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A Parmeggiani

Publications and source records attributed to A Parmeggiani.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mutant EF-Tu species reveal novel features of the enacyloxin IIa inhibition mechanism on the ribosome.

For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu. GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu.GDP. enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu.GDP.enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu.GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.

Escherichia coli↗
PubMed1999

Mutagenesis of three residues, isoleucine-60, threonine-61, and aspartic acid-80, implicated in the GTPase activity of Escherichia coli elongation factor Tu.

The properties of variants of elongation factor (EF) Tu mutated at three positions implicated in its GTPase activity are presented. Mutation I60A, which reduces one wing of a "hydrophobic barrier" screening off the nucleophilic water molecule found at the GTP gamma-phosphate, causes a reduction of the intrinsic GTPase activity contrary to prediction and has practically no influence on other properties. Mutation D80N, which in the isolated G-domain of EF-Tu caused a strong stimulation of the intrinsic GTPase, reduces this activity in the intact molecule. However, whereas for wild-type EF-Tu complex formation with aa-tRNA reduces the GTPase, EF-Tu[D80N] shows a strongly increased activity when bound to Phe-tRNA. Moreover, ribosomes or kirromycin can stimulate its GTPase up to the same level as for wild-type. This indicates that a local destabilization of the magnesium binding network does not per se cause an increased GTPase but does affect its tight regulation. Interestingly, mutant D80N sequestrates EF-Ts by formation of a more stable complex. Substitutions T61A and T61N induce low intrinsic GTPase, and the stimulation by ribosome is less for T61A than for T61N but still detectable, while kirromycin stimulates the GTPase of both mutants equally. This provides more evidence that stimulation by kirromycin and ribosomes follows a different mechanism. The functional implications of these mutations are discussed in the context of a transition state mechanism for catalysis. An alternative structural explanation for the strong conservation of Ile-60 is proposed.

Aspartic Acid↗
PubMed1999

Functional-structural analysis of threonine 25, a residue coordinating the nucleotide-bound magnesium in elongation factor Tu.

Elongation factor (EF) Tu Thr-25 is a key residue binding the essential magnesium complexed to nucleotide. We have characterized mutations at this position to the related Ser and to Ala, which abolishes the bond to Mg2+, and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S). Binding Phe-tRNAPhe to poly(U).ribosome needed a higher magnesium concentration for the latter two mutants but was comparable at 10 mM MgCl2. Whereas EF-Tu(T25S) synthesized poly(Phe), as effectively as wild type, the rate was reduced to 50% for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A). In contrast, protection of Phe-tRNAPhe against spontaneous hydrolysis by the latter two mutants was very low. The intrinsic GTPase in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses to ribosomes and kirromycin suggest that stimulation by these two agents follows different mechanisms. Of the mutants, only EF-Tu(T25A) forms a more stable complex with EF-Ts than wild type. This implies that stabilization of the EF-Tu.EF-Ts complex is related to the inability to bind Mg2+, rather than to a decreased nucleotide affinity. These results are discussed in the light of the three-dimensional structure. They emphasize the importance of the Thr-25-Mg2+ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu.

GTP Phosphohydrolase-Linked Elongation Factors↗
PubMed1999

Landau-Kleffner syndrome (LKS): long-term follow-up and links with electrical status epilepticus during sleep (ESES).

We describe 11 patients affected by Landau-Kleffner syndrome (LKS) with a mean follow-up of 9 years and 8 months. EEG recordings during wakefulness, NREM and REM sleep showed a bitemporal electrical status epilepticus during sleep (BTESES) in all cases; four of them presented a shift from a BTESES towards an 'intercalated electrical status epilepticus during sleep' (IESES) accompanied by a global regression of cognitive and behavioural functions in 3/4 of cases. At the last observation, only 18.2% of cases presented a complete language recovery and mental retardation was evident in 63.6%. The prognosis of LKS in our cases may depend on the interaction of different negative factors such as onset of aphasia before 4 years, its duration for longer than 1 year, long-lasting duration and continuity without fluctuations of BTESES/IESES, probably preexisting mild speech delay. It is important for the prognosis to utilize antiepileptic treatment and possibly neurosurgical techniques to eliminate EEG paroxysmal abnormalities. At present, no similar cases with clinical-EEG evolution from LKS to electrical status epilepticus during sleep (ESES) have ever been described. Our observation demonstrates that LKS and ESES classified as different clinical-EEG syndromes represent two aspects of the same brain dysfunction and they may exist separately or pass one into the other with a change in the clinical-EEG picture. The common origin of the two syndromes is confirmed by recent functional brain imaging, neurophysiological and neurosurgical techniques.

Adolescent↗
PubMed1999

Energy transduction of isothermal ratchets: generic aspects and specific examples close to and far from equilibrium.

