Biomedical subjects

H Zhou

Publications and source records attributed to H Zhou.

At least 109 records · Page 6Linked to original sources

PubMed2001

Identification of dipeptidyl nitriles as potent and selective inhibitors of cathepsin B through structure-based drug design.

Cathepsin B is a member of the papain superfamily of cysteine proteases and has been implicated in the pathology of numerous diseases, including arthritis and cancer. As part of an effort to identify potent, reversible inhibitors of this protease, we examined a series of dipeptidyl nitriles, starting with the previously reported Cbz-Phe-NH-CH(2)CN (19, IC(50) = 62 microM). High-resolution X-ray crystallographic data and molecular modeling were used to optimize the P(1), P(2), and P(3) substituents of this template. Cathepsin B is unique in its class in that it contains a carboxylate recognition site in the S(2)' pocket of the active site. Inhibitor potency and selectivity were enhanced by tethering a carboxylate functionality from the carbon alpha to the nitrile to interact with this region of the enzyme. This resulted in the identification of compound 10, a 7 nM inhibitor of cathepsin B, with excellent selectivity over other cysteine cathepsins.

Animals↗

PubMed2001

Radiation risk to low fluences of alpha particles may be greater than we thought.

Based principally on the cancer incidence found in survivors of the atomic bombs dropped in Hiroshima and Nagasaki, the International Commission on Radiation Protection (ICRP) and the United States National Council on Radiation Protection and Measurements (NCRP) have recommended that estimates of cancer risk for low dose exposure be extrapolated from higher doses by using a linear, no-threshold model. This recommendation is based on the dogma that the DNA of the nucleus is the main target for radiation-induced genotoxicity and, as fewer cells are directly damaged, the deleterious effects of radiation proportionally decline. In this paper, we used a precision microbeam to target an exact fraction (either 100% or < or =20%) of the cells in a confluent population and irradiated their nuclei with exactly one alpha particle each. We found that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a confluent mammalian cell population with a single alpha particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. This effect was significantly eliminated in cells pretreated with a 1 mM dose of octanol, which inhibits gap junction-mediated intercellular communication, or in cells carrying a dominant negative connexin 43 vector. The data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose, high linear-energy-transfer (LET) radiation exposure.

Alpha Particles↗

PubMed2001

A statistical model for the evaluation of barrier contraceptive efficacy.

This paper describes an approach for the analysis of barrier contraceptive efficacy trials that accounts for timing frequency of intercourse and compliance. We allow exposure variables to vary for each act of intercourse and we control for timing of each act through a specific parametric function of the day of the act relative to the last day of the follicular phase of the cycle. The model can be used to examine the level of protection provided by a barrier versus no contraceptive method even when no control group of non-users is studied, as long as there are acts with no barrier use during the fertile window. We present results of a simulation study which examines performance of estimators and power under a variety of scenarios, including situations where an accurate benchmark for ovulation day is not available. As compared to the survival analysis approach commonly used in this setting, simulation results show that the new approach yields considerable gains in power to detect differences between the efficacy of contraceptive methods. An application to data from the FemCap versus diaphragm trial show results consistent with previous findings suggesting superiority of the diaphragm but also provides new evidence of the per act protection provided by both methods.

Biometry↗

PubMed2001

Chaperone suppression of cellular toxicity of huntingtin is independent of polyglutamine aggregation.

Polyglutamine protein aggregation is associated with eight inherited neurodegenerative disorders. In Huntington's disease, N-terminal fragments of mutant huntingtin form intracellular aggregates and mediate cellular toxicity. Recent studies have shown that chaperones inhibit polyglutamine-mediated aggregation and cellular toxicity. Because chaperones also inhibit caspase activation to protect cells from death, it remains unclear whether the protective effect of chaperones on polyglutamine-mediated cellular toxicity is dependent on their inhibition of protein aggregation. In this study, we show that several chaperones including HSP 40, HSP 70, and N-ethylmaleimide-sensitive factor can inhibit cellular toxicity caused by N-terminal mutant huntingtin fragments. However, only HSP 40 is able to inhibit huntingtin aggregation. Furthermore, time-course study suggests that the protection of chaperones against huntingtin toxicity is not the result of their suppression of huntingtin aggregation. Chaperones inhibit caspase-3 and caspase-9 activation mediated by mutant huntingtin, and this inhibition is independent of huntingtin aggregation. We propose that the inhibition of caspase activity by chaperones is involved in their suppression of polyglutamine toxicity.

