Biomedical subjects

H C Wu

Publications and source records attributed to H C Wu.

At least 109 records · Page 6Linked to original sources

PubMed1991

Phosphatidylethanolamine is not essential for the N-acylation of apolipoprotein in Escherichia coli.

It has been postulated that the N-acyl fatty acid attached to the amino terminus of the major Escherichia coli lipoprotein is derived from the fatty acid at the 1-position of phosphatidylethanolamine (PtdEtn) (Jackowski, S., and Rock, C.O. (1986) J. Biol. Chem. 261, 11328-11333). To ascertain the role of PtdEtn in the conversion of apolipoprotein to the mature lipoprotein, the lipoprotein from E. coli strain AH930 (pss::kan) containing a null mutation in the phosphatidylserine synthase gene (pss) was studied. Pulse labeling with [35S]methionine for 30 s or 5 min revealed the formation of mature lipoprotein in both wild-type (W3110) and mutant (AH930) cells. [3H]Palmitate-labeled lipoproteins from both the mutant and wild-type cells were found to contain nearly identical amounts of alkali-resistant (amide-linked, 41-42%) and alkali-labile (ester-linked, 58-59%) fatty acids. Edman degradation and dansylation of the immuno-affinity-purified [35S]cysteine-labeled lipoprotein showed that the NH2 terminus of the lipoprotein in the mutant was blocked as in the wild type. In vitro assay of apolipoprotein N-acyltransferase using membranes either from the mutant or the wild-type strain as the source of both the enzyme and the acyl donor revealed that both membranes were equally active in the conversion of [35S]methionine-labeled apolipoprotein to lipoprotein. These data strongly suggest that PtdEtn is not essential for the N-acylation of apolipoprotein to form lipoprotein, and other major phospholipids such as phosphatidylglycerol and cardiolipin can serve as the donor of fatty acid in the N-acylation of apolipoprotein.

Acylation↗

PubMed1991

Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin.

We have studied the cytotoxicity of ricin in cells treated with brefeldin A (BFA), which dramatically disrupts the structure of the Golgi apparatus causing Golgi content and membrane to redistribute to the ER. BFA inhibits the cytotoxicity of ricin in Chinese hamster ovary, normal rat kidney, and Vero cells and abolishes the enhancement of ricin cytotoxicity by NH4Cl, nigericin, swainsonine, and tunicamycin or by a mutation in endosomal acidification. BFA protects cells from the cytotoxicities of modeccin and Pseudomonas toxin, but has no effect on the intoxication by diphtheria toxin. Pretreatment of BFA does not protect cells from ricin treatment in the absence of BFA. Our results suggest that ricin, modeccin, and Pseudomonas toxin share a common pathway of intracellular transport from endosomes to the Golgi region where they are released into the cytosol. In contrast, the lack of protection of Vero cells from diphtheria toxin by BFA indicates that diphtheria toxin is released from acidified endosomes without involving the Golgi region.

ADP Ribose Transferases↗

PubMed1991

Identification and subcellular localization of apolipoprotein N-acyltransferase in Escherichia coli.

Apolipoprotein N-acyltransferase, the enzyme catalyzing the conversion of apolipoprotein to mature lipoprotein, was detected by an in vitro assay using [35S]methionine-labeled apolipoprotein as the substrate. Triton X-100 solubilized the enzyme, and was required for its activity. The enzyme showed a broad pH optimum (pH 6.5-7.5). N-Acylation of apolipoprotein with ethanol-washed membranes was dependent on exogenous phospholipids, with phosphatidylethanolamine, phosphatidylglycerol and cardiolipin all showing about 10- to 20-times enhancement of the enzyme activity in the delipidated membranes. Incubation of apolipoprotein with [3H]palmitate-labeled membranes resulted in the incorporation of [3H]palmitate into lipoprotein. The enzyme was found to be enriched in the inner membrane and in the inner membrane/outer membrane mixed fractions of the E. coli cell envelope.

Acyltransferases↗

PubMed1991

Trypsin inhibitor from the seeds of Acacia confusa.

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.

Acacia↗

PubMed1991

Immunocytochemical Localization of Mandelonitrile Lyase in Mature Black Cherry (Prunus serotina Ehrh.) Seeds.

Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by alpha-methyl-d-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated lyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity.

Journal Article↗

PubMed1991

Renal angiomyolipoma: experience of 23 patients.

