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Biomedical subjects

J Liao

Publications and source records attributed to J Liao.

At least 127 records · Page 7Linked to original sources
PubMed1995

Digital angiographic impulse response analysis of myocardial perfusion: influence of heart rate and blood pressure changes on microcirculatory transit time measurement in the normal canine coronary circulation.

The study describes the effect of acute changes in aortic blood pressure and heart rate in the intact normal canine coronary circulation on digital angiographic measurements of the mean transit time of the microcirculation compartment and on direct flow meter measurement of resting and maximal hyperemic coronary blood flow. The mean transit time of the radiographic contrast material through the normal coronary circulation was calculated in 20 dogs by the impulse response analysis from serial digital coronary angiograms under systematically changed blood pressure and heart rate conditions. The mean aortic blood pressure (MAP) was controlled by the inflation of a balloon in the descending aorta (70-150 mmHg). The heart rate was altered by atrial pacing (90, 120, and 150/min). The mean transit times (T mu) of contrast material across the myocardial microcirculation which had been recently shown to correlate with coronary flow reserve (CFR) in stenosed arteries in the canine model were calculated under resting and hyperemic flow conditions from 428 selective coronary angiograms after a hand-injected contrast bolus, T mu was found to be heart rate independent. T mu-1 was linearly correlated with MAP (r = 0.7). There was no difference in T mu under resting and hyperemic flow conditions at comparable MAP. The corresponding distribution volume (V mu) of the microcirculation was calculated as the product of the mean transit time T mu and the measured coronary blood flow. Under resting flow conditions, V mu decreased with rising MAP. Furthermore, V mu increased with rising heart rate, V mu was constant during blood pressure or heart rate changes under hyperemic flow conditions. The data suggest that CFR whether by direct measurements of flow or by radiographic assessment of contrast material kinetics should be standardized with respect to hemodynamic conditions of heart rate and MAP.

Animals↗
PubMed1995

Heat stress or rotavirus infection of human epithelial cells generates a distinct hyperphosphorylated form of keratin 8.

The two major intermediate filament (IF) phosphoglycoproteins of human simple epithelia are keratins 8 and 18 (K8/18). Previous studies showed that heat stress and, in some cases, viral infection of cultured cells can be associated with alteration in IF organization. Here we show that heat stress of the human colonic cell line HT29 increased K8 and K18 phosphorylation and glycosylation and generated a distinct hyperphosphorylated form of K8 (HK8) that was previously noted upon G2/M arrest of epithelial cells. In contrast, rotavirus infection of HT29 cells resulted in phosphorylation changes similar to those induced by heat stress but did not alter K8/18 glycosylation. The identity of HK8 was determined using phosphatase treatment, tryptic phosphopeptide mapping, and K8-specific antibodies. A time course of heat stress showed that the increased phosphorylation and glycosylation of K8/18 occurred several hours after induction of heat shock protein 70 expression. Rotavirus altered the organization of the K8/18 network in most infected cells, whereas the effect of heat stress on K8/18 organization was less prominent. However, in vitro filament assembly of purified K8/18 was not significantly altered after isolation from heat-stressed or rotavirus-infected cells, despite having increased solubility and hyperphosphorylation. Our results indicate that increased glycosylation and/or phosphorylation of K8 and K18, in association with heat stress or rotavirus infection, does not significantly alter in vitro keratin filament assembly, whereas the association of these modifications with filament reorganization in cells is more dramatic. Generation of HK8 appears to be a late event and to occur in common with several forms of stress including heat, viral infection, and arrest of cells in G2/M.

Blotting, Western↗
PubMed1995

Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants.

To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3'-variable AChE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural 'atypical' BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the 'atypical' BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.

Acetylcholinesterase↗
PubMed1995

Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues.

