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J Zhou

Publications and source records attributed to J Zhou.

At least 181 records · Page 10Linked to original sources
PubMed2001

Pseudomonas sp. strain KC represents a new genomovar within Pseudomonas stutzeri.

Pseudomonas sp. strain KC (= ATCC 55595 = DSM 7136) is a denitrifying aquifer isolate that produces and secretes pyridine-2,6-bis(thiocarboxylate) (PDTC), a chelating agent that fortuitously transforms carbon tetrachloride without producing chloroform. Although KC has been used successfully for full-scale bioremediation of carbon tetrachloride, its taxonomy has proven difficult to resolve, as it retains properties of both Pseudomonas stutzeri and Pseudomonas putida. In the present work, a polyphasic approach was used to conclude that strain KC represents a new genomovar (genomovar 9) within the species P. stutzeri.

Biodegradation, Environmental↗
PubMed2001

The N-terminal portion of mature aldehyde dehydrogenase affects protein folding and assembly.

Human liver cytosolic (ALDH1) and mitochondrial (ALDH2) aldehyde dehydrogenases are both encoded in the nucleus and synthesized in the cytosol. ALDH1 must fold in the cytosol, but ALDH2 is first synthesized as a precursor and must remain unfolded during import into mitochondria. The two mature forms share high identity (68%) at the protein sequence level except for the first 21 residues (14%); their tertiary structures were found to be essentially identical. ALDH1 folded faster in vitro than ALDH2 and could assemble to tetramers while ALDH2 remained as monomers. Import assay was used as a tool to study the folding status of ALDH1 and ALDH2. pALDH1 was made by fusing the presequence of precursor ALDH2 to the N-terminal end of ALDH1. Its import was reduced about 10-fold compared to the precursor ALDH2. The exchange of the N-terminal 21 residues from the mature portion altered import, folding, and assembly of precursor ALDH1 and precursor ALDH2. More of chimeric ALDH1 precursor was imported into mitochondria compared to its parent precursor ALDH1. The import of chimeric ALDH2 precursor, the counterpart of chimeric ALDH1 precursor, was reduced compared to its parent precursor ALDH2. Mature ALDH1 proved to be more stable against urea denaturation than ALDH2. Urea unfolding improved the import of precursor ALDH1 and the chimeric precursors but not precursor ALDH2, consistent with ALDH1 and the chimeric ALDHs being more stable than ALDH2. The N-terminal segment of the mature protein, and not the presequence, makes a major contribution to the folding, assembly, and stability of the precursor and may play a role in folding and hence the translocation of the precursor into mitochondria.

Aldehyde Dehydrogenase↗
PubMed2001

Simultaneous recovery of RNA and DNA from soils and sediments.

Recovery of mRNA from environmental samples for measurement of in situ metabolic activities is a significant challenge. A robust, simple, rapid, and effective method was developed for simultaneous recovery of both RNA and DNA from soils of diverse composition by adapting our previous grinding-based cell lysis method (Zhou et al., Appl. Environ. Microbiol. 62:316-322, 1996) for DNA extraction. One of the key differences is that the samples are ground in a denaturing solution at a temperature below 0 degrees C to inactivate nuclease activity. Two different methods were evaluated for separating RNA from DNA. Among the methods examined for RNA purification, anion exchange resin gave the best results in terms of RNA integrity, yield, and purity. With the optimized protocol, intact RNA and high-molecular-weight DNA were simultaneously recovered from 19 soil and stream sediment samples of diverse composition. The RNA yield from these samples ranged from 1.4 to 56 microg g of soil(-1) dry weight), whereas the DNA yield ranged from 23 to 435 microg g(-1). In addition, studies with the same soil sample showed that the DNA yield was, on average, 40% higher than that in our previous procedure and 68% higher than that in a commercial bead milling method. For the majority of the samples, the DNA and RNA recovered were of sufficient purity for nuclease digestion, microarray hybridization, and PCR or reverse transcription-PCR amplification.

DNA, Bacterial↗
PubMed2001

Development and evaluation of functional gene arrays for detection of selected genes in the environment.

