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Biomedical subjects

H C Wu

Publications and source records attributed to H C Wu.

At least 127 records · Page 7Linked to original sources
PubMed1989

Comparison of penile vascular effect induced by intracavernous injection of papaverine and prostaglandin E1.

An intracavernous injection with papaverine or prostaglandin E1 (PGE1) combined with duplex ultrasonography is an objective technique to conduct a penile blood flow study (PBFS). Three hundred patients with impotence underwent papaverine (9-60 mg) induced PBFS at the University of California School of Medicine, San Francisco (UCSF), U.S.A. and another 80 patients with impotence received PGE1-induced (20 micrograms) PBFS at Veterans General Hospital-Taichung (VGH-TC), R.O.C. Preliminary evaluation of age, duration of disease and pre-injection diameter of cavernous arteries disclosed no significant difference in these 2 series. It was interesting to find that almost all parameters of vascular response to pavaverine vs PGE1 differed significantly, such as onset of response (6.5 +/- 6.5 vs 11.7 +/- 6.4 min, slower in the PGE1 group, p less than 0.001); post-injection diameter of cavernous arteries (right: 0.73 +/- 0.20 vs 0.79 +/- 0.18 mm, p = 0.03; left: 0.74 +/- 0.20 vs 0.82 +/- 0.21 mm, p = 0.005); diametral increment of cavernous arteries (right: 0.23 +/- 0.17 vs 0.33 +/- 0.17 mm, p less than 0.001; left: 0.24 +/- 0.17 vs 0.36 +/- 0.19 mm, p less than 0.001) and peak velocity (right: 27.5 +/- 16.1 vs 42.0 +/- 20.1 cm/sec, p less than 0.001; left: 28.9 +/- 15.9 vs 39.7 +/- 17.9 cm/sec, p less than 0.001). The side effects, primarily injection pain (23.8%, 19/80) in the PGE1 group and dizziness (3.0%, 9/300) in the papaverine group, were minor in these 2 series. Prolonged erection was not encountered in either series; however, immediate treatment was performed if a papaverine-induced erection lasted over 60 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Transrectal sonography of the prostate and seminal vesicles on patients with hemospermia.

Morphological change of the seminal tract of patients with hemospermia was determined by means of transrectal sonography on 38 patients. Twenty-seven (71%) had positive sonographic findings, including 20 prostatitis and/or seminal vesiculitis, three benigh prostatic hypertrophy, three prostate calculi and one seminal vesicle stone. Simultaneous bacteriologic study revealed positive cultures on 11 patients; 9 of whom (81%) had morphologic appearance on transrectal sonography suggesting inflammatory origin. Our experience indicates that transrectal sonography is capable of surveying the histopathologic change of the prostate and seminal vesicles, and is therefore effective in studying patients with organic hemospermia.

Adult↗
PubMed1988

Enhancement of cytotoxicity of modeccin by nigericin in modeccin-resistant mutant cell lines.

We have isolated a Chinese hamster ovary cell mutant (DMPR-2) simultaneously resistant to diphtheria toxin and modeccin. In addition to the increased resistance to these two toxins used in the selection, this mutant is more resistant to Pseudomonas toxin and hypersensitive to ricin than the parental cell line. In contrast to the wild-type cells in which nigericin protects cells from modeccin, the cytotoxicity of modeccin in the DMPR-2 mutant is enhanced by nigericin. We have also studied the effects of nigericin and NH4Cl on the cytotoxicity of modeccin in a modeccin-resistant mutant of HeLa cells (ModRI). The cytotoxicity of modeccin is enhanced by nigericin in ModRI mutant cells, in contrast to the protection of modeccin cytotoxicity by nigericin in the parental HeLa cells. Our results suggest that modeccin can reach the cytosol of mammalian cells by two distinct routes; the major route requires endosomal acidification and the minor route is activated by nigericin.

Ammonium Chloride↗
PubMed1988

Synthesis and export of the outer membrane lipoprotein in Escherichia coli mutants defective in generalized protein export.

