Biomedical subjects

D Muller

Publications and source records attributed to D Muller.

At least 127 records · Page 7Linked to original sources

PubMed1994

Inhibition of cathepsin L-like cysteine proteases by cytotoxic T-lymphocyte antigen-2 beta.

The protein sequence of cytotoxic T-lymphocyte antigen-2 beta (CTLA-2 beta) is 36% identical to the proregion of mouse cathepsin L (Denizot, F., Brunet, J.F., Roustan, P., Harper, K., Suzan, M., Luciani, M. F., Mattei, M. G., and Goldstein, P. (1989) Eur. J. Immunol. 19, 631-635). Here we report the expression, purification, and characterization of recombinant murine CTLA-2 beta. The protein was purified by consecutive gel-filtration, anion-exchange, and reverse-phase (C4) chromatography. Purified CTLA-2 beta exists in solution primarily as a dimer but also as a disulfide-linked tetramer as judged by size exclusion chromatography. Circular dichroism studies suggest that the dimeric form of the protein contains 8% alpha-helix, 67% beta-sheet, and 21% random coil and also indicates that there is a conformational change upon formation of the tetramer. The protein is a competitive inhibitor of certain cysteine proteases including papain (Ki = 25 nM), cathepsins L (Ki = 24 nM) and H (IC50 = 67 nM) but not cathepsin B. CTLA-2 beta forms a noncovalent complex with cathepsin L and has a stoichiometry of binding to papain of 1 mol of CTLA-2 beta/mol of papain. There is no homology between CTLA-2 beta and any of the known cysteine protease inhibitors, including the kininogens and cystatins. Therefore, CTLA-2 beta represents a novel class of cysteine protease inhibitor that is specific for the cathepsin L family of proteases.

Antigens↗

PubMed1994

Enumeration of IFN-gamma producing lymphocytes by flow cytometry and correlation with quantitative measurement of IFN-gamma.

We describe a flow cytometry method to simultaneously analyze IFN-gamma production and T cell surface phenotype in freshly isolated lymphocytes without requiring prior in vitro stimulation. We show that enumeration of intracytoplasmic IFN-gamma positive T cells correlates with quantitative measurement of IFN-gamma in culture supernatant fluids. This suggests that cytokines can be reliably measured using flow cytometry. Flow cytometry has the added advantage of simultaneous detection of cell surface markers. Furthermore, we suggest that analysis of ex vivo IFN-gamma production at the single cell level may reflect more accurately T cell IFN-gamma production, by avoiding the polyclonal stimulation of IFN-gamma production observed after short term in vitro stimulation.

Animals↗

PubMed1994

Are antioxidants or supplements protective for age-related macular degeneration?

OBJECTIVES: The relationships between fasting plasma levels of retinol, ascorbic acid, alpha-tochopherol, and beta-carotene and age-related macular degeneration (AMD) were studied in a population enrolled in the Baltimore Longitudinal Study of Aging (BLSA), in which most of the data were collected 2 or more years before assessment of macular status. DESIGN: A total of 976 participants in the study were scheduled for a biennial examination from January 1988 through January 1, 1990, which included taking lens and macular photographs. A total of 827 (85%) of the participants had fundus photographs taken, and most plasma data were available for 82% of those subjects with fundus photographs. Age-related macular degeneration was defined as neovascular changes, geographic and nongeographic atrophy, large or confluent drusen, or hyperpigmentation. A total of 226 cases of AMD were available for analysis. RESULTS: Logistic regression analyses suggested that alpha-tocopherol was associated with a protective effect for AMD, adjusted for age, sex, and nuclear opacity. An antioxidant index, including ascorbic acid, alpha-tocopherol, and beta-carotene, was also protective for AMD. Our conclusions must be tempered with the knowledge that the population under study was basically well nourished, and few individuals had any clinically deficient status. The study cannot exclude the possibility that quite low levels of micronutrients, lower than those observed in this study, might be risk factors for AMD. CONCLUSIONS: The data suggest a protective effect for AMD of high plasma values of alpha-tocopherol. An antioxidant index, composed of plasma ascorbic acid, alpha-tocopherol, and beta-carotene, was also protective. The use of vitamin supplements to prevent AMD is not supported by these data, which showed no protective effect of vitamin use.

