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↗

PubMed1994

Use of retroviral vectors to introduce and express the beta-galactosidase marker gene in cultured chicken primordial germ cells.

Three methods of isolating primordial germ cells (PGCs) from gonads of 5-day-old chick embryos were compared. PGCs were then cultured in vitro in DMEM/F12 medium containing 10% fetal calf serum. BrdU incorporation showed that at least 10% of the PGC population were dividing, under our culture conditions, during the 2nd day of in vitro culture. During this culture period, PGCs were exposed to avian leukosis sarcoma virus-based retroviral vector pseudotyped with subgroup A envelope, carrying the LacZ reporter gene. X-Gal staining showed that PGCs were permissive to infection, with more than 50% of PGCs expressing the beta-Gal protein. These data represent the first demonstration that PGCs, isolated from gonads of 5-day-old chick embryos, are able to divide in vitro and that it is possible to introduce and express exogenous DNA in chick PGCs maintained in vitro.

Animals↗

PubMed1994

Over-expression of human type I (placental) 3 beta-hydroxy-5-ene-steroid dehydrogenase/isomerase in insect cells infected with recombinant baculovirus.

Human type I placental 3 beta-hydroxy-5-ene-steroid dehydrogenase/steroid 5-->4-ene-isomerase (3 beta-HSD/isomerase) synthesizes androstenedione from fetal dehydroepiandrosterone and progesterone from pregnenolone. The full length cDNA that encodes type I 3 beta-HSD/isomerase was inserted into the baculovirus, Autographa californica multiple nucleocapsid polyhedrosis virus, and expressed in Spodoptera fungiperda (Sf-9) insect cells. Western blots showed that the baculovirus-infected Sf-9 cells produced an immunoreactive protein that co-migrated with purified placental 3 beta-HSD/isomerase. Ultracentrifugation localized the expressed enzyme activities in all the membrane-associated organelles of the Sf-9 cell (nuclear, mitochondrial and microsomal). Kinetic studies showed that the expressed enzyme has 3 beta-HSD and isomerase activities. The Michaelis-Menton constant is very similar for the 3 beta-HSD substrate, 5 alpha-androstan-3 beta- ol-17-one, in the Sf-9 cell homogenate (Km = 17.9 microM) and placental microsomes (Km = 16.7 microM). The 3 beta-HSD activity (Vmax = 14.5 nmol/min/mg) is 1.6-fold higher in the Sf-9 cell homogenate compared to placental microsomes (Vmax = 9.1 nmol/min/mg). The Km values are almost identical for the isomerase substrate, 5-androstene-3,17-dione, in the Sf-9 cell homogenate (Km = 14.7 microM) and placental microsomes (Km = 14.4 microM). The specific isomerase activity is 1.5-fold higher in the Sf-9 cells (Vmax = 25.7 nmol/min/mg) relative to placenta (Vmax = 17.2 nmol/min/mg). These studies show that our recombinant baculovirus system over-expresses fully active enzyme that is kinetically identical to native 3 beta-HSD/isomerase in human placenta.

Animals↗

PubMed1994

Membrane solubilization by a hydrophobic polyelectrolyte: surface activity and membrane binding.

We have previously observed that the hydrophobic polyelectrolyte poly(2-ethylacrylic acid) solubilizes lipid membranes in a pH-dependent manner, and we have exploited this phenomenon to prepare lipid vesicles that release their contents in response to pH, light, or glucose (Thomas, J. L., and D. A. Tirrell. Acc. Chem. Res. 25:336-342, 1992). The physical basis for the interaction between poly(2-ethylacrylic acid) and lipid membranes has been explored using surface tensiometry and fluorimetry. Varying the polymer concentration results in changes in surface activity and membrane binding that correlate with shifts in the critical pH for membrane solubilization. Furthermore, the binding affinity is reduced as the amount of bound polymer increases. These results are consistent with a hydrophobically driven micellization process, similar to those observed with apolipoproteins, melittin, and other amphiphilic alpha-helix-based polypeptides. The absence of specific secondary structure in the synthetic polymer suggests that amphiphilicity, rather than structure, is the most important factor in membrane micellization by macromolecules.

