Biomedical subjects

H Huang

Publications and source records attributed to H Huang.

At least 19 recordsLinked to original sources

PubMed1997

Reduced fecundity in insulin-like growth factor-binding protein-1 transgenic mice.

Transgenic mice that overexpress insulin-like growth factor-binding protein-1 (IGFBP-1) demonstrate a reduced litter size compared to nontransgenic, wild-type mice derived from the same genetic background. To determine the mechanism underlying this phenomenon, we examined the number of ovulatory follicles per cycle in naturally mated transgenic and wild-type mice by counting the corpora lutea and the blastocysts harvested by uterine flushing. In addition, we investigated the effects of insulin-like growth factor-I (IGF-I) on blastocyst DNA synthesis and examined the effects of a transgenic maternal environment on fetal outcome by cross transfer of blastocysts. Significantly fewer corpora lutea were observed in ovaries from transgenic vs. wild-type mice (7.6 +/- 1.8 vs. 11.8 +/- 1.5), and fewer blastocysts were harvested from transgenic mice compared to wild-type mice (7.6 +/- 2.3 vs. 10.1 +/- 2.0). DNA content and basal DNA synthesis were similar in blastocysts from nontransgenic and transgenic mice. However, unlike wild-type blastocysts, transgenic blastocysts did not respond to IGF-I with an increase in DNA synthesis. To determine the effects of maternal environment on fetal outcome, a mixture of equal numbers of transgenic and nontransgenic blastocysts was transferred into foster mothers. The ratio of transgenic to nontransgenic pups was not significantly different from the theoretically predicted value of 1. However, the litter size was significantly reduced in wild-type compared to transgenic foster mothers. These data suggest that the reduced fecundity is due to reduced ovulation and blastocyst number. Furthermore, expression of the transgene in neither the blastocyst nor the maternal tissues had any significant negative effect on implantation or fetal wastage.

Animals↗

PubMed1997

Expression of insulin-like growth factor binding proteins (IGFBPs) in IGFBP-1 transgenic mice.

The hepatic and renal expressions of the insulin-like growth factor binding proteins (IGFBPs) were examined in transgenic (Tg) mice which overexpress a rat IGFBP-1 transgene driven by the phosphoglycerate kinase-1 promoter. There were no significant difference in the abundance of serum IGFBPs in Tg and wild-type (Wt) mice. Although total hepatic IGFBP-1 mRNA mouse and transgene-derived) levels were similar in Tg mice to the levels of mouse IGFBP-1 mRNA in Wt mice on day 1 of life, in Tg mice only approximately 30% of the IGFBP-1 mRNA was derived from transcription of the mouse gene. An age-related decline in hepatic IGFBP-1 mRNA levels was apparent in both Tg and Wt mice. Food deprivation resulted in increased levels of mouse IGFBP-1 mRNA but the total IGFBP-1 mRNA levels were not significantly different in Tg and Wt mice. In the kidney, unlike the liver, IGFBP-1 mRNA levels in Tg mice were markedly elevated compared with Wt mice and no significant decline was seen with age. Northern blots of hepatic and renal RNA demonstrated similar levels of IGFBP-3, -4, -5 and -6 mRNAs in Tg and Wt mice. From these data we can conclude that in the liver expression of the transgene leads to a coordinated reduction in mouse IGFBP-1 mRNA levels.

Aging↗

PubMed1996

Amino acid-derived phthalimide and saccharin derivatives as inhibitors of human leukocyte elastase, cathepsin G, and proteinase 3.

Amino acid-derived phthalimide and saccharin derivatives have been investigated for their inhibitory activity toward the serine proteinases human leukocyte elastase, cathepsin G, and proteinase 3. The saccharin derivatives were found to be effective time-dependent inhibitors of elastase and proteinase 3 (kobs/[I] values ranged between 180 and 3620 M-1 S-1) and showed weak or no inhibition toward cathepsin G. The corresponding phthalimide derivatives were found to be inactive.

Amino Acid Sequence↗

PubMed1996

Structure and critical residues at the active site of spermidine/spermine-N1-acetyltransferase.

