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Biomedical subjects

B Roy

Publications and source records attributed to B Roy.

At least 55 records · Page 3Linked to original sources
PubMed1996

A case study on energy balance during an expedition through Greenland.

A 59-year-old man who had always been lean was submitted to overfeeding for two months in preparation for a 3-week expedition through Greenland, during which a considerable negative energy balance was expected. The overfeeding protocol consisted of an estimated energy surplus of 2000 kJ per day and this induced body weight and fat gains of 4.7 and 3.6 kg, respectively. Whole-body indirect calorimetry measurements following 4 and 8 weeks of overfeeding showed that there was no change in the subject's daily energy expenditure. Ad libitum energy intake measured during a buffet-type meal was substantially increased during overfeeding. As expected, the expedition induced a negative energy balance which reduced body weight below the pre-overfeeding level. Daily energy expenditure measured 12 days after the expedition, when the subject had recovered his initial body weight, was decreased by 1.4 MJ/day. The increase in ad libitum energy intake observed during the pre-expedition overfeeding persisted after the expedition. These adaptations tended to persist three weeks and four months after the end of the expedition. These results suggest that both energy expenditure and intake were modified after the expedition to prevent a subsequent body energy deficit.

Body Composition↗
PubMed1996

Identification of a set of protein species approximately 40 kDa as high-affinity DNA binding factor(s) to the cell cycle regulatory region of the human thymidine kinase promoter.

Promoter elements that are important for the G1-S induction of the human thymidine kinase (htk) promoter reside within the core of the cell cycle regulatory unit, positioned between -110 and -84 upstream of the TATA element. Within this 27-bp region are three GC-rich motifs, which resemble the E2F binding site. By site-directed mutagenesis, we identified a 14-bp region, between -97 and -84, critical for the htk promoter transcriptional activity. Methylation interference studies indicate that the sequences between -97 and -84 are major protein contact points, correlating with the functional significance of this sequence in vivo. Although the core of the cell cycle regulatory unit contains three E2F-like sites and can form minor S-phase-specific complexes containing p107, cyclin A, and cdk2, the major complex that binds to this region is not competed by E2F binding sites. Through DNA affinity chromatography, we identified a set of protein species of approximately 40 kDa that copurified with the htk DNA binding activity. From gel shift assays and Western blot analysis, this protein species is antigenically distinct from E2F-1, E2F-2, E2F-3, and E2F-4. Our studies raise the possibility that other members of the E2F protein family or a novel protein(s) with preferred binding affinity for the htk promoter exert(s) control on the G1 to S regulation of the htk promoter through their interactions with cyclins and kinases.

Animals↗
PubMed1995

Reexpression of retinoic acid receptor (RAR) gamma or overexpression of RAR alpha or RAR beta in RAR gamma-null F9 cells reveals a partial functional redundancy between the three RAR types.

Disruption of retinoic acid receptor (RAR) gamma in F9 embryonal carcinoma cells leads to aberrent differentiation and reduced activation of expression of several all-trans-retinoic acid (RA)-induced genes. We have analyzed the expression of several additional RA-responsive genes in RAR alpha- and RAR gamma-null F9 cells. The RA-induced activation of Cdx1, Gap43, Stra4, and Stra6 was specifically impaired in RAR gamma-null cells, supporting the idea that each RAR may regulate distinct subsets of target genes. To further investigate the role of RAR gamma in F9 cell differentiation, "rescue" cell lines reexpressing RAR gamma 2 or overexpressing either RAR alpha 1 or RAR beta 2 were established in RAR gamma-null cells. Reexpression of RAR gamma or overexpression of RAR alpha restored both target-gene activation and the differentiation potential. In contrast, over-expression of RAR beta only poorly restored differentiation, although it could replace RAR gamma for the activation of target genes. Functional redundancy between the various RARs is discussed.

Animals↗
PubMed1995

Effects of nifedipine and Bay K 8644 on myotropic responses in aortic rings of pregnant rats.