Acrylates↗

PubMed1994

Large-scale co-aggregation of fluorescent lipid probes with cell surface proteins.

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface of RBL cells results in the co-aggregation of a large fraction of the lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels the plasma membranes much more uniformly in the absence of receptor aggregation. Most of the diI molecules that are localized in patches of aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The diI outside of patches is mobile, and its mobility is similar to that in control cells without receptor aggregates. It is unlikely that the co-aggregation of diI with IgE receptors is due to specific interactions between these components, as two other lipophilic probes of different structures are also observed to redistribute with aggregated IgE receptors, and aggregation of two other cell surface antigens also results in the coredistribution of diI at the RBL cell surface. Quantitative analysis of CCD images of labeled cells reveals some differences in the spatial distributions of co-aggregated diI and IgE receptors. The results indicate that cross-linking of specific cell surface antigens causes a substantial change in the organization of the plasma membrane by redistributing pre-existing membrane domains or causing their formation.

Carbocyanines↗

PubMed1994

Two proteins associated with secretory granule membranes identified in chicken regulated secretory cells.

Lately, we have identified two polypeptides of 92-94 kDa (GRL1) and 45-60 kDa (GRL2), expressed in cytoplasmic granules of chicken granulocytes and thrombocytes. Here, we report that GRL1 and GRL2 are widely distributed in all exocrine and several endocrine cell types, but not in neurons of the central nervous system, during late stages of embryonic development, as well as in newly hatched and two-month-old chickens. Immunogold studies in ultrathin frozen sections of pancreatic acinar cells show that GRL1 and GRL2 are co-localized at the periphery of zymogen granules, in granules fused with apical acinar membranes and on apical membranes of acini, while the pregranular compartments of the secretory pathway are weakly or not labeled. Semiquantitative morphometric studies indicate that GRL1 and GRL2 are equally distributed in secretory granules. A variety of physical and metabolic studies reveal that GRL2, a highly N-glycosylated polypeptide, is an intrinsic membrane protein, while GRL1 is a peripheral membrane polypeptide released by Na2CO3 treatment of granulocyte membranes. In all hematopoietic, exocrine or endocrine cells examinated, GRL1 shows identical electrophoretic patterns, while GRL2 is identified as a diffuse band, at 40-65 kDa, in hematopoietic and pancreatic cells. Taken together, the morphological and biochemical studies indicate that GRL1 and GRL2 are components of the secretory granule membrane in chicken exocrine, endocrine and hemopoietic cell types.

Animals↗

PubMed1994

Dysplasia epiphysealis hemimelica affecting the subtalar joint.

The case presented here is unique because of the location of the lesions and the extent to which the foot was affected. A two-stage procedure was performed because of the degree of bone deformity. The first stage of the procedure was directed at reducing osseous proliferation; the second stage addressed the frontal and transverse plane deformities. One-stage procedures can be used in treating less severe deformities.

Adolescent↗

PubMed1993

Affinity radiolabeling identifies peptides and amino acids associated with substrate binding in human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase.

Purified human placental 3 beta-hydroxy-delta(5)-steroid dehydrogenase (3 beta-HSD) was affinity radiolabeled by 2 alpha-bromo[2'-14C]acetoxyprogesterone (2 alpha-BAP) in the presence or absence of 3 beta-HSD substrate, pregnenolone. The substrate steroid substantially protects 3 beta-HSD activity from inactivation by 2 alpha-BAP. Tryptic peptides of unprotected and substrate-protected radioalkylated enzyme were purified by high pressure liquid chromatography. The amino acid sequence of each radiolabeled peptide was determined and localized within the cDNA-derived primary structure of the enzyme. According to the percent total radioactivity associated with each of four radiolabeled peaks separated by high pressure liquid chromatography, two peptides were protected by substrate from affinity radioalkylation by 2 alpha-BAP. The first, 251GQFYYISDDTPHQSYDNLNYTLSK274, was produced by tryptic cleavage at Arg-250 and Lys-274 (the Arg-250 peptide) and contained radiolabeled His262. The second, 176NGGTLYTCALR186, was produced by tryptic cleavage at Lys-175 and Arg-186 (the Lys-175 peptide) and contained radiolabeled Cys183. Based on amino acid analysis to quantitate radioactivity incorporated per nmol of peptide, substrate steroid decreased the radiolabeling of His262 in the Arg-250 peptide by 3.6-fold and decreased the radiolabeling of Cys183 in the Lys-175 peptide by 3.7-fold. Three minor radiolabeled peptides (the NH2-terminal, Arg-71, and Arg-196 tryptic peptides) were also identified in the primary structure, but pregnenolone did not diminish their affinity radioalkylation. These observations indicate that the Arg-250 and Lys-175 peptides are involved in substrate binding and suggest that His262 and Cys183 are in close proximity in the three-dimensional structure of the enzyme.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Identification in the chicken of GRL1 and GRL2: two granule proteins expressed on the surface of activated leukocytes.

