Biomedical subjects

M Perego

Publications and source records attributed to M Perego.

At least 37 records · Page 2Linked to original sources

PubMed1997

A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

ATP-Binding Cassette Transporters↗

PubMed1997

Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning.

The Bacillus subtilis spo0J gene is required for accurate chromosome partitioning during growth and sporulation. We have characterized the subcellular localization of Spo0J protein by immunofluorescence and, in living cells, by use of a spo0J-gfp fusion. We show that the Spo0J protein forms discrete stable foci usually located close to the cell poles. The foci replicate in concert with the initiation of new rounds of DNA replication, after which the daughter foci migrate apart inside the cell. This migration is independent of cell length extension, and presumably serves to direct the daughter chromosomes toward opposite poles of the cell, ready for division. During sporulation, the foci move to the extreme poles of the cell, where they function to position the oriC region of the chromosome ready for polar septation. These observations provide strong evidence for the existence of a dynamic, mitotic-like apparatus responsible for chromosome partitioning in bacteria.

Amino Acid Sequence↗

PubMed1997

Capillary zone electrophoresis of oligonucleotides and peptides in isoelectric buffers: theory and methodology.

The use of isoelectric buffers in capillary zone electrophoresis is reviewed. Such buffers allow application of extremely high voltage gradients (up to 1000 V/cm in relatively high bore capillary, e.g. 75 to 100 microm internal diameter), permitting separations of the order of a few minutes and thus favoring high resolution due to minimal, diffusion-driven peak spreading. The fundamental properties of ampholytes are first discussed, such as buffering power (beta) as a function of delta pK, i.e. of the distance between the pI value and neighboring protolytic groups. The highest possible relative beta value (= 2) is obtained for amphoteres possessing a delta pK = 0.6, a condition not met by existing amphoteric species. A novel parameter for ampholyte evaluation is then proposed, namely the beta/lambda ratio, i.e. the ratio between the beta power and conductivity at the pI value. It is additionally shown that the pI is not a constant value, but depends on ampholyte concentration in solution. In addition, at constant concentration, the theoretical pI can change as a function of delta pK. Isoelectric His and, to a lesser extent, Lys have been found to offer unique separations of oligonucleotides in sieving liquid polymers. In the absense of sieving media, isoelectric Asp, in presence of 7 M urea (apparent pH 3.77), permits unique separations of oligonucleotides having the same length but different nucleotide composition. Isoelectric Asp (pI 2.77 at 50 mM concentration) provides a medium of high resolving power for generating peptide maps. In difficult cases, of coincident titration curves, the pH can be moved up to higher values (e.g. pH 3.0 for 30 mM Asp) thus eliciting separation of unresolved peptides at pH 2.77. This was illustrated by running peptide maps of tryptic digests of human beta-globin chains. Also imino diacetic acid (pI 2.33 at 50 mM concentration) allows generation of high resolution peptide maps.

Buffers↗

PubMed1997

Separation of oligonucleotides of identical size, but different base composition, by free zone capillary electrophoresis in strongly acidic, isoelectric buffers.

A novel method for analyzing oligonucleotides of the same length, but bearing a single base substitution, is reported, based on free zone capillary electrophoresis (CZE) under rather acidic pH values. For this purpose, a set of four 18-mers of fairly random base composition has been synthesized, bearing, in nucleotide 9, the following bases: T, C, G or A. Theoretical predictions, based on titration curves of single free nucleotides, allowed us to predict that the simultaneous separation of a mixture of all four oligonucleotides could be possible in a pH 3-4 window. In fact, electrophoresis at pH 5.7 gave a single, asymmetric peak, whereas CZE at pH 4.8 could resolve three out of four species (the T9 and G9 oligonucleotides co-migrating into a single zone). A unique separation power could be obtained at pH 3.3 in a buffer comprising an amphoteric species (isoelectric iminodiacetic acid, IDA) and 7 M urea. Although IDA exhibited a pI of 2.23 (for a 100 mM solution), the addition of 7 M urea (necessary to denature the oligonucleotides) raised the apparent pH of the solution to 3.3.

Acids↗

PubMed1997

Characterization of a family of bacterial response regulator aspartyl-phosphate (RAP) phosphatases.

