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F Martin

Publications and source records attributed to F Martin.

At least 127 records · Page 7Linked to original sources
PubMed1998

Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins [see comment].

The mitochondrial outer membrane contains machinery for the import of preproteins encoded by nuclear genes. Eight different Tom (translocase of outer membrane) proteins have been identified that function as receptors and/or are related to a hypothetical general import pore. Many mitochondrial membrane channel activities have been described, including one related to Tim23 of the inner-membrane protein-import system; however, the pore-forming subunit(s) of the Tom machinery have not been identified until now. Here we describe the expression and functional reconstitution of Tom40, an integral membrane protein with mainly beta-sheet structure. Tom40 forms a cation-selective high-conductance channel that specifically binds to and transports mitochondrial-targeting sequences added to the cis side of the membrane. We conclude that Tom40 is the pore-forming subunit of the mitochondrial general import pore and that it constitutes a hydrophilic, approximately 22 A wide channel for the import of preproteins.

Amino Acid Sequence↗
PubMed1998

Design of selective eglin inhibitors of HCV NS3 proteinase.

Hepatitis C virus (HCV) infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals, estimated to be 100-200 million worldwide. Unfortunately, immunotherapy or other effective treatments for HCV infection are not yet available, and interferon administration has limited efficacy. Different approaches to HCV therapy are being explored, and these include inhibition of the viral proteinase, helicase, and RNA-dependent RNA polymerase and development of a vaccine. Here we present the design of selective inhibitors with nanomolar potencies of HCV NS3 proteinase based on eglin c. These eglin c mutants were generated by reshaping the inhibitor active site-binding loop, and the results emphasize the role played by residues P5-P4' in enzyme recognition. In addition, alanine scanning experiments provide evidence that the N terminus of eglin c also contributes to NS3 binding. These eglin inhibitors offer a unique tool for accurately assessing the requirements for effective inhibition of the enzymatic activity of NS3 and at the same time can be considered lead compounds for the identification of other NS3 inhibitors in targeted design efforts.

Animals↗
PubMed1998

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat-shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by approximately 30% compared with wild-type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.

Adenosine Triphosphatases↗
PubMed1998

Bcl-2-mediated inhibition of apoptosis prevents immunogenicity and restores tumorigenicity of spontaneously regressive tumors.

Tumor cell clones from a rat colon carcinoma differ in their tumorigenicity and immunogenicity. The PRO clones give rise to progressive tumors, whereas the REG clones yield tumors that regress in a few weeks through a specific immune response. REG cells were more sensitive than PRO cells to apoptosis triggered by serum withdrawal in vitro. Furthermore, a fraction of REG cells, but no PRO cells, underwent apoptosis in the hours following injection into syngeneic rats. To further analyze the role of apoptosis, we overexpressed the antiapoptotic protein Bcl-2 in REG cells. Unlike parental or fake-transfected REG cells, Bcl-2-overexpressing REG cells resisted serum withdrawal-induced apoptosis, did not undergo apoptosis at 48 h postinjection into naive syngeneic rats, and gave rise to progressive, metastatic, and lethal tumors. Interestingly, REG-bcl2 cells were rejected by syngeneic hosts that had been preimmunized by an injection of parental REG cells, indicating that Bcl-2 overexpression did not alter tumor cell sensitivity to the effector cells of the immune response. Taken together, these observations indicate that tumor cell apoptosis may contribute to immunogenicity.

Adenocarcinoma↗
PubMed1998

Engineered peptides corresponding to segments of the H3 domain of syntaxin inhibit insulin release both in intact and permeabilized mouse pancreatic beta cells.

Syntaxin is one of the proteins involved in the exocytotic event through sequential binding to specific proteins, including SNAP25 and synaptobrevin. In a previous work in digitonin-permeabilized beta cells, we characterized the functional role of two segments: synA and synB of the H3 domain of syntaxin. As a continuation of these experiments in the present study we have initially outlined a zone of 17 residues as the very effective uncoupling element of the synA segment. Further functional studies have been accomplished in intact pancreatic beta cells with a specific myristoylated (myr) 13-mer peptide comprised in this active zone. These experiments showed a concentration-dependent inhibition of glucose-induced insulin release (IC50 = 4 microM) of this engineered peptide that was specific since a myristoylated random peptide with the same composition was ineffective. A second myristoylated 13-mer peptide comprised into the synB segment was shown to be even more potent promoting a selective inhibition of insulin release. These data show for the first time, that nutrient-induced secretory process can be specifically uncoupled in intact beta cells demonstrating at the same time that syntaxin plays a central role in this mechanism.

