Biomedical subjects

J L Thomas

Publications and source records attributed to J L Thomas.

At least 19 recordsLinked to original sources

PubMed1995

An NADH-induced conformational change that mediates the sequential 3 beta-hydroxysteroid dehydrogenase/isomerase activities is supported by affinity labeling and the time-dependent activation of isomerase.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and steroid delta-isomerase were copurified as a single protein from human placental microsomes. Because NADH is an essential activator of isomerase (Kact = 2.4 microM, Vmax = 0.6 mumol/min/mg), the affinity alkylating nucleotide, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-diphosphate (8-BDB-TADP), was synthesized. 8-BDB-TADP activates isomerase (Kact = 338 microM, Vmax = 2.1 mumol/min/mg) prior to inactivating the enzyme. The inactivation kinetics for isomerase fit the Kitz and Wilson model for time-dependent, irreversible inhibition by 8-BDB-TADP (KI = 314 microM, first order maximal rate constant kobs = 7.8 x 10(-3) s-1). NADH (50 microM) significantly protects isomerase from inactivation by 8-BDB-TADP (100 microM). The isomerase activity is inactivated more rapidly by 8-BDB-TADP as the concentration of the affinity alkylator increases from 67 microM (t1/2 = 8.4 min) to 500 microM (t1/2 = 2.4 min). In sharp contrast, the 3 beta-HSD activity is inactivated more slowly as the concentration of 8-BDB-TADP increases from 67 microM (t1/2 = 4.8 min) to 500 microM (t1/2 = 60.0 min). We hypothesized that the paradoxical kinetics of 3 beta-HSD inactivation is a consequence of the activation of isomerase by 8-BDB-TADP via a nucleotide-induced shift in enzyme conformation. Biophysical support for an NADH-induced conformational change was obtained using stopped-flow fluorescence spectroscopy. The binding of NADH (10 microM) quenches the intrinsic fluorescence of the enzyme protein in a time-dependent manner (rate constant kapp = 8.1 x 10(-3) s-1, t1/2 = 85 s). A time lag is also observed for the activation of isomerase by NADH. This combination of affinity labeling and biophysical data using nucleotide derivatives supports our model for the sequential reaction mechanism; the cofactor product of the 3 beta-HSD reaction, NADH, activates isomerase by inducing a conformational change in the single, bifunctional enzyme protein.

Adenosine Diphosphate↗

PubMed1995

Cardiovascular risk factors and combined estrogen-progestin replacement therapy: a placebo-controlled study with nomegestrol acetate and estradiol.

OBJECTIVE: To assess the effects of oral E2 replacement therapy combined with nomegestrol acetate, a 19-norprogesterone derivative, on cardiovascular risk factors. DESIGN: A double-blind randomized prospective study comparing the effect of a placebo and two oral E2-nomegestrol acetate combinations (1 mg-2.5 mg and 1.5 mg-3.75 mg) over a three-cycle trial. SETTING: Department of Internal Medicine and Nutrition, Hotel-Dieu, Paris, France. PATIENTS: Fifty-seven nonhysterectomized women with natural menopause. MAIN OUTCOME MEASURES: Blood pressure, renin substrate, glucose, total cholesterol, high-density and low-density lipoprotein cholesterol, triglycerides, apoproteins A1 and B, lipoprotein(a), antithrombin III, fibrinogen, plasminogen, prothrombin fragment 1 + 2, protein C, and total and free protein S. RESULTS: Both treatments significantly reduced menopausal complaints, total cholesterol, low-density lipoprotein cholesterol and lipoprotein(a). Treatment with the 1.5 mg-3.75 mg combination resulted in a significant increase in apolipoprotein A1. No significant change were observed in other parameters. CONCLUSIONS: Sequentially combined with oral E2 in hormone replacement therapy, nomegestrol acetate had favorable effects on plasma lipids and lipoproteins. This nonandrogenic progestin decreased lipoprotein(a) levels as observed previously with medroxyprogesterone acetate combined with conjugated equine estrogens.

Apoproteins↗

PubMed1992

Affinity labeling of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase: evidence for bifunctional catalysis by a different conformation of the same protein for each enzyme activity.

