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Biomedical subjects

M Martin

Publications and source records attributed to M Martin.

At least 271 records · Page 15Linked to original sources
PubMed1997

Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten.

BACKGROUND: One of the main limitations of radioimmunotherapy (RIT) is the secondary toxicity related to the poor therapeutic indices achieved with labeled whole immunoglobulin (Ig)G or F(ab')2 fragments. To overcome this problem, we have developed a two-step targeting method, which we refer to as the Affinity Enhancement System (AES), using a radiolabeled bivalent hapten and a bispecific antibody recognizing the hapten and a target cell antigen. This method has been applied successfully to immunoscintigraphy in carcinoembryonic antigen (CEA)-expressing carcinoma patients and increased tumor to normal tissue uptake ratios have been achieved. The aim of the current study was to evaluate the application of AES to RIT of CEA-expressing solid tumors in an animal model. METHODS: Nude mice grafted with LS174T human colorectal carcinoma were treated either with 111 megabecquerels (MBq) of iodine-131 labeled bivalent diethylenetriamine pentaacetic acid (DTPA) hapten 20 hours after pretargeting by anti-CEA x anti-DTPA-indium bispecific antibody or 12 MBq of iodine-131 labeled anti-CEA IgG. RESULTS: Treatment with the IgG induced only a growth delay of 53 +/- 5 days but all tumors progressed. Treatment with the AES was highly efficient because tumor growth inhibition was achieved over 150 days. Hematologic and overall toxicity of both treatments were equivalent. CONCLUSIONS: The long term tumor regression consecutive to AES RIT represents a very significant improvement over the use of directly labeled IgG. Toxicity consecutive to AES or IgG RIT were similar despite an administered activity nearly ten times higher with the AES. However, given the efficacy of the AES treatment, a lower dose may afford lower toxicity and significant antitumor effect.

Animals↗
PubMed1997

Coactivation of AP-1 activity and TGF-beta1 gene expression in the stress response of normal skin cells to ionizing radiation.

Activation of the AP-1 transcription factor and TGF-beta1 growth factor by ionizing radiation was studied both in vivo in pig skin, and in vitro in human fibroblasts and keratinocytes. Three and 6 h after irradiation, the Fos and Jun proteins and their binding activity to an AP-1 consensus sequence were strongly induced by high doses of gamma-rays. c-Fos, c-Jun and JunB proteins were found to be present in gel-shift complexes by probing with specific antibodies. Both keratinocytes and fibroblasts exhibited heightened AP-1 activity following irradiation. As we previously found that TGF-beta1 is involved in the development of skin lesions induced by radiation, TGF-beta1 gene expression was also examined. Two and 6 h after irradiation, the levels of TGF-beta1 transcripts were increased in skin. By immunostaining, TGF-beta1 protein levels were found to be increased in fibroblasts, keratinocytes and endothelial cells. As the TGF-beta1 promoter contains AP-1 binding sites, the relation between AP-1 activity and TGF-beta1 induction was addressed. The -365 TGF-beta1 promoter fragment, which contains a high affinity AP-1 site, exhibited increased binding to Jun and Fos proteins following irradiation. These results suggest that stress-inducible TGF-beta1 expression is mediated by the activation of AP-1 transcription factor.

Animals↗
PubMed1997

A dual involvement of the amino-terminal domain of ezrin in F- and G-actin binding.

Human recombinant ezrin, or truncated forms, were coated in microtiter plate and their capacity to bind actin determined. F-actin bound ezrin with a Kd of 504 +/- 230 nM and a molecular stoichiometry of 10.6 actin per ezrin. Ezrin bound both alpha- and beta/gamma-actin essentially as F-form. F-actin binding was totally prevented or drastically reduced when residues 534-586 or 13-30 were deleted, respectively. An actin binding activity was detected in amino-terminal constructs (ezrin 1-310 and 1-333) provided the glutathione S-transferase moiety of the fusion protein was removed. Series of carboxyl-terminal truncations confirmed the presence of this actin-binding site which bound both F- and G-actin. The F- and G-actin-binding sites were differently sensitive to various chemical effectors and distinct specific ezrin antibodies. The internal actin-binding site was mapped between residues 281 and 333. The association of ezrin amino-terminal fragment to full-length ezrin blocked F-actin binding to ezrin. It is proposed that, in full-length ezrin, the F-actin-binding site required the juxtaposition of the distal-most amino- and carboxyl-terminal residues of the ezrin molecule.

