo aging; therefore, genetic disruption of senescence effectors may impact lifespan. RNase-L-/- mice survived 31.7% (P<0.0001) longer than strain-matched RNase-L+/+ mice providing evidence for a physiological role for RNase-L in aging. These findings identify a novel role for RNase-L in senescence that may contribute to its tumor suppressive function and to the enhanced longevity of RNase-L-/- mice.

Aging↗
PubMed2006

Biophysical characterisation of electrofused giant HEK293-cells as a novel electrophysiological expression system.

Giant HEK293 cells of 30-65 microm in diameter were produced by three-dimensional multi-cell electrofusion in 75 mOsm sorbitol media. These strong hypotonic conditions facilitated fusion because of the spherical shape and smooth membrane surface of the swollen cells. A regulatory volume decrease (RVD), as observed at higher osmolalities, did not occur at 75 mOsm. In contrast to field-treated, but unfused cells, the increase in volume induced by hypotonic shock was only partly reversible in the case of fused giant cells after their transfer into isotonic medium. The large size of the electrofused cells allowed the study of their electrophysiological properties by application of both whole-cell and giant excised patch-clamp techniques. Recordings on giant cells yielded a value of 1.1+/-0.1 microF/cm2 for the area-specific membrane capacitance. This value was consistent with that of the parental cells. The area-specific conductivity of giant cells (diameter > 50 microm) was found to be between 12.8 and 16.1 microS/cm2, which is in the range of that of the parental cells. Measurements with patch-pipettes containing fluorescein showed uniform dye uptake in the whole-cell configuration, but not in the cell-attached configuration. The diffusion-controlled uniform uptake of the dye into the cell interior excludes internal compartmentalisation. The finding of a homogeneous fusion was also supported by expression of the yellow fluorescent protein YFP (as part of the fusion-protein ChR2-YFP) in giant cells since no plasma-membrane bound YFP-mediated fluorescence was detected in the interior of the electrofused cells. Functional expression and the electrophysiological characterisation of the light-activated cation channel Channelrhodopsin 2 (ChR2) yielded similar results as for parental cells. Most importantly, the giant cells exhibited a comparable expression density of the channel protein in the plasma membrane as observed in parental cells. This demonstrates that electrofused cells can be used as a heterologous expression system.

Bacterial Proteins↗
PubMed2006

Molecular pathology of X linked retinoschisis: mutations interfere with retinoschisin secretion and oligomerisation.

BACKGROUND/AIM: X linked retinoschisis (XLRS) is caused by mutations in RS1 which encodes the discoidin domain protein retinoschisin, secreted by photoreceptors and bipolar cells. Missense mutations occur throughout the gene and some of these are known to interfere with protein secretion. This study was designed to investigate the functional consequences of missense mutations at different locations in retinoschisin. METHODS AND RESULTS: The authors developed a structural model of the retinoschisin discoidin domain and used this to predict the effects of missense mutations. They expressed disease associated mutations and found that those affecting conserved residues prevented retinoschisin secretion. Most of the remaining mutations cluster within a series of loops on the surface of the beta barrel structure and do not interfere with secretion, suggesting this region may be a ligand binding site. They also demonstrated that wild type retinoschisin octamerises and associates with the cell surface. A subgroup of secreted mutations reduce oligomerisation (C59S, C219G, C223R). CONCLUSIONS: It is suggested that there are three different molecular mechanisms which lead to XLRS: mutations interfering with secretion, mutations interfering with oligomerisation, and mutations that allow secretion and oligomerisation but interfere with retinoschisin function. The authors conclude that binding of oligomerised retinoschisin at the cell surface is important in its presumed role in cell adhesion.

Amino Acid Sequence↗
PubMed2004

Charge translocation during cosubstrate binding in the Na+/proline transporter of E.coli.

Charge translocation associated with the activity of the Na(+)/proline cotransporter PutP of Escherichia coli was analyzed for the first time. Using a rapid solution exchange technique combined with a solid-supported membrane (SSM), it was demonstrated that Na(+)and/or proline individually or together induce a displacement of charge. This was assigned to an electrogenic Na(+)and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k>50s(-1) which preceeds the rate-limiting step. Based on the kinetic analysis of our electrical signals, the following characteristics are proposed for substrate binding in PutP. (1) Substrate binding is electrogenic not only for Na(+), but also for the uncharged cosubstrate proline. The charge displacement associated with the binding of both substrates is of comparable size and independent of the presence of the respective cosubstrate. (2) Both substrates can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails, while at sufficiently high concentrations, each substrate can bind in the absence of the other. (3) Both substrate binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate. (4) Proline binding proceeds in a two-step process: low affinity (approximately 1mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition.

