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Biomedical subjects

F Martin

Publications and source records attributed to F Martin.

At least 163 records · Page 9Linked to original sources
PubMed1997

The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44.

Preprotein import into mitochondria is mediated by translocases located in the outer and inner membranes (Tom and Tim) and a matrix Hsp70-Tim44 driving system. By blue native electrophoresis, we identify an approximately 90K complex with assembled Tim23 and Tim17 as the core of the inner membrane import site for presequence-containing preproteins. Preproteins spanning the two membranes link virtually all Tim core complexes with one in four Tom complexes in a stable 600K supercomplex. Neither mtHsp70 nor Tim44 are present in stoichiometric amounts in the 600K complex. Preproteins in transit stabilize the Tim core complex, preventing an exchange of subunits. Our studies define a central role for the Tim core complexes in mitochondrial protein import; they are not passive diffusion channels, but can stably interact with preproteins and determine the number of translocation contact sites. We propose the hypothesis that mtHsp70 functions in protein import not only by direct interaction with preproteins, but also by exerting a regulatory effect on the Tim channel.

Amino Acid Sequence↗
PubMed1997

Tumor-infiltrating dendritic cells are defective in their antigen-presenting function and inducible B7 expression in rats.

Tumors are tolerated by the immune system notwithstanding the expression of tumor-associated antigens. PROb tumor cells, derived from a rat colon carcinoma, are rejected by tumor-immune hosts but give rise to progressive tumors in naive hosts. Paradoxically, these tumors are heavily infiltrated by dendritic cells that express MHC class II and ICAM-1. These tumor-infiltrating dendritic cells (TiDCs) could be expected to process and present to T cells the antigens released by the adjacent tumor cells. Indeed, we report here that TiDCs, compared with splenic dendritic cells, are poor stimulators of primary allogeneic T-cell proliferation and cytokine [interleukin-2 (IL-2) and interferon-gamma] production. Most of them (89-97%) do not express B7, an essential co-stimulatory signal for T cells, even after a culture period allowing B7 up-regulation on epidermal Langerhans cells. GM-CSF in association with tumor necrosis factor-alpha or IL-4, or cell-associated CD40-ligand, all known to be potent stimulators of B7 expression on other dendritic cells, did not restore B7 expression by TiDCs. After a first exposure to TiDCs, allogeneic T-cell response to a second challenge to splenic dendritic cells was decreased. The failure of most dendritic cells infiltrating PROb tumors to express B7, even after stimulation, may contribute to their poor capacity to stimulate T cells and could play a role in the immune tolerance allowing tumor growth.

Adenocarcinoma↗
PubMed1997

Sensitization of cancer cells treated with cytotoxic drugs to fas-mediated cytotoxicity.

BACKGROUND: The transmembrane receptor Fas, together with its protein-binding partner (Fas ligand), is a key regulator of programmed cell death (i.e., apoptosis). Fas and Fas ligand also influence the ability of cytotoxic T lymphocytes and natural killer cells to eliminate tumor cells. However, by inducing apoptosis in activated T cells, the Fas/Fas ligand system may protect some tumor cells from clearance by the immune system. Anticancer drugs enhance Fas ligand expression on the surface of Fas receptor-expressing leukemia cells, thus suggesting that apoptosis caused by these drugs may be mediated via the Fas/Fas ligand system. PURPOSE: This study was conducted to further investigate the relationship between the modulation of Fas receptor gene and protein expression by treatment of cells with cytotoxic drugs and the immune clearance of tumor cells. METHODS: Fas expression on human HT29 colon carcinoma cells treated with a variety of anticancer drugs (cisplatin, doxorubicin, mitomycin C, fluorouracil, and camptothecin) was analyzed by use of quantitative flow cytometry. Human HCT8R and HCT116 colon carcinoma cells and human U937 leukemia cells were treated with cisplatin only and analyzed in the same way. Fas ligand messenger RNA and protein levels were studied by use of a reverse transcription-polymerase chain reaction assay and by flow cytometry. Fas gene expression and messenger RNA levels in cisplatin-treated HT29 cells were characterized by use of in vitro nuclear run-on and northern blot hybridization assays. The cytotoxic activities of agonistic anti-Fas antibodies, Fas ligand, and allogeneic peripheral blood leukocytes, in the absence or presence of Fas-blocking monoclonal antibodies, against tumor cells were assessed by methylene blue staining and chromium-51 release assays. RESULTS: Clinically relevant concentrations of cisplatin, doxorubicin, mitomycin C, fluorouracil, or camptothecin enhanced Fas receptor expression on the plasma membrane of HT29 cells. Cisplatin-mediated increases in Fas expression were confirmed in HCT8R, HCT116, and U937 cells. The enhancement of Fas protein expression was associated with an increased sensitivity of cisplatin-treated tumor cells to agonistic anti-Fas antibodies, to soluble Fas ligand, and to allogeneic peripheral blood leukocyte-mediated cytotoxicity. Each of these effects was blocked by co-treatment of the cells with antagonistic anti-Fas antibody. CONCLUSION AND IMPLICATIONS: In addition to their direct cytotoxic effects, chemotherapeutic drugs sensitize tumor cells to Fas-mediated cytotoxicity and Fas-dependent immune clearance. On the basis of these findings, new strategies might be developed to improve the efficacy of these drugs.

