Biomedical subjects

Y Zhou

Publications and source records attributed to Y Zhou.

At least 379 records · Page 21Linked to original sources

PubMed1999

Interpreting the folding kinetics of helical proteins.

The detailed mechanism of protein folding is one of the major problems in structural biology. Its solution is of practical as well as fundamental interest because of its possible role in utilizing the many sequences becoming available from genomic analysis. Although the Levinthal paradox (namely, that a polypeptide chain can find its unique native state in spite of the astronomical number of configurations in the denatured state) has been resolved, the reasons for the differences in the folding behaviour of individual proteins remain to be elucidated. Here a Calpha-based three-helix-bundle-like protein model with a realistic thermodynamic phase diagram is used to calculate several hundred folding trajectories. By varying a single parameter, the difference between the strength of native and non-native contacts, folding is changed from a diffusion-collision mechanism to one that involves simultaneous collapse and partial secondary-structure formation, followed by reorganization to the native structure. Non-obligatory intermediates are important in the former, whereas there is an obligatory on-pathway intermediate in the latter. Our results provide a basis for understanding the range of folding behaviour that is observed in helical proteins.

Computer Simulation↗

PubMed1999

Inhibition of Vpr-induced cell cycle abnormality by quercetin: a novel strategy for searching compounds targeting Vpr.

Vpr, an accessory gene product of HIV-1 which induces cell cycle abnormality leading to the increased HIV replication, is supposed to be a possible target for anti-AIDS drugs. We recently established a cell line (MIT-23) in which Vpr-induced cell cycle perturbation could be manipulated by a tetracycline promoter. Here, we screened anti-Vpr activity in 27 kinds of herb drugs using MIT-23 cells. One of the extracts prepared from Houttuyniae herba showed an inhibitory activity. Quercetin (QCT), a compound of this crude drug, efficiently inhibited Vpr function without affecting its expression. Furthermore, data suggested that Vpr-induced transcription from HIV-LTR was considerably abrogated by QCT. These data indicate that QCT, a flavonoid previously reported to inhibit HIV replication, also targets Vpr, implicating that MIT-23 cell provides a novel strategy for screening compounds possessing anti-Vpr activity which would be in turn utilized for clarifying the mechanism of Vpr function.

Anti-HIV Agents↗

PubMed1999

Changing single side-chains can greatly enhance the resistance of a membrane protein to irreversible inactivation.

The thermal inactivation rates of a set of 20 cysteine-substituted variants of the integral membrane protein diacylglycerol kinase were measured. Two of the mutations, I53C and I70C, were found to significantly prolong the half-life of the enzyme in detergent solution. By combining the single mutants to create a double mutant, I53C/I70C, the half-life of the enzyme was improved from less than a minute at 70 degrees C to 51 minutes. These results demonstrate that individual side-chain substitutions can significantly improve the properties of membrane proteins in detergent solution.

Amino Acid Substitution↗

PubMed1999

The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed1999

Antichymotrypsin interaction with chymotrypsin. Intermediates on the way to inhibited complex formation.

Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9') and of the serpin molecule in general. The "hinge" region of the reactive center loop, including residues P10-P14, shows facile movement in and out of beta-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the alpha1-antichymotrypsin.alpha-chymotrypsin encounter complex, E.I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of alpha1-antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates, EIa, EIb, and EIc, between E.I and E*I* and permit informed speculation regarding the nature of these intermediates. Partitioning between inhibitor and substrate pathways occurs late in the process of E*I* formation, most likely from a species occurring between EIc and E*I*.

Animals↗

PubMed1999

The mouse GATA-2 gene is expressed in the para-aortic splanchnopleura and aorta-gonads and mesonephros region.

