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Biomedical subjects

A Parmeggiani

Publications and source records attributed to A Parmeggiani.

At least 55 records · Page 3Linked to original sources
PubMed1997

Benign myoclonic epilepsy: long-term follow-up of 11 new cases.

The authors report a long-term follow-up of 11 new subjects with benign myoclonic epilepsy. There were some unusual clinical features such as the need for dual therapy in 45.5% of subjects, and the presence of non-epileptic myoclonus in 54.5%, neither of which influenced the prognosis. Neuropsychological and behavioral evolution was less favorable in 45.5% of patients (mental retardation, school learning problems, attention deficit disorder, hyperkinesia, aggressiveness, irritability, negativism). The less favorable neuropsychological outcome might be related to additional interacting factors such as personal antecedents, seizure onset and antiepileptic treatment.

Adolescent↗
PubMed1997

Familial Unverricht-Lundborg disease: a clinical, neurophysiologic, and genetic study.

PURPOSE: Progressive myoclonus epilepsies (PMEs) are a clinically and etiologically heterogeneous group of disorders. The authors report clinical, neurophysiological, and genetic findings of a family from Southern Italy with three members affected with PME. METHODS: All data about familial and personal antecedents, clinical history, neurologic examination, laboratory tests, neurophysiological findings, brain imaging studies, and DNA analysis were examined. RESULTS: All results were compatible with the features of Unverricht-Lundborg disease and patients were homozygous for the "Finnish" ancestral haplotype. CONCLUSIONS: Work is in progress to identify and characterize the common EPM1 mutation in the Finnish patients. Subsequently, it will be possible to verify the hypothesis on the existence of a common mutation in the Finnish patients and the Italian family under study, or even in other Mediterranean EPM1 families.

Adult↗
PubMed1996

Distal switch II region of Ras2p is required for interaction with guanine nucleotide exchange factor.

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25(Mm), yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF. The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant. The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt. Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase. Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase. This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1996

Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF-Tu-GTP is strongly retarded, causing the Kd of EF- Tu-GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP- and GDP-bound EF-Tu on native PAGE is increased. The stimulation of EF-Tu-GDP dissociation by EF-Ts is inhibited. EF- Tu-GTP can still form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no longer protects aa-tRNA against spontaneous deacylation, showing that the EF-Tu-GTP orientation with respect to the 3' end of aa-tRNA is modified. However, the EF-Tu-dependent binding of aa-tRNA to the ribosomal A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-site directly, inducing an anomalous positioning of aa-tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF-Tu and the ribosome.

Bacterial Proteins↗
PubMed1996

Determinants of Ras proteins specifying the sensitivity to yeast Ira2p and human p120-GAP.

Human and Saccharomyces cerevisiae Ras proteins and their regulators GAP (GTPase activating protein)and GEF (guanine nucleotide exchange factor) display structural similarities and are functionally interchangeable in vivo and in vitro, indicating that the molecular mechanism regulating Ras proteins has been conserved during evolution. As the only exceptions, the two S.cerevisiae GAPs, Ira1p and Ira2p, are strictly specific for yeast Ras proteins and cannot stimulate the GTPase of mammalian Ras. This study searches for the reasons for the different sensitivity to Ira2p of human H-ras p21 and yeast Ras2p. Construction of H-ras/Ras2p chimaeras showed that Gly18 of Ras2p (Ala11 of H-ras p21) is an important determinant for the specificity of Ira2p, revealing for the first time a function for this position. A second even more crucial determinant was found to be the 89-102 region of Ras2p (82-95 of H-ras p21) including the distal part of strand beta4, loop L6 and the proximal part of helix alpha3. It was possible to construct Ras2p's resistant to Ira2p but still sensitive to human p120-GAP and, conversely, a H-ras p21 sensitive to Ira2p. This work helps clarify specific aspects of the conserved molecular mechanism of interaction between Ras proteins and their negative GAP regulators.

Amino Acid Sequence↗
PubMed1996

[Intramuscular myxoma].

Intramuscolar mixoma is a rare benign neoplasm of mesenchymal origin consisting of stellate shaped cells in an abundant loose, mucopolysaccharide rich matrix. We report two cases of intramuscolar mixoma with review of the literature. Our objectives are to delineate the clinico pathologic characteristic, the nature of constituent cells and the features differing from others benign and malignant neoplasms.

Adult↗
PubMed1996

[Spindle-cell lipoma of the palatine tonsil].

Lipomatous tumor of palatine tonsil are rare. We report a case detected in a 72 year old male with a discussion about differential diagnosis of malignant polipoid lesions of the tonsil and some consideration about the problem of the growth of such neoformation.

