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Biomedical subjects

F Martin

Publications and source records attributed to F Martin.

At least 91 records · Page 5Linked to original sources
PubMed2000

GM-CSF expression by tumor cells correlates with aggressivity and with stroma reaction formation.

Granulation tissue involved in tissue repair and in the stroma reaction to epithelial tumors is characterized by the presence of myofibroblastic cells. It has been previously reported that granulocyte macrophage-colony stimulating factor (GM-CSF) induces a fibrotic reaction containing numerous myofibroblasts. This reaction results from a cascade of events, including stimulation of transforming growth factor-beta1 (TGF-beta1) production by macrophages which, in turn, promotes alpha-smooth muscle actin and collagen synthesis by fibroblasts. Moreover, GM-CSF is known to be expressed by many tumor cell types. In this study we have analyzed, by means of reverse transcription-polymerase chain reaction, GM-CSF mRNA expression in a progressive and a regressive rat colon carcinomas and in the corresponding cell lines, eliciting different degrees of desmoplastic reaction. We have also evaluated the expression of GM-CSF protein in selected cases. The expression of GM-CSF mRNA and, when tested, protein were higher in progressive compared to regressive cancer cells both in vivo and in vitro. We then investigated GM-CSF mRNA and protein expression in different human colon cancer cell lines known to exhibit different degrees of aggressivity in vivo. We found high levels of GM-CSF mRNA and protein in the most aggressive cell lines. Similar results were also obtained on human breast and cervical cancer cell lines. Our results are in agreement with the assumption that GM-CSF expression is correlated to tumor aggressivity. Conceivably, one of the GM-CSF actions affecting tumor progression is exerted through its influence on stroma reaction development.

Animals↗
PubMed1999

FAS(CD95) ligand expression by tumor cell variants can be unrelated to their capacity to induce tolerance or immune rejection.

According to the results of in vitro experiments, Fas(CD95) ligand expression by cancer cells might induce apoptosis of activated T cells and contribute to immune tolerance. However, Fas ligand expression had never been explored in vivo in tumor cell models yielding either immune response or tolerance. In the present study, we analyzed the expression and function of Fas ligand in 2 clones of tumor cells originating from the same rat colon carcinoma. REGb cells were immunogenic and yielded tumors that regressed in immunecompetent syngeneic hosts, whereas PROb cells induced active tolerance and yielded progressive tumors. Fas ligand was expressed on the plasma membrane of both REGb and PROb cells, and its cDNA sequencing showed no mutation. However, neither REGb nor PROb cells induced apoptosis of co-cultured Fas-sensitive target cells. Our results show that surface expression of Fas ligand by tumor cells does not always induce killing of adjoining Fas-sensitive cells and that tumor cells may induce a protective immune response or an active tolerance independently of Fas ligand expression.

Animals↗
PubMed1999

Protein-coding region discovery in organisms underrepresented in databases.

The prediction of coding sequences has received a lot of attention during the last decade. We can distinguish two kinds of methods, those that rely on training with sets of example and counter-example sequences, and those that exploit the intrinsic properties of the DNA sequences to be analyzed. The former are generally more powerful but their domains of application are limited by the availability of a training set. The latter avoid this drawback but can only be applied to sequences that are long enough to allow computation of the statistics. Here, we present a method that fills the gap between the two approaches. A learning step is applied using a set of sequences that are assumed to contain coding and non-coding regions, but with the boundaries of these regions unknown. A test step then uses the discriminant function obtained during the learning to predict coding regions in sequences from the same organism. The learning relies upon a correspondence analysis and prediction is presented on a graphical display. The method has been evaluated on a sample of yeast sequences, and the analysis of a set of expressed sequence tags from the Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza illustrates the relevance of the approach in its biological context.

Algorithms↗
PubMed1999

IgMhighCD21high lymphocytes enriched in the splenic marginal zone generate effector cells more rapidly than the bulk of follicular B cells.

