Biomedical subjects

M Perego

Publications and source records attributed to M Perego.

At least 73 records · Page 4Linked to original sources

PubMed1990

Decreased density of benzodiazepine receptors in lymphocytes of anxious patients: reversal after chronic diazepam treatment.

Peripheral-type benzodiazepine receptors were measured in human circulating lymphocytes using 3H-PK 11195 as specific ligand. In a group of outpatients with anxiety disorders a significant decrease of receptor density (-37%) was found compared with age-matched controls. In these patients long-term diazepam treatment restored binding density to normal levels: the effect persisted after drug withdrawal. Acute i.v. diazepam administration did not change receptor density. The observed receptor changes could reflect a down-regulation phenomenon and indicate that lymphocyte function reflect central nervous events.

Anxiety Disorders↗

PubMed1990

A study of heart-pineal interactions: atrial natriuretic peptide response to melatonin administration in healthy humans.

It is known that the pineal hormone melatonin plays a role in the regulation of several biological functions. In an attempt to investigate interactions between the pineal and the cardiac endocrine activity, in this preliminary study we have evaluated the effect of melatonin on the secretion of the cardiac hormone, atrial natriuretic peptide (ANP). The study included five healthy volunteers, and melatonin was given orally at a dose of 30 mg at 17:00. The results of the study show that the administration of melatonin does not influence ANP plasma concentrations. Further studies will be required to better define the cardiac-pineal interaction.

Administration, Oral↗

PubMed1990

Structural alterations in the Bacillus subtilis Spo0A regulatory protein which suppress mutations at several spo0 loci.

Secondary site mutations that restore sporulation to sporulation-defective spo0F or spo0B deletion mutants were found to reside in the spo0A gene. Sequence analysis of 23 such sof mutants showed that the sof mutations fell into six classes of missense codon changes, primarily in the conserved amino-terminal domain of the response regulator Spo0A protein. Changes were observed in codons 12, 14, 60, 92, and 121. The residues affected were predominantly located in the potential turn regions at one end of the amino-terminal conserved domain on the same topological face as the active site aspartate residues. The ability of sof mutations to suppress deficiencies in the transmitter kinases, KinA and KinB, of two-component regulatory systems was tested. All of the sof mutations suppressed the sporulation deficiency of kinA mutants but only two classes among five tested suppressed kinB mutations. sof mutants segregated Spo- colonies at high frequency. Five of these Spo- mutants were found to result from mutations in the spo0A locus that reversed the effect of the sof mutatation. One of these was sequenced and found to have the original sof mutation and a new mutation, sos, at codon 105. The accumulation of sos mutations in sof strains suggested that the sof mutations have a subtle, yet deleterious, effect on the growth of the cell. The results suggested that the sof mutations increase the avidity for or reactivity with transmitter kinases in an allele-specific manner, although in some cases it is possible that the sof mutations obviate the need for phosphorylation to activate the Spo0A protein. An alternative hypothesis is presented in which the sof mutations play the role of bypass mutations for kinases.

Amino Acid Sequence↗

PubMed1989

The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein.

The product of the abrB gene of Bacillus subtilis is an ambiactive repressor and activator of the transcription of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Purified AbrB protein binds specifically in a highly co-operative fashion to fragments of DNA containing the promoters it affects. DNase I footprints of the binding regions in these promoters revealed large protected areas of 50-120 nucleotides or more depending on the promoter. Methylation protection experiments gave protected guanine residues on only one face of the DNA helix. A consensus sequence could be deduced around these guanine residues that was not found around non-protected guanine residues in the footprint region. The results suggested that stationary phase functions and sporulation are repressed during active growth by AbrB and other transition state regulators by binding to the affected promoters in a concentration-dependent manner.

Bacillus subtilis↗

PubMed1989

The transition state transcription regulator AbrB of Bacillus subtilis is autoregulated during vegetative growth.

The DNA-binding AbrB protein of Bacillus subtilis is an ambiactive transcriptional regulator of genes expressed during the transition state between vegetative growth and the onset of stationary phase and sporulation. Studies on the transcriptional control of AbrB synthesis using abrB-lacZ fusions indicated that the abrB gene was autoregulated. This was consistent with the observation that purified AbrB protein bound specifically to the promoter region of its own gene in DNase I protection experiments. The structural gene mutation abrB4 abolished the autoregulation and purified AbrB4 protein did not have the promoter binding properties associated with the wild-type protein. Both AbrB and AbrB4 proteins were shown to be hexamers of 10,500 Dalton subunits and subunit exchange occurred between the proteins in vitro. However, the presence of only one or two mutant subunits dramaticaly altered the DNA-binding ability of the multimeric protein. The results support a model in which autoregulation of the abrB gene is an important factor in preventing sporulation-associated genes from being expressed during vegetative growth.

