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P Zuber

Publications and source records attributed to P Zuber.

71 records · Page 4Linked to original sources
PubMed1990

Molecular biology of antibiotic production in Bacillus.

Several species of the genus Bacillus produce peptide antibiotics which are synthesized either through a ribosomal or non-ribosomal mechanism. The antibiotics gramicidin, tyrocidine, and bacitracin are synthesized nonribosomally by the multienzyme thiotemplate mechanism. Surfactin and mycobacillin are also synthesized nonribosomally but by a mechanism that, apparently, is distinct from that of the multienzyme thiotemplate. Other antibiotics such as subtilin are gene encoded and are ribosomally synthesized. Molecular genetic and DNA sequence analysis have shown that biosynthesis genes for some antibiotics are clustered into polycistronic transcription units and are under the control of global regulatory systems that govern the expression of genes that are induced when Bacillus cells enter stationary phase of growth. Future experiments involving the molecular dissection of peptide antibiotic biosynthesis genes in Bacillus will be attempted in hopes of further examining the mechanism and regulation of antibiotic production.

Amino Acid Sequence↗
PubMed1989

Mutation changing the specificity of an RNA polymerase sigma factor.

We describe a mutation that changes the fine specificity of promoter selection by a secondary form of RNA polymerase holoenzyme in Bacillus subtilis. The product of regulatory gene spo0H is an RNA polymerase sigma factor called sigma H, which directs transcription of a sporulation gene known as spoVG. We show that the spo0H mutation spo0H81, which blocks transcription from the wild-type spoVG promoter, enhances transcription from a mutant form of the spoVG promoter (spoVG249) bearing a severe down-mutation (a G.C to A.T transition) at position -13 in the "-10 region." Suppression of the spoVG249 mutation is specific in the sense that the transcription from several other spoVG mutant promoters was not restored by the mutant sigma. Evidently, spo0H81 is a change-of-specificity mutation that alters sigma H-RNA polymerase in a way that decreases its capacity to use the wild-type spoVG promoter, while increasing its capacity to use the mutant promoter. Transcription experiments in vitro using RNA polymerase containing the wild-type or mutant sigma support this interpretation. The spo0H81 mutation causes a threonine (Thr100) to isoleucine substitution in a region of sigma H that is highly homologous among sigma factors of diverse origins. We discuss the possibility that Thr100 is an amino acid-base-pair contact site and that sigma factors contact the -10 region of their cognate promoters by means of amino acid residues in this highly conserved region.

Bacillus subtilis↗
PubMed1989

AbrB, a regulator of gene expression in Bacillus, interacts with the transcription initiation regions of a sporulation gene and an antibiotic biosynthesis gene.

The abrB gene of Bacillus subtilis is believed to encode a repressor that controls the expression of genes involved in starvation-induced processes such as sporulation and the production of antibiotics and degradative enzymes. Two such genes, spoVG, a sporulation gene of B. subtilis, and tycA, which encodes tyrocidine synthetase I of the tyrocidine biosynthetic pathway in Bacillus brevis, are negatively regulated by abrB in B. subtilis. To examine the role of abrB in the repression of gene transcription, the AbrB protein was purified and then tested for its ability to bind to spoVG and tycA promoter DNA. In a gel mobility shift experiment, AbrB was found to bind to a DNA fragment containing the sequence from -95 to +61 of spoVG. AbrB protein exhibited reduced affinity for DNA of two mutant forms of the spoVG promoter that had been shown to be insensitive to abrB-dependent repression in vivo. These studies showed that an upstream A + T-rich sequence from -37 to -95 was required for optimal AbrB binding. AbrB protein was also observed to bind to the tycA gene within a region between the transcription start site and the tycA coding sequence as well as to a region containing the putative tycA promoter. These findings reinforce the hypothesis that AbrB represses gene expression through its direct interaction with the transcription initiation regions of genes under its control.

Amino Acid Sequence↗
PubMed1989

Cloning and characterization of srfB, a regulatory gene involved in surfactin production and competence in Bacillus subtilis.