Animals↗
PubMed1997

Characterization of the calf thymus hairpin-binding factor involved in histone pre-mRNA 3' end processing.

Using ion exchange chromatography we have enriched the RNA hairpin-binding factor involved in histone pre-mRNA processing from calf thymus whole cell extract. We demonstrate that the interaction of the factor with its target RNA sequence, the hairpin structure located at the 3' end of mature histone mRNA, is sequence-specific and highly salt-resistant. We have developed a simple in vitro system which allows detection of activities stimulating histone pre-mRNA 3' end processing, based on mouse cell nuclear extract fractionated by Mono Q column chromatography. Using this system, we show that the bovine hairpin-binding factor participates in histone pre-mRNA 3' end processing in vitro. We have further purified the hairpin-binding factor in form of a RNA.protein complex by RNA-mediated elution from phosphocellulose. This led to a fraction highly enriched for 2 proteins of 40 and 43 kDa.

Animals↗
PubMed1997

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.

Amino Acid Sequence↗
PubMed1997

Mouse CD38 is down-regulated on germinal center B cells and mature plasma cells.

Germinal center formation is the result of antigenic stimulation of B cells in a T cell-rich area. B cells cycle through the germinal centers, and a small percentage survive to become plasma cells or memory B cells. The transformation from a mature B cell into a germinal center B cell and finally into a terminally differentiated B cell is not well understood. Human CD38 is highly expressed on both germinal center B cells and plasma cells, and is useful in delineating these B cell subsets and in understanding the signaling events involved in the development of these B cells. To determine whether CD38 expression on activated germinal center B cells and postgerminal center B cells influences germinal center differentiation, we studied the expression of CD38 in the mouse. CD38 is expressed on follicular B cells in the Peyer's patches but is down-regulated on germinal center B cells located within the Peyer's patches. CD38dim/-B220+ germinal center B cells are also found in the spleens of immunized but not control mice, suggesting that Ag-stimulated germinal center formation is involved in the production of CD38dim/-B220+ B cells. Furthermore, mature plasma cells isolated from in vitro LPS cultures do not express CD38, but do contain high levels of cytoplasmic Ig. These results are in contrast to studies in humans in which CD38 is not found on follicular B cells but is highly expressed on germinal center B cells and plasma cells.

ADP-ribosyl Cyclase↗
PubMed1997

Microglial cells induce cytotoxic effects toward colon carcinoma cells: measurement of tumor cytotoxicity with a gamma-glutamyl transpeptidase assay.

Activated macrophages have been shown to exert cytostatic and cytotoxic effects toward tumor cells via nitric oxide (NO) release. In the CNS, microglial cells are considered to be the main resident population of immune effector cells. In this study, cytotoxic activity of N11, an immortalized murine microglial cell line, toward rat progressive DHD/PROb and regressive DHD/REGb colon carcinoma cells was examined in parallel with NO production. Cytotoxicity was evaluated using a novel method, the gamma-glutamyl transpeptidase (gamma-GTP) assay, based on the fact that DHD tumor cells expressed high levels of gamma-GTP activity, while no gamma-GTP activity was found in cells of the monocyte/macrophage lineage. Results showed that activation of N11 cells by interferon-gamma plus either lipopolysaccharide or tumor necrosis factor-alpha induced high amounts of NO release and cytotoxic effects toward DHD/PROb as well as DHD/REGb cells. NO release by activated N11 cells was augmented by addition of tumor cell-conditioned medium. Both NO release by N11 cells and cytotoxicity were blocked by addition of N(G)-monomethyl-L-arginine (L-NMA), an inhibitor of NO synthase, suggesting that cytotoxicity was mediated by N11-derived NO. However, in the presence of L-NMA an increased production of interleukin-6 was also observed. In conclusion, in opposition to information obtained with brain-derived endothelial cells, brain-derived microglial cells did not differentiate between progressive and regressive clones of colon carcinoma cells. Our results point to a specific role for both endothelial and microglial cell types in the context of brain metastasis. Microglial cells can be cytotoxic for tumor cells, and this cytotoxicity is mediated by NO.

