Search PubMed⌕ Search

Biomedical subjects

M Forte

Publications and source records attributed to M Forte.

At least 37 records · Page 2Linked to original sources
PubMed1989

Probing the structure of the mitochondrial channel, VDAC, by site-directed mutagenesis: a progress report.

The voltage-dependent anion-selective channel (VDAC) of the mitochondrial outer membrane is formed by a small (approximately 30 kDa) polypeptide, but shares with more complex channels the properties of voltage-dependent gating and ion selectivity. Thus, it is a useful model for studying these properties. The molecular biology techniques available in yeast allow us to construct mutant versions of the cloned yeast VDAC gene in vitro, using oligonucleotide-directed mutagenesis, and to express the mutant genes in yeast cells in the absence of wild-type VDAC. We find that one substitution mutation (lys 61 to glu) alters the selectivity of VDAC.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of acetylcholinesterase from Drosophila.

The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila are described. Sequential extraction procedures indicated that approximately 40% of the activity was soluble and 60% membrane-bound and that virtually none (less than 4%) corresponded to collagen-tailed forms. The membrane-bound enzyme was extracted with Triton X-100 and purified over 4000-fold by affinity chromatography on acridinium resin. Hydrodynamic analysis by both sucrose gradient centrifugation and chromatography on Sepharose CL-4B revealed an Mr of 165,000 similar to that observed for dimeric (G2) forms of the enzyme in mammalian tissues. In contrast, the purified enzyme gave predominant bands of about 100 kDa prior to disulfied reduction and 55 kDa after reduction on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, values that are significantly lower than those reported for purified G2 enzymes from other species. However, the presence of a faint band at 70 kDa which could be labeled by [3H]diisopropyl fluorophosphate prior to denaturation suggested that the 55-kDa band as well as a 16-kDa species arose from proteolysis. This was confirmed by reductive radiomethylation and amine analysis of the 70-, 55-, and 16-kDa bands. All three contained ethanolamine and glucosamine residues that are characteristic of a C-terminal glycolipid anchor in other G2 acetylcholinesterases. The catalytic properties of the enzyme were examined by titration with a fluorogenic reagent which revealed a turnover number for acetylthiocholine that was 6-fold lower than eel and 3-fold lower than human erythrocyte acetylcholinesterase. Furthermore, the Drosophila enzyme hydrolyzed butyrylthiocholine much more efficiently than these eel or human enzymes, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetylcholinesterases and butyrylcholinesterases.

Acetylcholinesterase↗
PubMed1987

Molecular genetics of the VDAC ion channel: structural model and sequence analysis.

The voltage-dependent anion-selective channel of the outer mitochondrial membrane provides a unique system in which to study the molecular basis of voltage gating of ion flow. We have cloned and sequenced a cDNA coding for this protein in yeast. From the derived amino acid sequence, we have generated a preliminary model for the secondary structure of the protein which suggests that the protein forms a "beta-barrel" type structure. Comparison of the VDAC amino acid sequence with that of the bacterial porins has indicated that the two classes of molecules appear to be unrelated.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast.

The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins from Drosophila, Dictyostelium and Neurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout, Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1986

Mutations resulting in resistance to polyene antibiotics decrease voltage-sensitive calcium channel activity in Paramecium.

In this report, the isolation of Paramecium tetraurelia mutants resistant to the polyene antibiotics amphotericin B and filipin are described. These antibiotics are known to specifically interact with membrane sterols to produce a cytotoxic effect. Four mutants resistant to amphotericin B and two mutants resistant to filipin have been isolated. In each case, an individual mutant shows resistance to both amphotericin B and filipin. Genetic analysis indicates that all 6 mutations map to the same complementation group and are not allelic to any of the 3 pawn mutations or to either of the two "barium shy" mutations. The behavioral analysis suggests that these mutations cause an alteration of normal ion channel function. Direct electrophysiological analysis of one mutant indicates mutations to polyene antibiotic resistance cause a specific decrease in voltage-sensitive Ca2+ channel activity.

Amphotericin B↗
PubMed1986

Ketoconazole treatment in superficial mycoses.

Ketoconazole is a new antifungal drug, used for oral treatment, with a broad spectrum of activity. It is a member of the imidazole series and is active in superficial and deep mycotic diseases, in candidosis and pityriasis versicolor. This new imidazole derivative has been successfully administered in several cases of dermatophytosis, yeasts and pityriasis versicolor in the authors' clinic, as well as in many other research centres. Treatment has been given to patients with tinea corporis, tinea cruris, tinea pedis, with or without location in interdigital spaces, while the cases of tinea capitis are still under investigation. The following variants of candida infections have been treated: onychomycosis, intertrigo and mucocutaneous candidosis due to prolonged immunosuppressive therapy. No remarkable haematochemical disorders have been observed after treatment.

Adolescent↗
PubMed1985

Muzolimine vs. furosemide in nephrotic syndrome: further support of different carrier(s) operating for enteral absorption.

The authors have compared during 4 weeks of study the effects of furosemide (F) by oral route or i.v. and muzolimine (M)/os in 10 patients with nephrotic syndrome (NS) and normal renal function. A satisfactory diuretic response was observed with i.v. F (100 mg) and M/os (30-60 mg) with respect to the basal condition (P less than 0.001), while F/os (100 mg) was ineffective in these patients. This behaviour may be explained by the different pharmacological properties of M vs. F, by gut alterations due to the oedema of mucosa or loss of some "carrier(s)" operating for intestinal absorption of these two high ceiling diuretics.

Adult↗
PubMed1984

Voltage-dependent calcium channels from Paramecium cilia incorporated into planar lipid bilayers.

Two different divalent cation-selective channels from Paramecium cilia were incorporated into planar lipid bilayers. Both channels were much more permeable to divalent than univalent cations, and one of them discriminated significantly among the divalent cations. The selectivity and voltage dependence of the latter channel are comparable to those of voltage-dependent calcium channels found in a variety of cells. A combined biochemical, biophysical, and genetic study of calcium channels is now possible.

Barium↗
PubMed1984

Characterization and purification of a soluble protein controlling Ca-channel activity in paramecium.

The analysis of the voltage-sensitive Ca++ channel of the unicellular eucaryote, Paramecium has been extended to a biochemical level based on recent observations that the transfer of cytoplasm from wild-type cells into mutants lacking Ca++-channel function ("pawn" in P. tetraurelia and "CNR" in P. caudatum) causes mutant cells to regain Ca++-channel function. We have microinjected various cytoplasmic fractions into mutant cells and measured the restored Ca++-channel function using a convenient behavioral assay. Following the "curing" activity, we characterized and purified the component from wild-type cytoplasm that can restore the function missing in cells carrying mutations in the cnrC gene. The curing factor is not an RNA, but a heat-labile, -SH-containing protein that appears to affect existing mutant channels on the ciliary membrane. We have purified this factor over 500-fold from the soluble cytoplasm using conventional techniques. The protein is of low apparent molecular weight (less than 30,000 daltons), acidic, soluble, and does not have the properties of calmodulin.

Animals↗