Adult↗

PubMed1994

A new cytochemical method for the ultrastructural localization of calcium in the central nervous system.

We have developed a new cytochemical method for the localization of calcium at the ultrastructural level in the central nervous system (CNS). The method is based on the use of phosphate buffer in the primary fixation followed by a mixture of a complex of chromium(III)-trisoxalate and osmium tetroxide (OsO4) which precipitates calcium and results in the formation of a high electron-dense reaction product. Calcium selectivity was verified by reactions made in test tube, by EGTA treatment of the tissue, by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The technique was found to be reproducible, yielding similar results in acutely prepared hippocampal slices or organotypic cultures fixed by immersion and in brain areas fixed by perfusion. In hippocampal slices, calcium deposits were found to accumulate in different subcellular compartments such as endoplasmic reticulum, mitochondria and synaptic vesicles. Interestingly, electron-dense reaction products were also visualized in smooth endoplasmic reticulum structures localized in presynaptic terminals or post-synaptic spines as well as in synaptic clefts and active zones. This new method may thus be of interest for studying the metabolism of calcium, specifically with regard to synaptic activity, in the CNS.

Animals↗

PubMed1994

A role for polysialylated neural cell adhesion molecule in lesion-induced sprouting in hippocampal organotypic cultures.

By mediating cell-cell interactions, the neural cell adhesion molecule (N-CAM) has been implicated in various events such as axonal pathway formation, neurite outgrowth or synaptic remodelling. One mechanism by which N-CAM could contribute to these events has been proposed to involve modifications of the content of the molecule in polysialic acid. Here we have tested this possibility using an in vitro model of lesion-induced reactive synaptogenesis in hippocampal organotypic cultures. We present evidence that the sprouting reaction triggered by a section of CA3-CA1 connections in these cultures is associated with the expression of the highly sialylated form of N-CAM on regenerating neurites. In addition, we have examined the functional importance of this sialylation mechanism by analysing the effect of treating sectioned cultures with endo-neuraminidase-N which removes the polysialic acid portions of N-CAM. Measurements of the time course of recovery from the lesion, as assessed by the formation of new functional synaptic contacts across the section, showed that removal of the polysialic acid moieties of N-CAM significantly delays the sprouting reaction. The results support the idea that up regulations of highly sialylated forms of N-CAM are of functional importance in neurite sprouting and synapse regeneration in this in vitro model.

Animals↗

PubMed1994

Frequent amplification of 11q13 DNA markers is associated with lymph node involvement in human head and neck squamous cell carcinomas.

Amplification of 11q13 DNA markers, particularly hst-1/FGF4 oncogene and the bcl-1 locus, was evaluated in 178 head and neck squamous cell carcinomas (SCCs) by Southern blot and slot blot hybridisation. Coamplification of hst-1/FGF4 and bcl-1 genes was found in 57% of primary tumours and in 60% of the 89 metastatic lymph nodes tested. The pattern of amplification was significantly similar in matched sets of primary SCCs and metastatic lymph nodes. Levels of amplification, quantified by densitometric analysis of slot blots, ranged from 2 to 18-fold normal gene dosage. Also, c-myc oncogene (8q24) was found amplified less frequently, since 7% of 169 SCCs tested contained amplification of this gene, the level of which ranged from 2 to 8-fold. Hst-1/bcl-1 gene amplification was observed more frequently in the tumours arising from the hypopharynx. Coamplification of hst-1 and bcl-1 genes was significantly positively associated with tumours with nodal involvement (P = 0.001). Incidence of hst-1/bcl-1 gene amplification is higher in the tumours with a clinical stage III or IV. Hst-1/bcl-1 gene amplification was not related to tumour differentiation or local invasiveness. This prospective study shows that amplification of 11q13 DNA markers is a prominent event occurring in head and neck SCC and may contribute to the pathogenesis and evolution of a subset of patients bearing this type of cancer.

Blotting, Southern↗

PubMed1994

Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia.

