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Biomedical subjects

Y N Chen

Publications and source records attributed to Y N Chen.

At least 55 records · Page 3Linked to original sources
PubMed1991

Peptide inhibitors and the active site(s) of angiotensin converting enzyme.

Angiotensin converting enzyme (ACE) is a central participant in blood pressure regulation and is strategically located within the pulmonary vasculature in order to carry out this function. It is also present in kidney, brain and a variety of other tissues where its function is unknown. The molecular weight of ACE from these sources is approximately 185,000. A smaller form of the enzyme, Mr approximately 100,000, is found in mature testis; its function is also unknown. The lung form of human ACE contains 1277 amino acids and consists of two homologous repeated domains, each of which appears to have a potential catalytic site. The testis form of human ACE contains 701 amino acids and has only a single domain which is largely identical to the carboxy-terminal half of the lung enzyme. This raises important questions such as, why does lung ACE possess two possible active sites, do each of the two bind zinc, and are they both catalytically active? To answer these questions, we have examined the binding of potent peptide inhibitors to ACE, redetermined the zinc stoichiometry and chemically modified both lung and testicular ACE with fluorodinitrobenzene (FDNB). Peptide inhibitors bind with essentially a 1:1 stoichiometry, indicative of a single active site. The zinc content of lung ACE is 1.4-1.8 g-at/mol. For testicular ACE it is 0.8-1.1 g-at/mol. FDNB modifies a single tyrosine, and to a much lesser extent a lysine, with concomitant loss of all catalytic activity. Sequence analysis identifies the specific residues modified and indicates that they occur only in the carboxy terminal half of lung ACE.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

O6-methylguanine-DNA methyltransferase and human cancer chemotherapy.

Two kinds of human tumor cell strains having different activity of O6-methylguanine-DNA methyltransferase (O6-MT) were transplanted into nude mice. Then the mice were injected intraperitoneally with bifunctional alkylating agent 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU). The tumors with low O6-MT activity were quickly suppressed or cured. The result suggests that some tumors, if provisionally determined with low O6-MT activity, might be efficiently cured by treatment with ACNU. This probably opens a new way for human cancer chemotherapy.

Animals↗
PubMed1991

Filariasis and its control in Fujian, China.

Epidemiological survey of filariasis in Fujian Province, China showed that malayan filariasis, transmitted by Anopheles lesteri anthropophagus was mainly distributed in the northwest part and bancroftian filariasis with Culex quinquefasciatus as vector, in middle and south coastal regions. Both species of filariae showed typical nocturnal periodicity. Involvement of the extremities was not uncommon in malayan filariasis. In contrast, hydrocele was often present in bancroftian filariasis, in which limb impairment did not appear so frequently as in the former. Hetrazan treatment was administered to the microfilaremia cases identified during blood examination surveys, which were integrated with indoor residual spraying of insecticides in endemic areas of malayan filariasis when the vector mosquito was discovered and with mass treatment with hetrazan medicated salt in endemic areas of bancroftian filariasis. At the same time the habitation condition was improved. These factors facilitated the decrease in incidence. As a result malayan and bancroftian filariasis were proclaimed to have reached the criterion of basic elimination in 1985 and 1987 respectively. Surveillance was pursued thereafter and no signs of resurgence appeared.

Animals↗
PubMed1990

Identification of essential tyrosine and lysine residues in angiotensin converting enzyme: evidence for a single active site.

Inactivation of rabbit lung angiotensin converting enzyme (ACE) by 1-fluoro-2,4-dinitrobenzene (Dnp-F) has been shown to be due primarily to the modification of a tyrosine residue [Bünning, P., Kleeman, S.G., & Riordan, J.F. (1990) Biochemistry (preceding paper in this issue)]. Rabbit testicular ACE is also inactivated by Dnp-F. The specific site of modification has been identified by peptide mapping of tryptic digests of the Dnp-modified protein. Two principal 340-nm-absorbing peaks, not observed with protein modified in the presence of inhibitor, have been characterized. Amino acid and sequence analyses show that these peptides contain two distinct residues that have been selectively modified. The sequence of the major (greater than 90% of the total) modified peptide is YVEFTNK with the Dnp group on tyrosine. The sequence of the second, minor peptide is KVQDLQR with the Dnp group on lysine. Identical peptides were obtained from Dnp-modified rabbit lung ACE. These modified amino acids correspond to residues 200 and 118, respectively, in testicular ACE (human enzyme numbering). Both peptides are present only in the carboxy-terminal half-domain of lung ACE, corresponding to residues 776 and 694, respectively. These results indicate that the Dnp-F sensitive, catalytically functional active site is located in the "testicular" half of lung ACE.

