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Biomedical subjects

N Zhang

Publications and source records attributed to N Zhang.

At least 19 recordsLinked to original sources
PubMed2001

Dimeric DNA quadruplex containing major groove-aligned A-T-A-T and G-C-G-C tetrads stabilized by inter-subunit Watson-Crick A-T and G-C pairs.

We report on an NMR study of unlabeled and uniformly 13C,15N-labeled d(GAGCAGGT) sequence in 1 M NaCl solution, conditions under which it forms a head-to-head dimeric quadruplex containing sequentially stacked G-C-G-C, G-G-G-G and A-T-A-T tetrads. We have identified, for the first time, a slipped A-T-A-T tetrad alignment, involving recognition of Watson-Crick A-T pairs along the major groove edges of opposing adenine residues. Strikingly, both Watson-Crick G-C and A-T pairings within the direct G-C-G-C and slipped A-T-A-T tetrads, respectively, occur between rather than within hairpin subunits of the dimeric d(GAGCAGGT) quadruplex. The hairpin turns in the head-to-head dimeric quadruplex involve single adenine residues and adds to our knowledge of chain reversal involving edgewise loops in DNA quadruplexes. Our structural studies, together with those from other laboratories, definitively establish that DNA quadruplex formation is not restricted to G(n) repeat sequences, with their characteristic stacked uniform G-G-G-G tetrad architectures. Rather, the quadruplex fold is a more versatile and robust architecture, accessible to a range of mixed sequences, with the potential to facilitate G-C-G-C and A-T-A-T tetrad through major and minor groove alignment, in addition to G-G-G-G tetrad formation. The definitive experimental identification of such major groove-aligned mixed A-T-A-T and G-C-G-C tetrads within a quadruplex scaffold, has important implications for the potential alignment of duplex segments during homologous recombination.

Base Pairing↗
PubMed2001

V-shaped scaffold: a new architectural motif identified in an A x (G x G x G x G) pentad-containing dimeric DNA quadruplex involving stacked G(anti) x G(anti) x G(anti) x G(syn) tetrads.

We report the results of an NMR study of unlabeled and uniformly (13)C,(15)N-labeled d(G(3)AG(2)T(3)G(3)AT) in 100 mM NaCl, conditions under which it forms a dimeric quadruplex containing several new topological features. The DNA oligomer chain in each symmetry-related monomer subunit undergoes three sharp turns to form a compact domain, with all the purine bases involved in pairing alignments. The first turn is of the double chain reversal type, the second is of the edgewise type, and the third represents a new alignment, the V-shaped type. Each monomer of the dimeric quadruplex contains two stacked G(anti) x G(anti) x G(anti) x G(syn) tetrads, one of which forms a newly identified A x (G x G x G x G) pentad, through sheared G.A mismatch formation. There is a break in one of the four G-G columns that link adjacent G x G x G x G tetrads within each monomer. This architectural interruption is compensated by a new topological feature of quadruplex architecture, the V-shaped scaffold. The missing G-G column results in an opening that could facilitate insertion of planar ligands into the quadruplex. The dimeric interface contains stacked A.(G.G.G.G) pentads, with each pentad containing four bases from one monomer and a syn G1 from the partner monomer. Several potential ligand-binding pockets, positioned towards either end of the folded architecture, were identifiable in a surface view of the solution structure of the dimeric d(G(3)AG(2)T(3)G(3)AT) quadruplex.

Base Sequence↗
PubMed2001

A DNA polymorphism specific to Candida albicans strains exceptionally successful as human pathogens.