Amino Acid Sequence↗

PubMed2001

A water-soluble galactomannan from the seeds of Phoenix dactylifera L.

A water-soluble polysaccharide, isolated from the seeds of dates, has been investigated using methylation, periodate and CrO(3) oxidation, NMR spectroscopy, and reaction with Bandeiraea simplicifolia lectin and alpha-D-galactosidase. The polysaccharide consists of a backbone composed of (1-->4)-beta-D-mannopyranosyl residues and carries a single (1-->6)-alpha-linked D-galactopyranosyl residue.

Carbohydrate Conformation↗

PubMed2001

Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.

The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate.

Affinity Labels↗

PubMed2001

MMTV promoter hypomethylation is linked to spontaneous and MNU associated c-neu expression and mammary carcinogenesis in MMTV c-neu transgenic mice.

The erbB family of receptor tyrosine kinases is frequently implicated in neoplasia. Amplification and overexpression of erbB2/neu has been found in 20 to 40% of human breast cancers. Previous studies using MMTV/c-neu transgenic mice have linked rat neu overexpression to mammary tumor development. In this study, we provide evidence that rat neu overexpression in mammary tumors of MMTV/c-neu transgenic mice is always associated with demethylation of the MMTV promoter, whereas the normal mammary glands of these transgenic mice always contain specific methylated regions of the MMTV promoter. In addition, after exposure to N-methyl-N-nitrosourea (MNU), the latency of mammary tumor development is significantly reduced and again is also associated with MMTV promoter demethylation. Thus, the transition from methylation to hypomethylation of the MMTV promoter induces high-level expression of c-neu and appears to be a prerequisite for transformation from normal to malignant mammary epithelium, either spontaneously or after carcinogen exposure. Expression of transgenic c-neu from the demethylated MMTV promoter appears to be an early event that allows outgrowth of mammary epithelium predisposed to malignant transformation.

Alkylating Agents↗

PubMed2001

Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen.

Overcoming immune tolerance of the growth factors associated with tumor growth should be a useful approach to cancer therapy by active immunity. We used vascular endothelial growth factor (VEGF) as a model antigen to explore the feasibility of the immunogene tumor therapy with a vaccine based on a single xenogeneic homologous gene, targeting the growth factors associated with angiogenesis. To test this concept, we constructed a plasmid DNA encoding Xenopus homologous VEGF (XVEGF-p) and control vectors. We found that immunogene tumor therapy with a vaccine based on XVEGF was effective at both protective and therapeutic antitumor immunity in several tumor models in mice. VEGF-specific autoantibodies in sera of mice immunized with XVEGF-p could be found in Western blotting analysis and ELISA assay. The purified immunoglobulins were effective at the inhibition of VEGF-mediated endothelial cell proliferation in vitro, and at antitumor activity and the inhibition of angiogenesis by adoptive transfer in vivo. The elevation of VEGF in the sera of the tumor-bearing mice could be abrogated with XVEGF-p immunization. The antitumor activity and production of VEGF-specific autoantibodies, significantly elevated IgG1 and IgG2b, could be abrogated by the depletion of CD4(+) T lymphocytes. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factors associated with tumor growth in a cross reaction with single xenogeneic homologous gene and may be of importance in the further exploration of the applications of other xenogeneic homologous genes identified in human and other animal genome sequence projects in cancer therapy.

Animals↗

PubMed2001

Interaction and feedback regulation between STK15/BTAK/Aurora-A kinase and protein phosphatase 1 through mitotic cell division cycle.