Phosphorylation of keratin polypeptides 8 and 18 (K8/18) and other intermediate filament proteins results in their reorganization in vitro and in vivo. In order to study functional aspects of human K18 phosphorylation, we generated and purified a polyclonal antibody (termed 3055) that specifically recognizes a major phosphorylation site (ser52) of human K18 but not dephosphorylated K18 or a ser52-->ala K18 mutant. Pulse-chase experiments followed by immunoprecipitation and peptide mapping of in vivo 32PO4-labeled K8/18 indicated that the overall phosphorylation turnover rate is faster for K18 versus K8, and that ser52 of K18 is a highly dynamic phosphorylation site. Isoelectric focusing of 32PO4 labeled K18 followed by immunoblotting with 3055 showed that the major phosphorylated K18 species contain ser52 phosphorylation but that some K18 molecules exist that are preferentially phosphorylated on K18 sites other than ser52. Immunoblotting of total cell lysates obtained from cells at different stages of the cell cycle showed that ser52 phosphorylation increases three to fourfold during the S and G2/M phases of the cell cycle. Immunofluorescence staining of cells at different stages of mitosis, using 3055 or other antibodies that recognize the total keratin pool, resulted in preferential binding of the 3055 antibody to the reorganized keratin fraction. Staining of human tissues or tissues from transgenic mice that express human K18 showed that the phospho-ser52 K18 species are located preferentially in the basolateral and apical domains in the liver and pancreas, respectively, but no preferential localization was noted in other simple epithelial organs examined. Our results support a model whereby phosphorylated intermediate filaments are localized in specific cellular domains depending on the tissue type and site(s) of phosphorylation. In addition, ser52 of human K18 is a highly dynamic phosphorylation site that undergoes modulation during the S and G2/M phases of the cell cycle in association with filament reorganization.

Amino Acid Sequence↗
PubMed1995

Assessing horizontal transfer of nifHDK genes in eubacteria: nucleotide sequence of nifK from Frankia strain HFPCcI3.

The structural genes for nitrogenase, nifK, nifD, and nifH, are crucial for nitrogen fixation. Previous phylogenetic analysis of the amino acid sequence of nifH suggested that this gene had been horizontally transferred from a proteobacterium to the gram-positive/cyanobacterial clade, although the confounding effects of paralogous comparisons made interpretation of the data difficult. An additional test of nif gene horizontal transfer using nifD was made, but the NifD phylogeny lacked resolution. Here nif gene phylogeny is addressed with a phylogenetic analysis of a third and longer nif gene, nifK. As part of the study, the nifK gene of the key taxon Frankia was sequenced. Parsimony and some distance analyses of the nifK amino acid sequences provide support for vertical descent of nifK, but other distance trees provide support for the lateral transfer of the gene. Bootstrap support was found for both hypotheses in all trees; the nifK data do not definitively favor one or the other hypothesis. A parsimony analysis of NifH provides support for horizontal transfer in accord with previous reports, although bootstrap analysis also shows some support for vertical descent of the orthologous nifH genes. A wider sampling of taxa and more sophisticated methods of phylogenetic inference are needed to understand the evolution of nif genes. The nif genes may also be powerful phylogenetic tools. If nifK evolved by vertical descent, it provides strong evidence that the cyanobacteria and proteobacteria are sister groups to the exclusion of the firmicutes, whereas 16S rRNA sequences are unable to resolve the relationships of these three major eubacterial lineages.

Amino Acid Sequence↗
PubMed1995

Characterization of a human monoclonal immunoglobulin M (IgM) antibody (IgMBEN) specific for Vi capsular polysaccharide of Salmonella typhi.

A search for human monoclonal antibodies to protective antigens of bacteria revealed an immunoglobulin M lambda chain [IgM(lambda); designated IgMBEN] reactive with the Vi capsular polysaccharide of Salmonella typhi. Vi, a linear homopolymer of alpha(1-->4)GalApNAc that is O acetylated at C-3, is a licensed vaccine for typhoid fever. Immunologic properties of IgMBEN were compared to those of burro globulin prepared by intravenous injections of S. typhi (B339-340). IgMBEN and B339-340 yielded identical precipitin lines with Vi by double immunodiffusion. IgMBEN and B339-340 produced similar precipitation results with Vi and its derivatives prepared by de-O-acetylation, carboxyl reduction, and removal or replacement of the N-acetyl at C-2 with O-acetyl. B339-340 yielded maximal precipitation with Vi (0.41 mg of antibody per ml with 1.4 micrograms of Vi); next was carboxyl-reduced, O-acetylated Vi, which precipitated 0.325 mg of antibody per ml with 2.5 micrograms of Vi. IgMBEN yielded maximal precipitation with de-O-acetylated, carboxyl-reduced Vi (approximately 11.0 mg of antibody per ml with approximately 1.3 micrograms of antigen); next were de-O-acetylated Vi (9.89 mg/ml) and Vi (9.19 mg/ml). The precipitin curves and equivalence points of these three antigens were similar. Pneumococcus type 1, which contains GalApNAc, did not precipitate with Vi or its derivatives. These slight differences in specificity between IgMBEN and B339-340 were related to our proposed structure of Vi. We plan to use IgMBEN as a reference for measurement of vaccine-induced Vi antibodies.

