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M A Barbieri

Publications and source records attributed to M A Barbieri.

At least 55 records · Page 3Linked to original sources
PubMed1997

Uncoupling of membrane ruffling and pinocytosis during Ras signal transduction.

Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, our study demonstrates that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling via the small GTPase Rac. In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant, via a Sindbis virus vector, resulted in a dramatic stimulation of membrane ruffling without affecting the uptake of horseradish peroxidase. Expression of Ha-Ras(G12V), an activated Ras mutant, stimulated both membrane ruffling and horseradish peroxidase uptake. The Ha-Ras(G12V)-stimulated pinocytosis but not membrane ruffling was abolished by either wortmannin or co-expression with a dominant negative mutant of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis but not membrane ruffling. Our data indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent membrane ruffling.

Androstadienes↗
PubMed1997

[The consumption of psychoactive substances by adolescents in schools in Ribeiräo Preto, SP (Brazil). I--Prevalence of consumption by sex, age and substance].

INTRODUCTION: Concern over the consumption of psychoactive substances by teenagers has given rise to a great worldwide effort to produce information about this phenomenon. This study set out to investigate the prevalence of consumption of legal and illegal psychoactive substances, its distribution by age, sex and age at first experience of them, among teenage pupils in county, Ribeirão Preto, SP, Southeastern Brazil. MATERIAL AND METHOD: A self-applicable questionnaire duly adapted and submitted to a reliability test was applied to a proportional sample of 1,025 teenagers enrolled in 8th, 9th, 10th and 11th grades at public and private city schools. The questionnaire contained questions about the use of ten classes of psychoactive substances, demographic questions and validation information, as well as questions about the perception and intrinsic behavior related to drug consumption. RESULTS: The sample of 88.9% had consumed alcoholic beverages sometime in their lives, 37.7% had used tobacco, 31.1% solvents, 10.5% medicines, 6.8% marihuana, 2.7% cocaine, 1.6% hallucinogens, and 0.3% of the sample had consumed some opiate substance. The rates of consumption increased with age for all substances; however, the use of tobacco and of illegal substances was less intense during the later years of adolescence. As to sex distribution, boys consumed more than girls, except for medicines, with girls consuming barbiturates, amphetamines and tranquilizers in proportions similar to or higher than those observed among boys. Age at first experience showed that access to psychoactive substances occurred at very early ages. CONCLUSIONS: Experimenting with psychoactive substances, whether legal or illegal, is a frequent phenomenon during adolescence, both among boys and girls, often at very early ages.

Adolescent↗
PubMed1997

[Consumption of psychoactive substances by school-age adolescents of Ribeirão Preto, SP (Brazil). II--Distribution of consumption by social levels].

INTRODUCTION: Many of the epidemiological studies on the consumption of legal and illegal psychoactive substances have included the evaluation of the influence of social context on the levels of prevalence of this consumption using indirect social indicators such as family income, and educational and housing levels in an attempt to identify individuals or groups in different social contexts. The present study investigates the distribution of consumption of psychoactive substances according to social class in a sample of teenage pupils in Ribeirão Preto, SP, Southeastern Brazil. MATERIAL AND METHOD: A self-applicable questionnaire duly adapted and submitted to a reliability test was applied to a proportional sample of 1,025 teenagers enrolled in the 8th, 9th, 10th and 12th grades in public and private city schools. The questionnaires contained questions about the use of ten classes of psychoactive substances, demographic questions and validation information, as well as questions about the perception and intrinsic behavior related to drug consumption. The adaptation of a model that identifies 5 social class strata (business middle class, managerial middle class, lower middle class, proletariat and subproletariat) on the basis of indicators that situate the individuals within the social relations of production, was used. RESULTS: The 3 middle class strata were more often represented, whereas the proletariat and subproletariat were less frequently represented in this teenage pupil population than in the population in general. There was no difference in alcohol or tobacco consumption according to social class, although prevalence tended to be higher at the two extremes of the social ladder. In contrast, the consumption of illegal substances was higher in the middle class and lower in the proletariat. CONCLUSION: Although the consumption of legal substances did not differ among social classes, the higher consumption of illegal substances by the wealthier teenagers was probably due to the higher cost of these products as compared those of alcohol and tobacco.

Adolescent↗
PubMed1997

[Orbital cellulitis. A case report].

This study describes the case of a 6 years old child, male, with orbital cellulitis and underlines the importance of an early diagnosis and therapy to avoid severe complications often present in this disease. Swelling and redness of the eyelid, pain and ophthalmoplegia are the first sign of an orbital cellulitis and they require rapid diagnostic procedure such as ultrasound and TC scan of the orbital region to evaluate the integrity of the profound orbital tissues. The child was admitted at the Department of Pediatrics, University "La Sapienza" of Rome and underwent an ultrasound, TC scan and serum exams which demonstrated the elevation of the sedimentation rate, reactive C protein and WBC plus the interesting of the profound orbital tissues. The child was treated with antibiotic and antiinflammatory therapy showing a complete recovery within 7 days. An ultrasound performed 7 days later demonstrated a complete resolution of the inflammatory process. In summary, this study would like to stress the necessity of an early diagnosis and an appropriate therapy in order to avoid the severe complications often present in children with orbital cellulitis.