STK15 is an Aurora/Ipl-1 related serine/threonine kinase that is associated with centrosomes and induces aneuploidy when overexpressed in mammalian cells. It is well known that phosphorylation and dephosphorylation of kinases are important for regulation of their activity. But mechanisms by which STK15 activity is regulated have not been elucidated. We report that STK15 contains two functional binding sites for protein phosphatase type 1 (PP1), and the binding of these proteins is cell cycle-regulated peaking at mitosis. Activated STK15 at mitosis phosphorylates PP1 and inhibits PP1 activity in vitro. In vivo, PP1 activity co-immunoprecipitated with STK15 is also reduced. These data indicate that STK15 inhibits PP1 activity during mitosis. Also, PP1 is shown to dephosphorylate active STK15 and abolish its activity in vitro. Furthermore, we show that non-binding mutants of STK15 for PP1 are superphosphorylated, but their kinase activities are markedly reduced. Cells transfected with these non-binding mutants manifest aberrant chromosome alignment during mitosis. Our results suggest that a feedback regulation through phosphorylation/dephosphorylation events between STK15 kinase and PP1 phosphatase operates through the cell cycle. Deregulation of this balance may contribute to anomalous segregation of chromosomes during mitotic progression of cancer cells.

Aurora Kinase A↗

PubMed2001

Serine 331 is the major site of receptor phosphorylation induced by agents that activate protein kinase G in HEK 293 cells overexpressing thromboxane receptor alpha.

Human embryonic kidney (HEK)293 cells stably transfected with the His-tagged thromboxane receptor alpha (TPalpha) was used to study the phosphorylation and desensitization of the receptor induced by 8-bromo-cyclic GMP (8-Br-cGMP), sodium nitroprusside (SNP), or S-nitroso-glutathione (SNG). These agents are known to activate cGMP-dependent protein kinase (PKG). Pretreatment of cells with these agents attenuated significantly agonist I-BOP induced Ca(2+) release. These agents also induced dose-dependent phosphorylation of the TPalpha as demonstrated by increased (32)P-labeling of the receptor from cells prelabeled with (32)Pi. To facilitate the identification of the intracellular domains involved in phosphorylation, glutathione S-transferase (GST)-intracellular domain fusion proteins were used as substrates for the purified PKG. It was found that only the GST-C-terminal tail fusion protein could serve as a substrate for the PKG. To identify the specific serine/threonine residues in the C-terminal tail being phosphorylated, various alanine mutants of these serine/threonine residues were checked for their ability to serve as substrates. It was found that the Ser-331 of the C-terminal tail was primarily involved in the PKG-mediated phosphorylation. That Ser-331 is a predominant site of phosphorylation was supported by in vivo studies in which HEK293 cells expressing the S331A mutant receptor showed little phosphorylation induced by any of the above three agents. Furthermore, HEK293 cells expressing the S331A mutant receptor pretreated with any of the above three agents became responsive to the agonist I-BOP-induced Ca(2+) release. These results indicate that Ser-331 of the TPalpha is the primary site responsible for the phosphorylation and the desensitization of the receptor induced by agents that activate the PKG.

Base Sequence↗

PubMed2001

Heterogeneity of channel catfish CTL with respect to target recognition and cytotoxic mechanisms employed.

Two types of catfish alloantigen-dependent cytotoxic T cells were cloned from PBL from a fish immunized in vivo and stimulated in vitro with the allogeneic B cell line 3B11. Because these are the first clonal cytotoxic T cell lines derived from an ectothermic vertebrate, studies were undertaken to characterize their recognition and cytotoxic mechanisms. The first type of CTL (group I) shows strict alloantigen specificity, i.e., they specifically kill and proliferate only in response to 3B11 cells. The second type (group II) shows broad allogeneic specificity, i.e., they kill and proliferate in response to several different allogeneic cells in addition to 3B11. "Cold" target-inhibition studies suggest that group II CTL recognize their targets via a single receptor, because the killing of one allotarget can be inhibited by a different allotarget. Both types of catfish CTL form conjugates with and kill targets by apoptosis. Killing by Ag-specific cytotoxic T cells (group I) was completely inhibited by treatment with EGTA or concanamycin A, and this killing is sensitive to PMSF inhibition, suggesting that killing was mediated exclusively by the secretory perforin/granzyme mechanism. In contrast, killing by the broadly specific T cytotoxic cells (group II) was only partially inhibited by either EGTA or concanamycin A, suggesting that these cells use a cytotoxic mechanism in addition to that involving perforin/granzyme. Consistent with the presumed use of a secretory pathway, both groups of CTL possess putative lytic granules. These results suggest that catfish CTL show heterogeneity with respect to target recognition and cytotoxic mechanisms.

