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L Wang

Publications and source records attributed to L Wang.

At least 1,225 records · Page 68Linked to original sources
PubMed1998

Proteolysis as a probe of thermal unfolding of cytochrome c.

Recent hydrogen exchange experiments on native cytochrome c implicate a sequential unfolding pathway in contrast to a simple two-state process. We have studied the heat-induced unfolding of this protein by using spectroscopic measurements to detect changes in conformation and proteolytic enzyme digestion to identify regions of the protein that are labile. Several spectroscopic profiles were monitored: CD at 222 nm, a measurement of secondary structure change in the protein, the absorbance at 280 nm, involving the local environment of Trp 59, and absorbance at 420 nm, the Soret band of the heme. The apparent Tm values for these probes differ, consistent with an unfolding pathway containing intermediates. The limited digestion by proteinase K is consistent with population of an intermediate state in unfolding. We find a single strong region of cleavage at low temperature with retention of structure in each fragment.

Animals↗
PubMed1998

Apoptotic killing of CD4+ T lymphocytes in HIV-1-infected PHA-stimulated PBL cultures is mediated by CD8+ LAK cells.

In vitro infection of PHA-stimulated, normal CD4+ human peripheral blood T lymphocytes (PBLs) with several HIV-1 isolates did not result in cytopathology, despite high levels of virus replication and the fact that some of these isolates were cytopathic in certain cell lines. In contrast, infection of unfractionated PBLs (containing CD8+ as well as CD4+ lymphocytes) with these isolates always resulted in death of the infected CD4+ T lymphocytes. It has been well documented that PHA stimulation and culture of PBLs in medium containing IL-2 generates lymphokine-activated killer (LAK) cell activity which can destroy many transformed cells and virus-infected normal cells. When CD8+ T lymphocytes from PHA-stimulated PBLs were added to HIV-1-infected purified CD4+ T lymphocytes, significant lysis occurred. This cytotoxicity was not MHC class I-restricted, and depletion of CD8+ T lymphocytes from unfractionated PBL cultures shortly after HIV infection largely abolished the killing of the infected CD4+ T lymphocytes. These results demonstrated that CD8+ LAK cells were killing the CD4+ T lymphocytes in unfractionated PBL cultures infected with these noncytopathic HIV-1 strains. Care is thus warranted when studying HIV cytopathology in unfractionated PBL cultures. Morphological and DNA gel electrophoretic analyses of HIV-infected CD4+ T lymphocytes being killed by CD8+ LAK cells demonstrated that apoptosis was the predominant mechanism of LAK cell-mediated killing. In contrast, necrosis was the major mechanism involved in killing of purified CD4+ T lymphocytes by HIV-1 strains which were directly cytopathic. These findings may explain some of the discrepancies in the literature concerning reports of either apoptotic or necrotic killing of cells by HIV in vitro. Moreover, these data strongly suggest that direct killing by replicating HIV-1 in vivo should reveal necrotic cells and immune effector cell killing should reveal apoptotic cells. Since the latter are much more frequently observed in vivo, perhaps immune effector-mediated depletion of CD4+ T lymphocytes is more important as a pathogenic mechanism.

Apoptosis↗
PubMed1998

Time-resolved and nonlinear optical imaging for medical applications.

In this article, we have presented an overview of emerging novel techniques for early-light transillumination imaging as well as nonlinear optical tomography of body organs. The use of light for probing and imaging biomedical media offers the promise for development of safe, noninvasive, and inexpensive clinical imaging modalities with diagnostic ability. The strong scattering of light by biological tissues buries the shadowgram formed by forward-propatating image-bearing photons in the background noise of multiple-scattered light. Several methods for extraction of image-bearing light that capitalize on spatial, temporal and polarization characteristics of transmitted light are reviewed. More recently emerging nonlinear-optical histopathology methods for imaging subsurface structures of tissues in terms of its local spatial symmetry and molecular content are introduced. The progress made so far indicates that some of these techniques are apt to make a transition from laboratory to useful clinical modalities.