Antibodies, Monoclonal↗
PubMed1995

Role of corticosterone in TNF and IL-6 production in isolated perfused rat liver.

Using an isolated perfused rat liver (IPRL) preparation, we assessed whether corticosterone may contribute to the rise in tumor necrosis factor (TNF) and interleukin-6 (IL-6) in rats after injection with lipopolysaccharide (LPS) or exposure to psychological stress. Intravenous infusion of LPS into the IPRL led to dose-dependent increases in TNF and IL-6 concentrations in the effluent. Anisomycin, a protein synthesis inhibitor, completely blocked the rise in TNF and IL-6 concentration in the IPRL effluent, supporting the hypothesis that the synthesis (or release) of these cytokines was dependent on protein synthesis. Intravenous infusion of corticosterone at nonstressed (35 ng/ml) and stressed levels (350 ng/ml) increased TNF and/or IL-6 release. However, when LPS was combined with corticosterone, the lower dose of corticosterone facilitated the release of cytokines, whereas the higher dose of corticosterone suppressed the release of cytokines. We then showed that isolated Kupffer cells were capable of significant TNF and IL-6 production and that corticosterone decreased LPS-induced cytokine production in these cells. Our data support the hypothesis that the liver is an important source of circulating cytokines in response to LPS. In addition, although in vitro data generally support the hypothesis that corticosterone suppresses the production of cytokines, our in situ data support the hypothesis that physiological levels of corticosterone cause an increase in TNF and IL-6.

Animals↗
PubMed1995

Role of epinephrine in TNF and IL-6 production from isolated perfused rat liver.

A bidirectional communication exists between the nervous system and the immune system. Evidence has accumulated suggesting that cytokines-immune peptides influence sympathetic neuronal survival and that cytokines can promote the secretion of catecholamines. Using an isolated perfused rat liver (IPRL) preparation, we have shown that the liver is an important source of circulating cytokines in response to lipopolysaccharide (LPS) and that corticosterone dose dependently influenced LPS-induced production of tumor necrosis factor (TNF) and interleukin-6 (IL-6). In this study, we investigated the direct effect of epinephrine (another stress hormone) on the production of TNF and IL-6 in liver. We demonstrated that epinephrine (1 microM/ml) alone did not induce TNF bioactivity but significantly increased IL-6 bioactivity from IPRL effluent. When the IPRL was infused with LPS, epinephrine significantly decreased TNF bioactivity. Epinephrine in LPS-treated livers also significantly increased IL-6 bioactivity. Both responses were totally inhibited by the beta-blocker propranolol (10 microM/ml). Anisomycin, a protein synthesis inhibitor, infused into the IPRL completely blocked the rise in TNF and IL-6 concentrations in the effluent leaving the IPRL, supporting the hypothesis that the synthesis (or release) of these cytokines was dependent on protein synthesis. We then attempted to determine whether epinephrine exerts similar effects in vitro. Using isolated Kupffer cells and hepatocytes, we found that epinephrine alone had no effect on TNF and IL-6 production in Kupffer cells and hepatocytes but significantly decreased LPS-induced TNF bioactivity and increased LPS-induced IL-6 bioactivity in Kupffer cells. Our data support the hypothesis that epinephrine can promote IL-6 secretion from IPRL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

An empirical Bayes approach to smoothing in backcalculation of HIV infection rates.

Backcalculation is a methodology to reconstruct the past human immunodeficiency virus (HIV) infection rates from the AIDS incidence data and incubation distribution by deconvolution. Smoothing has proved important in backcalculation, and a key question is how to choose the amount of smoothing. This paper proposes an empirical Bayes approach in which the smoothing parameter is estimated from the data. We introduce a family of priors that reflect the notion of closeness of neighboring infection rates. The variance parameter in the prior family plays the role of the smoothing parameter and is estimated by a method similar to the residual maximum likelihood in linear random effects model through an efficient EM (expectation/maximization) algorithm. A number of penalized likelihood functions that have been used in backcalculation have an empirical Bayes formulation. A bootstrap confidence interval for the infection rates is proposed. The methodology is illustrated with United States AIDS incidence data.

