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K Tran

Publications and source records attributed to K Tran.

At least 37 records · Page 2Linked to original sources
PubMed1998

Functional analysis of disulfide linkages clustered within the amino terminus of human apolipoprotein B.

We tested the involvement of N-terminal six disulfide bonds (Cys-1 through Cys-12) of human apolipoprotein (apo) B in the assembly and secretion of lipoproteins using two C-terminal-truncated apoB variants, namely B50 and B18. In transfected rat hepatoma McA-RH7777 cells, B50 could assemble very low density lipoproteins (VLDL), and B18 was secreted as high density lipoproteins. When all 12 cysteine residues were substituted with alanines in B50, the mutant protein (B50C1-12) lost its ability to assemble lipid and was degraded intracellularly. However, mutation had no effect on B50C1-12 translation or translocation across the microsomal membrane. Post-translational degradation of B50C1-12 was partially inhibited by the proteasome inhibitor MG132. To determine which cysteines were critical in VLDL assembly and secretion, we prepared three additional mutant B50s, each containing four selected Cys-to-Ala substitutions in tandem (i.e. Cys-1 to Cys-4, Cys-5 to Cys-8, and Cys-9 to Cys-12). Expression of these mutants showed that disruption of disulfide bond formation within Cys-5 to Cys-8 diminished apoB secretion, whereas within Cys-1 to Cys-4 or Cys-9 to Cys-12 had lesser or no effect. In another two mutants in which only one disulfide bond (i.e. between Cys-5 and Cys-6 or between Cys-7 and Cys-8) was eliminated, only secretion of B50 with mutations at Cys-7 and Cys-8 was decreased. Thus, the disulfide bond involving Cys-7 and Cys-8 is most important for VLDL assembly and secretion. In addition, assembly and secretion of VLDL containing endogenous B100 or B48 were impaired in cells transfected with B50s containing Cys-7 and Cys-8 mutation. The Cys-to-Ala substitution abolished recognition of B50 by MB19, a conformational antibody with an epitope at the N terminus of human apoB. The Cys-to-Ala substitution also attenuated secretion of B18, but the effect of the mutation on B18 secretion was less evident than on B50.

Amino Acid Substitution↗
PubMed1998

Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 microM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.

Arachidonic Acid↗
PubMed1998

Detection of an interleukin-1 intracellular receptor antagonist mRNA variant.

At least two versions of interleukin-1 receptor antagonist generated by alternative splicing are found intracellularly, but their functions remain poorly characterized. During studies aimed at characterizing the expression of these transcripts in human articular cartilage, we detected a variant cDNA species that contained an additional 171 nucleotides within the type I interleukin-1 intracellular receptor antagonist cDNA which interrupted the coding region. This mRNA variant was also found to be expressed in keratinocytes. Translation likely initiates at an alternate methionine codon than that utilized for the previously reported interleukin-1 intracellular receptor antagonist isoforms, suggesting that this mRNA variant encodes a novel polypeptide that may play a role in interleukin-1 signaling.

Alternative Splicing↗
PubMed1998

Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes.

The release of arachidonic acid is the rate limiting step in eicosanoid synthesis. In mammalian cells, the release of arachidonic acid is catalyzed by several enzymes. The 85 kDa cytosolic phospholipase A2 (cPLA2) is the key enzyme for the release reaction because of its specific acyl selectivity in phospholipid substrates. We have previously reported that vitamin E enrichment potentiates the arachidonic acid release as well as the spontaneous prostacyclin release in human endothelial cells. In contrast, similar enrichment of diets caused a dose-dependent suppression of platelet thromboxane synthesis. Therefore, the present study was undertaken to determine the effect of vitamin E on arachidonate release and phospholipaseA2 activity in a platelet precursor cell, the MEG-01 megakaryocyte cell line. When these cells were incubated with different concentrations of vitamin E, cellular incorporation was linear with the dosages of this vitamin. Determination of arachidonate release after labeling cells with [3H]-arachidonate showed that vitamin E enrichment caused a dose-dependent increase in ionophore A23187-induced [3H]-arachidonic acid release. Analysis of PLA2 activity showed that activity was detected in the cytosol and this activity was completely abolished by the addition of anti-cPLA2, antibody. Determination of cPLA2 activity demonstrated that vitamin E enrichment caused an increase in enzyme activity. Analysis of cPLA2 protein by Western blot revealed that vitamin E caused an increase in enzyme protein. These data showed that the potentiation of arachidonic acid release and cPLA2, activity by vitamin E was mediated by the enhanced expression of cPLA2 protein.

Arachidonic Acid↗
PubMed1998

Quality control of white cell-reduced red cells: white cell preservation and simplified counting.

