Biomedical subjects

G Zhang

Publications and source records attributed to G Zhang.

At least 19 recordsLinked to original sources

PubMed1998

Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal domain.

The 2.5 angstrom resolution x-ray crystal structure of the Escherichia coli RNA polymerase (RNAP) alpha subunit amino-terminal domain (alphaNTD), which is necessary and sufficient to dimerize and assemble the other RNAP subunits into a transcriptionally active enzyme and contains all of the sequence elements conserved among eukaryotic alpha homologs, has been determined. The alphaNTD monomer comprises two distinct, flexibly linked domains, only one of which participates in the dimer interface. In the alphaNTD dimer, a pair of helices from one monomer interact with the cognate helices of the other to form an extensive hydrophobic core. All of the determinants for interactions with the other RNAP subunits lie on one face of the alphaNTD dimer. Sequence alignments, combined with secondary-structure predictions, support proposals that a heterodimer of the eukaryotic RNAP subunits related to Saccharomyces cerevisiae Rpb3 and Rpb11 plays the role of the alphaNTD dimer in prokaryotic RNAP.

Amino Acid Sequence↗

PubMed1998

Locomotor activity in D2 dopamine receptor-deficient mice is determined by gene dosage, genetic background, and developmental adaptations.

Locomotor activity is a polygenic trait that varies widely among inbred strains of mice (). To characterize the role of D2 dopamine receptors in locomotion, we generated F2 hybrid (129/Sv x C57BL/6) D2 dopamine receptor (D2R)-deficient mice by gene targeting and investigated the contribution of genetic background to open-field activity and rotarod performance. Horizontal activity of D2R-/- mice was approximately half that of drug-naive, strain-matched controls but was significantly greater than haloperidol-treated controls, which were markedly hypokinetic. Wild-type 129/SvEv and C57BL/6 mice with functional D2 receptors had greater interstrain differences in spontaneous activity than those among the F2 hybrid mutants. Incipient congenic strains of D2R-deficient mice demonstrated an orderly gene dosage reduction in locomotion superimposed on both extremes of parental background locomotor activity. In contrast, F2 hybrid D2R-/- mice had impaired motor coordination on the rotarod that was corrected in the congenic C57BL/6 background. Wild-type 129/SvEv mice had the poorest rotarod ability of all groups tested, suggesting that linked substrain 129 alleles, not the absence of D2 receptors per se, were largely responsible for the reduced function of the F2 hybrid D2R-/- and D2R+/- mice. Neurochemical and pharmacological studies revealed unexpectedly normal tissue striatal monoamine levels and no evidence for supersensitive D1, D3, or D4 dopamine receptors in the D2R-/- mice. However, after acute monoamine depletion, akinetic D2R+/- mice had a significantly greater synergistic restoration of locomotion in response to SKF38393 and quinpirole compared with any group of D2R+/+ controls. We conclude that D2R-deficient mice are not a model of Parkinson's disease. Our studies highlight the interaction of multiple genetic factors in the analysis of complex behaviors in gene knock-out mice.

Adaptation, Physiological↗

PubMed1998

Predictors of delay in starting radiation treatment for patients with early stage breast cancer.

PURPOSE: To describe the factors predicting waiting time for radiation treatment in early breast cancer. MATERIALS AND METHODS: Between January 1992 and December 1993, 739 patients with Stage I and II breast cancer were treated with conservative treatment at three McGill University Hospitals. Waiting time was defined as the interval between the date of surgery and the date of the first radiation treatment. Delay was defined as a waiting time of more than 7 weeks for women who did not receive chemotherapy (Group NC, n = 478), and as a waiting time of more than 24 weeks for those who received chemotherapy (Group C, n = 261). We analyzed predictive factors related to the patient (age, stage, treatment on protocol, income by postal code) and to the referring hospital (university or community hospital). RESULTS: For the entire population, 54% of patients were delayed, 72% in Group NC and 21.4% in Group C. Univariate analysis showed an impact of referring hospital in both groups, and of stage and treatment on protocol in Group C (all p = 0.001). Multivariate analysis showed that delays were significantly less in Group NC for women referred from a community hospital (p = 0.001) and in Group C for women with Stage I disease (p = 0.06), those treated on protocol, and those referred from a university hospital (p = 0.001). CONCLUSION: More than half of patients with early breast cancer waited more than the recommended intervals for radiation therapy. However, lower income breast cancer patients did not wait longer for treatment than higher income patients, possibly a result of the Canadian Medicare system which provides universal access to health care.