4-Chloromercuribenzenesulfonate↗
PubMed2004

Latent antithrombin and its detection, formation and turnover in the circulation.

It is now apparent that the inactivated latent and cleaved conformers of antithrombin (AT) are of pathological significance. Using a single-run electrophoretic technique that allows the quantitative assessment of these conformers in 2 microL plasma, we show that near 3% of the total AT in the circulations of normal individuals is in latent conformation. Only trace amounts of cleaved AT were observed. The slow decline in AT activity on incubation of plasma at 37 degrees C was shown to be almost wholly due to a transition of native AT to its inactive latent form. Also initial studies in the rabbit indicate that the latent form, like the cleaved, has an identical circulatory half-life to that of native AT. We deduce that the steady concentration of latent AT in the circulation is due to the transition of some 10(12) molecules of AT per second balanced by an equivalent clearance of the latent form. Examples of clinical applications of the new technique include its use as a comprehensive single-step screen for genetic variants associated with AT deficiency, and notably the potential it provides to monitor the changes responsible for the loss of AT in the shock syndromes.

Animals↗
PubMed2001

Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma.

It has been reported that interferons (IFNs) may have antitumor activity in multiple myeloma (MM). The mechanism for their effect on MM, however, remains elusive. This study shows that IFN-alpha and -beta, but not -gamma, induce apoptosis characterized by Annexin V positivity, nuclear fragmentation and condensation, and loss of clonogenicity in 3 MM cell lines (U266, RPMI-8266, and NCI-H929), and in plasma cells from 10 patients with MM. Apo2 ligand (Apo2L, also TRAIL) induction was one of the earliest events following IFN administration in U266 cells. Treatment of these cells with TRAIL, but not with Fas agonistic antibodies, induces apoptosis. Cell death induced by IFNs and Apo2L in U266 cells was partially blocked by a dominant-negative Apo2L receptor, DR5, demonstrating the functional significance of Apo2L induction. This study shows that IFNs activate caspases and the mitochondrial-dependent apoptotic pathway, possibly mediated by Apo2L production. Thus, IFN-alpha and -beta induce cytochrome c release from mitochondria starting at 12 hours, with an amplified release seen at 48 hours. Moreover, Bid cleavage precedes the initial cytochrome c release, whereas the late, amplified cytochrome c release coincides with changes in levels of Bcl-2, Bcl-X(L), and reduction of mitochondrial membrane potential. These results link the Apo2L induction and modulation of Bcl-2 family proteins to mitochondrial dysfunction. Furthermore, IFNs and Apo2L induce cell death of CD38(+)/CD45(-/dim) plasma cells, without significant effect on nonplasma blood cells, in a caspase and Bcl-2 cleavage-dependent manner. These results warrant further clinical studies with IFNs and Apo2L in MM.

Apoptosis↗
PubMed2001

Severe defect in proglucagon processing in islet A-cells of prohormone convertase 2 null mice.

Mice homozygous for a deletion in the gene encoding prohormone convertase 2 (PC2) are generally healthy but have mild hypoglycemia and flat glucose-tolerance curves. Their islets show marked alpha (A)-cell hyperplasia, suggesting a possible defect in glucagon processing (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). In this report we have examined the biosynthesis and processing of proglucagon in isolated islets from these mice via pulse-chase labeling and find that proglucagon undergoes essentially no processing in chase periods up to 8 h in duration. Only a small percent of cleavage at the sensitive interdomain site (residues 71 and 72) appears to occur. These observations thus conclusively demonstrate the essentiality of PC2 for the production of glucagon in the islet A-cells. Ultrastructural and immunocytochemical studies indicate the presence of large amounts of proglucagon in atypical appearing secretory granules in the hyperplastic and hypertrophic A-cells, along with morphological evidence of high rates of proglucagon secretion in PC2 null islets. These findings provide strong evidence that active glucagon is required to maintain normal blood glucose levels, counterbalancing the action of insulin at all times.