We have characterized a novel family of response regulator aspartyl-phosphate (RAP) phosphatases found exclusively in gram-positive bacteria. The family consists of 15 members, 12 of which are from Bacillus subtilis. The N-terminal domains proved to be more highly conserved than the C-terminal domains, and a signature sequence for the family was derived from the former domains. Phylogenetic analyses revealed clustering patterns showing that all Bacillus proteins are closely related. Most of the Bacillus RAP phosphatase genes are followed by and are translationally coupled to small nonhomologous phosphatase regulator (phr) genes that encode exported peptides with regulatory functions. Most of the paralogous RAP phosphatases of B. subtilis may serve related functions in signal transduction systems. They appear to have arisen by relatively recent gene duplication events that occurred after the divergence of major groups within the gram-positive bacterial kingdom. We suggest that the N-terminal domains of the RAP phosphatases function in catalysis, whereas the C-terminal domains function in regulation.

Amino Acid Sequence↗

PubMed1997

[Gangliocytic paraganglioma. A case report and review of the literature].

Gangliocytic paraganglioma is an uncommon and usually benign lesion although some cases with metastasis to regional lymph nodes have been described. It is usually located in the descending duodenum submucosa and more frequently arises in male patients. It comes to clinical attention for discomfort, gastrointestinal bleeding, incidental finding and rarely for obstructive jaundice when it involves the papilla. Even if its histologic and immunocytochemical features have been thoroughly described, its histogenesis is still debated although hyperplastic and amartoma-choristoma theories are well considered. We report a case of duodenal gangliocytic paraganglioma in 46 years old chronic alcoholic man who underwent previous upper endoscopy for discomfort without diagnosing the lesion. This one was observed during a second upper endoscopy that was made for hemathemesis. But it was only after an episode of melena that a third upper endoscopy showed the paraganglioma that still kept hemorrhage stigmata and no other lesions. We describe this case of duodenal gangliocytic paraganglioma with review of the literature.

English Abstract↗

PubMed1997

Rapid detection of 21-hydroxylase deficiency mutations by allele-specific in vitro amplification and capillary zone electrophoresis.

A quick diagnosis of the classic form of 21-hydroxylase deficiency (simple virilizing and salt wasting) is of great importance, especially for prenatal diagnosis and treatment in pregnancies at risk. A method for simultaneous detection of common point mutations in the P450c21 B gene is here proposed by combining a nested PCR amplification refractory mutation system (ARMS) with capillary zone electrophoresis (CZE) in sieving liquid polymers. In the first PCR, B genes are selectively amplified. In the nested reaction, ARMS-detected wild-type and mutated alleles are separately pooled and resolved by CZE. CZE is performed in coated capillaries in the presence of 30 g/L hydroxyethyl cellulose in the background electrolyte for size separation of the DNA analytes. For high-sensitivity detection the electrophoresis buffer contains the fluorescent dye SYBR Green I. Laser-induced fluorescence detection is obtained by excitation at 488 nm and signal collection at 520 nm. Specificity and reproducibility of the protocols were established by using samples from 75 Italian families with 21-hydroxylase deficiency already genotyped by allele-specific oligonucleotide hybridization or direct sequencing. Whereas dot-blot is time consuming because of the high number of hybridizations with radioactive probes, this present protocol is more rapid, giving sufficient separation on CZE after PCR reactions without preconcentration or desalting of samples.

Adrenal Hyperplasia, Congenital↗

PubMed1997

Helicobacter pylori eradication in the healing and recurrence of benign gastric ulcer: a two-year, double-blind, placebo controlled study.

