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Biomedical subjects

L Liao

Publications and source records attributed to L Liao.

At least 73 records · Page 4Linked to original sources
PubMed1996

Selective induction of a cationic amino acid transporter by tumor necrosis factor-alpha in vascular endothelium.

Treatment of bovine aortic endothelial cells with tumor necrosis factor-alpha (TNF-alpha) resulted in a concentration-dependent increase in L-arginine transport. The stimulatory effect of TNF-alpha was time-dependent, requiring at least 6 hr of exposure. Both actinomycin D and cycloheximide inhibited the TNF-alpha mediated increase in L-arginine transport, indicating that de novo RNA and protein synthesis were required. Ribonuclease protection analysis revealed the presence of cationic amino acid transporter (CAT)-1 and CAT-2 mRNA. Treatment of bovine aortic endothelial cells with TNF-alpha selectively increased the levels of CAT-2 mRNA, whereas message for CAT-1 remained unchanged. These results demonstrate that TNF-alpha stimulates L-arginine transport in endothelial cells by selectively inducing the expression of CAT-2 mRNA. The capacity of TNF-alpha to stimulate the expression of CAT-2 protein may provide an important mechanism by which increases in substrate are provided to endothelial cells during periods of elevated L-arginine metabolism.

Animals↗
PubMed1995

Mapping of a serotype specific epitope of the major capsid protein VP2 of infectious pancreatic necrosis virus.

A serotype-specific, conformational epitope of VP2 of infectious pancreatic necrosis virus (IPNV) (Jasper), has been mapped by restriction enzyme site-specific deletions of cloned viral cDNA. Subclones encoding fragments of VP2 were expressed in Escherichia coli followed by polyacrylamide gel electrophoresis and Western blot analysis. The epitope (between amino acid residues 242 and 325) reacted with monoclonal anti-VP2 antibodies that neutralized the homologous (Jasper), but not the heterologous (Sp) serotype of IPNV.

Amino Acid Sequence↗
PubMed1995

Antibody-mediated autoimmune myocarditis depends on genetically determined target organ sensitivity.

Injury to cardiac myocytes often leads to the production of anti-myosin antibodies. While these antibodies are a marker of myocardial injury, their contribution to pathogenesis in diseases such as autoimmune myocarditis or rheumatic fever is much less clear. We demonstrate in this report that monoclonal anti-myosin antibodies can mediate myocarditis in a susceptible mouse strain. Additionally, we show disease susceptibility depends on the presence of myosin or a myosin-like molecule in cardiac extracellular matrix. This study demonstrates that susceptibility to autoimmune heart disease depends not only on the activation of self-reactive lymphocytes but also on genetically determined target organ sensitivity to autoantibodies.

Animals↗
PubMed1995

Isolation and characterization of two genes encoding proteases associated with the mycelium of Streptomyces lividans 66.

A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-beta-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-beta-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.

Amino Acid Sequence↗
PubMed1995

Differential regulation of L-arginine transport and inducible NOS in cultured vascular smooth muscle cells.

Experiments were performed to characterize the uptake of L-arginine in rat aortic smooth muscle cells (SMC) and to examine whether inducers of nitric oxide synthase (NOS) could regulate the transport of L-arginine into these cells. L-Arginine transport by SMC was saturable, Na+ independent, strongly inhibited in the presence of other cationic amino acids, and could be stimulated by preloading the cells with cationic amino acids. Kinetic studies revealed the presence of both a high [Michaelis constant (Km) approximately 125 microM] and low (Km approximately 1.4 mM) affinity L-arginine transporter. Treatment of vascular SMC with interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) resulted in parallel increases in L-arginine transport and nitric oxide (NO) synthesis, as measured by nitrite production. Increasing the concentration of IL-1 beta and TNF-alpha caused a progressive elevation in nitrite production but did not further stimulate L-arginine uptake. Treatment of SMC with the combination of TNF-alpha and interferon-gamma (IFN-gamma) synergistically enhanced nitrite release without having any additional effect on L-arginine transport. Both induction of L-arginine transport and NOS activity by these cytokines were blocked by cycloheximide. Finally, treatment of SMC with interferon-gamma and N6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate selectively stimulated the formation of nitrite by SMC but had no effect on L-arginine transport. These results demonstrate that L-arginine transport by cultured vascular SMC is mediated by the system y+ carrier and that inducers of NOS differentially regulate the activity of this transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1995

Modulation of oxidized low-density lipoprotein-induced microvascular dysfunction by nitric oxide.