Spermidine/spermine-N1-acetyltransferase (SSAT) is a key enzyme in the degradation of polyamines. Alanine-scanning mutagenesis of all eight arginine residues was used to investigate the arginine residues involved in acetyl-CoA binding. The results indicate that Arg101, Arg142 and Arg143 are important for such binding. The apparent Km values for acetyl-CoA were significantly increased when any one of these residues was replaced by an alanine residue. These mutations also abolished the ability of acetyl-CoA to protect the protein from digestion by trypsin. Co-expression of the inactive R101A (Arg101 --> Ala) mutant and an E152K (Glu152 --> Lys) mutant, previously known to inactivate SSAT, led to restoration of activity, showing that the active enzyme is a dimer with residues contributed by both subunits. The double mutant R101A/E152K acted as a dominant negative when co-expressed with the wild-type SSAT. Transfection of COS-7 cells with a plasmid producing this mutant greatly attenuated the increase in SSAT activity brought about by N1, N12-bis(ethyl)spermine. These results indicate that the double mutant R101A/E152K-SSAT protein can be used to evaluate the importance of SSAT activity in response to exogenous polyamines or polyamine analogues.

Acetyl Coenzyme A↗

PubMed1996

Interactions between ifenprodil and the NR2B subunit of the N-methyl-D-aspartate receptor.

Ifenprodil is an atypical noncompetitive modulator of the N-methyl-D-aspartate (NMDA) receptor (NR) which demonstrates a 140-fold preference for NR2B over NR2A subunits, although the molecular basis for this subunit specificity is unknown. We have made chimeric receptors by fusing the murine forms of NR2A (epsilon 1) and NR2B (epsilon 2) to localize the high affinity determinants of ifenprodil inhibition on the 2B subunit. Binding experiments with 125I-MK-801 implicated the region between amino acids 198 and 356 of NR2B for high affinity ifenprodil interaction. Site-directed mutants at Arg-337 showed that this residue is absolutely required for high affinity ifenprodil inhibition. Polyamines also modulate the NMDA receptor with a preference for NR2B subunits, and the pharmacology of these agents overlaps with ifenprodil. Although the determinants of the polyamine enhancement of iodo-MK-801 binding also localize to the NH2 terminus of NR2B, the point mutants at Arg-337 form receptors that are polyamine-stimulated at wild type levels. In addition, polyamine stimulation depends on the expression of NR1 splice variants, whereas high affinity ifenprodil inhibition is independent of NR1 isoform expression. These studies provide evidence that ifenprodil and polyamines interact at discrete sites on the NR2B subunit.

Alternative Splicing↗

PubMed1996

Arsenite induces apoptosis in Chinese hamster ovary cells by generation of reactive oxygen species.

Arsenic, a human carcinogen, possesses a serious environmental threat but the mechanism of its toxicity remains unclear. Knowledge of how arsenic induces cell death and how cells escape the death path may help to understand arsenic carcinogenesis. We have investigated the nature of sodium arsenite-induced cell death in Chinese hamster ovary K1 cells. Following phosphate-citric acid buffer extraction, apoptotic cells with lower DNA content than the G1 cells were detected by flow cytometry. Immediately after 4 h of 40 microM arsenite treatment, no appreciable fraction of cells with sub-G1 DNA content was detected; however, the sub-G1 cell fraction increased with postarsenite incubation time, and detectable increase started at 8 h of incubation, whereas the intracellular peroxide level as measured by the fluorescent intensity of 2',7'-dichlorofluorescein increased immediately following a 4-h arsenite treatment. Simultaneous treatment with arsenite plus antioxidant (N-acetyl-cysteine, Trolox, and Tempo); copper ion chelator (neocuproine); protein kinase inhibitor (H-7) or protein synthesis inhibitor (cycloheximide) reduced the fraction of sub-G1 cell and internucleosomal DNA degradation. Trolox, neocuproine, or cycloheximide given after arsenite treatment also effectively reduced apoptosis. These results lead to a working hypothesis that arsenite-induced apoptosis in CHO-K1 cells is triggered by the generation of hydrogen peroxide, followed by a copper-mediated Fenton reaction that catalyzes the production of hydroxyl radicals, which selectively activates protein kinase through de novo synthesis of macromolecules.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Hematopoietic stem cells in the mouse embryonic yolk sac.