We report the production of two monoclonal antibodies reacting, respectively, with a 92-kDa protein (GRL1) and a 40- to 65-kDa membrane glycoprotein (GRL2), both present in chicken thrombocyte and myelocyte granules. We examined the expression of GRL1 and GRL2 during the development of the hematopoietic system: GRL1 is restricted to thrombocytes and myelocytes, whereas GRL2 is present in thrombocytes, myelocytes, myeloid progenitors, and a subpopulation of erythroid progenitors. In the lymphoid lineages, neither GRL1 nor GRL2 is expressed during thymus and bursa ontogeny or on resting peripheral blood lymphocytes. However, CD3+ T lymphoblasts obtained by mitogenic stimulation of GRL2-negative quiescent T lymphocytes are stained on their surface by anti-GRL2 Mab. In vitro stimulation of thrombocytes and granulocytes with their specific secretagogues results in the expression of GRL1 and in the overexpression of GRL2 on the cell surface. These observations are consistent with the following two conclusions: the presence on the cell surface of GRL1 epitope is a marker of thrombocyte and myelocyte activation; GRL2 epitope is present on the granule membrane of leukocytes, including T cells. In that respect, GRL2 appears to share certain features with leukocyte activation antigens recently described in human.

Animals↗

PubMed1993

Affinity labeling identifies histidine at the active site of human placental 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase.

OBJECTIVE: Our aim was to determine if the multifunction enzyme, 3 beta-hydroxysteroid dehydrogenase and steroid 5-->4-ene-isomerase has one or more active sites to effect dehydrogenase and isomerase activities. STUDY DESIGN: This steroid, which we have purified to homogeneity from human placental microsomes, was inactivated by the affinity labeling steroid, 2 alpha-bromo[2'-14C]acetoxyprogesterone. The amino acids that were radioalkylated in the absence and presence of the dehydrogenase substrate pregnenolone were identified. RESULTS: Pregnenolone completely abolished the inactivation of dehydrogenase. Histidine was localized in the active site of 3 beta-hydroxysteroid dehydrogenase because the radiolabel disappeared from enzyme inactivated in the presence of pregnenolone. Cysteine, a major radiolabeled product (80%) in the absence of pregnenolone, was decreased twofold in incubations that contained pregnenolone. Neither pregnenolone nor the isomerase substrate 5-androstene-3,17-dione protected isomerase from inactivation by the affinity alkylator. CONCLUSION: This observation contradicts coexisting, separate binding sites, one for each activity. Rather, a conformation shift around one binding region prompted by products of the dehydrogenase reaction may create the isomerase activity.

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Enzyme immunoassay for nomegestrol acetate in human plasma.

