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Biomedical subjects

J Sun

Publications and source records attributed to J Sun.

At least 451 records · Page 25Linked to original sources
PubMed1997

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.

Amino Acid Sequence↗
PubMed1997

Binding of monoclonal antibody 4B1 to homologs of the lactose permease of Escherichia coli.

The conformationally sensitive epitope for monoclonal antibody (mAb) 4B1, which uncouples lactose from H+ translocation in the lactose permease of Escherichia coli, is localized in the periplasmic loop between helices VII and VIII (loop VII/VIII) on one face of a short helical segment (Sun J, et al., 1996, Biochemistry 35;990-998). Comparison of sequences in the region corresponding to loop VII/VIII in members of Cluster 5 of the Major Facilitator Superfamily (MFS), which includes five homologous oligosaccharide/H+ symporters, reveals interesting variations. 4B1 binds to the Citrobacter freundii lactose permease or E. coli raffinose permease with resultant inhibition of transport activity. Because E. coli raffinose permease contains a Pro residue at position 254 rather than Gly, it is unlikely that the mAb recognizes the peptide backbone at this position. Consistently, E. coli lactose permease with Pro in place of Gly254 also binds 4B1. In contrast, 4B1 binding is not observed with either Klebsiella pneumoniae lactose permease or E. coli sucrose permease. When the epitope is transferred from E. coli lactose permease (residues 245-259) to the sucrose permease, the modified protein binds 4B1, but the mAb has no significant effect on sucrose transport. The studies provide further evidence that the 4B1 epitope is restricted to loop VII/VIII, and that 4B1 binding induces a highly specific conformational change that uncouples substrate and H+ translocation.

Amino Acid Sequence↗
PubMed1997

Effects of cyclosporin A on proliferation of cultured rat mesangial cells.

The proliferation of mesangial cells on cyclosporin (CsA) test medium was studied by MTT assay and TNF-alpha in cultured supernatant was examined by using ELISA. The results showed that cyclosporin A significantly inhibited the proliferation of mesangial cells at the concentration between 0.25-15 micrograms/ml (IC50 1 microgram). This action appeared to be dose-dependent. Release of TNF-alpha from mesangial cells stimulated by LPS was also dose-dependently suppressed. It is suggested that cyclosporin A play an important role in antiproliferation mechanism of mesangial cells in vitro.

Animals↗
PubMed1997

Sequence analysis of pig switch mu, C mu, and C mu m.

The complete sequence of pig switch mu (S mu) and the various domains of C mu, a total of ca 8 kilobases (kb) of sequence, are presented and comparatively analyzed. S mu starts about 2.5 kb downstream of the single pig JH gene and ends 0.4 kb upstream of C mu; the length of S mu is 3.2 kb. Three major pentameric repeats, gagct, gggct, and gggtt, comprise more than half of the entire S mu sequence. The two most frequent decamers are gagctgagct and gagctgggct. The longest single repeated sequence is 93 base pairs. The frequency of the various pentamers indicates that pig S mu is more similar to human S mu than to mouse S mu. The sequence of genomic pig C mu and its deduced amino acid sequence are similar to the C mu sequences published for other vertebrates and are most similar to those of sheep, another artiodactyl. The membrane and secreted tailpiece exons are highly conserved (ca > 90% similarity) and are actually identical to those of the sheep. The degree of similarity to other mammalian IgMs generally decreases from the carboxyl to the amino terminal domains, with the exception of C mu 2, which shows the lowest similarity among species (61% DNA, 44% aa). As in human C mu, we identified five potential N-linked glycosylation sites, two cysteines involved in forming interchain disulfide bonds, and the two additional cysteines involved in the linkage of IgM pentamers. This is only the third species for which a S mu sequence has been reported but the first from a species lacking the second virgin B-cell receptor, i.e., IgD. IgD in mice and humans lacks a switch region and its expression depends on the splicing of C mu-bearing mRNA with C delta. However, we found no evidence that the lack of C delta influenced the genomic structure of S mu, C mu, or C mu m.

Amino Acid Sequence↗
PubMed1997

Adenosine receptor blockade enhance glycolysis in hypoperfused guinea-pig myocardium.