A large proportion of infection-causing isolates of the yeast Candida albicans belong to a general-purpose genotype, identifiable by fingerprinting with the moderately repetitive sequence Ca3. The high prevalence of this group -- up to 70% in some patient categories -- suggests that its members possess genetic determinants, which enhance their success as pathogens compared to other strains. To find such determinants we are comparing the genomes of representatives of the general-purpose genotype cluster with the genomes of other strains. In this paper we describe the identification of a 985 bp HpaII fragment (MU13-4) specific to general-purpose genotype strains. The fragment was present in 90% of these strains, but only in 10% of other strains. The fragment did not hybridize with probe Ca3, used to define the general-purpose cluster. It contains elevated levels of repetitive DNA. Sequences homologous to MU13-4 are dispersed throughout the chromosomes of general-purpose strains but are rarer or absent in other strains, as judged by Southern hybridization. Using the Stanford C. albicans genome database, we have placed the MU13-4 fragment next to a CARE-1 element. We also found 79 significant homologies between parts of MU13-4 and 19 other contigs. Attempts to amplify the region surrounding the polymorphic fragment in non-general-purpose genotype strains suggest, as do the hybridization data, that the polymorphism is created by a deletion in non-cluster strains. These results show that it is possible to identify polymorphisms specific to general-purpose genotype strains. Primers against the fragment will allow PCR-based discrimination between general-purpose genotype strains and other strains, facilitating investigations aimed at determining morbidity and mortality caused by general-purpose genotype strains compared to other strains.

Base Sequence↗
PubMed2001

Two-layer sample preparation method for MALDI mass spectrometric analysis of protein and peptide samples containing sodium dodecyl sulfate.

Sodium dodecyl sulfate (SDS) is widely used in protein sample workup. However, many mass spectrometric methods cannot tolerate the presence of this strong surfactant in a protein sample. We present a practical and robust technique based on a two-layer matrix/sample deposition method for the analysis of protein and peptide samples containing SDS by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The two-layer method involves the deposition of a mixture of sample and matrix on top of a thin layer of matrix crystals. It was found that for SDS-containing samples, the intensity of the MALDI signals can be affected by the conditions of sample preparation: on-probe washing, choice of matrix, deposition method, solvent system, and protein-to-SDS ratio. However, we found that, under appropriate conditions, the two-layer method gave reliable MALDI signals for samples with levels of SDS up to approximately 1%. The applications of this method are demonstrated for MALDI analysis of hydrophobic membrane proteins as well as bacterial extracts. We envision that this two-layer method capable of handling impure samples including those containing SDS will play an important role in protein molecular weight analysis as well as in proteome identification by MALDI-MS and MS/MS.

Bacterial Proteins↗
PubMed2001

MEK (MAPKK) inhibitors. Part 2: structure-activity relationships of 4-anilino-3-cyano-6,7-dialkoxyquinolines.

A series of 4-anilino-3-cyano-6,7-dialkoxyquinolines with different substituents attached to the 4-anilino group has been prepared that are potent MEK (MAP kinase kinase) inhibitors. The best activity is obtained when a phenyl or a thienyl group is attached to the para-position of the aniline through a hydrophobic linker, such as an oxygen, a sulfur, or a methylene group. The most active compounds show low nanomolar IC(50)'s against MEK (MAP kinase kinase), and have potent growth inhibitory activity in LoVo cells (human colon tumor line).

Aniline Compounds↗
PubMed2001

Functional Analysis of six novel ORFs on the left arm of Chromosome XII of Saccharomyces cerevisiae reveals three of them responding to S-starvation.

Six novel Open Reading Frames (ORFs) located on the left arm of the chromosome XII (YLL061w, YLL060c, YLL059c, YLL058w, YLL057c and YLL056c) have been analysed using either short-flanking homology (SFH) or long-flanking homology (LFH) gene replacement. Sporulation and tetrad analysis showed none of these ORFs to be essential for vegetative growth. The standard EUROFAN growth tests failed to reveal any obvious phenotypes resulting from deletion of each of the ORFs. Bioinformatic analysis revealed that YLL061w is probably an amino acid permease for S-methylmethionine and that YLL060c encodes a glutathione transferase which is involved in cellular detoxification, while YLL058w may play a role in sulphur-containing amino-acid metabolism, YLL057c in sulphonate catabolism and YLL056c in stress response. The transcription of three ORFs (YLL061w, YLL057c and YLL056c) has been shown to increase more than 10-fold under sulphate starvation. Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7. All the cognate clones, were generated using direct PCR products amplified from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Amino Acid Transport Systems↗
PubMed2001

Synthesis and Src kinase inhibitory activity of a series of 4-phenylamino-3-quinolinecarbonitriles.