Ag encounter will recruit Ag-specific cells from the pool of mature B lymphocytes in the spleen and activate them to perform effector functions: generation of Ab-forming cells (plasma cells) and presentation of Ag to T cells. We have compared the ability of mature follicular and marginal zone cells to develop into effector B cells. The generation of marginal zone B cells and their localization in the marginal sinus area are T cell and CD40 ligand independent, suggesting that they do not represent a postgerminal center population. Compared with mature recirculating follicular B cells, they express several characteristics of previous antigenic experience, including higher levels of B7.1 (CD80) and B7.2 (CD86) when freshly isolated and following in vitro stimulation. After a brief 6- to 8-h in vitro stimulation with LPS or anti-CD40 Abs, marginal zone B cells become potent APCs. In addition, their ability to proliferate and differentiate into plasma cells in response to low doses of T-independent polyclonal stimuli (LPS) is far greater than that of follicular B cells. These findings indicate a functional heterogeneity within splenic mature B lymphocytes, with marginal zone B cells having the capacity to generate effector cells in early stages of the immune response against particulate Ags scavenged efficiently in this special anatomical site.

Animals↗
PubMed1999

[Early signs of systemic disease in patients taking minocycline chlorhydrate].

BACKGROUND: Search for etiology in young subjects with joint disease must take into consideration causes other than reactional arthropathy or systemic disease. CASE REPORTS: Two young subjects consulted for joint pain involving various joints. Physical examination did not reveal any other abnormality. Serum tests were positive for antinuclear antibodies and/or showed moderately and persistently elevated transaminases. History taking revealed that these subjects had been taking minocyclin chlorhydrate for several months, or even years, for acne. Joint pain subsided with drug withdrawal and serum tests returned to normal. DISCUSSION: Long-term treatment with minocylin chlorhydrate should always be included in the search for an etiology in patients presenting joint pain.

Adolescent↗
PubMed1999

Retroviral display of functional binding domains fused to the amino terminus of influenza hemagglutinin.

We have previously shown that retroviral vector particles derived from Moloney murine leukemia virus (Mo-MuLV) can efficiently incorporate influenza hemagglutinin (HA) glycoproteins from fowl plague virus (FPV), thus conferring a broad tropism to the vectors. To modify its host range, we have engineered the FPV HA to display four different polypeptides on its N terminus: the epidermal growth factor, an anti-human MHC class I molecules scFv (single-chain antibody), an anti-melanoma antigen scFv, and an IgG Fc-binding polypeptide. All recombinant HA glycoproteins were correctly expressed and processed, and efficiently incorporated into Mo-MuLV retroviral particles, indicating that amino-terminal insertion of large polypeptides did not alter the conformation of HA chimeras. Virions carrying the different chimeras bound specifically to cells expressing the targeted cell surface molecules of each ligand. In addition, all virion types were infectious but exhibited various degrees of specificity regarding the use of the targeted cell surface molecule versus the wild-type FPV HA receptor for cell entry and infection. For some ligands tested, infectivity was significantly increased on cells that express the targeted receptor, compared with cells that express only the wild-type HA receptor. Furthermore, some polypeptides could abolish infectivity via the wild-type FPV HA receptor. Our data therefore indicate that it is possible to engineer the HA envelope glycoprotein by fusing ligands to its amino-terminal end without affecting its fusion activity.

3T3 Cells↗
PubMed1999

Both familial Parkinson's disease mutations accelerate alpha-synuclein aggregation.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies, the major component of which are filaments consisting of alpha-synuclein. Two recently identified point mutations in alpha-synuclein are the only known genetic causes of PD, but their pathogenic mechanism is not understood. Here we show that both wild type and mutant alpha-synuclein form insoluble fibrillar aggregates with antiparallel beta-sheet structure upon incubation at physiological temperature in vitro. Importantly, aggregate formation is accelerated by both PD-linked mutations. Under the experimental conditions, the lag time for the formation of precipitable aggregates is about 280 h for the wild type protein, 180 h for the A30P mutant, and only 100 h for the A53T mutant protein. These data suggest that the formation of alpha-synuclein aggregates could be a critical step in PD pathogenesis, which is accelerated by the PD-linked mutations.