Animals↗
PubMed1998

Frequent homozygous deletions in the FRA3B region in tumor cell lines still leave the FHIT exons intact.

FRA3B at human chromosomal band 3p14.2 is the most active common fragile site in the human genome. The molecular mechanism of fragility at this region remains unknown but does not involve expansion of a trinucleotide or minisatellite repeat as has been observed for several of the cloned rare fragile sites. Deletions and rearrangements at FRA3B have been observed in a number of distinct tumors. The recently identified putative tumor suppressor gene FHIT spans FRA3B, and various groups have reported identifying deletions in this gene in different tumors. Using a high density of PCR amplifiable markers within FRA3B searching for deletions in the FRA3B region, we have analysed 21 tumor cell lines derived from renal cell, pancreatic, and ovarian carcinomas. We found a commonly deleted region in the renal cell and ovarian carcinoma cell lines located in the middle of an HPV16 viral integration site. Despite the presence of deletions in the FRA3B region in most of the cell lines, we did not detect alterations in FHIT exons in any of the cell lines examined. Thus, deletions of 3p14.2 in these carcinoma cell lines may simply reflect instability of the FRA3B region during tumor progression.

Carcinoma, Renal Cell↗
PubMed1998

Synergistic kinetic interactions between components of the phosphorelay controlling sporulation in Bacillus subtilis.

The four individual phosphotransfer steps in the multicomponent phosphorelay system controlling sporulation in Bacillus subtilis have been characterized kinetically using highly purified samples of the individual protein components in vitro. The autophosphorylation of KinA is the initial occurrence, and a divalent metal ion is required. KinA-mediated phosphotransfer, which displays a 57,000-fold preference (kcat/Km) for catalysis of Spo0F-P formation relative to Spo0A-P formation, is shown to proceed via a hybrid ping-pong/sequential mechanism with pronounced (> or = 40-fold) substrate synergism by Spo0F of KinA autophosphorylation. In addition, evidence is presented for formation of an abortive KinA.Spo0F complex. Kinetic parameters derived for Spo0F-P and Spo0A as substrates for Spo0B, the second phosphotransferase in the phosphorelay chain, indicate that Spo0B-mediated production of Spo0A-P is 1.1-million-fold more efficient (kcat/KSpo0A) than the direct KinA-mediated process. A rationale is presented for a four component cascade as the means for controlling sporulation, which focuses on the utility of synergistic interactions among the phosphorelay components that may be modulated by environmental stimuli.

Adenosine Diphosphate↗
PubMed1998

Elimination reactions in the medium-chain acyl-CoA dehydrogenase: bioactivation of cytotoxic 4-thiaalkanoic acids.