A Tn917 insertion mutation srfB impairs the production of the lipopeptide antibiotic surfactin in Bacillus subtilis. srfB is located between aroG and ald in the B. subtilis genome, as determined by phage PBS1 transduction mapping, and is not linked to the previously described surfactin loci sfp or srfA. A srfB mutant was found to be also deficient in the establishment of competence. SP beta phage-mediated complementation analysis showed that both competence and surfactin production were restored in the srfB mutant by a single DNA fragment of 1.5 kilobase pairs. The sequence of the complementing DNA revealed that the srfB gene is comA, an early competence gene which codes for a product similar to that of the activator class of bacterial two-component regulatory systems. The srfB mutation impaired the expression of a srfA-lacZ fusion, suggesting that surfactin production is positively regulated at the transcriptional level by the srfB (comA) gene product.

Bacillus subtilis↗
PubMed1989

The implications of the heterogeneous distribution of Ki-67 labelled cells in meningiomas.

Ki-67 monoclonal antibody reacts with a nuclear protein only expressed in proliferating cells. In order to evaluate the applicability of Ki-67 in meningiomas we determined the index of labelled cells in 52 biopsies. Two counting methods were used to calculate the labelling index. The mean Ki-67 index was determined by the number of positive Ki-67 cells in three randomly chosen fields divided by the total number of cells in the three fields. The preparations were also screened for the area with the highest percentage of positive Ki-67 cells, yielding the 'highest' Ki-index. The three patients who developed a recurrence did not show an elevated Ki-67 index by any of the two counting methods. Because of the differences observed between the two counting methods and the differences of the Ki-67 indices in the three randomly chosen fields we performed a statistical analysis to evaluate the possibility of a heterogeneous distribution of Ki-67 positive cells in meningiomas. Homogeneity was rejected if the data showed no Poisson distribution. We found that five of the 13 tumours which had a mean Ki-67 index greater than 1.00% were heterogeneous (38.4%). One of the highest differences between the two counting methods (5.05%) was noted in the only tumour with local signs of malignancy. Our results indicate a heterogeneity of the labelling of Ki-67 in histologically benign meningiomas and emphasise the importance of representative sampling. More attention should be paid to this heterogeneity for cell kinetic studies in brain tumours, and one should be aware that regrowth is not only determined by the proliferative capacity.

Antibodies, Monoclonal↗
PubMed1988

Effects of recombinant human tumor necrosis factor-alpha on the surface phenotype and the growth of human malignant glioma cell lines.

Human malignant glioma cell lines and clones were incubated with various concentrations of recombinant human TNF-alpha, either alone or in combination with recombinant human IFN-gamma. The surface expression of HLA-ABC (class I) antigens and beta 2-microglobulin, was significantly enhanced by TNF-alpha alone on every cell line and clone tested. After incubation with both TNF-alpha and IFN-gamma, the surface expression of HLA-ABC antigens was only slightly higher than that observed with each cytokine alone. In contrast to IFN-gamma, TNF-alpha had no effect on the surface expression of HLA-DR (class II) antigens. Moreover, the surface expression of HLA-DR induced by IFN-gamma was unaffected by TNF-alpha. The increased expression of HLA-ABC antigens after treatment with TNF-alpha or IFN-gamma correlated with increased levels of HLA-ABC-specific mRNA. In addition, TNF-alpha, like IFN-gamma, selectively enhanced the surface expression of a tumor-associated antigen, Me14-D12, while it had no effect on the expression of various other surface antigens. In the absence of actinomycin D, TNF-alpha exhibited no direct cytotoxic/cytostatic effect on the glioma cell lines tested. These results indicate that TNF-alpha can enhance the surface expression of HLA-ABC antigens on human glioma cells in the absence of a direct cytotoxic/cytostatic effect.

Animals↗
PubMed1988

Transforming growth factor-beta 2 down-regulates HLA-DR antigen expression on human malignant glioma cells.

