Biomedical subjects

J Xu

Publications and source records attributed to J Xu.

At least 559 records · Page 31Linked to original sources

PubMed1999

Efficiency of a novel non-Shine-Dalgarno and a Shine-Dalgarno consensus sequence to initiate translation in Escherichia coli of genes with different downstream box composition.

The efficiency of a novel non-Shine-Dalgarno translational initiator (ACCUACUCGAGUUAG, denoted PL) to promote translation in Escherichia coli was compared with that of the Shine-Dalgarno (SD) consensus sequence (AAGGAGGU) using four reporter genes. The obtained results showed that the genes of pokeweed antiviral protein (PAP I) and human calcitonin (CT) were poorly expressed under the conventional SD and were better expressed under the PL sequence. On the contrary, the genes of human interferon gamma (hIFN gamma) and chloramphenicol acetyltransferase (CAT) were highly expressed under SD and poorly expressed under the PL sequence. Computer search revealed a great diversity between the four reporter genes in respect to their complementarity to E. coli 16S rRNA. PAP I and CT genes were rich in nucleotides matching 16S rRNA (called downstream boxes) whereas the complementary domains in the other two (hIFN-gamma and CAT) genes were much shorter. The different behavior of the four reporter genes when placed under the translational control of SD and PL sequences was explained by the different binding energy of their mRNAs to the 30S ribosomal subunit.

Calcitonin↗

PubMed1999

Structural analysis of 14-3-3 phosphopeptide complexes identifies a dual role for the nuclear export signal of 14-3-3 in ligand binding.

We have solved the high-resolution X-ray structure of 14-3-3 bound to two different phosphoserine peptides, representing alternative substrate-binding motifs. These structures reveal an evolutionarily conserved network of peptide-protein interactions within all 14-3-3 isotypes, explain both binding motifs, and identify a novel intrachain phosphorylation-mediated loop structure in one of the peptides. A 14-3-3 mutation disrupting Raf signaling alters the ligand-binding cleft, selecting a different phosphopeptide-binding motif and different substrates than the wild-type protein. Many 14-3-3: peptide contacts involve a C-terminal amphipathic alpha helix containing a putative nuclear export signal, implicating this segment in both ligand and Crm1 binding. Structural homology between the 14-3-3 NES structure and those within I kappa B alpha and p53 reveals a conserved topology recognized by the Crm1 nuclear export machinery.

14-3-3 Proteins↗

PubMed1999

Differential effect of cycloheximide on neuronal and glioma cells treated with chemotherapy and radiation.

Dividing cells and non-dividing cells are distinct in their cell cycle kinetics, and react differently when facing cytotoxic stimuli. A protein synthesis inhibitor, cycloheximide (CHX), has recently been found to protect neuronal cells from oxidative stress. We investigated whether CHX exerts differential effects on dividing and non-dividing cells in the brain under cytotoxic stimuli. Mitotic C6 rat glioma cells and postmitotic neuronal cells were studied with a cytotoxic regimen combining gamma-irradiation (RT) and 1,3-bis,2-chloroethyl-1-nitrosourea (BCNU). Cells were exposed to BCNU (1 g/ml) for 15 h before gamma-irradiation and incubated with CHX (1 g/ml) from 30 min before and until 5 h after irradiation. Clonogenic assay was used to assess cytotoxic effects on C6 glioma cells. LDH assay was used for the viability of H19-7 postmitotic neuronal cells. A 2.27-3.75 fold enhancement of cytotoxicity was noticed with the addition of CHX to BCNU and 2-10 Gy of radiation. Our data demonstrated that CHX enhanced cytotoxicity of RT plus BCNU, while no additional toxicity was incurred to the postmitotic neuronal cells when CHX was added. We further studied whether the inhibition of DNA repair, assayed by single-cell DNA electrophoresis (comet assay), is a contributing factor for the enhanced cytotoxicity on C6 glioma cells. Interestingly, the initial DNA damage after RT plus BCNU was equivalent; whereas DNA repair was significantly less at 5 h after radiation in CHX-treated C6 glioma cells. Protecting non-dividing neuronal cells to avoid excessive functional deficit is an integral part of a successful brain tumor treatment regimen. Taking advantage of the differential effect of CHX on glioma and neuronal cells may improve tumor control without excessive neural toxicity.

