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A Bernardi

Publications and source records attributed to A Bernardi.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1996

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

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

Adenylyl Cyclases↗
PubMed1996

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

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

Amino Acid Sequence↗
PubMed1996

DNA sequence analysis of the VPH1-SNF2 region on chromosome XV of Saccharomyces cerevisiae.

The nucleotide sequence of a 37 000 base pair region from the left arm of chromosome XV of Saccharomyces cerevisiae has been determined and analysed. This region contains 21 open reading frames (ORFs) coding for proteins of more than 100 amino acids. Six ORFs correspond to the genes PAC1, VPH1, MOD5, CAP20, ORF1 and SNF2 already described. Eight ORFs show some similarities to known genes from yeast and other organisms. They include genes coding for serine/threonine protein kinases, a multidrug resistance family homologue, a protein related to dihydrofolate reductase, a cluster of heat shock-like proteins and a gene coding for an enzyme related to protein disulfide isomerase. Finally seven ORFs do not show any similarities with a known gene. In addition we found a new ala-tRNA (UGC) gene located next to a sigma sequence.

Amino Acid Sequence↗
PubMed1995

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

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

Binding Sites↗
PubMed1995

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

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

Catalysis↗
PubMed1994

Properties of the catalytic domain of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor: comparison of its activity on full-length and C-terminal truncated RAS2 proteins.

Two C-terminal fragments (334 and 509 amino acid residues) of CDC25, a Saccharomyces cerevisiae GDP/GTP exchange factor, and the RAS2 protein were purified from E. coli, using the pGEX system. With this method it was possible to avoid in part the proteolytic phenomena that usually convert full-length RAS2 (42kDa) into 37 and 30kDa forms. Of the two CDC25 fragments containing the conserved catalytic domain, only CDC25-509 could enhance the guanine nucleotide exchange on RAS2. Comparison of the activities of RAS2-42/37kDa and RAS2-30kDa showed that the C-terminal region (112 residues) influences neither the intrinsic GDP/GTP exchange nor its stimulation by CDC25-509. RAS2-42/37kDa was somewhat more effective in enhancing the adenylylcyclase activity of a yeast membrane reconstituted system. CDC25-509 displayed a higher specific activity than the catalytic domains of the two CDC25-like proteins: S. cerevisiae SDC25 and mouse CDC25Mm.

Adenylyl Cyclases↗
PubMed1994

Evidence for a second tumor suppressor gene on 17p linked to high S-phase index in primary human breast carcinomas.

The short area of chromosome 17 is a frequent target for deletions in human tumors, including breast cancer. We have investigated by restriction fragment polymorphism analysis the pattern of loss of heterozygosity (LOH) at four loci on 17p13.1-17pter in a panel of 110 primary human breast carcinomas. A copy of the p53 gene was lost in 23% of the informative cases. Point mutations in the p53 gene were statistically associated with LOH at the same locus (p = 0.003) but not at other loci on 17p13.3-17pter. A second region bordered by the loci D17S5/D17S28 (17p13.3) and D17S34 (17pter) is also affected by LOH, independent of point mutations in the p53 gene. We propose the presence of a second tumor suppressor gene within this region. In support of this hypothesis is the significant association (p = 0.005) between LOH at the D17S5/D17S28, but not at the TP53 or D17S34 loci, and tumors having a high S-phase index.

Alleles↗
PubMed1993

Properties of the SDC25 C-domain, a GDP to GTP exchange factor of RAS proteins and in vitro modulation of adenylyl cyclase.