Animals↗

PubMed2001

Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A(2).

Calcium-/calmodulin-dependent protein kinase II (CaM kinase II), a decoder of Ca(2+) signals, and cytosolic phospholipase A(2) (cPLA(2)), an enzyme involved in arachidonate release, are involved in many physiological and pathophysiological processes. Activation of CaM kinase II in norepinephrine-stimulated vascular smooth muscle cells leads to activation of cPLA(2) and arachidonic acid release. Surface plasmon resonance, mass spectrometry, and kinetic studies show that CaM kinase II binds to cPLA(2) resulting in cPLA(2) phosphorylation on Ser-515 and an increase in its enzymatic activity. Phosphopeptide mapping studies with cPLA(2) from norepinephrine-stimulated smooth muscle cells indicates that phosphorylation of cPLA(2) on Ser-515, but not on Ser-505 or Ser-727, occurs in vivo. This novel signaling pathway for arachidonate release is shown to be cPLA(2)-dependent by use of a recently described and highly selective inhibitor of this enzyme.

Animals↗

PubMed2001

Catalytic Meerwein-Pondorf-Verley reduction by simple aluminum complexes.

[reaction: see text] Catalytic MPV reduction was successfully carried out using simple aluminum precatalysts. Alkylaluminum reagents were converted to a low-aggregation aluminum alkoxide that was highly active for the MPV reduction of several carbonyl substrates in high yield (50-99%) using (i)PrOH as the reducing agent. A high degree of cis/trans selectivity was achieved in the reduction of 2-methylcyclohexanone (cis/trans = 20/80) by (i)PrOH. When chiral hydride sources were utilized in the reduction of 2-chloroacetophenone, high enantioselectivity (68-80% ee) was observed.

Journal Article↗

PubMed2001

Association between maternal serum concentration of the DDT metabolite DDE and preterm and small-for-gestational-age babies at birth.

BACKGROUND: DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) is highly effective against most malaria-transmitting mosquitoes and is being widely used in malaria-endemic areas. The metabolite, DDE (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene), has been linked to preterm birth in small studies, but these findings are inconclusive. Our aim was to investigate the association between DDE exposure and preterm birth. METHODS: Our study was based on the US Collaborative Perinatal Project (CPP). From this study we selected a subset of more than 44000 eligible children born between 1959 and 1966 and measured the DDE concentration in their mothers' serum samples stored during pregnancy. Complete data were available for 2380 children, of whom 361 were born preterm and 221 were small-for-gestational age. FINDINGS: The median maternal DDE concentration was 25 mg/L (range 3-178)-several fold higher than current US concentrations. The adjusted odds ratios (OR) of preterm birth increased steadily with increasing concentrations of serum DDE (ORs=1, 1.5, 1.6, 2.5, 3.1; trend p<0.0001). Adjusted odds of small-for-gestational-age also increased, but less consistently (ORs=1, 1.9, 1.7, 1.6, 2.6; trend p=0.04). After excluding preterm births, the association of DDE with small-for-gestational-age remained. INTERPRETATION: The findings strongly suggest that DDT use increases preterm births, which is a major contributor to infant mortality. If this association is causal, it should be included in any assessment of the costs and benefits of vector control with DDT.

Adolescent↗

PubMed2001

Imidazolinium salts as catalysts for the ring-opening alkylation of meso epoxides by alkylaluminum complexes.