The objectives of this study were to determine 1) whether the leukocyte-endothelial cell adhesion in postcapillary venules elicited by copper-oxidized low-density lipoproteins (Cu-LDL) is accompanied by enhanced vascular albumin leakage and mast cell degranulation and 2) whether nitric oxide (NO) donors attenuate the Cu-LDL-induced microvascular dysfunction. Infusion of Cu-LDL, but not normal LDL, caused significant increases in leukocyte rolling, adherence, emigration, mast cell degranulation, and an enhanced albumin leakage in rat mesenteric venules. Treatment with the NO donors sodium nitroprusside and spermine-NO or pretreatment with superoxide dismutase or L-arginine significantly reduced the Cu-LDL-induced leukocyte adherence, emigration, mast cell degranulation, and albumin leakage, whereas spermine and D-arginine had no effect. These results indicate that NO protects the microvasculature against the deleterious effects of oxidized LDL, an effect that may be related to NO's ability to reduce leukocyte-endothelial cell adhesion and/or prevent mast cell degranulation.

Animals↗
PubMed1995

Mechanisms of oxidized chylomicron-induced leukocyte-endothelial cell adhesion.

The objectives of this study were to determine whether oxidatively modified chylomicrons (oxCM) can elicit leukocyte-endothelial cell adhesion in the mesenteric microcirculation and to define the mechanisms underlying the oxCM-induced adhesive interactions. Oxidation of chylomicrons (CM) with the peroxyl radical generator 2,2'-azobis(2-amidinopropane)hydrochloride was associated with the formation of thiobarbituric acid-reactive substances and lipid hydroperoxides. Leukocyte rolling, adherence, and emigration as well as erythrocyte velocity were monitored in rat mesenteric venules infused with either native CM or oxCM. oxCM, but not native CM, increased the numbers of rolling, adherent, and emigrated leukocytes. The oxCM-induced leukocyte adherence was significantly blunted by pretreating the animals with either superoxide dismutase, a platelet-activating factor (PAF) receptor antagonist, or monoclonal antibodies (MAb) directed against either CD11/CD18 or intracellular adhesion molecule 1. A MAb against P-selectin reduced oxCM-induced leukocyte rolling but not adherence. These findings suggest that the increased plasma oxCM levels associated with ingestion of oxidized lipids may promote leukocyte adhesion through a mechanism that involves the superoxide anion, PAF, and adhesion receptors on leukocytes and endothelial cells.

Animals↗
PubMed1995

Oxidized LDL-induced microvascular dysfunction. Dependence on oxidation procedure.

Human LDLs oxidized with Cu2+ are known to promote leukocyte-endothelial cell adhesion (LECA) and albumin leakage in postcapillary venules. The objective of this study was to compare the ability of LDL oxidized with Cu2+ (Cu-LDL), phospholipase A2 plus lipoxygenase (PLA2-LDL), horseradish peroxidase plus H2O2 (HRP-LDL), or -OCl (-OCl-LDL) to promote (1)neutrophil-endothelial cell adhesion (NECA) in vitro and (2)LECA and albumin leakage in rat mesenteric venules. In vitro adhesion assays revealed that only Cu-LDL elicited a dose-dependent NECA response, whereas PLA2-LDL but not normal (N-LDL), HRP-LDL, or -OCl-LDL increased NECA at the highest concentration studied (670 micrograms/mL). The magnitude of the NECA responses elicited by the different forms of oxidized LDL was related to the degree of lipid peroxidation but unrelated to the level of protein oxidation. Local intra-arterial infusion of Cu-LDL, PLA2-LDL, or -OCl-LDL but not N-LDL elicited significant increases in leukocyte adherence and emigration, mast cell degranulation, and albumin leakage in rat mesenteric venules. The LECA induced by all forms of oxidized LDL was not accompanied by significant alterations of venular shear rate.