An ex vivo approach to gene therapy for familial hypercholesterolaemia (FH) has been developed in which the recipient is transplanted with autologous hepatocytes that are genetically corrected with recombinant retroviruses carrying the LDL receptor. We describe the treatment of a 29 year old woman with homozygous FH by ex vivo gene therapy directed to liver. She tolerated the procedures well and in situ hybridization of liver tissue four months after therapy revealed evidence for engraftment of transgene expressing cells. The patient's LDL/HDL ratio declined from 10-13 before gene therapy to 5-8 following gene therapy, improvements which have remained stable for the duration of the treatment (18 months). This represents the first report of human gene therapy in which stable correction of a therapeutic endpoint has been achieved.

Adult↗

PubMed1994

Safety and toxicity of catheter gene delivery to the pulmonary vasculature in a patient with metastatic melanoma.

One approach to gene therapy for human cancer is transcatheter injection of DNA liposomes into tumor masses. To determine the feasibility of selective delivery of recombinant genes by a catheter to the pulmonary vasculature in humans, a patient with melanoma received two treatments of HLA-B7 plasmid DNA complexed to cationic liposomes into a right posterior basal pulmonary artery associated with a mass lesion. The treatments were well tolerated. No adverse respiratory, cardiac, immunologic, or other organ toxicities were detected. The delivery of recombinant genes by catheter may be a useful modality to treat human malignancy and other diseases.

Adenocarcinoma↗

PubMed1994

Concentrations of vitamins A and E in amniotic fluid, extraembryonic coelomic fluid, and maternal serum in the first trimester of pregnancy.

Paired samples of amniotic fluid and coelomic fluid were obtained by transvaginal ultrasound guided amniocentesis from 15 women with an ultrasonographically normal pregnancy between 8 and 12 weeks' gestation. Vitamins A and E were measured in the two pregnancy fluids and in maternal serum by high performance liquid chromatography with detection by ultraviolet absorption and fluorimetry respectively. Concentrations of vitamins A and E were higher in maternal serum than in coelomic fluid and were generally undetectable in amniotic fluid. All differences in concentration were significant. The vitamin E/cholesterol ratio was similar in maternal serum and coelomic fluid. No correlation was shown between the vitamin concentrations in the three fluids. These findings suggest that the coelomic cavity plays a part in the maternofetal exchange of these vitamins.

Amniotic Fluid↗

PubMed1994

Tyrosyl sulfamide derivatives as ligands for affinity adsorption of anti FVIII antibodies.

Human anti factor VIII (anti FVIII) antibodies neutralize factor VIII procoagulant activity. These antibodies appear in about 5-10% of severely affected haemophiliac A patients treated with FVIII concentrates. In order to obtain non-thrombogenic materials able to interact specifically with anti FVIII, we have grafted amino acid residues which mimic part of the epitope of the FVIII molecule recognized by the anti FVIII. For this purpose, crosslinked polystyrenes functionalized with sulfonate and tyrosyl or methyl ester tyrosyl sulfamide groups have been synthesized and characterized. The in vitro removal of anti FVIII from haemophiliac patient plasma with antibodies, was performed on these different active supports. These polymers exhibit strong and selective affinity for the anti FVIII. The adsorption of the antibodies vary with the percentage of units bearing methyl ester tyrosyl sulfamide groups and present a maximum at 25% grafting rate. For the more efficient resin, the affinity constant, determined for the adsorption isotherm for the anti FVIII is about 10(9) M-1, whereas the affinity constant for the IgG in the same experimental conditions, is low (10(5) M-1). The influence of different chemical groups on the polymeric phase, on their affinity for the inhibitors was also studied. The most active resins can be selected and used in an extracorporeal circulation to reduce the concentration of anti FVIII in a blood epuration process.

Adsorption↗

PubMed1994

Body mass index and mortality in Seventh-day Adventist men. A critique and re-analysis.

The aim of this work was to determine if the relationship between weight-adjusted-for-height (expressed as body mass index or BMI) using the BMI-at-entry and age-at-entry as opposed to BMI at entry and age-at-event (i.e. death, loss to follow-up, or end of the study) would alter the results previously reported from a population of Seventh-day Adventist men. The subjects were 8828 non-smoking, non-drinking Seventh-day Adventist men, ages 30-89 and older on entry, mean follow-up 15 years (maximum 26 years). The BMI and age reported by subjects when they were enrolled into the study were used to calculate the relationship between BMI and mortality. Mortality rates in each of five BMI quintiles were computed by dividing the number of deaths in each quintile by the number of person years of follow-up in the quintile. Rate ratios were computed by dividing each mortality rate by the rate in the reference quintile. The mortality rate ratios were then adjusted for the age difference between each quintile and the reference quintile. Calculations based upon age-at-enrollment rather than 'age-at-event' (as used in the original paper) demonstrate no increase in mortality until a BMI of 27.5 kg/m2 or greater is reached rather than a progressive increase in mortality with increasing BMI.