To determine the potential of DNA array technology for assessing functional gene diversity and distribution, a prototype microarray was constructed with genes involved in nitrogen cycling: nitrite reductase (nirS and nirK) genes, ammonia mono-oxygenase (amoA) genes, and methane mono-oxygenase (pmoA) genes from pure cultures and those cloned from marine sediments. In experiments using glass slide microarrays, genes possessing less than 80 to 85% sequence identity were differentiated under hybridization conditions of high stringency (65 degrees C). The detection limit for nirS genes was approximately 1 ng of pure genomic DNA and 25 ng of soil community DNA using our optimized protocol. A linear quantitative relationship (r(2) = 0.89 to 0.94) was observed between signal intensity and target DNA concentration over a range of 1 to 100 ng for genomic DNA (or genomic DNA equivalent) from both pure cultures and mixed communities. However, the quantitative capacity of microarrays for measuring the relative abundance of targeted genes in complex environmental samples is less clear due to divergent target sequences. Sequence divergence and probe length affected hybridization signal intensity within a certain range of sequence identity and size, respectively. This prototype functional gene array did reveal differences in the apparent distribution of nir and amoA and pmoA gene families in sediment and soil samples. Our results indicate that glass-based microarray hybridization has potential as a tool for revealing functional gene composition in natural microbial communities; however, more work is needed to improve sensitivity and quantitation and to understand the associated issue of specificity.

Bacteria↗
PubMed2001

Pseudomonas stutzeri nitrite reductase gene abundance in environmental samples measured by real-time PCR.

We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primer-probe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 10(6) gene copies (r2 = 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier.

DNA, Bacterial↗
PubMed2001

Evaluation of PCR-generated chimeras, mutations, and heteroduplexes with 16S rRNA gene-based cloning.

To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.

Artifacts↗
PubMed2001

DNA microarray analysis of the hyperthermophilic archaeon Pyrococcus furiosus: evidence for anNew type of sulfur-reducing enzyme complex.

DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S(primary)) on the levels of gene expression in cells grown at 95 degrees C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S(primary) and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S(primary). Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S(primary)-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S(primary)-grown cells, and these are also thought be involved in S(primary) reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S(primary)-grown cells, a finding consistent with the fact that growth on peptides is a S(primary)-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S(primary), although the reason for this is as yet unknown. Of the 20 S(primary)-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S(primary)-independent ORFs that are not expressed above the detection limit have been characterized. These results represent the first derived from the application of DNA microarrays to either an archaeon or a hyperthermophile.

Archaeal Proteins↗
PubMed2001

Identification and antimicrobial susceptibility of Alcaligenes xylosoxidans isolated from patients with cystic fibrosis.

In the past decade, potential pathogens, including Alcaligenes species, have been increasingly recovered from cystic fibrosis (CF) patients. Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF. We examined the frequency of correct identification of Alcaligenes spp. by microbiology laboratories affiliated with American CF patient care centers. Selective media, an exotoxin A probe for Pseudomonas aeruginosa, and a commercial identification assay, API 20 NE, were used for identification. The activity of antimicrobial agents against these clinical isolates was determined. A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied. Most (89%) were correctly identified by the referring laboratories as Alcaligenes xylosoxidans. However, 12 (11%) strains were misidentified; these were found to be P. aeruginosa (n = 10), Stenotrophomonas maltophilia (n = 1), and Burkholderia cepacia (n = 1). Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited. High concentrations of colistin (100 and 200 microg/ml) inhibited 92% of strains. Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains. Selective media and biochemical identification proved to be useful strategies for distinguishing A. xylosoxidans from other CF pathogens. Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of A. xylosoxidans should be determined.

Alcaligenes↗
PubMed2001

Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network.

The emergence of disease caused by penicillin-resistant and multidrug-resistant pneumococci has become a global concern, necessitating the identification of the epidemiological spread of such strains. The Pneumococcal Molecular Epidemiology Network was established in 1997 under the auspices of the International Union of Microbiological Societies with the aim of characterizing, standardizing, naming, and classifying antimicrobial agent-resistant pneumococcal clones. Here we describe the nomenclature for 16 pneumococcal clones that have contributed to the increase in antimicrobial resistance worldwide. Guidelines for the recognition of these clones using molecular typing procedures (pulsed-field gel electrophoresis, BOX-PCR, and multilocus sequence typing) are presented, as are the penicillin-binding profiles and macrolide resistance determinants for the 16 clones. This network can serve as a prototype for the collaboration of scientists in identifying clones of important human pathogens and as a model for the development of other networks.

