Biomedical subjects

J Hu

Publications and source records attributed to J Hu.

At least 19 recordsLinked to original sources

PubMed1995

The inhibitory effects of boldine, glaucine, and probucol on TPA-induced down regulation of gap junction function. Relationships to intracellular peroxides, protein kinase C translocation, and connexin 43 phosphorylation.

The naturally occurring antioxidant boldine and its di-methoxy analogue glucine, as well as the drug antioxidant probucol, all inhibit TPA-induced downregulation of gap junctional intercellular communication in WB-F344 rat liver epithelial cells in dose-dependent manners. The compounds were essentially 100% inhibitory to the effect of TPA (10 nM) at 50 microM each. Analysis of the mechanism of the antitumor promotive action of these agents in vitro revealed that boldine and probucol (both at 10 microM) totally inhibited the TPA-induced accumulation of intracellular oxidants. Additionally, boldine, glaucine, and probucol, each at 50 microM, inhibited TPA-induced translocation of protein kinase C (PKC) to the particulate fraction of the cells, with concomitant inhibition of TPA-induced hyperphosphorylation of gap junctional connexin 43 (cx43) and TPA-induced internalisation of cx43 protein from the plasma membrane of the cells. None of the compounds inhibited the binding of (3H)-PDBu to TPA-specific binding sites in the cells. The results indicate that antioxidant molecules, irrespective of structure, possess common antitumor promotive potential in this model of gap junctional intercellular communication. The data also indicate that the compounds may interfere with the promotive function of TPA, at least in part, by the destruction of oxidants within the cells. Xanthine oxidase was excluded as a major source of such intracellular oxidants because allopurinol (50 microM) did not significantly affect either the accumulation of oxidants in the cells or the downregulation of gap junctional communication in response to TPA. Taken together, these data also suggest that TPA-induced oxidants play a role in the translocation of PKC to cellular membranes and it is at this level where the antioxidants may interfere in TPA-induced downregulation of gap junctional function.

Allopurinol↗

PubMed1995

A transcriptional role for conserved footprinting sequences within the larval promoter of a Drosophila alcohol dehydrogenase gene.

All Drosophila alcohol dehydrogenase (Adh) genes that are expressed in larvae display strong transcription in the larval fat body. To identify and characterize elements needed for Adh promoter function, footprinting analysis of the Drosophila affinidisjuncta Adh gene was performed with stage-specific nuclear proteins from embryos and larvae. Multiple sites upstream of the larval promoter were protected from deoxyribonuclease digestion by both embryonic and larval extracts. Comparison with foot-printing results for Adh genes from other Drosophila species revealed only one nuclease-protected region that is conserved in both sequence and position. Clustered point mutations in this sequence were analyzed by footprinting analysis, transient transformation and in vitro transcription. Two separate sequences in this footprinting region exerted positive effects on transcription from the Adh proximal promoter in the larval fat body. The effects of these sequences on gene expression were synergistic. One of these sequences, TGATAA, bound in vitro to Drosophila melanogaster box A binding factor protein, as shown by gel mobility shift assays. This is the first direct demonstration of specific protein-DNA interactions influencing transcription of a Drosophila Adh gene in the larval fat body.

Alcohol Dehydrogenase↗

PubMed1995

Cyclohexylamine inhibits the adhesion of lymphocytic cells to human syncytiotrophoblast.

We have previously shown that lymphocytic cells adhere to cultured syncytiotrophoblast and that this may be important in the lymphocyte-mediated infection of trophoblast with the human immunodeficiency virus (HIV). During the course of studies aimed at investigating the role of cell surface carbohydrates in adhesion, it was discovered that a contaminant of commercial fucose-1-phosphate, dicyclohexylamine, inhibited MOLT-trophoblast adhesion. Dicyclohexylamine and the related compounds, cyclohexylamine and hexylamine, inhibited adhesion in a dose-responsive manner with half-maximal inhibition seen at about 4 mM. While the pressor effects of cyclohexylamine, the principal metabolite of cyclamate, are well known, this is the first report of an effect of this and related compounds on cell adhesion activity. The inhibitory effect was reversible and, at concentrations less than 25 mM, did not result in loss of cell viability. Several possible mechanisms of action of cyclohexylamine were examined in an attempt to explain the effect on adhesion. No evidence was found to suggest that the effects of cyclohexylamine were due to inhibition of polyamine synthesis, increase in intracellular Ca2+ concentration or to a lysosomotropic effect. The concentrations of cyclohexylamine used are within the range of plasma concentrations attainable in humans, raising the possibility that the in vitro effects described here may also occur in vivo. The results also suggest that caution should be used in the interpretation of results obtained from experiments where cell adhesion is blocked using exogenous monosaccharides that are in the form of dicyclohexylammonium salts. Appropriate controls must be included or, if possible, sodium, potassium or barium salts should be chosen.