3 beta-Hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase copurify from human placental microsomes as a single enzyme protein. The affinity-alkylating secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivates the dehydrogenase and isomerase reactions in a time-dependent manner, but which of the two activities is targeted depends on the concentration of secosteroid. At 2-5 microM secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (KI = 4.2 microM, k3 = 1.31 x 10(-2) min-1). As the secosteroid level increases from 11 to 30 microM, dehydrogenase is paradoxically inactivated at progressively slower rates, and pregnenolone no longer protects against the alkylator. The inactivation of isomerase exhibits the expected first-order kinetics (KI = 31.3 microM, k3 = 6.42 x 10(-2) min-1) at 11-30 microM secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15 microM secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5 microM). A shift from a dehydrogenase to an isomerase conformation in response to rising secosteroid levels explains these results. Analysis of the ligand-induced conformational change along with cofactor protection data suggests that the enzyme expresses both activities at a bifunctional catalytic site. According to this model, the protein begins the reaction sequence as 3 beta-hydroxysteroid dehydrogenase. The products of the first step (principally NADH) promote a change in protein conformation that triggers the isomerase reaction.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

Effects of protein concentration on IgE receptor mobility in rat basophilic leukemia cell plasma membranes.

The ability of variations of membrane protein concentrations to modulate the lateral diffusion rate of an exemplary membrane protein has been studied in healthy and osmotically shocked cultured cells of the rat basophilic leukemia cell line, 2H3 subclone. Cell surface protein was redistributed by the method of in situ electrophoresis; exposure to electric fields of 1.25-5 V/cm results in cathodal migration of the majority of the surface proteins on this cell type (Ryan, T. A., J. Myers, D. Holowka, B. Baird, and W. W. Webb. Science [Wash. DC]. 239:61-64). Even in these small fields, the steady-state distribution becomes "crowded" with more than an 80% protein occupancy of accessible membrane area at the cathodal end of these spheroidal cells, and the anodal end becomes significantly depleted. We have employed fringe pattern fluorescence photobleaching with CCD imaging detection to measure lateral diffusion coefficients of the liganded IgE receptor on both crowded and uncrowded regions of individual rat basophilic leukemia cells. We find no significant difference in lateral diffusion rates in these regions. Cells swollen by hypoosmotic stress exhibit faster diffusion overall, with the uncrowded regions having a significantly greater increase in diffusion coefficient than the crowded regions. These results are consistent with the partial or total release of cytoskeletal constraints to membrane protein diffusion induced by osmotic stress.

Animals↗

PubMed1992

In situ expression of helper-free avian leukosis virus (ALV)-based retrovirus vectors in early chick embryos.

Defective avian leukosis-based vectors expressing the bacterial lacZ gene were used as helper-free preparations to infect early stage Brown-Leghorn embryos. Both in toto X-gal staining and DNA analysis using Southern blot technique were applied to detect virus integration and expression. Our results demonstrate a low efficiency of in vitro infection in early stages of embryonic development. Southern blot analysis reveals that only 1% of embryonic cells integrate the vector genome after infection using 2 to 12 virus particle per embryonic cell. In situ expression of the lacZ marker gene was detected in only 0.06% of embryonic cells. These results lead us to conclude that only 6% of infected cells express efficiently the lacZ marker gene. This low level of expression could result from avian leukosis virus LTRs inhibition in chicken embryonic cells at an early stage of development. In spite of the low efficiency of infection, no evidence for tissue restrictive expression was observed. However, vector containing LTRs from RAV-2 virus allows preferential expression of provirus vector in neural tube tissue, whereas cardiac localization of the preferential expression was observed using vector containing the RAV-1 LTRs. The chronological analysis of the marker gene expression in terms of location of expression foci and sizes of these foci, lead us to hypothesize the putative regulation of retrovirus expression linked to embryonic development.

Animals↗

PubMed1991

Effects of nomegestrol acetate (5 mg/d) on hormonal, metabolic and hemostatic parameters in premenopausal women.

The effects of nomegestrol acetate on circulating hormone levels, metabolic and hemostatic parameters and blood pressure were studied in 36 premenopausal women. The progestogen was administered from day 7 to 25 of the cycle during six cycles at a dosage (5 mg/d) known to inhibit ovulation. Analysis were performed before and in the third and sixth cycles. Estradiol and progesterone levels decreased significantly (p less than 0.001) during treatment. Body weight, fasting blood glucose and insulin, total HDL and LDL cholesterol, apolipoprotein B, fibrinogen and plasminogen did not change significantly. Triglycerides in the third cycle (p less than 0.05) and apolipoprotein A1 levels (p less than 0.01) in both periods of sampling decreased significantly. There was a significant increase in antithrombin III (p less than 0.01). These results indicate that nomegestrol acetate has no deleterious effect on blood glucose and lipids. The decrease in apolipoprotein A1 and increase in antithrombin III may be related either to the decrease in estradiol levels induced by the treatment or to the effect of the progestogen itself.

