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Biomedical subjects

D Xu

Publications and source records attributed to D Xu.

At least 217 records · Page 12Linked to original sources
PubMed1998

Cloning of the canine beta-glucuronidase cDNA, mutation identification in canine MPS VII, and retroviral vector-mediated correction of MPS VII cells.

Mucopolysaccharidosis type VII (MPS VII) is an inherited disease resulting from deficient activity of the lysosomal acid hydrolase beta-glucuronidase (GUSB) and has been reported in humans, mice, cats, and dogs. To characterize canine MPS VII, we have isolated and sequenced the canine GUSB cDNA from normal and affected animals. A single nucleotide substitution was detected in the GUSB cDNA derived from MPS VII dogs. This guanosine to adenine base change at nucleotide position 559 in the canine cDNA sequence causes an arginine to histidine substitution at amino acid position 166. Introduction of the G to A substitution at position 559 in a mammalian expression vector containing the normal canine GUSB cDNA nearly eliminated the GUSB enzymatic activity, demonstrating that this mutation is the cause of canine MPS VII. A retroviral vector expressing the full-length canine beta-glucuronidase cDNA corrected the deficiency in MPS VII cells.

Amino Acid Sequence↗
PubMed1998

Protective effect on Leishmania major infection of migration inhibitory factor, TNF-alpha, and IFN-gamma administered orally via attenuated Salmonella typhimurium.

The genes encoding murine macrophage migration inhibitory factor (MIF), IL-2, IFN-gamma or TNF-alpha were cloned individually into an expression plasmid under the control of the inducible promoter nirB and transfected into the aroA- aroD- deletion mutant strain of Salmonella typhimurium (BRD509). These S. typhimurium derivatives (henceforward called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) expressed their respective cytokines in vitro under anaerobic conditions and stably colonized BALB/c mice up to 14 days after oral administration. The highly susceptible BALB/c mice that had received the constructs orally and that had been subsequently infected via the footpad with Leishmania major, developed significantly reduced disease compared with control mice administered the untransfected Salmonella strain (BRD509). Importantly, a combination of GIDMIF, GIDIFN, and GIDTNF administered orally after L. major infection was able to significantly limit lesion development and reduced parasite loads by up to three orders of magnitude. Spleen and lymph node cells of mice administered this combination expressed markedly higher levels of inducible nitric oxide synthase (iNOS) compared with those from mice receiving an equivalent dose of the control strain of Salmonella (BRD509). These data therefore demonstrate the feasibility of therapeutic treatment in an infectious disease model using cytokines delivered by attenuated Salmonella. The protective effect observed correlates with the induction of inducible nitric oxide synthase in vivo.

Administration, Oral↗
PubMed1998

Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages.

We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2-deficient mice infected with Leishmania major in vivo or stimulated with IFN-gamma or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL-12 than those from heterozygous or wild-type mice. A macrophage cell line, J774, produced significant amounts of IL-12 following activation with LPS, or LPS plus IFN-gamma. This could be markedly enhanced by the NOS inhibitor L-NG monomethyl arginine (L-NMMA), but profoundly inhibited by the NO-generating compound S-nitroso-N-acetyl-penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L-NMMA decreased TNF-alpha synthesis by LPS-activated J774 cells. The differential effect of NO on IL-12 and TNF-alpha is at the transcriptional level and is activation dependent. Since IL-12 is a major inducer of Th1 cells which produce IFN-gamma that can activate macrophages to produce IL-12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.

Animals↗
PubMed1998

Mechanism and evolution of protein dimerization.

We have investigated the mechanism and the evolutionary pathway of protein dimerization through analysis of experimental structures of dimers. We propose that the evolution of dimers may have multiple pathways, including (1) formation of a functional dimer directly without going through an ancestor monomer, (2) formation of a stable monomer as an intermediate followed by mutations of its surface residues, and (3), a domain swapping mechanism, replacing one segment in a monomer by an equivalent segment from an identical chain in the dimer. Some of the dimers which are governed by a domain swapping mechanism may have evolved at an earlier stage of evolution via the second mechanism. Here, we follow the theory that the kinetic pathway reflects the evolutionary pathway. We analyze the structure-kinetics-evolution relationship for a collection of symmetric homodimers classified into three groups: (1) 14 dimers, which were referred to as domain swapping dimers in the literature; (2) nine 2-state dimers, which have no measurable intermediates in equilibrium denaturation; and (3), eight 3-state dimers, which have stable intermediates in equilibrium denaturation. The analysis consists of the following stages: (i) The dimer is divided into two structural units, which have twofold symmetry. Each unit contains a contiguous segment from one polypeptide chain of the dimer, and its complementary contiguous segment from the other chain. (ii) The division is repeated progressively, with different combinations of the two segments in each unit. (iii) The coefficient of compactness is calculated for the units in all divisions. The coefficients obtained for different cuttings of a dimer form a compactness profile. The profile probes the structural organization of the two chains in a dimer and the stability of the monomeric state. We describe the features of the compactness profiles in each of the three dimer groups. The profiles identify the swapping segments in domain swapping dimers, and can usually predict whether a dimer has domain swapping. The kinetics of dimerization indicates that some dimers which have been assigned in the literature as domain swapping cases, dimerize through the 2-state kinetics, rather than through swapping segments of performed monomers. The compactness profiles indicate a wide spectrum in the kinetics of dimerization: dimers having no intermediate stable monomers; dimers having an intermediate with a stable monomer structure; and dimers having an intermediate with a stable structure in part of the monomer. These correspond to the multiple evolutionary pathways for dimer formation. The evolutionary mechanisms proposed here for dimers are applicable to other oligomers as well.