A range of 4-thiaacyl-CoA derivatives has been synthesized to study the bioactivation of cytotoxic fatty acids by the mitochondrial medium-chain acyl-CoA dehydrogenase and the peroxisomal acyl-CoA oxidase. Both enzymes catalyze alpha-proton abstraction from normal acyl-CoA substrates with elimination of a beta-hydride equivalent to the FAD prosthetic group. In competition with this oxidation reaction, 4-thiaacyl-CoA thioesters undergo dehydrogenase-catalyzed beta-elimination, providing that the corresponding thiolates are sufficiently good leaving groups and can be accommodated by the active site of the enzyme. Thus, the dehydrogenase catalyzes the elimination of 2-mercaptobenzothiazole and 4-nitrothiophenolate from 4-(2-benzothiazole)-4-thiabutanoyl-CoA and 4-(4-nitrophenyl)-4-thiabutanoyl-CoA, respectively. However, the 2,4-dinitrophenyl-analogue appears too bulky and the unsubstituted thiophenyl-derivative is insufficiently activated for significant elimination. Molecular modeling shows that steric interference from the flavin ring dictates a syn rather than an anti elimination. Acryloyl-CoA, the other product of 4-thiaacyl-CoA elimination reactions, is not a significant inactivator of the medium-chain dehydrogenase. In contrast, the irreversible inactivation observed during beta-elimination using 5,6-dichloro-4-thia-5-hexenoyl-CoA (DCTH-CoA), 5,6-dichloro-7,7,7-trifluoro-4-thia-5-heptenoyl-CoA (DCTFTH-CoA), and 6-chloro-5,5,6-trifluoro-4-thiahexanoyl-CoA (CTFTH-CoA) is caused by release of cytotoxic thiolate products within the active site of the dehydrogenase. The double bond between C5 and C6 found in the vinylic analogues DCTH- and DCTFTH-CoA is not essential for enzyme inactivation, although CTFTH-CoA is a weaker inhibitor of the dehydrogenase. Mechanism-based inactivation with CTFTH-CoA requires elimination, is unaffected by exogenous nucleophiles, and is strongly protected by octanoyl-CoA. The peroxisomal acyl-CoA oxidase efficiently oxidizes 4-thiaacyl-CoA analogues, but is only rapidly inactivated by DCTFTH-CoA. The variable ratio of elimination to oxidation observed for DCTH-, DCTFTH-, and CTFTH-CoA may influence the metabolism of the corresponding cytotoxic alkanoic acids in vivo.

Acyl-CoA Dehydrogenase↗
PubMed1998

Liver-derived CTL in hepatitis C virus infection: breadth and specificity of responses in a cohort of persons with chronic infection.

Hepatitis C virus (HCV)-specific CTL have been found within the inflammatory infiltrate of the liver of chronically infected individuals, but the breadth and specificity of the CTL response in relation to viral load are less well characterized. In this study, we analyzed the intrahepatic CTL response in liver biopsy specimens from 44 chronically infected subjects. Liver-infiltrating lymphocytes were expanded polyclonally in bulk cultures, and multiple clones were derived by limiting dilution. HCV-specific CTL responses directed at genotype 1a structural proteins were assessed in all subjects, and 22 subjects were tested more comprehensively using vectors expressing all structural and nonstructural HCV Ags. CTL responses were further characterized to determine the HLA restriction and optimal epitopes recognized. In those persons screened for recognition of all HCV Ags, HLA class I-restricted CTL were detected in 45%. Nineteen different CTL epitopes were identified, which were distributed throughout the genome; only one epitope was targeted by more than one person. In those persons with CTL responses, the breadth of response ranged from one to five epitopes. There was no correlation between the presence of a detectable CTL response and viral load. These results indicate considerable heterogeneity in detectable HCV-specific CTL responses in chronically infected persons. The mechanisms by which HCV persists during chronic infection remain to be clarified.

Cells, Cultured↗
PubMed1998

Conversion of pancreas allograft rejection to acceptance by liver transplantation.

BACKGROUND: Liver allografts of PVG(RT1c)-->DA(RT1a) are spontaneously accepted, whereas pancreas, heart, and kidney allografts are rejected. Our previous studies have shown that simultaneous liver and pancreas transplantation prevents pancreas allograft rejection. The aim of this study was to examine the effect of liver transplantation on subsequent pancreas allografts and on ongoing pancreas rejection. METHODS: Heterotopic, duct-ligated segmental pancreas grafts were transplanted into streptozotocin-induced diabetic recipients (60 mg/kg body weight i.p.) with or without orthotopic liver grafting at different times. Experimental design was as follows. Group 1 received PVG-->PVG pancreas syngrafts (n=6); group 2, PVG-->DA pancreas allografts (n=7); groups 3-5, PVG-->DA pancreas allografts followed by liver transplantation on day 2 (n=6), on day 4 (n=5), and on day 6 (n=5), respectively, group 6, PVG-->DA pancreas allografts after liver transplantation at 4 weeks (n=6). RESULTS: The results showed that pancreas allografts in group 2 were rejected from postoperative day 7 to 13. Liver transplantation prevented subsequent pancreas allograft rejection in group 6. Ongoing pancreas rejection was reversed by liver transplantation with subsequent graft acceptance in groups 3-5. Significant graft-infiltrating lymphocyte apoptosis was demonstrated at 2 weeks in pancreas transplants associated with liver grafting. Graft-versus-host disease was not detected in the pancreas recipients. CONCLUSIONS: We conclude that pancreas allografts in the PVG-->DA combination are rejected rapidly with median survival time of 9 days. Liver transplantation can protect subsequent pancreas grafts from rejection and reverse ongoing pancreas graft rejection with subsequent pancreatic acceptance. Graft-infiltrating lymphocyte apoptosis may be associated with the process of graft acceptance.