BACKGROUND: Helicobacter pylori infection is associated with idiopathic gastric ulcer in about 90% of the cases, but only a few controlled studies aimed at evaluating gastric ulcer healing and the natural history after Helicobacter pylori-eradication have been carried out. OBJECTIVE: The aim of the present study was to evaluate the efficacy of omeprazole coupled with amoxicillin in the eradication of Helicobacter pylori and healing and prevention of gastric ulcer recurrence. PATIENTS: Fifty-nine patients with active gastric ulcer were randomized under double-blind conditions to receive either omeprazole 20 mg twice daily for four weeks plus amoxicillin 3 g daily during the first and second week (29 patients, Group A) or omeprazole .20 mg twice daily for 4 weeks plus placebo for two weeks (30 patients, Group B). METHODS: Endoscopic studies were carried out at the end of the 4 weeks treatment (or after 8 weeks in non-healed patients) as well as 2, 6 and 12 months later. A total of 3 biopsies in the antrum, 3 in the gastric body and at least seven at the edge of the crater were taken at each endoscopic control for exclusion, of malignancy, histological detection of Helicobacter pylori and for evaluation of gastric histology according to the Sydney system. RESULTS: With intention to treat analysis, the percentage of healing after 4 and 8 weeks was 86% and 100% in Group A patients and 86% and 93% in Group B, respectively. Two patients dropped out in Group B for non medical reasons. The percentage of eradication was 63% in Group A and 7% in Group B. During a 12-month follow-up gastric ulcer relapsed in 20/32 (63%) of the persistently Helicobacter pylori positive patients. Only two out 20 (10%) Helicobacter pylori cured patients showed a gastric ulcer relapse and Helicobacter pylori reinfection. Twenty out of 30 patients, still healed after 12 months, underwent endoscopic control after two years. A gastric ulcer relapse was observed in three out of nine (33%) patients with persisting infection after treatment. No gastric lesions, but one case of erosive oesophagitis were observed in the 11 Helicobacter pylori-eradicated patients. CONCLUSIONS: In our experience, Helicobacter pylori eradication does not favour gastric ulcer healing but does positively influence the subsequent natural history.

Amoxicillin↗

PubMed1996

Cell-cell communication regulates the effects of protein aspartate phosphatases on the phosphorelay controlling development in Bacillus subtilis.

Rap phosphatases are a recently discovered family of protein aspartate phosphatases that dephosphorylate the Spo0F--P intermediate of the phosphorelay, thus preventing sporulation of Bacillus subtilis. They are regulators induced by physiological processes that are antithetical to sporulation. The RapA phosphatase is induced by the ComP-ComA two-component signal transduction system responsible for initiating competence. RapA phosphatase activity was found to be controlled by a small protein, PhrA, encoded on the same transcript as RapA. PhrA resembles secreted proteins and the evidence suggests that it is cleaved by signal peptidase I and a 19-residue C-terminal domain is secreted from the cell. The sporulation deficiency caused by the uncontrolled RapA activity of a phrA mutant can be complemented by synthetic peptides comprising the last six or more of the C-terminal residues of PhrA. Whether the peptide controls RapA activity directly or by regulating its synthesis remains to be determined. Complementation of the phrA mutant can also be obtained in mixed cultures with a wild-type strain, suggesting the peptide may serve as a means of communication between cells. Importation of the secreted peptide required the oligopeptide transport system. The sporulation deficiency of oligopeptide transport mutants can be suppressed by mutating the rapA and rapB genes or by introduction of a spo0F mutation Y13S that renders the protein insensitive to Rap phosphatases. The data indicate that the sporulation deficiency of oligopeptide transport mutants is due to their inability to import the peptides controlling Rap phosphatases.

ATP-Binding Cassette Transporters↗

PubMed1996

Comparison of behavior of N-substituted acrylamides and celluloses on double-stranded DNA separations by capillary electrophoresis at 25 degrees and 60 degrees C.

The behavior of N-substituted poly(acrylamides) and of cellulose in the separation of double-stranded DNA by capillary zone electrophoresis (CZE) is evaluated. Contrary to previously held belief, which attributed best separations in poly(acrylamides) to small DNA fragments (typically in the 50-1000 bp size range) and in celluloses to large DNA fragments, we show that also celluloses can achieve fine sieving of short DNA sizes provided they are used at much higher concentrations than previously reported. For example, in the case of hydroxyethylcellulose (HEC), typically used at 0.2-0.8% concentrations, levels of 3% produce excellent patterns, at 25 degrees C, in the 50-600 bp size range. If separations are conducted at 60 degrees C, sieving is lost in most liquid polymers. However, if the concentration of HEC is raised to 6% and that of hydroxypropylmethylcellulose (HPMC) above 1%, sieving is fully restored. Also, N-substituted acrylamido derivatives, notably N-acryloylaminoethoxyethanol (AAEE) and N-acryloylaminopropanol (AAP) offer excellent performance at both 25 degrees and 60 degrees C, whereas plain poly(acrylamide) irreversibly collapses at high temperatures, possibly due to intrinsic instability of the amido bond under such harsh conditions. Among all the polymers investigated, an 8% solution of poly(AAP) offers the best performance and highest theoretical plates in the 25 degree-60 degree C interval. Separations at high temperatures are necessary when dealing with detection of point mutations in temperature-programmed CZE and are preferred in DNA sequencing. Two additional advantages are evident when running DNA separations at 60 degrees C: a marked reduction of analysis times and a linearization of the transit times of the larger (434-587 bp) DNA fragments, in all polymers tested.

