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Biomedical subjects

H Liang

Publications and source records attributed to H Liang.

At least 181 records · Page 10Linked to original sources
PubMed1995

Comparison of recombinant cyclooxygenase-2 to native isoforms: aspirin labeling of the active site.

The search for isoform-specific enzyme inhibitors has been the focus of much recent research effort. Towards this goal, human recombinant cyclooxygenase-2 (EC 1.14.99.1, prostaglandin H synthase) was expressed in insect cells and purified to > 98% purity. Recombinant enzyme was characterized both by physical methods and activity measurements and shown to be fully active with kinetic properties similar to native COX-2 and COX-1. After detergent extraction, the enzyme had hydrodynamic properties indistinguishable from native bovine COX-1 and corresponded to the enzyme dimer as measured with size-exclusion chromatography. Peptide mapping via Lys-C protease identified a site of N-linked glycosylation and the aspirin covalent modification site. In the presence of heme, aspirin-specifically acetylated Ser-516. The enzyme will be suitable for biophysical studies and may lead to isoform-specific enzyme inhibitors.

Amino Acid Sequence↗
PubMed1995

pp125FAK in human melanocytes and melanoma: expression and phosphorylation.

Focal adhesion kinase (pp125FAK) is a nonreceptor tyrosine kinase which colocalizes with integrins to focal contacts, sites where multiple proteins interact to regulate the assembly of the actin cytoskeleton. Autophosphorylation and activation of pp125FAK occur after integrin clustering or cell adhesion to ligands through cognate integrin receptors and are postulated to mediate integrin signaling events. In this report we examined pp125FAK expression and phosphorylation in normal human melanocytes, an adherent human metastatic melanoma cell line (SKMEL28), and a nonadherent human metastatic melanoma cell line (SKMEL1). We show that SKMEL28 cells express constitutively phosphorylated pp125FAK and that pp125FAK phosphorylation in melanocytes is induced by phorbol esters and growth factors present in melanocyte growth medium. Focal adhesion kinase phosphorylation could be enhanced by b1 integrin-activating antibodies in human melanocytes, but not in SKMEL28 cells. In contrast with SKMEL28 cells, constitutive phosphorylation of pp125FAK was not observed in SKMEL1 cells, and incubation with activating b1 integrin antibodies had no effect on pp125FAK phosphorylation. Absence of pp125FAK phosphorylation in SKMEL1 cells was not due to lack of expression of pp125FAK, as shown by immunoprecipitation of the pp125FAK protein from cell lysates. However, b1 integrin expression was significantly less in SKMEL1 cells than in human melanocytes and SKMEL28 cells. This study further supports the importance of integrins in pp125FAK-mediated signaling and indicates that transformation-related changes in pp125FAK phosphorylation exist in human melanocytes and melanoma cells.

Antibodies↗
PubMed1995

Quantitation of beta 1 triiodothyronine receptor mRNA in human tissues by competitive reverse transcription polymerase chain reaction.

Thyroid hormones act by binding to nuclear receptor proteins, the thyroid hormone receptors (TR) alpha and beta. Data from cell culture and animal studies indicate that TR expression may be regulated to modulate target organ responsiveness to thyroid hormone. To investigate whether such adaptive changes in TR expression occur in humans, we determined the mRNA levels of the hTR beta 1 in various thyroid states. Patients with overt hypo- or hyperthyroidism were enrolled in the study. Total RNA was isolated from peripheral blood mononuclear cells and hTR beta 1 mRNA levels determined by quantitative competitive reverse transcription PCR. For comparison, hTR beta 1 mRNA levels were determined in lymphocytes and normal thyroid tissue of euthyroid patients. Human TR beta 1 mRNA levels in lymphocytes were 1.8 +/- 0.4, 1.9 +/- 0.5, 1.1 +/- 0.4 10(-18) mol/microgram RNA in hypo-, eu- and hyperthyroid patients, respectively, corresponding to an estimated 0.5 - 2 molecules per cell. Although the mean hTR beta 1 mRNA levels were 40% lower in hyperthyroid than in euthyroid subjects, this difference did not reach statistical significance. Similar levels of hTR beta 1 mRNA levels were detected in thyroid gland from euthyroid patients. In summary, we developed an assay for the quantitative determination of hTR beta 1 mRNA levels in small human tissue samples, containing as little as 50 ng of total RNA. Absolute hTR beta 1 mRNA levels are very low with an estimated one molecule of mRNA being present in a mononuclear blood cell or thyrocyte. No up-regulation of hTR beta 1 was seen in hypothyroid relative to euthyroid patients. However, there is a non-significant trend towards a down-regulation of hTR beta 1 mRNA levels in hyperthyroid patients.