Cell Adhesion↗

PubMed1995

Glutathione depletion potentiates 12-O-tetradecanoyl phorbol-13-acetate(TPA)-induced inhibition of gap junctional intercellular communication in WB-F344 rat liver epithelial cells: relationship to intracellular oxidative stress.

Treatment of WB-F344 liver epithelial cells with buthionine sulfoximine (BSO, 100 microM) for 24 h caused a greater than 95% depletion in cellular glutathione (GSH) and potentiated the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA) to inhibit gap junctional intercellular communication (GJIC) between the cells (IC50 shifted from 5 microM to 2 microM). Similarly, acute depletion of GSH by up to 30%, either with the thiol oxidant diamide or with BSO, also potentiated the inhibitory effect of the phorbol ester on GJIC. The treatment of the control cells with TPA caused a concomitant increase in the accumulation of oxidation products of 2',7'-dichlorofluorescein (DCF), indicating elevated production of oxidants in the cells during the blockade of GJIC. The depletion of GSH over a 24 h period with BSO itself increased the flux of oxidants in the cells but did not inhibit GJIC. Treatment of these GSH-depleted cells with TPA caused an additive elevation in the accumulation of oxidised DCF metabolites. Direct application of H2O2 (25-200 microM) or benzoyl peroxide (25-150 microM) to the control cells for 60 min caused weak, dose-dependent inhibitions of gap junctional communication in these cells but these responses were accompanied by the induction of acute, sub-lethal cytotoxicity. The depletion of GSH from the cells did not potentiate these responses to the peroxides but did facilitate synergistic inhibition of gap junctional communication in response to both TPA and sub-toxic doses of either peroxide. The results of the above studies indicate that oxidants are produced in WB-F344 cells in response to TPA and that these function in a co-operative manner with other cellular responses to the phorbol ester in the inhibition of gap junctional communication. This may explain why priming the cells for the induction of oxidative stress by the depletion of GSH potentiates the inhibitory activity of TPA on gap junctional communication.

Animals↗

PubMed1995

Hybrid three dimensional (1D-Hadamard, 2D-chemical shift imaging) phosphorus localized spectroscopy of phantom and human brain.

A hybrid of two localized spectroscopy techniques, chemical shift imaging (CSI) and Hadamard spectroscopic imaging (HSI), is used to obtain an array of 16 x 16 x 4 (3 x 3 x 3 cm3 voxels) proton-decoupled phosphorus (31P) spectra of human brain. For equal spatial resolution, this organ's oblate shape requires fewer axial than coronal or sagittal slices. These different spatial requirements are well suited to 1D, 4th order, transverse HSI in the axial direction, combined with 2D 16 x 16 CSI in the other two orientations. The reduced localization matrix (16 x 16 x 4 over just the brain versus a cubic-16 x 16 x 16 matrix of equal resolution, over the entire head) may proportionally shorten data acquisition if the voxel size is not signal-to-noise limited. In addition, the use of Hadamard encoding can improve the intervoxel spectral isolation.

Aged↗

PubMed1995

A fast, reliable, automatic shimming procedure using 1H chemical-shift-imaging spectroscopy.

The importance and the difficulty of achieving good B0 homogeneity over the volume of interest in in vivo NMR spectroscopy are well known. A fast, reliable, versatile, and fully automatic shimming procedure has been developed, using 3D chemical-shift imaging to measure the field distribution using only the water peak in the sample of interest. The procedure minimizes the mean-square error in the field distribution with respect to a constant field where the signal exists (e.g., the head). This produces a set of linear equations that can be solved by standard matrix methods. The procedure has been applied on a commercial imager producing water linewidths from the entire head as low as 8 Hz at 1.5 T. The mean linewidth and standard deviation from 94 head studies were 11.7 +/- 1.9 Hz.