Amino Acid Sequence↗
PubMed1990

Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein.

Development of multidrug resistance due to overexpression of P-glycoprotein (Pgp), a cell membrane drug efflux pump, occurs commonly during in vitro selections with adriamycin (Adr). Pgp-mediated drug resistance can be overcome by the calcium channel blocker verapamil (Vp), which acts as a competitive inhibitor of drug binding and efflux. In order to identify other mechanisms of Adr resistance, we isolated an Adr-resistant subline by selecting the human breast cancer cell line MCF-7 with incremental increases of Adr in the presence of 10 microgram/ml verapamil. The resultant MCF-7/AdrVp subline is 900-fold resistant to Adr, does not overexpress Pgp, and does not exhibit a decrease in Adr accumulation. It exhibits a unique cross-resistance pattern: high cross-resistance to the potent Adr analogue 3'-deamino-3'-(3-cyano-4-morpholinyl)doxorubicin, lower cross-resistance to the alkylating agent melphalan, and a sensitivity similar to the parental cell line to vinblastine. The levels of glutathione and glutathione S-transferase are similar in the parental line and the Adr-resistant subline. Topoisomerase II-DNA complexes measured by the potassium-sodium dodecyl sulfate precipitation method shows a 2-3 fold decrease in the resistant subline. The MCF-7/AdrVp cells overexpress a novel membrane protein with an apparent molecular mass of 95 kDa. Polyclonal antibodies raised against the P-95 protein demonstrate a correaltion between the level of expression and Adr resistance. Removal of Adr but not verapamil from the selection media results in a decline in P-95 protein levels that parallels a restoration of sensitivity to Adr. Immunohistochemistry demonstrates localization of the P-95 protein on the cell surface. The demonstration of high levels of the protein in clinical samples obtained from patients refractory to Adr suggests that this protein may play a role in clinical drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Human hepatic regenerative stimulator substance: partial purification and biological characterization of hepatic stimulator substance from human fetal liver cells.

Current support or replacement therapies for fulminant acute hepatic failure are frequently very disappointing. In this study, human hepatic stimulator substance--a liver-specific growth factor--was partially purified from human fetal liver cells and characterized by its biological effects. Almost 70-fold protein content was purified with an approximately 80-fold increase in specific growth stimulator activity. Human hepatic stimulator substance proved to be heat-stable, protease-sensitive, organ-specific and species-nonspecific. Human hepatic stimulator substance produced a two- to threefold increase of 3H-thymidine incorporation into hepatic DNA when injected intraperitoneally into growing weanling mice (nonhepatectomized) or regenerating rats (34% hepatectomy). The effects of hHSS in reversing the lethality of D-galactosamine (1.6 gm/kg body weight)-induced hepatic necrosis in rats were further evaluated. A survival rate of 4% (n = 24), 41% (n = 12, p less than 0.05), 33% (n = 12, p less than 0.05), 31% (n = 13, p less than 0.05) and 18% (n = 11, p greater than 0.05) was observed when the rats were injected with 4 ml of saline intraperitoneally, 4 ml of human intact fetal hepatocytes (2.4 x 10(8] intraperitoneally, 4 ml of human hepatic stimulator substance intraperitoneally, 2 ml of twofold concentrated human hepatic stimulator substance intravenously and 1 ml of fourfold human hepatic stimulator substance intramuscularly, respectively, 20 hr after poisoning.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

[Application of thoracoscopy in the diagnosis of pleuro-pulmonary disease].

Thoracoscopic examination was performed in 20 cases of pleuro-pulmonary diseases of uncertain etiology, 16 patients (80%) obtained positive thoracoscopic biopsy results while 4 false negative. Needle pleural biopsy was carried out in 10 cases and four of them gave correct diagnosis. After thoracoscopy thoracotomy were performed in 7 cases and all lesions were resected. Thirteen cases with advanced tumor or benign pleural diseases were treated conservatively. It was concluded that thoracoscopy is as an important technique in diagnosis of pleuro-pulmonary diseases.