Currently available chromatographic assays of the progestative drug nomegestrol acetate in human plasma are not suitable for monitoring drug kinetics more than 24 h after clinical dosage. A specific and sensitive enzyme immunoassay was therefore developed. A 3(O-carboxymethyl)oxime derivative of nomegestrol acetate was synthesized and coupled to bovine serum albumin in order to raise polyclonal antibodies in rabbits. The enzymatic tracer was obtained by coupling the 3(O-carboxymethyl)oxime derivative to acetylcholinesterase (E.C.3.1.1.7.). HPLC fractionation of human plasma samples followed by enzyme immunoassay revealed the presence of cross-reacting metabolites. An automated procedure of metabolite separation was developed using silica bonded with diol groups (Diol Bakerbond column). This procedure ensured assay specificity. The quantification limit in human plasma was 0.1 ng/ml. Mean repeatability (intra-assay variation) and reproducibility (inter-assay variation) were 9 and 15%, respectively. The enzyme immunoassay allowed monitoring of the kinetics of nomegestrol acetate 144 h after oral administration of a single 5 mg dose. Values for human samples were in excellent agreement with those assayable by HPLC followed by u.v. detection.

Chromatography, High Pressure Liquid↗

PubMed1993

3 beta-hydroxysteroid dehydrogenase activity in tissues of the human fetus determined with 5 alpha-androstane-3 beta,17 beta-diol and dehydroepiandrosterone as substrates.

3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD)/delta 5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3 beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3 beta-HSD/delta 5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3 beta-HSD activity. For this purpose, we compared the efficiencies of a 3 beta-hydroxy-5-ene steroid (DHEA) and a 3 beta-hydroxy-5 alpha-reduced steroid (5 alpha-androstane-3 beta,17 beta-diol, 5 alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (Km) for 5 alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent Km of placental 3 beta-HSD for 5 alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5 alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5 alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to Kms and rates of product formation was obtained by use of purified placental 3 beta-HSD with DHEA, pregnenolone, and 3 beta-hydroxy-5 alpha-androstan-17-one (epiandrosterone) as substrates: the Km of 3 beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein.min and, with epiandrosterone, 127 nmol/mg protein.min. With placental homogenate as the source of 3 beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [3H]5 alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [3H]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5 alpha-A-diol (40 mumol/l).(ABSTRACT TRUNCATED AT 400 WORDS)

3-Hydroxysteroid Dehydrogenases↗

PubMed1993

Concerns regarding adult children's assistance: a comparison of young-old and old-old parents.

This study describes differences between young-old (ages 60-74) and old-old (ages 75-90) parents' concerns regarding assistance provided by adult children. Old-old parents expressed more concern regarding amounts of help than did younger parents; there were no age differences in levels of concern regarding help outcomes. The percentage of children receiving parents' help and reliance on formal services predicted level of concern regarding help outcomes for the young-old parents, but not the old-old parents. Familism values and the extent of help received predicted level of concern regarding help amounts for the old-old parents, but not for younger parents. Implications for interventions to minimize parents' concerns and for further study of family assistance to the older people are discussed.

Aged↗

PubMed1993

Hospital Outcomes Project for the Elderly (HOPE): rationale and design for a prospective pooled analysis.

OBJECTIVE: To describe a collaborative investigation that is based on a series of six clinical studies aimed at reducing functional decline in the acutely-ill hospitalized elderly. DESIGN: A prospective, multicenter pooled analysis project involving collection of a common set of data from a group of related but distinct intervention trials with similar objectives. SETTING: Five university-affiliated hospitals and one community hospital. PARTICIPANTS: Elderly patients (age minimums from 65 to 75 years) admitted for a range of acute illnesses. INTERVENTIONS: Site-specific interventions include exercise and physical therapy; developing and implementing methods to improve detection and evaluation of delirious patients; a multidisciplinary geriatric care unit; a multidisciplinary intervention implemented in-hospital that includes some post-discharge care; and a nursing-centered geriatric care program. MAIN OUTCOME MEASURE: Functional status. CONCLUSION: The prospective, multicenter design of the Hospital Outcomes Project for the Elderly (HOPE) provides an innovative approach for analysis of hospital outcomes in the elderly. Although differences in study populations and interventions exist, qualitative comparisons across sites will enhance generalizability and will provide a great opportunity to examine consistency among the sites. The HOPE pooled analysis project will impart greater statistical power to detect the primary and secondary outcomes compared with previous single-center trials that have assessed interventions related to functional decline in the hospitalized elderly.

Activities of Daily Living↗

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↗