Animals↗
PubMed1998

Proximal colon distention increases Fos expression in the lumbosacral spinal cord and activates sacral parasympathetic NADPHd-positive neurons in rats.

Fos expression induced by nociceptive mechanical distention of the proximal colon was examined in the lumbosacral spinal cord in freely moving rats equipped with a chronic balloon in the proximal colon. Fos protein in lumbosacral neurons was detected immunocytochemically, and colocalization with nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity was determined histochemically at 1 hour after distention. Distention of the proximal colon (10 ml, 30 seconds on/off for 10 minutes, about 90 mm Hg) increased the number of Fos-positive cells in the lumbar 6 (L6) and sacral 1 and 2 (S1, S2) segments, whereas no change was observed in the L1-L5 and S3 segments compared with the sham distended group or with animals that received no treatment. In L6-S2 segments, Fos-positive neurons were increased by two-fold in laminae I-VII (mainly in laminae I and outer II) and area X (surrounding the central canal) and by nine-fold in the sacral parasympathetic nucleus. Results of time course studies indicate that the maximal increase in Fos expression observed at 1 hour after distention returns to basal levels within 4 hours. In the S1 segment, distention of the proximal colon increased the percentage of NADPHd/Fos-positive neurons selectively in the parasympathetic nucleus by 40% compared with less than 4% in the sham distention group; the number and pattern of NADPHd-stained cells were not modified. These results indicate that noxious distention of the proximal colon for a short duration in awake rats selectively activates neurons in the L6-S2 segments of the dorsal horn mainly in laminae involved in nociceptive and autonomic processing. The marked activation of NADPHd-positive neurons in the sacral parasympathetic nucleus suggests a possible role of nitric oxide in the visceroautonomic reflexes induced by distention of the proximal colon.

Animals↗
PubMed1998

Hyperosmolarity associated with diabetes insipidus alters hepatocyte structure and function but not survival after orthotopic liver transplantation in rats.

BACKGROUND: This study was designed to evaluate the effect of donor hyperosmolarity secondary to diabetes insipidus, an almost universal occurrence among brain-dead patients, on hepatic function. METHODS: In vitro (isolated liver perfusion) and in vivo (hyaluronic acid and indocyanine green uptake, arterial ketone body ratio, orthotopic liver transplantation) experiments were conducted using Brattleboro rats, with hereditary hypothalamic diabetes insipidus, and Sprague-Dawley rats, with normal pituitary function. ATP content and recovery after cold preservation were measured during the perfusion. RESULTS: Cold-preserved livers from hyperosmolar rats were observed to have elevated hepatic enzyme release and decreased bile production compared with normosmolar controls. Moreover, in these livers, the recovery of ATP after cold preservation was completely absent. Transmission electron microscopy of liver biopsies collected from hyperosmolar rats demonstrated profound ultrastructural changes, particularly in the mitochondria, that were not evident in the biopsies from normosmolar rats. All the experimental groups demonstrated similar hyaluronic acid uptake, whereas indocyanine green uptake was markedly impaired in the hyperosmolar group, suggesting that hepatocyte and not sinusoidal endothelial cell function is adversely affected by hyperosmolarity. The arterial ketone body ratio was profoundly compromised by chronic and, to an even greater degree, by acute hyperosmolarity. Survival after transplantation using hyperosmolar donors was not affected in this study. CONCLUSIONS: These results are an important step toward understanding the mechanism whereby brain death, a complicated pathophysiologic phenomenon, adversely affects the hepatic allograft.