Biological Evolution↗
PubMed1998

Detection of circulating antibodies to malondialdehyde-acetaldehyde adducts in ethanol-fed rats.

BACKGROUND & AIMS: Malondialdehyde and acetaldehyde react together with proteins and form hybrid protein conjugates designated as MAA adducts, which have been detected in livers of ethanol-fed rats. The aim of this study was to examine the immune response to MAA adducts and other aldehyde adducts during long-term ethanol exposure. METHODS: Rats were pair-fed for 7 months with a liquid diet containing either ethanol or isocaloric carbohydrate. Circulating antibody titers against MAA adducts and acetaldehyde adducts were measured and characterized in these animals. RESULTS: A significant increase in antibody titers against MAA-adducted proteins was observed in the ethanol-fed animals. Competitive inhibitions of antibody binding indicated that the circulating antibodies against MAA-modified proteins in the ethanol-fed rats recognized mainly a specific, chemically defined MAA epitope. Antibody titers to reduced and nonreduced acetaldehyde adducts were very low, and no significant differences were observed between ethanol-fed and control animals. Significant plasma immunoreactivity to not only MAA-adducted but also unmodified rat liver proteins (cytosol, microsomes, and especially plasma membrane) were also observed in the ethanol-fed rats. CONCLUSIONS: Long-term ethanol feeding generates circulating antibodies not only against MAA epitopes but possibly also against unmodified, native (self) protein epitopes, suggesting that MAA adducts could trigger harmful autoimmune responses.

Acetaldehyde↗
PubMed1998

Alcohol-related diols cause acute insulin resistance in vivo.

Epidemiological studies suggest that alcohol consumption is an independent risk factor for the development of non-insulin-dependent diabetes mellitus (NIDDM). Alcoholism is known to be associated with increased plasma levels of two novel diols, 2,3-butanediol and 1,2-propanediol, metabolites known to impair insulin action in isolated adipocytes. This study examines whether 2,3-butanediol and 1,2-propanediol have the capacity to impair insulin action acutely in vivo in the rat. Using the euglycemic-hyperinsulinemic clamp, it is shown that the two diols reduce whole-body glucose utilization (by approximately 30%), with the onset of insulin resistance in vivo occurring at plasma concentrations of 2,3-butanediol (33 micromol/L) at least one order of magnitude (P < .001) lower than 1,2-propanediol (432 micromol/L). Tracer methodologies using [U-14C]glucose and 2-deoxy[1-(3)H]glucose indicate that the reduction in whole-body glucose utilization is accompanied by a reduction in glucose uptake and glycogen synthesis in the skeletal muscle and heart. The association between elevated plasma diol levels and insulin resistance demonstrated in this report raises the question of whether there is a link between the high plasma diol levels in alcohol abusers and their increased susceptibility to NIDDM.

Alcoholism↗
PubMed1998

Favorable domain size in proteins.

BACKGROUND: It has been observed that single-domain proteins and domains in multidomain proteins favor a chain length in the range 100-150 amino acids. To understand the origin of the favored size, we construct an empirical function for the free energy of unfolding versus the chain length. The parameters in the function are derived by fitting to the energy of hydration, entropy and enthalpy of unfolding of nine proteins. Our energy function cannot be used to calculate the energetics accurately for individual proteins because the energetics also depend on other factors, such as the composition and the conformation of the protein. Nevertheless, the energy function statistically characterizes the general relationship between the free energy of unfolding and the size of the protein. RESULTS: The predicted optimal number of residues, which corresponds to the maximum free energy of unfolding, is 100. This is in agreement with a statistical analysis of protein domains derived from their experimental structures. When a chain is too short, our energy function indicates that the change in enthalpy of internal interactions is not favorable enough for folding because of the limited number of inter-residue contacts. A long chain is also unfavorable for a single domain because the cost of configurational entropy increases quadratically as a function of the chain length, whereas the favorable change in enthalpy of internal interactions increases linearly. CONCLUSIONS: Our study shows that the energetic balance is the dominant factor governing protein sizes and it forces a large protein to break into several domains during folding.

