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Biomedical subjects

K Zhou

Publications and source records attributed to K Zhou.

At least 19 recordsLinked to original sources
PubMed2000

Authentication of an animal crude drug, Zaocys, by diagnostic PCR.

A pair of diagnostic primers for distinguishing the Chinese crude drug Zaocys (Zaocys dhumandes) from its substitutes was designed based on the sequence data of the original animal of the drug and substitutes. Total DNAs were extracted from genuine crude drug and 5 of its substitutes, as well as from 12 species of original animal of the snake crude drug. Diagnostic PCRs were performed using the primers with these total DNAs as a template, annealing at 60-65 degrees C. Positive amplifications were obtained from all DNA templates of Zaocys, whereas negative amplifications were obtained from that of others. The results indicate that Zaocys samples could be definitely distinguished from its substitutes by diagnostic PCR, and no incorrect discrimination was found under the same reaction conditions. The advantages of the method in the authentication of crude drugs are also discussed in the present paper.

Animals↗
PubMed2000

Fas activation reduces neutrophil adhesion to endothelial cells.

Polymorphonuclear neutrophils (PMN) express apoptotic markers and lose effector functions including adhesion, chemotaxis, and phagocytosis when cultured overnight. Although the loss of function correlates with apoptosis, it is not clear if functions are lost before an early marker of apoptosis, the display of phosphatidylserine (PS), targets PMN for removal by phagocytic cells. To address this question, freshly isolated PMN were treated with Fas-activating antibodies to induce apoptosis rapidly. Early markers of apoptosis and PMA-stimulated adhesion to endothelial cells were measured. After 1 h of Fas exposure, only 16% PMN had externalized PS. In contrast, Fas activation reduced PMA-stimulated adhesion between 68 and 27% depending on PMA concentration. The loss of adhesion was accompanied by a reduction in beta2 integrin expression and receptor clustering. These results indicate that the Fas-induced loss of adhesion may precede PS externalization and could limit participation in the inflammatory response before PS externalization targets PMN for removal.

Animals↗
PubMed1999

The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold.

Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5' untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains.

Base Sequence↗
PubMed1999

[Evaluation of the arterial phase of biphase enhanced SCT in the diagnosis of small HCC].

OBJECTIVE: To evaluate the value of the arterial phase (AP) of biphase enhanced SCT in the diagnosis of small HCC and investigate the criteria, initial time, ending time and duration of AP. METHODS: From May 1995 to March 1999, patients with small HCC proved by surgicopathology including 49 cases (N = 53) in the 1st group, 148 cases (N = 186) in the 2nd group and 52 cases (N = 52)n in the 3rd group were collected. Biphase dynamic enhanced SCT scans were performed in all patients of three groups and additional single-level dynamic scans only done in the 3rd group. The detectability, diagnostic accuracy of the lesions and enhancement of the lesion in AP were analyzed statistically. In addition, the initial time, ending time and duration of AP were measured. RESULTS: The results of the 1st group showed the detectability of small HCC was 88.68% in AP and 90.57% in both phases, higher than those by US. Markedly enhanced lesion in AP accounted for 76% and 78% in the 2nd and 3rd groups respectively. The initial, ending and duration times of AP measured on single-level dynamic scans were 16.9 s, 39.6 s and 22.7 s in average respectively. CONCLUSION: The biphase dynamic SCT especially its arterial phase appears to be very valuable in diagnosing small HCCs. In light of short duration of AP, understanding and strict control of AP is obviously imperative.

Adult↗
PubMed1998

A novel, putative MEK kinase controls developmental timing and spatial patterning in Dictyostelium and is regulated by ubiquitin-mediated protein degradation.

We have identified a developmentally regulated, putative MEK kinase (MEKKalpha) that contains an F-box and WD40 repeats and plays a complex role in regulating cell-type differentiation and spatial patterning. Cells deficient in MEKKalpha develop precociously and exhibit abnormal cell-type patterning with an increase in one of the prestalk compartments (pstO), a concomitant reduction in the prespore domain, and a loss of the sharp compartment boundaries, resulting in overlapping prestalk and prespore domains. Overexpression of MEKKalpha or MEKKalpha lacking the WD40 repeats results in very delayed development and a severe loss of compartment boundaries. Prespore and prestalk cells are interspersed throughout the slug. Analysis of chimeric organisms suggests that MEKKalpha function is required for the proper induction and maintenance of prespore cell differentiation. We show that the WD40 repeats target MEKKalpha to the cortical region of the cell, whereas the F-box/WD40 repeats direct ubiquitin-mediated MEKKalpha degradation. We identify a UBC and a UBP (ubiquitin hydrolase) that interact with the F-box/WD40 repeats. Our findings indicate that cells lacking the ubiquitin hydrolase have phenotypes similar to those of MEKKalpha null (mekkalpha-) cells, further supporting a direct genetic and biochemical interaction between MEKKalpha, the UBC, and the UBP. We demonstrate that UBC and UBP differentially control MEKKalpha ubiquitination/deubiquitination and degradation through the F-box/WD40 repeats in a cell-type-specific and temporally regulated manner. Our results represent a novel mechanism that includes targeted protein degradation by which MAP kinase cascade components can be controlled. More importantly, our findings suggest a new paradigm of spatial and temporal control of the kinase activity controlling spatial patterning during multicellular development, which parallels the temporally regulated degradation of proteins required for cell-cycle progression.