Adenosine Triphosphate↗
PubMed1998

Molecular cloning and characterization of decay-accelerating factor deficiency in Cromer blood group Inab phenotype.

An additional decay-accelerating factor (DAF) mutation, designated as Inab phenotype in the Cromer blood group system, was recently identified in a 28-year-old Japanese woman (H.A.). The red blood cells of H.A., like those of other Inab phenotype individuals, were negative for Cromer system antigens, Cra, Tca, Dra, UMC, and IFC. The deficiency of DAF on the red blood cells of H.A. has been shown by immunoblotting with a murine monoclonal antibody to DAF. Molecular analysis has shown that H.A. is homozygous for a single nucleotide substitution, C1579-->A, at the position 24 bp upstream of the 3'-end of exon 2 of the DAF gene. This substitution causes the activation of a novel cryptic splice site and results in the production of mRNA with a 26 bp deletion. The deletion introduces a reading frame shift and creates a stop codon immediately downstream of the deletion. Translation of mRNA would be terminated at the first amino acid residue of the second short consensus repeat (SCR2) domain (exon 3) of DAF. The functional domains of DAF's complement regulatory activity and the carboxy-terminal signal domains for glycosylphosphatidylinositol (GPI) anchoring are predicted to be lacking in H.A. Thus, there would be no DAF present on the cell surface.

Adult↗
PubMed1998

Strategies for analyzing nerve conduction data: superiority of a summary index over single tests.

We compared three strategies for diagnosing carpal tunnel syndrome: using a single test; requiring one, two, or three of three tests to be abnormal; or utilizing a single summary variable incorporating data from three tests. Sixty-five hands of subjects without clinical carpal tunnel syndrome (CTS) were compared with 66 hands with clinical CTS. Three latency differences were measured: median-ulnar (8 cm) midpalmar orthodromic (palmdiff); median-ulnar ring finger (14 cm) antidromic (ringdiff); and median-radial thumb (10 cm) antidromic (thumbdiff). The combined sensory index (CSI) was the sum of these three differences. Sensitivity for the tests was palmdiff 69.7%, ringdiff 74.2%, thumbdiff 75.8%, and CSI 83.1%. Specificity was 95.4-96.9%. Requiring one, two, or three of three tests to be abnormal yielded sensitivities of 84.8%, 74.2%, or 56.1%, respectively, but specificities of 92.3%, 98.5%, and 100%, respectively. We conclude that a combined index improves diagnostic classification over use of single test results.

Carpal Tunnel Syndrome↗
PubMed1998

Methylation dynamics, epigenetic fidelity and X chromosome structure.

DNA methylation of the X chromosome is reviewed and discussed, with emphasis on the partial methylation seen in the mouse X-linked Pgk1 promoter region. A new study of partial methylation is presented in which the methylation of CpG site H3 in the mouse Igf2 upstream region was quantitatively measured during growth of subcloned cells in tissue culture. Before subcloning the average methylation level was 50%. After subcloning, methylation was highly variable in early stage clones. With continued passage, clones initially having high methylation lost methylation, whereas clones initially having low methylation gained methylation. By about the 25th generation, all clones had returned to a steady-state methylation level of 50%. These findings are discussed in the context of epigenetic mechanisms and epigenetic fidelity. Interpretation of the results is made according to a model that assumes stochastic methylation and demethylation, with rate parameters influenced by local chromatin structure. A second type of study is reported in which we have measured chromatin accessibility differences between the active X chromosome (Xa) and the inactive X chromosome (Xi). We found that Xa/Xi differences in accessibility to DNase I are surprisingly labile. Relatively infrequent DNA nicks rapidly eliminate differential accessibility.