Adolescent↗
PubMed1995

A role for photoreceptor outer segments in the induction of deprivation myopia.

An ultrastructural examination of the outer retina and choroid of hatchling chicks reared for periods of 1, 2 or 4 weeks with opaque occluders (MD) covering one eye, was instigated to elucidate the mechanism of deprivation myopia. Refractive myopia (approximately 20 D), retinal and choroidal thinning were induced in all deprived eyes. Electron microscopy showed significant changes in the MD eyes compared to normals. Cone inner segments were markedly thicker and outer segment lamellae more damaged. The rod outer segments were elongated and thicker than normal, such that their distal tips either directly apposed the basal lamina of the retinal pigment epithelium, or indented the cell nuclei. We hypothesize that this "rod-push" mechanism leads to thinning of the choroid in deprived eyes, and may directly contribute to axial myopia.

Animals↗
PubMed1995

Formoguanamine-induced inhibition of deprivation myopia in chick is accompanied by choroidal thinning while retinal function is retained.

Twenty hatchling chickens were injected intravitreally every 4 days from day 2 to day 16 with dimethyl sulphoxide (DS) in one eye and DS or formoguanamine dissolved in DS (FG.DS) with or without occlusion in the other (FG.DS.MD, DS.MD, FG.DS). At day 16, the FG.DS.MD eyes failed to show the high refractive myopia and showed less axial elongation than that developed by the DS.MD eyes. Electroretinograms indicated that at the dosage used, FG.DS does not eliminate phototransduction. Light microscopy showed choroidal and retinal thinning in DS.MD and FG.DS.MD eyes but less than in FG.DS eyes, suggesting that change in choroidal thickness is unlikely to be the primary cause of form deprivation myopia.

Animals↗
PubMed1995

Developmental regulation of focal contact protein expression in human melanocytes.

Focal contacts are transmembrane links between the extracellular matrix and the actin cytoskeleton that play a critical role in directed cell migration, adhesion, and normal growth. Several different component proteins of the focal contact show developmentally dependent changes in expression, suggesting that this is an important mechanism by which focal contact formation is controlled during embryogenesis. In this report we examine the expression of focal contact-associated proteins in human fetal and neonatal melanocytes using Western blotting. We show that expression of paxillin, a 69-kDa vinculin binding protein, is fourfold higher in neonatal melanocytes than in fetal melanocytes. Further, we show that talin, a high molecular weight structural protein that links integrins to the actin cytoskeleton, is proteolytically cleaved in fetal, but not in neonatal melanocytes. Immunofluorescence microscopy of cells grown on fibronectin confirmed the presence of paxillin, talin, and vinculin at the ends of actin stress fibers at presumptive focal contacts in melanocytes. Adhesion experiments to extracellular matrix ligands revealed significant differences in adhesion of fetal and neonatal melanocytes to fibronectin. The developmentally specific changes in focal contact protein expression observed suggest that this may be an important mechanism by which focal contact assembly is controlled in human melanocytes during development.

Actinin↗
PubMed1995

Human papillomavirus (HPV) type distribution and serological response to HPV type 6 virus-like particles in patients with genital warts.

