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Biomedical subjects

A Bernardi

Publications and source records attributed to A Bernardi.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1990

A rapid inhibition micro ELISA for detecting antibodies to Plasmodium falciparum sporozoites in human blood.

A sensitive and specific micro ELISA, named MONOPLATE ELISA, for the detection of antibodies against P. falciparum sporozoites was developed. It can be applied to many kinds of samples including serum, plasma, whole blood, eluted bloodspot and mosquito bloodmeal as well. The method makes use of a single microtiter plate and the chemically synthesized (Asn-Ala-Asn-Pro)20 (NANP20) antigen both as coating material and as competitive (binding) inhibitor in the samples. The specific value of each sample is obtained as the absorbance difference between the uninhibited and the fully inhibited sample. Using appropriate conditions, the results can be evaluated by simple visual inspection of the plate, without any instrument. A rapid procedure, where the incubation times for sample and conjugate are just 15 minutes, is also described. When unknown samples from a P. falciparum endemic area were tested, a close correlation was found between our results and those obtained with the only commercial ELISA kit now available (Sclavo S.p.A). For screening purposes, as many as 48 samples per plate can be tested by this method.

Amino Acid Sequence↗
PubMed1988

Transcription of the target is required for IS102 mediated deletions.

We have constructed several plasmids to test the specificity of target selection for IS102 associated deletions. We had previously shown that the cIII region of phage lambda is a target for IS102 mediated deletions and this region was therefore introduced in pSC101, where IS102 resides, in such a way that it was silent, transcribed or fully expressed. The deletion selection was provided by the galactokinase system. The results we have obtained show that transcription of the target region is required for deletion formation. The frequency of deletions in this target depends on its position along the transcript and is also influenced by translation. Implications of these results on the regional specificity of transposition are discussed.

Bacteriophage lambda↗
PubMed1988

Different substrates influence the expression of intermediate filaments and the deposition of basement membrane proteins.

A primary culture of serous cystadenocarcinoma of the ovary was used to study the expression of intermediate filament proteins and the deposition of basal lamina proteins. It was found that cells grown on type I and IV collagens or in collagen gels failed to express vimentin, which was readily demonstrable in cultures of the same cells grown on plastic or glass. Furthermore cells grown in collagen gels formed colonies demonstrating a cystic architecture. Unlike what is commonly observed on glass or plastic, where laminin and fibronectin are deposited as disorganized fibrils in the extracellular space, in or on collagen these proteins appear solely at the interface between the epithelial cells and matrix. The results suggest that the extracellular matrix influences the cytoskeletal organization of the intermediate filaments and determines cell polarity. They confirm that collagen substrates permit epithelial cell cultures to progress toward a more differentiated state.

Basement Membrane↗
PubMed1987

Role of replication in IS102-mediated deletion formation.

Intramolecular transposition produces replicon dissociation in a bireplicon; this reaction is homologous to the well-characterized IS-associated deletions in the case of a monoreplicon. However the frequencies at which these two reactions occur differ by a factor of more than 10(2) in favor of deletion formation. This raises the question of how these deletions occur. We show that the presence of a productive replication on the fragment to be deleted interferes with deletion formation. Our results also suggest that the deleted fragment is not degraded during deletion formation.

Chromosome Deletion↗
PubMed1987

Pollution episodes at Venice related to weather types: an analysis for a better predictability.

In order to obtain a rational strategy for the control and management of emissions from the industrial area of Venice, determination of the air quality is made from different perspectives. A source-receptor oriented approach shows a remarkable scatter of data due to the complex source field and variability of atmospheric conditions at a coastal site. As many pollution episodes are initiated by local diffusion potentials induced by the interaction between topographic irregularities (i.e. Alpine chain and Adriatic Sea) and the mesoscale atmospheric circulation system, a correlation between pollution events and synoptic conditions is made. Two periods of two years each were considered, the former characterized by very frequent, moderate episodes, the latter by more rare but potentially more severe episodes, although the emission rate was reduced. Four different criteria (i.e. weather types as classified by the U.K. Meteorological Office, by Borghi and Giuliacci, and by Urbani, and the passage of fronts) are considered and compared in order to describe the synoptic conditions and to make a reasonable prediction of the local dynamic climatology. A risk index (RI) is computed for each weather type. The U.K. classification appears to be the most suitable as it classifies the maximum number of synoptic maps and has the greatest spread of RI, thus making a more sound prediction. The analysis shows that the pollution episodes are associated with instability phenomena, i.e. passage of fronts or the onset of moderate to fresh winds, and not with stationary conditions. Also, the areal extension over which the highest pollution levels are recorded is related to particular weather types. The statistics of the meteorological weather types occurring during the pollution events give satisfactory results for some weather types, especially for those correlating highly or poorly with pollution events.

Air Pollution↗
PubMed1987

Expression of the monoclonal antibody-defined CAR-3 epitope on neoplastic and preneoplastic lesions of the colon mucosa.

The AR-3 monoclonal antibody, which defines the tumor-associated antigen CAR-3, was previously found to be able to discriminate between neoplastic cells in gastric, pancreatic, colonic, ovarian and endometrial carcinomas and their normal counterparts. In fact, it strongly reacts with carcinomatous cells at the level of both the glycocalix and the cytoplasm, while its reactivity with normal tissues is restricted to the glycocalix of few mucin-producing epithelial cells. We have now investigated the reactivity of this antibody with immunohistochemical techniques on a series of formalin-fixed paraffin-embedded specimens, from precancerous and cancerous lesions of the large bowel which were classified as adenomas with mild, moderate or severe dysplasia, adenomas with cancer and adenocarcinomas, respectively. It was found that the intensity and extent of the staining correlated with the degree of dysplasia and that the highest expression of the CAR-3 epitope was detectable in adenocarcinomas. Also the localization of the staining in the lesions displayed an increasingly complex pattern, going from linear in adenomas with mild dysplasia to a very strong intracytoplasmic and/or intraluminal expression in adenomas with severe dysplasia or adenocarcinomas.

Adenocarcinoma↗
PubMed1986

Intramolecular transposition of IS102.

It has been postulated that deletions mediated by transposable elements are intramolecular transposition events. An implication of this hypothesis is that the deleted fragment may be recovered if it is capable of autonomous replication. We report here the characterization of the products of intramolecular transposition of the element IS102 in bireplicons. We show that when two origins (ori's) (of pSC101 and R6-5) generate the same copy numbers, two dissociated replicons are recovered as well as the inversions. On the contrary, when two ori's (of pSC101 and pBR322) have different copy numbers, intramolecular transposition results essentially in inversions. However, the very low frequency (5 X 10(-8)) at which intramolecular transpositions in the bireplicons occurs, as compared to the single replicon (10(-4)), suggests that a complete transposition reaction may not be necessary to generate deletions.

Chromosome Deletion↗