The yolk sac is the first site of hematopoiesis during mammalian development. The yolk sac is also the first site of blood vessel development. Development of the blood islands in the yolk sac is an integrated process in which these two developmental events, hematopoiesis and vasculogenesis, proceed in concert. This review focuses on mouse yolk sac hematopoietic stem cells (YS-HSC), describing their differentiation in vitro and in vivo. YS-HSC go through a progressive series of changes prior to the initiation of lineage-specific differentiation. Experiments tracing their origins from postulated hemangioblasts, and the subsequent interaction between these stem cells and yolk sac endothelial cells are described. Differences between the extraembryonic YS-HSC and HSC found later within the embryo, perinatally or in adults, are described. YS-HSC have greater reproductive capability than HSC obtained from fetal liver, umbilical cord blood or adult bone marrow; they do not yet express major histocompatibility complex-associated antigens and they are able to reconstitute adult immunocompromised animals even when introduced in small numbers (< 100 cells/mouse). With recent results demonstrating the feasibility of expanding YS-HSC in vitro as well as of introducing new genes into these cells by transfection, the YS-HSC shows promise both as a means of achieving long-term restitution of hematopoiesis across histocompatibility barriers and as a self-renewing vehicle for gene transfer.

Animals↗

PubMed1996

Organization of the mouse cardiac natriuretic peptide locus encoding BNP and ANP.

The genes encoding the mouse atrial natriuretic peptide and B-type natriuretic peptide were previously shown to be physically linked on mouse chromosome 4 (Steinhelper ME, 1993, Structure, expression, and genomic mapping of the mouse natriuretic peptide type-B gene. Circ Res 72: 984-992). In the present study the spatial relationship and orientation of the mouse atrial natriuretic peptide and B-type natriuretic peptide transcription units were identified and a physical map of the mouse cardiac natriuretic peptide locus was obtained. To this end, genomic clones encoding atrial natriuretic peptide and B-type natriuretic peptide were isolated from a mouse genomic library in bacteriophage P1. Three independent clones encoding atrial natriuretic peptide were isolated and two of these also encode B-type natriuretic peptide. Both transcripts were shown to arise from the same DNA strand, with B-type natriuretic peptide encoded approximately 15 kb 5'-of atrial natriuretic peptide based on field inversion gel electrophoresis of fragments amplified with specific oligonucleotides. This finding was confirmed by isolation of subclones comprising the entire locus and by blot hybridization analysis of mouse genomic DNA. The results show that the genes encoding the two natriuretic peptides expressed predominantly in mammalian cardiac myocytes are organized in tandem on mouse chromosome 4. This information provides a physical framework for investigating mechanisms that regulate transcription of the cardiac natriuretic peptide locus.

Animals↗

PubMed1996

A genetic linkage map of the rat derived from recombinant inbred strains.

We have constructed a genetic linkage map in the rat by analyzing the strain distribution patterns of 500 genetic markers in a large set of recombinant inbred strains derived from the spontaneously hypertensive rat and the Brown-Norway rat (HXB and BXH recombinant inbred strains). 454 of the markers could be assigned to specific chromosomes, and the amount of genome covered by the mapped markers was estimated to be 1151 centimorgans. By including a variety of morphologic, biochemical, immunogenetic, and molecular markers, the current map integrates and extends existing linkage data and should facilitate rat gene mapping and genetic studies of hypertension and other complex phenotypes of interest in the HXB and BXH recombinant inbred strains.

Animals↗

PubMed1996

Synthesis and evaluation of a polyamine phosphinate and phosphonamidate as transition-state analogue inhibitors of spermidine/spermine-N1-acetyltransferase.

Polyamine analogues such as bis(ethyl)norspermine and N1-ethyl-N11-[(cyclopropyl)methyl]-4,8-diazaundecane (CPENSpm) act as inhibitors of the enzyme spermidine/spermine-N1-acetyltransferase (SSAT) in vitro and possess impressive antitumor activity against a number of cell lines. However, the propensity of these compounds to superinduce SSAT in intact cells limits their usefulness in studies aimed at elucidating the role of SSAT in cellular metabolism. The recently synthesized alkylpolyamine analogue N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm, 3) is also an effective inhibitor of SSAT and has potent antitumor activity, but does not appear to superinduce SSAT. These findings suggest that it is possible to synthesize polyamine analogues that can be used for selective inhibition of the enzyme in cellular metabolic studies. Along these lines, the phosphate-based transition state analogues 4 and 5 were synthesized and evaluated as inhibitors of isolated SSAT. Phosphonamidate 4 was rapidly hydrolyzed under the assay conditions, and thus did not inhibit the enzyme. However, the phosphinate analogue 5 was an effective inhibitor of purified human SSAT, with a Ki value of 250 microM. The inhibitory activity of 5 was also compared with that of CHENSpm (IC50 = 13 microM), as well as a series of bis-substituted alkylpolyamine analogues. The unsymmetrically substituted polyamine analogue CHENSpm (3) and the phosphinate transition state analogue 5 represent the first functional, nonsuperinducing inhibitors of human SSAT.