Amino Acid Sequence↗
PubMed1998

Crystal structure of a hepatitis delta virus ribozyme.

The self-cleaving ribozyme of the hepatitis delta virus (HDV) is the only catalytic RNA known to be required for the viability of a human pathogen. We obtained crystals of a 72-nucleotide, self-cleaved form of the genomic HDV ribozyme that diffract X-rays to 2.3 A resolution by engineering the RNA to bind a small, basic protein without affecting ribozyme activity. The co-crystal structure shows that the compact catalytic core comprises five helical segments connected as an intricate nested double pseudoknot. The 5'-hydroxyl leaving group resulting from the self-scission reaction is buried deep within an active-site cleft produced by juxtaposition of the helices and five strand-crossovers, and is surrounded by biochemically important backbone and base functional groups in a manner reminiscent of protein enzymes.

Base Sequence↗
PubMed1998

Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42.

The Rac and Cdc42 GTPases regulate diverse cellular behaviors involving the actin cytoskeleton, gene transcription, and the activity of multiple protein and lipid kinases. All of these pathways can potentially become activated when GTP-Rac or GTP-Cdc42 is formed in response to external cell signals, yet it is evident that each activity must also be able to be controlled individually. The mechanisms by which such specificity of GTPase signaling in response to upstream stimuli is achieved remains unclear. We investigated the action of several well characterized guanine nucleotide exchange factors (GEFRho) to activate Rac- and/or Cdc42-dependent kinase pathways. Coexpression studies in COS-7 cells revealed that the ability of individual guanine nucleotide exchange factors (GEFs) to activate the p21-activated kinase PAK1 could be dissociated from activation of c-Jun amino-terminal kinase, even though activation of both pathways requires the action of the GEFs on Rac and/or Cdc42. In contrast, expression of constitutively active forms of Rac or Cdc42 effectively stimulated both downstream kinases. We conclude that GEFs can be important determinants of downstream signaling specificity for members of the Rho GTPase family.

Animals↗
PubMed1998

A general module for RNA crystallization.

Crystallization of RNA molecules other than simple oligonucleotide duplexes remains a challenging step in structure determination by X-ray crystallography. Subjecting biochemically, covalently and conformationally homogeneous target molecules to an exhaustive array of crystallization conditions is often insufficient to yield crystals large enough for X-ray data collection. Even when large RNA crystals are obtained, they often do not diffract X-rays to resolutions that would lead to biochemically informative structures. We reasoned that a well-folded RNA molecule would typically present a largely undifferentiated molecular surface dominated by the phosphate backbone. During crystal nucleation and growth, this might result in neighboring molecules packing subtly out of register, leading to premature crystal growth cessation and disorder. To overcome this problem, we have developed a crystallization module consisting of a normally intramolecular RNA-RNA interaction that is recruited to make an intermolecular crystal contact. The target RNA molecule is engineered to contain this module at sites that do not affect biochemical activity. The presence of the crystallization module appears to drive crystal growth, in the course of which other, non-designed contacts are made. We have employed the GAAA tetraloop/tetraloop receptor interaction successfully to crystallize numerous group II intron domain 5-domain 6, and hepatitis delta virus (HDV) ribozyme RNA constructs. The use of the module allows facile growth of large crystals, making it practical to screen a large number of crystal forms for favorable diffraction properties. The method has led to group II intron domain crystals that diffract X-radiation to 3.5 A resolution.

Base Sequence↗
PubMed1998

Disruption of Dictyostelium PI3K genes reduces [32P]phosphatidylinositol 3,4 bisphosphate and [32P]phosphatidylinositol trisphosphate levels, alters F-actin distribution and impairs pinocytosis.

To understand how phosphatidylinositol 3-kinase (PI3K) modulates cell structure and function, we examined the molecular and cellular defects of a Dictyostelium mutant strain (pik1(Delta)2(Delta)) missing two (DdPIK1 and 2) of three PI3K genes, which are homologues of the mammalian p110 subunit. Levels of [32P]phosphatidylinositol 3, 4 bisphosphate (PI(3,4)P2) and [32P]phosphatidylinositol trisphosphate (PIP3) were reduced in pik1(Delta)2(Delta), which had major defects in morphological and functional correlates of macropinocytosis. This was accompanied by dramatic deficits in a subset of F-actin-enriched structures such as circular ruffles, actin crowns and pseudopodia. Although pik1(Delta)2(Delta) were mobile, they failed to aggregate into streams. Therefore we conclude that PIK1 and 2, possibly through modulation of the levels of PIP3 and PI(3,4)P2, regulate the organization of actin filaments necessary for circular ruffling during macropinocytosis, the extension of pseudopodia and the aggregation of cells into streams, but not the regulation of cell motility.