Analysis of Variance↗

PubMed1998

Molecular cloning and tissue expression of porcine beta-defensin-1.

Beta-defensins constitute an emerging family of cysteine-rich antimicrobial peptides, which are particularly prominent at mucosal epithelial sites in mammals. Here we report the identification of a novel beta-defensin from porcine tissues, porcine beta-defensin-1 (pBD-1). The cDNA sequence of pBD-1 encoded a 64 amino acid prepro-peptide, which contained the beta-defensin consensus sequence of six invariantly spaced cysteine residues. Northern blot analysis showed that pBD-1 was expressed abundantly in tongue epithelia and that the expression was regulated developmentally. Using RT-PCR, pBD-1 mRNA was detected throughout the respiratory and digestive tracts and also in thymus, spleen, lymph node, brain, liver, kidney, urinary bladder, testis, skin, heart, muscle, bone marrow, peripheral blood neutrophils, alveolar macrophages, and umbilical cord. The wide expression of pBD-1 suggests that this endogenous peptide antibiotic may contribute to both mucosal and systemic host defenses in pigs, which may have implications for the use of porcine tissues and organs in xenotransplantation.

Amino Acid Sequence↗

PubMed1998

Orphanin FQ is the major OFQ1-17-containing peptide produced in the rodent and monkey hypothalamus.

In order to investigate the processing of OFQ containing peptides in the hypothalamus we have developed a sensitive and quantitative radioimmunoassay for OFQ. We fractionated rodent and monkey hypothalamic extracts by reversed-phase high performance liquid chromatography and found that the extracts contained multiple peaks of OFQ immunoreactivity with the major peak co-eluting with synthetic OFQ1-17. Mouse hypothalamic extracts were also fractionated by SDS-PAGE to determine the apparent molecular weights of molecules containing the OFQ peptide. Multiple peaks of OFQ immunoreactivity, ranging in size from approximately 1 to 30 kilodaltons, were detected by this method. These results suggest that OFQ1-17 is processed to smaller peptides in mouse and monkey hypothalamic neurons.

Amino Acid Sequence↗

PubMed1998

DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA.

The nuclear entry of exogenous DNA in mammalian cells is critical for efficient gene transfer. A novel technique was developed for the covalent attachment of cationic peptides to double-stranded DNA using a cyclo-propapyrroloindole cross-linker. The attachment of the SV40 large T antigen nuclear localization signal peptide induced the nuclear accumulation of the conjugated DNA in digitonin-permeabilized cells via the classical pathway for the nuclear transport of karyophilic proteins. Increased nuclear uptake of the modified DNA, however, did not occur after it was microinjected into the cytoplasm of cultured cells. This demonstration that the covalent modification of DNA with a signal peptide alters its behavior and interaction with other cellular factors portends the potential of DNA vector chemistry to enhance the efficiency of cellular gene transfer.

Antigens, Polyomavirus Transforming↗

PubMed1998

The efficient expression of intravascularly delivered DNA in rat muscle.

Previous studies have demonstrated that muscle can take up and express naked DNA or RNA. This study demonstrates that the pDNA can be delivered to and expressed within skeletal muscle when injected rapidly, in a large volume and when all blood vessels leading into and out of the hindlimb are occluded. The additional use of collagenase, papaverine and ischemia raised expression moderately but was not critical. These results demonstrate that a nonviral method can lead to high levels of expression in the muscles of adult animals large than mice.

Animals↗

PubMed1998

Characterization of a second transcription initiation element (STIE) in the human interleukin-1 beta (IL-1beta) gene.

Interleukin-1-beta (IL-1beta) is a significant mediator in the inflammation process. Although the human IL-1beta genomic sequence has been known for several years, our understanding of its molecular regulation of transcription remains incomplete. We are reporting a new transcription initiation element that is located within intron 1 and exon 2 of the human IL-1beta gene. Different lengths of the human IL-1beta gene fragment (-685 to +550) were cloned upstream from a chloramphenical acetyltransferase (CAT) gene to make the IL-1beta promoter/CAT reporter constructs. Transient CAT expression with these constructs in the human monocytic leukemia cell line THP1 illustrates an important positive regulatory element exists within the region from +387 to +550. Using electromobility shift assays and by DNase I footprinting analysis, we identified three nuclear factor binding sites (+448 to +502, +513 to +531, and +539 to +548). Functional studies show that CAT production is undetectable when the 19 bp region (+513 to +531) is removed, and CAT production is diminished when the 10-bp region (+539 to +548) is deleted. The region containing these sites is likely to initiate a new transcript starting at +559 of exon 2. This second transcript of the IL-1beta gene shares the same reading frame with the previously recognized cDNA transcript.

