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G Cecchini

Publications and source records attributed to G Cecchini.

At least 37 records · Page 2Linked to original sources
PubMed1998

Protein phosphatase 1 delta is associated with focal adhesions.

In all mammalian cells protein phosphatase-1 (PP1) exists in three isoforms, defined as alpha, gamma 1 and delta. Immunofluorescence studies with isoform-specific antibodies indicated that delta, but not alpha or gamma 1, is enriched at focal adhesions in HeLa cells, fibroblasts, endothelial cells and keratinocytes. This was confirmed also by interference reflection microscopy, which indicated that PP1 delta was in areas of tight adhesion of the membrane to the extracellular matrix at sites where the microfilament cytoskeleton is organized. In all the cell types so far considered the PP1 delta in focal adhesions represented only a small aliquot of the total PP1 delta, which was predominantly localized to the nucleus. The association of PP1 delta to focal adhesions was confirmed by the co-immunoprecipitation of PP1 delta with the focal adhesion kinase pp125FAK and with the alpha v integrin. Comparison between the amount of PP1 delta associated with focal adhesion proteins and that of PP1 delta recovered in an anti-PP1 delta immunoprecipitate confirmed that only a minor amount of the enzyme was associated with the focal adhesions. Since some focal adhesion proteins are phosphorylated on Ser/Thr, it is likely that PP1 delta may be involved in the regulation of focal adhesion functions and particularly in the signaling pathway generated by cell-substratum adhesion.

3T3 Cells↗
PubMed1996

Inhibitor probes of the quinone binding sites of mammalian complex II and Escherichia coli fumarate reductase.

The structural and catalytic properties of beef heart succinate dehydrogenase (succinate-ubiquinone oxidoreductase, complex II) and Escherichia coli fumarate reductase are remarkably similar. One exception is that whereas electron exchange between the mammalian enzyme and its quinone pool is inhibited by thenoyltrifluoroacetone and carboxanilides, the enzyme from E. coli is not sensitive to these inhibitors. The lack of good inhibitors has seriously hampered the elucidation of the mechanism of quinone oxidation/reduction in the E. coli enzyme. We have previously reported (Tan, A. K., Ramsay, R. R., Singer, T. P., and Miyoshi, H. (1993) J. Biol. Chem. 268, 19328-19333) that 2-alkyl-4,6-dinitrophenols inhibit mammalian complexes I, II, and III, but with different potencies and kinetic characteristics. Based on these studies we have selected a series of 2-alkyl-4,6-dinitrophenols which proved to be very effective noncompetitive inhibitors of mammalian complex II, particularly when acting in the direction of quinone reduction, the physiological event. These compounds turned out to be even more potent inhibitors of E. coli fumarate reductase, particularly when acting in the direction of quinol oxidation, again, the physiological event. Kinetic analysis revealed that with both enzymes 2 inhibitor binding sites seem to be involved in the oxidation of succinate by quinone, but one seems to be functioning when fumarate is reduced by external quinol. Since the E. coli enzyme can be modified by site-directed mutagenesis, these studies were extended to four mutants of fumarate reductase, impaired by single amino acid substitutions at either of the putative quinone binding sites (QA or QB) of the enzyme. The results were analyzed in terms of the model of these dual sites of quinone binding in fumarate reductase, as well as the nature of the substituent in the 2-position of the dinitrophenol inhibitors.

Animals↗
PubMed1996

Primary lymphoma of the mandible with diffuse widening of the mandibular canal: report of a case.

A case of primary non-Hodgkin's lymphoma (NHL) of the mandible with unusual radiographic presentation is reported. The panoramic radiography revealed diffuse widening of the right mandibular canal with ill-defined margins and absence of edge sclerosis. Computed tomography showed infiltration of the superior portion of the canal with involvement of the vestibular cortex of the bone and of the alveolar ridge.