The hypothesis that Ca2+ channel function is altered during pregnancy was tested by comparing responses to potassium chloride (KCl) and phenylephrine in aortic rings of virgin and term-pregnant rats under the influence of nifedipine and Bay K 8644. Maximum response to KCl was progressively reduced by increasing nifedipine concentrations (1.0-100 nM) in both groups of tissues. Nifedipine produced a smaller inhibition of KCl-induced contraction in aortic rings of pregnant than of virgin rats. It exerted little inhibition on the concentration-response curve to phenylephrine. The Ca2+ channel antagonist (100 nM) reduced the maximum response to the alpha-adrenoceptor agonist in rings from virgin rats, but had no effect in pregnant rats. Bay K 8644, a Ca2+ channel activator, potentiated the responses to low concentrations of both phenylephrine and KCl in the tissues of both virgin and pregnant rats, but did not affect maximum responses. It also induced concentration-dependent contractions in rings of virgin but not of pregnant rats. The effects of Bay K 8644 were markedly potentiated by precontracting the aorta with 10mM KCl. Nevertheless tissues from pregnant rats were still less responsive to Bay K 8644. However, when the strips were precontracted to the same level by different concentrations of KCl, the concentration-response curves to Bay K 8644 were identical in both groups. [3H]Nitrendipine binding to membrane preparations of the thoracic aorta was similar in virgin and pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Inhibition of ribonucleotide reductase by nitric oxide derived from thionitrites: reversible modifications of both subunits.

Thionitrites are spontaneous nitric oxide (NO) donors in neutral aqueous solutions. Consequently, they inhibit ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis, from Escherichia coli and murine adenocarcinoma TA3 cells. They also inhibit tumor cell proliferation. Reaction of thionitrites with protein R1, the large subunit, results in the nitrosation of cysteines, as shown from the formation of a chromophore with a characteristic absorption at 340 nm. EPR spectroscopy both on whole murine R2-overexpressing L1210 cells and on the pure protein showed that the tyrosyl radical of protein R2, the small subunit, reversibly couples to the NO radical, presumably leading to nitrosotyrosine adducts. Both molecular events might be at the origin of the inhibition of ribonucleotide reductase by NO, since a number of cysteines and the tyrosyl radical are essential for catalysis. These results identify NO donors as a new class of inhibitors of ribonucleotide reductase with potential applications as anticancer or antiviral chemotherapy agents.

Adenocarcinoma↗
PubMed1995

NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli.

5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates. The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.4.1) has been studied with 19F-NMR. The product analogue is shown to be in fast exchange with substrate binding sites on protein subunit 1 (R1) of ribonucleoside-diphosphate (NDP) reductase. The number of binding sites is reduced to half when the complete holoenzyme R1R2 is formed. The temperature dependence of the line broadening of 5-FdUDP was studied using 19F-NMR, and of dCDP and dUDP using 1H-NMR. The temperature dependences are complex and a molecular model in which R1 is in a temperature dependent equilibrium between at least two conformations is suggested in order to explain the observed behaviour. Binding of a ligand to the substrate binding sites affects the conformational equilibrium in a ligand specific way. Formation of the holoenzyme R1R2 also affects the equilibrium.

Binding Sites↗
PubMed1995

Inducible expression of Mad accelerates growth arrest of serum deprived human glioblastoma cells.

Mad is a basic-helix-loop-helix leucine zipper protein that heterodimerizes with Max. Mad:Max heterodimers recognize the same DNA binding sites as Myc:Max heterodimers. However, Myc and Mad are thought to influence transcription and cell proliferation in opposite ways through interaction with Max. While Myc activates transcription and cell proliferation Mad represses these activities (Ayer et. al., Cell 72, 211-222, 1993). We have constructed an expression vector containing the mad cDNA cloned downstream of an inducible viral promoter. Cell lines carrying the construct were derived by transfection into human glioblastoma cells and the effect of Mad induction on their growth rate was studied. When the cells were cultured in low serum concentrations (0.1%) Mad induction resulted in accelerated growth arrest. These findings are consistent with the proposed growth repression function of the Mad protein.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed1995

Some aspects of the causes of enhanced immune response of in vitro frozen ascites fibrosarcoma tumor cells in mice.