Amino Acid Sequence↗
PubMed1998

Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells.

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.

Amyloid Precursor Protein Secretases↗
PubMed1998

Erythroid maturation and globin gene expression in mice with combined deficiency of NF-E2 and nrf-2.

NF-E2 binding sites, located in distant regulatory sequences, may be important for high level alpha- and beta-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2-related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2-deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of alpha- and beta-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2-deficient mice is not the result of compensation by Nrf-2.

Amino Acid Sequence↗
PubMed1998

Generation of the germline peripheral B cell repertoire: VH81X-lambda B cells are unable to complete all developmental programs.

The generation of VH81X heavy chain lambda-light chain-expressing B cells (VH81X-lambda+ B cells) was studied in VH81X heavy chain transgenic mice as well as in VH81X JH (-/-) and VH81X JH (-/-) Ck (-/-) mice, in which competition resulting from expression of heavy and light chains from the endogenous heavy and kappa light chain loci was prevented. We show that although lambda light chain gene rearrangements occur normally and give rise to light chains that associate with the transgenic heavy chain to form surface and soluble IgM molecules, further B cell development is almost totally blocked. The few VH81X-lambda+ B cells that are generated progress into a mature compartment (expressing surface CD21, CD22, CD23, and low CD24 and having a relatively long life span) but they also have reduced levels of surface Ig receptor and express higher amounts of Fas Ag than VH81X-kappa+ B cells. These VH81X-lambda+ B cells reach the peripheral lymphoid organs and accumulate in the periarteriolar lymphoid sheath but are unable to generate primary B cell follicles. In other heavy chain transgenic mice (MD2, M167, and M54), lambda+ B cells are generated. However, they seem to be preferentially selected in the peripheral repertoire of some transgenic heavy chain mice (M54) but not in others (MD2, M167). These studies show that a crucial selection step is necessary for B cell survival and maintenance in which B cells, similar to T cells, receive signals depending on their clonal receptors.

Animals↗
PubMed1998

Retroviral vector targeting to melanoma cells by single-chain antibody incorporation in envelope.

Two strategies for targeting recombinant retroviruses to melanoma cells were compared. One was to extend the tropism of an ecotropic envelope to human melanoma cells, the other was to enhance the tropism of an amphotropic envelope for melanoma cells. Chimeric retroviral envelopes, incorporating a single-chain antibody (ScFv) directed against high-molecular-weight melanoma-associated antigen (HMWMAA) at the amino terminus are correctly processed and incorporated into virions. ScFv-ecotropic envelope chimeras allow specific, but low-titer, targeting of HMWMAA-positive cells, when co-expressed with ecotropic envelopes. ScFv-amphotropic envelope chimeras bind specifically to HMWMAA-positive cells and allow preferential infection at high titer.

Amino Acid Sequence↗
PubMed1998

On the 5d 1Pig --> 2 (1)Sigma+ u and 5d 1Pig --> C 1Piu Fluorescence in 7Li2.

Following excitation of the 5d 1Pig Rydberg state of 7Li2 by optical-optical double resonance, fluorescence has been observed in the infrared region to the 2 (1)Sigma+ u and C 1Piu states. Analysis of high-resolution Fourier transform spectra yields term energies and rotational constants of the lowest seven vibrational levels of the inner well of the 2 (1)Sigma+ u "double minimum" state. The equilibrium term value and dissociation energy have been determined to be Te = 30101.45 +/- 0.12 cm-1 and De = 5621.3 +/- 0.2 cm-1. The v = 0 and 1 levels of the C 1Piu state have been analyzed, resulting in new values of Te = 30551.0 +/- 0.1 cm-1 and De = 7773.3 +/- 0.2 cm-1. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Coexpression of G-CSF with an unglycosylated G-CSF receptor mutant results in secretion of a stable complex.