Thirty-nine patients with condylomas (12 women and 27 men) attending a dermatology clinic were tested for genital human papillomavirus (HPV) DNA and for seroprevalence to HPV type 6 (HPV6) L1 virus-like particles. The L1 consensus PCR system (with primers MY09 and MY11) was used to determine the presence and types of HPV in sample specimens. All 37 (100%) patients with sufficient DNA specimens were positive for HPV DNA, and 35 (94%) had HPV6 DNA detected at the wart site. Three patients (8%) had HPV11 detected at the wart site, and one patient had both HPV6 and -11 detected at the wart site. Thirteen additional HPV types were detected among the patients; the most frequent were HPV54 (8%) and HPV58 (8%). Baculovirus-expressed HPV6 L1 virus-like particles were used in enzyme-linked immunosorbent assays to determine seroprevalence among the patients with warts. Seronegativity was defined by a control group of 21 women who were consistently PCR negative for HPV DNA. Seroprevalence was also determined for reference groups that included cytologically normal women who had detectable DNA from either HPV6 or HPV16 and women with HPV16-associated cervical intraepithelial neoplasia. Among the asymptomatic women with HPV6, only 2 of 9 (22%) were seropositive, compared with 12 of 12 (100%) female patients with warts. A similar trend in increased HPV6 seropositivity with increased grade of disease was found with the HPV16 DNA-positive women, whose seroprevalence increased from 1 in 11 (9%) in cytologically normal women to 6 in 15 (40%) among women with cervical intraepithelial neoplasia 1 or 3. However, only 4 of 25 (16%) male patients were seropositive. No factors examined, such as age, sexual behavior, or a history of warts, were found to definitively account for the gender difference in seroresponse.

Antibodies, Viral↗
PubMed1995

Long-lasting effects of Triac and thyroxine on the control of thyrotropin and hepatic deiodinase type I.

The purpose of this study was to investigate the relation between the serum levels of thyroid hormones and their biological effects. For this purpose, hypothyroid rats were studied after stopping treatment with a long-acting thyroid hormone, thyroxine (T4) and a short-acting one, triiodothyroacetic acid (Triac). Based on preliminary experiments with different doses of T4 and Triac, hypothyroid rats (N = 84) received over 6 days' injections of 10 nmol Triac or 2 nmol T4/100 g body wt per day. Biological effects of Triac and T4 were measured in the pituitary, liver and kidney up to 8 days after stopping treatment. With Triac, serum thyrotropin (TSH) levels were inhibited completely 6 h after injection, yet after 24 h they were 4.9 +/- 1.8 micrograms/l (hypothyroid 14.5 +/- 0.8 micrograms/l). The rapid changes in serum TSH levels were followed by a more gradual increase in serum TSH levels were followed by a more gradual increase in serum TSH, which was similar to that after T4 injection. Even 8 days after Triac treatment, serum TSH levels did not reach the hypothyroid control levels. Changes in beta-TSH mRNA levels also showed a prolonged inhibition after both treatments and a slow return to hypothyroid values, which was not complete 8 days after stopping treatment. A second parameter was hepatic 5'-deiodinase type I (5'D-I). The 6-day treatment with Triac had a markedly stronger effect on 5'D-I enzyme activity and mRNA levels than treatment with T4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

A model for the formation of ring mitochondria in retinal pigment epithelium.

PURPOSE: To investigate the mechanism and sequence of formation of ring-shaped mitochondria in retinal pigment epithelial cells of a chick model of gyrate atrophy. METHODS: Electron microscopic analysis of the ultrastructure of retinal pigment epithelial (RPE) mitochondria was carried out in chicks injected intravitreally with formoguanamine regularly (every 4 days) over the first 2 weeks or 4 weeks post-hatching. Formoguanamine is a triazine drug which induces hyperornithinemic symptoms in the chick eye similar to those seen in human gyrate atrophy. RESULTS: A large population of irregularly shaped mitochondria was observed in the RPE of both peripheral and central retina. They showed extensive morphological changes. At 2 wk, the mitochondria appeared enlarged and abnormal in shape with vacuolisation, partial loss of their double membrane and reduced mitochondrial cristae. By 4 wk, the mitochondria had assumed a rounder, almost circular profile, many with central holes, so-called ring mitochondria. CONCLUSION: The appearance of ring-shaped mitochondria has been previously ascribed to the section of cupshaped three-dimensional structures. We present evidence that ring-shaped mitochondrial profiles arise through at least two different mechanisms of membrane breakdown and intraorganelle vacuolisation. The nature of the three dimensional structures of these abnormal mitochondria is re-examined.

Animals↗
PubMed1995

[Effects of Astragalus polysaccharides and ginsenosides of Ginseng stems and leaves on lymphocytes membrane fluidity and lipid peroxidation in traumatized mice].

From days 0 to 3 posttrauma, daily administration of Astragalus polysaccharides (250mg/kg,ip) and ginsenosides of ginseng stems and leaves (50mg/kg,sc) can elevate significantly the lymphocytes membrane fluidity of plasmalemma, mitochondria and microsome from spleen,thymus and mesenteric lymph nodes in traumatized mice, reduce lipid peroxide levels, and increase superoxide dismutase activities in serum and lymphocytes from traumatized mice.