Base Sequence↗

PubMed1998

Identification and characterization of Escherichia coli DNA helicase II mutants that exhibit increased unwinding efficiency.

Using a combination of both ethyl methanesulfonate and site-directed mutagenesis, we have identified a region in DNA helicase II (UvrD) from Escherichia coli that is required for biological function but lies outside of any of the seven conserved motifs (T. C. Hodgman, Nature 333:22-23, 1988) associated with the superfamily of proteins of which it is a member. Located between amino acids 403 and 409, alterations in the amino acid sequence DDAAFER lead to both temperature-sensitive and dominant uvrD mutations. The uvrD300 (A406T) and uvrD301 (A406V) alleles produce UV sensitivity at 44 degrees C but do not affect sensitivity to methyl methanesulfonate (MMS). In contrast, the uvrD303 mutation (D403AD404A) causes increased sensitivity to both UV and MMS and is dominant to uvrD+ when present at six to eight copies per cell. Several of the alleles demonstrated a strong antimutator phenotype. In addition, conjugal recombination is reduced 10-fold in uvrD303 strains. Of all of the amino acid substitutions tested, only an alanine-to-serine change at position 406 (uvrD302) was neutral. To determine the biochemical basis for the observed phenotypes, we overexpressed and purified the UvrD303 protein from a uvrD delta294 deletion background and characterized its enzymatic activities. The highly unusual UvrD303 protein exhibits a higher specific activity for ATP hydrolysis than the wild-type control, while its Km for ATP binding remains unchanged. More importantly, the UvrD303 protein unwinds partial duplex DNA up to 10 times more efficiently than wild-type UvrD. The DNA binding affinities of the two proteins appear comparable. Based on these results, we propose that the region located between amino acids 403 and 409 serves to regulate the unwinding activity of DNA helicase II to provide the proper balance between speed and overall effectiveness in the various DNA repair systems in which the protein participates.

Adenosine Triphosphatases↗

PubMed1997

HIV-1 infection in women is associated with severe nutritional deficiencies.

Nutritional deficiencies may contribute to immune dysregulation, and have been shown to be sensitive markers of HIV-1 disease progression. Only limited information exists, however, regarding the nutritional profile of HIV-1-seropositive drug abusers. Immune and nutritional measurements were obtained in a subsample of 125 subjects from a larger cohort of drug users being followed for HIV-1 infection and cofactors of disease progression. Nutritional deficiencies, particularly vitamins A, E, and zinc, were widespread with up to 86% of the drug users exhibiting at least one nutritional alteration. Although immune parameters (CD4 count, CD8 count, beta2-microglobulin) were similar in the HIV-1-infected men and women, women had significantly poorer overall nutritional status, as measured by plasma proteins, which are considered to be sensitive markers of malnutrition. A comparison of individuals with advanced disease (CD4 count <200/mm3) revealed significantly lower levels of plasma prealbumin (p < .01), selenium, (p < .05), and greater deficiency of vitamins A (p < .01) and E (p < .05) in women than in men. The greater severity of nutritional deficiencies noted in HIV-1-infected women may be an important determinant of disease progression and survival.

Adult↗

PubMed1997

Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence.

The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a widely used marker for gene expression and protein localization studies. Dissection of the structure of the protein would be expected to shed light on its potential applications to other fields such as the detection of protease activity. Using deletion analysis, we have defined the minimal domain in GFP required for fluorescence to amino acids 7-229. This domain starts at the middle of the first small alpha helix at the N terminus of GFP and ends immediately following the last beta sheet. Studies of the amino acids at both termini of the minimal domain revealed that positions 6 and 7 at the N terminus are Glu-specific. Change of the Glu residues to other amino acids results in reduction of GFP fluorescence. Position 229 at the C terminus of GFP, however, is nonspecific: the Ile can be replaced with other amino acids with no measurable loss of fluorescence. A total of only 15 terminal amino acids can be deleted from GFP without disrupting fluorescence, consistent with findings of a previous study of GFP crystal structure (Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., Remington, S. J. (1996) Science 273, 1392-1395 and Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nat. Biotechnol. 14, 1246-1251) that a tightly packed structure exists in the protein. We also generated internal deletions within the loop regions of GFP according to its crystal structure and found that all such deletions eliminated GFP fluorescence.