OBJECTIVE: This study tested the hypothesis that endogenous adenosine depresses anaerobic glycolysis in preischaemic and moderately ischaemic myocardium. METHODS: Isolated, working guinea-pig hearts, perfused with glucose-fortified Krebs-Henseleit buffer, were subjected to 15 min mild hypoperfusion (coronary flow 60% of baseline) followed by 10 min ischaemia (coronary flow 20% of baseline). Adenosine A1 receptors were blocked with 8-p-sulfophenyl theophylline (8-SPT; 20 microM). Glucose oxidation and lactate production from exogenous glucose were assessed from 14CO2 and [14C]lactate formation, respectively, from [U-14C]glucose. Energy metabolites, glycolytic intermediates and glycogen were measured in extracts of stop-frozen preischaemic, mildly hypoperfused and ischaemic myocardium. RESULTS: Adenosine receptor blockade did not affect left ventricular function assessed from heart rate x pressure product and pressure x volume work although coronary flow was slightly reduced. Adenosine receptor blockade increased glucose uptake (P < 0.05) by 100% during preischaemia and by 74% during mild hypoperfusion, and increased lactate production from exogenous glucose (P < 0.05) by 89% during preischaemia and fourfold during mild hypoperfusion, but did not stimulate glucose oxidation under any condition. Glycogen degradation was not increased by adenosine receptor blockade during ischaemia. Crossover plots of glycolytic intermediates revealed that phosphofructokinase was activated by adenosine receptor blockade at all three levels of perfusion. CONCLUSION: Endogenous adenosine attenuates anaerobic glycolysis in normally perfused, hypoperfused and ischaemic myocardium by blunting phosphofructokinase activity; this effect is mediated by adenosine A1 receptors.

Adenosine↗
PubMed1997

An efficient SAS program for extract stratification of person-years.

An efficient Statistical Analysis System computer program for the exact stratification of person-years is described, in order to determine the expected number of deaths or cases of diseases in a cohort for the calculation of standardized mortality ratios or standardized morbidity ratios. A comparison of this program with Macaluso's "Method B" program shows that the speed of the former is considerably faster than that of the latter, when the widths of the categories defined by all of the time factors are identical.

Age Distribution↗
PubMed1997

Assessment of retrograde cardioplegia with magnetic resonance imaging and localized 31P spectroscopy in isolated pig hearts.

OBJECTIVE: This study was done to determine whether retrograde delivery of cardioplegic solution provides uniform blood flow to the myocardium supplied by an occluded coronary artery and whether it maintains myocardial energy levels beyond the coronary occlusion. METHODS: Isolated pig hearts were used. A hydraulic occluder was placed at the origin of the left anterior descending coronary artery. The perfusion pressure for retrograde delivery of cardioplegic solution was controlled at 40 to 50 mm Hg. Magnetic resonance imaging and localized 31P magnetic resonance spectroscopy were used to assess myocardial perfusion and energy metabolism, respectively. RESULTS: Magnetic resonance perfusion images (n = 7) showed that the perfusion defect that occurred during antegrade delivery of cardioplegic solution (as a result of the occlusion of the left anterior descending coronary artery) resolved during retrograde delivery of cardioplegic solution. Retrograde perfusion delivered similar amounts of flow to the jeopardized myocardium as it did to other areas of the myocardium. However, the distribution of cardioplegic solution by the retrograde route was heterogeneous (cloudlike) across both ventricular walls. 31P magnetic resonance spectra showed that the ischemic changes induced by occlusion of the left anterior descending artery during antegrade perfusion were greatly alleviated by retrograde perfusion; however, it took longer for retrograde cardioplegia (n = 7, 17.08 minutes) to restore the levels of inorganic phosphate/phosphocreatine relative to the effect of releasing the left anterior descending artery occluder during antegrade delivery of cardioplegic solution (n = 7, 5.3 minutes). CONCLUSIONS: First, retrograde delivery of cardioplegic solution provides sufficient flow to the myocardium beyond a coronary occlusion to maintain near normal levels of energy metabolites, and second, the efficacy of the retrograde route of cardioplegic solution delivery (in terms of distribution of the solution and rate of myocardial energy recovery) is significantly lower than that of the antegrade route.

Animals↗
PubMed1997

Aspartate/glutamate-enriched blood does not improve myocardial energy metabolism during ischemia-reperfusion: a 31P magnetic resonance spectroscopic study in isolated pig hearts.