Estimation of 3 M KCl-extracted ascites fibrosarcoma (AFS) tumor cell membrane peripheral proteins in native and frozen tumor cells showed approximately a three-, four-, and fivefold increase per 1 x 10(6) cells of single-, three-, and programmed three-cycle frozen AFS tumor cells, respectively, compared to the same number of native cells, indicating an increase in surface membrane protein concentration with freezing. The 10% gel (homogeneous) electrophoretic (SDS-PAGE) study of 3 M KCl-extracted native and frozen cell membrane proteins showed (i) membrane proteins of native cells resolve into many more components compared to those of any frozen membranes, i.e., single, three, and programmed three cycle, the components decreasing in that order; (ii) the concentration of larger-molecular-weight protein fractions (> or = 75 kDa) decreases while those of smaller fractions (14 to 24 kDa) increase in frozen cells, with the maximum being in the programmed three-cycle frozen group. In contrast, the native cell membrane is rich in higher-molecular-weight proteins (> or = 75 kDa) with concentrations slowly decreasing toward lower-molecular weight fractions. Thus, the probable reasons for increased immune response of animals immunized with frozen AFS tumor cells are (i) absolute increase in cell surface protein concentrations as given by 3 M KCl extraction of cell membrane peripheral protein estimation in AFS tumor cells postfreeze; (ii) cell-surface protein pattern which is heterogeneous before freezing becomes relatively more homogeneous following freezing of AFS tumor cells; and (iii) depolymerization and breaking of higher-molecular-weight components which increase the concentration of terminal-sequence antigenic determinants and increase accessibility of determinant grouping by removing steric hindrance following freezing.

Animals↗
PubMed1995

Effect of drafting on work intensity in classical cross-country skiing.

The purpose of this study was to compare the heart rate responses during cross-country skiing as a leading skier, as well as in a drafting situation, three meters behind the leader. Eight male and two female cross-country ski racers, paired for skiing ability, skied a 2 km course (two loops of 1 km) using the diagonal stride and double-poling techniques at a fixed speed (4.75 m.s-1 and 4.45 m.s-1 for males and females respectively) on two different occasions, once as a leading skier, the other as a drafter. A recovery period of 30 minutes was allowed between the two trials. Heart rates (HR) were registered every five seconds during all performances. Results revealed that HR were significantly lower (165 vs 172 beats.min-1) when skiing behind another skier as opposed to leading. Results also revealed that projected frontal areas appeared to influence the effects of drafting such that the HR differences between the leading and the drafting situations were larger for smaller skiers drafting behind larger skiers. These results showed that skiing behind another skier in a classical cross-country ski race would be very advantageous when the situation is encountered and could help racers using this energy saving strategy.

Adult↗
PubMed1995

FK506 immunosuppression to control the immune reactions triggered by first-generation adenovirus-mediated gene transfer.

Despite good initial success in vivo, gene transfer using first-generation replication-defective adenovirus has been reported to lead to transient reporter gene expression and to trigger inflammatory reactions in various organs and animal models. To gain more knowledge on this phenomenon, immune reactions were investigated following in vivo transfection of adult immunocompetent mouse muscle using a delta E1/E3a adenoviral vector encoding a beta-galactosidase (beta-Gal) expression cassette. Cellular and humoral immune reactions, and rejection of beta-Gal-positive muscle fibers, occurred within 3 weeks. The muscles showed massive infiltration by macrophages, natural killer cells, and CD8+ leukocytes. The mRNA levels of granzyme B and interferon-gamma were increased 6 days after vector injection, indicating that the infiltrating lymphocytes were activated. Antibodies were formed against the adenovirus group antigen and the beta-Gal gene product 2 weeks after construct injection. The immunosuppressant FK506, however, blocked the cellular infiltration and the humoral response and allowed strong, stable transgene expression over 1 month. These data emphasize the immune problems related to the use of delta E1/E3a adenoviruses as vectors for gene therapy, and they underline the potential of FK506 as an immunosuppressant adjunct treatment for adenovirus-mediated gene transfer.