Acquired Immunodeficiency Syndrome↗
PubMed1995

[The effect of anti basic fibroblast growth factor on the development of experimental silicosis bacillus].

Anti-bFGF serum was used to treat rabbit experimental silicosis. At 90 days, when experimental group was compared with control group, the number and size of the nodules in experimental group were much less and smaller than those in control group. Nodules in control group were mainly consist of fibroblast mixed with a small amount of collagen fiber, whereas nodules in experimental group were made up of macrophages. At 180 days, in addition to fibroblasts, there was II to III degree of collagen fiber in nodules of control group. Most of nodules in experimental group mainly contained macrophages. Fibroblasts of lung in fetal mice were cultured in vitro. The results showed that bFGF stimulated proliferation of fibroblast, whereas anti-bFGF serum inhibited fibroblast growth. It appeared that anti-bFGF serum actually blocked proliferation of fibroblast and fibrosis in rabbit experimental silicosis.

Animals↗
PubMed1994

The membrane form of acetylcholinesterase from rat brain contains a 20 kDa hydrophobic anchor.

Rat brain acetylcholinesterase (AChE, EC 3.1.1.7) consists of about 80% amphiphilic detergent-soluble (DS-) AChE and 20% hydrophilic salt-soluble (SS-) AChE. DS-AChE contains about 65% tetrameric, 20% dimeric and 10% monomeric, SS-AChE about 40% tetrameric and 60% monomeric forms. N-terminal sequencing of DS- and SS-AChE gave identical N-termini corresponding to the published cDNA sequence of the mature enzyme. The band pattern on SDS-gels is similar to that of AChE from human and bovine brain. SDS-PAGE of hydrophobically labeled DS-AChE revealed the presence of a disulfide bonded hydrophobic membrane anchor of about 20 kDa. Monoclonal antibodies (mAbs) recognizing the anchor-containing subunits of mammalian brain DS-AChE, crossreacted with rat brain DS-AChE but not with SS-AChE. DS- and SS-AChE also reacted with antibodies raised against a peptide comprising the last 10 amino acids of the sequence of bovine brain AChE. Our results led us to conclude that both DS- and SS-AChE from rat brain contain T-type catalytic subunits, and DS-AChE in addition a P-type hydrophobic anchor similar to other mammalian brain DS-AChE.

Acetylcholinesterase↗
PubMed1994

Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos.

Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DNA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2- and 3-day-old embryos within the same subcellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.

Acetylcholinesterase↗
PubMed1994

Characterization of salt-soluble forms of acetylcholinesterase from bovine brain.

The hydrophilic, salt-soluble (SS) form of acetylcholinesterase (AChE) from bovine brain caudate nucleus exists mainly as a tetramer sedimenting at 10.3S (approximately 40%), and a monomer sedimenting at 3.4S (approximately 60%). The enzyme is N-glycosylated and contains similar HNK-1 carbohydrates as detergent-soluble (DS) AChE. No O-linked carbohydrates could be detected. Amino acid sequencing showed that the N terminus of SS-AChE is identical to that of DS-AChE. In tetrameric SS-AChE, two pairs of disulfide-linked dimers are associated by hydrophobic forces located in the C terminus. Antibodies were raised against a peptide identical to the last 10 amino acid residues of bovine brain DS-AChE. The peptide included the sequence of residues 574-583 (H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser- Asp-Leu-OH) of the enzyme. The antibodies cross-reacted with tetrameric, but not with monomeric, SS-AChE, showing that in the latter form, the C terminus is truncated. Limited proteolysis of tetrameric SS-AChE at the C terminus led to the formation of an enzymatically active monomer, which did not react with anti-C-terminal antibody. Although the DS form of AChE contains a structural subunit that serves as membrane anchor, no anchor was detected in SS-AChE. Enzyme antigen immunoassays showed that SS-AChE reacted with all monoclonal antibodies directed against the catalytic subunit of DS-AChE, but not with monoclonal antibodies targeting the membrane-anchored subunits. From our results, we conclude that SS-AChE utilizes the same alternative splicing pattern as DS-AChE, leading to tetrameric SS-AChE devoid of the membrane anchor.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗
PubMed1994

[Chemical constituents of Corydalis decumbens (Thunb.) Pers].

