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Biomedical subjects

D Cheng

Publications and source records attributed to D Cheng.

At least 55 records · Page 3Linked to original sources
PubMed1999

Interaction in vivo and in vitro between the yeast fimbrin, SAC6P, and a polymerization-defective yeast actin (V266G and L267G).

A mutant yeast actin (GG) has decreased hydrophobicity in a subdomain 3/4 hydrophobic plug believed to be involved in a hydrophobic cross-strand "plug-pocket" interaction necessary for actin filament stability. This actin will not polymerize in vitro but is compatible with cell viability. We have assessed the ability of Sac6p, the yeast homologue of the actin filament stabilizing and bundling protein fimbrin, to restore polymerization in vitro and to facilitate GG-actin function in vivo. Sac6p rescues GG-actin polymerization at 25 degrees C but not at 4 degrees C. The actin polymerizes into bundles at room temperature with a fimbrin:actin molar ratio of 1:4. At this ratio, every actin monomer contacts a Sac6p actin binding domain. Following cold-induced depolymerization, actin/Sac6p mixtures repolymerize beginning at 15 degrees C instead of the 25 degrees C required for de novo assembly, because of the presence of residual actin-Sac6p nuclei. Generation of haploid Deltasac6/GG-actin cells from either diploid or haploid cells was unsuccessful. The facile isolation of cells with either mutation alone indicates a synthetic lethal relationship between this actin allele and the SAC6 gene. Sac6p may allow GG-actin function in vivo by stabilizing the actin in bundles thereby helping maintain sufficient levels of an otherwise destabilized actin monomer within the cell.

Actins↗
PubMed1999

Human acyl-CoA:cholesterol acyltransferase-1 in the endoplasmic reticulum contains seven transmembrane domains.

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis and is involved in atherosclerosis. ACAT-1 protein is located mainly in the ER. The hydropathy plot suggests that ACAT-1 protein contains multiple transmembrane segments. We inserted either the hemagglutinin tag or the HisT7 tag at various hydrophilic regions within the human ACAT-1 protein and used immunofluorescence microscopy to determine the topography of the tagged proteins expressed in mutant Chinese hamster ovary cells lacking endogenous ACAT. All of the tagged proteins are located mainly in the ER and retain full or partial enzyme activities. None of the tagged proteins produces detectable intracellular degradation intermediates. Treating cells with digitonin at 5 micrograms/ml permeabilizes the plasma membranes while leaving the ER membranes sealed; in contrast, treating cells with 0.25% Triton X-100 or with cold methanol permeabilizes both the plasma membranes and the ER membranes. After appropriate permeabilization, double immunostaining using antibodies against the N-terminal region and against the inserted tag were used to visualize various regions of the tagged protein. The results show that human ACAT-1 in the ER contains seven transmembrane domains.

Algorithms↗
PubMed1999

Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.

Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH(2)-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P. Protease protection assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH(2)-terminal signal sequence of S1P generates S1P-A (amino acids 23-1052), which is inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138-1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Site-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2. S1P-B is further cleaved at an internal RRLL sequence to yield S1P-C (amino acids 187-1052). Mutational analysis suggests that S1P-B and S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur. These data provide the initial description of the processing of a subtilisin-related protease that controls the level of cholesterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805-22812), we develop an in vitro assay to characterize the activity of purified recombinant S1P.

Amino Acid Sequence↗
PubMed1999

Secreted site-1 protease cleaves peptides corresponding to luminal loop of sterol regulatory element-binding proteins.