Animals↗
PubMed1998

Comparison of the single-dose pharmacokinetics and tolerability of modafinil and dextroamphetamine administered alone or in combination in healthy male volunteers.

An open-label, randomized, crossover study was performed in healthy male volunteers to evaluate the potential pharmacokinetic and pharmacodynamic interactions and tolerability of single oral doses of modafinil (200 mg) and dextroamphetamine (10 mg). Blood samples were collected for determination of plasma levels of modafinil, the acid and sulfone metabolites of modafinil, and dextroamphetamine at intervals through 48 hours after administration for each treatment. Vital signs (blood pressure and pulse rate) were measured through 48 hours, and electrocardiograms were measured through 24 hours after administration. Pharmacokinetic parameters were determined using noncompartmental methods. The data collected in this study of 24 healthy volunteers suggest that concomitant administration of single oral doses of modafinil and dextroamphetamine has no clinically significant effects on the pharmacokinetic profile of either agent. Although there was a slightly greater incidence of adverse events when modafinil and dextroamphetamine were administered together, the concomitant administration of the two drugs was well tolerated.

Adult↗
PubMed1998

Correction for variations in MRI scanner sensitivity in brain studies with histogram matching.

Quantitative comparisons of abnormalities in MRI scans between patients or within patients serially are affected by variations in MR scanner performance. A histogram matching method is proposed to correct for variation in scanner sensitivity. It is demonstrated that this histogram matching method reduced the variation in white matter intensities across normal subjects from 7.5 to 2.5% and provided a method to remove the threshold dependency in lesion volume measurement with global thresholding in patients with multiple sclerosis (MS). The effectiveness of the method was compared with three other possible correction schemes. The histogram matching method was shown to be 2 to 5 times better.

Algorithms↗
PubMed1998

Stabilizing the subtilisin BPN' pro-domain by phage display selection: how restrictive is the amino acid code for maximum protein stability?

We have devised a procedure using monovalent phage display to select for stable mutants in the pro-domain of the serine protease, subtilisin BPN'. In complex with subtilisin, the pro-domain assumes a compact structure with a four-stranded antiparallel beta-sheet and two three-turn alpha-helices. When isolated, however, the pro-domain is 97% unfolded. These experiments use combinatorial mutagenesis to select for stabilizing amino acid combinations at a particular structural locus and determine how many combinations are close to the maximum protein stability. The selection for stability is based on the fact that the independent stability of the pro-domain is very low and that binding to subtilisin is thermodynamically linked to folding. Two libraries of mutant pro-domains were constructed and analyzed to determine how many combinations of amino acids at a particular structural locus result in the maximum stability. A library comprises all combinations of four amino acids at a structural locus. Previous studies using combinatorial genetics have shown that many different combinations of amino acids can be accommodated in a selected locus without destroying function. The present results indicate that the number of sequence combinations at a structural locus, which are close to the maximum stability, is small. The most striking example is a selection at an interior locus of the pro-domain. After two rounds of phagemid selection, one amino acid combination is found in 40% of sequenced mutants. The most frequently selected mutant has a deltaG(unfolding) = 4 kcal/mol at 25 degrees C, an increase of 6 kcal/mol relative to the naturally occurring sequence. Some implications of these results on the amount of sequence information needed to specify a unique tertiary fold are discussed. Apart from possible implications on the folding code, the phage display selection described here should be useful in optimizing the stability of other proteins, which can be displayed on the phage surface.

Amino Acid Sequence↗
PubMed1998

Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives.

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2.0 kcal mol(-1) less stable than the reduced form, and 0.07 kcal mol(-1) is more stable than the Cyt c: azide complex at 25 degrees C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.

Amino Acid Sequence↗