Anti-Bacterial Agents↗
PubMed2001

Nef enhances human immunodeficiency virus type 1 infectivity resulting from intervirion fusion: evidence supporting a role for Nef at the virion envelope.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by facilitating an early event in the HIV-1 life cycle. Although no structural or biochemical defects in Nef-defective HIV-1 particles have been demonstrated, the Nef protein is incorporated into HIV-1 particles. To localize the function of Nef within the virus particle, we developed a novel technology involving fusion of enveloped donor HIV-1 particles bearing core defects with envelope-defective target virions bearing HIV-1 receptors. Although neither virus alone was capable of infecting CD4(+) target cells, the incubation of donor and target virions prior to addition to target cells resulted in infection. This effect, termed "virion transcomplementation," required a functional Env protein on the donor virus and CD4 and an appropriate coreceptor on target virions. To provide evidence for intervirion fusion as the mechanism of complementation, experiments were performed using dual-enveloped HIV-1 particles bearing both HIV-1 and ecotropic murine leukemia virus (E-MLV) Env proteins as donor virions. Infection of CD4-negative target cells bearing E-MLV receptors was prevented by HIV-1 entry inhibitors when added before, but not after, incubation of donor and target virions prior to the addition to cells. When we used Nef(+) and Nef(-) donor and target virions, Nef enhanced infection when present in donor virions. In contrast, no effect of Nef was detected when present in the target virus. These results reveal a potential mechanism for enhancing HIV-1 diversity in vivo through the rescue of defective viral genomes and provide a novel genetic system for the functional analysis of virion-associated proteins in HIV-1 infection.

CD4-Positive T-Lymphocytes↗
PubMed2001

SAP97 interacts with Kv1.5 in heterologous expression systems.

PDZ domain-containing proteins such as SAP97 and ZO-1 have been implicated in the targeting and clustering of ion channels. We have explored the interactions of these polypeptides with a cardiac voltage-gated potassium channel. Immunocytochemistry in cardiac myocytes revealed colocalization of SAP97 and Kv1.5, both at the intercalated disks and the lateral membranes. Transient transfection experiments in COS-7 cells revealed that SAP97 and Kv1.5 polypeptides formed perinuclear clustered complexes that could be coimmunoprecipitated. Mutation of the three COOH-terminal amino acid residues of Kv1.5 (T-D-L to A-A-A) abolished these interactions. Whereas in most COS-7 cells the SAP97-Kv1.5 complexes were retained in the ER, functional analyses in Xenopus oocytes showed that Kv1.5-encoded outward potassium currents were augmented by coexpression with SAP97. By contrast, cotransfected ZO-1 and Kv1.5 polypeptides in COS-7 cells could not be coprecipitated nor did the coinjection of ZO-1 augment the Kv1.5-encoded currents in oocytes. Collectively, our results suggest that SAP97 may play an important role in the modulation of Kv1.5 channel function in cardiac myocytes.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Dietary supplementation with methionine and homocysteine promotes early atherosclerosis but not plaque rupture in ApoE-deficient mice.

Hyperhomocysteinemia is an independent risk factor for atherothrombosis. However, causality is unproven, and it remains unknown whether hyperhomocysteinemia promotes atherosclerosis, plaque rupture, and/or thrombosis. We evaluated the short- and long-term effects of hyperhomocysteinemia on plaque size and structure in 99 atherosclerosis-prone apolipoprotein E-deficient mice. Hyperhomocysteinemia was induced by methionine (Met) or homocysteine (HcyH) supplementation: low Met (+11 g Met/kg food), high Met (+33 g Met/kg food), low HcyH (0.9 g HcyH/L drinking water), and high HcyH (1.8 g HcyH/L drinking water). Met and HcyH supplementation significantly raised plasma total homocysteine levels by 4- to 16-fold above those observed in mice fed a control diet (up to 146.1 micromol/L). Compared with controls, aortic root plaque size was significantly larger in supplemented groups after 3 months (56% and 173% larger in high-Met and high-HcyH, respectively) but not after 12 months. Hyperhomocysteinemia was associated with an increase in the amount of collagen in plaques after both 3 and 12 months. Mechanical testing of the tail tendons revealed no weakening of collagen after 12 months of hyperhomocysteinemia. Many plaques in both control and supplemented mice appeared rupture prone morphologically, but all aortic root plaques and all but 1 coronary plaque had an intact surface without rupture or thrombosis. Thus, diet-induced hyperhomocysteinemia promotes early atherosclerosis and plaque fibrosis but does not, even in the long term, weaken collagen or induce plaque rupture.

Animals↗
PubMed2001

Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways.

Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine beta-synthase. However, the mechanism by which homocysteine promotes the development and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regulatory element-binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intracellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by overexpression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the activation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secretion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia.

Animals↗
PubMed2001

Changes in rat adipocyte and liver glucose metabolism following repeated restraint stress.