We describe a permanent line of Chinese hamster ovary cells transfected with a cDNA encoding a truncated form of Site-1 protease (S1P) that is secreted into the culture medium in an enzymatically active form. S1P, a subtilisin-like protease, normally cleaves the luminal loop of sterol regulatory element-binding proteins (SREBPs). This cleavage initiates the two-step proteolytic process by which the NH(2)-terminal domains of SREBPs are released from cell membranes for translocation to the nucleus, where they activate transcription of genes involved in the biosynthesis and uptake of cholesterol and fatty acids. Truncated S1P (amino acids 1-983), produced by the transfected Chinese hamster ovary cells, lacks the COOH-terminal membrane anchor. Like native S1P, this truncated protein undergoes normal autocatalytic processing after residue 137 to release an NH(2)-terminal propeptide, thereby generating an active form, designated S1P-B. Prior to secretion, truncated S1P-B, like native S1P-B, is cleaved further after residue 186 to generate S1P-C, which is the only form that appears in the culture medium. The secreted enzyme, designated S1P(983)-C, cleaves a synthetic peptide that terminates in a 7-amino-4-methyl-coumarin fluorochrome. This peptide, RSLK-MCA, corresponds to the internal propeptide cleavage site that generates S1P-B as described in the accompanying paper (Espenshade, P. J., Cheng, D., Goldstein, J. L., and Brown, M. S. (1999), J. Biol. Chem. 274, 22795-22804). The secreted enzyme does not cleave RSVL-MCA, a peptide corresponding to the physiologic cleavage site in SREBP-2. However, S1P(983)-C does cleave after this leucine when the RSVL sequence is contained within a 16-residue peptide corresponding to the central portion of the SREBP-2 luminal loop. The catalytic activity of S1P(983)-C differs from that of furin/prohormone convertases, two related proteases, in its more alkaline pH optimum (pH 7-8), its relative resistance to calcium chelating agents, and its ability to cleave after lysine or leucine rather than arginine. These data provide direct biochemical evidence that S1P is the protease that cleaves SREBPs and thereby functions to control lipid biosynthesis and uptake in animal cells.

Amino Acid Sequence↗
PubMed1999

Large pleural effusions occurring after coronary artery bypass grafting. Cardiovascular Surgery Associates, PC.

BACKGROUND: Large pleural effusions sometimes occur after coronary artery bypass grafting (CABG), but their characteristics and clinical course are largely unknown. OBJECTIVE: To describe the clinical course and pleural fluid findings in patients with large pleural effusions occurring after CABG. DESIGN: Retrospective case series. SETTING: Tertiary care, university-affiliated, nonprofit teaching hospital. PATIENTS: 3707 patients who had CABG between 1 February 1996 and 1 August 1997. MEASUREMENTS: Chest radiographs were reviewed, and information on pleural fluid findings, pleural effusion treatment, and cardiac surgery was obtained from medical records and a cardiac surgery database. RESULTS: Pleural effusions that occupied more than 25% of the hemithorax were found in 29 patients (0.78%). Seven of the effusions were attributed to congestive heart failure, 2 were attributed to pericarditis, and 1 was attributed to pulmonary embolism. The explanation for the remaining 19 effusions was unclear. All but 2 effusions were predominantly left-sided. Of these 19 effusions, 8 were bloody and 11 were nonbloody. Bloody effusions usually occurred earlier, contained higher lactic acid dehydrogenase levels, and were frequently eosinophilic. Nonbloody effusions tended to be more difficult to manage. CONCLUSIONS: Large pleural effusions may develop in a small proportion of patients after CABG. The cause of many of these effusions is unclear. Most bloody effusions can be managed with one to three therapeutic thoracenteses. Resolution of nonbloody effusions may require anti-inflammatory agents, tube thoracostomy, or intrapleural injection of sclerosing agents.

Aged↗
PubMed1999

Etiology of preoperative anemia in patients undergoing scheduled cardiac surgery.

PURPOSE: Ten percent of our cardiac surgical patients have preoperative anemia. Anemia diagnosed before scheduled cardiac surgery is a strong predictor of the need for homologous blood transfusion (RBC) perioperatively but the cause of this preoperative anemia is not known. The purpose of this study was to evaluate the etiology of preoperative anemia. METHODS: Seventy-five consecutive anemic cardiosurgical patients (Hb = < 120 g x L(-1) the day before surgery) were studied prospectively. All had multiple diagnostic blood tests done in the preoperative period to diagnose the cause of the anemia and subsequently underwent non-emergency cardiac surgery. Anesthesia and RBC transfusion were standardized according to the protocol. Data in respect to operation, RBC and other blood product transfusion during operation and hospital stay were recorded. RESULTS: Hospital-acquired anemia was present in 37.3% of anemic patients (hemoglobin decrease during hospitalization before surgery > or =9 g x l(-1)). The second most common diagnosis was iron deficiency anemia (29.3% patients) followed by anemia of chronic renal disease (10.7% patients). When coronary angiography was performed close to operation time, patients had a higher decrease in hemoglobin concentration during hospitalization --suggesting that blood loss during angiography was, in part, responsible for anemia. Seventy-five percent of anemic patients were transfused with RBC perioperatively compared with our overall transfusion rate of 30% of cardiac surgery patients. CONCLUSIONS: In the majority of patients, preoperative anemia is potentially preventable. Investigation and treatment of anemia before cardiac surgery should be a priority in preparing the patient for surgery.