Adult↗
PubMed1989

Heme prosthetic group required for acetylation of prostaglandin H synthase by aspirin.

The ability of aspirin to acetylate PGH synthase was determined by reacting [3H-acetyl]-aspirin with purified enzyme followed by high pressure liquid chromatography analysis of the protein components of the reaction mixture. Heme-reconstituted enzyme incorporated approximately one acetyl group per 70-kDa subunit, whereas apoprotein incorporated 0.1 acetyl group per subunit. The ability of the heme prosthetic group to enhance acetylation of the protein was correlated with its ability to protect the Arg253-Gly254 peptide bond from cleavage by trypsin. Thus, heme-induced alteration of protein conformation may contribute to the enhanced labeling of Ser506 by aspirin. The present results indicate that irreversible inactivation of prostaglandin H synthase by aspirin occurs only when the heme prosthetic group is bound to the protein. Considering its short in vivo half-life, it is likely that aspirin inactivates only the steady-state fraction of PGH synthase in a cell that is active but not newly synthesized apoprotein. This may contribute to the differential kinetics of inactivation and recovery of PGH synthase activity in platelets and vascular endothelial cells after administration of low dose aspirin as a prophylactic agent against cardiovascular disease.

Acetylation↗
PubMed1989

Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation.

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.

ADP Ribose Transferases↗
PubMed1989

[Clinicopathologic analysis of condyloma acuminatum of the vulva in 165 cases].

Condyloma acuminatum of the vulva was diagnosed in 165 cases, of which 117 were identified during mass screening and 48 discovered in the outpatient clinic. The gross appearance and histologic characteristics were described. The lesions were removed surgically in 5 cases, and treated with topical 5-FU ointment in 4. The remaining cases were treated by cryosurgery. A permanent cure was achieved in all cases. The authors emphasize that the importance of condyloma acuminata should not be overlooked on account of its close relationship to vulvar cancer.

Adult↗
PubMed1989

Observations on the clinical manifestations and treatment of an experimental infection with Brugia malayi in man.

This paper reports clinical observations made during experimental Brugia malayi infection in man. This was induced by 52 infective larvae-carrying mosquitoes, Anopheles sinensis. A monkey, Maccaca mulatta, was inoculated with infective larvae from the mosquitoes infected with nocturnally periodic type of B. malayi from patients. Six months later, the monkey became a microfilaria carrier. Nine days later, the subject was experimentally bitten by mosquitoes infected from biting the monkey, with an estimated delivery of over 200 infective larvae. Seven days after the infection, general (but fluctuating) pruritus developed, followed by lymphadenitis and swelling of the hand and forearm, with a mild pyrexia for nearly 3 months. The eosinophil counts continuously increased. There were four separate attacks of lymphangitis. On day 142, microfilaraemia was first detected. After two more weeks, the subject was treated with a total dose of 15 g diethylcarbamazine (DEC) over 30 days. During this period, 28 small tender subcutaneous nodules appeared on his arms. After treatment, microfilaraemia gradually disappeared and the swelling of his arms improved. Three months later, the eosinophil count of his blood returned to normal but some temporary and scattered small pruritic erythematous patches still persisted over his left forearm. Lymphangitis has not recurred.

Animals↗
PubMed1989

[Thoracoscopy using flexible fiberoptic bronchoscopy--a preliminary report of experimental and clinical studies].

The preliminary experience on thoracoscopy with fiberoptic bronchoscope through a trocar designed by the authors for the purpose of studying its diagnostic implication and safety was reported. Structures in the pleural cavity of 8 dogs, being examined for 16 times, could be clearly perceived and taken for biopsy with exact localization. This technique was also carried out in 5 patients suffering from pleural effusion, mediastinal mass and periphery lung disease of unknown cause. After the examination, diagnosis was made as mesothelioma in 2 cases and non-specific pleurisy, malignant lung tumor with mediastinal lymphatic node metastasis and pulmonary benign tumor in the remaining 3 cases respectively. It was observed that its respiratory and circulatory effects were negligible as there were only transient fever in all the cases and local subcutaneous emphysema in one case with prompt recovery without treatment. Thoracoscopy is a technique easy to operate, safe, less traumatic and gives accurate diagnosis in clinical practice.