Animals↗

PubMed1999

Analysis of Philadelphia chromosome-negative BCR-ABL-positive chronic myelogenous leukemia by hypermetaphase fluorescence in situ hybridization.

BACKGROUND: In 5%-10% of patients with of chronic myelogenous leukemia (CML), the Philadelphia chromosome (Ph) is not identified, despite the presence of the associated BCR-ABL molecular abnormality (Ph-negative, BCR-ABL-positive CML) because of sub-microscopic rearrangements. PATIENTS AND METHODS: Six patients with Ph-negative, BCR-ABL-positive CML were investigated. The Ph chromosome detection via fluorescence in situ hybridization after 24-hour mitotic arrest of bone marrow cultures resulting in several hundreds of metaphases (hypermetaphase FISH or HMF) was useful in explaining the nature of the six cases. RESULTS: Four patients had a low frequency of Ph-positive cells by HMF (5.7%, 4.8%, 3.9%, 0.2%), i.e., a typical Ph translocation. However, two cases involved a 9q34 inserted into chromosome 22q11 (74.2% and 92%), without a deletion from chromosome 22 and reciprocal translocation onto 9, i.e., not a typical Ph translocation. The pattern of UBCR gene rearrangement was characterized by the same genomic recombination of 5-BCR and c-ABL, both in the four cases of typical translocation (9;22) and in the two cases of insertion of 9q34 into chromosome 22q11. CONCLUSIONS: The HMF identified two different bases for Ph-negative, BCR-ABL-positive cells in CML-presence of low frequency of cells with typical Ph translocations or presence of cells with ABL insertions into the BCR gene on chromosome 22.

Adult↗

PubMed1999

Studies on the cytosolic phospholipase A2 in immortalized astrocytes (DITNC) revealed new properties of the calcium ionophore, A23187.

Besides playing an important role in the maintenance of cell membrane phospholipids, phospholipases A2 (PLA2) are responsible for the release of arachidonic acid (AA) which is a precursor for prostaglandin biosynthesis. The cytosolic PLA2 has been the focus of recent studies, probably due to its ability to respond to protein kinases and changes in intracellular calcium levels. In this study, we examined agents for stimulation of the cytosolic phospholipase A2 in immortalized astrocytes (DITNC). Incubation of DITNC cells with [14C]arachidonic acid (AA) resulted in a time-dependent uptake of the label into phospholipids (PL) and neutral glycerides. In prelabeled cells, release of labeled AA could be stimulated by calcium mobilizing agents such as calcium ionophore A23187 (4-20 microM) and thimerosal (100 microM), and by phorbol myristate acetate (PMA, 100 nM), an agent for activation of protein kinase C. The release of AA could also be stimulated by ATP (200 microM), probably through activation of the purinergic receptor but not by glutamate (1 mM). The stimulated release of AA was dependent on extracellular Ca2+ and was inhibited by mepacrine (50 microM), a non-specific PLA2 inhibitor. Western blot analysis further confirmed the presence of an 85 kDa cPLA2 in both membrane and cytosol fractions of these cells and stimulation by A23187 resulted in translocation of this protein to the membrane fraction. Besides labeled fatty acids, A23187 also stimulated the concomitant release of labeled PL into the culture medium and this event was accompanied by the increased release in lactate dehydrogenase (LDH). Results thus revealed that besides activation of cPLA2, the calcium ionophore A23187 is capable of perturbating cell membrane integrity.

Animals↗

PubMed1999

Engineered detoxification confers resistance against a pathogenic bacterium.