Aged↗
PubMed1999

Activation of acyl-CoA cholesterol acyltransferase: redistribution in microsomal fragments of cholesterol and its facilitated movement by methyl-beta-cyclodextrin.

Acyl-CoA cholesterol acyltransferase (ACAT) (EC 2.3.1.26) in the yolk sac membrane of chicken eggs plays an important role in the transport of lipids, which serve as both structural components and as an energy source during embryogenesis. ACAT from the yolk sac membrane of chicken eggs 16 d after fertilization has higher activity and better stability than its mammalian liver counterpart. During our study of the avian enzyme, ACAT was found to be activated up to twofold during storage at 4 degrees C. The activation was investigated, and data suggest that redistribution of cholesterol within microsomal vesicles leads to the increase. Methyl-beta-cyclodextrin (MbetaCD) increases activation an additional twofold, possibly by facilitating the movement of cholesterol within microsomal fragments and allowing redistribution of cholesterol in lipid bilayers to a greater extent. Treatment of microsomes with MbetaCD removes cholesterol from the membranes. Controlled amounts of cholesterol can be restored to the membranes by mixing them with cholesterol-phosphatidylcholine liposomes in the presence of MbetaCD. Under these conditions, the plot of ACAT vs. cholesterol mole fraction in the liposomes is sigmoidal. The finding that MbetaCD can enhance cholesterol transfer between liposomes and microsomes and reduce the limitation of slow movement of nonpolar molecules in aqueous media should make cyclodextrins more useful in in vitro studies of apolar molecule transport between membrane vesicles.

Animals↗
PubMed1999

Duration of preoperative amiodarone treatment may be associated with postoperative hospital mortality in patients undergoing heart transplantation.

OBJECTIVE: To assess the effect of preoperative amiodarone treatment on patient mortality after heart transplantation. DESIGN: Retrospective study. SETTING: Single-institution university hospital. PARTICIPANTS: One hundred six consecutive patients with heart transplants. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Patients were grouped according to duration of preoperative amiodarone treatment, and posttransplant mortality before hospital discharge was compared with patients not treated with amiodarone. The authors collected cardiovascular data in the preoperative and postoperative periods. There was a significant increase in posttransplant mortality before hospital discharge in patients treated with amiodarone for more than 4 weeks in the preoperative period (p < 0.05). Patients treated with amiodarone had significantly lower (p < 0.05) heart rates (mean heart rate, 103+/-19 beats/min) in the early postoperative period than patients not treated with amiodarone (mean heart rate, 111+/-15 beats/min), but there was no relationship with mortality (p = not significant). Patients who died had a significantly lower (p < 0.05) postoperative cardiac index (2.2+/-0.7 to 2.5+/-0.7) in the first 24 hours after cardiopulmonary bypass compared with patients who survived to hospital discharge (3.0+/-0.7 to 3.1+/-0.9), but there was no relationship to amiodarone treatment (p = not significant). CONCLUSION: Preoperative amiodarone treatment for more than 4 weeks may be associated with a significant increase in postoperative mortality in patients undergoing heart transplantation. Therefore, the indications for amiodarone must be carefully considered and, if needed, the maintenance dose should be kept to a minimum.

Amiodarone↗
PubMed1999

Expression and translocation of Rac2 in eosinophils during superoxide generation.