Animals↗

PubMed1997

Orphanin FQ has an inhibitory effect on the guinea pig ileum and the mouse vas deferens.

The activity of the recently isolated endogenous opioid-like peptide orphanin FQ (OFQ) was measured in two classical bioassays of opioid action. OFQ potently and concentration-dependently suppressed the electrically stimulated contractions of the guinea pig ileum (GPI) and the mouse vas deferens (MVD) with EC50 values of 1.82 +/- 0.16 and 2.97 +/- 0.01 nM, and Emax values of 56 +/- 3% and 96 +/- 4%, respectively. This effect of OFQ, in both the GPI and MVD, was insensitive to the opioid receptor antagonist naloxone. OFQ competed with [3H]diprenorphine binding to mu-, delta- or kappa-opioid receptors stably expressed in Chinese hamster ovary cell lines with IC50 values of 2.1 +/- 0.4, 2.2 +/- 0.3, 0.75 +/- 0.3 microM, respectively. Low affinity for the classical opioid receptors together with the inability of naloxone to antagonize its effect suggest that the inhibitory action of OFQ is mediated via a distinct OFQ receptor in the GPI and MVD. Consequently, the MVD could serve as a valuable bioassay of potential OFQ receptor antagonists.

Animals↗

PubMed1997

Expression of naked plasmid DNA injected into the afferent and efferent vessels of rodent and dog livers.

A variety of reporter genes within plasmid constructs were injected into the afferent and efferent vessels of the liver in mice, rats, and dogs. Efficient plasmid expression was obtained following delivery via the portal vein, the hepatic vein, and the bile duct. The use of hyperosmotic injection solutions and occlusion of the blood outflow from the liver substantially increased the expression levels. Combining these surgical approaches with improved plasmid vectors enabled uncommonly high levels of foreign gene expression in which over 15 microg of luciferase protein/liver was produced in mice and over 50 microg in rats. Equally high levels of beta-galactosidase (beta-Gal) expression were obtained, in that over 5% of the hepatocytes had intense blue staining. Expression of luciferase or beta-Gal was evenly distributed in hepatocytes throughout the entire liver when either of the three routes were injected. Peri-acinar hepatocytes were preferentially transfected when the portal vein was injected in rats. These levels of foreign gene expression are among the highest levels obtained with nonviral vectors. Repetitive plasmid administration through the bile duct led to successive events of foreign gene expression. The integration of these findings into laboratory and clinical protocols is discussed.

Animals↗

PubMed1997

Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine.

The human dopamine D4 receptor (D4R) has received considerable attention because of its high affinity for the atypical antipsychotic clozapine and the unusually polymorphic nature of its gene. To clarify the in vivo role of the D4R, we produced and analyzed mutant mice (D4R-/-) lacking this protein. Although less active in open field tests, D4R-/- mice outperformed wild-type mice on the rotarod and displayed locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Biochemical analyses revealed that dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from D4R-/- mice. Based on these findings, we propose that the D4R modulates normal, coordinated and drug-stimulated motor behaviors as well as the activity of nigrostriatal dopamine neurons.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1997

Smectic ordering in solutions and films of a rod-like polymer owing to monodispersity of chain length.

Solutions and melts of stiff ('rod-like') macromolecules often exhibit nematic liquid crystalline phases characterized by orientational, but not positional, molecular order. Smectic phases, in which macromolecular rods are organized into layers roughly perpendicular to the direction of molecular orientation, are rare, owing at least in part to the polydisperse nature (distribution of chain lengths) of polymers prepared by conventional polymerization processes. Bacterial methods for polypeptide synthesis, in which artificial genes encoding the polymer are expressed in bacterial vectors, offer the opportunity to make macromolecules with very well defined chain lengths. Here we show that a monodisperse derivative of poly(gamma-benzyl alpha,L-glutamate) prepared in this way shows smectic ordering in solution and in films. This result suggests that methods for preparing monodisperse polymers might provide access to new smectic phases with layer spacings that are susceptible to precise control on the scale of tens of nanometres.