Adolescent↗

PubMed1991

Analysis of coenzyme binding by human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase using 5'-[p-(fluorosulfonyl)benzoyl]adenosine, an affinity labeling cofactor analog.

3 beta-Hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase copurify as a single, homogeneous protein from human placental microsomes. Affinity alkylation with 2 alpha-bromoacetoxyprogesterone suggests that the dehydrogenase and isomerase substrate steroids bind at different sites on the same protein. However, the coenzyme, NADH, completely abolishes the alkylation of both enzyme activities by the progestin analog [Thomas J .L., Myers R. P., Rosik L. O. and Strickler R. C., J. Steroid Biochem. 36 (1990) 117-123]. Unlike bacterial 3-keto-5-ene-steroid isomerase, the human isomerase reaction is stimulated by diphosphopyridine nucleotides (NADH, NAD+). The affinity labeling nucleotide analog, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSA), inactivates the dehydrogenase and isomerase activities at similar rates in an irreversible manner which follows first order kinetics with respect to both time and alkylator concentration (0.2-0.6 mM). FSA is a cofactor site-directed reagent that binds with similar affinity as a competitive inhibitor of NAD+ reduction by dehydrogenase (Ki = 162 microM) or as a stimulator of isomerase (Km = 153 microM). Parallel plots derived from Kitz and Wilson analysis indicate that FSA inactivates the two enzyme activities with equal alkylation efficiency (k3/Ki = 1/slope = 0.51/mol-s for both). The 3 beta-hydroxysteroid substrate, pregnenolone, protects isomerase as well as dehydrogenase from inactivation by FSA. These observations are evidence for a single cofactor binding region which services both enzyme activities.

3-Hydroxysteroid Dehydrogenases↗

PubMed1991

Improvement of avian leukosis virus (ALV)-based retrovirus vectors by using different cis-acting sequences from ALVs.

Production and expression of double-expression vectors which transduce both Neo(r) and lacZ genes and are based on the structure of avian leukosis virus were enhanced by using cis-acting sequences (long terminal repeats and noncoding sequences) from Rous-associated virus-1 and Rous-associated virus-2 rather than those of avian erythroblastosis virus previously used in our constructs. Polyclonal producer cells obtained after transfection of these vectors into the Isolde packaging cell line gave rise to titers as high as 3 x 10(5) lacZ CFU/ml, whereas it was possible to isolate clones of producer cells giving rise to titers of more than 10(6) resistance focus-forming units per ml.

Animals↗

PubMed1991

Reevaluation of transient ischemic attacks as a risk factor for early mortality.

The prevailing belief that transient ischemic attack is a risk factor for cardiovascular morbidity and mortality is based primarily on comparisons of survival of patients after transient ischemic attacks to that of an age-, race-, and sex-adjusted general population. Concomitant conditions that carry a high risk of premature mortality or morbidity, such as ischemic heart disease, hypertension, and diabetes, are very prevalent among patients with transient ischemic attacks. Hence, the poor prognosis of such patients may be attributable to these factors rather than their transient ischemic attack per se, which may only serve to bring patients into the medical system. We compared the survival of 336 patients after transient ischemic attack to that of a control group with a similar risk factor profile consisting of 6,710 patients evaluated for cardiac catheterization. Survival estimates, both unadjusted and adjusted for risk factors, did not differ between the two groups. Three-year survival estimates, after adjustment to the mean value of covariates, were 94% for the patients with transient ischemic attacks and 91% for the controls. These results suggest that the transient ischemic attack may not be an independent risk factor for mortality, although it may identify patients already at increased risk from coexisting conditions.

Cardiac Catheterization↗

PubMed1991

Preoperative and intraoperative predictors of inotropic support and long-term outcome in patients having coronary artery bypass grafting.