Bacillus subtilis↗

PubMed1989

Characterization of the gene for a protein kinase which phosphorylates the sporulation-regulatory proteins Spo0A and Spo0F of Bacillus subtilis.

The kinA (spoIIJ) locus contains a single gene which codes for a protein of 69,170 daltons showing strong homology to the transmitter kinases of two component regulatory systems. The purified kinase autophosphorylates in the presence of ATP and mediates the transfer of phosphate to the Spo0A and Spo0F sporulation regulatory proteins. Spo0F protein was a much better phosphoreceptor for this kinase than Spo0A protein in vitro. Mutants with deletion mutations in the kinA gene were delayed in their sporulation. They produced about a third as many spores as the wild type in 24 h, but after 72 h on solid medium, the level of spores approximated that found for the wild-type strain. Such mutations had no effect on the regulation of the abrB gene or on the timing of subtilisin expression and therefore did not impair the repression function of the Spo0A protein. Placement of the kinA locus on a multicopy vector suppressed the sporulation-defective phenotype of spo0B, spo0E, and spo0F mutations but not of spo0A mutations. The results suggest that the spo0B-, spo0E-, and spo0F-dependent pathway of activation (phosphorylation) of the Spo0A regulator may be by-passed through the kinA gene product if it is present at sufficiently high intracellular concentration. The results suggest that multiple kinases exist for the Spo0A protein.

Amino Acid Sequence↗

PubMed1989

[A thioridazine-dihydroergotoxine combination in the treatment of senile pruritus].

Senile itching, a peculiar clinical situation consisting of a cutaneous senile involution associated with a relevant neurogenic component, still keeps being a difficult therapeutic problem. For the purpose, a clinical trial, carried out with a combination of thioridazine and dihydroergotoxine on 19 patients carriers of the affection, could show a relevant decrease in all parameters assessed, ie itching, muscle tension and skin temperature measured by biofeedback. The combination also showed a good tolerance.

Administration, Oral↗

PubMed1988

Structure of the gene for the transition state regulator, abrB: regulator synthesis is controlled by the spo0A sporulation gene in Bacillus subtilis.

Sporulation begins coincidentally with the expression of several stationary-phase-associated gene products during the transition state of a culture from exponential to stationary phase. Mutations in the stage 0 sporulation genes prevent the expression of these gene products in addition to blocking sporulation. Suppressor mutations in the abrB gene, in a spo0 background, restore stationary-phase-associated gene expression but not sporulation. The nature of the abrB gene product was investigated by isolating and sequencing the abrB gene. The abrB gene coded for a 96-amino-acid protein (molecular weight 10773) and contained a helix-turn-helix structure common to DNA binding proteins. Analysis of expression of the abrB gene using lacZ transcription fusions and direct measurement of mRNA content by hybridization showed that the spo0A gene repressed transcription of the abrB gene. Primer extension analysis of abrB gene mRNA revealed two initiation sites. The downstream site was dramatically repressed in spo0A+ strains, while the upstream site appeared not to be regulated by spo0A. Five abrB mutant alleles were cloned and sequenced. One mutation, abrB4, resided within the structural gene and continued to overexpress abrB messenger RNA from both promoters. A promoter mutation, abrB15, reduced transcription from the downstream promoter but not the upstream promoter. Thus, the phenotype of abrB mutations results from inactivation of the abrB gene product or by prevention of its overexpression. The results suggest that the abrB gene codes for a regulator which controls several genes whose products are normally produced during the transition phase between active growth and sporulation. The level of this regulator is, in turn, controlled by the spo0A gene. The pleiotropic phenotypes of spo0A mutants result from uncontrolled overexpression of the abrB regulator.

Amino Acid Sequence↗

PubMed1988

Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants.

The start point for transcription of the subtilisin (aprE) gene was determined by primer extension analysis and was found to be at a point significantly different from that identified in a previously published report (S. L. Wong, C. W. Price, D. S. Goldfarb, and R. H. Doi, Proc. Natl. Acad. Sci. USA 81:1184-1188, 1984). An aprE-lacZ fusion was used to analyze expression of the promoter. Deletion analyses of the promoter were performed to determine the extent of the upstream region necessary for activity. This was found to be between -52 and -41 with respect to the transcription start site. Expression of the aprE-lacZ fusion was unimpaired in a mutant deleted for the sigma B subunit of RNA polymerase. Mutations in the gene for the sigma H subunit of RNA polymerase decreased expression of the aprE-lacZ fusion to approximately 25% of that of the wild type. These results leave the identity of the sigma factor responsible for transcription of this gene in question. Mutations in the spo0A gene drastically decreased the activity of the aprE promoter and its upstream deletion derivatives, while the abrB gene, a phenotypic suppressor of spo0 mutations, restored activity of the aprE promoter in all of the deletion derivatives. Thus, inhibition of transcription by the spo0A mutation and its restoration by an abrB mutation could not be separated from the promoter of the aprE gene.