BACKGROUND: White cell (WBC) degradation restricts the interval between the filtration process and the assay for residual WBCs. Maintaining WBC integrity would permit extended sample storage for batching and/or shipment to centralized laboratories. The usual quality control assay for WBC-reduced red cell units requires determining the number of WBCs in the entire counting area of a Nageotte hemocytometer, which consists of 40 rows. Reducing the counting area would simplify the quality control procedure. STUDY DESIGN AND METHODS: Adsol red cell units were prepared either on the day of collection (Day 0) or on Day 1 and WBC reduced by filtration on the same day. By using prefiltration and postfiltration red cells, samples containing WBC concentrations of 15, 10, and 3 WBCs per microL were prepared by serial dilution. Identical samples were treated with glutaraldehyde and stored at either 20 to 24 degrees C or 1 to 6 degrees C. All samples were assayed on the day of component preparation and on Days 7 and 14. The numbers of WBCs corresponding to 10- and 40-row areas of the Nageotte hemocytometer were determined. RESULTS: For the conditions and WBC concentration range studied, no significant changes in WBC concentrations were observed through Day 14 for glutaraldehyde-treated samples stored at either temperature, although there were substantial decreases in untreated samples. A 10-row measurement was determined to be sufficient for identifying WBC-reduced red cell units passing the present limit of 5 x 10(6) residual WBCs. CONCLUSION: Glutaraldehyde treatment can preserve WBCs in red cell samples at least up to Day 14, which provides increased efficiency in quality control for laboratories. Current red cell WBC-reduction filters produce components that, when assayed, contain fewer than 10 WBCs per full counting area. The simplified procedure would allow reduction of the counting area by 75 percent.

Blood Preservation↗
PubMed1997

An NF-kappaB-like transcription factor in axoplasm is rapidly inactivated after nerve injury in Aplysia.

We found a protein in Aplysia neurons that has many characteristics of the transcription factor NF-kappaB. Thus, the protein recognized a radiolabeled probe containing the kappaB sequence from the human interferon-beta gene enhancer element (PRDII), and the binding was not affected by PRDIV, an ATF-2 enhancer sequence from the same gene. Binding was efficiently inhibited, however, by nonradioactive oligonucleotides containing H2, the kappaB site from the major histocompatibility complex I gene promotor. In addition, recombinant mammalian IkappaB-alpha, which associates specifically with the P65 subunit of NF-kappaB, inhibited the binding to the PRDII probe in a dose-dependent manner. The nuclear form of the Aplysia protein was constitutively active. Axoplasm, however, contained the constitutively active form as well as a latent form. The latter was activated by treatment with deoxycholate under the same conditions as mammalian NF-kappaB. Based on these findings, we believe the protein to be a homolog of NF-kappaB. To investigate the role of apNF-kappaB in the axon, we crushed the peripheral nerves to the body wall. Surprisingly, there was a rapid loss of apNF-kappaB binding at the crush site and, within 15 min, as far as 2.5 cm along the axon. In contrast, exposing either the intact animal or the nervous system in situ to levels of 5-HT that induce synaptic facilitation did not affect apNF-kappaB activity.

Animals↗
PubMed1997

Influence of Parks' anal retractor on anal sphincter pressures.

PURPOSE: The effects of the Parks' anal retractor on anal sphincter function were studied in a prospective, randomized trial. A closed hemorrhoidectomy was performed intra-anally in 20 patients using the Parks' anal retractor; in 20 other patients, the procedure was done perineally without the use of a retractor. METHODS: Anal manometry was performed before and at 6 and 12 weeks after hemorrhoidectomy. RESULTS: Mean squeeze pressure decreased by 4 percent whether or not a retractor was used. Mean resting pressure decreased by 23 percent after use of Parks' anal retractor (P = 0.01) compared with 8 percent when it was not used (P > 0,05). CONCLUSIONS: The internal anal sphincter is easily damaged with the use of the Parks' anal retractor. When possible, its use should be avoided to obtain better manometric and functional results.

Adult↗
PubMed1997

Distinct functional properties of IkappaB alpha and IkappaB beta.

The biological activity of the transcription factor NF-kappaB is controlled mainly by the IkappaB alpha and IkappaB beta proteins, which restrict NF-kappaB to the cytoplasm and inhibit its DNA binding activity. Here, we carried out experiments to determine and compare the mechanisms by which IkappaB alpha and IkappaB beta inhibit NF-kappaB-dependent transcriptional activation. First, we found that in vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Second, we showed that both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. Third, the inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes.

Animals↗
PubMed1997

Oxidative modification of low density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation.