We generated transgenic sugarcane plants that express an albicidin detoxifying gene (albD), which was cloned from a bacterium that provides biocontrol against leaf scald disease. Plants with albicidin detoxification capacity equivalent to 1-10 ng of AlbD enzyme per mg of leaf protein did not develop chlorotic disease symptoms in inoculated leaves, whereas all untransformed control plants developed severe symptoms. Transgenic lines with high AlbD activity in young stems were also protected against systemic multiplication of the pathogen, which is the precursor to economic disease. We have shown that genetic modification to express a toxin-resistance gene can confer resistance to both disease symptoms and multiplication of a toxigenic pathogen in its host.

Anti-Bacterial Agents↗

PubMed1999

PCR-restriction fragment length polymorphism (RFLP) analyses reveal both extensive clonality and local genetic differences in Candida albicans.

Using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to obtain genotypes for the diploid pathogenic yeast, Candida albicans, we analysed 204 C. albicans isolates from three populations of the Duke University community: two from clinical sources [one from patients infected with human immunodeficiency virus (HIV) and the other from patients without HIV infection], and the third from healthy student volunteers. The results indicated: (i) extensive evidence for clonality within and between populations of C. albicans; and (ii) greater genotypic and gene diversities in the nonclinical population than those derived from clinical specimens, regardless of HIV status. The two clinical populations were genetically more similar to each other than either was to the population consisting of isolates from healthy people. Within each population sample there was a general lack of heterozygotes, and random associations of alleles within and between loci were found in less than 50% of the loci or pairs of loci. These findings were consistent between the two sets of samples analysed: those including all isolates and those including only clone-corrected isolates. Possible mechanisms are presented to explain the observed patterns of genetic variation within and between C. albicans populations.

AIDS-Related Opportunistic Infections↗

PubMed1999

Mechanisms of bronchial hyperresponsiveness: the interaction of endothelin-1 and other cytokines.

Bronchial hyperresponsiveness (BHR) is a fundamental characteristic of asthma and has become part of a more recent definition of asthma. Data show a close link between bronchial hyperresponsiveness (both transient and persistent) and airway inflammation. Airway inflammation is orchestrated by a complex network of cytokines. It has been considered that endothelin-1 is involved in bronchoconstriction and airway structural remodelling and has several pro-inflammatory properties of potential relevance to asthma. We focus on endothelin-1, its activities and interaction with other cytokines in the pathogenesis of asthma.

Airway Resistance↗

PubMed1999

Pyrrolidine dithiocarbamate induces bovine cerebral endothelial cell death by increasing the intracellular zinc level.

The antioxidant and metal-chelating effects of pyrrolidine dithiocarbamate (PDTC) have been extensively studied. PDTC prevents cell death induced by various insults. However, PDTC itself may cause cell death in selected experimental paradigms. PDTC induced bovine cerebral endothelial cell death. However, in serum-depleted medium, PDTC did not affect the cell viability, suggesting that certain factors in serum may mediate the cytotoxic effect of PDTC. The metal chelators bathocuproine disulfonic acid, o-phenanthroline, bathophenanthroline disulfonic acid, and N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine (TPEN) prevented the cell death induced by PDTC. In a serum-deprived condition, addition of exogenous metals, copper or zinc, restored the cytotoxic effect of PDTC. These data indicate that metals such as copper or zinc in serum may mediate the cytotoxic effect of PDTC. The potency of zinc for PDTC-induced endothelial cell death was greater than that of copper. Zn-EDTA did not block PDTC-induced cell death, whereas Ca-EDTA and Cu-EDTA were able to prevent this PDTC effect. PDTC increased the intracellular fluorescence of the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide, which was quenched by TPEN or various EDTA preparations but not by Zn-EDTA. Results suggest that an increase in intracellular zinc concentration is required in PDTC-induced cerebral endothelial cell death.

Animals↗

PubMed1999

Chronic electrical stimulation of the auditory nerve using non-charge-balanced stimuli.