Algorithms↗

PubMed1995

Regulation of neuronal nitric oxide synthase by histone, protamine, and myelin basic protein.

We examined the effects of endogenous basic proteins rich in the amino acid L-arginine on neuronal NO synthase activity by monitoring cyclic GMP formation in intact neuron-like neuroblastoma N1E-115 cells. Histone, protamine and myelin basic protein significantly stimulated cyclic GMP formation, both in a time- and concentration-dependent manner. These effects were blocked by hemoglobin and NO synthase inhibitors. Removal of the extracellular/intracellular Ca2+ gradient by a Ca2+ chelator completely abolished the cyclic GMP responses elicited by histone and protamine, suggesting that influx of extracellular Ca2+ might be involved in their activation of NO synthase. The effects of myelin basic protein on cyclic GMP formation, however, appeared to be due to Ca2+ release from intracellular stores. In cytosolic preparations of rat cerebellum, these basic proteins inhibited the metabolism of L-arginine into L-citrulline by NO synthase. We conclude from our findings that endogenous basic proteins might be involved in the regulation of neuronal NO synthase activity. Their effects on the enzyme could be either stimulatory or inhibitory, depending on whether the basic proteins exert their effects extracellularly or intracellularly, respectively.

Amino Acid Oxidoreductases↗

PubMed1995

Anomalous increase in nitric oxide synthase activity by certain nitric oxide-generating compounds in intact neuronal cells.

It has been shown that nitric oxide (NO) regulates NO synthase (NOS) activity through negative feedback in cytosolic enzyme preparations in various cell types. We compared the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) on NOS activity in intact neuroblastoma N1E-115 cells and in the cytosol obtained from the same cells. Enzyme activity was measured by the conversion of L-[3H]arginine into L-[3H]citrulline. At concentrations that elicit almost complete inhibition of NOS activity in cytosolic enzyme preparations of these cells, SIN-1 and SNP did not cause significant attenuation of enzyme activity measured at 45 min in intact cells. It is surprising that SIN-1 and SNP markedly stimulated L-[3H]citrulline formation in a time- and concentration-dependent manner when cells were incubated with the compounds for > 1.5 h. Neither inhibitory nor stimulatory effects of SNAP on NOS were observed in intact N1E-115 cells. This is in contrast to the inhibitory effects of SNAP in cytosolic preparations of the enzyme. The increased NOS activity by SIN-1 or SNP in intact cells was dependent on the presence of extracellular Ca2+, suggesting that it might be due to increased Ca2+ influx. On the other hand, measurements of the activity of lactate dehydrogenase showed that there was no generalized increase in cell permeability in response to SIN-1 or SNP. There was no agreement in the rank order of potencies of these compounds in activating guanylate cyclase and in affecting NOS activity, both in broken-cell preparations and in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMed1995

Redox-active chalcogen-containing glutathione peroxidase mimetics and antioxidants inhibit tumour promoter-induced downregulation of gap junctional intercellular communication between WB-F344 liver epithelial cells.