The elevated level of plasma low density lipoprotein (LDL) in hyperlipidemic patients is an important risk factor for the production of atherosclerosis. Plasma LDL must be modified before it can produce an impairment of endothelium-dependent relaxation in aortic rings or enhancement of uptake by macrophages. The dramatic increase in lysophosphatidylcholine (lysoPC) content in oxidatively modified LDL has been touted as an important biochemical factor for the impairment of endothelium-dependent relaxation. The present study was designed to examine the lysoPC composition of oxidized LDL samples from normal and hyperlipidemic subjects, and their effects on the impairment of endothelium-dependent relaxation. Oxidatively modified LDL from hyperlipidemic patients contained a slightly higher level (17%) of lysoPC, but produced a disproportionately greater impairment of endothelium-dependent relaxation than that from normal subjects. As lysoPC is composed of many molecular species, its composition in oxidized LDL samples was analyzed. In hyperlipidemic patients, lysoPC samples were found to contain a higher proportion of long-chain acyl groups. Subsequent studies revealed that only long-chain lysoPC (C > 16:0) were effective in impairing endothelium-dependent relaxation. Experimental loading of oxidized LDL from normal subjects with long chain lysoPC to mimic levels observed in oxidized LDL from hyperlipidemic patients resulted in further impairment of endothelium-dependent relaxation. We conclude that the greater proportion of long-chain lysoPC found in the oxidized LDL of hyperlipidemic subjects is responsible for the increased impairment of endothelium-dependent vascular relaxation. We propose that the high level of LDL found in the plasma of hyperlipidemic patients, coupled with its enhanced ability to generate long chain species of lysoPC during oxidative modification, are important factors for the development of atherosclerosis in these patients.

Adult↗
PubMed1997

Intracellular degradation of newly synthesized apolipoprotein B.

Intracellular degradation of newly synthesized apolipoprotein (apo) B can occur at every stage of the secretory pathway, from the protein translation, polypeptide translocation across the membrane of endoplasmic reticulum (ER), to vesicular transport. The prevalence of apoB degradation at each stage varies in different hepatic cell systems examined. Proteolysis of nascent apoB can be catalyzed by the ubiquitin-proteasome system in the cytosol, and probably by unidentified ER resident proteases as well. Cytosolic and ER lumenal molecular chaperones that facilitate apoB translocation and folding may also assist in the degradation of misfolded apoB proteins. Factors affecting the synthesis and mobilization of lipids during lipoprotein assembly exert important regulatory effects on apoB degradation in trans, and specific hydrophobic amino acid sequence elements within the apoB-100 molecule may play roles in apoB degradation in cis. This review summarizes the current understanding of the cellular and molecular mechanisms responsible for intracellular degradation of apoB in hepatocytes. The emphasis centers primarily on the topology of apoB with respect to the ER membrane during and after apoB translation and its relationship to proteolytic mechanisms potentially involved in apoB degradation.

Animals↗
PubMed1996

Vitamin E potentiates arachidonate release and phospholipase A2 activity in rat heart myoblastic cells.

Cytosolic phospholipase A2 (cPLA2) selectively catalyses the release of arachidonic acid from the sn-2 position of glycero-phospholipids to produce prostaglandins and leukotrienes. In this study, vitamin E enrichment of rat heart myoblastic H9c2 cells caused an increase in the release of arachidonate during ionophore (A23187) stimulation. PLA2 activity in the cytosolic fraction was also enhanced but enzyme activity in the particulate fraction was not affected by this treatment. Immunoblotting analysis with a polyclonal anti-cPLA2 antibody showed an increased level of the enzyme in vitamin E-treated cells. Direct incorporation of vitamin E into lipid vesicles in the assay mixture resulted in modulation of enzyme activity in a biphasic manner. Pretreatment of cells with phorbol 12-myristate 13-acetate, a known activator of protein kinase C, synergistically potentiated the ionophore-induced arachidonate release in both the control and vitamin E-treated cells. However, vitamin E treatment by itself did not affect the protein kinase C activity, indicating that the vitamin E-induced activation of cPLA2 was independent of the protein kinase C cascade. Collectively, these results suggest that vitamin E potentiates arachidonate release through the direct and/or indirect modulation of cPLA2 activity.

Animals↗
PubMed1996

The modulation of choline phosphoglyceride metabolism in human colon cancer.

Colorectal cancer has a high incidence of morbidity and mortality in the North American population. Elevated levels of plasmalogens have been reported in some neoplastic tissues including colon tumors, but the mechanism for this increase has not been defined. Since changes in plasmalogen level are usually associated with changes in the other phospholipid subclasses, a general increase in all phospholipid subclasses may also be found in colonic neoplasms. In this study, the levels of the major phospholipids, including their plasmalogen and diacylphospholipid subclasses, were found to be elevated in human malignant colonic tissues. Since phosphatidylcholine is the most prominent type of phospholipid found in both malignant and control tissues, the mechanism for its accumulation during malignancy was investigated. Decreases in phospholipase C and D activities were observed in tumor samples, but an enhancement of the CTP: phosphocholine cytidylyltransferase activity was also detected. Immunoblotting analysis revealed that the elevated cytidylyltransferase activity was caused by a three-fold increase in the level of enzyme protein during tumor development. Based on these enzyme studies, we conclude that the high level of phosphatidylcholine in colon tumors resulted from a decrease in its turnover and an increase in its expression.