Adult↗

PubMed1993

Time-dependent reversal of long-term potentiation by brief cooling shocks in rat hippocampal slices.

Using a recording chamber built with peltier elements, we studied the effects of fast and brief reductions in temperature on synaptic transmission and plasticity in area CA1 of rat hippocampal slices. Cooling shocks consisted of a drop in temperature from 33 degrees C to 30 degrees C, 27 degrees C or 24 degrees C for 2-5 min. Equilibrium to the new temperature was reached in about 30 s. During these cooling episodes, marked modifications of the size and time course of synaptic responses were observed. Changing the temperature for 4-5 min from 33 degrees C to 24 degrees C resulted in a 75% reduction in amplitude and 158% prolongation of the rise time of excitatory postsynaptic potentials (EPSPs). These changes were followed by a complete recovery of synaptic transmission. This recovery was very fast for the EPSP rise time (about 30 s), but much slower for the amplitude or initial slope (20-30 min). This slow recovery was correlated with changes in size of the presynaptic fiber volley, thereby indicating transient modifications of cell excitability. Application of cooling episodes of 4-5 min from 33 degrees C to 24 degrees C during the first 20 min that followed induction of long-term potentiation resulted in a complete reversal of synaptic potentiation. The LTP abolished by a cooling shock could be reinstated by re-applying high frequency trains. Several sequential induction/abolition effects could thus be obtained. In contrast, cooling episodes applied later than 25 min after LTP induction did not affect synaptic potentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Enhancement of AMPA-mediated synaptic transmission by the protein phosphatase inhibitor calyculin A in rat hippocampal slices.

Using the phosphatase inhibitor calyculin A, we have examined the influence of phosphorylation on synaptic transmission and plasticity in rat CA1 hippocampal slices. Bath application of 0.5-1 microM of calyculin A resulted in an increase of 42.6 +/- 2.9% in synaptic responses. The effect produced by calyculin A was not accompanied by changes in fibre volley, was not associated with changes in paired-pulse facilitation, and could be reproduced by intracellular injection of the compound, thereby indicating a postsynaptic action. Also, the synaptic enhancement produced by calyculin A was expressed only by potentials mediated by amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, but not by the NMDA responses recorded in the presence of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and low magnesium. The effect of calyculin A could be prevented by KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II. Long-term potentiation could still be induced in the presence of calyculin A, but the effect of the compound was slightly reduced on potentiated compared with control pathways. These results indicate that calyculin A can selectively increase the efficacy of AMPA receptor-mediated synaptic transmission at excitatory synapses.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1993

Affinity chromatography of sulphated polysaccharides separately fractionated on antithrombin III and heparin cofactor II immobilized on concanavalin A--sepharose.

Three sulphated polysaccharides, dermatan sulphate, fucan and heparin, were fractionated according to their affinity towards antithrombin III (ATIII) and heparin cofactor II (HCII), the two main physiological thrombin (IIa) inhibitors. Both inhibitors were immobilized on concanavalin A-Sepharose, which binds to the glycosylated chains of the proteins while the protein-binding site for the polysaccharide remains free. Each polysaccharide was fractionated into bound and unbound fractions either for ATIII or HCII. The eluted fractions were tested for their ability to catalyse ATIII/IIa and HCII/IIa interactions. The possible presence of a unique binding site for ATIII and HCII, on each sulphated polysaccharide, was also studied.

Animals↗

PubMed1993

Long-term potentiation is associated with an increased activity of Ca2+/calmodulin-dependent protein kinase II.