Animals↗
PubMed1995

[Changes in free calcium concentration and protein kinase C activity in activated T cells of burn mice and their significance].

TBSA 10%III degrees burn mice model was used. The changes in free calcium concentration ([Ca2+]i) and protein kinase C (PKC) activity in activated T cells from burn mice, and their relationship with T cell functions was studied. The results showed that [Ca2+]i and PKC activity in activated T cells were reduced after burn and these changes were closely related to reduced interleukin 2(IL-2) mRNA and IL-2 receptor alpha (IL-2R alpha) mRNA levels, decreased IL-2 production, suppressed IL-2R alpha expression, reduced T lymphocytes transformation in T cells of burn mice. Calcium cation ionophore A 23187 and PKC activator TPA could in vitro elevate respectively [Ca2+]i and PKC activity in activated T cells of burn mice. They also increased significantly IL-2 and IL-2R alpha gene expression in T cells of burn mice, but not up to the normal control. It is suggested that reduced [Ca2+]i, PKC activity in activated T cells may be one of the causes which produce suppression of T cell functions after burns.

Animals↗
PubMed1995

[A study on the simultaneous HPLC determination of chlorhexidine and its impurity 4-chloroaniline].

This paper reports a new RP-HPLC method using phenacetin internal standard (IS) for simultaneous determination of chlorhexidine (CH) and its impurity 4-chloroaniline (4-CA). We use the LC-3A HPLC instrument, a Zorbax-C8 column (25cm x 4.6mm id, 10 microns), a variable wave length UV detector and C-RIA chromatographic data processor. A mixture of methanol and 0.2 mol/L NaH2PO4 (50.5 : 49.5, pH 3.0) is used as mobile phase, whose flow rate is 1.0 ml/min. Sample solution of 15 microliters is injected onto the column, which is maintained at 30 degrees C. The eluent is monitored at 240nm. The retention times are 16.72 min for CH and 5.54 min for 4-CA. The standard curves of CH and 4-CA are established using peak area ratio of CH/IS and 4-CA/IS to concentration respectively. They are both linear (r = 0.9999) within 15-200 micrograms/ml for CH and 60-1200 micrograms/ml for 4-CH. The limits of detection are 1.33 micrograms/ml for CH AND 7.5 micrograms/ml for 4-CA (S/N = 3). The analytical recovery rates are 100.1% for CH and 103.1% for 4-CA. This method has been used for investigating the stability of CH preparation.

Aniline Compounds↗
PubMed1994

The secondary structure of the ets domain of human Fli-1 resembles that of the helix-turn-helix DNA-binding motif of the Escherichia coli catabolite gene activator protein.

The ets family of eukaryotic transcription factors is characterized by a conserved DNA-binding domain of approximately 85 amino acids for which the three-dimensional structure is not known. By using multidimensional NMR spectroscopy, we have determined the secondary structure of the ets domain of one member of this gene family, human Fli-1, both in the free form and in a complex with a 16-bp cognate DNA site. The secondary structure of the Fli-1 ets domain consists of three alpha-helices and a short four-stranded antiparallel beta-sheet. This secondary structure arrangement resembles that of the DNA-binding domain of the catabolite gene activator protein of Escherichia coli, as well as those of several eukaryotic DNA-binding proteins including histone H5, HNF-3/fork head, and the heat shock transcription factor. Differences in chemical shifts of backbone resonances and amide exchange rates between the DNA-bound and free forms of the Fli-1 ets domain suggest that the third helix is the DNA recognition helix, as in the catabolite gene activator protein and other structurally related proteins. These results suggest that the ets domain is structurally similar to the catabolite gene activator protein family of helix-turn-helix DNA-binding proteins.

Amino Acid Sequence↗
PubMed1994

Identification of a novel mammalian member of the NSF/CDC48p/Pas1p/TBP-1 family through heterologous expression in yeast.