Animals↗
PubMed2001

The serpin inhibitory mechanism is critically dependent on the length of the reactive center loop.

The recent crystallographic structure of a serpin-protease complex revealed that protease inactivation results from a disruption of the catalytic site architecture caused by the displacement of the catalytic serine. We hypothesize that inhibition depends on the length of the N-terminal portion of the reactive center loop, to which the active serine is covalently attached. To test this, alpha(1)-antitrypsin Pittsburgh variants were prepared with lengthened and shortened reactive center loops. The rates of inhibition of factor Xa and of complex dissociation were measured. The addition of one residue reduced the stability of the complex more than 200,000-fold, and the addition of two residues reduced it by more than 1,000,000-fold, whereas the deletion of one or two residues lowered the efficiency of inhibition and increased the stability of the complex (2-fold). The deletion of more than two residues completely converted the serpin into a substrate. Similar results were obtained for the alpha(1)-antitrypsin variants with thrombin and for PAI-1 and PAI-2 with their common target tissue plasminogen activator. We conclude that the length of the serpin reactive center loop is critical for its mechanism of inhibition and is precisely regulated to balance the efficiency of inhibition and stability of the final complex.

Amino Acid Sequence↗
PubMed2001

A piezoelectric quartz crystal impedance study on Cu(2+)-induced precipitation of bovine serum albumin in aqueous solution.

Piezoelectric quartz crystal impedance (QCI) technique was used for monitoring the Cu(2+)-induced precipitation of bovine serum albumin onto the gold electrode. The critical precipitate concentration of Cu(2+) reflected by the significant decrease in the resonant frequency was estimated to be 9.98 x 10(-5) mol x l(-1), and the saturated adherence of the precipitate on the electrode occurred when the Cu(2+) concentration was greater than 9.79x10(-3) mol x l(-1). The frequency shift in air was about 85.5% of that in liquid, and the Deltaf(0)/DeltaR(1) ratio found in solution was 82.67 Hz Omega(-1), suggesting that the frequency response was predominated by the mass change due to precipitate adherence to the electrode surface. The response of the resonant frequency was analyzed using an equation Deltaf=a(0) + a(1) e(-t/tau(1)) + a(2) e(-t/tau(2)). The relationship between the total a(0) values and the Cu(2+) concentration was discussed.

Animals↗
PubMed2001

Analysis of chromosomal and organellar DNA of somatic hybrids between Triticum aestiuvm and Haynaldia villosa Schur.

Intergeneric somatic hybridization between wheat (cv. Jinan 177) protoplasts that have 24-28 chromosomes and Haynaldia villosa protoplasts containing 11-14 chromosomes was carried out by the polyethylene glycol (PEG) method. A high frequency of hybrid calli and plants were obtained from the fusion products, as revealed by cytological and biochemical techniques and by PCR analysis of 5S rDNA spacer sequences. GISH (genomic in situ hybridization) analysis confirmed the presence of chromosomes from both parents in the hybrid clones and the common occurrence of translocations between them. The RFLP analysis of the organellar DNA using mitochondrion- and chloroplast-specific probes revealed that mitochondria from both parents existed in the cells of hybrid calli and their recombination, whereas chloroplasts segregated and recombined randomly. The gross morphology of hybrid plants resembled that of wheat, but the gross morphology of their ovaries and anthers were intermediate between those of the two parents. The relationship between hybrid plant regeneration and the balance of genetic materials in hybrid clones is discussed.

Blotting, Southern↗
PubMed2001

Application of double-impedance system and cyclic voltammetry to study the adsorption of fullerols (C60(OH)n) on biological peptide-adsorbed gold electrode.