This study was designed to evaluate the pathophysiological response of the cochlea following long-term intracochlear electrical stimulation using a poorly charge-balanced stimulus regime, leading to direct current (DC) levels >0.1 microA. Four normal-hearing adult cats were bilaterally implanted with scala tympani electrode arrays and unilaterally stimulated for periods up to 2200 h. Stimuli consisted of 50 micros monophasic current pulses presented at 2000 pulses per second (pps) per channel, and resulted in DC levels of 0.4-2.8 microA. Both acoustic and electrical (EABR) evoked potentials were periodically recorded during the stimulation program. Frequency-specific stimuli indicated that an extensive and widespread hearing loss occurred over the 4-24 KHz region in all stimulated cochleae, although the 2 KHz region exhibited thresholds close to normal in some animals, despite long-term implantation and chronic stimulation. Longitudinal EABRs showed a statistically significant increase in threshold for three of the four animals. Histopathological evaluation of the cochleae revealed a highly significant reduction in ganglion cell density in stimulated cochleae compared with their controls. Spiral ganglion cell loss was significantly correlated with the degree of inflammation, duration of electrical stimulation, and the level of DC. In conclusion, the present study highlights the potential for neural damage following stimulation using poorly charge-balanced stimuli.

Action Potentials↗

PubMed1999

The isoprostanes: unique prostaglandin-like products of free-radical-initiated lipid peroxidation.

The discovery of IsoPs as products of nonenzymatic lipid peroxidation has opened up new areas of investigation regarding the role of free radicals in human physiology and pathophysiology. The quantification of IsoPs as markers of oxidative stress status appears to be an important advance in our ability to explore the role of free radicals in the pathogenesis of human disease. An important need in the field of free-radical medicine is information regarding the clinical pharmacology of antioxidant agents. Because of the evidence implicating free radicals in the pathogenesis of a number of human diseases, large clinical trials are planned or underway to assess whether antioxidants can either prevent the development or ameliorate the pathology of certain human disorders. However, data regarding the most effective doses and combination of antioxidant agents to use in these clinical trials is lacking. As mentioned previously, administration of antioxidants suppresses the formation of IsoPs, even in normal individuals. Thus, measurement of IsoPs may provide a valuable approach to define the clinical pharmacology of antioxidants. In addition to being markers of oxidative stress, several IsoPs possess potent biological activity. The availability of additional IsoPs in synthetic form should broaden our knowledge concerning the role of these molecules as mediators of oxidant stress. Despite the fact that considerable information has been obtained since the initial report of the discovery of IsoPs [6], much remains to be understood about these molecules. With continued research in this area, we believe that much new information will emerge that will open up additional important new areas for future investigation.

Dinoprost↗

PubMed1999

In Swedish families with hereditary prostate cancer, linkage to the HPC1 locus on chromosome 1q24-25 is restricted to families with early-onset prostate cancer.

Prostate cancer clusters in some families, and an estimated 5%-10% of all cases are estimated to result from inheritance of prostate cancer-susceptibility genes. We previously reported evidence of linkage to the 1q24-25 region (HPC1) in 91 North American and Swedish families each with multiple cases of prostate cancer (Smith et al. 1996). In the present report we analyze 40 (12 original and 28 newly identified) Swedish families with hereditary prostate cancer (HPC) that, on the basis of 40 markers spanning a 25-cM interval within 1q24-25, have evidence of linkage. In the complete set of families, a maximum two-point LOD score of 1.10 was observed at D1S413 (at a recombination fraction [theta] of.1), with a maximum NPL (nonparametric linkage) Z score of 1.64 at D1S202 (P=.05). The evidence of linkage to this region originated almost exclusively from the subset of 12 early-onset (age <65 years) families, which yielded a maximum LOD score of 2.38 at D1S413 (straight theta=0) and an NPL Z score of 1.95 at D1S422 (P=.03). Estimates from heterogeneity tests suggest that, within Sweden, as many as 50% of early-onset families had evidence of linkage to the HPC1 region. These results are consistent with the hypothesis of linkage to HPC1 in a subset of families with prostate cancer, particularly those with an early age at diagnosis.

Age of Onset↗

PubMed1999

Preliminary evaluation of human immunodeficiency virus type 1 (HIV-1) immunogen in children with HIV-1 infection.

