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Biomedical subjects

B Roy

Publications and source records attributed to B Roy.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1994

Activation function 2 (AF-2) of retinoic acid receptor and 9-cis retinoic acid receptor: presence of a conserved autonomous constitutive activating domain and influence of the nature of the response element on AF-2 activity.

A motif essential for the transcriptional activation function 2 (AF-2) present in the E region of retinoic acid receptor (RAR) alpha and 9-cis retinoic acid receptor (RXR) alpha has been characterized as an amphipathic alpha-helix whose main features are conserved between transcriptionally active members of the nuclear receptor superfamily. This conserved motif, which can activate autonomously in the absence of ligand in animal and yeast cells, can be swapped between nuclear receptors without affecting the ligand dependency for activation of transcription, thus indicating that a ligand-dependent conformational change is necessary to reveal the AF-2 activation potential within the E region of the nuclear receptor. Interestingly, we show that the precise nature of the direct repeat response element to which RAR/RXR heterodimers are bound can affect the activity of the AF-2s of the heterodimeric partners, as well as the relative efficiency with which all-trans and 9-cis retinoic acids activate the RAR partner.

Amino Acid Sequence↗
PubMed1994

Erp61 is GRP58, a stress-inducible luminal endoplasmic reticulum protein, but is devoid of phosphatidylinositide-specific phospholipase C activity.

Using antibody raised against putative Form I phosphatidylinositide-specific phospholipase C (PI-PLC) and direct amino acid sequencing of the protein recognized by this antibody, we have shown that the antibody reacts with luminal endoplasmic reticulum (ER) proteins, including ERp61. ERp61 possesses a COOH-terminal QEDL sequence that acts as an ER retention signal. Additional experiments have shown, however, that PI-PLC activity is separable from ERp61 and that rat or murine ERp61 expressed in COS cells failed to produce an increase in PI-PLC activity in the COS cells. Finally, we have identified ERp61 as GRP58, a 58-kDa protein inducible by glycosylation block and treatment with the Ca2+ ionophore, A23187.

Amino Acid Sequence↗
PubMed1994

Transactivation of the human p53 tumor suppressor gene by c-Myc/Max contributes to elevated mutant p53 expression in some tumors.

Elevated levels of mutant forms of the p53 tumor suppressor are a hallmark of many transformed cells. Multiple mechanisms such as increased stability of the protein and increased transcription of the gene can account for elevated p53 expression. Recent findings indicate that c-Myc/Max heterodimers can bind to an essential CA(C/T)GTG-containing site in the p53 promoter and elevate its expression. We have addressed the possibility that elevated mutant p53 expression is due to deregulated c-Myc expression. Here we demonstrate that the human p53 promoter is transactivated by high c-Myc expression and repressed by high Max expression. In examining the relative levels of c-Myc and p53 in human Burkitt's lymphomas and other B-lymphoid lines, we found that there is a correlation between the levels of c-Myc protein and p53 mRNA expression. In particular, cells that express very low levels of c-Myc protein also express low levels of p53 mRNA, while cells that express high levels of c-Myc tend to express high levels of p53 mRNA. To determine whether the p53 gene can be a target for c-Myc in vivo, we assayed the effects of antisense c-myc RNA on the levels of endogenous p53 mRNA. The results indicate that the presence of antisense c-myc RNA leads to a reduction in the levels of c-Myc protein, p53 mRNA, and expression from the p53 promoter. Taken together, our findings support a direct role for c-Myc in elevating expression of the mutant p53 gene in some tumors.

Animals↗
PubMed1994

The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications.

The knowledge of GRPs as molecular chaperones is rapidly evolving. It is anticipated that the GRPs will make special contributions in the areas of basic cell biology, biotechnology, and cancer biology. In particular, they may play a role as the prototype of a class of genes that are regulated by signal transduction pathways originating in the ER and traveling to the nucleus. GRP78 and GRP94 function as molecular chaperones and can bind to malfolded proteins and unassembled complexes. They are induced in response to stress, but once the stress is removed the GRPs are posttranscriptionally modified into biologically inactive forms. The promoters of the grp genes are highly conserved, with several CCAAT-like motifs and GC-rich regions. The high level of redundancy that exists in the mammalian grp promoters may act to ensure that the expression of the genes, both of which are single copy, is unlikely to be significantly lowered in the event of mutation. These genes are thought to be controlled by several transcription factors whose complex interactions with the grp promoters allow variable patterns of grp induction. The promoters of the grp genes constitutively express their gene products, and their promoter activities can be further enhanced in cellular environments of low glucose or oxygen. The grp78 promoter is known to retain its strong activity in differentiated and undifferentiated tissues. These features make it an attractive alternative to viral promoters for use in gene therapy. Gene therapy may also be useful in treating cancer in some cases, especially solid tumors. In these instances, GRP levels are already likely to be quite high. These high levels of GRPs may inhibit the efficacy of several anti-cancer treatments. Suppression of GRP induction, perhaps by anti-sense or ribozyme technology, may prove to be useful in conjunction with anti-cancer drugs to treat tumors.

Animals↗
PubMed1994

Effect of drafting on heart rate in 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, 2 m behind the leader. Six male cross-country ski racers, paired for VO2max skied a 2-km course (two loops of 1 km) using the skating techniques at a fixed speed (5.6 m.s-1) on two different occasions, once as a leading skier, the other as a drafter. A recovery period of 30 min was allowed between the two trials. Heart rates (HR) were monitored every 5 s during all performances. Capillary blood lactate concentrations were taken before and at 5 min posttrial. Results revealed that HR were significantly lower (154 vs 163 beats.min-1) when skiing behind another skier as opposed to leading. Lactate concentrations were not significantly different between the two trials. 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 ski race would be very advantageous when the situation is encountered and that coaches should use the drafting situation in training so that athletes can practice this energy-saving strategy.

Adult↗