The adsorption of fullerols (C60(OH)n) on glutathione-adsorbed gold electrode was characterized by using double-impedance system, i.e., electrochemical quartz crystal impedance and electrochemical impedance spectroscopy, and cyclic voltammetry. The time courses of piezoelectric parameters were used to reflect the changes of interfacial physical properties, such as mass, density-viscosity, and dielectric constant, during the adsorptions of peptide and fullerols onto electrode. The electrochemical impedance based on the simple equivalent electric network were also simultaneously measured and provided electrochemical interface information, e.g., double-layer capacitance and charge-transfer resistance. It was found that the double-impedance responses were varied with the forms of glutathione. It was also shown that the frequency curves due to the adsorption of oxidized (GSSG) and reduced (GSH) glutathione could be exhibited as different kinetic equations. The heterogeneous charge-transfer rate constants of ferricyanide/ferrocyanide before and after the peptide and fullerols adsorption were determined by CV and EIS methods. The results showed that the proposed method has potential applications in interfacial studies of biomaterials, since these combined techniques have advantages in real time providing multidimensional piezoelectric and electrochemical impedance information.

Adsorption↗
PubMed2001

Altered expression of the neuropeptide-processing enzyme carboxypeptidase E in the rat brain after global ischemia.

Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemia-induced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brain's response to ischemia.

Animals↗
PubMed2001

Gravidity-dependent production of antibodies that inhibit binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate proteoglycan during pregnancy.

During pregnancy, Plasmodium falciparum-infected erythrocytes sequester in the placenta by adhering to chondroitin 4-sulfate, creating a risk factor for both the mother and the fetus. The primigravidae are at higher risk for placental malaria than the multigravidae. This difference in susceptibility has been attributed to the lack of antibodies that block the adhesion of infected erythrocytes to placental chondroitin 4-sulfate in primigravid women. However, recent results show that many primigravidae at term have antibody levels similar to those of multigravidae, and thus the significance of antiadhesion antibodies in providing protection against malaria during pregnancy remains unclear. In this study, we analyzed plasma samples from women of various gravidities at different gestational stages for antiadhesion antibodies. The majority of women, regardless of gravidity, had similar levels of antibodies at term. Most primigravidae had low levels of or no antiadhesion antibodies prior to ~20 weeks of pregnancy and then produced antibodies. Multigravidae also lacked antibodies until ~12 weeks of pregnancy, but thereafter they efficiently produced antibodies. In pregnant women who had placental infection at term, higher levels of antiadhesion antibodies correlated with lower levels of placental parasitemia. The difference in kinetics of antibody production between primigravidae and multigravidae correlated with the prevalence of malaria in these groups, suggesting that antibodies are produced during pregnancy in response to placental infection. The early onset of efficient antibody response in multigravidae and the delayed production to antibodies in primigravidae appear to account for the gravidity-dependent differential susceptibilities of pregnant women to placental malaria.

Adult↗
PubMed2001

Increased severity of HSV-1 keratitis and mortality in mice lacking the 2-5A-dependent RNase L gene.

PURPOSE: The2',5'-oligoadenylate-dependent RNase L gene functions in the interferon-inducible RNA decay pathway known as the 2-5A system. The purpose of this study was to determine whether the absence of this gene affects the pathogenesis of herpes simplex virus type 1 (HSV-1) ocular infection in the mouse. METHODS: HSV-1 (strain McKrae) was applied bilaterally to unscarified corneas of RNase L-null mice and congenic controls. To evaluate the severity of herpetic keratitis, slit lamp examinations (SLE) were performed every other day for 14 days. To study corneal histology and apoptosis, HSV-1-inoculated RNase-L-null and congenic control mice, as well as mock-inoculated mice (apoptosis negative control), were killed at 6 and 18 hours postinoculation (PI). Uninoculated mice that underwent corneal scarification (apoptosis positive control) were killed 2 hours after scarification. Eyes were dissected and the corneas processed for light and transmission electron microscopy and the TUNEL assay. RESULTS: In comparison with the congenic control mice, RNase L-null mice showed significantly more severe herpetic keratitis (PI day 8, SLE score, mean +/- SEM: 3.27 +/- 0.10 vs. 2.34 +/- 0.06; P: < 0.001) and significantly higher mortality (PI day 14, 70% vs. 20%; P: < 0.001). Few apoptotic cells were seen in HSV-1-infected RNase L-null mice, although DNA fragmentation consistent with apoptosis was detected in the corneas of congenic control mice 6 and 18 hours after HSV-1 inoculation and in uninfected mice with scarified corneas. Signs of apoptosis were not present in the mock-infected corneas. Electron microscopic evidence of keratocytic apoptosis was detected only in the uninfected scarified corneas and the HSV-1-infected congenic control corneas. CONCLUSIONS: The increased severity of ocular disease and increased mortality in the RNase L-null mice provides evidence, for the first time, that the 2-5A system contributes to protection during ocular herpetic infection. The reduced frequency of apoptosis in these mice suggests that one possible mechanism for this protective effect could be the induction of apoptosis in corneal cells as a means of reducing the spread of infectious virus.