We study the energetics of isothermal ratchets which are driven by a chemical reaction between two states, and operate in contact with a single heat bath of constant temperature. We discuss generic aspects of energy transduction such as Onsager relations in the linear response regime as well as the efficiency and dissipation close to and far from equilibrium. In the linear response regime where the system operates reversibly, the efficiency is in general nonzero. Studying the properties for specific examples of energy landscapes and transitions, we observe in the linear response regime that the efficiency can have a maximum as a function of temperature. Far from equilibrium in the fully irreversible regime, we find a maximum of the efficiency with values larger than in the linear regime for an optimal choice of the chemical driving force. We show that the corresponding efficiencies can be of the order of 50%. A simple analytic argument allows us to estimate the efficiency in this irreversible regime for small external forces.

Energy Metabolism↗
PubMed1999

Posterior fossa malformations and epilepsy.

The association between posterior fossa malformations and epilepsy is rarely reported in the literature. We describe 54 cases with posterior fossa malformations, according to embryogenesis classification, divided into two groups on the basis of presence or absence of epilepsy. Epilepsy occurred in 22 cases (40.7%) and was not related to the type of posterior fossa malformation or to supratentorial cerebral lesions associated with the malformation. Familial antecedents for epilepsy and/or febrile convulsions influenced the presence of epilepsy in patients with posterior fossa malformations (P < .01). Epilepsy was mainly partial (77.3%); benign partial/generalized epilepsies and febrile convulsions occurred in 27.3% of cases. Seizures disappeared for 2 or more years at the end of follow-up in 36.4% of patients. Good epilepsy prognosis was not related to the age at onset of seizures, familial antecedents for epilepsy and/or febrile convulsions, supratentorial associated lesions, or age of patients at the last observation. Profound mental retardation prevailed in patients with epilepsy (P < .01), as did pathologic electroencephalograms (EEG) (P < .0001), with paroxysmal abnormalities (P < .001) and asymmetry (P < .01). In our 54 cases of posterior fossa malformation, we identified two risk factors for epilepsy: familial antecedents for epilepsy and/or febrile convulsions and the involvement of the cerebellum in the malformation.

Adolescent↗
PubMed1999

Niaprazine in the treatment of autistic disorder.

Niaprazine is a histamine H1-receptor antagonist with marked sedative properties. It has been employed in subjects with behavior and sleep disorders. No data concerning the use of niaprazine in subjects with autistic disorder are reported in the literature. The authors performed an open study to assess niaprazine efficacy in a sample of 25 subjects with autistic disorder and associated behavior and sleep disorders. Niaprazine was administered at 1 mg/kg/day for 60 days. A positive effect was found in 52% of patients, particularly on hyperkinesia, unstable attention, resistance to change and frustration, mild anxiety signs, heteroaggressiveness, and sleep disorders. Statistical comparison between responders and nonresponders showed no influence on niaprazine effect by age over or under 12 years, presence of neurologic signs, epilepsy, or abnormalities seen on brain imaging. Niaprazine was more efficacious in subjects with a mild or moderate degree of mental retardation. No side effects were observed. Because of its sedative effects and good tolerability, niaprazine can be used as a first-choice drug to improve behavior and sleep disorders in patients with autistic disorder.

Adolescent↗
PubMed1998

Raf-1 is involved in the regulation of the interaction between guanine nucleotide exchange factor and Ha-ras. Evidences for a function of Raf-1 and phosphatidylinositol 3-kinase upstream to Ras.

The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1-149) and Raf (51-131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras.Raf complex, showed that the binding of Raf and GEF to Ha-Ras.GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras.GTP by Raf. Consistent with our results, the GEF-stimulated exchange of Ha-Ras.GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha. Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell.

Animals↗
PubMed1998

Interaction of EF-Tu with EF-Ts: substitution of His-118 in EF-Tu destabilizes the EF-Tu x EF-Ts complex but does not prevent EF-Ts from stimulating the release of EF-Tu-bound GDP.

Elongation factor Tu from Escherichia coli with His-118 substituted by glycine (EF-TuH118G) was found to be defective in complex formation with EF-Ts. EF-Ts in excess failed to dissociate kirromycin from the EF-TuH118G x kirromycin complex and to form a stable complex with EF-TuH118G on column chromatography. However, the stimulatory effect of EF-Ts on GDP dissociation from EF-TuH118G x GDP and on poly(U)-directed poly(Phe) synthesis catalyzed by EF-TuH118G was only partially influenced. These results indicate that His-118, while very important for the formation of a stable EF-Tu-EF-Ts complex, is not essential for the transmission of the EF-Ts-dependent signal accelerating the release of the EF-Tu-bound GDP.

Amino Acid Substitution↗
PubMed1998

Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands.

Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP. EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was >90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF-Tu-specific antibiotics [kirromycin, pulvomycin, GE2270 A (=MDL 62 879), and enacyloxin IIa] retarded significantly the dissociation of EF-Tu(DeltaC).GTP, showing the same kind of effect as on EF-Tu.GTP, but they were little active on EF-Tu(DeltaM). GTP. Like EF-Tu(DeltaC).GTP, EF-Tu(DeltaM).GTP was, however, able to bind efficiently kirromycin and enacyloxin IIa, as determined via competition with EF-Ts. Together, these results enlight selective functions of domains 2 and 3, particularly toward the interaction with EF-Ts and antibiotics, and emphasize their functional cooperativity for an efficient interaction of EF-Tu with ribosomes and aa-tRNA and for maintaining the differential affinity for GTP and GDP.

Circular Dichroism↗
PubMed1998

Cortical reflex myoclonus in Rett syndrome.

Rett syndrome (RS) is one of the most frequent causes of mental retardation in females. As there are no known biochemical, genetic, or morphological markers, diagnosis is based on clinical phenotype including severe dementia, autism, truncal ataxia/apraxia, loss of purposeful hand movements, breathing abnormalities, stereotypies, seizures, and extrapyramidal signs. Myoclonus, although reported in some series, has never been characterized. We studied 10 RS patients, age 3 to 20 years, and observed myoclonus in 9. Severity of myoclonus did not correlate with that of the other symptoms or with age. Multifocal, arrhythmic, and asynchronous jerks mainly involved distal limbs. Electromyographic bursts lasted 48 +/- 12 msec. Burst-locked electroencephalographic averaging generated a contralateral centroparietal premyoclonus transient preceding the burst by 34 +/- 7.2 msec. Motor evoked potentials showed normal latencies, indicating integrity of the corticospinal pathway. Somatosensory evoked potentials were enlarged. The C-reflex was hyperexcitable and markedly prolonged (62 +/- 4.3 msec), mainly due to increase in cortical relay time (28.4 +/- 4.5 msec). We conclude that RS patients show a distinctive pattern of cortical reflex myoclonus with prolonged intracortical delay of the long-loop reflex.

Adolescent↗
PubMed1998

Exacerbation of epileptic seizures by carbamazepine: report of 10 cases.

Carbamazepine (CBZ) is an effective anticonvulsant agent. Current literature reports describe several cases of seizure exacerbation and/or EEG worsening due to CBZ with a high prevalence in children and adolescents; we report 10 new cases. Nine patients had epilepsy; one showed delayed psychomotor development and frequent EEG paroxysmal abnormalities. Four patients were on monotherapy, six on polytherapy. All but one had therapeutic CBZ plasma concentrations. Seizures increased in frequency in nine, and in eight patients new seizure types appeared, mostly absences. Cognitive functions/behaviour worsened in eight; EEG recordings showed slowing background activity and increased paroxysmal abnormalities, in six cases diffuse/generalized spike waves were seen and in two continuous spike wave discharges. The mean time of clinical EEG worsening was 1-2 days after introduction of CBZ at therapeutic doses. After CBZ withdrawal clinical EEG improvement was evident in a few days. The underlying pathogenetic mechanism is not yet understood. However, the pathophysiology of seizure exacerbation might be related to the interaction between age-related alterations in the balance of excitation and inhibition in the developing thalamocortical circuitry and the essential activity of CBZ that tends to induce interictal discharges.

Age Factors↗
PubMed1998

Multilobar polymicrogyria, intractable drop attack seizures, and sleep-related electrical status epilepticus.

BACKGROUND AND OBJECTIVE: Patients with cortical malformations often have intractable seizures and are candidates for epilepsy surgery. Within an unselected series of patients with various forms of cortical malformation, nine patients with multilobar polymicrogyria had electrical status epilepticus during sleep (ESES) accompanied by infrequent focal motor seizures. Eight patients also had intractable atonic drop attack seizures. Because ESES usually is accompanied by a good long-term seizure prognosis, the objective of this study was to examine ESES outcome among patients with a structural lesion that is usually highly epileptogenic and has a low seizure remission trend. METHODS: The nine patients had follow-up periods lasting 4 to 19 years. All underwent brain MRI, serial sleep EEG recordings, and cognitive testing during and after ESES. RESULTS: ESES and drop attack seizures appeared between the ages of 2 and 5 years (mean, 4 years) and ceased between the ages of 5 and 12 years (mean, 8 years). At the last visit patients were 8 to 23 years of age (mean, 14.5 years) and were either seizure free or had very infrequent focal motor seizures during sleep. Three patients were free from antiepileptic drugs. In no patient was definite cognitive deterioration apparent after ESES in comparison with earlier evaluations. CONCLUSIONS: Age-related secondary bilateral synchrony underlying ESES may be facilitated in multilobar polymicrogyria. The good seizure outcome contrasts with that usually found in the presence of cortical malformations. For children with polymicrogyria and drop attack seizures, surgical treatment of the epilepsy should be considered cautiously, and sleep EEG recordings should be performed systematically.