Eosinophils induce tissue injury by releasing granule-associated cytotoxic proteins, lipid mediators and superoxide anions in response to appropriate stimuli. Superoxide generation associated with respiratory burst is largely dependent on the assembly of the NADPH oxidase complex in the membrane, consisting of membrane-bound cytochrome b558 and translocated p47phox and p67phox. The activation of this complex is critically dependent on the translocation of GTP-bound Rac1, or its homologue Rac2, from the cytosol to the membrane in neutrophils. Rac expression has not yet been fully characterized in eosinophils. We proposed that eosinophils translate and express Rac2 and its GDP-dissociation inhibitor, RhoGDI. Furthermore, we hypothesized that Rac2 translocates along with p47phox and p67phox proteins from the cytosol to the plasma membrane during respiratory burst. By reverse transcription-polymerase chain reaction analysis and sequencing of the amplified product, guinea-pig eosinophils were found to express Rac2 mRNA, exhibiting 93% homology with the human Rac2 sequence. Rac1 mRNA was also detected in eosinophils but not its translated product. In contrast, Rac2 protein expression was detected using a specific antibody. In subcellular fractions, Rac2 was found to translocate, along with p47phox and p67phox, from cytosol to plasma membrane-associated fractions following phorbol myristate acetate stimulation, while RhoGDI remained within cytosolic fractions. These findings suggest that Rac2 is preferentially expressed and activated in eosinophils, and is likely to be a crucial regulator of the release of reactive oxygen species from these cells during inflammatory reactions.

Amino Acid Sequence↗
PubMed1999

SNARE complex proteins, including the cognate pair VAMP-2 and syntaxin-4, are expressed in cultured oligodendrocytes.

Myelin membrane synthesis in the CNS by oligodendrocytes (OLs) involves directed intracellular transport and targeting of copious amounts of specialized lipids and proteins over a relatively short time span. As in other plasma membrane-directed fusion, this process is expected to use specific trafficking and vesicle fusion proteins characteristic of the SNARE model. We have investigated the developmental expression of SNARE proteins in highly enriched primary cultures of OLs at discrete stages of differentiation. VAMP-2/synaptobrevin-2, syntaxin-2 and -4, nsec-1/munc-18-1, Rab3a, synaptophysin, and synapsin were expressed. During differentiation, expression of the vesicular SNARE VAMP-2, the small GTP-binding protein Rab3a, and the target SNARE syntaxin-4 were up-regulated. VAMP-2 and Rab3 proteins detected immunocytochemically in cultured OLs were localized within the developing process network; in situ anti-VAMP-2 antibody stained the perikarya of rows of cells with the distribution and appearance of OLs. We discuss the potential involvement of SNARE complex proteins in a plasma membrane-directed transport mechanism targeting nascent myelin vesicles to the forming myelin sheath.

Animals↗
PubMed1999

Vascular endothelial growth factor in pleural fluid.

STUDY OBJECTIVES: The purpose of this study was to analyze the relationship of the pleural fluid vascular endothelial growth factor (VEGF) level with the diagnostic category and with the pleural fluid characteristics in a group of 70 patients. DESIGN: The VEGF levels of consecutive patients undergoing therapeutic thoracentesis were determined with an enzyme-linked immunosorbent assay. SETTING: University-affiliated tertiary care center. RESULTS: The median level of pleural fluid VEGF in the patients with congestive heart failure (150 pg/mL) was significantly (p < 0.05) lower than the median level in the patients with coronary artery bypass grafting (357 pg/mL), which in turn was significantly lower (p < 0.05) than the median levels in the patients with malignancy (1,097 pg/mL). The overlap between groups, however, limits the diagnostic usefulness of pleural fluid VEGF levels. The VEGF level was most closely correlated with the lactate dehydrogenase level (r = 0.42, p < 0.001) and was also significantly correlated with the total pleural fluid protein level. The median VEGF levels in the pleural fluid of patients with breast cancer were significantly lower (p = 0.017) than in those with lung cancer. The VEGF level was very high (3,294 pg/mL) in the one patient with pulmonary embolism. CONCLUSIONS: We conclude that the VEGF levels in pleural fluid differ significantly from one diagnostic category to another with the highest median levels occurring in patients with malignant pleural effusions. We speculate that VEGF may be responsible for the pleural fluid accumulation in at least some situations.

Breast Neoplasms↗
PubMed1999

Mitomycin chemotherapeutic pleurodesis to palliate malignant pleural effusions secondary to gynecological cancer.

