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Biomedical subjects

B Huang

Publications and source records attributed to B Huang.

At least 181 records · Page 10Linked to original sources
PubMed1993

Molecular cloning and centrosomal localization of human caltractin.

Caltractin, a 20-kDa calcium-binding protein, was previously purified and cloned at the DNA level from the unicellular green alga Chlamydomonas. It is a structural component of the basal body complex, the major microtubule-organizing center in Chlamydomonas and the functional homolog of the centrosome in the animal cell. Here we report the characterization of a cDNA encoding a human caltractin that shares a high degree of amino acid identity (70%) with its algal counterpart. Caltractin was identified in both HeLa and BHK cells as a 21-kDa polypeptide specifically localized to the centrosome of interphase and mitotic cells. The high level of conservation in the amino acid sequence of caltractin from algae to humans and its association with the major microtubule-organizing center in the cell suggest that caltractin plays a fundamental role in microtubule-organizing center structure and function.

Amino Acid Sequence↗
PubMed1993

Effects of rhynchophylline on motor activity of mice and serotonin and dopamine in rat brain.

Rhynchophylline (Rhy) reduced the spontaneous motor activity and enhanced the sedative and hypnotic effects of sodium pentobarbital in mice. The effects of Rhy on serotonin (5-HT) and dopamine (DA) concentrations in rat brain, and the release of 5-HT and DA from the regional brain slices were studied by a fluorescence detector. Rhy increased the 5-HT content in the hypothalamus and cortex, but reduced the DA concentrations in the cortex, amygdala, and spinal cord. Rhy promoted the release of endogenous DA from 4 brain regions. The release of 5-HT was increased in 2 brain regions and decreased in hypothalamus slice. However, Rhy inhibited the release of both 5-HT and DA evoked by high potassium.

Alkaloids↗
PubMed1992

Crystal structure of the Y52F/Y73F double mutant of phospholipase A2: increased hydrophobic interactions of the phenyl groups compensate for the disrupted hydrogen bonds of the tyrosines.

The enzyme phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 ester bond of membrane phospholipids. The highly conserved Tyr residues 52 and 73 in the enzyme form hydrogen bonds to the carboxylate group of the catalytic Asp-99. These hydrogen bonds were initially regarded as essential for the interfacial recognition and the stability of the overall catalytic network. The elimination of the hydrogen bonds involving the phenolic hydroxyl groups of the Tyr-52 and -73 by changing them to Phe lowered the stability but did not significantly affect the catalytic activity of the enzyme. The X-ray crystal structure of the double mutant Y52F/Y73F has been determined at 1.93 A resolution to study the effect of the mutation on the structure. The crystals are trigonal, space group P3(1)21, with cell parameters a = b = 46.3 A and c = 102.95 A. Intensity data were collected on a Siemens area detector, 8,024 reflections were unique with an R(sym) of 4.5% out of a total of 27,203. The structure was refined using all the unique reflections by XPLOR to a final R-factor of 18.6% for 955 protein atoms, 91 water molecules, and 1 calcium ion. The root mean square deviation for the alpha-carbon atoms between the double mutant and wild type was 0.56 A. The crystal structure revealed that four hydrogen bonds were lost in the catalytic network; three involving the tyrosines and one involving Pro-68. However, the hydrogen bonds of the catalytic triad, His-48, Asp-99, and the catalytic water, are retained. There is no additional solvent molecule at the active site to replace the missing hydroxyl groups; instead, the replacement of the phenolic OH groups by H atoms draws the Phe residues closer to the neighboring residues compared to wild type; Phe-52 moves toward His-48 and Asp-99 of the catalytic diad, and Phe-73 moves toward Met-8, both by about 0.5 A. The closing of the voids left by the OH groups increases the hydrophobic interactions compensating for the lost hydrogen bonds. The conservation of the triad hydrogen bonds and the stabilization of the active site by the increased hydrophobic interactions could explain why the double mutant has activity similar to wild type. The results indicate that the aspartyl carboxylate group of the catalytic triad can function alone without additional support from the hydrogen bonds of the two Tyr residues.