Adenoviridae↗
PubMed1995

Synergistic activation of retinoic acid (RA)-responsive genes and induction of embryonal carcinoma cell differentiation by an RA receptor alpha (RAR alpha)-, RAR beta-, or RAR gamma-selective ligand in combination with a retinoid X receptor-specific ligand.

Retinoic acid receptor (RAR)-retinoid X receptor (RXR) heterodimers bind to cognate response elements in vitro more efficiently than do RAR or RXR homodimers, and both RAR and RXR partners have been shown to activate various promoters in transiently transfected cells. We have now investigated whether ligand-dependent activation of both heterodimeric partners is involved in induced expression of endogenous RA-responsive genes and in P19 and F9 cell differentiation. On their own, low concentrations of retinoids selective for either RAR alpha, RAR beta, or RAR gamma did not induce or very inefficiently induced the expression of several RA target genes or triggered differentiation. An RXR-specific synthetic retinoid was similarly inefficient at any concentration. In contrast, at the same concentrations, various combinations of RAR (RAR alpha, RAR beta, or RAR gamma) and RXR selective retinoids resulted in synergistic induction of all retinoic acid (RA) target genes examined, as well as in cell differentiation. However, the magnitude of this synergistic activation varied depending on both the RAR-RXR combination and the promoter context of the responsive genes. Promiscuous activation of the three RARs, or concomitant activation of RAR alpha and RAR gamma, at selective retinoid concentrations also resulted in induction of gene expression and cell differentiation. Taken together, our results are consistent with the conclusion that the RAR and RXR partners of RAR-RXR heterodimers can synergistically activate transcription of RA-responsive genes and can induce differentiation of P19 and F9 cells. Our results also indicate that there is a significant degree of functional redundancy between the three RAR types which, however, varies with the nature of the RA target genes.

Animals↗
PubMed1995

Transduction of calcium stress through interaction of the human transcription factor CBF with the proximal CCAAT regulatory element of the grp78/BiP promoter.

Mammalian GRP78/BiP is a stress-inducible 78-kDa endoplasmic reticulum (ER) protein with molecular chaperone and calcium-binding properties. The transactivation of grp78 by the calcium ionophore A23187 provides a model system with which to study the signal transduction that allows mammalian cells to sense calcium depletion in intracellular stores and activate transcription of specific genes. Linker-scanning mutation analysis of the grp78 promoter reveals that the single most important regulatory element is C1, which contains a CCAAT motif most proximal to the TATA sequence. The C1 element is crucial for mediating the stimulatory effects by the upstream regulatory elements under normal and stress conditions. In this report, we establish that the heteromeric CCAAT-binding factor CBF is the major component of the C1-binding factor (C1F) in human cells. A GGAGG motif flanking the CCAAT sequence also contributes to high-affinity C1F/CBF binding. We show here that the binding of C1F in vitro is sensitive to the concentration of calcium ions. At high calcium ion concentrations, the C1F-binding activity is lower because of a higher dissociation rate. This binding characteristic correlates with the induction of grp78 transcription in response to the depletion of intracellular calcium stores. The strikingly similar behavior of C1F from nuclear extracts of control and A23187-treated cells further suggests that C1F itself does not undergo any major inherent changes after calium depletion stress. Rather, its binding property could be modulated by the immediate calcium ionic environment in stressed and nonstressed cells. On the basis of the in vitro and in vivo site occupancies of C1F and other stress-inducible changes of upstream regulatory complexes, we present a model to explain how C1F and other upstream factors can synergistically activate grp78 transcription in calcium-depleted cells.

Animals↗
PubMed1995

Survivability and leucocyte migration inhibition in mice immunized with cryotreated ascites fibrosarcoma cells using different freeze-thaw cycles.