Animals↗
PubMed1997

Dual phosphorylation of the T-loop in cdk7: its role in controlling cyclin H binding and CAK activity.

A cyclin-dependent kinase (cdk)-activating kinase (CAK) has been shown previously to catalyze T-loop phosphorylation of cdks in most eukaryotic cells. This enzyme exists in either of two forms: the major one contains cdk7, cyclin H and an assembly factor called MAT-1, whilst the minor one lacks MAT-1. Cdk7 is unusual among cdks because it contains not one but two residues (S170 and T176 in Xenopus cdk7) in its T-loop that are phosphorylated in vivo. We have investigated the role of S170 and T176 phosphorylation in the assembly and activity of cyclin H-cdk7 dimers. In the absence of MAT-1, phosphorylation of the T-loop appears to be required for cdk7 to bind cyclin H. Phosphorylation of both residues does not require cyclin H binding in vitro. Phosphorylation of S170 is sufficient for cdk7 to bind cyclin H with low affinity, but high affinity binding requires T176 phosphorylation. By mutational analysis, we demonstrate that in addition to its role in promotion of cyclin H binding, S170 phosphorylation plays a direct role in the control of CAK activity. Finally, we show that dual phosphorylation of S170 and T176, or substitution of both phosphorylatable residues by aspartic residues, is sufficient to generate CAK activity to one-third of its maximal value in vitro, even in the absence of cyclin H and MAT-1, and may thus provide further clues as to how cyclins activate cdk subunits.

Animals↗
PubMed1997

Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis.

The DAO1 gene of Trigonopsis variabilis encoding a D-amino acid oxidase (EC 1.4.3.3) was isolated from genomic clones selected for their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that have been conserved through evolution. The nucleotide sequence of the gene predicts a protein with similarities to human, pig, rabbit, mouse and Fusarium solani D-amino acid oxidases. The open reading frame of the T. variabilis DAO1 gene was interrupted by an intron. The Dao1p sequence displays two regions, one in the N-terminal section--the FAD binding site--and the other near the C-terminal region that contains conserved signatures found in all the D-amino acid oxidases. The three C-terminal amino acids suggest that the enzyme may be located in peroxisomes. Northern blot experiments showed that no transcriptional activation occurred in the presence of D-methionine. The cDNA encoding Dao1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of the GAL1 promoter. In K. lactis, up to six times more enzyme units per gram of dry weight are produced with a multicopy plasmid in comparison with the wild-type strain of T. variabilis. The yeast expression system we describe may constitute an alternative source for the production of D-amino acid oxidases at industrial level.

Amino Acid Sequence↗
PubMed1997

Retroperitoneal fibrosis and infection of an aortic graft prosthesis: diagnosis and therapeutic problems.

The occurrence of a retroperitoneal fibrosis (RPF) after an aorto-bifemoral bypass is unusual. A case of RPF as a complication of an infection of the graft prosthesis is reported. Computed tomography and magnetic resonance imaging prove useful in diagnosing graft infection: they may reveal periprosthetic gas or perigraft fluid persisting for several months after surgery. However, they may fail when they are performed too early, and repeat performance is suggested.

Aged↗
PubMed1997

Marginal zone B cells exhibit unique activation, proliferative and immunoglobulin secretory responses.