Previously, we have shown that the entire extracellular domain of the granulocyte-colony stimulating factor receptor (sG-CSFr) produced in Chinese hamster ovary (CHO) cells forms a stable complex with its ligand G-CSF, at a stoichiometry of 2:2. A truncated receptor molecule consisting of the cytokine receptor homology domain and N-terminus Ig-like domain (Ig CRH) behaves quite similarly. Both of these forms of the receptor are highly glycosylated. To address the importance of glycosylation toward receptor activity and stability, and possibly obtain nonglycosylated receptor for crystallization, mutations were made to replace four Asn residues which are N-glycosylated in the truncated receptor. Virtually no receptor was recovered from conditioned media of CHO cells transfected with this mutant construct, although a high-level of mRNA coding for receptor was detected; this mRNA was translated as determined by Western blots of cell lysates. These results indicate that the translated product is apparently not secreted from these cells. Cells transfected with mutant receptor cDNA were cotransfected with a cDNA construct expressing G-CSF in which the single O-glycosylation site was eliminated by mutation. Upon fermentation of the cotransfectants, we observed a large amount of receptor-ligand complex in the conditioned media. The purified unglycosylated complex appeared to be of the same binding stoichiometry and approximate binding affinity as that of complex formed by addition of purified ligand and unmutated receptor. These results show that while glycosylation of sG-CSFr is not necessary for ligand binding, it appears to be crucial in folding and export from the cell.

Animals↗
PubMed1998

Quadratic dose-response relationship between caffeine (1,3,7-trimethylxanthine) and EEG correlation dimension.

Many studies have failed to determine a systematic dose-response relationship across different cognitive tasks between caffeine and EEG power spectra. However, a nonlinear approach to EEG analysis, which reconstructs a multi-dimensional state space from each electrode recording, can be used to compute the number of active degrees of freedom in the signal (the correlation dimension, D2), and can be interpreted as a measure of signal complexity. This study attempted to determine a consistent dose-response relationship between caffeine and EEG D2, across six oral caffeine doses (100-600 mg), with each subject acting as their own control, to create a probabilistic bias against finding any consistent linear or nonlinear dose-response relationship across different cognitive tasks. The experiment (n = 10) was conducted with three within-subjects explanatory variables, 2 (experimental, placebo) x 8 (caffeine level) x 4 (type of cognitive task performed), with EEG D2 as the response variable, measured from Fz, F3, F4 and Cz. A significant three-way interaction was found [F(21,245.3) = 3.65, P = 0.001]. Regression analyses revealed a linear trend for the response variable across trials for the placebo condition (average R2 = 0.54), whereas linear+quadratic trends explained an average 30% of the variance for the experimental condition, compared to 0.01% for the linear fit, indicating a robust quadratic dose-response relationship between caffeine and EEG D2. Three conditions had positive quadratic co-efficients, and one condition had a negative quadratic co-efficient. These results are discussed in terms of the implications for brain dynamics, and with respect to recent criticisms of the computation of D2 from EEG.

Adult↗
PubMed1998

The expression of a symbiosis-regulated gene in eucalypt roots is regulated by auxins and hypaphorine, the tryptophan betaine of the ectomycorrhizal basidiomycete Pisolithus tinctorius.

A full-length cDNA coding for a symbiosis-regulated transcript, EgHypar, was isolated by differential screening from a Eucalyptus globulus bicostata--Pisolithus tinctorius ectomycorrhiza. The sequence of this clone revealed a protein with an estimated molecular mass of 25.5 kDa that exhibited a high degree of homology (66%) with plant auxin-induced glutathione-S-transferases. Expression of the EgHypar gene in seedlings was confined largely in roots and it is drastically increased by ectomycorrhiza development. The concentration of EgHypar transcripts was similarly up-regulated in roots incubated in media supplemented with P. tinctorius cell-free extracts, indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid or hypaphorine (tryptophan betaine), the major indolic compound secreted by P. tinctorius. The latter fungal alkaloid concomitantly induced a decrease in root hair elongation in eucalypt seedlings. Up-regulation of EgHypar expression by auxins and fungal metabolites suggests that this symbiosis-regulated gene could be involved in the morphological changes taking place in plants roots upon symbiosis development. To our knowledge, these results provide the first molecular evidence that gene expression of the host plant is altered by molecules produced by the ectomycorrhizal mycobiont.

Amino Acid Sequence↗
PubMed1998

Oral contraceptive use and smoking are risk factors for relapse in Crohn's disease. The Canadian Mesalamine for Remission of Crohn's Disease Study Group.