Animals↗
PubMed1995

[A preliminary study on etiology of atrophic rhinitis in Zunyi].

200 cases (households with atrophic rhinitis (AR) diagnosed from outpatients and mobile medical team as the AR group were studied. Another 200 cases (households) served as the control group, who were residents of countryside and members of a factory in Zunyi, and they have lived in Zunyi for more than 20 years. The subjects between the two groups were similar in age and sex. Each of these two groups was further divided into two groups, the opentype stove group (100 cases) and the closetype stove group (100 cases). The heritability (h2, 59.4 +/- 0.032%) was estimated by using the method of Falconer liability threshold. The concentration of SO2 in living environment was monitored with Parafuchsin colorimetry. The daily average SO2 concentrations have been drawn for comparison. The result showed that the SO2 concentration of living environment in AR group was significantly higher than that in control group (P < 0.01). We believe that the contraction and development of AR were determined by the combination of inheritance with environment. This fact suggests that AR may be transmitted in a multigenic and multifacterial modes.

Adolescent↗
PubMed1995

[CT examination for renal vein thrombosis in nephrotic syndrome and the effect of thrombolytic therapy].

Renal vein thrombosis (RVT) was diagnosed in 12 of 60 patients with nephrotic syndrome with CT scan and confirmed by selective renal angiography. Of the 60 patients, 50 had primary glomerulonephritis with various pathological findings and 10 lupus nephritis. CT is valuable in screening renal vein thrombosis. Renal vein and cubital vein blood in the 12 patients were drawn for assay of FDP, AT III, VIIIR: AG, fibrinogen; the results indicated the presence of a state of hypercoagulation. Of these, 7 were given 200,000 units of urokinase (UK) in divided doses into renal vein within one hour and 5,200,000 units UK into renal artery in the same way. Patients also received 2.5mg/d warfarin and 75mg/d persantine. Except for the 3 with focal sclerosis, patients received 40mg/d prednisone. After 1 month CT and blood fibrinogen, FDP, AT III, VIIIR: AG studies were repeated. RVT in patients with intraarterial UK had complete dissolution of their thrombi. Complete dissolution occurred in 2 of the 7 receiving UK by renal vein and there was partial dissolution in the other 5. It seems that intraarterial injection yielded better results. Hypercoagulation state was alleviated in all patients with UK therapy.

Adult↗
PubMed1994

Identification and characterization of alpha-protein kinase C binding proteins in normal and transformed REF52 cells.

Immunocytofluorescence studies demonstrated that alpha-PKC is concentrated in focal contacts of REF52 cells but not in their SV40-transformed derivatives [Jaken et al. (1989) J. Cell Biol. 109, 697-704; Hyatt & Jaken (1990) Mol. Carcinog. 3, 45-53]. Discrete localizations imply that PKC is targeted to these areas possibly via protein-protein interactions. We have used an overlay assay to detect alpha-PKC binding proteins. The molecular interactions between alpha-PKC and the binding proteins depended on phospholipid and either calcium or phorbol esters. Unlike the kinase activity, binding activity was detected in the absence of added calcium, indicating that calcium, which is necessary for phosphorylation of most substrates, is not required for binding. Vinculin and talin, two focal contact proteins, bound alpha-PKC. REF52 cells express several annexins (I, II, and VI) which bind PKC. Both annexin I expression and vinculin expression were decreased in SV40-REF52 cells. The two major REF52 cell binding proteins (p71 and p > 200 kDa) were also down-regulated in the transformed cells, indicating transformation-sensitive regulation of PKC binding protein activity.

Amino Acid Sequence↗
PubMed1994

Protein kinase C domains involved in interactions with other proteins.