Amino Acid Sequence↗
PubMed1992

Cellular reaction to the Vascugraft polyesterurethane vascular prosthesis: in vivo studies in rats.

The biocompatibility of Vascugraft, a polyesterurethane vascular prosthesis manufactured by Braun-Melsungen AG, was assessed by immunofluorescence and histological studies. Discs, 1 cm2, of Vascugraft prosthesis were implanted into the peritoneal cavity of rats. Results were compared with Impra, GORE-TEX and Mitrathane prostheses and a control group. Animals were killed at 1, 2, 6, 9 and 12 wk. Total T cells, T helper cells, T suppressor cells and activated T lymphocytes expressing Interleukin-2 receptors were quantified by a cytofluorometric technique in the peripheral blood of rats. For each period of implantation, all vascular prostheses showed no significant change in the percentage of total T cells, T subsets and T cells expressing Interleukin-2 receptors when compared to the control group. Histological examination of the tissue reaction surrounding the Vascugraft revealed a mild inflammatory reaction, similar to the one observed with both polytetrafluoroethylene grafts. However, the rate and the degree of encapsulation were different between grafts. The Vascugraft prosthesis was well encapsulated 2 wk after implantation, whereas inhibition of fibroblastic proliferation into the graft wall and surrounding both polytetrafluoroethylene grafts was observed for each period of implantation. The Mitrathane prosthesis exhibited a moderate inflammatory response, characterized by a high level of activation on fibroblasts compared to other grafts.

Animals↗
PubMed1992

Evaluation of transgenic canola plants under field conditions.

Eleven independent transgenic canola (Brassica napus ssp. oleifera L. cv. Westar and Regent) lines were evaluated in the field. The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation. NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene. A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns. These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.

Base Sequence↗
PubMed1992

Insulin and insulinlike growth factors in embryonic development. Effects of a biologically inert insulin (guinea pig) on rat embryonic growth and development in vitro.

Congenital anomalies occur up to four times more frequently in diabetic pregnancy than in the nondiabetic population. Although past work has shown that maternal hyperglycemia and hyperketonemia may increase embryonic abnormalities, recent experimental evidence suggests that low insulin levels may also contribute to diabetic embryopathy. This study investigated the effects of guinea pig serum (whose insulin is inactive in rat systems) on rat embryonic growth and development in culture. Supplementation of guinea pig serum with pork insulin at low (1 ng/ml) and high (5 ng/ml) physiological concentrations and insulinlike growth factors (IGF) I and II were also studied. Culture of rat embryos from the early headfold stage in guinea pig serum resulted in poor embryonic growth and development with a 92% rate of anomalies. Supplementation of guinea pig serum with zinc-binding pork insulin significantly improved rat embryonic growth and development (46% anomaly rate) especially between the first 5 and 21 h of the period of organogenesis. This evidence supports our most recent findings that low insulin levels, as encountered in untreated diabetic pregnancy, may contribute to the increased risk of congenital abnormality. Insulin at low physiological concentrations improved growth, whereas higher physiological concentrations were required to increase growth and development. IGF-I or IGF-II supplementation improved rat embryonic growth and development but failed to match that of the controls, indicating that other growth factors including insulin may also be required.

Abnormalities, Drug-Induced↗
PubMed1992

The production of hybridoma cell line secreting monoclonal antibodies against Xanthomonas campestris pv. oryzae and its application in the classification of strains.

By the fusion of mouse myeloma cells (SP2/0-Ag14) and spleen cells derived from BALB/c mice immunized with the preparation of Xanthomonas campestris pv. oryzae Ks-6-6, Os-213, Yz-32 and Yz-24, we obtained 12 hybridoma cell lines secreting monoclonal antibodies. None of the McAbs cross-reacted with the other varieties of plant pathogenetic and non-pathogenetic bacteria. The McAbs could distinguish three variant serotypes of strains. Antibody titers of ascites were about 1:10(3)-1:10(6) when measured by ELISA method. The McAbs could differentiate 6 epitopes. Based on the epitopes, the 63 strains of X. campestris pv. oryzae we collected were grouped into nine groups.