Aged↗
PubMed1995

Properties and regulation of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein of Ras2p.

This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products. A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-383) and highly purified (> 90%) by affinity chromatography. The affinity of Ras2p for the GST-fused Ira2p-383 was 18 microM and the maximal stimulation of the Ras2p GTPase activity 6,000 times. The Ira2p activity was confirmed to be strictly specific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase being detectable. Comparison with the GAP-like domain of mammalian p120-GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2p-383 has an affinity and turnover intermediary between GAP-334 and NF1-414. The activity of Ira2p-383 was strongly inhibited by monovalent and divalent salts. The simultaneous presence of the catalytic domains of Ira2p and the yeast GDP/GTP exchange factor Cdc25p induced on Ras2p a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, showing that it is possible to reconstitute in vitro a S. cerevisiae system suitable for the study of the regulation of the Ras2p GDP/GTP cycle. The tubulin partially inhibited (25%) the GAP activity of the Ira2p-383. A larger Ira2p catalytic fragment, Ira2p-505 (amino acids 1549-2053), that showed the same Km for Ras2p as Ira2p-383, was also inhibited by tubulin to the same extent but with a higher affinity than Ira2p-383.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Characterization of mammalian C-CDC25Mm exchange factor and kinetic properties of the exchange reaction intermediate p21.C-CDC25Mm.

This work characterizes several aspects of the interaction between H-ras p21 and the catalytic domain of the mammalian GDP/GTP exchange factor (GEF) CDC25Mm (C-CDC25Mm), isolated in pure form as recombinant protein from Escherichia coli. Formation of a complex with nucleotide-free H-ras p21 could be analyzed on native gel electrophoresis by combining C-CDC25Mm and p21.GDP, as the result of the fast separation of GDP from p21. Therefore, in order to obtain highly purified heterodimer in preparative amounts, p21.GDP and C-CDC25Mm were exposed to an electric field and the complex purified by anionic chromatography. The rate of the C-CDC25Mm-mediated nucleotide exchange on p21 depended on the nature of the bound nucleotide (6 times faster for p21.GDP than p21.GTP) but was uninfluenced by the nature of the free nucleotide acting as second substrate. No saturating concentration of p21.GDP could be determined by measuring the C-CDC25Mm-mediated increase in the nucleotide exchange rate with a nitrocellulose binding assay. On this basis the Km and the kcat of the reaction were calculated to be > 11 microM and > 0.8 s-1, respectively. The dramatic increase in the nucleotide exchange rate was found to be almost exclusively based on the strong stimulation of the "off-rate". The "on-rate" of the nucleotide on the p21.C-CDC25Mm complex was similar for GDP and GTP and was little increased vs that on p21 alone. The observation that the apparent affinity of both nucleotides for the p21.C-CDC25Mm complex was lower than for p21 alone stressed the great affinity of this complex, whose Kd was calculated to be approximately 3 pM. These results are discussed and compared with the properties of other GEFs, from which C-CDC25Mm differs for a number of specific properties.

Animals↗
PubMed1995

Relevance of histidine-84 in the elongation factor Tu GTPase activity and in poly(Phe) synthesis: its substitution by glutamine and alanine.

Substitution of His-84 (-->Gln and -->Ala), a residue of the switch II region of E. coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP. The activity in poly(Phe) synthesis and GTP hydrolysis of EF-Tu H84Q were both reduced to about 35%, as compared to EF-Tu wt, whereas EF-Tu H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity. Phe-tRNAPhe exerted a similar inhibitory effect on the GTPase activity of EF-Tu wt and EF-Tu H84Q while abolishing that of EF-Tu H84A. Ribosomes enhanced the GTPase activity of EF-Tu H84Q, but not that of EF-Tu H84A, on which they even seemed to exert an inhibitory effect. The one-round GTP hydrolysis associated with the EF-TuH84Q-dependent binding of Phe-tRNAPhe to poly(U)-programmed ribosomes was less efficient than with EF-Tu wt. Kirromycin stimulated the GTPase activities of both mutants less than EF-Tu wt. The results of this work do not support a catalytic role of His-84 in the intrinsic GTPase of EF-Tu, but they emphasize the importance of its side-chain for polypeptide synthesis and GTP hydrolysis.

Alanine↗
PubMed1995

Substitution of Arg230 and Arg233 in Escherichia coli elongation factor Tu strongly enhances its pulvomycin resistance.

Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu.GTP). Recently, three pulvomycin-resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF-Tu substitutions at positions 230, 333 or 334. In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl-tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis. We have purified two mutants species, [R233S]EF-TuHis and [R230V, R233F]EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affinity chromatography. In this study, we describe the in vitro characterization of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rather than by EF-Tu.GTP.pulvomycin. Moreover, the mutant [R230V, R233F]EF-TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.