Acetyltransferases↗

PubMed1996

Abrogation of P53 function by transfection of HPV16 E6 gene does not enhance resistance of human tumour cells to ionizing radiation.

Suppression of wild-type p53 expression has been shown to enhance the radiation resistance of human diploid fibroblasts, but results concerning the role of p53 expression in the sensitivity of human tumour cells have been conflicting. In order to address this question, we transfected four human tumour cell lines with the human papilloma virus 16 E6 gene and compared the radiosensitivity of subclones expressing E6 with that of subclones transfected with the neo gene alone. E6 binds to wild-type p53 promoting its degradation and abrogating its function. Two of these cell lines, one derived from a squamous cell carcinoma and the other an osteogenic sarcoma, expressed wild-type p53. The other two cell lines were of similar origins and histologies but expressed mutant or no p53 (null). Insertion of E6 into the cell was accomplished by two techniques: (1) to-transfection of plasmid vectors containing neo and E6; (2) infection with a retroviral vector containing neo and E6. Multiple transfected subclones were examined for each cell line. Transfection with E6 and abrogation of p53 function had no significant influence on the radiosensitivity of any of the cell lines tested. In particular, there was no evidence that loss of wild-type p53 function increased the resistance of these human tumour cell lines to ionizing radiation.

Base Sequence↗

PubMed1996

Fidelity and predominant mutations produced by deep vent wild-type and exonuclease-deficient DNA polymerases during in vitro DNA amplification.

Denaturing gradient gel electrophoresis (DGGE) was used to examine error rates and mutations induced by native (wt) and exonuclease-deficient (exo-) Deep Vent DNA polymerases during DNA amplification by polymerase chain reaction (PCR), in the presence or absence of the T4 bacteriophage gene 32 protein (gp32).gp32 was found to decrease the error rate of the wt, but not that of the exo-, Deep Vent. The average errors per base duplication for the native form were 8.0 x 10(-5) and 6.0 x 10(-5) in the absence and presence of gp32, respectively. For the exo- form, the error rates were 2.0 x 10(-4) and 2.2 x 10(-4) errors per base duplication in the absence and presence of gp32, respectively. Examination of mutations produced by native Deep Vent showed that A/T to G/C transition predominated, consistent with the results of our earlier studies with DNA polymerases derived from other thermophilic bacteria. These results indicate that PCR with high fidelity can be achieved by using wt Deep Vent in combination with gp32.

Base Sequence↗

PubMed1996

Lowering extracellular calcium content protects cells from arsenite-induced killing and micronuclei formation.

The present study demonstrated that calcium ions were accumulated in nuclei of Chinese hamster ovary (CHO)-K1 cells after arsenite treatment. This process was enhanced by verapamil (a calcium channel blocker). Verapamil also significantly increased the cytotoxic effects of arsenite. In contrast, ethylene glycol-bis[beta-aminoethylether] N,N, N1,N1,-tetraacetic acid (EGTA, a calcium-specific chelator), or calcium-free conditions significantly reduced the cytotoxicity or arsenite. Similarly, the strategy of lowering extracellular calcium concentration by modifying the medium or lowering intracellular calcium concentration by administration of the intracellular calcium chelator quin 2, protected cells from arsenite-induced micronuclei formation. These data indicate that the disturbances in intracellular calcium homeostasis maybe involved in arsenite-induced cytotoxicity and micronuclei formation.

Animals↗

PubMed1996

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.

Amino Acid Sequence↗

PubMed1996

Functional domains of the floral regulator AGAMOUS: characterization of the DNA binding domain and analysis of dominant negative mutations.

The Arabidopsis MADS box gene AGAMOUS (AG) controls reproductive organ identity and floral meristem determinacy. The AG protein binds in vitro to DNA sequences similar to the targets of known MADS domain transcription factors. Whereas most plant MADS domain proteins begin with the MADS domain, AG and its orthologs contain a region N-terminal to the MADS domain. All plant MADS domain proteins share another region with moderate sequence similarity called the K domain. Neither the region (I region) that lies between the MADS and K domains nor the C-terminal region is conserved. We show here that the AG MADS domain and the I region are necessary and sufficient for DNA binding in vitro and that AG binds to DNA as a dimer. To investigate the in vivo function of the regions of AG not required for in vitro DNA binding, we introduced several AG constructs into wild-type plants and characterized their floral phenotypes. We show that transgenic Arabidopsis plants with a 35S-AG construct encoding an AG protein lacking the N-terminal region produced apetala 2 (ap2)-like flowers similar to those ectopically expressing AG proteins retaining the N-terminal region. This result suggests that the N-terminal region is not required to produce the ap2-like phenotype. In addition, transformants with a 35S-AG construct encoding an AG protein lacking the C-terminal region produced ag-like flowers, indicating that this truncated AG protein inhibits normal AG function. Finally, transformants with a 35S-AG construct encoding an AG protein lacking both K and C regions produced flowers with more stamens and carpels. The phenotypes of the AG transformants demonstrate that both the K domain and the C-terminal region have important and distinct in vivo functions. We discuss possible mechanisms through which AG may regulate downstream genes.