We have used a blot overlay assay to detect protein kinase C (PKC) interactions with other proteins. In many cases, the PKC binding proteins are also PKC substrates [Chapline et al. (1993) J. Biol. Chem. 268, 6858]. The purpose of the current studies was to characterize the PKC domains involved in the interactions with other proteins. alpha, beta, and epsilon isoforms of PKC interact with the same binding proteins in fibroblast cell extracts. These results indicate that constant rather than isozyme-specific (variable) regions are the major determinants of the interactions studied. PKC binding required phosphatidylserine (PS), indicating that the PS binding regulatory domain of PKC is involved in the interactions. The PKC pseudosubstrate peptide sequence, which is contained within the regulatory domain, also showed PS-dependent binding to the PKC binding proteins. To further investigate the role of the pseudosubstrate peptide in promoting PKC-protein interactions, an N-terminal truncation mutant lacking the pseudosubstrate sequence was prepared. Binding of the mutant alpha-PKC was diminished compared to wild-type alpha-PKC, although some binding was still apparent. These results indicate that the pseudosubstrate sequence contributes to, but is not the sole determinant of, PKC binding activity.

Amino Acid Sequence↗
PubMed1994

Protein kinase C isozymes in progressively transformed rat embryo fibroblasts.

The role of individual protein kinase C (PKC) isoforms in progressive transformation of a rat embryo fibroblast cell line (REF52 cells) has been evaluated. Normal (REF A) cells were transfected with SV40 to produce the partially transformed REF B cell line. REF B cells are morphologically transformed but have only limited capacity for growth in soft agar. Message and protein levels for PKC-alpha and PKC-delta were similar in REF A and B cells, indicating that expression of SV40 large T did not directly influence the amounts of PKCs. However, PKC-alpha localization was influenced. PKC-alpha was associated with focal contacts of REF A but not REF B cells, indicating that changes in location rather than content are an early event in REF52 cell transformation. Clones of REF B cells were selected for growth in soft agar (REF C cells). Levels of PKC-delta, but not PKC-alpha or epsilon, were increased in several of these clones, suggesting that increased PKC-delta content may facilitate anchorage-independent growth. In other studies, we have determined that PKCs interact with their binding proteins/substrates through their regulatory domains (RD; L. Liao et al., Biochemistry, 33: 1229-1233, 1994). These results predict that RDs could potentially inhibit PKC signaling by competing with endogenous wild-type PKCs for binding protein/substrate interactions. Overexpression of the RD of PKC-delta inhibited growth in soft agar of one representative REF C clone, whereas overexpression of the RD of PKC-alpha promoted growth in soft agar. These results suggest that RD expression may be a useful approach for dominant negative PKC inhibitors with potential isozyme specificity.

Animals↗
PubMed1994

Correlation between protein kinase C binding proteins and substrates in REF52 cells.

We have used a blot overlay assay to identify phosphatidylserine-dependent interactions between protein kinase C (PKC) and PKC binding proteins. The purpose of the present studies was to compare the properties of PKC binding proteins and PKC substrates detected by in vivo and in vitro phosphorylation assays. The major binding proteins and substrates in REF52 cells shared similar properties including enrichment by calmodulin-Sepharose chromatography, binding to phosphatidylserine, and resistance to heat denaturation. In addition, several of the major binding proteins and substrates were coordinately down modulated in SV40-transformed REF52 cells. The major PKC substrate, MARCKS, was also detected as a PKC binding protein. These results emphasize that the phosphatidylserine-dependent interactions between PKC and several substrates are of sufficient affinity to be detected in a blot overlay. Down modulation of binding proteins/substrates in transformed cells may reflect either decreased expression or increased basal phosphorylation of the target proteins and is likely to be involved in maintenance of the transformed phenotype.

Blotting, Western↗
PubMed1993

Cardiac alpha-myosin heavy chains differ in their induction of myocarditis. Identification of pathogenic epitopes.