Animals↗
PubMed1992

Light-evoked expansion of subretinal space volume in the retina of the frog.

The retina of the frog was superfused with a Ringer solution containing impermeant "probe" cations and anions. Light-evoked concentration changes in these probe ions were measured in the subretinal space (SRS) with ion-selective microelectrodes. A decrease in probe ion concentration was found, and several observations suggest that this is caused by a light-evoked expansion of the SRS. The probe ion decrease was not seen in the isolated retina; thus, the pigment epithelial (PE) cells are important for its generation. Pharmacological studies suggest that K+ channels in the PE cells are important--perhaps the PE cells shrink in response to the light-evoked decrease in SRS [K+]. The light-evoked decrease of SRS volume may be important in the understanding of SRS solute concentrations, retina-PE adhesivity, photoreceptor-PE cell interactions, and the interphotoreceptor matrix.

Animals↗
PubMed1991

Genomic structure of Chlamydomonas caltractin. Evidence for intron insertion suggests a probable genealogy for the EF-hand superfamily of proteins.

A clone containing the gene locus for Chlamydomonas caltractin, a 20,000 Mr calcium-binding protein that is a member of the EF-hand superfamily of calcium-modulated proteins, was isolated and the structural organization of the gene was determined. The intron-exon organization was resolved by direct comparison of the genomic sequence with a caltractin cDNA. The promoter region does not contain the typical TATA or CCAAT boxes, but the sequences at the splice junctions are similar to those of other eukaryotes. The positions of the six introns in the caltractin gene do not typically define unit structures, nor do they coincide with those in genes for other members of the EF-hand superfamily. An analysis of exon sequences at the splice junctions in the genes of this multigene family was undertaken; evidence was obtained that supports the hypothesis that introns arose at proto-splice sites. A probable evolutionary history for the EF-hand superfamily based on intron insertion is offered.

Amino Acid Sequence↗
PubMed1991

Comparison of pharmacological agents (aspartate vs. aminophosphonobutyric plus kynurenic acids) to block synaptic transmission from retinal photoreceptors in frog.

The combination of aminophosphonobutyric plus kynurenic acids (APB/Kyn) was compared to aspartate with respect to its ability to block synaptic transmission from photoreceptors. Like aspartate, APB/Kyn blocks photoreceptor synaptic transmission, as monitored by the b- and d-waves of the electroretinogram, by the proximal negative response and M-wave of the proximal retina, and by the light-evoked increase in extracellular K+ concentration in the inner plexiform layer. Unlike aspartate, APB/Kyn has relatively minor effects on retinal resistance, light-evoked changes in K+ and Ca2+ concentrations in the subretinal space, light-evoked changes in subretinal space volume, resting extracellular concentrations of K+ and Ca2+ in the proximal and distal retina, and the c-wave. Effects of APB/Kyn are generally more reversible than effects of Asp. A disadvantage of APB/Kyn is that the a-wave usually becomes smaller and slower. Overall, APB/Kyn disrupts the retina less than aspartate. Therefore, in some situations in which blockade of photoreceptor synaptic transmission is desired, the use of APB/Kyn may be preferable to that of aspartate.

Aminobutyrates↗
PubMed1991

The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.

Acetylation↗
PubMed1990

Light-evoked changes in extracellular calcium concentration in frog retina.

Light-evoked changes in extracellular Ca2+ concentration were recorded with Ca2(+)-selective microelectrodes in the retina of the frog eyecup. A Ca-decrease at light onset and offset was found in the inner plexiform layer, and its properties are consistent with it resulting from Ca2+ influx into activated neuronal terminals. In the subretinal space, a Ca-increase at light onset and a Ca-decrease at offset were observed, and these likely arise directly from photoreceptors. A slower ON Ca-decrease was also seen here. Because it survives pharmacological isolation of the photoreceptors from post-synaptic interactions, but not physical isolation of the retina from the pigment epithelium, this component probably depends on pigment epithelial activity.

Animals↗
PubMed1990

Missense mutations at lysine 350 in beta 2-tubulin confer altered sensitivity to microtubule inhibitors in Chlamydomonas.