We previously reported that the mouse GATA-2 gene is regulated by two alternative promoters (Minegishi et al, J Biol Chem, 273:3625, 1998). Although the more proximal IG (general) promoter is active in almost all GATA-2-expressing cells, the distal IS (specific) promoter activity was selectively detected in hematopoietic tissues but not in other mesodermal tissues. We report here in vivo analysis of the GATA-2 locus and its regulatory characteristics in hematopoietic tissues of transgenic mice. Transgenes containing 6 or 7 kbp of sequence flanking the 5' end of the IS first exon direct expression of beta-galactosidase or green fluorescent protein (GFP) reporter genes specifically to the para-aortic splanchnopleura, aorta-gonads, and mesonephros (AGM) region, and in the neural tissues. In situ hybridization analysis showed that reporter gene expression specifically recapitulates the endogenous expression profile of GATA-2 in these tissues. The flk-1, CD34, c-kit, and CD45 antigens were identified in the GFP-positive cells from the AGM region and fetal liver, indicating that GATA-2 is expressed in immature hematopoietic cells. Deletion of 3.5 kbp from the 5' end of the 6.0 kbp IS promoter construct, including one of the DNase I hypersensitive sites, completely abolished hematopoietic expression. These experiments describe an early developmental GATA-2 hematopoietic enhancer located between 6.0 and 2.5 kbp 5' to the IS exon.

Animals↗

PubMed1999

Electrochemical studies of cytochrome c disulfide at gold electrodes.

The electrochemistry of disulfide in cytochrome c on gold electrodes was reported. The observed electrochemical response was used to explain why the electrochemical reaction of cytochrome c is irreversible at gold electrodes. Disulfide bonds in cytochrome c were strongly adsorbed onto the surface of gold electrodes and caused slow rate of electron transfer of the heme group. It was found that the presence of disulfides in cytochrome c was responsible for the lack of electrochemical response of the heme group on a gold electrode. The mechanisms for this effect were studied using electrochemistry and photoelectron spectroscopy.

Amino Acids↗

PubMed1999

Simultaneous binding of two protein kinases to a calcium-dependent potassium channel.

Large-conductance calcium-dependent potassium channels are subject to modulation by protein kinases, phosphatases, and other signaling proteins, and it has been inferred from electrophysiological experiments that signaling proteins sometimes can be intimately associated with these channels in a regulatory complex. We show here that endogenous protein kinase activity coimmunoprecipitates with both native and recombinant Drosophila Slowpoke (dSlo) calcium-dependent potassium channels. Coimmunoprecipitation experiments using antibodies against several protein kinases demonstrate that dSlo can bind simultaneously to the Src tyrosine kinase and to the catalytic subunit of the cAMP-dependent protein kinase (PKAc). Both kinases can phosphorylate the channel in Drosophila heads and in heterologous host cells. The PKAc binds directly to a 172-amino acid region in the C-terminal domain of dSlo, without the intervention of regulatory subunits or anchoring proteins, and channel phosphorylation by PKAc is not required for this binding interaction. In contrast, several phosphorylatable tyrosine residues in dSlo are important for Src binding. The results are consistent with the idea that an ion channel can act as a scaffold for its own specific set of modulatory enzymes.

Animals↗

PubMed1999

Inactivation of misselected CD8 T cells by CD8 gene methylation and cell death.

Misselected CD8 cells that express T cell receptors (TCRs) that do not recognize class I major histocompatibility complex (MHC) protein can emerge from thymic selection. A postthymic quality control mechanism that purges these cells from the repertoire is defined here. The failure of mature CD8 cells to simultaneously engage their TCR and CD8 coreceptor triggers an activation process that begins with inhibition of CD8 gene expression through remethylation and concludes with up-regulation of surface Fas and Fas ligand and cellular apoptosis. Thus, inhibition of a death signal through continued TCR-CD8 coengagement of MHC molecules is a key checkpoint for the continued survival of correctly selected T cells. Molecular defects that prevent delivery of the death signal to mistakenly selected T cells underlie the expansion of double-negative T cells, which is the cellular signature of a subset of systemic autoimmune diseases.

Adoptive Transfer↗

PubMed1999

Structural basis for the design of antibiotics targeting peptide deformylase.