Transforming growth factor-beta (TGF-beta) is known to have a potent inhibitory influence on several immune functions. It has recently been demonstrated that TGF-beta 2 is identical to the glioblastoma-derived T cell suppressor factor (G-TsF). In the present study, human malignant glioma cell lines were incubated with various concentrations of TGF-beta 2. An optimal concentration of 1 ng/ml TGF-beta 2 produced a partial but significant decrease of HLA-DR (class II) surface antigen expression on glioma cells expressing this antigen, as well as decreased levels of HLA-DR-specific mRNA. The surface expression of other HLA-related molecules, such as HLA-ABC (class I) and beta 2-microglobulin, was not influenced by TGF-beta 2. The suppressive effect of TGF-beta 2 on HLA-DR expression, both at the surface antigenic and cytoplasmic mRNA levels, could be completely overcome by adding relatively high concentrations (500 U/ml) of interferon (IFN)-gamma to the culture system. However, TGF-beta 2 inhibited the enhancement of HLA-DR surface expression produced by low concentrations of IFN-gamma on some cells which initially did not express these antigens. These results show that TGF-beta 2 can act as a regulator of HLA-DR antigen expression on human glioma cells.

Antigens, Neoplasm↗
PubMed1988

Effect of levocabastine, a new H1 antagonist, in a conjunctival provocation test with allergens.

Levocabastine is a new, highly potent, and specific H1 antagonist. The effects of this drug, administered topically, were evaluated in a conjunctival provocation test (CPT) with allergens. CPT was performed by the instillation of one drop of allergen at increasing concentrations in the inferior conjunctival sac of each eye, alternatively, and stopped when both itching and redness of the conjunctiva were present. The concentration of allergen at this step was considered as the reaction threshold. Eleven patients, allergic to grass pollen, underwent, in winter, a first CPT without pretreatment (screening test); the CPT was then repeated twice after a 24-hour treatment, once, with a placebo, and once, with levocabastine (one drop twice a day, 0.5 mg/ml), administered in a double-blind fashion and in random order. The minimal interval between the two tests was 1 week. There was no significant difference between the thresholds determined in the two CPTs performed without medication (screening test and placebo), whereas the threshold was significantly increased (p less than 0.001) after pretreatment with levocabastine. Individually, the threshold increased in 10/11 patients (p less than 0.01). Levocabastine prevented both redness and itching. A late allergic reaction was observed by the patient in 6/11 CPTs performed after placebo treatment and 8/11 after levocabastine treatment. We conclude that, in this model of allergic conjunctivitis, levocabastine increased the conjunctival tolerance to an allergen. Further studies should help to determine the true place of this H1 antagonist in the treatment of allergic conjunctivitis.

Allergens↗
PubMed1988

Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis.

Surfactin is a lipopeptide antibiotic produced by the cells of Bacillus subtilis ATCC 21332. A genetic locus responsible for surfactin production (sfp) was transferred from ATCC 21332 to JH642, a derivative of the standard B. subtilis 168. To study the sfp locus at the molecular level, a Tn917 insertion mutant that was blocked in surfactin production (srf) was isolated. The srf::Tn917 mutation was found to be closely linked to sfp, and both loci mapped by PBS1 phage transduction to the chromosomal region between aroI and mtlB. These studies suggest that JH642, a strain which is not a producer of surfactin (genotypically sfp0), contains at least some of the genes encoding surfactin production. Expression of the srf gene(s) was examined in both sfp and sfp0 cells by assaying beta-galactosidase activity encoded by a promoterless lacZ gene that was fused to the srf::Tn917 insertion. In cells of both strains, srf-directed beta-galactosidase activity increased when cells entered the stationary phase of the growth curve, but the activity in sfp cells was higher than that in sfp0 cells. srf-lacZ expression was partially impaired by a mutation in spo0A. In sfp0 cells, this dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation. In the sfp cells, the abrB mutation could not restore the defect conferred by the spo0A mutation. These data suggest that the sfp locus, which is responsible for surfactin production, alters the transcriptional regulation of srf in JH642 cells.

Anti-Bacterial Agents↗
PubMed1988

Identification of proliferating cells in human gliomas using the monoclonal antibody Ki-67.