Screening of a directed compound library in a yeast-based assay identified 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (2a) as a Src inhibitor. An enzymatic assay established that 2a was an ATP-competitive inhibitor of the kinase activity of Src. We present here SAR data for 2a which shows that the aniline group at C-4, the carbonitrile group at C-3, and the alkoxy groups at C-6 and C-7 of the quinoline are crucial for optimal activity. Increasing the size of the C-2 substituent of the aniline at C-4 of 2a from chloro to bromo to iodo resulted in a corresponding increase in Src inhibition. Furthermore, replacement of the 7-methoxy group of 2a with various 3-heteroalkylaminopropoxy groups provided increased inhibition of both Src enzymatic and cellular activity. Compound 25, which contains a 3-morpholinopropoxy group, had an IC(50) of 3.8 nM in the Src enzymatic assay and an IC(50) of 940 nM for the inhibition of Src-dependent cell proliferation.

Aniline Compounds↗
PubMed2001

Cyclosporine A as a potential neuroprotective agent: a study of prolonged hypothermic circulatory arrest in a chronic porcine model.

OBJECTIVE: To assess whether Cyclosporine A (CsA) or cycloheximide (CHX) can reduce ischemia-induced neurological damage by blocking apoptotic pathways, we assessed their effects on cerebral recovery in a chronic animal model of hypothermic circulatory arrest (HCA). METHODS: Twenty-eight pigs (28-33 kg) underwent 90 min of HCA at 20 degrees C. In this blinded study, animals were randomized to placebo (n=12), 5 mg/kg CsA (n=8), given intravenously before and subcutaneously for 7 days after HCA, or a single dose of 1 mg/kg CHX (n=8), given after weaning from cardiopulmonary bypass. Hemodynamics, intracranial pressure (ICP) and neurophysiological data (EEG, SSEP) were assessed for 3 h after HCA; early behavioral recovery was scored, and neurological/behavioral evaluation (9=normal) was carried out daily until elective sacrifice on postoperative day (POD) 7. Brains were selectively perfused and evaluated histopathologically for apoptosis. RESULTS: Basic hemodynamic data revealed no differences between CsA or CHX and control groups. ICP was significantly lower throughout rewarming (P=0.009) and reperfusion (P=0.05) in the CsA group. EEG recovery 3 h after HCA was observed in four of eight CsA animals but in only 1 of 12 controls (P=0.11) and one of eight CHX animals; cortical SSEP recovery also seemed faster in CsA animals, but failed to reach significance. Some early recovery scores were significantly better in the CsA group, and daily behavioral scores were consistently and significantly higher in the CsA-treated animals from POD1 through POD4. CONCLUSIONS: The data indicate that treatment with Cyclosporine A but not cycloheximide has a positive effect on cerebral recovery following HCA. Whether CsA results in inhibition of neuronal apoptosis, and/or inhibits release of cytokines and thereby reduces postischemic cerebral edema remains to be elucidated. The neuroprotective effect of CsA, if confirmed in further studies, would make its clinical application conceivable.

Animals↗
PubMed2001

The ties that bind what is known to the recognition of what is new.

Recognition success varies with how information is encoded (e.g., level of processing) and with what is already known as a result of past learning (e.g., word frequency). This article presents the results of experiments showing that preexisting connections involving the associates of studied words facilitate their recognition regardless of whether the words are intentionally encoded or are incidentally encoded under semantic or nonsemantic conditions. Words are more likely to be recognized when they have either more resonant connections coming back to them from their associates or more connections among their associates. Such results occur even though attention is never drawn to these associates. Regression analyses showed that these connections affect recognition independently of frequency, so the present results add to the literature showing that prior lexical knowledge contributes to episodic recognition. In addition, equations that use free-association data to derive composite strength indices of resonance and connectivity were evaluated. Implications for theories of recognition are discussed.

Adult↗
PubMed2001

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations.

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are "silent, " that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing "metabolic snapshots," can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY-an abbreviation for functional analysis by co-responses in yeast.