Cell Line↗
PubMed1999

Kinetic analysis of the effect on Fab binding of identical substitutions in a peptide and its parent protein.

Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs.

Antibody Affinity↗
PubMed1999

Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells.

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.

Amino Acid Sequence↗
PubMed1999

Recombinant rat fibroblast growth factor-16: structure and biological activity.

Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties.

3T3 Cells↗
PubMed1999

The High-Lying Vibrational Levels and Dissociation Energy of the a3Sigma+u State of 7Li2.

Perturbation-facilitated optical-optical double resonance (PFOODR) has been used to access the 2(3)Pig state of 7Li2 via the excitation scheme using two single-mode tunable lasers. The selected () mixed level provides a gateway through which the triplet manifold can be accessed. Fluorescence from single rovibrational levels of 2(3)Pig to the state was detected at high resolution using a Fourier transform spectrometer. Transitions to v = 0-9 in the state were observed, covering the potential well almost to the dissociation limit. The data were analyzed using a near dissociation expansion (NDE) technique and the resulting vibrational and rotational parameters were used to calculate a new RKR potential curve which reproduced the observed energy levels to within a rms error of 0.02 cm-1. The following parameters were obtained for the state: D0 = 301.829 +/- 0.015 cm-1, De = 333.69 +/- 0.10 cm-1, Te = 8183.12 +/- 0.12 cm-1. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

CD21high IgMhigh splenic B cells enriched in the marginal zone: distinct phenotypes and functions.

Collectively, these findings suggest that MZ B cells have unique signaling and subsequent differentiative capabilities that permit them to react much more vigorously than the majority of splenic B cells (FO) in the earliest stages of an in vivo immune response. This is particularly evident with limiting T cell help, low concentration of thymus-independent mitogens or low amounts of particulate blood-borne antigen in the spleen (Fig. 4). They are uniquely situated adjacent to the marginal sinuses and a rich array of antigen trapping macrophages. Because of this location the MZ B cells are ideally positioned for immediate exposure to blood-borne antigens. In contrast, the FO B cells are in juxtaposition to the PALS which may expedite the interactions of FO B cells with T cells and antigen presenting cells. Collectively these properties point to a role for FO B cells in antibody responses to T dependent antigens generated in germinal centers. These responses occur temporally later in immune responses and may be involved principally in the response to protein antigens. [figure: see text].

Animals↗
PubMed1999

Laparoscopic treatment vs open surgery in the solution of major incisional and abdominal wall hernias with mesh.

BACKGROUND: Despite being one of the most exact indications, laparoscopic treatment of eventrations and ventral hernias is barely known among the array of laparoscopic techniques. METHODS: A total of 60 patients were assigned at random over a 3-year period to two homogeneous groups to be operated on for major ventral hernias with mesh. Half of them were operated upon laparoscopically and the rest with open surgery. Early and longer-term complications were analyzed, as were operative time and postoperative hospital stays. RESULTS: The two groups were homogeneous in terms of demographic and clinical characteristics. The group that was operated on laparoscopically presented a lower rate of postoperative and longer-term complications; similarly, surgery time was significantly lower (p < 0.05). Hospitalization time was also significantly lower than in the group undergoing conventional open surgery (p < 0.05). CONCLUSIONS: Laparoscopic treatment of postoperative eventration and primary ventral hernia reduces complications and relapse rates, eliminates reintervention through mesh infection, reduces operative time, and considerably shortens the hospital stay.