Actins↗
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

Lack of interleukin 10 expression in monocyte-derived macrophages in response to in vitro infection by HIV type 1 isolates.

Interleukin 10 is a pleiotropic cytokine that is overexpressed in HIV-infected patients. Here, we investigated IL-10 expression in primary cultures of monocyte-derived macrophages (MDMs) in response to in vitro infection by HIV-1/Ba-L or two macrophage-tropic HIV-1 primary isolates. Whatever the multiplicity of infection used, and in spite of high replication levels and an increase in HIV-infected cell frequency, neither significant IL-10 secretion nor IL-10 mRNA overexpression was induced in HIV-1-infected MDMs. Moreover, identical results were obtained with HIV-1-infected 1-day monocytes. These results show that MDM infection by HIV is not sufficient by itself for inducing IL-10 synthesis.

Cells, Cultured↗
PubMed1997

Calcium release from intracellular stores and excitation-contraction coupling in intestinal smooth muscle.

Calcium release from intracellular stores plays a central role in excitation-contraction coupling of striated and smooth muscle cells. Two main intracellular calcium pools have been identified in phasic smooth muscle: (1) the inositol 1,4,5-trisphosphate-sensitive and the (2) ryanodine-sensitive calcium stores. We studied the contribution of the ryanodine-sensitive calcium stores to the excitation-contraction coupling in the intestine. The intracellular calcium concentration was measured in cultured intestinal smooth muscle cells using the fluorescent probe fura-2-AM. Isometric tension generated by the murine jejunum was recorded in vitro using force displacement transducers. The cytosolic calcium level increased significantly on cholinergic stimulation. The rise persisted in the absence of extracellular calcium. Depletion of ryanodine-sensitive calcium stores with caffeine or ryanodine blunted the response to a cholinergic agonists. Similarly, the ryanodine receptor channel blocker dantrolene significantly decreased the carbachol-induced calcium increase. We subsequently tested the effects of these pharmacological tools on the spontaneous and carbachol-induced contractions of the murine jejunum. Depletion of the ryanodine-sensitive stores and calcium release channel block both significantly decreased the contractile activity of the circular and longitudinal layer of the muscularis propria. Our data confirm the importance of intracellular calcium stores in excitation-contraction coupling of intestinal smooth muscle cells. The effects of different pharmacological tools on the intracellular calcium signal and the contractile function are consistent with other observations in phasic smooth muscle. They suggest a significant contribution of calcium release from ryanodine-sensitive stores to the calcium signal that triggers contraction.

Animals↗
PubMed1997

Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase.

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 degrees C and pH7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1. 4x10(-3) M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin-Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

Cell Membrane↗
PubMed1997

Attitudes toward environmental hazards: where do toxic wastes fit?

The public is continually faced with making decisions about the risks associated with environmental hazards, and, along with managers and government officials, must make informed decisions concerning possible regulation, mitigation, and restoration of degraded sites or other environmental threats. We explored the attitudes regarding several environmental hazards of six groups of people: undergraduate science majors, undergraduate nonscience majors, and graduate students in environmental health, in ecological risk assessment, and in nonscience disciplines, as well as nonstudents over 35 yr of age. We had predicted that there would be significant differences in attitudes between science and nonscience majors and as a function of age. Relative concerns could be divided into three discrete classes (in descending order of concern): (1) general ecological problems (cutting tropical forests, polluting groundwater, trash along the coasts, lead in drinking water, and acid rain), (2) radon and nuclear wastes, and finally (3) specific nuclear waste facilities, chromium, fertilizers and pesticides, and electromagnetic waves. For any hazard, attitudes were consistent across groups with regard to ranking the severity of the environmental problem and willingness to expend funds to solve the problems. Attitudes about spending money to develop methods to evaluate risk fell in the middle level of concern. There were no major differences among classes of college-age students, or between them and older nonstudents.