Export of the outer membrane lipoprotein in Escherichia coli was examined in conditionally lethal mutants that were defective in protein export in general, including secA, secB, secC, and secD. Lipoprotein export was affected in a secA(Ts) mutant of E. coli at the nonpermissive temperature; it was also affected in a secA(Am) mutant of E. coli at the permissive temperature, but not at the nonpermissive temperature. The export of lipoprotein occurred normally in E. coli carrying a null secB::Tn5 mutation; on the other hand, the export of an OmpF::Lpp hybrid protein, consisting of the signal sequence plus 11 amino acid residues of mature OmpF and mature lipoprotein, was affected by the secB mutation. The synthesis of lipoprotein was reduced in the secC mutant at the nonpermissive temperature, as was the case for synthesis of the maltose-binding protein, while the synthesis of OmpA was not affected. Lipoprotein export was found to be slightly affected in secD(Cs) mutants at the nonpermissive temperature. These results taken together indicate that the export of lipoprotein shares the common requirements for functional SecA and SecD proteins with other exported proteins, but does not require a functional SecB protein. SecC protein (ribosomal protein S15) is required for the optimal synthesis of lipoprotein.

Alleles↗
PubMed1988

Effect of a mutation preventing lipid modification on localization of the pCloDF13-encoded bacteriocin release protein and on release of cloacin DF13.

The pCloDF13-encoded bacteriocin release protein (BRP; Mr 2,871) is essential for the translocation of cloacin DF13 across the cell envelope of producing Escherichia coli cells. Overproduction of this BRP provokes lysis (quasilysis) of cells. Construction and analysis of a hybrid BRP-beta-lactamase protein (BRP-Bla) demonstrated that the BRP contains a lipid modified cysteine residue at its amino terminus and is mainly located in the outer membrane. The significance of lipid modification for the localization and functioning of the BRP was investigated. Site-directed mutagenesis was used to substitute the cysteine residue for a glycine residue in the lipobox of the BRP and the BRP-Bla protein. The mutated BRP was unable to bring about the release of cloacin DF13 and could not provide the lysis (quasilysis) of host cells. However, the mutated BRP strongly inhibited the colony-forming ability of the cells, indicating that induction of the mutated protein still affected cell viability. In contrast to the wild-type BRP-Bla protein, the mutated BRP-Bla protein was mainly located in the cytoplasmic membrane, indicating that the mutation prevented the proper localization of the protein. The results indicated that lipid modification of the BRP is required for its localization and release of cloacin DF13, but not for its lethality to host cells.

Bacterial Proteins↗
PubMed1987

Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells.

Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.

Animals↗
PubMed1987

Identification of the genes in the Escherichia coli ileS-lsp operon. Analysis of multiple polycistronic mRNAs made in vivo.

The genes encoding isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) of Escherichia coli were previously shown to be co-transcribed (Miller, K. W., and Wu, H. C. (1987) J. Biol. Chem. 262, 389-393). However, the boundaries of this transcriptional unit have not been established. In this regard, DNA sequence determination has shown that ileS and lsp are closely flanked by four open reading frames, i.e. x-ileS-lsp-orf149-orf316-orf304. To define the boundaries of the operon, we applied Northern blotting hybridization and mRNA 5'-end mapping to analyze mRNA from a wild-type strain (SM31) and a mutant strain (SM31-2B4) that exhibits an increased expression of prolipoprotein signal peptidase. Four ileS-lsp co-transcripts were detected in RNA from the strain SM31. In addition to these four mRNAs, two new, highly abundant co-transcripts were also detected in RNA from the mutant. Based upon the determination of the 5'-ends of the mRNAs and analysis of their coding sequences, we conclude that the six mRNAs actually are comprised of three pairs of related mRNAs. The two mRNAs in a given pair have the same 5'-termini (all located upstream of or within gene x), but vary with respect to the identity of their 3'-terminal coding sequence (lsp or orf316). In conclusion, the ileS-lsp operon contains five genes, x-ileS-lsp-orf149-orf316, whose transcription probably is dependent upon promoter(s) located upstream of or within gene x. The next gene downstream, orf304, apparently does not reside in the operon.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Cotranscription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes. Fine-structure mapping of the lsp internal promoter.