While protein synthesis in bacteria begins with a formylated methionine, the formyl group of the nascent polypeptide is removed by peptide deformylase. Since eukaryotic protein synthesis does not involve formylation and deformylation at the N-terminus, there has been increasing interest in peptide deformylase as a potential target for antibacterial chemotherapy. Toward this end and to aid in the design of effective antibiotics targeting peptide deformylase, the structures of the protein-inhibitor complexes of both the cobalt and the zinc containing Escherichia coli peptide deformylase bound to the transition-state analogue, (S)-2-O-(H-phosphonoxy)-L-caproyl-L-leucyl-p-nitroanilide (PCLNA), have been determined. The proteins for both deformylase-inhibitor complexes show basically the same fold as for the native enzyme. The PCLNA inhibitor adopts an extended conformation and fits nicely into a hydrophobic cavity located near the metal site. On the basis of these structures, guidelines for the design of high-affinity deformylase inhibitors are suggested. As our results show that the protein residues which interact with the PCLNA inhibitor are conserved over a wide variety of species, we suggest that antibiotics targeting deformylase could have wide applicability.

Amidohydrolases↗

PubMed1999

Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas.

A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Agkistrodon↗

PubMed1999

Value of sublingual isosorbide dinitrate before isoproterenol tilt test for diagnosis of neurally mediated syncope.

Head-up tilt-table test is valuable in diagnosing type of syncope. Sensitivity varies in reports and is mostly poor. Isoproterenol or nitroglycerin is regularly used to increase head-up tilt-table test sensitivity. The aim of this study was to assess the value of sublingual isosorbide dinitrate administration before isoproterenol tilt test as a simple, practical test for diagnosis of neurally mediated syncope. Ninety-six patients (45 men and 51 women, mean age 42.5 years) with recurrent (median 4 times) loss of consciousness that remained unexplained after clinical and noninvasive assessment and 72 healthy volunteers (36 men and 36 women, mean age 40.1 years) were randomly divided into groups: isosorbide-isoproterenol test and isoproterenol test. During isosorbide-isoproterenol test, a positive response (syncope in association with sudden hypotension or bradycardia) occurred in 35 patients (72.9%) with unexplained syncope, an exaggerated response (minor symptoms in association with slowly increasing hypotension alone) occurred in 7 (14.6%), a negative response in 2 (4.2%), and drug intolerance in 4 (8.3%). During isoproterenol testing, these percentages were 52.1%, 16.7%, 25.0%, and 6.2%, respectively. Only 3 control volunteers (8.3%) had a positive response to isosorbide-isoproterenol test and 2 (5.6%) to isoproterenol test. The duration of the test and the time for syncope induction in the isosorbide-isoproterenol test were shorter than those in the isoproterenol test (24.84 +/- 5.15 vs 35.70 +/- 6.28 minutes [p <0.01]; 4.53 +/- 2.86 vs 6.27 +/- 4.11 minutes [p <0.05]). This study concluded that isosorbide-isoproterenol test could be valuable in diagnosing unexplained syncope for its high sensitivity, powerful specificity, and short duration.

Administration, Sublingual↗

PubMed1999

NH2-terminal fragments of the 130 kDa subunit of myosin phosphatase increase the Ca2+ sensitivity of porcine renal artery.

1. The effects of the NH2-terminal fragments of M130, a 130 kDa regulatory subunit of smooth muscle myosin phosphatase, on contraction and myosin light chain phosphorylation were investigated in Triton X-100-permeabilized porcine renal artery. 2. Incubation of the permeabilized fibres with M1301-633 (a fragment containing amino acid residues 1-633) or M13044-633 enhanced the Ca2+-induced contraction and shifted the [Ca2+]i-force relationship to the left (EC50 of Ca2+: 330 nM, control, without fragment; 145 nM, M1301-633; 163 nM, M13044-633). Pre-incubation for 1-3 h was needed for these long constructs. 3. M1301-374, M130304-511 and M130297-374, i.e. relatively short constructs compared with M1301-633 and M13044-633, also induced leftward shifts of the [Ca2+]i-force relationship (EC50 of Ca2+: 65 nM, 72 nM and 180 nM, respectively). However, these required no pre-incubation. 4. Deletion of residues 304-374 from the most potent construct, M1301-374, abolished the Ca2+-sensitizing effect. 5. Wortmannin inhibited the enhancement of contraction induced by M130 fragments when added before contraction was initiated and partially inhibited the effects when added after steady-state contraction. 6. M1301-374 slowed the rate of relaxation in Ca2+-free medium. The time for 50 % relaxation with this fragment was 510 +/- 51 s, compared with 274 +/- 14 s for control. 7. The levels of myosin light chain phosphorylation (22.4 %) and force (34. 5 %) obtained with 300 nM Ca2+ were increased by 3 microM M1301-374 to 35.7 and 92.2 %, respectively. However, M1301-374 had no effect on the phosphorylation-force relationship. 8. In conclusion, the NH2-terminal M130 fragments containing residues 304-374 inhibited myosin phosphatase, increased myosin light chain phosphorylation and increased the Ca2+ sensitivity of the contractile apparatus in permeabilized porcine renal artery.