The prognostic value of preoperative symptoms, preoperative left ventricular function, and intraoperative factors as related to postoperative outcome in coronary artery bypass grafting is unclear. This study was performed to identify risk factors that could be used as markers to predict immediate and long-term outcome, knowledge of which might allow physicians to modify these factors to decrease the likelihood of an adverse outcome. We retrospectively evaluated preoperative factors (including age, sex, New York Heart Association [NYHA] classification of symptoms, ejection fraction [EF], wall motion abnormalities, baseline left ventricular end-diastolic pressure [LVEDP], postradiographic contrast injection LVEDP, change in LVEDP with contrast injection, cardiac enlargement, and collateral vessels) and intraoperative factors (duration of bypass and aortic cross-clamp time) in 128 patients. The need for inotropic drug support was used as a marker of immediate outcome. A 36-mo follow-up used death and the postoperative NYHA classification of symptoms as markers of long-term outcome. The various factors associated with the use of inotropes and immediate outcome were analyzed by logistic regression. The factors related to inotrope use (and presumed adverse short-term outcome) in order of decreasing significance were lower EF, older age, cardiac enlargement, female sex, and higher baseline and postcontrast LVEDP. Patients with EF greater than or equal to 55%, but also having wall motion abnormalities and LVEDP change greater than or equal to 10 mm Hg, and all patients with EF less than 55% were more likely to require inotropic drug stimulation after cardiopulmonary bypass. Neither the change in LVEDP nor the presence of wall motion abnormalities independently predicted the need for postoperative inotropic support.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1990

Affinity alkylation of human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase by 2 alpha-bromoacetoxyprogesterone: evidence for separate dehydrogenase and isomerase sites on one protein.

We have copurified human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, which synthesize progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate, from microsomes as a homogeneous protein based on electrophoretic and NH2-terminal sequencing data. The affinity alkylator, 2 alpha-bromoacetoxyprogesterone, simultaneously inactivates the pregnene and androstene dehydrogenase activities as well as the C21 and C19 isomerase activities in a time-dependent, irreversible manner following first order kinetics. At four concentrations (50/1-20/1 steroid/enzyme M ratios), the alkylator inactivates the dehydrogenase activity (t1/2 = 1.5-3.7 min) 2-fold faster than the isomerase activity. Pregnenolone and dehydroepiandrosterone protect the dehydrogenase activity, while 5-pregnene-3,20-dione, progesterone, and androstenedione protect isomerase activity from inactivation. The protection studies and competitive kinetics of inhibition demonstrate that the affinity alkylator is active site-directed. Kitz and Wilson analyses show that 2 alpha-bromoacetoxyprogesterone inactivates the dehydrogenase activity by a bimolecular mechanism (k3' = 160.9 l/mol.s), while the alkylator inactivates isomerase by a unimolecular mechanism (Ki = 0.14 mM, k3 = 0.013 s-1). Pregnenolone completely protects the dehydrogenase activity but does not slow the rate of isomerase inactivation by 2 alpha-bromoacetoxyprogesterone at all. NADH completely protects both activities from inactivation by the alkylator, while NAD+ protects neither. From Dixon analysis, NADH competitively inhibits NAD+ reduction by dehydrogenase activity. Mixed cofactor studies show that isomerase binds NAD+ and NADH at a common site. Therefore, NADH must not protect either activity by simply binding at the cofactor site. We postulate that NADH binding as an allosteric activator of isomerase protects both the dehydrogenase and isomerase activities from affinity alkylation by inducing a conformational change in the enzyme protein. The human placental enzyme appears to express the pregnene and androstene dehydrogenase activities at one site and the C21 and C19 isomerase activities at a second site on the same protein.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

Establishment of an IL-2-dependent, antigen nonspecific chicken T-cell line.

In this article, we describe a-chicken T helper-activated cell line, III-C5, which is non-antigen-specific but IL-2-dependent. By virtue of its absolute requirement for IL-2, the III-C5 cell line is a useful tool for quantifying IL-2 production by any chicken-activated T lymphocytes in culture. The III-C5 line will be used to quantify IL-2 production in mixed lymphocyte reaction in vitro, in order to study the functional activity of T lymphocytes from avian chimeras constructed in our laboratory and particularly for studying their state of tolerance.

Animals↗

PubMed1990

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

Animals↗

PubMed1990

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland.

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.