Acrylamides↗

PubMed1996

Capillary electrophoresis of oligonucleotides in sieving liquid polymers in isoelectric buffers.

Analysis of oligonucleotides (especially in regard to assessing the purity of antisense compounds) is typically performed in 18% T sieving liquid polyacrylamide, in 30% formamide and 7 M urea. Up to 600 V/cm have been reported, with transit times, for 20 to 25 oligomers, of 15-20 min. We show that the same analysis can be performed in isoelectric buffers, typically histidine (His), and in more dilute linear polyacrylamides, e.g. 10% T (at 0% C), with much reduced analysis times. A series of His concentrations has been explored, ranging from 25 to 150 mM. Best performance is obtained in 100 mM His (at pH = pI, i.e., 7.47 at 25 degrees C), dissolved in 7 M urea, in presence of 10% sieving liquid linear polyacrylamide. Such a buffer allows delivering 800 V/cm without any loss of resolution due to Joule heating, with retention of very high resolving power down to fragments as short as tetranucleotides. Under these conditions, the analysis time for an antisense oligonucleotide containing fragments from a 10-mer to 18-mer is in a time window of 4-5 min. It is shown that the smallest fragment (10-mer) migrates in the capillary at the remarkable speed of 5 cm/min.

Buffers↗

PubMed1996

Modifications of diazepam binding inhibitor and peripheral benzodiazepine receptors in the lymphocytes of epileptic patients.

Recent experimental evidence has suggested that peripheral benzodiazepine receptors (PBR) may play a role in epilepsy and antiepileptic drug action. Since PBR are also present in circulating lymphocytes, and may interact with anticonvulsant drugs, this study was designed to look for possible modifications of these receptors and their endogenous ligand diazepam binding inhibitor (DBI) in the lymphocytes of epileptic patients treated with various drugs. PBR levels were 50% to 80% higher in patients treated with carbamazepine, phenobarbital and valproic acid than in controls and untreated epileptics. DBI levels were significantly increased in the lymphocytes of untreated patients, and showed only a slight further increase after anticonvulsant therapy. The possibility that PBR and DBI modifications in the lymphocytes of epileptic patients may be linked to the immunological alterations reported in these patients and/or may represent possible markers of neurochemical modifications in the central nervous system is discussed.

Adolescent↗

PubMed1996

Protein aspartate phosphatases control the output of two-component signal transduction systems.

Phosphorylation or dephosphorylation of an aspartate regulates the output activity of the response regulator of two-component signaling systems. Signal input in these systems is dependent on signal-transducing kinases, which can respond to a variety of signal ligands and, in some cases, to small phosphorylated metabolic intermediates. The kinase component of many two-component signaling systems also displays a response regulator-phosphate phosphatase activity that inactivates the response regulator in response to signals. Newly discovered kinase-independent phosphatases allow additional signals to influence the extent of response-regulator phosphorylation. Such phosphatases are prevalent in signal transduction systems controlling complex processes, such as the initiation of development in microorganisms.

ATP-Binding Cassette Transporters↗

PubMed1996

D-alanine deprivation of Bacillus subtilis teichoic acids is without effect on cell growth and morphology but affects the autolytic activity.

Using insertional inactivation of the different genes of the dlt operon in Bacillus subtilis, we searched for metabolic and morphological changes caused by D-alanine ester deprivation of lipoteichoic acid and wall teichoic acid. There were no alterations of cell growth, basic metabolism, cellular content of phosphorus-containing compounds, ultrastructure, cell separation, and surface charge. The only alteration observed was an enhancement of endogenous and beta-lactam-induced cell lysis. Since this enhancement is doubtless correlated with the D-alanine ester deprivation of the teichoic acids, the present view based on in vitro experiments, that negatively charged LTA is inhibitory to autolysins, may be questioned. We propose that negatively charged lipoteichoic acid and/or wall teichoic acid serve in vivo to fix the cationic autolysins within the cell wall-membrane complex by electrostatic interaction. Positively charged D-alanine ester substituents decrease the binding capacity of the teichoic acids for autolysins by charge compensation.