We have applied the technique of Northern blotting hybridization analysis to examine the transcription of the Escherichia coli isoleucyl-tRNA synthetase (ileS) and prolipoprotein signal peptidase (lsp) genes. RNA samples from the wild-type strain CS412 and CS412 containing the plasmids pMT521 and pSYC890 were examined with ileS- and lsp-specific 32P-labeled probes. pMT521 is a pBR322 derivative into which a chromosomal DNA fragment containing the ileS and lsp genes has been cloned downstream of the pBR322 tet promoter. pSYC890 is a derivative of pMT521 which lacks the tet promoter, but nevertheless is active in lsp mRNA transcription due to the presence of a weak lsp internal promoter located in the distal portion of the upstream ileS DNA, RNA from CS412(pMT521) exhibited a high level of a 5500-nucleotide ileS-lsp cotranscript that hybridized to both probes. RNA from CS412(pSYC890) contained a high level of low molecular weight lsp-specific transcripts from the lsp internal promoter. However, only high molecular weight (5000-6500 nucleotides) cotranscripts were detected in RNA from CS412 by Northern blotting analysis. A trace level of lsp-specific mRNA was detected in CS412 by S1 nuclease mapping. The 5'-end of this transcript was mapped using RNA from CS412(pSYC890). The mRNA begins 192/193 base pairs upstream of the lsp translation initiation codon at a site within ileS DNA that codes for the aspartate residue located 61 amino acids from the carboxyl-terminal of ILES. In conclusion, most of the transcription of lsp in wild-type E. coli originates from upstream of ileS and yields ileS-lsp cotranscripts.

Amino Acyl-tRNA Synthetases↗
PubMed1987

Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase.

A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells.

Bacterial Proteins↗
PubMed1987

Isolation and characterization of a new globomycin-resistant dnaE mutant of Escherichia coli.

We isolated a globomycin-resistant, temperature-sensitive mutant of Escherichia coli K-12 strain AB1157. The mutation mapped in dnaE, the structural gene for the alpha-subunit of DNA polymerase III. The in vivo processing of lipid-modified prolipoprotein was more resistant to globomycin in the mutant strain 307 than in its parent. The prolipoprotein signal peptidase activity was also increased twofold in the mutant, and there was a threefold increase in the activity of isoleucyl-tRNA synthetase. The results suggest that a mutation in dnaE may affect the expression of the ileS-lsp operon in E. coli. In addition, strain 307 showed a reduced level of streptomycin resistance compared with its parental strain AB1157 (rpsL31). Strain 307 was killed by streptomycin at a concentration of 200 micrograms/ml, which did not affect the rate of bulk protein synthesis in this mutant. A second mutation which was involved in the reduced streptomycin resistance in strain 307 was identified and found to be closely linked to or within the rpsD (ramA, ribosomal ambiguity) gene. Both dnaE and rpsD were required for the reduced streptomycin resistance in strain 307.

Anti-Bacterial Agents↗
PubMed1986

Processing of Bacillus licheniformis penicillinases lacking a lipoprotein modification site in Escherichia coli.

We have previously shown that the penP Ser-27 prepenicillinase is processed into two forms, Ser-35-penicillinase and Asn-29 penicillinase. Two new penicillinase mutants, penP Ser-27 Pro-28 and penP Ser-27,23' (Pro-Asp)24', were derived from the penP Ser-27 mutant by oligonucleotide-directed site-specific mutagenesis. The penP Ser-27 Pro-28 mutant prepenicillinase was also processed into two forms, Ser-35-penicillinase and Gly-26-penicillinase. On the contrary, the penP Ser-27,23' (Pro-Asp)24' mutant prepenicillinase is unprocessed.

Bacillus↗
PubMed1985

Identification of prolipoprotein signal peptidase and genomic organization of the lsp gene in Escherichia coli.

The product of the lsp gene of Escherichia coli, i.e. the prolipoprotein signal peptidase, was identified by both in vivo pulse labeling experiments using a high expression lambda PL promoter vector and by an in vitro transcription/translation coupled system. The molecular weight of prolipoprotein signal peptidase was estimated to be approximately 18,000 by its mobility on polyacrylamide gel electrophoresis and was found to be the same as that of SPase II purified from the wild-type cells. Analysis of SPase II activities in strains containing various subclones, deletion derivatives generated from plasmid pMT521, and analysis of protein products in a strain harboring an ileS-lsp-fused gene indicated that ileS and lsp genes are transcribed on the same mRNA. This was further supported by the observation that Tn5 insertions in the ileS gene resulted in a reduced expression of the lsp gene. In addition to an upstream promoter shared by the ileS and lsp genes, these analyses also revealed the presence of an internal promoter for the lsp gene within the coding region of the ileS gene.

Bacterial Outer Membrane Proteins↗
PubMed1985

Characterization of the ileS-lsp operon in Escherichia coli. Identification of an open reading frame upstream of the ileS gene and potential promoter(s) for the ileS-lsp operon.