Rats exposed to repeated restraint weigh less than controls even 8 weeks after stress. Stress-induced weight loss is lean tissue, but the post-stress difference in weight between control and restrained rats is lean and fat mass. Whole-body glucose clearance is enhanced 1 day after stress, but adipocyte glucose utilization is inhibited and muscle glucose transport is unchanged. The studies described here demonstrated that glucose transport was increased in both restrained and pair-fed rats, but that glycogen synthesis was increased only in restrained rats, which may account for the improved whole-body glucose clearance. Adipocyte glucose transport was inhibited and adipose plasma membrane beta-adrenergic receptor number was increased 1 day post-stress in restrained rats when weight loss was lean tissue, but were not different from control rats 5 days post-stress, when both fat and lean tissue were reduced. Thus, repeated restraint induces reversible changes in adipocyte metabolism that may represent a transition from the catabolic state of stress to a new energetic equilibrium in rats that maintain a reduced body weight for an extended period of time.

Adipocytes↗
PubMed2001

The role of thrombospondin-1 in tumor progression.

The role of thrombospondin-1 (TSP-1) in tumor progression is both complex and controversial. It is clear from the literature that the function of TSP-1 in malignancy depends on the presence of other factors and the level of TSP-1 expression in the tumor tissue. High levels of TSP-1 secreted by tumors, which were engineered to overexpress TSP-1, inhibit tumor growth, while anti-sense inhibition of TSP-1 production in certain tumors also inhibits growth. Clearly, the presence of other factors in these experimental systems must be important. The role of TSP-1 in angiogenesis also depends on the levels of TSP-1, the presence and level of angiogenic stimulators such as basic fibroblast growth factor (bFGF), and the localization of TSP-1 in the tissue. Matrix-bound TSP-1 promotes capillary tube formation in the rat aorta model of angiogenesis, while TSP-1 inhibits bFGF- induced angiogenesis in the rat cornea model. The inhibitory effect also depends on the proteolytic state of TSP-1 since the amino terminus promotes angiogenesis in the cornea model, while the remaining 140-kDa fragment inhibits bFGF-induced angiogenesis. Both the stimulatory and inhibitory effects of TSP-1 are likely due to upregulation of matrix-degrading enzymes and their inhibitors. These enzymes are critical for maintaining optimal matrix turnover during angiogenesis. These varied TSP-1-dependent mechanisms offer new targets for the development of anti-angiogenic therapeutics for the treatment of a variety of cancers, as well as other pathologies involving inappropriate angiogenesis such as diabetic retinopathy.

Animals↗
PubMed2001

Glucagon-like peptide-1 causes pancreatic duodenal homeobox-1 protein translocation from the cytoplasm to the nucleus of pancreatic beta-cells by a cyclic adenosine monophosphate/protein kinase A-dependent mechanism.

Glucagon-like peptide-1 (GLP-1) enhances insulin secretion and synthesis. It also regulates the insulin, glucokinase, and GLUT2 genes. It mediates increases in glucose-stimulated insulin secretion by activating adenylyl cyclase and elevating free cytosolic calcium levels in the beta-cell. In addition, GLP-1 has been shown, both in vitro and in vivo, to be involved in regulation of the transcription factor, pancreatic duodenal homeobox-1 protein (PDX-1), by increasing its total protein levels, its translocation to the nucleus and its binding and resultant increase in activity of the insulin gene promoter in beta-cells of the pancreas. Here we have investigated the role of protein kinase A (PKA) in these processes in RIN 1046-38 cells. Three separate inhibitors of PKA, and a cAMP antagonist, inhibited the effects of GLP-1 on PDX-1. Furthermore, forskolin, (which stimulates adenylyl cyclase and insulin secretion), and 8-Bromo-cAMP, (an analog of cAMP which also stimulates insulin secretion), mimicked the effects of GLP-1 on PDX-1. These effects were also prevented by PKA inhibitors. Glucose-mediated increases in nuclear translocation of PDX-1 were not prevented by PKA inhibitors. Our results suggest that regulation of PDX-1 by GLP-1 occurs through activation of adenylyl cyclase and the resultant increase in intracellular cAMP, in turn, activates PKA, which ultimately leads to increases in PDX-1 protein levels and translocation of the protein to the nuclei of beta-cells.

Animals↗
PubMed2001

Effects of two saponins extracted from the polygonatum Zanlanscianense pamp on the human leukemia (HL-60) cells.

Two saponins, methyl protodioscin and dioscin, were extracted from the root of Polygonatum Zanlanscianense Pamp. One of them, dioscin exerted significant inhibitory effects on the growth of the human leukemia cell HL-60, inducing differentiation and apoptosis. HL60 cells were induced mainly along the granulocytic lineage. In addition, we have found that dioscin affects many cancer cells. These studies may have important significance in treating related cancers.

Apoptosis↗