Adult↗
PubMed1997

Surface plasmon resonance analysis of gp17, a natural CD4 ligand from human seminal plasma inhibiting human immunodeficiency virus type-1 gp120-mediated syncytium formation.

We have previously isolated from human seminal plasma a CD4 ligand, the gp17 glycoprotein, which shares sequence identity with three previously identified proteins: secretory actin-binding protein (SABP) from seminal plasma, gross-cystic-disease fluid protein-15 (GCDFP-15) and prolactin-inducible protein (PIP) from breast tumor cells. Functions of these glycoproteins are unknown. To further characterize the physical interaction between gp17 and CD4 we used surface plasmon resonance and demonstrated that gp17-CD4 binding affinity is high. Competition experiments indicated that gp17 interferes with human immunodeficiency virus (HIV) envelope protein/CD4 binding, although it binds to a site distinct from but close to the gp120-binding site. We observed moreover that gp17 inhibits syncytium formation between transfected cells expressing the wild-type HIV-1 envelope glycoprotein and CD4, respectively. Our results suggest that gp17, which may function as an immunomodulatory CD4-binding factor playing a role at insemination, may also play a role in controlling HIV spread in the sexual tract.

Apolipoproteins↗
PubMed1997

A Polycomb-group gene regulates homeotic gene expression in Arabidopsis.

Cell fate is determined when the commitment of cells to a particular fate is autonomously maintained, irrespective of their environment. In Drosophila, fate determination is maintained through the action of the Polycomb-group and trithorax-group genes, which are required so that states of homeotic gene activity are inherited through cell division. It is shown here that the CURLY LEAF gene of Arabidopsis is necessary for stable repression of a floral homeotic gene and encodes a protein with homology to the product of the Polycomb-group gene Enhancer of zeste. We suggest that Polycomb-group genes have a similar role in fate determination in plants and animals.

AGAMOUS Protein, Arabidopsis↗
PubMed1997

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus.

Amino Acid Sequence↗
PubMed1997

Zinc inhibits interleukin-1-dependent T cell stimulation.

Zinc is a trace element which is essential for immune functions. It directly induces monokine secretion by monocytes; however, effects of zinc on T cells appear contradictory. Apart from enhanced lymphocyte proliferation in peripheral blood mononuclear cells (PBMC), inhibitory properties of high zinc dosages have also been described. In this study, PBMC failed to produce lymphokines like interferon (IFN)-gamma after stimulation with zinc in a serum- and LPS-free cell culture system, whereas monokine secretion [interleukin (IL)-1 beta] occurred. Zinc-uptake studies with the zinc-specific fluorescent probe zinquin revealed that zinc is taken up by PBMC within a few minutes, reaching nearly equal levels in PBMC, isolated monocytes, and T cells. However, if zinc was depleted 1 h after monocyte induction, zinc-free pre-cultured T cells were stimulated to secrete IFN-gamma by zinc-induced monokines. Furthermore, the necessity for a cell-cell interaction between monocytes and T cells for IFN-gamma induction was elucidated. Zinc ions inhibited the proliferation of the IL-1-dependent T cell line D 10N in a dose-dependent manner, suggesting a direct inhibitory effect of zinc. By immunoprecipitation we revealed a specific inhibition of IL-1 receptor-associated protein kinase (IRAK) by zinc ions. Therefore, in contrast to an indirect stimulation of T cells due to zinc-induced monokines, higher concentrations of zinc directly inhibit T cell functions by means of specific inhibition of IRAK and subsequent signaling events such as NF kappa B activation. The divergent effects of zinc on different cell populations, depending on the zinc concentration, could explain contradictory results of zinc stimulation. Furthermore, our data suggest new strategies of specific zinc-mediated immune modulation.

Animals↗
PubMed1997

Inducible expression of Na+/myo-inositol cotransporter mRNA in anterior epithelium of bovine lens: affiliation with hypertonicity and cell proliferation.