Mouse splenic B cells can be separated based on their distinctive expression of cell surface antigens. Marginal zone (MZ) B cells are CD38high CD21high CD23low/-, while follicular (FO) B cells are CD21int CD23int and newly formed (NF) B cells are CD21dim/- CD23-. Exploiting these phenotypic distinctions, we isolated the three B cell subsets and assessed their other phenotypic differences and functional capabilities in vitro. FO B cells proliferate more than the other B cell subsets in response to either IgM or CD38 cross-linking. MZ B cells proliferate better than FO B cells when stimulated with lipopolysaccharide (LPS), sub-optimal levels of LPS and CD38 cross-linking or CD40 ligation. When NF, FO and MZ B cells were stimulated with either LPS or LPS and interleukin-4, MZ B cells secreted more IgM and IgG3 than the other two subsets. Similarly, calcium fluxes measured within MZ B cells are larger in amplitude and more sustained after B cell receptor cross-linking than those found in the other two subsets. Collectively, these results indicate that CD38high CD21high CD23low/- MZ B cells are specially suited to play a unique role in response to antigens delivered to the MZ area.

Animals↗
PubMed1997

A randomized, placebo-controlled, double-blind trial of mesalamine in the maintenance of remission of Crohn's disease. The Canadian Mesalamine for Remission of Crohn's Disease Study Group.

BACKGROUND & AIMS: The efficacy of mesalamine for the maintenance of remission in patients with Crohn's disease is controversial. The aim of this study was to conduct a double-blind, placebo-controlled study of mesalamine (750 mg four times a day for 48 weeks) in maintaining remission in 293 patients with Crohn's disease. Patients were stratified according to the method of induction of remission (medical or surgical). METHODS: Patients were assessed at weeks 4, 12, 24, 36, and 48. Relapse was defined as a Crohn's Disease Activity Index of >150 (+60 points over baseline). RESULTS: Of the 293 patients, 246 (84%) returned for at least 4 weeks of follow-up and were included in the final analysis. Thirty of the 118 (25%) who received mesalamine had a relapse compared with 47 of 128 (36%) receiving placebo (P = 0.056). Among those with relapse, the time to relapse was 119 days for the mesalamine-treated patients compared with 109 days for placebo-treated patients (P = NS). However, 25% of mesalamine-treated patients had relapsed by 249 days of follow-up compared with 154 days for placebo-treated patients. Subgroup analysis showed that patients with ileocecal-colonic disease or patients who were women had fewer relapses on mesalamine therapy than placebo-treated patients (21% vs. 41%, P = 0.018; and 19% vs. 41%, P = 0.003, respectively). CONCLUSIONS: Mesalamine treatment reduced relapse compared with placebo treatment, although conventional statistical significance was not achieved.

Adult↗
PubMed1997

The heat shock stress response after brain lesions: induction of 72 kDa heat shock protein (cell types involved, axonal transport, transcriptional regulation) and protein synthesis inhibition.

The cerebral stress response is examined following a variety of pathological conditions such as focal and global ischemia, administration of excitotoxins, and hyperthermia. Expression of 72 kDa heat shock protein (Hsp70) and hsp70 mRNA, the mechanism underlying induction of hsp70 mRNA involving activation of heat shock factor 1, and inhibition of cerebral protein synthesis are different aspects of the stress response considered here. The results are compared with those in the literature on induction, transcriptional regulation, expression, and cellular location of Hsp70, with a view to getting more insight into the function of the stress response in the injured brain. The present results illustrate that Hsp70 can be expressed in cells affected at various degrees following an insult that will either survive or dic as the brain lesion develops, depending on the severity of cell injury. This indicates that, under certain circumstances, synthesized Hsp70 might be necessary but not sufficient to ensure cell survival. Other situations involve uncoupling between synthesis of hsp70 mRNA and protein, probably due to very strict protein synthesis blockade, and often result in cell loss. Cells eventually will die if protein synthesis rates do not go back to normal after a period of protein synthesis inhibition. The stress response is a dynamic event that is switched on in neural cells sensitive to a brain insult. The stress response is, however, tricky, as affected cells seem to need it, have to deal transiently with it, but eventually be able to get rid of it, in order to survive. Putative therapeutic treatments can act either selectively, potentiating the synthesis of Hsp70 protein and recovery of protein synthesis, or preventing the stress response by deadening the insult severity.

Animals↗
PubMed1997

Responsiveness of human male volunteers to immunization with ovine follicle stimulating hormone vaccine: results of a pilot study.