We reported 29 kidneys with angiomyolipoma in 23 patients (14 women and 9 men). One of these cases was associated with tuberous sclerosis. Their age ranged from 20 to 82 years (mean 48.9). Six patients had bilateral renal involvement and 7 had multiple lesions in one kidney. Synchronous renal tumors were noted in three patients. One patient was associated with renal cell carcinoma, and the other two patients with transitional cell carcinoma. Nineteen tumors in 16 patients were larger than 4 cm. Among them 93.8% (15/16 patients) were symptomatic and tumor hemorrhage was found in 57.9% (11/19 kidneys). Of these patients, 17 renal units underwent surgical intervention (including 8 total nephrectomy, 7 partial nephrectomy and 2 enucleation of tumor). Preoperative diagnosis was made in 82.4% (14/17) with combined imaging of sonogram and CT scan. One patient received embolization only. There were two mortalities: one patient with tumor rupture who received emergent nephrectomy but died of irreversible hypovolemic shock, and the other with tuberous sclerosis and pulmonary fibrosis who received embolization for spontaneous rupture of tumor but expired due to respiratory failure. Among nine patients receiving renal preserving operation, the post-operative effective renal plasma flow of the affected kidneys ranged from 96 to 184 ml/min (mean 131.7 ml/min). There was no evidence of recurrence during a mean followup of 2.6 years. Eight asymptomatic patients with 9 renal masses had incidental angiomyolipoma and did not undergo exploration. We did not note any progression of the lesions in the cases under observation for a mean followup of 2.6 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Hormonal screening in impotent patients.

Of a total 260 impotent patients entered in a hormonal study, serum testosterone, prolactin and hormonal abnormalities were detected in 30 (11.5%) patients (18 with hypotestosteronemia and 12 with hyperprolactinemia). The 18 cases of hypotestosteronemia included: 8 cases of hypergonadotropic hypogonadism due to klinefelter's syndrome (1), orchitis (2), chronic alcoholism (1), and idiopathic primary gonadal failure (4) and 10 cases etiologically unknown hypotestosteronemia. Nine of the 18 patients also displayed other organic etiologies (6 vasculogenic and 3 diabetic impotence). After replacement of testosterone propionate by intramuscular injection, the improvement in impotence was significant in 12 patients (excellent in 1, good in 11) and insignificant in 6 (poor in 3, no response in 3). The positive response rate was 89% (8/9) for sole hypotestosteronemia and 44% (4/9) for hypotestosteronemia accompanied by other organic causes of impotence. In the 12 patients with hyperprolactinemia, 4 had prolactinomas. Of these, 3 were treated by surgery and 1 with bromocriptine. Three (excellent in 2, good in 1) of the 4 showed a positive erection response afterwards, but the other patient's response was poor because of postoperative residual tumor. Two patients had drug-induced hyperprolactinemia (haloperidol and methyldopa) and for one, impotence was improved after withdrawal of haloperidol. However, the other responded poorly due to vasculogenic impotence. Six patients with hyperprolactinemia of unknown etiology (2 accompanied by vasculogenic impotence, 2 by diabetic impotence and 2 by sole hyperprolactinemia) were treated with bromocriptine and improvement was noted in 3. The positive response rate was 71.4% (5/7) for sole hyperprolactinemia and 40% (2/5) for hyperprolactinemia accompanied by other organic causes of impotence. Good results were achieved in prolactinoma, sole hyperprolactinemia and hypotestosteronemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Primary retroperitoneal sarcomas--a report of 16 cases.

From November 1982 to August 1990, 16 patients with primary retroperitoneal sarcoma were treated in our hospital. The most frequent types of tumor were liposarcoma (6), malignant fibrous histiocytoma (3). The commonest clinical findings were palpable abdominal mass (14/16). In management of these patients; nine patients underwent complete resection of the tumor; three patients underwent partial resection of the tumor and four patients underwent biopsy only. The histology of tumor appeared to have prognostic significance. Patients suffering from liposarcoma (3/5) and malignant fibrous histiocytoma (1/2) survived the longest. With a mean follow up of 25.0 months, the three year overall survival rate of complete resection group was 50 per cent. No patients with partial resection and biopsy only group survive three years and the total three-years overall survival rate was 29%. The mean survival for patients after adjuvant chemotherapy and/or radiotherapy was similar to that after a radical operation alone. The prognosis of primary retroperitoneal sarcoma is grave and complete resection of the tumor as initial treatment seems to provide the only chance of cure for these patients.

Adolescent↗

PubMed1991

Enhancement of the cytotoxicity of mistletoe lectin-1 (ML-1) by high pH or perturbation in Golgi functions.