Colon↗
PubMed1996

Ag(I)-binding to phytochelatins.

Phytochelatins (PCs) are glutathione-derived peptides with the general structure (gamma-Glu-Cys)nGly, where n varies from 2 to 11. A variety of metal ions such as Cu(II), Cd(II), Pb(II), Zn(II), and Ag(I) induce PC synthesis in plants and some yeasts. It has generally been assumed that the inducer metals also bind PCs. However, very little information is available on the binding of metals other than Cu(I) and Cd(II) to PCs. In this paper, we describe the Ag(I)-binding characteristics of PCs with the structure (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly. The Ag(I)-binding stoichiometries of these three peptides were determined by (i) UV/VIS spectrophotometry, (ii) luminescence spectroscopy at 77 K, and (iii) reverse-phase HPLC. The three techniques yielded similar results. ApoPCs exhibit featureless absorption in the 220-340 nm range. The binding of Ag(I) to PCs induced the appearance of specific absorption shoulders. The titration end point was indicated by the flattening of the characteristic absorption shoulders. Similarly, luminescence at 77 K due to Ag(I)-thiolate clusters increased with the addition of graded Ag(I) equivalents. The luminescence declined when Ag(I) equivalents in excess of the saturating amounts were added to the peptides. At neutral pH, (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly, and (gamma-Glu-Cys)4Gly bind 1.0, 1.5, and 4.0 equivalents of Ag(I), respectively. The Ag(I)-binding capacity of (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly was increased at pH 5.0 and below so that Ag(I)/-SH ratio approached 1.0. A similar pH-dependent binding of Ag(I) to glutathione was also observed. The increased Ag(I)-binding to PCs at lower pH is of physiological significance as these peptides accumulate in acidic vacuoles. We also report lifetime data on Ag(I)-PCs. The relatively long decay-times (approximately 0.1-0.3 msec) accompanied with a large Stokes shift in the emission band are indicative of spin-forbidden phosphorescence.

Amino Acid Sequence↗
PubMed1996

Mice lacking the myotonic dystrophy protein kinase develop a late onset progressive myopathy.

Myotonic dystrophy (DM) is an autosomal dominant disorder resulting from the expansion of a CTG repeat in the 3' untranslated region of a putative protein kinase (DMPK). To elucidate the role of DMPK in DM pathogenesis we have developed Dmpk deficient (Dmpk-/-) mice. Dmpk-/-mice develop a late-onset, progressive skeletal myopathy that shares some pathological features with DM. Muscles from mature mice show variation in fibre size, increased fibre degeneration and fibrosis. Adult Dmpk-/-mice show ultrastructural changes in muscle and a 50% decrease in force generation compared to young mice. Our results indicate that DMPK may be necessary for the maintenance of skeletal muscle structure and function and suggest that a decrease in DMPK levels may contribute to DM pathology.

Animals↗
PubMed1996

Imaging of complications in liver transplantation.

Hepatic transplantations are being performed with increasing frequency, leading to greater demand for accurate evaluation of related complications. Ultrasonography (US) is the primary screening technique for detection of vascular complications of hepatic transplantation: angiography is used to confirm the US findings or when the US study is suboptimal. Hepatic artery thrombosis, the most common (as high as 42% of pediatric cases; 4%-12% of adult cases) and important vascular complication, may be associated with bilomas, infarcts, or abscesses at gray-scale US and absence of proper hepatic and intrahepatic arterial flow at Doppler analysis. Hepatic artery stenosis (seen in 11% of cases) is suspected if a focal accelerated velocity of greater than 2-3 m/sec with turbulence is seen at or distal to the stenosis or if a tardus parvus pattern of intrahepatic arterial flow is seen. In cases of inferior vena cava thrombosis and stenosis, US may show echogenic thrombus or obvious narrowing, with a substantially increased flow velocity through the stenosis or reversal of flow in the hepatic veins. Biliary complications occur relatively often (13%-25% of cases) after liver transplantation; bile leakage and biliary stricture, the most common biliary complications, are seen as a fluid collection and a stricture, respectively. Although acute rejection is one of the most serious complications affecting graft survival, it cannot be reliably detected with available diagnostic tests or radiologic methods.

Anastomosis, Surgical↗