Animals↗
PubMed2001

The conformational basis of thrombosis.

Antithrombin readily undergoes a spontaneous transition from its active five-stranded form to a six-stranded inactive latent form. The recognition of this change in plasma has been obscured by the immediate linkage of newly formed latent antithrombin to a molecule of active antithrombin to give a dimer with an electrophoretic mobility readily confused with that of native active antithrombin. A new micromethod now allows unequivocal identification of latent antithrombin in whole plasma. This shows that at 37 degrees C some 10% of plasma antithrombin is converted to the latent form in 24 h. The rate of conversion is greatly accelerated at increased temperatures, as occurs in the pasteurisation of plasma concentrates that should now be checked for efficacy. But increased transition also occurs in the plasma at the slightly increased temperatures that accompany incidental infections. This is of particular significance if there is a conformationally unstable variant of antithrombin; here fever can provoke a sudden transition with the onset of a characteristically severe episode of thromboembolism. Such variants are not rare and include those previously classified as pleiotropic. The precise structural pathway, now known with antithrombin, provides a model of the changes occurring in other conformational diseases, including Alzheimer's and the prion dementias.

Antithrombins↗
PubMed2001

The role of hypertension-related gene in aortic vascular smooth muscle cells from mice and rats.

OBJECTIVE: To study the role of hypertension-related gene (HRG-1) in cardiovascular disease. METHODS: The expression of HRG-1 was analyzed with RT-PCR and Northern blotting. Vascular smooth muscle cell (VSMC) proliferation was measured with 3H-TdR incorporation and was confirmed with histological analysis. RESULTS: Northern blot analysis showed that HRG-1 mRNA was expressed not only in VSMC, but also in various rat tissues (heart, brain, lung, kidney, and liver). In addition, the expression of HRG-1 mRNA in heart, brain, kidney and liver of spontaneously hypertensive rat (SHR) was lower than that in the same tissues of Wistar-Kyotorat (WKY). Semi-quantitative RT-PCR and histological analysis showed that the expression of HRG-1 mRNA in ApoE-knockout mice and in animal models of restenosis was decreased and neointimal formation was observed in both models. ET, AII, and IL-1 stimulating VSMC proliferation reduced the expression of HRG-1 mRNA of VSMC. Atrial natriuretic factor (ANF), calcitonin gene-related peptide and adrenomedullin, inhibited VSMC proliferation and elevated the expression of HRG-1 mRNA. These effects could be blocked or attenuated by their corresponding antagonists or antibodies. CONCLUSION: HRG-1 is a gene related to VSMC proliferation. It may play an important role in several occlusive cardiovascular diseases including atherosclerosis, restenosis and hypertension.

Animals↗
PubMed2001

Percutaneous balloon aortic valvuloplasty in the treatment of congenital valvular aortic stenosis in children.

OBJECTIVE: To assess the effect of the balloon valvuloplasty for for congenital valvular aortic stenosis (AS) in children. METHODS: A total of 27 (mean age 6.09 years) children with AS accepted the treatment of percutaneous balloon aortic valvuloplasty (PBAV). The ratios of balloon/valve were 0.95 +/- 0.08 for 19 cases of typical AS and 1.00 +/- 0.11 for 8 cases of hypoplastic AS. The patients were evaluated by the gradients across aortic valves in pre- and post-PBAV and by echocardiogram during the follow-up period. RESULTS: Fifteen of 19 (78.9%) cases of typical AS had a better outcome and the gradient of the remaining 4 cases (26.7%) had increased after follow-up (delta P > 50 mm Hg). Four of 8 (50.0%) cases of hypoplastic AS had satisfactory responses and the gradient of the remaining 3 cases (75.0%) rose. There was no moderate to severe aortic insufficiency (AI). CONCLUSION: The balloon aortic valvuloplasty provides safe and significant hemodynamic and clinical improvement in pediatric patients. The outcome of PBAV for typical AS is better than for hypoplastic AS.

Adolescent↗