Protein Folding↗
PubMed1998

Protein folding via binding and vice versa.

The terms intermolecular and intramolecular recognition are often used when referring to binding and folding, highlighting the common ground between the two processes. Most studies, however, are aimed at either one process or the other. Here, we show how knowledge from binding can aid in understanding folding and vice versa.

Apolipoproteins↗
PubMed1998

Multidrug resistance phenotype in leukaemic cells from patients with acute myelocytic leukaemia can be detected with 99Tc(m)-MIBI.

The aim of the study was to investigate whether 99Tc(m)-MIBI (Cardiolite), recently shown to be a substrate for P-glycoprotein, has the potential to be used as a marker for mdr1 gene expression and whether cyclosporin A (CyA) can modify its accumulation in vivo. Leukaemic cells from ten patients with acute myelocytic leukaemia (AML) were used, five with undetectable mdr1 gene expression and five with mdr1 mRNA levels ranging from 1.0 to 3.8 mdr1 mRNA transcripts per cell. Cells were incubated with 99Tc(m)-MIBI, or with daunorubicin (Dnr), with and without 3 microM CyA. The median 99Tc(m)-MIBI accumulation (% of added radioactivity) in mdr1-negative cells was 0.89% and in the mdr1-positive cells 0.34%, P = 0.01. In mdr1-negative cells, the median increase in 99Tc(m)-MIBI accumulation with CyA was 30% compared with the mdr1-positive cells with a median increase of 242%, P = 0.009. CyA had no significant effect on Dnr accumulation in four of the mdr1-negative samples. The median increase of Dnr accumulation in the mdr1-positive cells was 40%. The results show that 99Tc(m)-MIBI with a high sensitivity can detect rather low levels of mdr1 gene expression in clinical samples. Consequently, 99T(c)m-MIBI scintigraphy has the potential to be used for monitoring the effect of resistance modifiers on the accumulation and retention of cytostatic drugs in human tumours in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Forced expression of Genesis, a winged helix transcriptional repressor isolated from embryonic stem cells, blocks granulocytic differentiation of 32D myeloid cells.

We recently isolated and characterized a novel member of the winged helix (formerly HNF-3/Forkhead) transcriptional regulatory family, termed Genesis. Genesis was found to be a transcriptional repressor expressed almost exclusively in embryonic stem cells or embryonal carcinoma cells. This expression rapidly declined when these cells were stimulated to differentiate. This expression pattern suggested that Genesis may play a role in cellular differentiation. To explore that possibility in a heterologous system of differentiation, Genesis was stably transduced into the IL-3-dependent myeloid cell line 32D using a retroviral expression vector. 32D cells overexpressing Genesis failed to mature normally when stimulated with G-CSF but continued to proliferate and maintained a primitive phenotype. This finding implicates Genesis in the regulation of development, and also implies that there may be common transcription pathways for many developing tissues.

Blotting, Northern↗
PubMed1998

Telomerase activity and the expression of telomerase components in acute myelogenous leukaemia.

In 95 leukaemic cell samples from 66 patients with acute myelogenous leukaemia (AML) (47 de novo and 19 secondary AML) telomerase activity was determined and the expression of the telomerase components: telomerase reverse transcriptase (hTERT), telomerase RNA template (hTR) and telomerase-associated protein (TP1) evaluated by RT-PCR. Compared to peripheral blood mononuclear cells (PBMC) from normal adult 87% (82/95) of patient samples exhibited elevated telomerase activity hTERT, but not hTR and TP1 expression strongly correlated with the levels of telomerase activity (r=0.47, P<0.0001). The levels of telomerase activity were significantly higher at time of relapse or progression than at time of diagnosis (P=0.003), and correlated to CD34 expression and chromosomal abnormalities of leukaemic cells (P=0.01 and P=0.001 respectively). The rate and duration of complete remission (CR) did not correlate with the levels of telomerase activity at diagnosis. Among eight patients in first relapse, however, two of three with low levels of telomerase activity re-entered CR. whereas none of five patients with high telomerase activity achieved a second CR. Taken together, telomerase activation/up-regulation in AML is a disease progression-associated event. Undifferentiated status and chromosomal aberration also lead to the up-regulation of telomerase activity in AML.

Adult↗