The SDC25 C-domain, the product encoded by the 3'-terminal part of the Saccharomyces cerevisiae SDC25 gene, acts as a GDP dissociation stimulator on RAS proteins (Créchet, J.B., Poullet, P., Mistou, M. Y., Parmeggiani, A., Camonis, J., Boy-Marcotte, E., Damak, F., and Jacquet, M. (1990b) Science 248, 866-868). To define further its role in the RAS-adenylyl cyclase pathway, an in vitro system was used, which utilized cell membranes from yeast strains with appropriate genotypes carrying alterations in the positive regulators of adenylyl cyclase activity. The SDC25 C-domain was able to stimulate the adenylyl cyclase activity of membranes from RAS2 cdc25 strains. Our results indicate that the SDC25 C-domain activates adenylyl cyclase by rapidly recycling the active RAS2. or RAS1.GTP complex from the respective GDP complex. This is also supported by the observation that the stimulation of adenylyl cyclase activity by RAS2T152I, a mutant characterized by a constitutively fast GDP to GTP exchange, was insensitive to the action of the SDC25 C-domain. No direct influence of this GDP dissociation stimulator on adenylyl cyclase was detected. Biochemical evidence was obtained, showing that in the presence of the functional target of RAS, the adenylyl cyclase, the effects of SDC25 C-domain and the catalytic domain of GTPase-activating protein are antagonistic. This in vitro system allowed a quantitative evaluation of the effects of positive and negative effectors of RAS on adenylyl cyclase and the biochemical analysis of conditions inducing a phenotype of permanently activated adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1993

In primary human breast carcinomas mutations in exons 5 and 6 of the p53 gene are associated with a high S-phase index.

A series of 121 human breast tumors was screened for point mutations in exons 5 through 8 of the p53 gene, by SSCP analysis. On the same tumor samples, the S-phase index (SPI) was determined by the incorporation of BUdR in fresh tissue. p53 mutations were observed in 29% of the cases. The frequency of point mutations for the individual exons was: exon 5, 10.0%; exon 6, 9.9%; exon 7, 7.1% and exon 8, 5.5%. Two mutations detected by SSCP were confirmed by sequencing the p53 cDNA. The presence of a p53 mutation, irrespective of its location, correlates (p = 0.003) with a high SPI. This association appears to primarily reflect mutations in exon 5 (p = 0.0002) and exon 6 (p = 0.05), since mutations in exons 7 and 8 failed to show any association. These results indicate that mutations in the p53 gene identify highly proliferating tumors, and that the position of the p53 mutation may have different effects upon the proliferative activity of tumor cells in vivo.

Base Sequence↗
PubMed1993

Cell proliferation of breast cancer evaluated by anti-BrdU and anti-Ki-67 antibodies: its prognostic value on short-term recurrences.

The prognostic value of breast cancer proliferative activity was evaluated in 385 women operated for primary, non-metastasised mammary carcinoma. Cell kinetics was measured using two immunohistochemical techniques. Cells in S-phase of cell cycle were labelled in vitro by incubation of fresh tissue fragments with 5-bromo 2-deoxyuridine (BrdU), a thymidine analogue. Nuclei of cells in active DNA synthesis were stained by an anti-BrdU monoclonal antibody (Mab). Cells in interphase and mitosis were detected with Ki-67, a Mab that is known to react with a nuclear antigen present in G1/S/G2/M phases of cell cycle, but not in resting cells. This reagent provides a means of evaluating the growth fraction of neoplastic cells. BrdU was incorporated in a proportion of tumour cells ranging from 0.1 to 65.5% (median 6.8%). In the panel of tumours presented in this report the median percentage of Ki-67 positive cells (Ki-67 score) was 9.0% (range 0.1-77%). The relationship between disease-free survival (DFS), BrdU labelling index, Ki-67 score and 13 different clinico-pathological variables was investigated by multivariate analysis, using the Cox proportional hazards model. Axillary node status (P = 0.009) and Ki-67 score (P = 0.038) emerged as independent prognostic factors. Nodal status and tumour growth fraction allowed division of patients into groups at different risk of relapse: tumours with a proliferative index below the median value showed a lower recurrence rate than tumours with a high proliferative activity (P < 0.001). In particular, no relapse occurred in pN0 patients bearing carcinomas with a Ki-67 labelling < 9.0% 4 years after surgery. These findings suggest that the evaluation of proliferative activity in breast cancer enhances the probability of correctly predicting outcome after surgery and could be of assistance in the planning of adjuvant therapies.

Adult↗
PubMed1992

Loss of heterozygosity on chromosome 17p13 in breast carcinomas identifies tumors with high proliferation index.