Adenine Nucleotides↗
PubMed2001

Cerebral effects of cold reperfusion after hypothermic circulatory arrest.

OBJECTIVES: This study was undertaken to explore whether an interval of cold reperfusion can improve cerebral outcome after prolonged hypothermic circulatory arrest. METHODS: Sixteen pigs (27-30 kg) underwent 90 minutes of circulatory arrest at a brain temperature of 20 degrees C. Eight animals were rewarmed immediately after hypothermic circulatory arrest (controls), and 8 were reperfused for 20 minutes at 20 degrees C and then rewarmed (cold reperfusion). Electrophysiologic recordings, fluorescent microsphere determinations of cerebral blood flow, calculations of cerebral oxygen consumption, and direct measurements of intracranial pressure (millimeters of mercury) were obtained at baseline (37 degrees C), before hypothermic circulatory arrest, after discontinuing circulatory arrest at 37 degrees C deep brain temperature, and at 2, 4, and 6 hours thereafter. Histopathologic features and percent brain water were determined after the animals were sacrificed. RESULTS: Cerebral blood flow and oxygen consumption decreased during cooling: cerebral oxygen consumption returned to baseline levels after 4 hours, but cerebral blood flow remained depressed until 6 hours in both groups. Cold reperfusion failed to improve electrophysiologic recovery or to reduce brain weight, but median intracranial pressure increased significantly less after cold reperfusion than in controls (P =.02). Although no significant difference in the incidence of histopathologic abnormalities between groups was found, all 3 animals with an intracranial pressure of more than 15 mm Hg after immediate rewarming had histopathologic lesions, and high intracranial pressure was more prevalent among all animals with subsequent histopathologic lesions (P =.03). CONCLUSIONS: Cold reperfusion significantly inhibited the rise in intracranial pressure seen in control pigs after 90 minutes of circulatory arrest at 20 degrees C, suggesting that cold reperfusion may decrease cerebral edema and thereby improve outcome after prolonged hypothermic circulatory arrest.

Animals↗
PubMed2001

Retrograde cerebral perfusion provides negligible flow through brain capillaries in the pig.

OBJECTIVES: Although retrograde cerebral perfusion is being used clinically during aortic arch surgery, whether retrograde flow perfuses the brain effectively is still uncertain. METHODS: Fourteen pigs were cooled to 20 degrees C with cardiopulmonary bypass and perfused retrogradely via the superior vena cava for 30 minutes: 7 underwent standard retrograde cerebral perfusion and 7 underwent retrograde perfusion with occlusion of the inferior vena cava. Antegrade and retrograde cerebral blood flow were calculated by quantitating fluorescent microspheres trapped in brain tissue after the animals were put to death; microspheres returning to the aortic arch, the inferior vena cava, and the descending aorta were also analyzed during retrograde cerebral perfusion. RESULTS: Antegrade cerebral blood flow was 16 +/- 7.7 mL. min(-1). 100 g(-1) before retrograde cerebral perfusion and 22 +/- 6.3 mL. min(-1). 100 g(-1) before perfusion with caval occlusion (P =.14). During retrograde perfusion, calculations based on the number of microspheres trapped in the brain showed negligible flows (0.02 +/- 0.02 mL. min(-1). 100 g(-1) with retrograde cerebral perfusion and 0.04 +/- 0.02 mL. min(-1). 100 g(-1) with perfusion with caval occlusion; P =.09): only 0.01% and 0.02% of superior vena caval inflow, respectively. Less than 13% of retrograde superior vena caval inflow blood returned to the aortic arch with either technique. During retrograde cerebral perfusion, more than 90% of superior vena caval input was shunted to the inferior vena cava and was then recirculated, as indicated by rapid development of an equilibrium in microspheres between the superior and inferior venae cavae. With retrograde perfusion and inferior vena caval occlusion, less than 12% of inflow returned to the descending aorta and only 0.01% of microspheres. CONCLUSIONS: The paucity of microspheres trapped within the brain indicates that retrograde cerebral perfusion, either alone or combined with inferior vena caval occlusion, does not provide sufficient cerebral capillary perfusion to confer any metabolic benefit. The slightly improved outcome previously reported with retrograde cerebral perfusion during prolonged circulatory arrest in this model may be a consequence of enhanced cooling resulting from perfusion of nonbrain capillaries and from venoarterial and venovenous shunting.