A study of 140 days duration was performed to examine if human male volunteers (n = 5) respond to ovine follicle stimulating hormone (oFSH) immunization (administered adsorbed on Alugel on days 1, 20, 40 and 70) by producing antibodies capable of both binding and neutralizing bioactivity of human FSH. The kinetics of antibody production for both the immunogen (oFSH) and the cross-reactive antigen (hFSH) were essentially similar. The volunteers responded only to the first two immunizations. The boosters given on days 40 and 70 were ineffective, probably because of the presence of substantial amounts of circulating antibody to oFSH. Of the antibodies generated to oFSH, 25-45% bound hFSH with a mean binding affinity of 0.65 x 10(9) +/- 0.53 M(-1). The binding capacities at the time of high (30-80 days of immunization) and low (>110 days) titres were 346 +/- 185 and 10.5 +/- 5.8 ng hFSH/ml respectively. During the period of high titre, free serum FSH (value in normal males 1-5 ng/ml) was not monitorable. A 50 microl aliquot of the antiserum obtained from different volunteers between days 30 and 80 and on day 140 blocked binding of (125)I-labelled hFSH to its receptor by 82 +/- 9.7 and 53 +/- 12.2% respectively. The antibody produced was specific for FSH, and no significant change in the values of related glycoprotein hormones (luteinizing hormone/testosterone and thyroid stimulating hormone/thyroxine) were recorded. Seminal plasma transferrin, a marker of Sertoli cell as well as of seminiferous tubular function, showed marked reduction (30-90%) following immunization with oFSH. Considering that endogenous FSH remained neutralized for approximately one sperm cycle only (65 days), the reduction in sperm counts (30-74%) exhibited by some volunteers is encouraging. Immunization with oFSH did not result in any significant changes in haematology, serum biochemistry or hormonal profiles. There was no production of antibodies capable of interacting with non-specific tissues. It is concluded that it should be possible to obtain a sustained long-term blockade of endogenous FSH action in men by using oFSH as an immunogen. This is a prerequisite for obtaining significant reduction in the quality and quantity of spermatozoa produced, thus leading to infertility.

Adult↗
PubMed1997

Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone.

Antibody reactivity to self-antigens is a normal component of the immune system. To study the mechanism by which self-reactive B cells are generated and maintained, we analyzed B cell development in transgenic mice that express a rearranged VH81X heavy chain from the pre-immune repertoire. In these mice, > 95% of B cells express the transgene in association with a variety of kappa light chains but V kappa 1 C being the dominant light chain. These transgenic B cells with identical V kappa 1C-J kappa 5 joins do not normally secrete IgM in vivo, but antibodies derived from these B cells, through LPS activation in vitro or after hybridoma immortalization, are self-reactive and recognize an ubiquitous epitope(s) on intracytoplasmic proteins from different tissues. They have the phenotype and localization pattern of long-lived marginal zone B cells and their development in vivo is blocked by injection of soluble VH81X-V kappa 1CJ kappa 5 IgM antibody. The observations in this transgenic mouse provide evidence for positive selection of a population of self-reactive B cells. These B cells enter the peripheral pool of B cells where they localize in the marginal zone of the spleen and, in contrast to other transgene-expressing B cells, do not secrete IgM antibody.

Animals↗
PubMed1997

Development of VH81X transgene-bearing B cells in fetus and adult: sites for expansion and deletion in conventional and CD5/B1 cells.

The most D-proximal functional VH gene, VH81X, is preferentially expressed in the mouse fetal B cell repertoire; however, it is expressed in few B cells in the adult. To determine when VH81X gene expression affects size and phenotype of particular stages in B cell differentiation, transgenic mice have been developed expressing a germline fetal liver-derived VH81X-mu rearrangement. Comparative analysis of B lymphopoiesis reveals similarities and differences between fetal liver and adult bone marrow which pinpoint developmental stages in mice during which VH81X-expressing B cell progenitors expand or deplete compartment sizes. These include a similar reduction in c-kitR+ and establishment of a predominant CD43low/HSAhigh phenotype within the B220+ CD43+ compartment which is dependent on the association of the transgene with lambda 5. In contrast, the CD43- pre-B and immature B cell compartments are expanded in the fetus but not in the adult. In addition, there are other factors that later disfavor the survival of VH81X-expressing B1 and B2 cells. Thus the failure to detect VH81X-bearing B cells in the adult is the result of a multistep selection process occurring at all stages during B repertoire expansion.

Aging↗