The capacity of breast tumor cells to proliferate is considered a potential prognostic factor together with other histopathologic parameters. The authors determined the proliferation index on a large panel of human primary breast tumors by measuring the levels of incorporation of bromodeoxyuridine (BrdU) by fresh tumor specimens in culture. Previous analysis showed that the percentage of cells entering the S-phase of the cell cycle strongly correlates with tumor grade, tumor size, and estrogen and progesterone receptor status. The capacity of tumor cells to proliferate might be associated with specific genetic mutations in primary tumors. To test this hypothesis, a panel of 96 human breast carcinomas, for which the BrdU labeling index (LI) was known, were tested for loss of heterozygosity (LOH) or increased copy number (ICN) at chromosomes 1q, 3p, 13q, 17p, and 18q. On chromosome 17p, LOH and ICN were observed in 27% and 12%, respectively, of the informative breast tumors. The LOH on chromosome 17p was significantly associated with tumors having an elevated BrdU proliferation index (P = 0.022). No association (P = 0.45) was observed between BrdU LI and tumor size (T2 + T3 compared with T1), tumor grade, and lymph node status. Increased copy number on chromosome 17p, LOH or ICN on 1q, and LOH on 13q14, 18q, and 3p also showed no significant correlation with cell kinetic parameters. These data are consistent with the presence of a gene or genes on chromosome 17p13 near the YNZ22.1 locus whose normal functioning is necessary for controlling breast tumor cells proliferation in vivo.

Blotting, Southern↗
PubMed1991

Inter- and intramolecular transposition of Tn903.

We have performed a detailed analysis of intra- and intermolecular endproducts of transposition of the compound transposon Tn903 and we show that, in our system, the transposition activity is almost entirely driven by one of the flanking insertion sequences, IS903L. The relatively inactive state of IS903R can be conferred on IS903L by changing the orientation of the internal Tn region. IS903L mediates the formation of the majority of adjacent deletions, insertion/inversions and cointegrates, all of which are representative of replicative transposition; only a very low level of conservative transposition can be observed. Our results are discussed in relation to those showing that Tn903 uses predominantly the conservative pathway.

Chromosome Deletion↗
PubMed1991

Introduction of a UV-damaged replicon into a recipient cell is not a sufficient condition to produce an SOS-inducing signal.

Three models have been proposed for the nature of the SOS-inducing signal in E. coli. One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein. We tested the models by measuring SOS induction produced by introducing into recipient cells the UV-damaged DNA of 2 constructed phagemids. We used phagemids since they transferred DNA to the recipients with 100% efficiency. The origin of replication of the phagemids was either oriC from the E. coli chromosome, or oriF from F plasmid. Replication of the oriC phagemid was dependent on methylation. A UV-damaged oriC phagemid failed to induce SOS functions in a recipient cell whereas an oriF phagemid did induce them. Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal. The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E. coli chromosome after UV damage.

Bacteriophage lambda↗
PubMed1991

Biological and molecular studies of endogenous retrovirus-like genes in Chinese hamster cell lines.

Two types of endogenous retroviral-like sequences related to type C or Intracisternal A Particle (IAP) genes respectively were detected in the Chinese hamster (CH) genome. Their structure and expression were analysed by virological and molecular approaches in different CH cell lines. Several clones were isolated from a CHO genomic library by screening--in stringent conditions--with type C (Gross-MuLV) or IAP (pMIA1) specific probes. These clones were further characterized by restriction mapping and sequencing. Transcripts of type C and IAP-like retroviral sequences were revealed either by PCR or by Northern blot analysis and hybridization with the respective specific probes. However only a few retroviral-like type C particles and no IA particles were detectable by electron microscopy. Only weak activation of type C viral production by halogenated pyrimidines or other classical viral activation agents was observed. No replication of this virus could be obtained in rodent, canine, simian or human cell lines.

Animals↗
PubMed1990

[Biopsy of the minor salivary glands in Sjögren's syndrome].

Twenty subjects, 17 females and 3 males, mean age 55.9, with Sjögren's syndrome (13 cases: classical or secondary form--7 cases: sicca syndrome) were submitted to biopsy of the minor salivary glands of the lower lip. The histopathological findings from the salivary glands and the clinical aspects of each patient were separately evaluated, in order to find a possible connection between the degree of glandular impairment and the severity of the disease. As a result of their findings, the authors conclude that the biopsy of the salivary glands, while constituting an important element for diagnosis, cannot be considered a determining factor for making a prognosis, or for therapy, because of the extreme polymorphism in the histopathological findings, and for the relative nature of the respondents with the severity of the clinical aspects.

Adult↗