The safety and preliminary activity of human immunodeficiency virus type 1 (HIV-1) immunogen were evaluated in 10 HIV-1-infected children with disease stage N1,2 or A1,2. Multiple inoculations of 2. 5 or 10 units (U) of HIV-1 immunogen were safe and well tolerated without an acceleration of disease progression. When antiretroviral agents were coadministered, the 10 U dose appeared to be associated with more sustained reduction in plasma HIV-1 RNA than the 2.5 U dose (median log10 HIV-1 RNA at month 18, 3.07 vs. 4.01 copies/mL in 10 U [n=4] vs. 2.5 U [n=3], respectively; P=.034). Levels of regulated-on-activation, normal T cell-expressed and -secreted chemokine produced from HIV-1 immunogen-stimulated lymphocytes in vitro were increased in the children who had HIV-1 immunogen-specific antibody responses (P<.02) and appeared to be inversely correlated with levels of plasma HIV-1 RNA (P<.01). These preliminary data warrant larger studies to determine the effectiveness of adjunctive therapy with HIV-1 immunogen in children with HIV-1 infection.

AIDS Vaccines↗

PubMed1999

Mouse monoclonal antibodies against Phytolacca americana antiviral protein PAP I.

Four hybridoma lines are constructed producing monoclonal antibodies against the pokeweed (Phytolacca americana) antiviral protein PAP I. Two of the antibodies, 4E8 and 5D3, are characterized in more detail. They recognize amino acid sequences rather than conformational changes and their epitopes are 65% distinct. One of these antibodies (5D3) is used to study localization of recombinant PAP I in Escherichia coli cells by immuno-gold electron microscopy.

Animals↗

PubMed1999

A role for Tlg1p in the transport of proteins within the Golgi apparatus of Saccharomyces cerevisiae.

Members of the syntaxin protein family participate in the docking-fusion step of several intracellular vesicular transport events. Tlg1p has been identified as a nonessential protein required for efficient endocytosis as well as the maintenance of normal levels of trans-Golgi network proteins. In this study we independently describe Tlg1p as an essential protein required for cell viability. Depletion of Tlg1p in vivo causes a defect in the transport of the vacuolar protein carboxypeptidase Y through the early Golgi. Temperature-sensitive (ts) mutants of Tlg1p also accumulate the endoplasmic reticulum/cis-Golgi form of carboxypeptidase Y at the nonpermissive temperature (38 degrees C) and exhibit underglycosylation of secreted invertase. Overexpression of Tlg1p complements the growth defect of vti1-11 at the nonpermissive temperature, whereas incomplete complementation was observed with vti1-1, further suggesting a role for Tlg1p in the Golgi apparatus. Overexpression of Sed5p decreases the viability of tlg1 ts mutants compared with wild-type cells, suggesting that tlg1 ts mutants are more susceptible to elevated levels of Sed5p. Tlg1p is able to bind His6-tagged Sec17p (yeast alpha-SNAP) in a dose-dependent manner and enters into a SNARE complex with Vti1p, Tlg2p, and Vps45p. Morphological analyses by electron microscopy reveal that cells depleted of Tlg1p or tlg1 ts mutants incubated at the restrictive temperature accumulate 40- to 50-nm vesicles and experience fragmentation of the vacuole.

Adenosine Triphosphatases↗

PubMed1999

Null mutation of the murine ATP7B (Wilson disease) gene results in intracellular copper accumulation and late-onset hepatic nodular transformation.