Actins↗
PubMed1997

Evidence that intramolecular interactions are involved in masking the activation domain of transcriptional activator Leu3p.

The Leu3 protein of Saccharomyces cerevisiae regulates the expression of genes involved in branched chain amino acid biosynthesis and in ammonia assimilation. It is modulated by alpha-isopropylmalate, an intermediate in leucine biosynthesis. In the presence of alpha-isopropylmalate, Leu3p is a transcriptional activator. In the absence of the signal molecule, the activation domain is masked, and Leu3p acts as a repressor. The recent discovery that Leu3p retains its regulatory properties when expressed in mammalian cells (Guo, H., and Kohlhaw, G. B. (1996) FEBS Lett. 390, 191-195) suggests that masking and unmasking of the activation domain occur without the participation of auxiliary proteins. Here we present experimental support for this notion and address the mechanism of masking. We show that modulation of Leu3p is exceedingly sensitive to mutations in the activation domain. An activation domain double mutant (D872N/D874N; designated Leu3-dd) was constructed that has the characteristics of a permanently masked activator. Using separately expressed segments containing either the DNA binding domain-middle region or the activation domain of wild type Leu3p (or Leu3-dd) in a modified yeast two-hybrid system, we provide direct evidence for alpha-isopropylmalate-dependent interaction between these segments. Finally, we use the phenotype of Leu3-dd-containing cells (slow growth in the absence of added leucine) to select for suppressor mutations that map to the middle region of Leu3-dd. The properties of nine such suppressors further support the idea that masking is an intramolecular process and suggest a means for mapping the surface involved in masking.

Amino Acid Sequence↗
PubMed1997

[Apoptosis of nasopharyngeal carcinoma cells induced by topoisomerase I inhibitor].

PURPOSE: To determine if the topoisomerase I inhibitor Camptothecin (CPT) can induce apoptosis in vitro in a human nasopharyngeal carcinoma (NPC) cell line with low differentiation (CNE-2Z). METHODS: The light microscopy, flow cytometry and agarose gel electrophoresis were used to examine the morphological changes, cell cycle distribution, hypodiploid cells and DNA fragmentation. RESULTS: After exposure to CPT for a certain period, CNE-2Z cells underwent obvious morphological changes with characteristics of apoptosis such as decrease in cell volumes condensation of chromatin and the formation of apoptotic bodies. Flow cytometry (FCM) test showed that when CNE-2Z cells were treated with 2-10 mumol/L CPT for 12 or 24 hours, hypodiploid cells accounted for 30% and 50% respectively. Cell cycle analysis by FCM revealed that changes in CNE-2Z cell cycle distribution were apparent 24 hours after treatment with various doses of CPT, showing no obvious dose-dependent relationship. Compared with controls, the percentage of cells in G2/M phase decreased markedly while those in G1 and S phases increased moderately. CONCLUSION: The results demonstrated that DNA topoisomerase I inhibitor Camptothecin can induce apoptosis in CNE-2Z cells in vitro.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Crystal structure of a group I ribozyme domain: principles of RNA packing.

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

Adenine↗
PubMed1996

RNA tertiary structure mediation by adenosine platforms.

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.

Adenosine↗
PubMed1996

Experimental asthma developed by room air contamination with cockroach allergen.

To study the asthmatogenic effect of certain airborne elements of the home environment, we studied a group of guinea pigs exposed to aerosolized cockroach allergen (CRa) and side-stream cigarette (S-SC) smoke. Four groups of guinea pigs were exposed to aerosols, either saline or CRa, for 4 weeks, after a sham or S-SC smoke pretreatment. Anaphylactic antibodies were measured by passive cutaneous anaphylaxis (PCA) assay and by skin test. Animals were challenged with aerosol CRa on day 35, and lung function and leukotrienes (LTB4 and LTC4/D4) were measured. Skin tests were positive on days 21 and 29. The antibodies were heat-stable, IgG1a-like antibodies (PCA titers 1:2-18). The CRa challenge caused an immediate reduction in both the maximal expiratory flow rate at 50% of the lung capacity and respiratory compliance. The decreased lung function continued for up to 6 h (p < 0.0001). LTB4 and LTC4/D4 were elevated (p < 0.0001) in the sensitized animals at the corresponding times of reduced lung function. S-SC smoke did not affect the CRa sensitization; instead, a protective effect on the CRa-induced bronchospasms was noted. Thus, the study indicates that a simple airborne CRa exposure without an adjuvant sensitizes guinea pigs, and that the animals respond to antigen challenge with CRa-specific airway obstructions.

Aerosols↗
PubMed1995

A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5 encodes a functional homolog of yeast Vps34p. These observations indicate that in D. discoideum, different PI kinases regulate distinct cellular processes, including cell growth, development, and protein trafficking.

1-Phosphatidylinositol 4-Kinase↗