Attempts have been made to assess as to what extent in vitro assay of cellular immunity, e.g. leucocyte migration inhibition (LMI) in mice immunized with different freeze-thaw cycles could reflect host resistance in vivo. While survivability of animals improved significantly by immunization with single cycle (P < 0.05) to three cycle (P < 0.001) and programmed three cycle (P < 0.001) cryo-treated tumor cells compared to controls, the percentage LMI in the same groups of animals decreased progressively. The KCl(3M) extracted tumor cell protein (antigen) of both viable and cryo-treated cells showed a progressively increased protein concentrations per 1 x 10(6) tumor cells with viable cells being least and programmed three cycle cryo-treated cells highest. The apparent discrepancy observed between percentage migration inhibition and survivability may be due to the fact that (1) survivability is a function of body's total immune response while LMI represents the response of one effector limb only; (2) immuno-regulatory mechanisms depend on a balance between activation and suppression and suppressor cells being more sensitive and of shorter life span, affect migration inhibition but not the survivability; (3) cryo-treatment alters tumor cell surface antigen affecting immunological balance; and (4) suppressor and antitumor activities against antigenic stimulation develop simultaneously in different organs and LMI performed with sensitized splenic cells, where, perhaps, suppressor cell activity dominates.

Animals↗
PubMed1995

The Tuskegee Syphilis Experiment: biotechnology and the administrative state.

The central issue of the Tuskegee Syphilis Experiment was property: property in the body and intellectual property. Once removed from the body, tissue and body fluids were not legally the property of the Tuskegee subjects. Consequently, there was not a direct relationship between a patient and research that used his sera. The Public Health Service (PHS) was free to exercise its property right in Tuskegee sera to develop serologic tests for syphilis with commercial potential. To camouflage the true meaning, the PHS made a distinction between direct clinical studies and indirect studies of tissue and body fluids. This deception caused all reviews to date to limit their examination to documents labeled by the PHS as directly related to the Tuskegee Syphilis Experiment. This excluded other information in the public domain. Despite the absence of a clinical protocol, this subterfuge led each to falsely conclude that the Tuskagee Syphilis Experiment was a clinical study. Based on publications of indirect research using sera and cerebrospinal fluid, this article conceives a very history of the Tuskagee Syphilis Experiment. Syphilis could only cultivate in living beings. As in slavery, the generative ability of the body made the Tuskegee subjects real property and gave untreated syphilis and the sera of the Tuskegee subjects immense commercial value. Published protocols exploited the Tuskegee Syphilis Experiment to invent and commercialize biotechnology for the applied science of syphilis serology.

Alabama↗
PubMed1995

Upper-body testing of cross-country skiers.

The purpose of this study was to evaluate maximal oxygen uptake during treadmill running and double-poling with a ski ergometer twice during the competitive season. A progressive double-poling power test was also performed to measure maximal power output. The athletes were five male and five female cross-country ski racers. Testing was done in December and April. Results showed that for both male and female skiers, no difference was observed during the competition season. Athletes were then divided into two subgroups according to whenever they had their peaking period early (December-January) or late (March-April) during the season. Skiers peaking early had significantly higher values for all measurements during the upper-body VO2peak test in December compared to April. Values for the upper-body VO2peak test of the skiers peaking late significantly increased from December to April, in accordance with their fitness state. Furthermore, results of the upper-body power test showed that there was no difference for the skiers peaking early for both testing periods, while power output increased significantly from December to April for the skiers peaking late. Thus specific upper-body tests appear to be more related to the fitness state of cross-country skiers than a running VO2max test.

Adult↗
PubMed1995

Deduced amino acid sequence of 2S storage protein from Brassica species and their conserved structural features.

2S seed storage albumin coding regions from five Brassica species, namely Brassica campestris, B. oleracea, B. nigra, B. juncea, and B. carinata have been cloned by PCR amplification of genomic DNA using oligonucleotide primers and their nucleotide sequences have been determined. These sequences showed more than 85% homology amongst themselves and considerable homology with some other crucifer 2S protein coding sequences. The deduced amino acid sequences showed more homology due to some inconsequential mutations in codons without changing the amino acids. Computer analysis of the protein sequences for possible secondary structure revealed a high degree of conservation of hydrophilic and hydrophobic domains and the invariant positions of cysteine residues. Unrooted phylogenic tree based on the coding region of 2S albumin from different Brassica species cloned by us and published sequences from other Cruciferae indicated that these genes originated before the evolutionary divergence of different Brassica species and were conserved due to some stringent structural and functional features required for seed metabolism.

Amino Acid Sequence↗