Adult↗
PubMed1988

Functional differentiation of cyclooxygenase and peroxidase activities of prostaglandin synthase by trypsin treatment. Possible location of a prosthetic heme binding site.

Treatment of prostaglandin (PG)H synthase purified from ram seminal vesicle microsomes with trypsin cleaves the 70-kDa subunits into 33- and 38-kDa fragments (Chen, Y.-N. P., Bienkowski, M. J., and Marnett, L. J. (1987) J. Biol. Chem. 262, 16892-16899). In contrast to a minimal decrease in cyclooxygenase activity, peroxidase activity declines rapidly following trypsin treatment. The time course for loss of guaiacol peroxidase activity corresponds closely to the time course for protein cleavage. The ability of trypsin-treated enzyme to support catalytic reduction of 5-phenyl-4-pentenyl-1-hydroperoxide in the presence of reducing substrates is significantly reduced. The products of metabolism of 10-hydroperoxy-8,12-octadecadienoic acid indicate that trypsin-treated enzyme catalyzes homolytic scission of the hydroperoxide bond in contrast to the heterolytic scission catalyzed by intact enzyme. Spectrophotometric titrations of hematin addition to trypsin-treated PGH synthase indicate approximately a 50% reduction in heme binding. These observations suggest that trypsin treatment of PGH synthase decreases the ability of the protein to bind prosthetic heme at a site that controls peroxidase activity. Comparison of the N-terminal sequence of the 38-kDa fragment of trypsin-treated PGH synthase to the amino acid sequence of the intact protein indicates that cleavage occurs between Arg253 and Gly254. Based on literature precedents and the results of the present investigations, we propose that the heme prosthetic group that controls the peroxidase activity of PGH synthase binds to the His residue of the sequence His250-Tyr251-Pro252-Arg253 located immediately adjacent to the trypsin cleavage site.

Animals↗
PubMed1987

Controlled tryptic digestion of prostaglandin H synthase. Characterization of protein fragments and enhanced rate of proteolysis of oxidatively inactivated enzyme.

Treatment of prostaglandin H (PGH) synthase (70 kDa) with trypsin generates fragments of 33 and 38 kDa. Each of the fragments was purified by reverse-phase high performance liquid chromatography (HPLC) using acetonitrile/water/trifluoroacetic acid gradients. Amino acid sequence analysis indicates that the 33-kDa protein contains the NH2 terminus of PGH synthase. Neither the 33- nor 38-kDa fragment isolated by HPLC exhibits any PGH synthase activity; however, cleavage of intact enzyme to 33- and 38-kDa fragments to the extent of 90% only reduces cyclooxygenase activity by 40%. This implies that the cleaved proteins or a complex formed between them retains the conformation necessary for enzyme activity. Extensive attempts to resolve active fragments from each other or from intact enzyme were unsuccessful; intact enzyme and digestion fragments cochromatograph under all conditions employed. Treatment of PGH synthase with [3H]acetylsalicylic acid followed by trypsin digestion introduces [3H]acetyl moieties into the intact protein and the 38-kDa fragment (0.8-0.9 acetyl group/subunit). Nearly complete conversion of PGH synthase to 33- and 38-kDa fragments by exposure to high concentrations of trypsin prior to [3H]acetylsalicylic acid treatment results in labeling of the 38-kDa fragment, but not the 33-kDa fragment. The present findings are consistent with the presence of a membrane-binding domain (33 kDa) and an active site domain (38 kDa) in the 70-kDa subunit of PGH synthase. They also suggest that, following cleavage, the 38-kDa fragment retains the structural features responsible for the cyclooxygenase activity and selective aspirin labeling of PGH synthase. PGH synthase undergoes self-catalyzed inactivation by oxidants generated during its catalytic turnover. When PGH synthase, inactivated by treatment with arachidonic acid or hydrogen peroxide, was treated with trypsin it was cleaved two to three times faster than unoxidized enzyme. Addition of heme to oxidized PGH synthase did not reconstitute cyclooxygenase activity or resistance to trypsin cleavage. Spectrophotometric studies demonstrated that oxidatively inactivated enzyme did not bind heme. This implies that oxidation of protein residues as well as the heme prosthetic group is an important determinant of proteolytic sensitivity. Oxidative modification may mark PGH synthase for proteolytic cleavage and turnover.

Amino Acids↗