Aminoglycosides↗
PubMed1995

Properties of the catalytic domain of sdc25p, a yeast GDP/GTP exchange factor of Ras proteins. Complexation with wild-type Ras2p, [S24N]Ras2p and [R80D, N81D]Ras2p.

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia coli recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95%) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc25p-C was stronger for Ras2p.GDP than Ras2p.GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p.GDP (GTP) complex was also enhanced by Sdc25p-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc25p-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc25p-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc25p-C (truncated Sdc25p-C) showing the same specific activity as the 64-kDa Sdc25p-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1:1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc25p-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc25p.

Catalysis↗
PubMed1995

The minimal active domain of the mouse ras exchange factor CDC25Mm.

The minimal active domain of the mouse CDC25Mm, a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein. The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro. A C-terminal domain of 256 residues (CDC25Mm 1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25Mm 1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm 1030-1260 corresponding to the most conserved domain) led to a complete loss of expression. The results in vivo were supported by experiments in vitro. Highly purified CDC25Mm 1005-1260, expressed in E. coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S. cerevisiae Ras2p and its activity was nearly as high as that of CDC25Mm 974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one.

Animals↗
PubMed1995

Possible interaction between acyclovir and antiepileptic treatment.

We report a clinically relevant suspected interaction between acyclovir and the antiepileptic drugs phenytoin (PHT) and valproic acid (VPA). In a child receiving PHT and VPA therapy, a 6-day acyclovir treatment reduced PHT and VPA plasma concentrations to subtherapeutic values. This probably worsened both clinical status and electroencephalographic recordings observed in this patient. We suggest that the interaction occurs at gastrointestinal level with a reduction of PHT and VPA oral bioavailability during antiviral treatment.

Acyclovir↗
PubMed1994

Pulvomycin-resistant mutants of E.coli elongation factor Tu.

This paper reports the generation of Escherichia coli mutants resistant to pulvomycin. Together with targeted mutagenesis of the tufA gene, conditions were found to overcome membrane impermeability, thereby allowing the selection of three mutants harbouring elongation factor (EF)-Tu Arg230-->Cys, Arg333-->Cys or Thr334-->Ala which confer pulvomycin resistance. These mutations are clustered in the three-domain junction interface of the crystal structure of the GTP form of Thermus thermophilus EF-Tu. This result shares similarities with kirromycin resistance; kirromycin-resistant mutations cluster in the domain 1-3 interface. Since both interface regions are involved in the EF-Tu switch mechanism, we propose that pulvomycin and kirromycin both act by specifically disturbing the allosteric changes required for the switch from EF-Tu-GTP to EF-Tu-GDP. The three-domain junction changes dramatically in the switch to EF-Tu.GDP; in EF-Tu.GDP this region forms an open hole. Structural analysis of the mutation positions in EF-Tu.GTP indicated that the two most highly resistant mutants, R230C and R333C, are part of an electrostatic network involving numerous residues. All three mutations appear to destabilize the EF-Tu.GTP conformation. Genetic and protein characterizations show that sensitivity to pulvomycin is dominant over resistance. This appears to contradict the currently accepted model of protein synthesis inhibition by pulvomycin.

Aminoglycosides↗
PubMed1994

Elongation factor Tu D138N, a mutant with modified substrate specificity, as a tool to study energy consumption in protein biosynthesis.

Substitution Asp138-->Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP [Hwang & Miller (1987) J. Biol. Chem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle [Weijland & Parmeggiani (1993) Science 259, 1311-1313]. Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process. In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N.XTP was 30% higher than with EF-Tu wild type (wt).GTP. However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems. The K'd values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5-40 degrees C). With rate-limiting amounts of XTP the K'm of its XTPase activity corresponded to the K'm for XTP of poly(phenylalanine) synthesis (0.3-0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Energy Metabolism↗
PubMed1994

Histidine-118 of elongation factor Tu: its role in aminoacyl-tRNA binding and regulation of the GTPase activity.

The function of His118 in elongation factor (EF)-Tu from Escherichia coli was investigated by its substitution with glycine. The substitution had a differential effect on individual functions of the protein. The affinity for aminoacyl (aa)-tRNA and the intrinsic GTPase activity of the mutant EF-Tu were decreased whereas the response of its GTPase center to aa-tRNA was strongly increased. These results suggest that the region around His118 is involved in the binding of aa-tRNA and in the transmission of a turn-off signal generated by the interaction with aa-tRNA and directed to the GTPase center of EF-Tu.

Animals↗