Biofilms↗

PubMed1997

Fluorometric and colorimetric detection of caspase activity associated with apoptosis.

The caspase (ICE/CED-3) family of proteases has been implicated to play a crucial role in apoptosis. However, the mechanisms by which caspase activity mediates apoptosis are not fully understood. Progress in this area has been limited due to the lack of a convenient and reliable system to quantify these protease activities. In this report, we describe a quantitative assay for the activity of caspase-3, a member of the caspase family thought to mediate apoptosis in most mammalian cell types. This assay utilizes a synthetic tetrapeptide, Asp-Glu-Val-Asp (DEVD), labeled with either a fluorescent molecule, 7-amino-4-trifluoromethyl coumarin (AFC), or a colorimetric molecule, p-nitroanilide (pNA) as substrates. DEVD-dependent protease activity is assessed by detection of the free AFC or pNA cleaved from the substrates. We demonstrate the utility of the assay for rapid quantification of caspase-3 activity in the onset of apoptosis. Using the assay, we show that apoptosis induced in 32D cells under various conditions is associated with an increase in the DEVD-dependent protease activity. These studies suggest that induction of the DEVD-dependent protease activity is an indicator of apoptosis and demonstrate the utility of the assays for assessment of the role of caspase-family proteases in apoptotic cell progression.

Amino Acid Sequence↗

PubMed1997

High risk of HIV-related mortality is associated with selenium deficiency.

To determine the independent contribution of specific immunologic and nutritional factors on survival in HIV-1 disease, CD4 cell count, antiretroviral treatment, plasma levels of vitamins A, E, B6, and B12 and minerals selenium and zinc were considered in relation to relative risk for HIV-related mortality. Immune parameters and nutrients known to affect immune function were evaluated at 6-month intervals in 125 HIV-1-seropositive drug-using men and women in Miami, FL, over 3.5 years. A total of 21 of the HIV-1-infected participants died of HIV-related causes during the 3.5-year longitudinal study. Subclinical malnutrition (i.e., overly low levels of prealbumin, relative risk [RR] = 4.01, p < 0.007), deficiency of vitamin A (RR = 3.23, p < 0.03), vitamin B12 deficiency (RR = 8.33, p < 0.009), zinc deficiency (RR = 2.29.1, p < 0.04), and selenium deficiency (RR = 19.9, p < 0.0001) over time, but not zidovudine treatment, were shown to each be associated with HIV-1-related mortality independent of CD4 cell counts <200/mm3 at baseline, and CD4 counts over time. When all factors that could affect survival, including CD4 counts <200/mm3 at baseline, CD4 levels over time, and nutrient deficiencies were considered jointly, only CD4 counts over time (RR = 0.69, p < 0.04) and selenium deficiency (RR = 10.8, p < 0.002) were significantly associated with mortality. These results indicate that selenium deficiency is an independent predictor of survival for those with HIV-1 infection.

Adult↗

PubMed1997

Isolation and characterization of a gene from the DiGeorge chromosomal region homologous to the mouse Tbx1 gene.

DiGeorge syndrome, velocardiofacial syndrome, conotruncal anomaly face syndrome, and isolated and familial forms of conotruncal cardiac defects have been associated with deletions of chromosomal region 22q11.2. This report describes the identification, cloning, and characterization of the human TBX1 gene, which maps to the center of the DiGeorge chromosomal region. Further, we have extended the mouse cDNA sequence to permit comparisons between human and mouse Tbx1. TBX1 is a member of a phylogenetically conserved family of genes that share a common DNA-binding domain, the T-box. T-box genes are transcription factors involved in the regulation of developmental processes. There is 98% amino acid identity between human and mouse TBX1 proteins overall, and within the T-box domain, the proteins are identical except for two amino acids. Expression of human TBX1 in adult and fetal tissues, as determined by Northern blot analysis, is similar to that found in the mouse. Additionally, using 3 'RACE, we obtained a differentially spliced message in adult skeletal muscle. Mouse Tbx1 has been previously shown to be expressed during early embryogenesis in the pharyngeal arches, pouches, and otic vesicle. Later in development, expression is seen in the vertebral column and tooth bud. Thus, human TBX1 is a candidate for some of the features seen in the 22q11 deletion syndrome.

Adult↗