We have studied the cytotoxicity of Mistletoe lectin-1 (ML-1), a cytotoxic protein produced by Viscum album, in CHO and V79 cells and in mutant cell lines altered in Golgi functions or in endosomal acidification. In wild-type CHO cells, cytotoxicity of ML-1 was greatly enhanced by ammonium chloride or nigericin. A CHO mutant defective in endosomal acidification (DMPR-2), which is resistant to diphtheria toxin, modeccin and Pseudomonas aeruginosa exotoxin A and hypersensitive to ricin, showed increased sensitivity to ML-1. MonR-31 and MF-1 are monensin- and compactin-resistant mutants derived from CHO and V79 cell lines, respectively, and are presumably altered in Golgi functions. The cytotoxicity of ML-1 was found to be increased in both MonR-31 and MF-1 cells as compared with their parental cells. These results indicate that the effects of chemicals or mutations altering endosomal acidification and Golgi functions on the cytotoxicity of ML-1 are similar to those on ricin cytotoxicity. Our results suggest that the cytotoxicity of ML-1 is enhanced by an increase in endosomal pH, as well as by chemicals or mutations altering the structure/functions of the Golgi regions. Like ricin, the intoxication process of ML-1 may involve the Golgi regions.

Ammonium Chloride↗

PubMed1990

Lipoproteins in bacteria.

Covalent modification of membrane proteins with lipids appears to be ubiquitous in all living cells. The major outer membrane (Braun's) lipoprotein of E. coli, the prototype of bacterial lipoproteins, is first synthesized as a precursor protein. Analysis of signal sequences of 26 distinct lipoprotein precursors has revealed a consensus sequence of lipoprotein modification/processing site of Leu-(Ala, Ser)-(Gly, Ala)-Cys at -3 to +1 positions which would represent the cleavage region of about three-fourth of all lipoprotein signal sequences in bacteria. Unmodified prolipoprotein with the putative consensus sequence undergoes sequential modification and processing reactions catalyzed by glyceryl transferase, O-acyl transferase(s), prolipoprotein signal peptidase (signal peptidase II), and N-acyl transferase to form mature lipoprotein. Like all exported proteins, the export of lipoprotein requires functional SecA, SecY, and SecD proteins. Thus all precursor proteins are exported through a common pathway accessible to both signal peptidase I and signal peptidase II. The rapidly increasing list of lipid-modified proteins in both prokaryotic as well as eukaryotic cells indicates that lipoproteins comprise a diverse group of structurally and functionally distinct proteins. They share a common structural feature which is derived from a common biosynthetic pathway.

Bacterial Proteins↗

PubMed1990

Increased cytotoxicity of ricin in a putative Golgi-defective mutant of Chinese hamster ovary cell.

We have studied the cytotoxicity of ricin in a monensin-resistant mutant (MonR-31) of Chinese hamster ovary (CHO) cell line which is presumably altered in Golgi functions/structures. The cytotoxicity of ricin was increased in MonR-31 mutant cells compared with that in its parental CHO cells. In wild-type CHO cells, the cytotoxicity of ricin was enhanced by HN4Cl, bafilomycin A1, or nigericin. The enhancement of ricin cytotoxicity by these compounds was greatly reduced in MonR-31 mutant cells. Brefeldin A (BFA), which disrupts the structure of the Golgi apparatus, inhibits the cytotoxicity of ricin in both CHO and MonR-31 cells. We have also examined the effects of glycosylation inhibitors and the removal of high mannose oligosaccharide chains in ricin on the ricin hypersensitivity in MonR-31 cells. The hypersensitivity of MonR-31 cells to ricin is apparently not due to any difference in glycosylation between CHO and MonR-31 cells or in the processing of oligosaccharides on ricin by the target cells. Nigericin at low concentration (10 nM), which has no effect on the cytotoxicity of diphtheria toxin, enhances the ricin cytotoxicity, but inhibits the modeccin cytotoxicity. Our results suggest that important step(s) in the intoxication process of CHO cells by ricin and modeccin take place in the Golgi region.

Alkaloids↗

PubMed1990

Cloning and nucleotide sequence of the Enterobacter aerogenes signal peptidase II (lsp) gene.

In Escherichia coli, prolipoprotein signal peptidase is encoded by the lsp gene, which is organized into an operon consisting of ileS, lsp, and three open reading frames, designated genes x, orf-149, and orf-316. The Enterobacter aerogenes lsp gene was cloned and expressed in E. coli. The nucleotide sequence of the Enterobacter aerogenes lsp gene and a part of its flanking sequences were determined. A high degree of homology was found between the E. coli ileS-lsp operon and the corresponding genes in Enterobacter aerogenes. Furthermore, the same five genes which constitute an operon in E. coli were found in Enterobacter aerogenes in the same order.

Amino Acid Sequence↗

PubMed1990

Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.

Amino Acid Sequence↗

PubMed1990

Metabolic evaluation of urolithiasis.