Female↗
PubMed1999

High genetic diversity in a population of the ectomycorrhizal basidiomycete laccaria amethystina in a 150-year-old beech forest

The genetic structure of a population of the ectomycorrhizal basidiomycete Laccaria amethystina (Bolt. ex Hooker) Murr. was assessed in a closed 150-year-old beech (Fagus sylvatica L.) forest in the Vosges Mountains in northeastern France. During the autumn of 1994 and 1997, sporophores were collected from three 100-m2 sampling plots located along a 120-m transect crossing the beech stand. The genetic variation of 676 sporophores was initially estimated using heteroduplex analysis of the ribosomal DNA intergenic spacer (IGS1). Ten unique IGS1 heteroduplex/homoduplex patterns were identified, although three types represented most of the sporophores analysed. Each group of IGS1 type was then analysed using random amplified microsatellite analysis (RAMS). RAMS resolved 388 different genotypes amongst the 634 sporophores analysed from the three plots during the autumn of 1994 and 1997. Density as high as 130 genets per 100 m2 was observed during the autumn of 1994. The largest clone covered approximately 1 m2, but most genets covered a few cm2 and produced only one to three sporophores. Only eight genotypes identified in 1994 were found in 1997. Although L. amethystina has the capacity for vegetative persistence, the present study indicates that its populations maintain a genetic structure more consistent with a high frequency of sexual reproduction. This suggests that beech trees could be recolonized by new genotypes each year. Alternatively, this spatial distribution may also arise from erratic fruiting of underground persistent genets. These features (i.e. numerous genets of small size), typical of ruderal species, contrast with studies carried out on other ectomycorrhizal basidiomycetes occurring in mature closed forests.

Journal Article↗
PubMed1999

A loop-mimetic inhibitor of the HCV-NS3 protease derived from a minibody.

We have been interested for some time in establishing a strategy for deriving lead compounds from macromolecule ligands such as minibody variants. A minibody is a minimized antibody variable domain whose two loops are amenable to combinatorial mutagenesis. This approach can be especially useful when dealing with 'difficult' targets. One such target is the NS3 protease of hepatitis C virus (HCV), a human pathogen that is believed to infect about 100 million individuals worldwide and for which an effective therapy is not yet available. Based on known inhibitor specificity (residues P6-P1) of NS3 protease, we screened a number of minibodies from our collection and we were able to identify a competitive inhibitor of this enzyme. We thus validated an aspect of recognition by HCV NS3 protease, namely that an acid anchor is necessary for inhibitor activity. In addition, the characterization of the minibody inhibitor led to the synthesis of a constrained hexapeptide mimicking the bioactive loop of the parent macromolecule. The cyclic peptide is a lead compound prone to rapid optimization through solid phase combinatorial chemistry. We therefore confirmed that the potential of turning a protein ligand into a low molecular weight active compound for lead discovery is achievable and can complement more traditional drug discovery approaches.

Binding Sites↗
PubMed1999

A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus tinctorius.

Development of the ectomycorrhizal symbiosis leads to the aggregation of fungal hyphae to form the mantle. To identify cell surface proteins involved in this developmental step, changes in the biosynthesis of fungal cell wall proteins were examined in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis. Enhanced synthesis of several immunologically related fungal 31- and 32-kDa polypeptides, so-called symbiosis-regulated acidic polypeptides (SRAPs), was observed. Peptide sequences of SRAP32d were obtained after trypsin digestion. These peptides were found in the predicted sequence of six closely related fungal cDNAs coding for ectomycorrhiza up-regulated transcripts. The PtSRAP32 cDNAs represented about 10% of the differentially expressed cDNAs in ectomycorrhiza and are predicted to encode alanine-rich proteins of 28.2 kDa. There are no sequence homologies between SRAPs and previously identified proteins, but they contain the Arg-Gly-Asp (RGD) motif found in cell-adhesion proteins. SRAPs were observed on the hyphal surface by immunoelectron microscopy. They were also found in the host cell wall when P. tinctorius attached to the root surface. RNA blot analysis showed that the steady-state level of PtSRAP32 transcripts exhibited a drastic up-regulation when fungal hyphae form the mantle. These results suggest that SRAPs may form part of a cell-cell adhesion system needed for aggregation of hyphae in ectomycorrhizas.

Amino Acid Sequence↗