Adult↗
PubMed1995

Effect of cysteine to serine mutations on the properties of the [4Fe-4S] center in Escherichia coli fumarate reductase.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys148, Cys151, Cys154, and Cys158 are replaced individually by Ser have been constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters have been investigated. The Cys154Ser and Cys158Ser FrdB mutations result in enzymes with negligible activity that have largely dissociated from the cytoplasmic membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters. In contrast the Cys148Ser and Cys151Ser FrdB mutations result in functional membrane bound enzymes that are able to support growth under anaerobic and aerobic conditions. EPR studies of these mutants indicate that all three of the constituent Fe-S clusters are assembled, and the redox and spectroscopic properties of the [2Fe-2S] and [3Fe-4S] clusters are unchanged compared to the wild-type enzyme. In both mutants the [4Fe-4S] cluster is assembled with one non-cysteinyl ligand, and the available data suggest serinate coordination. The physicochemical consequences are perturbation of the intercluster spin interaction between the S = 1/2 [4Fe-4S]+ and S = 2 [3Fe-FS]0 clusters and a 60-mV decrease in redox potential for the [4Fe-FS]2+,+ cluster in the FrdB Cys148Ser mutant, and a S = 1/2 to S = 3/2 spin state conversion for the [4Fe-4S]+ cluster and a 72-mV decrease in redox potential for the [4Fe-4S]2+,+ cluster in the FrdB Cys151Ser mutant. Taken together with the previous FrdB Cys to Ser mutagenesis results [Werth, M. T., Cecchini, G., Manodori, A., Ackrell, B. A. C., Schröder, I., Gunsalus, R. P., & Johnson, M. K. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 8965-8969; Manodori, A., Cecchini, G., Schröder, I., Gunsalus, R. P., Werth, M. T., & Johnson, M. K. (1992) Biochemistry 31, 2703-2712], the results provide strong support for the proposal that all three clusters are located in the FrdB subunit with Cys57, Cys62, Cys65, and Cys77 ligating the [2Fe-2S] cluster, Cys148, Cys151, Cys154, and Cys214 ligating the [4Fe-4S] cluster, and Cys158, Cys204, and Cys210 ligating the [3Fe-4S] cluster. The role of the low potential [4Fe-4S] cluster in mediating electron transfer from menaquinol to the FAD active site is discussed in light of these mutagenesis results.

Amino Acid Sequence↗
PubMed1995

Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain.

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found n fumarate reductase and succinate dehydrogenase.

Aerobiosis↗
PubMed1994

Cloning, sequencing, and expression of the structural genes for the cytochrome and flavoprotein subunits of p-cresol methylhydroxylase from two strains of Pseudomonas putida.

The structural genes for the flavoprotein subunit and cytochrome c subunit of p-cresol (4-methylphenol) methylhydroxylase (PCMH) from Pseudomonas putida NCIMB 9869 (National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland) and P. putida NCIMB 9866 were cloned and sequenced. The genes from P.putida NCIMB 9869 were for the plasmid-encoded A form of PCMH, and the genes from P.putida NCIMB 9866 were also plasmid encoded. The nucleotide sequences of the two flavoprotein genes from P.putida NCIMB 9869 and P.putida NCIMB 9866 (pchF69A and pchF66, respectively) were the same except for 5 bases out of 1,584, and the translated amino acid sequences were identical. The nucleotide sequences of the genes for the cytochrome subunits of PCMH from the two bacteria (pchC69A and pchC66) varied by a single nucleotide in their 303-base sequences, and the translated amino acid sequences differed by a single residue at position 41 (Asp in PchC69A and Ala in PchC66). Both cytochromes had 21-residue signal sequences, as expected for periplasmic proteins, and these sequences were identical. On the other hand, no signal sequences were found for the flavoproteins.pchF69A and pchC69A were expressed, separately or together, in Escherichia coli JM109 and P.putida RA4007, with active PCMH produced in both bacteria. The E. coli-expressed flavocytochrome was purified. Our studies indicated that the E.coli-expressed subunits were identical to the subunits expressed in P.putida NCIMB 9869: molecular weights, isoelectric points, UV-visible spectra, and steady-state kinetic parameters were the same for the two sets of proteins. The subunits readily associated upon mixing two crude extracts of E.coli, one extract containing PchC69A and the other containing PchF69A. The courses of association of PchC69A and PchF69A were essentially identical for pure E. coli-expressed subunits and pure P. putida 9869-expressed subunits. E. coli-expressed PchC69A and PchF69A contained covalently bound heme and covalently bound flavin adenine dinucleotide, respectively, as the proteins expressed in nature.

Amino Acid Sequence↗
PubMed1993

Escherichia coli fumarate reductase frdC and frdD mutants. Identification of amino acid residues involved in catalytic activity with quinones.