Evidence is mounting supporting a role for oxidative stress in the mechanism of tumour promotion in response to agents such as 12-O-tetradecanoylphorbol-13-acetate (TPA). In this paper we demonstrate that glutathione peroxidase-mimetic xenobiotics, ebselen, ebselen-glutathione, alpha-(phenylselenenyl) acetophenone and bis-(4-aminophenyl) telluride (at concentrations between 10 microM and 50 microM) all demonstrate protective effects on TPA-induced downregulation of gap-junctional intercellular communication (GJIC) between WB-F344 rat liver epithelial cells. These effects were, in each case, diminished if the cells were depleted of their intracellular glutathione, and potentiated if glutathione was supplemented into the incubations. Additionally, bis-(4-aminophenyl) selenide and several N-substituted analogues, possessing potent antioxidant activity but being devoid of GSH peroxidase-mimetic activity, demonstrated remedial activity against TPA-induced downregulation of GJIC. Structure-activity relationships between these molecules showed a strong correlation to the oxidation potential of the selenium atom in the compound as the bis-(4-nitrophenyl)- and bis-(4-cyanophenyl)- derivatives, which possess poor antioxidant capacity and a half-wave redox potential well above +1.0 V, did not affect TPA-induced effects on GJIC. Examination of the mechanism of action of these redox-active compounds demonstrated correlations between their abilities to (i) prevent TPA-induced downregulation of GJIC, (ii) abolish the accumulation of intracellular oxidants and (iii) prevent the hyper-phosphorylation and internalization of connexin 43 in the cells. The active compounds were also able to prevent the rapid, TPA-induced translocation of protein kinase C to the particulate fraction of the cells, without affecting phorbol ester binding. These data support a synergistic role for oxidants and other TPA-dependent responses within the cell in mediating the downregulation of GJIC. Such oxidative metabolism may play a role in the control of translocation of protein kinase C from the cytosol to membranes in response to TPA within these cells. Despite the nature of the in vitro test system studied, the data also clarify the molecular basis for a potential anti-tumour promotive effect of antioxidants, based on established redox chemistries of several series of structurally-related molecules.

Animals↗

PubMed1995

Development of an ELISA to detect MX virus, a human calicivirus in the snow Mountain agent genogroup.

MX virus is a Snow Mountain agent (SMA) genogroup human calicivirus (HuCV) identified in a Mexican child with diarrhoea. An ELISA using hyperimmune antisera to the recombinant MX virus (rMX) capsid was developed to detect SMA genogroup HuCVs in stool specimens. The rMX ELISA detected the prototype MX virus, SMA, and Hawaii agent (HA), but not Norwalk virus (NV) or Sapporo virus. Twenty-three diarrhoea stool specimens from children attending day care centres in Norfolk, Virginia, were positive by the rMX ELISA and results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR). Eight of 20 diarrhoea stool specimens from children in the United Kingdom previously shown to contain small round structured viruses (SRSVs) or HuCVs by electron microscopy were also positive by the rMX ELISA and RT-PCR. Sequence analysis of the RT-PCR products showed that all the rMX ELISA-positive viruses belong to the SMA genogroup. These data also showed that the SMA genogroup can be further divided into two subgroups: subgroup 1 includes prototypes SMA and HA, and subgroup 2 includes MX virus, minireovirus, Oth-25 and Bristol virus.

Animals↗

PubMed1995

Generation of DNA-based markers in specific genome regions by two-primer RAPD reactions.

Random amplified polymorphic DNA (RAPD) markers offer quick screening of different regions of the genome for genetic polymorphisms. The standard RAPD procedure uses a single 10-base-long random oligonucleotide as a primer to amplify short stretches of the genome by PCR. We modified the procedure by using two primers in each reaction in a Brassica napus mapping project. We found that the two-primer RAPD tends to amplify more and smaller fragments than the standard RAPD technique. These new bands were always amplified in the two-primer reactions, and Southern analysis revealed that they had no homology to the bands amplified in single-primer reactions involving the same primers. Furthermore, these new markers were not linked to markers amplified with the same primers in the standard RAPD reactions, suggesting that they were amplified from different genomic regions. The advantage of the two-primer RAPDs is that it allows more reactions to be carried out with a limited number of primers to generate more markers. Using a single primer, the number of reactions is equal to the number of primers (n), which in turn limits the total number of markers. When using two primers in all possible combinations, the total number of reactions increases to n x (n-1/2). This method could be useful in conjunction with bulked segregant analysis to develop high density maps of certain chromosomal regions. We used this approach to map a second marker linked to a gene governing low linolenic acid concentration in a B. napus F2 population.

Brassica↗

PubMed1995

Expression, self-assembly, and antigenicity of a snow mountain agent-like calicivirus capsid protein.

Virus-like particles were produced in insect cells infected with a recombinant baculovirus containing the capsid gene of MX virus, a Mexican strain of human calicivirus. These recombinant MX (rMX) particles were morphologically similar to recombinant Norwalk virus (rNV) particles as observed under an electron microscope and contained a single capsid protein with a molecular weight of 57,000, which was slightly smaller than that of rNV. This protein was immunoprecipitated by sera from volunteers infected with the Snow Mountain agent, but it reacted weakly with sera from volunteers infected with NV. This protein did not react with hyperimmune antisera from animals immunized with rNV in the rNV antigen enzyme immunoassay (EIA). Seroresponses were detected from volunteers infected with Snow Mountain agent and Hawaii agent when the rMX particles were used as antigen in an EIA. This EIA also detected an immune response in the sera of child from whom the MX virus was isolated, and a high prevalence of antibody to MX virus was found in the sera of a cohort of Mexican children.