OBJECTIVE: Our objective was to test the effects of exogenous L-aspartate and L-glutamate on myocardial energy metabolism during ischemia-reperfusion. METHODS: Phosphorus 31-magnetic resonance spectroscopy was used to observe cellular energetics and intracellular pH in isolated pig hearts perfused with blood (group A, n = 8) or blood enriched with 13 mmol/L each of L-aspartate and L-glutamate (group B, n = 6). The hearts were subjected to 30 minutes of total normothermic ischemia and then reperfused for 40 minutes. Two hearts from each group were inotropically stimulated by titration with calcium after normokalemic reperfusion. Left ventricular function was measured with the use of a compliant balloon and oxygen consumption was calculated. RESULTS: Magnetic resonance spectroscopy showed no decrease in the rate of energy decline during ischemia for group B versus group A. No significant differences were observed between the two groups in terms of myocardial function, oxygen consumption, or the rate or extent of high-energy phosphate recovery after normokalemic reperfusion or inotropic stimulation. Inotropic stimulation of postischemic hearts, however, led to dramatic improvement in myocardial function in both groups (p < 0.05 for all parameters) and significant improvement in oxygen consumption (p = 0.01). CONCLUSIONS: In a normal, isolated, blood-perfused pig heart subjected to 30 minutes of total normothermic ischemia, (1) enrichment of the perfusate with aspartate/glutamate before and after ischemia affects neither myocardial energy metabolism during ischemia-reperfusion nor postischemic recovery of myocardial function or oxygen consumption and (2) inotropic stimulation can recruit significant postischemic function and sufficient aerobic respiration to support it, irrespective of aspartate/glutamate enrichment.

Adenosine Triphosphate↗
PubMed1997

Airway inflammation despite loss of bronchial hyper-responsiveness after multiple ozone exposures.

The effect of single and multiple exposures to ozone (O3) on airway responsiveness and inflammation was examined in guinea pigs. Airway responsiveness, measured as acetylcholine concentration needed to increase baseline airway resistance (RL) by 250% (PC250), increased 1 h after exposure to ozone at 3 ppm for 3 h (-log PC250 from 3.88 +/- 0.17 to 4.78 +/- 0.18; P < 0.05), but returned to baseline at 8 h. An increase in neutrophil numbers was found at 8 h in bronchoalveolar lavage fluid (BALF). After O3 exposure on 4 successive days, baseline RL increased and airway responsiveness decreased at 1, 8 and 72 h (-log PC250 = 2.88 +/- 0.17, 2.83 +/- 0.10 and 3.12 +/- 0.08, respectively, compared to control value of 3.48 +/- 0.05). Repeated exposures to O3 also increased neutrophil numbers in bronchoalveolar lavage fluid and in bronchial submucosa. Thus, single exposure to O3 caused a rapid and transient increase in airway responsiveness, while multiple exposures induced a rapid but prolonged decrease in airway responsiveness associated with persistent bronchoconstriction. Both single and multiple exposures induced airway inflammation as evidenced by an increase in neutrophil influx. These studies demonstrated a dissociation between ozone-induced changes in airway responsiveness and neutrophil influx, and indicate that multiple exposures to O3 induce persistent bronchoconstriction with airway hyporesponsiveness.

Adaptation, Physiological↗
PubMed1997

Specific recognition of thymic self-peptides induces the positive selection of cytotoxic T lymphocytes.

To understand how thymic selection gives rise to T cells that are capable of major histocompatibility complex (MHC)-restricted recognition of antigen but are tolerant of self, we directly examined how peptide/MHC ligands expressed on thymic epithelial cells trigger the positive selection of immature thymocytes. We demonstrate that abundant self-peptides, purified from the H-2D(b) molecules of thymic epithelial cells, are specifically recognized during the positive selection of CD8+ T cells, implying that positive selection generates a repertoire of T cells that is weakly self-reactive. We also found that this recognition is somewhat cross-reactive, thereby providing an explanation for how the specific recognition of a limited repertoire of thymic self-peptides can select a diverse repertoire of T cells.

Animals↗
PubMed1997

Self-consistency estimation of distributions based on truncated and doubly censored survival data with applications to AIDS cohort studies.