BACKGROUND & AIMS: Lifestyle factors have been shown to influence prognosis in Crohn's disease. The purpose of this study was to prospectively assess the effects of smoking and oral contraceptive use on clinical relapse rates. METHODS: Placebo-treated patients formed a prospective cohort, followed up for 48 weeks or until relapse. The influence of smoking and the use of oral contraceptives on relapse risk was examined by life-table analysis (log rank tests) and Cox proportional hazards modeling, taking into account demographic and disease characteristics. RESULTS: Of 152 patients, 61 (40%) had a relapse. Univariate analysis showed unfavorable outcomes for women (P = 0.05), current smokers (P = 0.005), and use of oral contraceptives (P = 0.001). Recent surgery was associated with a decreased risk of relapse (P = 0.02). The Cox model retained current smoking vs. never smoking (hazard ratio, 2.1; 95% confidence interval, 1.1-4.2), oral contraceptive use (hazard ratio, 3.0; 95% confidence interval, 1.5-5.9), and medical compared with surgical induction of remission (hazard ratio, 2.1; 95% confidence interval, 1.0-4.2) as predictors of relapse. Ex-smokers did not have an increased risk. Finally, sex, age, time in remission, disease location, and disease duration were not significant predictors. CONCLUSIONS: Oral contraceptive use and smoking are associated with an increased risk of relapse in patients with Crohn's disease.

Adult↗
PubMed1998

A novel immunoglobulin superfamily junctional molecule expressed by antigen presenting cells, endothelial cells and platelets.

The architecture of lymphoid microenvironments depends upon complex interactions between several stromal cell types. We describe in this report the cloning of a cDNA which encodes a novel membrane molecule containing two external Ig-like domains. It is expressed at the junction between endothelial cells including HEV. It is also expressed by platelets and MHC class II+ antigen presenting cells in thymic medulla and T-cell areas in peripheral lymphoid organs. These cells which lack in RelB-deficient mice include tissue-derived dendritic, epithelial cells and macrophages. Thus, this molecule might contribute to the organization of cell junctions in different microenvironments.

Amino Acid Sequence↗
PubMed1998

Expression of bovine activin-A and inhibin-A in recombinant baculovirus-infected Spodoptera frugiperda Sf 21 insect cells.

Currently, bioactive activin and inhibin for investigative purposes are obtained either by purification from bovine or porcine follicular fluid or have been kindly supplied in limited amounts by Genentech. The latter are recombinant formulations produced in cultured monkey kidney CV-1 cells. The aims of this study were to assess the potential of the baculovirus expression system as an alternative means to produce recombinant activin and inhibin. Towards these goals, two recombinant baculoviruses, AcBovACTA and AcBovINHA, were constructed. AcBovACTA contains a contiguous copy of the bovine beta A-inhibin/activin structural gene encoding the beta A-preproprotein whereas AcBovINHA contains contiguous copies of the bovine alpha-inhibin and beta A-inhibin/activin structural genes encoding the alpha- and beta A-preproproteins, respectively. Western blot analyses, using monoclonal antibodies specific for the mature portions of the alpha-inhibin and beta A-inhibin/activin subunits, demonstrated that Spodoptera frugiperda Sf21 cells infected with either recombinant virus secreted mature homodimeric activin-A into the medium. In addition, Sf21 cells infected with the recombinant AcBovINHA virus were found also to produce substantial amounts of the alpha-inhibin precursor protein. However, the mature portion of the latter is not secreted into the medium but is retained within infected cells in an incompletely processed form(s). The recombinant activin-A secreted by Sf21 cells infected with the AcBovACTA virus was shown to possess activin bioactivity when analysed by in vitor bioassay and, therefore, provides an alternative route to mammalian cell expression for the production of recombinant activin-A.

Activins↗
PubMed1998

Functional coupling of a recombinant human 5-HT5A receptor to G-proteins in HEK-293 cells.

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT5A receptor. 2. We have shown that ligand activation of the Human 5-HT5A receptor results in functional coupling to G-proteins in HEK-293 cells. 3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [35S]-GTPgammaS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT5A receptor in any cell. 4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT5A receptor cell line, suggesting a functional coupling to a G alpha i, G-protein in the HEK-293 cell line. 5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

Amino Acid Sequence↗