Ki-67 is a monoclonal antibody directed against a nuclear antigen present only in proliferating cells in the G1, S, G2, and M phases of the cell cycle. Fifty-one frozen glioma specimens were stained with Ki-67 using the avidin-biotin immunoperoxidase system. For each tumor, six different randomly selected fields were examined. The percentage of Ki-67-positive cells in the total number of cells in the five fields counted with counterstaining has been calculated. The areas of necrosis and the vascular endothelial cells when they were distinguishable were not included in the calculation. The indices determined on this material ranged from 0% to 4.5% (mean, 1.0; SD, 1.5) for 16 low grade astrocytomas; from 0.7% to 7.4% (mean, 3.5; SD, 2.2) for 8 anaplastic astrocytomas; and from 1.7% to 32.2% (mean, 11.1; SD, 8.2) for 27 glioblastomas. The differences among the means of each group are statistically significant. Five patients with malignant gliomas with an index of less than 2.5 had survival times of more than 40 weeks. These results show that the Ki-67 index of proliferating cells in human gliomas correlates with the usual histological classification of these tumors. There is a potential interest in using this technique in routine histopathology because it is simple and more rapid than the classic methods of evaluation of proliferating cells.

Adolescent↗
PubMed1987

Effects of plasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subtilis.

Transcription of the sporulation gene spoVG of Bacillus subtilis is induced at the onset of spore formation and depends on the products of the regulatory genes spoOA, spoOB, and spoOH. We describe two effects of propagating the promoter region of spoVG on a multicopy plasmid replicon in B. subtilis cells. One effect is that transcription from the plasmid-borne spoVG promoter is altered with respect to the time of its induction and the dependence on spoO gene products. An example of this effect is that plasmid propagation was observed to relieve substantially the inhibitory effect of a mutation in spoOH, the spoO gene upon which spoVG promoter activity is most strongly dependent. We present results which suggest that propagation on a plasmid replicon causes an alteration in the conformation of spoVG promoter DNA which somehow compensates for the defective spoOH gene product. Plasmid propagation did not, however, entirely eliminate the requirement for the spoOH gene product; little or no spoVG-directed RNA synthesis was observed in cells bearing a putative spoOH deletion mutation, a finding which indicates that SpoOH protein plays an indispensable role in spoVG promoter utilization. Another effect of propagating the promoter region of spoVG on a multicopy plasmid is to inhibit sporulation. S1 nuclease mapping experiments suggest that amplification of spoVG on a multicopy plasmid causes the titration of a transcription factor or minor form of RNA polymerase holoenzyme required for utilization of one of the two overlapping promoters which comprise the spoVG transcription initiation region.

Bacillus subtilis↗
PubMed1987

Identification of the promoter for a peptide antibiotic biosynthesis gene from Bacillus brevis and its regulation in Bacillus subtilis.

Tyrocidine is a cyclic decapeptide antibiotic which is produced and secreted by stationary-phase cells of the sporeforming bacterium Bacillus brevis. We identified the promoter for the B. brevis structural gene (tycA) for tyrocidine synthetase I, the enzyme catalyzing the first step in tyrocidine biosynthesis, and studied its regulation in cells of B. brevis and Bacillus subtilis. Transcription from the tycA promoter was induced at the end of the exponential phase of the growth cycle in B. brevis cells growing in sporulation medium. To study the regulation of tycA in B. subtilis, we constructed a derivative of the B. subtilis bacteriophage SP beta containing a transcriptional fusion of the tycA promoter to the lacZ gene of Escherichia coli and introduced the tycA-lacZ operon fusion by means of specialized transduction into sporulation mutants known to be blocked in sporulation-associated antibiotic production. Our principal finding was that tycA-directed lacZ expression was impaired in the stage-0 mutants with mutations spo0A, spo0B, and spo0E but not in spo0C, spo0F, spo0H, or spo+ bacteria. The dependence on the spo0A gene product could be entirely bypassed by an abrB suppressor mutation, which caused tycA-lacZ to be transcribed constitutively at all stages of growth. A simple model is proposed for the mechanism of tycA induction based on the Spo0A-dependent inactivation of Ab-B protein, which is proposed to be a negative regulator of tycA transcription.