BALB/c mice develop autoimmune myocarditis after immunization with mouse cardiac myosin, whereas C57B/6 mice do not. To define the immunogenicity and pathogenicity of cardiac myosin in BALB/c mice, we immunized mice with different forms of cardiac myosin. These studies demonstrate the discordance of immunogenicity and pathogenicity of myosin heavy chains. The cardiac alpha-myosin heavy chains of BALB/c and C57B/6 mice differ by two residues that are near the junction of the head and rod in the S2 fragment of myosin. Myosin preparations from both strains are immunogenic in susceptible BALB/c as well as in nonsusceptible C57B/6 mice; however, BALB/c myosin induces a greater incidence of disease. To further delineate epitopes of myosin heavy chain responsible for immunogenicity and disease, mice were immunized with fragments of genetically engineered rat alpha cardiac myosin. Epitopes in the region of difference between BALB/c and C57B/6 (residues 735-1032) induce disease in both susceptible and nonsusceptible mice. The data presented here demonstrate that pathogenic epitopes of both mouse and rat myosin residue in the polymorphic region of the S2 subunit. In addition, these studies suggest that polymorphisms in the autoantigen may be part of the genetic basis for autoimmune myocarditis.

Animals↗
PubMed1993

Effect of alpha-protein kinase C neutralizing antibodies and the pseudosubstrate peptide on phosphorylation, migration, and growth of REF52 cells.

REF52 cells are a line of rat embryo fibroblasts that express alpha-, delta-, epsilon-, and zeta-protein kinase Cs (PKCs). In this report, we have used neutralizing antibodies to alpha-PKC to study the role of this specific PKC isozyme in REF52 cell functions. Effects of the more general PKC inhibitor, the pseudosubstrate peptide, were also studied. Previous work demonstrated that alpha-PKC is concentrated in focal contacts of REF52 cells (Jaken, S., Leach, K., and Klauck, T. J. Cell Biol., 109: 697-704, 1989). alpha-PKC redistributed to the leading lamellopodia of cells stimulated to migrate into an artificial wound, indicating that alpha-PKC activation may be coupled to migratory stimuli. The effects of the alpha-PKC neutralizing antibodies and the pseudosubstrate peptide on responses associated with focal contact functions, namely attachment, migration, and growth, were studied. The data demonstrate that the antibodies and the pseudosubstrate peptide were all efficient inhibitors of phorbol ester-stimulated phosphorylation. However, only the pseudosubstrate peptide efficiently inhibited the migration and growth responses necessary to repopulate an artificial wound. These results indicate that PKCs, but probably not alpha-PKC in particular, are important in these responses. However, because the neutralizing antibodies did not completely inhibit phorbol 12,13-dibutyrate-stimulated phosphorylation, a potential role for alpha-PKC in migration and growth cannot be excluded.

Animals↗
PubMed1992

Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets.

Human platelets undergo a rapid, major reorganization of the cytoskeletal matrix upon exposure to thrombin, and accumulate 3-phosphorylated phosphoinositides in a protein kinase C (PKC)-dependent manner. These phosphoinositides have been suggested to be involved in actin polymerization/depolymerization. We reasoned that, if newly generated 3-phosphorylated phosphoinositide modulates cytoskeletal reorganization, a prerequisite for such action would be generation near cytoskeletal proteins. We have found that, after platelet activation, phosphatidylinositol 3-kinase and phosphatidylinositol(4)P 3-kinase activities, antibody-detectable phosphoinositide 3-kinase, and PKC become markedly and specifically enriched in a Triton X-100-insoluble cytoskeletal fraction that contains GPIIb/IIIa (integrin) and pp60c-src. The cytoskeletal fraction then accounts for up to 70% of total phosphoinositide 3-kinase activity, a function of recruited activated enzyme. These proteins are not occluded or directly associated with newly polymerized actin, since blockage by cytochalasin D of actin polymerization, and consequent inhibition of accumulation of about 40% of incremental protein and actin in this fraction, has no effect on its content of phosphoinositide 3-kinase, GPIIb/IIIa, pp60c-src, or PKC. Depolymerization of actin with DNase I, or inhibition of ligand binding to GPIIb/IIIa by RGDS, however, in combination with cytochalasin D, further depletes actin and significantly decreases sedimentability of GPIIb/IIIa as well as phosphoinositide 3-kinase, pp60c-src, and PKC, without inhibiting total 3-kinase activity. Our results suggest that, as a function of platelet activation, enzymes that regulate the synthesis of 3-phosphorylated phosphoinositides rapidly associate with the membrane skeleton and that skeletally associated phosphoinositide 3-kinase is more active than the Triton-soluble form.

1-Phosphatidylinositol 4-Kinase↗