Among the molecular mechanisms that have been proposed to contribute to long-term potentiation in hippocampus are the activation and autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). Here we report that high, but not low frequency stimulation applied to two groups of CA1 afferents resulted in a long lasting increase in the Ca(2+)-independent and total activities of the enzyme as well as an increase in the ratio of Ca(2+)-independent to total activity. The effect was obtained using two different CaM kinase II substrates, it was observed in hippocampal slices and in hippocampal organotypic cultures, and it could be blocked by preincubation of slices with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonopentanoate. Treatment of slices with calyculin A, a phosphatase inhibitor, modified the activity of the enzyme, but long term potentiation could still be induced and a further increase in Ca(2+)-independent CaM kinase II activity still observed.

Animals↗

PubMed1993

Structural modifications associated with synaptic development in area CA1 of rat hippocampal organotypic cultures.

Using morphological techniques, we characterized the developmental reorganization that takes place during the first weeks after explanation in area CA1 of organotypic hippocampal cultures maintained at the interface between medium and a CO2-enriched atmosphere. Pyramidal neurones redistributed from a vertical into an horizontal cell layer in the middle of a three-dimensional culture, with apical dendrites running above the pyramidal layer. Glial cells redistributed into a thin layer at the bottom of the culture, forming an interface between tissue and culture medium. Astrocytes were identified as the most numerous non neuronal cells. No sign of glial proliferation could be observed, except for a transient increase during the first days after explanation. The density of synaptic contacts in the stratum radiatum decreased immediately after explanation and then increased by about 20-fold to reach values in the proximal part of the apical layer after 4 weeks in culture which were only slightly smaller than those measured in 1-month-old rats. The synaptic density in the most distal part of the dendritic layer which receives connections extrinsic to the hippocampus remained significantly lower than in vivo. The ratio of spine to shaft contacts was comparable to that found in vivo. These results indicate that interface type of organotypic cultures can be used as an interesting model for studies of synaptic development in vitro.

Animals↗

PubMed1993

Time course of synaptic development in hippocampal organotypic cultures.

Using electrophysiological recordings of field potentials, we investigated the time course of synapse formation and maturation in organotypic cultures prepared from neonate animals of different ages. Following explanation, the size of the maximal synaptic responses elicited in area CA1 by stimulation of a small group of CA3 neurons increased progressively during the first three weeks in culture in a way that corresponded to the changes observed in synaptic contact density. Growth of synaptic responses was found to occur much more rapidly in cultures prepared from 8-day-old as compared with 2-day-old rats. Development of synaptic connections between CA3 and CA1 neurones was also faster than between granule cells and CA3 neurones. Acquisition of mature synaptic properties occurred in vitro as indicated by changes in degree of paired-pulse facilitation and the onset of long-term potentiation (LTP) after a few days in culture. The onset of LTP was much faster in cultures prepared from 8-day-old as compared with 2-day-old neonates and corresponded approximately to the 12-14th postnatal day. It is concluded that development proceeds in the cultures with a time course that resembles the in situ situation.

Animals↗

PubMed1993

Increased stromelysin 3 gene expression is associated with increased local invasiveness in head and neck squamous cell carcinomas.

Matrix metalloproteinases are believed to play an important role in tumor invasion and metastasis. To examine the expression of the stromelysin 3 (ST3) gene, a new member of the matrix metalloproteinase gene family, 111 head and neck squamous cell carcinomas and 21 metastatic lymph nodes were analyzed by Northern blot. ST3 gene expression was observed in 106 carcinomas and 19 metastatic nodes, but in only 2 of 60 samples of corresponding normal tissue tested in parallel. ST3 RNA, by in situ hybridization, and ST3 protein, by immunohistochemical analysis, were specifically detected in fibroblastic cells immediately surrounding invasive cancer cells. This fibroblastic expression of the ST3 gene is characteristic among the matrix metalloproteinase genes known to be overexpressed in head and neck carcinomas, since stromelysin 2 transcripts were specifically detected in neoplastic cells, and type I collagenase transcripts in both neoplastic cells and stromal fibroblasts. Furthermore, there was a highly significant positive correlation (P < 0.0001) between ST3 RNA levels and local invasiveness by the cancer cells, suggesting that enhanced expression of the ST3 gene may contribute to the neoplastic phenotype in head and neck carcinomas.

Carcinoma, Squamous Cell↗