Bacillus↗
PubMed1987

Role of AbrB in Spo0A- and Spo0B-dependent utilization of a sporulation promoter in Bacillus subtilis.

Transcription of the Bacillus subtilis gene spoVG is induced at the onset of sporulation and is dependent on the products of the stage-0 regulatory genes spo0A, spo0B, and spo0H. We show here that the dependence of spoVG transcription on Spo0A and Spo0B (but not Spo0H) can be bypassed by a mutation at abrB, a previously identified locus at which mutations that suppress some of the phenotypes of spo0A are often located, or by a cis-acting mutation within the spoVG promoter. To explain the epistatis of abrB to spo0A and spo0B mutations, we propose that AbrB acts, directly or indirectly, to block transcription of spoVG and that Spo0A and Spo0B cause inactivation of the abrB gene product(s). Spo0A-Spo0B-dependent inactivation of AbrB could be a general explanation for the pleiotropic effects of spo0A and spo0B mutations on B. subtilis gene expression.

Bacillus subtilis↗
PubMed1987

Effect of a new selective H1 receptor antagonist (levocabastine) in a nasal and conjunctival provocation test.

Levocabastine is a new selective H1 receptor antagonist. The effect of the drug administered locally was compared to placebo in a quantified nasal and conjunctival provocation test with allergens performed in patients allergic to grass pollen. In the nasal provocation test, levocabastine was able to increase the 'reaction threshold' (dose of allergen necessary to trigger allergic symptoms) in 9 out of 12 patients; the drug inhibited rhinorrhea and sneezing, but not nasal obstruction. In the conjunctival provocation test, the 'reaction threshold' clearly increased in 10 out of 11 patients. The local administration of levocabastine might be useful in allergic rhinitis and conjunctivitis.

Administration, Topical↗
PubMed1987

[Immunobiology of the normal and tumor astrocyte].

Immunobiology of the normal and tumoral astrocytes studies interactions between these cells and the immune system. Their antigenic characterization defines 3 classes of antigens: glial antigens, tumor associated antigens (neuroectodermal and gliomatous) and lymphoid differentiation antigens which can be modulated by gamma interferon and other cytokines. Glioma associated antibodies could be used for radiolocalization of tumours and for immunotherapy. The enhancement or induction of the Major Histocompatibility Complex antigen expression by interferon gamma could enhance tumour-antigen presentation by glioma cells to helper and cytotoxic T cells and thus activate the host's immune response. The presence of oncogenes and their products in glioma cells, mainly growth factor receptors, brings new potential therapies using oncogenes products as tumoral markers or as targets for monoclonal antibodies blocking their mitogenic activity. Normal and tumoral astrocytes produce lymphokines: interleukin 1, interleukin 3, prostaglandin E as well as a suppressor factor inhibiting interleukin 2 mediated effects and probably responsible for the suppression of glioma infiltrating T cells. The interaction of astrocytes with several humoral factors related to the immune system and their capacity to function as antigen presenting cells underline their importance for immune reactions within the central nervous system.

Animals↗
PubMed1983

Use of a lacZ fusion to study the role of the spoO genes of Bacillus subtilis in developmental regulation.

A mutation in any one of eight spoO genes of Bacillus subtilis blocks the process of spore formation at its earliest stage. To investigate how the products of the spoO genes may be involved in developmental gene expression, we fused the lacZ gene of E. coli to spoVG, a sporulation gene whose induction at the onset of sporulation is under spoO control. In cells of Spo+ bacteria containing a single copy of the gene fusion, conditions leading to the onset of sporulation resulted in the induction of beta-galactosidase synthesis. This induction was moderately to severely impaired by mutations in any of seven spoO genes. Deletion and hybridization analysis demonstrated that this sporulation-induced enzyme synthesis was exclusively expressed from the two overlapping promoters, which comprise the spoVG transcription-initiation region, and that a small DNA segment (157 bp) containing the spoVG promoters was sufficient to cause spoO-dependent induction of the fused lacZ gene.

Bacillus subtilis↗