After detailed instruction, 62 patients with urolithiasis treated at Taichung Veterans General Hospital entered this program, which ran from September 1987 to November 1988. Based on Pak's classification, there were 13 cases (21.0%) of absorptive hypercalciuria, type I (AH-I); 12 cases (19.4%) of absorptive hypercalciuria, type II (AH-II); 16 cases (25.8%) of renal hypercalciuria (RH); 3 cases (4.8%) of hyperuricosuric calcium urolithiasis (HUCU); 11 cases (17.7%) of hypocitraturia (Hypocit); 3 cases (4.8%) of hyperoxluria (HO); one case (1.6%) of primary hyperparathyroidism (PHPT) and one case (1.6%) of infectious lithiasis. Two cases (3.2%) with no metabolic abnormalities were found. Hypocitraturia, HUCU, and HO can be the primary abnormal findings, but more often coexist with various forms of hypercalciuria as a second factor. If the coexistence is considered, hypocitraturia (33 cases, 53.2%) and HUCU (24 cases, 38.7%) were the most prevalent categories. Meanwhile, 24 cases (38.7%) had only one physiological derangement, 25 cases (40.3%) had two derangements, and 13 cases (21.0%) had three. This study indicates that metabolic evaluation can elucidate the physiological derangements of urolithiasis, so that further medical treatment can be administered selectively.

Adult↗

PubMed1989

Identification of the carboxyl-terminal amino acids important for the ADP-ribosylation activity of Pseudomonas exotoxin A.

The ADP-ribosylation domain of Pseudomonas exotoxin A (PE) has been identified to reside in structural domain III (residues 405-613) and a portion of domain Ib (residues 385-404) of the molecule (Hwang, J., FitzGerald, D. J., Adhya, S., and Pastan, I. (1987) Cell 48, 129-136). To further determine the carboxyl end region essential for ADP-ribosylation activity, we constructed sequential deletions at the carboxyl-terminal of PE. Our results show that a clone with a deletion of the carboxyl-terminal amino acid residues from Arg-609 to Lys-613 and replaced with Arg-Asn retained wild-type PE ADP-ribosylation activity. Deletion of the terminal amino acid residues from Ala-596 to Lys-613 and replaced with Val-Ile-Asn reduced ADP-ribosylation activity by 75%, while deletions of 36 or more amino acids from the carboxyl terminus completely lose their ADP-ribosylation activity. These modified PEs were also examined for their ability to block PE cytotoxicity. Our results shown that modified PEs which lost their ADP-ribosylation activity correspondingly lost their cytotoxicity. Furthermore, extracts containing PE fragments without ADP-ribosylation activity were able to block the cytotoxic activity of intact PE. Our results thus indicate that carboxyl-terminal amino acids in the Ser-595 region are crucial for ADP-ribosylation activity and, consequently, cytotoxicity of PE. The modified PEs which have lost their ADP-ribosylation activity may also be a route to new PE vaccines.

ADP Ribose Transferases↗

PubMed1989

Fatty acylation of yeast glycoproteins proceeds independently of N-linked glycosylation.

The relationship between protein glycosylation and fatty acylation of glycoproteins was studied in the wild-type and asparagine-linked glycosylation-deficient mutants (alg1 and alg2) of Saccharomyces cerevisiae. At the non-permissive temperature (37 degrees C), both mutant cells exhibited increased incorporation of [3H]palmitate into five polypeptides based on SDS-PAGE. In contrast, the wild-type yeast cells contained [3H]palmitate-labeled polypeptides of higher molecular weights, which were converted to the bands seen in the mutant cells upon treatment of the cell extract with endoglycosidase H prior to SDS-PAGE. In addition, labeling of the wild-type yeast cells with [3H]palmitate in the presence of tunicamycin revealed the incorporation of [3H]palmitate into the same five bands as found in the alg1 and alg2 mutants at the non-permissive temperature without tunicamycin. These results indicate that fatty acylation of glycoproteins proceeds independently of protein N-glycosylation in yeast cells.

Acylation↗

PubMed1989

Impotence evaluated by the use of prostaglandin E1.

We screened 80 patients at our hospital for the differential diagnosis of impotence using intracavernous injection of prostaglandin E1 (20 micrograms). The rate of positive response was 78.8 per cent (63 patients). Neither systemic reactions nor priapism occurred. However, a considerable incidence (23.8 per cent, 19 of 80 patients) of tolerable injection pain was encountered. The 133xenon penile washout study was conducted routinely in impotent men for hemodynamic evaluation of penile vascularity. In 80 patients a positive correlation between the response of intracavernous prostaglandin E1 injection and the result of the washout study was found (r equals 0.381, p less than 0.0002). We selected 14 subjects randomly to receive additional intravenous infusions of prostaglandin E1 (6 ampules, 120 micrograms total) for 3 days, after which another 133xenon washout study was done. The washout studies before and after intravenous prostaglandin E1 infusion were compared, and 10 patients (71.4 per cent) appeared to obtain improvement in half-time clearance and penile blood flow. However, only 3 patients noticed improvement subjectively. We suggest that prostaglandin E1 could be a desirable alternative for the diagnosis and treatment of impotence.

Adult↗

PubMed1989

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).

Adenosine Triphosphate↗