Gomez and Lagakos (1994) propose a nonparametric method for estimating the distribution of a survival time when the origin and end points defining the survival time suffer interval-censoring and right-censoring, respectively. In some situations, the end point also suffers interval-censoring as well as truncation. In this paper, we consider this general situation and propose a two-step estimation procedure for the estimation of the distribution of a survival time based on doubly interval-censored and truncated data. The proposed method generalizes the methods proposed by DeGruttola and Lagakos (1989) and Sun (1995) and is more efficient than that given in Gomez and Lagakos (1994). The approach is based on self-consistency equations. The method is illustrated by an analysis of an AIDS cohort study.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Mutational analysis of slyD, an Escherichia coli gene encoding a protein of the FKBP immunophilin family.

slyD encodes a 196 amino acid polypeptide that is a member of the FKBP family of cis-trans peptidyl-prolyl isomerases (PPlases). slyD mutations affect plaque formation by the phage phiX174 by blocking the action of the phage lysis protein E. Here we describe the selection of a set of spontaneous slyD mutations conferring resistance to the expression of gene E from a plasmid. These mutations occur disproportionately in residues of SlyD that, based on the structure of the prototype mammalian FKBP12, make ligand contacts with immunosuppressing drug molecules or are conserved in other FKBP proteins. A wide variation in the plating efficiency of phiX174 on these E(R) strains is observed, relative to the parental, indicating that these alleles differ widely in residual SlyD activity. Moreover, it is found that slyD mutations cause significant growth rate defects in Escherichia coli B and C backgrounds. Finally, overexpression of slyD causes filamentation of the host. Thus, among the FKBP genes found in organisms across the evolutionary spectrum, slyD is unique in having three distinct drug-independent phenotypes.

Alleles↗
PubMed1997

Shading and stereo in early perception of shape and reflectance.

Recent experiments involving shaded 2-D stimuli have shown that early-vision mechanisms are capable of interpreting 3-D shape from shading. In particular, target discrimination tasks suggest that a target 'pops out' when background distractors, but not the target, can be interpreted as convex and lit from above or top-left. Since the problem of extracting 3-D shape from shading is intrinsically ill-defined, early vision may need to make these twin assumptions of convexity and top-left lighting in order to constrain the problem. Would these assumptions be recognized as unnecessary and consequently discarded when 3-D shape could be unambiguously defined by some other cue, like stereo disparity? A 2AFC stimulus onset asynchrony paradigm with masking was used in target discrimination experiments. The performance of five naive subjects on tasks where only shading cues were present was compared with that on tasks involving shading as well as stereo cues that define shape unambiguously. The results show that although stereo disparity information is incorporated by early-vision 3-D mechanisms, it is not used to overturn the default assumptions of lighting and shape. Stereo information is interpreted within the framework of top-left lighting, and a consistent preference for convexity is seen over concavity.

Contrast Sensitivity↗
PubMed1997

Interaction of ozone exposure with airway hyperresponsiveness and inflammation induced by trimellitic anhydride in sensitized guinea pigs.

The effect of prior ozone (O3) exposure on airway hyperresponsiveness and inflammation induced by trimellitic anhydride (TMA) has been investigated in TMA-sensitized guinea pigs. Airway responsiveness was measured as the concentration of acetylcholine needed to increase baseline lung resistance (RL) by 300% (PC300). Ozone (3 ppm for 3 h) caused an increase in -log PC300 at 1 h after exposure, with return of -log PC300 to control levels at 8 h. Ozone also increased baseline RL at 8 h. TMA challenge increase -log PC300 in TMA-sensitized guinea pigs at 8 h after challenge from 3.85 +/- 0.09 to 4.11 +/- 0.09. Ozone exposure prior to TMA challenge prevented the induction of airway hyperresponsiveness with a mean -log PC300 of 3.51 +/- 0.20, which was not different from that of control TMA-sensitized group. Baseline RL was significantly higher in ozone-pretreated animals after TMA challenge when compared to those of either control or challenged with TMA alone. Ozone had no effect on TMA challenge-induced BAL eosinophilia and neutrophilia. We conclude that a single exposure to ozone inhibits the increase in airway responsiveness but increases the bronchoconstrictor response induced by TMA in TMA-sensitized guinea pigs; however, the inflammatory airway response to TMA is unchanged by preexposure to ozone.

Acetylcholine↗