The preceding paper presents evidence for the co-transcriptional expression of the ileS and lsp genes in Escherichia coli. To identify the promoter for the ileS-lsp operon, we have determined the nucleotide sequence of an 1.8-kilobase DNA fragment between the rpsT and IleS genes. The sequence data have revealed an open reading frame, designated gene X, which encodes a polypeptide with 312 amino acid residues. Both in vivo and in vitro expressions of the x gene result in the synthesis of a soluble protein with an apparent Mr of 35,000. The x gene is transcribed in the same direction as that of the ileS-lsp operon and opposite to that of the upstream adjacent rpsT gene. No transcription termination sequence can be discerned in the intercistronic region between the x and ileS genes. DNase I footprinting experiment revealed a RNA polymerase binding site at 170-151 base pairs upstream of the x gene.

Bacterial Proteins↗
PubMed1985

Modification and processing of internalized signal sequences of prolipoprotein in Escherichia coli and in Bacillus subtilis.

We have cloned the Escherichia coli lipoprotein structural gene (lpp) into a shuttle vector and studied its expression in both E. coli and in Bacillus subtilis. Using in vitro gene fusion techniques, the lpp gene was placed under the control of the promoter for the erythromycin-resistance (ery) gene. This fusion gene directed the synthesis of Braun's prolipoprotein which can be subsequently processed into the mature lipoprotein. In addition to the prolipoprotein, two ery-lpp hybrid proteins containing a 45- and a 22-amino acid extension preceding the NH2 terminus of prolipoprotein, respectively, are also synthesized in E. coli. The synthesis of these three proteins appears to involve the utilization of three distinct translation initiation sites. In B. subtilis, only two proteins are synthesized, the hybrid protein with a 45-amino acid extension and the prolipoprotein. In both E. coli and B. subtilis, the precursor forms of the hybrid proteins are lipid-modified, and they are processed to mature lipoprotein in vivo. These results indicate that internalized signal sequence containing the prolipoprotein modification and processing site (Leu-Ala-Glys-Cys) can function normally and permit the modification of hybrid proteins to lipid-modified precursors which can be subsequently processed by the globomycin-sensitive prolipoprotein signal peptidase.

Amino Acid Sequence↗
PubMed1985

Enhancement of ricin cytotoxicity in Chinese hamster ovary cells by depletion of intracellular K+: evidence for an Na+/H+ exchange system in Chinese hamster ovary cells.

Depletion of intracellular K+ has been reported to result in an arrest of the formation of coated pits in human fibroblasts (Larkin, J.M., M.S. Brown, J.L. Goldstein, and R.G.W. Anderson, 1983, Cell, 33:273-285). We have studied the effects of K+ depletion on the cytotoxicities of ricin, Pseudomonas exotoxin A, and diphtheria toxin in Chinese hamster ovary (CHO) cells. The cytotoxicities of ricin and Pseudomonas toxin were enhanced in K+-depleted CHO cells whereas the cytotoxicity of diphtheria toxin was reduced by K+ depletion. The effects of NH4Cl on the cytotoxicities of ricin, Pseudomonas toxin, and diphtheria toxin were found to be similar to those of K+ depletion, and there were no additive or synergistic effects on ricin cytotoxicity by NH4Cl in K+-depleted medium. The enhancement of ricin cytotoxicity by K+ depletion could be completely reversed by the addition of K+, Rb+, and partially by the addition of Cs+, before the ricin treatment, whereas Li+ was ineffective. These protective effects of K+ or Rb+ requires a functional Na+/K+ ATPase. CHO cells grown in K+-depleted media were found to contain 6.3-fold increase in intracellular Na+ level, concomitant with a 10-fold reduction in intracellular K+ level. The enhanced cytotoxicity of ricin in K+-free medium and the increased uptake of Na+ could be abolished by amiloride or amiloride analogues, which are known to be potent inhibitors of the Na+/H+ antiport system. Our results suggest that a depletion of intracellular K+ results in an influx of Na+, which is accompanied by the extrusion of H+. Consequently, there is an alkalinization of the cytosol and the ricin-containing endosomes. As a result, ricin is more efficiently released from the endosomes in-K+-depleted cells. Results from the studies of the binding, internalization, and degradation of 125I-ricin, and the kinetics of inhibition of protein synthesis by ricin in K+-depleted cells are consistent with this working hypothesis.

ADP Ribose Transferases↗