The Atp7b protein is a copper-transporting ATPase expressed predominantly in the liver and to a lesser extent in most other tissues. Mutations in the ATP7B gene lead to Wilson disease, a copper toxicity disorder characterized by dramatic build-up of intracellular hepatic copper with subsequent hepatic and neuro-logical abnormalities. Using homologous recombination to disrupt the normal translation of ATP7B, we have generated a strain of mice that are homozygous mutants (null) for the Wilson disease gene. The ATP7B null mice display a gradual accumulation of hepatic copper that increases to a level 60-fold greater than normal by 5 months of age. An increase in copper concentration was also observed in the kidney, brain, placenta and lactating mammary glands of homo-zygous mutants, although milk from the mutant glands was copper deficient. Morphological abnormalities resembling cirrhosis developed in the majority of the livers from homozygous mutants older than 7 months of age. Progeny of the homozygous mutant females demonstrated neurological abnormalities and growth retardation characteristic of copper deficiency. Copper concentration in the livers of the newborn homozygous null mutants was decreased dramatically. In summary, inactivation of the murine ATP7B gene produces a form of cirrhotic liver disease that resembles Wilson disease in humans and the 'toxic milk' phenotype in the mouse.

Adenosine Triphosphatases↗

PubMed1999

Dietary supplementation of grape polyphenols to rats ameliorates chronic ethanol-induced changes in hepatic morphology without altering changes in hepatic lipids.

Increase in oxidative stress after chronic ethanol consumption can result in hepatic injury. Because polyphenolic compounds can offer antioxidant protection to the cardiovascular system, this study was designed to investigate whether dietary supplementation of polyphenols from grapes may ameliorate hepatic injury resulting from chronic ethanol consumption. Male Sprague-Dawley rats were administered the following diets for 2 mo: 1) Lieber-DeCarli (L-D) diet with isocaloric amount of maltose instead of ethanol (Basal), 2) the L-D diet with 50g/L ethanol (EtOH); 3) L-D diet with 50 mg/L of grape polyphenols (GP) and 4) ethanol diet with GP (EtOH + GP). Rats given EtOH or EtOH + GP diets had significantly more hepatic triacylglycerols (P < 0.0001) and lipid peroxidation products (P < 0.01) compared with those given the Basal and GP diets. In addition, ethanol ingestion also decreased significantly (P < 0.01) the proportion of 16:0 and increased 18:0 and 18:1 in hepatic phospholipids, suggesting a perturbation of the de novo fatty acid biosynthesis pathways. However, GP supplementation alone and GP added to the ethanol diet did not alter the lipid changes mediated by ethanol except for the levels of 22:6(n-3) which were significantly (P < 0.05) higher in the EtOH + GP group than in the EtOH group. Despite a lack of gross lipid changes, histologic assessment showed significantly (P < 0.05) less hepatic damage in the GP + EtOH group compared with the EtOH group. These results clearly distinguished ethanol-mediated changes in hepatic morphology from the changes in hepatic lipids and further demonstrated the ability of GP to ameliorate hepatic damage resulting from chronic ethanol consumption.

Animals↗

PubMed1999

Salvage of free flaps after secondary venous ischemia by local delivery of heparin.

Recent studies have demonstrated that heparin may protect against reperfusion injury through a direct effect on the microvascular endothelium that is independent of its effect on systemic coagulation. The purpose of this study was to determine whether local delivery of low-dose heparin has a role in the salvage of musculocutaneous flaps after secondary venous ischemia and revascularization. Cutaneous maximus musculocutaneous flaps were transplanted to the contralateral groin in adult Sprague-Dawley rats. All flaps were subjected to 2 hours of primary arteriovenous ischemia followed by 20 hours of reperfusion. The flaps were then subjected to a 6-hour secondary venous ischemic insult followed by anastomotic revision and reperfusion. Animals in group I received no adjunctive treatment. Those in group II were treated with low-dose heparin (5-6 U per kilogram per hour) infused systemically via the inferior epigastric vein. Those in group III received the same dose of heparin infused locally into the flap via the inferior epigastric artery. The dose of heparin used in groups II and III was insufficient to prolong the activated partial thromboplastin time above normal values. At 7 days, mean flap necrosis was 60.8% in group I and 62.1 in group II. Local heparin delivery (group III) resulted in complete survival of all flaps. Histological examination after 48 hours of reperfusion demonstrated improved microvascular patency and reduced neutrophilic infiltration in the flaps of group III animals. Thus, local infusion of low-dose heparin resulted in significantly improved flap salvage through a mechanism independent of its effect on systemic coagulation.

Animals↗