Bovine lenses were pretreated with physiological (314 +/- 6 mosm) or hypertonic medium (> 450 mosm) for 20 hours prior to introduction of the lenses into modified Ussing chambers (providing a leakproof seal at the lens equator). The anterior (epithelial-containing) and posterior (cell-free) aspects of the mounted intact lens could thus be independently bathed with myo-[3H]inositol. Myo-inositol uptake as a function of concentration across the anterior aspect was indicative of an active carrier-mediated component and at high concentration, significant myo-inositol influx due to passive diffusion. Myo-inositol uptake was markedly stimulated across the anterior aspect and inhibitable by ouabain but not stimulated across the posterior aspect, when lenses were exposed to hyperosmotic medium by the addition of raffinose or sodium chloride. Myo-inositol rapidly desaturates across both aspects of the lens consistent with its efflux being largely a function of passive diffusional leakout from the extracellular space. In order to corroborate the apparent hypertonic-induced upregulation of gene expression inferred by kinetic analysts, the level of Na+/myo-inositol cotransporter mRNA was determined by reverse transcription and quantitative PCR using harvested anterior epithelial cells from intact lenses exposed to physiologic or hypertonic medium for 20 hours. RNA samples were microextracted from both the central and equatorial regions of the lenticular epithelium of the anterior lens capsule. The abundance of Na+/myo-inositol cotransporter mRNA was similar from epithelium of the central zone irrespective of medium tonicity but was markedly elevated from the equatorial zone of osmotically-stressed lenses. While both the quiescent epithelial cells of the central region and actively dividing epithelial cells of the equatorial zone of the intact lens express Na+/myo-inositol contransporter mRNA, only the equatorial epithelium responds to hypertonic insult with enhanced uptake of myo-inositol due to increased transcription of the Na+/myo-inositol cotransporter gene, suggesting a possible role for the state of cell proliferation.

Animals↗
PubMed1997

Increase in oxidative key enzymes in a case of muscle ubiquinol-cytochrome c reductase deficiency.

In a 29-year-old patient suffering from exertional muscle intolerance with a ubiquinol-cytochrome c reductase deficiency related to a cytochrome b gene point mutation of the mitochondrial DNA, we conducted a study of the aims of which were: (1) to test whether changes in the maximum activities of muscle key enzymes of the main energy-producing pathways occur, (2) to address the issue of whether fibers of different types are equally affected in their enzymatic machinery involved in energy production, and (3) to correlate the results obtained with histochemical and 31P NMR spectroscopy data. When compared to results obtained in six normal subjects, our study clearly shows that the type I fibers of the patient virtually all contained subsarcolemmal mitochondrial aggregates and increased activities of succinate dehydrogenase and cytochrome c oxidase; microdissected type I fibers also displayed a significant increase in both citrate synthase and beta-hydroxyacyl-CoA dehydrogenase, two key enzymes of mitochondrial oxidative metabolism. Despite these changes in the patient's muscle, its whole energy-producing machinery remained impaired as revealed by a slowed post-exercise recovery of phosphocreatine.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1997

Differential expression of the HsKin17 protein during differentiation of in vitro reconstructed human skin.

In eukaryotic cells, various proteins homologous to the E. coli RecA protein are involved in the elimination of DNA damage. These proteins contribute to the repair of double-strand breaks and to genetic recombination. The mouse Kin17 protein is recognised by antibodies directed against the RecA protein. Kin17 has a zinc-finger domain allowing binding to curved DNA stretching over illegitimate recombination junctions. In the present study, we identified the human counterpart of the mouse Kin17 protein (named HsKin17) in skin cells. We employed an in vitro reconstructed skin model composed of an epidermal sheath lying on a dermal matrix with human fibroblasts embedded in rat collagen type I. The maturation programme (proliferation versus differentiation) of keratinocytes was highly dependent on stromal cells. Immunohistochemical staining of frozen sections obtained from skin specimens was monitored by an interactive laser cytometer. In this way we analysed protein levels in both dermal and epidermal compartments. After having characterised the epithelium, we focused our attention on HsKin17 expression. We detected HsKin17 in human keratinocytes. HsKin17 protein levels increased in proliferating epithelial keratinocytes after 7 days of culture. After 2 weeks of culture, epidermal sheaths acquired most of the differentiated features of mature epithelium. At this time, HsKin17 protein dropped below measurable levels in the stratum corneum, and diminished in nucleated cells. This study showed that HsKin17 is expressed in human reconstructed epithelium under conditions of hyperproliferation.

Adult↗