Analysis of Variance↗
PubMed2001

Ggamma in dictyostelium: its role in localization of gbetagamma to the membrane is required for chemotaxis in shallow gradients.

G-protein-mediated signal transduction pathways play an essential role in the developmental program of the simple eukaryotic organism Dictyostelium discoideum. Database searches have yielded 11 Galpha-subunits, a single Gbeta-subunit, but no Ggamma-subunits. We report here the purification, cDNA isolation, and functional analysis of a Ggamma-subunit. Like Gbeta, the Ggamma appears to be unique and hybridization studies show that Ggamma and Gbeta are expressed in parallel during development. Species-wide sequence comparisons of Ggamma-subunits and gamma-like domains of RGS proteins reveal short stretches of highly conserved residues as well as the common CXXL motif at the COOH-terminal of Ggammas that target Gbetagammas to plasma membrane. Overexpression of a CSVL-deleted Ggamma (GgammaDelta) in wild-type cells shifts Gbetagamma to the cytosol and selectively impairs certain G-protein-mediated signal transduction pathways. These cells are able to respond to increments in the stimulus, but are unable to sense chemoattractant gradients. They neither move directionally nor recruit PH-domains to their leading edge. Thus, a full complement of membrane-tethered Gbetagamma is required for sensing shallow gradients, but is not essential for responses to increments in extracellular stimuli.

Animals↗
PubMed2001

Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms.

Cyclin E is a G(1) cyclin essential for S-phase entry and has a profound role in oncogenesis. Previously this laboratory found that cyclin E is overexpressed and present in lower-molecular-weight (LMW) isoforms in breast cancer cells and tumor tissues compared to normal cells and tissues. Such alteration of cyclin E is linked to poor patient outcome. Here we report that the LMW forms of cyclin E are hyperactive biochemically and they can more readily induce G(1)-to-S progression in transfected normal cells than the full-length form of the protein can. Through biochemical and mutational analyses we have identified two proteolytically sensitive sites in the amino terminus of human cyclin E that are cleaved to generate the LMW isoforms found in tumor cells. Not only are the LMW forms of cyclin E functional, as they phosphorylate substrates such as histone H1 and GST-Rb, but also their activities are higher than the full-length cyclin E. These nuclear localized LMW forms of cyclin E are also biologically functional, as their overexpression in normal cells increases the ability of these cells to enter S and G(2)/M. Lastly, we show that cyclin E is selectively cleaved in vitro by the elastase class of serine proteases to generate LMW forms similar to those observed in tumor cells. These studies suggest that the defective entry into and exit from S phase by tumor cells is in part due to the proteolytic processing of cyclin E, which generates hyperactive LMW isoforms whose activities have been modified from that of the full-length protein.

Amino Acid Sequence↗
PubMed2000

Differential processing of UV mimetic and interstrand crosslink damage by XPF cell extracts.

We have recently developed a mammalian cell free assay in which interstrand crosslinks induce DNA synthesis in both damaged and undamaged plasmids co-incubated in the same extract. We have also shown using hamster mutants that both ERCC1 and XPF are required for the observed incorporation. Here, we show that extracts from an XPF patient cell line differentially process UV mimetic damage and interstrand crosslinks in vitro. XPF extracts are highly defective in the stimulation of repair synthesis by N:-acetoxy-N:- acetylaminofluorene, but are proficient in the stimulation of DNA synthesis by psoralen interstrand crosslinks. In addition, we show that extracts from the hamster UV140 mutant, which has high UV sensitivity, but moderate mitomycin C sensitivity, are similar in both assays to XPF cell extracts. These findings support the hypothesis that the activities of XPF in nucleotide excision repair (NER) and crosslink repair are separable, and that mutations in XPF patients result in the abolition of NER, but not recombinational repair pathways, which are likely to be essential as has been observed in ERCC1 homozygous -/- mice.

Animals↗