Age of Onset↗
PubMed1997

A new function of p120-GTPase-activating protein. Prevention of the guanine nucleotide exchange factor-stimulated nucleotide exchange on the active form of Ha-ras p21.

This work studies the coordination of the action of GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) on activated human c-Ha-Ras p21. Purified human p120-GAP was obtained with a new efficient procedure. To distinguish the GTPase-activating effect of p120-GAP from other effects dependent on the interaction with activated Ha-Ras, the nonhydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was used. The results showed that the GTPgammaS/GTPgammaS exchange enhanced by the C-terminal catalytic domain of the yeast GEF Sdc25p (C-Sdc25p) is prevented by p120-GAP. This effect is strictly specific for the activated form of Ha-Ras, the target of GAP; no effect on Ha-Ras.GDP was detectable. The GAP catalytic domain also inhibited C-Sdc25p but to a lower extent. The interfering effect by p120-GAP was also evident in a homologous mammalian system, using full-length mouse RasGEF, its C-terminal half-molecule, or C-terminal catalytic domain. As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of Ha-Ras.GTPgammaS by GEF at a GDP:GTPgammaS ratio mimicking the in vivo GDP:GTP ratio. Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras.GTP in vivo against unproductive exchanges. This constrain is likely involved in the regulation of the physiological GDP/GTP cycle of Ras and in the action of p120-GAP as downstream effector of Ras. Helix alpha3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras.

Animals↗
PubMed1997

Hearing loss in the Sjögren syndrome.

BACKGROUND: Ear involvement is not unusual in autoimmune diseases, but few data on this problem in the Sjögren syndrome are available. OBJECTIVES: To determine whether the incidence of hearing loss is increased in patients with the Sjögren syndrome and to determine what factors might be involved in the pathogenesis of Sjögren syndrome-related hearing loss. DESIGN: Cross-sectional study. SETTING: Secondary referral center in Italy. PATIENTS: 30 women with the Sjögren syndrome were evaluated for evidence of audiovestibular disorder. Their results were compared with those of 40 age-matched healthy women. MEASUREMENTS: Evaluation techniques included pure tone audiometry thresholds at octave frequencies of 125 Hz to 8000 Hz, tympanometry, and the stapedial reflex test in both ears. The presence of vasculitis, the Raynaud phenomenon, nervous system involvement, and serum anticardiolipin antibodies was recorded. RESULTS: 14 patients with the Sjögren syndrome (46% [95% CI, 28% to 66%]) had sensorineural hearing loss. Only one control (2.5% [CI, 0.06% to 13%]) had a similar hearing impairment (P < 0.001). Nine of the 14 patients who had the Sjögren syndrome and sensorineural hearing loss (64% [CI, 36% to 87%]) had anticardiolipin antibodies compared with only 3 controls (18% [CI, 4% to 45%]) (P = 0.02). CONCLUSION: The high prevalence of hearing loss in the Sjögren syndrome supports the value of doing audiometric studies before excluding cranial nerve involvement in this disease. The correlation of sensorineural hearing loss with anticardiolipin antibodies must be investigated further.

Adult↗
PubMed1997

The N-terminal moiety of CDC25(Mm), a GDP/GTP exchange factor of Ras proteins, controls the activity of the catalytic domain. Modulation by calmodulin and calpain.

This work describes the in vitro properties of full-length CDC25(Mm) (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21. CDC25(Mm), isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21.GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25(Mm285)) and 5 times lower than the activity of the C-terminal half-molecule (631 residues). This reveals a negative regulation of the catalytic domain by other domains of the molecule. Accordingly, the GEF activity of CDC25(Mm) was increased severalfold by the Ca2+-dependent protease calpain that cleaves around a PEST-like region (residues 798-853), producing C-terminal fragments of 43-56 kDa. In agreement with the presence of an IQ motif on CDC25(Mm) (residues 202-229), calmodulin interacted functionally with the exchange factor. Depending on the calmodulin concentration an inhibition up to 50% of the CDC25(Mm)-induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca2+ ions. Calmodulin also inhibited C-CDC25(Mm285) but with a approximately 100 times higher IC50 than in the case of CDC25(Mm) ( approximately 10 microM versus 0.1 microM, respectively). Together, these results emphasize the role of the other domains of CDC25(Mm) in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25(Mm) activity.

Animals↗
PubMed1997

Complex brain malformation and drug resistant epilepsy.

A 6-year-old girl presented with severe epileptic encephalopathy and delayed psychomotor development. MRI was normal but during the clinical course disclosed a complex brain malformation involving the gray and white matter. Clinical and brain imaging data are discussed in light of relevant reports in the literature.

Brain↗