Arabidopsis↗

PubMed1996

Protection of asparagine and glutamine during N alpha-Bpoc-based solid-phase peptide synthesis.

In this paper we describe the synthesis and properties of Bpoc-Asn(Trt)-OH, Bpoc-Asn(Trt)-OPfp, Bpoc-Gln(Trt)-OH and Bpoc-Gln(Trt)-OPfp. These derivatives are highly soluble in CH2Cl2 and can be coupled efficiently in solid-phase peptide synthesis. The peptides, acetyl-Ala-Phe-Asn(Trt)-Gly-Leu-Ala-O-Dbf-SH and Boc-Cys(Acm)-Ala-Phe-Gln(Trt)-Gly-Leu-Ala-O-Dbf-SH (where O-Dbf-SH is the peptide ester of 4-mercapto-6-hydroxydibenzofuran) were synthesized by stepwise solid-phase peptide synthesis using N alpha-Bpoc amino acids. We have observed that less than 0.1% of the trityl group is removed from the carboxamide of Gln and Asn during a standard 15 min N alpha-Bpoc deprotection in 0.5% TFA in CH2Cl2.

Asparagine↗

PubMed1996

The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa.

Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem across the outer membrane. In this study, we examined the effects of deletions in six of the proposed eight surface loops of OprD on the in vivo and in vitro functions of this protein. Native OprD formed very small channels in planar lipid bilayers, with an average single-channel conductance in 1.0 M KCl of 20 pS. When large numbers of OprD channels were incorporated into lipid bilayer membranes, addition of increasing concentrations of imipenem to the bathing solutions resulted in a progressive blocking of the membrane conductance of KCl, indicating the presence of a specific binding site(s) for imipenem in the OprD channel. From these experiments, the concentration of imipenem value of resulting in 50% inhibition of the initial conductance was calculated as approximately 0.6 microM. In contrast, no decrease in channel conductance was observed for the OprDdeltaL2 channel upon addition of up to 2.4 microM imipenem, confirming that external loop 2 was involved in imipenem binding. Deletion of four to eight amino acids from loops 1 and 6 had no effect on antibiotic susceptibility, whereas deletion of eight amino acids from loops 5, 7, and 8 resulted in supersusceptibility to beta-lactams, quinolones, chloramphenicol, and tetracycline. Planar lipid bilayer analysis indicated that the OprDdeltaL5 channel had a 33-fold increase in single-channel conductance in 1 M KCl but had retained its imipenem binding site. The disposition of these loop regions in the interior of the OprD channel is discussed.

Bacterial Outer Membrane Proteins↗

PubMed1996

Isolation, mapping, and regulated expression of the gene encoding mouse C-type natriuretic peptide.

Genomic sequences encoding mouse C-type natriuretic peptide (CNP) were isolated from bacteriophage libraries and characterized by restriction enzyme and sequence analysis. The mouse CNP gene (Nppc) comprised at least two exons and one intron and included several cis-regulatory elements in the 5'-flanking sequence. The deduced amino acid sequence of mouse CNP-22 was identical to other mammalian CNPs. Analysis of allele distributions in interspecific back-cross and recombinant inbred strains assigned Nppc to chromosome 1. CNP transcripts were detected by ribonuclease protection analysis in brain, ovary, and uterus, with lower levels in testes and epididymus. Uterine CNP transcripts and protein were low in sexually immature mice and adults at estrus and increased at proestrus, but similar variations in ovarian CNP expression were not statistically significant. Atrial natriuretic peptide and B-type natriuretic peptide transcripts were not detected in mouse ovary or uterus. Thus CNP gene expression is regulated by tissue-specific and inducible mechanisms in female reproductive organs. Correlations between CNP expression and uterine fluid content suggest that CNP may regulate uterine fluid balance in mice and other mammals.

Alleles↗