Animals↗

PubMed1995

Mutational analysis of Saccharomyces cerevisiae U4 small nuclear RNA identifies functionally important domains.

U4 small nuclear RNA (snRNA) is essential for pre-mRNA splicing, although its role is not yet clear. On the basis of a model structure (C. Guthrie and B. Patterson, Annu. Rev. Genet. 22:387-419, 1988), the molecule can be thought of as having six domains: stem II, 5' stem-loop, stem I, central region, 3' stem-loop, and 3'-terminal region. We have carried out extensive mutagenesis of the yeast U4 snRNA gene (SNR14) and have obtained information on the effect of mutations at 105 of its 160 nucleotides. Fifteen critical residues in the U4 snRNA have been identified in four domains: stem II, the 5' stem-loop, stem I, and the 3'-terminal region. These domains have been shown previously to be insensitive to oligonucleotide-directed RNase H cleavage (Y. Xu, S. Petersen-Bjørn, and J. D. Friesen, Mol. Cell. Biol. 10:1217-1225, 1990), suggesting that they are involved in intra- or intermolecular interactions. Stem II, a region that base pairs with U6 snRNA, is the most sensitive to mutation of all U4 snRNA domains. In contrast, stem I is surprisingly insensitive to mutational change, which brings into question its role in base pairing with U6 snRNA. All mutations in the putative Sm site of U4 snRNA yield a lethal or conditional-lethal phenotype, indicating that this region is important functionally. Only two nucleotides in the 5' stem-loop are sensitive to mutation; most of this domain can tolerate point mutations or small deletions. The 3' stem-loop, while essential, is very tolerant of change. A large portion of the central domain can be removed or expanded with only minor effects on phenotype, suggesting that it has little function of its own. Analysis of conditional mutations in stem II and stem I indicates that although these single-base changes do not have a dramatic effect on U4 snRNA stability, they are defective in RNA splicing in vivo and in vitro, as well as in spliceosome assembly. These results are discussed in the context of current knowledge of the interactions involving U4 snRNA.

Base Sequence↗

PubMed1995

Cardioselective derivatives of 2,2-diphenyl-2-ethylthioacetate do not discriminate between m2 and m3 muscarinic receptors expressed in CHO cells.

Characteristics of interaction of two derivatives of 2,2-diphenyl-2-ethylthioacetate with muscarinic receptors were studied in Chinese hamster ovary (CHO) cells stably transfected with the genes of human m2 and m3 muscarinic receptors. Data from radioligand-receptor binding assays and measurements of m2 receptor-inhibited cyclic AMP formation and m3 receptor-stimulated phosphoinositide (PI) hydrolysis showed that this new series of muscarinic receptor antagonists exhibited a middle range of affinities in binding to muscarinic receptors (Ki = 0.2-0.7 mumol/l), without being able to discriminate between m2 and m3 receptors. They completely displaced [3H]N-methylscopolamine ([3H]NMS) binding at equilibrium and inhibited receptor-mediated increase in PI turnover in m3 CHO cells and decrease in cyclic AMP synthesis in m2 CHO cells in an apparent competitive manner. However, higher concentrations of the compounds (> 10 mumol/l) decelerated the kinetics of atropine-induced dissociation of [3H]NMS at m2 and m3 receptors, indicating an allosteric interaction. Collectively, our results demonstrate that these derivatives of 2,2-diphenyl-2-ethylthioacetate display a mixed mechanism of interaction with muscarinic receptors, being competitive at low concentrations and allosteric at higher concentrations. In contrast to previous reports of a significantly higher affinity at cardiac M2 as compared to ileal M3 receptor, these compounds do not exhibit such selectivity when the two receptor subtypes are expressed in the same type of cells.

Animals↗

PubMed1995

[Establishment and biological characteristics of the nude mice xenograft model from human hepatocellular carcinoma].