Escherichia coli fumarate reductase (FRD) is a four-subunit enzyme that catalyzes the terminal step in anaerobic respiration to fumarate. The hydrophobic FrdC and FrdD subunits anchor the FrdA and FrdB catalytic subunits to the inner surface of the cytoplasmic membrane and are required for the enzyme to interact with quinones. Thirty-five single-site mutations were constructed in the FrdC and FrdD polypeptides by site-directed mutagenesis. Each mutant enzyme was characterized for its ability to catalyze quinone oxidation and reduction and to support growth of E. coli DW35 (delta frdABCD sdhC::kan) under selective conditions requiring functional enzyme. Replacement of FrdCE29 with Asp, Leu, Lys, or Phe had a deleterious effect both on quinol oxidase and quinone reductase activities. Substitution of FrdCH82 with Arg, Leu, Tyr, or Glu also decreased menaquinol oxidase activity, but had variable effects on the reverse reaction, the reduction of ubiquinone. Data are presented to support the hypothesis that the positive charge at FrdCH82 is required for stabilization of the quinone radical intermediate and the negative charge at FrdCE29 for deprotonation of menaquinol. Other critical amino acids identified in FrdC included Ala-32, Phe-38, Trp-86, Phe-87, and in FrdD residues Phe-57, Gln-59, Ser-60, and His-80. The established roles of such residues in the QA and QB sites of the photosynthetic reaction center would suggest a similar type of structure operative in the FRD complex. In such a model, Glu-29, Ala-32, His-82, Trp-86 of FrdC and His-80 of FrdD are considered participants in a QB-type site, and FrdD Phe-57, Gln-59, and Ser-60 components in an apolar QA-type site.

Amino Acid Sequence↗
PubMed1992

Evidence for non-cysteinyl coordination of the [2Fe-2S] cluster in Escherichia coli succinate dehydrogenase.

The consequences of replacing Cys65 in the FrdB subunit of Escherichia coli fumarate reductase by Asp or Ala have been investigated in terms of bacterial growth, enzymatic activity, and the ERP/redox properties of the [2Fe-2S] cluster. An aspartic acid residue occupies the equivalent position in E. coli succinate dehydrogenase, and the FrdBCys65Asp mutation has little effect on cell growth, enzyme activity or the physical properties of the Frd [2Fe-2S] cluster. In contrast, the [2Fe-2S] cluster was not observed in the FrdBCys65Ala mutant showing that a coordinating residue is required at this position for assembly of this cluster and significant levels of enzymatic activity. These results support the presence of one non-cysteinyl, oxygenic ligand for the [2Fe-2S] cluster in E. coli succinate dehydrogenase.

Cysteine↗
PubMed1992

[3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.

Amino Acid Sequence↗
PubMed1992

Urinary excretion of glycated albumin in insulin-dependent diabetic patients with micro- and macroalbuminuria.

Non-enzymatic glycosylation (glycation) involves both circulating proteins, such as albumin and structural proteins, such as the components of the glomerular basement membrane. Glycated albumin is more anionic than unmodified plasma albumin at physiologic pH. Preferential urinary excretion of glycated proteins has occasionally been reported in diabetes. We therefore investigated the selectivity index (renal clearance of non-glycated/glycated albumin) in 25 insulin-dependent diabetic patients (17 with microalbuminuria and 8 with macroalbuminuria), and 19 healthy subjects. The selectivity index was significantly higher (p less than 0.01) in the microalbuminuric patients than in the macroalbuminuric patients and the control subjects: 1.11 +/- 0.03 SEM vs 0.94 +/- 0.08 vs 0.98 +/- 0.02. These results are not consistent with preferential urinary excretion of glycated albumin in insulin-dependent diabetic patients with increased urinary albumin excretion.

Adult↗
PubMed1991

Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis.

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.

Amino Acid Sequence↗
PubMed1991

The increase in albuminuria induced by exercise is not associated with preferential excretion of glycated albumin in type 1 (insulin-dependent) diabetic patients.

The increase in urinary albumin excretion induced by acute exercise in Type 1 (insulin-dependent) diabetic patients is associated with the urinary excretion of cationic proteins. To test whether the renal excretion of glycated albumin (more anionic than non-glycated albumin) is affected by exercise, we submitted seven normoalbuminuric (albumin excretion rate less than 30 mg/24 h) Type 1 diabetic patients and six well-matched healthy subjects to an exercise test (600 kpm/min for 20 min) on a bicycle ergometer, preceded and followed by a 1-h resting period. The selectivity index (renal clearance of non-glycated/glycated albumin) was not significantly different among the pre-exercise, exercise and post-exercise periods, either in the normal subjects (1.01 +/- 0.03 vs 1.08 +/- 0.06 vs 1.08 +/- 0.05) or in the diabetic patients (1.25 +/- 0.09 vs 1.20 +/- 0.07 vs 1.20 +/- 0.06), whereas it was significantly higher (p less than 0.05) in diabetic patients compared to healthy subjects during pre-exercise. These results are not consistent with the hypothesis that acute exercise may induce a preferential excretion of glycated albumin.