Alanine↗

PubMed1996

Analysis of antisense oligonucleotides by capillary electrophoresis, gel-slab electrophoresis, and HPLC: a comparison.

Three techniques are evaluated for assessing the purity of synthetic oligodeoxyribonucleotides: reversed phase high-performance liquid chromatography (RP-HPLC), polyacrylamide gel-slab electrophoresis (PAGE), and capillary zone electrophoresis highly concentrated (18% T) entangled polymer networks (CZE). RP-HPLC does not seem to be able to discriminate and resolve the spectrum of failed sequences expected to accompany an oligonucleotide of a given length. The purity data, as given by the manufacturer, are most often close to 100%. PAGE in 20% T matrices, followed by ethidium bromide staining, gives a good resolution of failure sequences and purity assessments decidedly more realistic. CZE in 18% liquid polyacrylamide is able to resolve to baseline all shorter fragments and to give a precise evaluation of the amount of impurities, based on the intrinsic DNA absorbance at 254 nm. Most of the 18 mer oligonucleotides (and of their phosphorothioate derivatives) analyzed by us, as supplied by three different manufacturers, were found to be contaminated by a spectrum of failed and truncated sequences, ranging in size from 7 mer to 17 mer. The purity data rarely exceeded 80% and most often were of the order of 60%-70%. Conditions for a good routine performance of the CZE technique are described.

Base Sequence↗

PubMed1996

The Bacillus subtilis genes for ribonucleotide reductase are similar to the genes for the second class I NrdE/NrdF enzymes of Enterobacteriaceae.

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria.

Amino Acid Sequence↗

PubMed1996

Bacillus subtilis mutS mutL operon: identification, nucleotide sequence and mutagenesis.

The Bacillus subtilis mutS and mutL genes, involved in the DNA mismatch repair system, have been cloned and characterized. From sequence analysis the two genes appear to be organized in a single operon, located immediately downstream of the cotE gene (approximately 150 degrees on the genetic map). The deduced MutS protein is 49% identical to HexA and MutL is 46% identical to HexB of Streptococcus pneumoniae. Deletion of both mutS and mutL resulted in an increase in the frequency of spontaneous mutations and abolished the marker effect observed in transformation. The expression of the mut operon was studied with the use of a mutSL-lacZ transcriptional fusion. An increase in expression was observed during late exponential growth.

Adenosine Triphosphatases↗

PubMed1996

Aspartyl-phosphate phosphatases deactivate the response regulator components of the sporulation signal transduction system in Bacillus subtilis.

Bacteria use two-component signal transduction systems to sense and respond to their environment. A sensor kinase and a response-regulator transcription factor work in concert by phosphorylation/dephosphorylation through kinase and phosphatase activities to maintain a level of phosphorylated response regulator commensurate with the level of signal input. Signal input can be accommodated through stimulation of the kinase activity or the phosphatase activity of the two-component system. With some notable exceptions, the sensor kinases recognize a single stimulatory ligand. A new dimension in the regulation of two-component signal transduction systems was discovered in the Rap phosphatases which dephosphorylate the SpoOF response-regulator of Bacillus subtilis independent of the sensor kinases. This family of phosphatases is encoded by at least six chromosomal genes. Although not all of the phosphatases of the family have activity on phosphorylated SpoOF, the two best-characterized members, RapA and RapB, prevent sporulation by dephosphorylating this response regulator component of the phosphorelay. Phosphatase activity of RapA is regulated by a gene, phrA, in the same transcriptional unit, that encodes a peptide secreted from the cell which may serve as a quorum sensor. Most of the Rap phosphatase operons have a gene coding for a protein with some similarity to PhrA in their transcription units, but it is uncertain whether all of these play a role in regulation. The Rap phosphatases are postulated to be a mechanism for allowing signals other than those that affect the sensor kinases to regulate the signal transduction pathway. They may have been recruited to help regulate sporulation because the multiple signals regulating this process may outstrip the recognition capacity of the kinases.

ATP-Binding Cassette Transporters↗