The human hepatocellular carcinoma was successfully heterotrasplanted into nude mice and 10 passages were made seriously during 11 months. Initial take rate was 14.3% and then the take rate increased to 100% after the 3rd passage. Histological, morphological, ultrastructural and biological investigation and chromosome analysis showed that the characteristic of transplanted tumor was identical to that of characteristics of human donor tumor. Through serial passages no metastatic signs were found grossly and under the microscope in the lymph nodes, liver and lung of the nude mice. Result also showed that the model retained some functions of the original tumor. The transplanted tumor could secret a-fetoprotein (AFP), r-glufamyl transpeptidase (r-GT). Acidic isoferritin (AIF) also could be detected. The tumor tissue of xenograft model was cultured in vitro in order to develop a permanent cell line. We found that the tumor specimen of the later passages grew more rapidly than the earlier ones. Up to now 3 cell passages were made. In this paper, a reliable animal model of human hepatocellular carcinoma is provided for studies of clinical diagnosis, treatment, antitumor drug screen test, etc.

Aged↗

PubMed1995

Quantitative cleaning efficacy evaluation methods with protein and lipid lens soiling models.

We developed a nondestructive, quantitative method for evaluating the cleaning efficacy of rigid gas permeable contact lens care systems. Simulated tears prepared with lysozyme, albumin, and calcium ions were applied to rigid gas permeable lenses as a protein soiling model, and a mixture of grease and unsaturated fat was applied as a lipid soiling model. The ComfortCare system (Pilkington Barnes Hind) and the Boston Cleaner and Boston Conditioning Solution (Polymer Technology) were evaluated for their efficacy in removing protein deposits; water was used as a control. The ComfortCare system and the Boston Advance Cleaner and Boston Advance Conditioning Solution were evaluated for their effectiveness in removing lipid deposits; saline was used as a control. Protein deposit level was detected by a densitometer at the absorbance of 280 nm. The lipid deposit level was evaluated by the absorbance of the fluorescence at 430 nm using the same densitometer with a fluorescence accessory. The two test systems demonstrated a significantly higher cleaning efficacy than the control for both soiling models. The methods provide quantitative measurements and therefore can be analyzed statistically for screening or comparison purposes.

Contact Lens Solutions↗

PubMed1995

Gentle Touch, a lens material resistant to protein deposition.

We investigated the ability of the Gentle Touch, a non-ionic contact lens material containing 35% netrafilcon A and 65% water, to repel protein deposits. Studies were conducted in vivo (lenses worn by patients) and in vitro (lenses coated by artificial tears). Acuvue soft contact lenses, an ionic lens material containing 42% etafilcon A and 58% water, were used as controls. After 1 to 2 weeks of wear, protein deposits on Gentle Touch lenses averaged 1.5-2.8 micrograms per lens versus 338-444 micrograms per lens for Acuvue. Gentle Touch lenses that were heat-coated with an FDA tear solution recommended for testing protein deposition averaged 6.5 micrograms of protein deposits per lens, whereas almost 100 micrograms of protein was deposited on each Acuvue lens. Lenses treated with an artificial tear solution deposited a non-detectable amount of protein on each Gentle Touch lens versus more than 358 micrograms on each Acuvue lens. These studies demonstrated that Gentle Touch lenses are highly resistant to protein deposits.

Biocompatible Materials↗

PubMed1994

Synergy in the spectral tuning of retinal pigments: complete accounting of the opsin shift in bacteriorhodopsin.

UV-visible and solid-state NMR studies of a series of 6-s-trans protonated Schiff bases of retinal with aniline show that the bathochromic shift induced by weakening the imine counterion is significantly greater in the 6-s-trans conformation than in the 6-s-cis conformation. Based on the observed magnitude of this coupling between the electronic effects of 6-s isomerization and imine counterion strength in the model compounds, the large opsin shift and unusual chemical shifts in light-adapted bacteriorhodopsin can be fully explained. These phenomena therefore do not require a negative point charge or polarizability effects in the chromophore binding pocket. The results are consistent with an effective center-to-center distance between the Schiff base and its counterion of about 4 A in light-adapted bacteriorhodopsin.

Aniline Compounds↗