Two suppressors of the growth deficiency of a potassium transport mutant of Saccharomyces cerevisiae were isolated from a mouse cDNA expression library. These suppressors, SKD1 and SKD2 (suppressor of K+ transport growth defect), were cDNAs encoding members of a family of ATPases involved in membrane fusion (N-ethylmaleimide-sensitive fusion protein, NSF), cell division cycle regulation (CDC48p), peroxisome assembly (Pas1p), and transcriptional regulation (TBP-1). The SKD1 protein constitutes a novel member of this family with 49-58% amino acid sequence similarity with other family members, and contains a single ATP binding site. The SKD2 polypeptide is the mouse homolog of NSF.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1994

Production of IgE antibody and allergic sensitization of intestinal and peripheral tissues after oral immunization with protein Ag and cholera toxin.

Cholera toxin (CTX) is a potent oral adjuvant for the induction of mucosal IgA Ab responses protein Ags. We examined the Ab responses and allergic sensitization of several strains of mice to protein Ags, administered orally with CTX. The mice made strong IgA and IgG1 serum Ab responses, but little IgG2a Ab to Ags such as hen egg lysozyme (HEL) and OVA. However, when given a subsequent i.p. challenge with Ag alone, the same mice had immediate hypersensitivity reactions that included respiratory distress and death. Within 10 min of i.p. challenge, immunized mice had high levels of plasma histamine and extensive degranulation of mast cells in target tissues. These mice had detectable serum IgE Ab. Ag administered orally with the B subunit (CTB) of CTX did not sensitize mice. Intestinal tissues taken from these mice had Ag-specific ion-secretory responses in vitro, typical of intestinal anaphylaxis. Ag given s.c. without adjuvant could also sensitize for systemic and intestinal anaphylaxis. Sensitization with HEL given s.c. was dose dependent and correlated with a critical amount of HEL in the circulation. HEL was detected in the circulation after oral immunization, but CTX did not increase the uptake of HEL. Thus, oral immunization with a protein Ag in the presence of CTX can sensitize an animal for systemic and intestinal anaphylaxis. These results suggest a cautious approach to the use of CTX as an adjuvant in oral vaccines, and provide a new model to study immediate hypersensitivity reactions to intestinal Ag.

Administration, Oral↗
PubMed1994

Molecular modelling of the inclusion complexes between beta-cyclodextrin and (R)/(S)-methylphenobarbitone and its application to HPLC.

Molecular modelling of beta-cyclodextrin and optimisation of its potential energy suggests that a favoured conformation is that distorted from a symmetrical torus. The inclusion of water molecules into the torus cavity simulates the increased stability in an aqueous solvent. Complexes of beta-cyclodextrin with (R)- and (S)-enantiomers of methylphenobarbitone have been modelled and energetically optimised by the application of molecular mechanics. The simulations suggests that the guest molecules adopt an orientation in which the phenyl ring is projected into the torus cavity, with in each case the plane of the ring parallel to a longer axis of the distorted torus and slightly displaced from the axis through the torus cavity. It is suggested that the asymmetry in the macrocyclic ring contributes to chiral recognition as a result of additional discriminatory binding to the barbiturate ring residue of each enantiomer, which occupy different 3D geometries. The enantiomers form complexes of different minimum potential energies. The resulting difference in complex stability can be related to the behaviour of beta-cyclodextrin, as a mobile phase additive in reverse-phase HPLC to effect chiral separation of rac-methylphenobarbitone during chromatography.

Chromatography, High Pressure Liquid↗
PubMed1994

Directed movement of chromosome arms and fragments in mitotic newt lung cells using optical scissors and optical tweezers.

A pulsed-laser microbeam at 532 nm wavelength (optical scissors) and a laser-induced optical trap at 1064 nm wavelength (optical tweezers) have been successively combined to dissect and manipulate chromosomes in live newt lung epithelial cells. These preliminary experimental results demonstrated that chromosome fragments dissected by laser microbeam surgery, regardless of their size, could be easily pulled or rotated by optical forces when positioned at the periphery of the mitotic spindle. In addition, chromosome arms which were not subjected to laser microsurgery also could be moved with the optical tweezers at the spindle periphery. In our previous study on rat kangaroo kidney cells (PTK2), this degree of facilty in manipulating chromosome movement was not possible, most likely due to the close proximity of the intermediate filament "cage" to the spindle. It is concluded herein that optical scissors and tweezers can be used in combination to study the interaction of chromosomes with the mitotic spindle in cells where the peripheral regions of the spindle are unobstructed by intermediate filaments. This can be performed on newt cells, where the diameter of the cage can be substantially larger than the diameter of the spindle.

Animals↗