Six compounds were isolated from the quaternary ammoruinn parts of the bulbs of Corydalis decumbenbs in Shangrao District. The structures of menisperine and ferulic acid obtained for the first time from this plant were identified. Other compounds were found identical with the literature so far reported.

Aporphines↗
PubMed1993

Monoclonal antibodies against brain acetylcholinesterases which recognize the subunits bearing the hydrophobic anchor.

Monoclonal antibodies were raised against amphiphilic detergent-soluble (DS) acetylcholinesterase (AChE) from human brain caudate nucleus. Three mAb, 132-4 (IgG1), 132-5 (IgG1) and 132-6 (IgG3), specific for brain DS-AChE were selected and subcloned. These mAb reacted with native as well as heat-denatured and SDS-denatured DS-AChE, indicating that the epitopes to which mAb bound are continuous determinants. The mAb cross-reacted with DS-AChE from bovine and mouse brain and with brain DS-AChE from river trout (Salmo trutta forma fario) and lake trout (Salmo trutta forma lacustris). No cross-reaction was detected with the following antigens: salt-soluble (SS) AChE from bovine brain, glycophospholipid-anchored AChE from human and bovine erythrocytes, DS-butyrylcholinesterase and SS-butyrylcholinesterase (BtChE) from the brains of human and bovine, DS-BtChE from chicken and BtChE from human serum. Deglycosylation of brain DS-AChE with N-glycosidase F did not abolish the binding of mAb to DS-AChE. After reduction of brain DS-AChE by dithiothreitol, the mAb no longer reacted with the antigen, indicating that a disulfide bridge is important for the epitope. Monomerization of brain DS-AChE by trypsin and limited proteinase K treatment also abolished the binding of mAb to DS-AChE. Sucrose-density-gradient centrifugation showed that mAb reacted only with native tetrameric forms, but not with dimeric and monomeric forms. Western blot, after SDS/PAGE under non-reducing conditions, showed that mAb reacted with those subunits carrying the hydrophobic anchor (i.e. tetramers, trimers and heavy dimers) but not with those devoid of it (light dimers or monomers). Since mAb 132-4, 132-5 and 132-6 recognized DS-AChE from fish up to mammalian brain in the evolutionary tree, it is concluded that the epitope to which these mAb bind, is conserved in nature.

Acetylcholinesterase↗
PubMed1993

A monoclonal IgM kappa from a blood group B individual with specificity for alpha-galactosyl epitopes on partially hydrolyzed blood group B substance.

We have characterized a human monoclonal IgM kappa, designated IgMDON, from a blood group B individual. IgMDON is specific for alpha-galactosyl residues on blood group B substance; its fine specificity as defined by hemagglutination, quantitative precipitin, and inhibition ELISA assays was for the defucosylated terminal Gal(alpha 1-3)Gal epitope. Gal(alpha 1-3)Gal epitopes are also found on a variety of normal and pathogenic intestinal bacteria, and polyclonal IgG antibodies with the same specificity are found in the serum of nearly all normal individuals. The specificity of IgMDON was also quite similar to that of a human antiserum, serum 262, obtained by immunizing an individual with blood group B substance that had been subjected to mild acid hydrolysis (BP1). The possible ways whereby IgMDON might have arisen are discussed.

ABO Blood-Group System↗
PubMed1993

Subunit association and glycosylation of acetylcholinesterase from monkey brain.

Cercopithecus monkey brain acetylcholinesterase (AChE; EC 3.1.1.7) consists of about 15% hydrophilic, salt-soluble enzyme and 83% amphiphilic, detergent-soluble enzyme. Sucrose density gradient centrifugation showed that hydrophilic, salt-soluble AChE was composed of about 85% tetramer (10.3S) and 15% monomer (3.3S). In amphiphilic, detergent-soluble AChE, 85% tetramer (9.7S), 10% dimer (5.7S), and 5% monomer (3.2S) were seen. The enzyme is N-glycosylated, and no O-linked carbohydrate could be detected. Use of two monoclonal antibodies, one directed against the catalytic subunit and the other against the hydrophobic anchor, gave new insights into the subunit assembly of brain AChE. It is shown that in tetrameric AChE, not all of the subunits are disulfide-bonded and that two populations of tetramers exist, one carrying one and the other carrying two hydrophobic anchors.

Acetylcholinesterase↗