Two beta-tubulin mutants of Chlamydomonas reinhardtii, colR4 and colR15, were previously isolated in our laboratory. Each mutant expressed an acidic beta-tubulin variant as a result of an alteration in the coding sequence of one of the two beta-tubulin genes in C. reinhardtii, which in the wild type encode identical proteins. In this report, we describe the identity of the specific beta-tubulin altered in the colR mutants and the precise nature of the genetic lesions. Hybrid selection of mutant poly(A)+ RNA with cDNA probes specific for the two beta-tubulins in C. reinhardtii indicated that both mutations resided in the beta 2-tubulin gene. cDNA libraries were constructed with mutant poly(A)+ RNA, and beta 2-tubulin cDNA clones were isolated. Results of in vitro transcription of cloned beta 2-tubulin cDNAs confirmed the identity of the altered genes. Sequencing of the entire coding regions of the beta 2-tubulin cDNA clones revealed that the mutants carried different single-base substitutions in the same codon for the amino acid at position 350 in the beta 2-tubulin sequence, effecting a change from a lysine to a glutamic acid in the colR4 variant and to a methionine in the colR15 variant. These changes in amino acids are consistent with the difference in the charge of the two variant polypeptides observed in isoelectric focusing. Because both the colR4 and colR15 mutations confer an altered sensitivity to a number of different microtubule inhibitors and herbicides, lysine 350 appears to be of functional importance in the structure of the tubulin molecule.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and characterization of senescence-related genes from carnation flower petals.

The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased production of ethylene which plays an important role in regulating this developmental event. Three senescence-related cDNA clones were isolated from a cDNA library prepared from mRNA isolated from senescing petals. These cDNAs are representative of two classes of mRNAs which increase in abundance in senescing petal tissue. The mRNA for one class is present at low levels during the early stages of development and begins to accumulate in mature petals prior to the increase in ethylene production. The accumulation of this mRNA is reduced, but not eliminated, in petals treated with aminooxyacetic acid, an inhibitor of ethylene biosynthesis, or silver thiosulfate, an ethylene action inhibitor. In contrast, expression of the second class of mRNAs appears to be highly regulated by ethylene. These mRNAs are not detectable prior to the rise in ethylene production and increase in abundance in parallel with the ethylene climacteric. Furthermore, expression of these mRNAs is significantly inhibited by both aminooxyacetic acid and silver thiosulfate. Expression of these mRNAs in vegetative and floral organs was limited to floral tissue, and predominantly to senescing petals.

Journal Article↗
PubMed1989

Current-evoked transcellular K+ flux in frog retina.

1. Changes in extracellular K+ concentration (delta[K+]o) evoked by electrical current were measured with K+-selective microelectrodes (K-ISMs) in the retina of the frog eyecup. 2. In the superfusate at 20 microns above the inner limiting membrane (ILM), current-evoked delta[K+] was a function of current polarity and strength; its amplitude decreased as the K-ISM was moved higher above the ILM. Responses were similar whether measured with K-ISMs containing the Corning exchanger or a valinomycin-based liquid membrane. No current-evoked delta[Ca2+] could be detected with Ca-selective microelectrodes (Ca-ISMs). 3. Within the retina, a complex spatiotemporal profile of current-evoked delta[K+]o was observed. Strophanthidin abolished responses in the proximal retina, but had little effect on the response in the superfusate. A blocker of K+ channels (Ba2+) depressed responses in the superfusate, but not in the proximal retina. 4. Quantitative analysis of these responses indicates a transport number for K+ of 0.18 at onset of current, and that decreases over a few seconds. In contrast, a transport number of approximately 0.01 is predicted from the expected ionic concentrations within extracellular space. 5. These findings are compatible with the delta[K+] above the ILM being due to transcellular movement of K+ through Müller cells. The results suggest that K+ spatial buffering may be particularly potent in the retina. Furthermore, determinations of tissue characteristics by passage of electrical current must take into account that at least 17% of the current does not travel through extracellular space.

Action Potentials↗