Bacillus subtilis↗

PubMed1988

Location of the targets of the hpr-97, sacU32(Hy), and sacQ36(Hy) mutations in upstream regions of the subtilisin promoter.

A number of mutations have been described with pleiotropic effects on the expression of genes for degradative enzymes in Bacillus subtilis. The sacU32(Hy) and sacQ36(Hy) mutations increase the expression of a wide variety of enzymes that degrade biological polymers. The phenotypes caused by mutations at the hpr locus are more restricted; they are known to increase expression of the alkaline and neutral proteases. The alkaline protease (aprE) promoter was analyzed to determine the target site for stimulation by these loci. Deletion of upstream regions of the aprE promoter could abolish or greatly reduce stimulation by mutations at these loci. A region upstream of -200 was necessary for full stimulation by an hpr-97 mutation, whereas a region between -141 and -164 was necessary for full stimulation by the sacU32(Hy) and sacQ36(Hy) mutations. Northern analyses of mRNA preparations showed that the levels of aprE mRNA were increased in strains carrying the sacU32(Hy) or hpr-97 mutation. Moreover, primer extension analysis of these mRNA preparations revealed that the transcription start point was identical to that in a wild-type strain. We hypothesize that upstream activation of the subtilisin promoter mediated by these genes is a mechanism for global responses to a variety of nutritional conditions.

Bacillus subtilis↗

PubMed1988

Sequence analysis and regulation of the hpr locus, a regulatory gene for protease production and sporulation in Bacillus subtilis.

The hyperproduction of alkaline and neutral proteases is a phenotype of mutation at the hpr locus. This locus has been cloned and sequenced and has been found to code for a protein of 23,718 Mr. The mutations hpr-1, scoC4, and catA7 were identified by sequencing as mutations within the hpr gene. The phenotype of mutations in the hpr gene is due to loss of the hpr gene product, and therefore we suggest that the hpr gene encodes a negative regulator of protease production. This negative regulator must control genes other than protease genes, and these genes must include at least one gene required for sporulation, since overproduction of the hpr gene product by cloning the locus on a multicopy vector results in the inhibition of sporulation as well as protease production. Truncated fragments of the hpr gene or its promoter do not have this phenotype. Transcription of the hpr locus is controlled by the spoOA gene. In an spoOA mutant the hpr gene transcript is constitutively overproduced, as determined by a transcription fusion to beta-galactosidase. The results are consistent with the view that the spoOA gene may control sporulation and transcription by modulating the level and activity of several regulatory proteins.

Alleles↗

PubMed1987

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism.

Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A lambda recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a lambda Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.

Bacillus subtilis↗

PubMed1987

Isolation and sequence of the spo0E gene: its role in initiation of sporulation in Bacillus subtilis.

The pleiotropic stage 0 sporulation locus spo0E was isolated and sequenced. The spo0E gene was found to code for a protein of 9791 molecular weight. Two spo0E mutations were identified by sequence analysis and were found to give rise to nonsense codons within the gene. The results indicated that it is the lack of the spo0E gene product that is responsible for the sporulation-defective phenotype. The DNA fragment containing the spo0E locus was inhibitory to sporulation when present on a multicopy plasmid. Since DNA fragments containing only the upstream region of the gene were also inhibitory, this effect was not due to over-production of the spo0E gene product. Coupling the transcription of the spo0E gene to beta-galactosidase in an integrative plasmid vector revealed that active transcription of this gene begins at the end of exponential growth and continues through the early part of sporulation. Studies of the regulation of this gene have allowed the generation of a hypothesis to explain the interactions of those five stage 0 genes involved in the activation of sporulation-specific transcription.

Amino Acid Sequence↗

PubMed1984

Expression of human dihydrofolate reductase cDNA and its induction by chloramphenicol in Bacillus subtilis.

A bifunctional plasmid (pMP358) able to replicate and to express cloned human dihydrofolate reductase cDNA (cDHFR) in both Escherichia coli and Bacillus subtilis was constructed. The expression of cDHFR in B. subtilis was the result of a deletion that placed the cDNA fragment under the control of the chloramphenicol acetyltransferase (CAT) gene promoter of Staphylococcus aureus plasmid pC194. By sequence analysis of plasmid pMP358, we observed a gene fusion occurring between the cDHFR and the 32nd codon of the CAT gene. We report that such a "hybrid" gene is able to direct the synthesis of a 25-kDal "hybrid" protein, which was found to be inducible by supplementing B. subtilis cells with sublethal doses of chloramphenicol.

Acetyltransferases↗

PubMed1982