BACKGROUND: To assess the efficacy of mitomycin pleurodesis in women with end stage malignant effusion. METHODS: Retrospective analysis. RESULTS: Of the 13 patients treated six had ovarian, six had endometrial and one had cervical adenocarcinoma. The median time of onset of effusion from diagnosis of primary disease was 12 months (range 1-75). Ten patients were evaluable for response. The median age of the patients was 59 years (range 42 to 74). Ten (77%) of them had repeated thoracocentesis prior to mitomycin pleurodesis. The median duration and volume of drainage was 4 days (range 2-11) and 3.1 liters (range 1.2-10.2) respectively. One of two patients developed a pneumothorax and required the insertion of an additional chest drain. Overall, seven patients (70%) responded - one completely (CR) and six partially (PR), while three did not respond. Four of five patients with ovarian cancer had a partial response, two of four patients with endometrial cancer (one CR and one PR) responded. The only patient with cervical cancer had a partial response. Nine patients (69%) had enough symptomatic improvement to be discharged home. The drug cost of bleomycin 60 units (US$191), another commonly used agent, was more than twice as expensive as mitomycin 30 mg (US$84). CONCLUSIONS: Mitomycin pleurodesis can palliate 70% of patients for at least 1 month. It offers cheap and effective palliation. There is a suggestion that patients' quality of life is improved.

Adult↗
PubMed1999

Wide local excision (WLE) for vaginal intraepithelial neoplasia (VAIN).

BACKGROUND: To assess the use of wide local excision in the treatment of VAIN. METHODS: A retrospective review on 40 WLE procedures for VAIN. RESULTS: The mean age was 60 years. Thirty-six (90%) patients had previous treatment for genital tract cancer or pre-cancer. The median duration and blood loss during operation was 45 minutes and 50 mls respectively. Fifteen complications affected 11 patients. Only one of five patients with 'vaginal cancer' was diagnosed prior to WLE. Of the 35 patients treated for VAIN 3, 12 (34%) developed abnormal cytology during follow up three had residual VAIN 3, five had recurrent VAIN 3 and four had invasive cancer diagnosed. The remaining 23 (66%) patients were disease free at a median follow up of 44 months. CONCLUSIONS: Ablative therapy for VAIN 3 is unsafe as occult invasive foci can be missed by biopsy. WLE is efficacious in treating high grade VAIN 3. Long term surveillance of the lower genital tract is needed to diagnose metachronous lesions.

Aged↗
PubMed1999

Family planning training in Maryland family practice and obstetrics/gynecology residency programs.

OBJECTIVE: To examine the extent of family planning and abortion training in Maryland family practice (FP) and obstetrics/gynecology (OB) residency training programs. METHODS: All final-year residents in every FP and OB residency training program in Maryland were asked in 1998 how many cases (0, 1-10, > 10) of ten methods of contraception and abortion they had managed. RESULTS: Seventy-five percent (55) of the 73 residents responded. Fifty percent of FP residents had never inserted an intrauterine device (IUD), 43% had never inserted an implant (Nor-plant), 37% had never prescribed emergency contraceptive pills, and 30% had never fitted a diaphragm. Ninety-seven percent of FP residents had no experience with elective termination of pregnancy, and 83% had no experience with sterilization. Twenty percent of OB residents had never inserted an IUD, 16% had never inserted implants or prescribed emergency contraceptive pills, 20% had never fitted a diaphragm, and 36% had no experience in elective termination of pregnancy. Not one FP resident had inserted or fitted more than ten IUDs, implants, diaphragms, or cervical caps. Except for oral and injectable contraception, the majority of OB residents had not managed more than ten cases of any other reversible contraceptive method: 80% had not inserted more than ten IUDs, 72% had not inserted more than ten implants, 88% had not fitted more than ten diaphragms, and 100% had not fitted more than ten cervical caps. CONCLUSION: These survey results indicate a need for more formal instruction in most contraceptive methods for OB and FP residency programs in Maryland. This study concurs with previous national studies showing deficits in family planning training.

Contraceptive Devices, Female↗
PubMed1999

[Demonstration for periodicity of leptospirosis in Yichang City of Hubei Province during 1960-1997].

OBJECTIVE: To study the periodicity of leptospirosis incidence. METHODS: Data of leptospirosis epidemics in Yichang City collected during the past 38 years from 1960 to 1997 were analyzed with periodic graphics method. RESULTS: Cause-specific incidence rate of leptospirosis was 15.43 per 100,000 in average, with an epidemic interval of 10 years, with statistical significance in periodic vibration (J = 4.179, P < 0.05). CONCLUSION: The results mentioned above provided scientific basis for the effective control and prevention of the disease.

China↗