3-Hydroxysteroid Dehydrogenases↗

PubMed1990

A widely distributed antigen developmentally regulated in the nervous system.

We have identified a glycoprotein (BEN) of 95-100 x 10(3) Mr using a monoclonal antibody. This protein is transiently expressed at the cell surface of the peripherally projecting neurons, i.e. motoneurons of the spinal cord and cranial nuclei, sensory neurons of the dorsal root and cranial sensory ganglia and sympathetic, parasympathetic and enteric neurons. In vitro cultures of dorsal root and sympathetic ganglia have shown that BEN is expressed on neurons but not on glial cells. On motor and sensory neurons, BEN first appears at the level of the cell body just after withdrawal from the cell cycle. Soon afterwards, expression of the antigen extends to the elongating axon. After a few days, BEN is no longer expressed by the motor and sensory neurons, disappearing first from the cell body and then progressively from the fibres. The loss of expression is concomitant with the onset of intense proliferation of satellite and Schwann cells. This modulated expression within the nervous system is unlike that of any surface glycoprotein so far described in vertebrates. Preliminary biochemical analysis indicates that, although it bears the adhesion-associated epitope HNK-1, BEN does not share characteristics with any previously described axonal glycoprotein. Consequently, we speculate that this glycoprotein might be a novel molecule implicated in selective adhesion phenomena, such as axonal fasciculation.

Animals↗

PubMed1990

The grandparent role: a double bind.

In response to open-ended questions, married and single mothers described advantages and disadvantages of having grandparents in the family. Single mothers emphasized the value of grandparents' practical and moral support in childrearing; married mothers more often stressed grandparents' value in providing family heritage and stability. Married and single mothers agreed that grandparents' interference in childrearing could pose threats to family harmony. It is proposed that grandparents may face a double bind in attempting to meet parents' expectations: parents may expect grandparents to simultaneously be supportive without interfering. This double bind may be particularly poignant for grandparents in single mother families.

Adult↗

PubMed1989

Human placental 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase: purification from mitochondria and kinetic profiles, biophysical characterization of the purified mitochondrial and microsomal enzymes.

In human placenta, 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase, an enzyme complex found in microsomes and mitochondria, synthesizes progesterone from pregnenolone and androstenedione from fetal dehydroepiandrosterone sulfate. The dehydrogenase and isomerase activities of the mitochondrial enzyme were copurified (733-fold) using sequential cholate solubilization, ion exchange chromatography (DEAE-Toyopearl 650S), and hydroxylapatite chromatography (Bio-Gel HT). Enzyme homogeneity was demonstrated by a single protein band in SDS-polyacrylamide gel electrophoresis (monomeric Mr = 41,000), gel filtration at constant specific enzyme activity (Mr = 77,000), and a single NH2-terminal sequence. Kinetic constants were determined for the oxidation of pregnenolone (Km = 1.6 microM, Vmax = 48.6 nmol/min/mg) and dehydroepiandrosterone (Km = 2.4 microM, Vmax = 48.5 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione (Km = 9.3 microM, Vmax = 914.2 nmol/min/mg) and 5-androstene-3,17-dione (Km = 27.6 microM, Vmax = 888.4 nmol/min/mg. Mixed substrate studies showed that the dehydrogenase and isomerase activities utilize their respective pregnene and androstene substrates competitively. Dixon analysis demonstrated that the product steroids, progesterone and androstenedione, are competitive inhibitors of the C-21 and C-19 dehydrogenase activities. Enzyme purified from mitochondria and microsomes had similar kinetic profiles with respect to substrate utilization, product inhibition, and cofactor (NAD+) reduction (mean Km +/- SD using C-19 and C-21 dehydrogenase substrates = 26.4 +/- 0.8 microM, mean Vmax = 73.2 +/- 1.3 nmol/min/mg). Pure enzyme from both organelles exhibited identical biophysical properties in terms of molecular weight and subunit composition, pH optima (pH 9.8, dehydrogenase; pH 7.5, isomerase), temperature optimum (37 degrees C), stability in storage and solution, effects of divalent cations, and the single NH2-terminal sequence of 27 amino acids. These results suggest that the mitochondrial and microsomal enzymes are the same protein localized in different organelles.

3-Hydroxysteroid Dehydrogenases↗

PubMed1989

Generation of a helper cell line for packaging avian leukosis virus-based vectors.

We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides.

Animals↗