Androstadienes↗

PubMed1999

Acute intermittent morphine increases preprodynorphin and kappa opioid receptor mRNA levels in the rat brain.

We determined the effects of morphine on mRNA levels for the opioid ligands preprodynorphin (PPD) and preproenkephalin (PPE) and the kappa opioid receptor (KOR). Rats received six injections of morphine (6.25 mg/kg/injection) every 2 h, and were sacrificed 30 min later. mRNA levels were measured in brain tissue after removal of the cortex, cerebellum and brainstem. There were increases in PPD and KOR mRNA levels (P<0.05 and P<0.005, respectively), with no alteration of PPE. These alterations in the kappa/dynorphin system may counter morphine-induced effects on the brain.

Animals↗

PubMed1999

Cerebral metabolic profile, selective neuron loss, and survival of acute and chronic hyperglycemic rats following cardiac arrest and resuscitation.

Cortical metabolites and regional cerebral intracellular pH (pHi) were measured in normoglycemic (NM), acute hyperglycemic (AH), and chronic hyperglycemic (CH, 2 week duration, streptozotocin-induced) Wistar rat brains during cardiac arrest and resuscitation. During total ischemia in AH and CH rats (plasma glucose approximately 30 mM), cortical ATP, PCr, glucose, and glycogen all fell significantly as expected. Lactate levels increased dramatically in association with a concomitant intracellular acidosis. Although lactate reached higher concentrations in AH and CH than NM, pHi was significantly lower only in the AH group. With 5 min of reperfusion, all groups recovered to near baseline in all variables, though lactate remained elevated. In a separate aspect of the study, animals from each experimental group were allowed to recover for 4 days following resuscitation, with outcome being gauged by mortality rate and hippocampal CA1 neuron counts. NM survival rate was significantly better than AH and CH. In particular, no CH rats survived for 4 days despite rapid initial recovery. After 4 days, the AH group had suffered significantly greater CA1 neuron loss than the NM rats. In summary, our research identified differences in intra-ischemic acid-base status in the two hyperglycemic groups, suggesting that chronic hyperglycemia may alter the brain's buffering capacity. These observations may account for differences between acutely and chronically hyperglycemic subjects regarding outcome, and they suggest that factors other than hydrogen ion production during ischemia are responsible for modulating outcome.

Acidosis, Lactic↗

PubMed1999

TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs.

The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta.

Cell Line↗

PubMed1999

cDNA cloning and expression of acutin.

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.

Agkistrodon↗

PubMed1999

Native proteins are surface-molten solids: application of the Lindemann criterion for the solid versus liquid state.

Since the internal motions of proteins play an essential role in their biological function, it is important to characterize them in a fundamental way. The Lindemann criterion for the solid state is applied to molecular dynamics simulations and temperature-dependent X-ray diffraction data of proteins. It is found that the interior of native proteins is solid-like, while their surface is liquid-like. When the entire protein becomes solid-like at low temperature ( approximately 220 K), the protein is inactive. Thus, the surface-molten solid nature of proteins in their native state permits the dynamics required for function, while preserving their stability. Comparison with rare gas clusters and polymer models indicates that their thermodynamic phase diagrams have many elements in common with those of proteins.

Bacterial Proteins↗