Adult↗
PubMed1991

[Hysteroscopy as a method of evaluation of endometrial pathology during climacteric ].

The Authors analyse 207 hysteroscopies performed in the outpatient clinic in women during menopause, without the use of dilatation or local anesthetic. The test was completed in 175 patients (84.6%). Tolerability was acceptable in 95% of cases. The main indication (80%) was atypical secretions during menopause or changes in the menstrual cycle. Hyperplastic lesions were confirmed in 12% of the tests carried out. An endometrial K was diagnosed in 5 cases (2.9%). The method showed a good level of specificity of diagnosis but it not always sufficiently sensitive. It is therefore necessary to complete the test by taking a selected histological sample using Novak's cannula.

Adult↗
PubMed1990

Electron transfer from menaquinol to fumarate. Fumarate reductase anchor polypeptide mutants of Escherichia coli.

Fumarate reductase (FRD) of Escherichia coli is a four-subunit membrane-bound complex that is synthesized during anaerobic growth when fumarate is available as a terminal oxidant. The two subunits that comprise the catalytic domain, FrdA and FrdB, are anchored to the cytoplasmic membrane surface by two small hydrophobic polypeptides, FrdC and FrdD, which are also required for the enzyme to interact with quinone. To better define the individual roles of the FrdC and FrdD polypeptides in FRD complex formation and quinone binding, we selectively mutagenized the frdCD genes. Frd- strains were identified by their inability to grow on restrictive media, and the resulting mutant FRD complexes were isolated and biochemically characterized. The majority of the frdC and frdD mutations were identified as single base deletions that caused premature termination in either FrdC or FrdD and resulted in the loss of one or more of the predicted transmembrane helices. Two additional frdC mutants were characterized that contained single base changes resulting in single amino acid substitutions. All mutant enzyme complexes were incapable of oxidizing the physiological electron donor, menaquinol-6, in the presence of fumarate. Additionally, the ability of the mutant complexes to oxidize reduced benzyl viologen or reduce the ubiquinone analogue 2,3-dimethoxy-5-methyl-6-pentyl-1,4-benzoquinone and phenazine methosulfate with succinate as electron donor were also affected but to varying degrees. The separation of oxidative and reductive activities with quinones suggests there are two quinone binding sites in the fumarate reductase complex and that electron transfer occurs in two le- steps carried out at these separate sites.

Amino Acid Sequence↗
PubMed1990

Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster.

Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers. In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., center 1, [2Fe-2S]; center 2, [4Fe-4S]; center 3, [3Fe-4S]) have been assembled. The mutations affect the properties of center 1 only and demonstrate that the anomalously high redox potential of this [2Fe-2S] center is essential for optimal enzymatic activity. The results are consistent with cysteines 57, 62, 65, and 77 providing the ligands to center 1 but leave open the possibility of noncysteinyl coordination for the localized valence Fe(III) site of the reduced cluster. The implications of the results for the role of center 1 in the electron-transfer pathway and the valence localization of reduced center 1 are discussed.

Cysteine↗
PubMed1989

Fumarate reductase mutants of Escherichia coli that lack covalently bound flavin.

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The catalytic domain of fumarate reductase consists of the FrdA subunit, which contains the active site, and a FAD prosthetic group covalently attached to His44, plus the FrdB subunit which contains at least two of the three nonidentical iron-sulfur clusters of the enzyme. To examine the role of covalently bound FAD in enzyme activity and electron transfer during anaerobic cell growth, site-directed mutagenesis was used to alter His44 of the FrdA subunit to a Ser, Cys, or Tyr residue. The resulting mutant enzyme complexes that were synthesized associated normally with the cytoplasmic membrane, but had decreased ability (greater than 70%) to reduce fumarate with reduced benzyl viologen, an artificial electron donor of low redox potential (Em = -359 mV; Clark, W. M. (1972) Oxidation-Reduction Potentials of Organic Systems, Robert E. Kreiger Publishing Co., Melbourne, FL). Even lower activities were measured when the higher potential, natural electron donor menaquinol was used, which, however, correlated with the slower growth rates of the different mutant complexes. In contrast to the normal enzyme, the mutant enzyme complexes were unable to oxidize succinate. Substitution of Arg for His44 produced a totally inactive enzyme complex that permitted no cell growth on nonfermentable substrates with fumarate as electron acceptor. All four mutant complexes contained noncovalently bound FAD in stoichiometric amounts. These data indicate a unique role of the 8 alpha-[N(3)-histidyl] FAD linkage in enzyme activity, by raising the redox potential of free FAD to permit reduction by both menaquinol and succinate.

Amino Acid Sequence↗