[reaction: see text] Imidazolinium salts and their N-heterocyclic carbene (NHC) derivatives catalyze the alkylation of a variety of meso epoxides in the presence of triethylaluminum (yield = 70-90%), under mild conditions. Imidazolinium salts are better catalysts than their NHC derivatives but can lead to dimerization side reactions under extended reaction time. Preformed NHC.AlEt(3) complexes and Wanzlick-type olefins, which are dimers of free NHCs, are also catalysts for this reaction.

Journal Article↗

PubMed2001

Distinct gene expression profiles of human type 1 and type 2 T helper cells.

BACKGROUND: The development and activation of CD4+ helper T cell (Th) subsets with distinct patterns of unbalanced production of cytokines play an important part in infectious, allergic and autoimmune diseases. Human neonatal cord blood CD4+ Th cells can be polarized into type 1 or type 2-like effector cells in vitro by culturing them in the presence of interleukin (IL)-12 or IL-4, respectively. We have exploited this experimental system to identify marker genes that are differentially expressed by polarized Th1 and Th2 cells. An oligonucleotide microarray specifically designed to screen for inflammation-related candidate genes was used and the differential expression was further validated with a quantitative real-time RT-PCR method. RESULTS: In addition to the previously described marker genes of Th cells, we report subtle changes in the expression of several other genes that represent growth factors, receptors and other signaling molecules in polarized Th1 and Th2 cell subsets. Additionally, we describe a novel set of genes as Th1/Th2 differentiation markers for cells activated by anti-CD3 and anti-CD28 antibodies. CONCLUSIONS: This study demonstrates the power of the targeted use of microarrays in combination with quantitative real-time RT-PCR in identifying and validating new marker genes for gene expression studies.

CD4 Antigens↗

PubMed2001

A general high-yield route to bis(salicylaldimine) zinc(II) complexes: application to the synthesis of pyridine-modified salen-type zinc(II) complexes.

A general, direct, and high-yield synthesis of bis(salicylaldimine) zinc complexes from the ligands and Et(2)Zn is reported. This synthetic method is particularly valuable, not only because it allows the efficient preparation of salen-type complexes of zinc but also because it can be used to prepare bifunctional pyridine-modified zinc(II) bis(salicylidene) complexes, which are potentially useful compounds for applications in asymmetric catalysis and materials chemistry. The synthesis and complete structural characterization of a new series of pyridine-modified zinc(II) bis(salicylidene) ligands is discussed.

Journal Article↗

PubMed2001

Rapid and accurate typing of Dichelobacter nodosus using PCR amplification and reverse dot-blot hybridisation.

Here we describe an approach to genotyping D. nodosus, based on variation in the fimbrial subunit gene (fimA), which uses polymerase chain reaction (PCR) amplification and hybridisation to immobilised oligonucleotides (PCR/oligotyping). The variable region of D. nodosus fimA, amplified and labelled with digoxigenin (DIG) in a single multiplex PCR amplification, was hybridised to a panel of group- and type-specific poly-dT tailed oligonucleotides that were immobilised on a nylon membrane strip. A mixture of positive control poly-dT tailed oligonucleotides was also included on the membrane. After hybridisation the membrane was washed to a defined specificity, and DIG-labelled fragments hybridising were detected with nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (SCIP). The specificity of the oligonucleotides was verified by the lack of cross-reactivity with D. nodosus fimA sequences that had a single base difference. DNA from 14 footrot samples previously genotyped by PCR-SSCP/sequencing [Vet. Microbiol. 71 (2000) 113], was assayed using the PCR/oligotyping technique. All types of D. nodosus which had been detected previously with a PCR-SSCP/sequencing method were detected by this procedure. However, for three of the 14 footrot samples, PCR/oligotyping detected additional types of D. nodosus. Further PCR amplification using type-specific primers, confirmed that these types of the bacterium were present in the footrot samples. These results indicate that PCR/oligotyping is a specific, accurate, and useful tool for typing footrot samples. In combination with a rapid DNA extraction protocol, D. nodosus strains present in a footrot sample can be accurately identified in less than 2 days.

Amino Acid Sequence↗