Anti-Bacterial Agents↗
PubMed1996

Characterization of Rab5:Q79L-stimulated endosome fusion.

Fusion of intracellular membrane-bound compartments is a common step in the transport of macromolecules along the endocytic and secretory pathways. Previous work has shown that GTP gamma S stimulates endosome fusion in the presence of low concentrations of cytosol. In this study, we have characterized the effect of rab5:Q79L, a mutant with reduced GTPase activity, on endosome fusion in a cell-free assay. rab5:Q79L stimulates in vitro endosome fusion. The stimulatory effects required ATP, were blocked by N-ethylmaleimide (NEM) and anti-NEM-sensitive fusion (NSF) protein antibody, but could proceed in the absence of cytosol. Stimulation of fusion with rab5:Q79L led to rapid inactivation of the vesicles when tested in a second incubation for fusogenic activity. By electron microscopy, endosomes connected by tubular structures were frequently observed in the presence of rab5:Q79L. Rab5:Q79L promoted fusion only among early endosomes; when the ligands were chased into more mature endocytic compartments, fusion was not observed. Phospholipase A2 inhibitors blocked rab5:Q79L-stimulated fusion. The results indicate that rab5:Q79L promotes fusion by activating factors already present in the membranes and that NSF and phospholipase A2 activities are required downstream of rab5.

Adenosine Triphosphate↗
PubMed1996

Trypanosoma cruzi: involvement of intracellular calcium in multiplication and differentiation.

The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Electrophoretic analysis of protein selectively extracted with chaotropic agents from membranes of rat sperm.

Sperm from rat cauda epididymis was washed, sonicated and centrifuged to obtain fractions sedimenting at 600 x g for 5 min, 27.000 x g for 5 min, and 100.000 x g for 40 min. All fractions were observed with the electron microscopy and assayed for cytochrome c oxidase activity. The 100.000 x g fraction contained only small membranous vesicles and less than 0.5% of the total enzymatic activity. This fraction was considered to represent sperm plasmalemma and it was extracted with Tris-HCl buffer before treating it with one of the following chemicals: acetate buffer, pH: 4.5; 0.6 M KCl; bicarbonate buffer, pH 11.0; Triton X-100, and Sodium Dodecyl Sulfate (SDS). After centrifuging, the residual sediments were solubilized in hot 2% SDS. The extracts and the solubilized sediments (hot SDS) were analyzed in SDS-PAGE. The extracts obtained with the first three chemicals contained 11,9, and 25% of total proteins respectively. The bicarbonate buffer solubilized 45%, and the detergents 55% and 65% respectively. A total of 30 bands were seen in the extracts and sediments. Acid pH extracted a low number of bands of high mobility and low molecular weight. Instead, the KCl and bicarbonate buffer, extracted a great number of bands over a wide range of molecular weights (23, 38.5, 55, 100, and 140 KD). The detergents had similar effects: both solubilized four new bands. In residual sediments there were no new proteins and the bands corresponded to those extracted with the detergents, but they varied in staining intensity. According to the results obtained with the mild chaotropic agents of 0.6 M KCl and bicarbonate buffer, 50% of the mass of membraneous proteins may be peripheric. Proteins partially extracted with the detergents were also found in the residual sediment, and they may constitute the skeleton of sperm membrane.

Animals↗
PubMed1995

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

Androstadienes↗
PubMed1995

Myristoylation cannot functionally replace the isoprenylation of Rab5.

C-terminal isoprenylation is necessary for the small GTPase Rab5 to associate with early endosomes and to exert its regulatory function in endocytosis. In this study, we tested whether Rab5 could retain its membrane association and biological function if the isoprenylation were replaced by another type of lipid modification (myristoylation). Rab5 mutants were constructed that contained both isoprenylation and myristoylation (Gag-Rab5), myristoylation only (Gag-Rab5 delta C4), and neither of the modifications (Rab5 delta C4), respectively. These mutants and wild-type Rab5 were expressed, via a Sindbis virus vector, in cultured BHK-21 cells and their membrane association and biological activity (stimulation of endocytosis) were examined. Wild-type Rab5 was isoprenylated, membrane associated, and biologically active. With additional myristoylation, Gag-Rab5 showed increased affinity for membranes but decreased biological activity. Rab5 delta C4 contained no lipid modifications, failed to associate with membranes, and had no biological activity. With myristoylation (Gag-Rab5 delta C4), there was a significant increase in membrane association (approximately 30%). However, this increased membrane association did not result in any recovery of Rab5 activity. In light of these data, we conclude that N-terminal myristoylation cannot functionally replace the C-terminal isoprenylation of Rab5. Furthermore, myristoylation of Rab5 (Gag-Rab5) interferes with its normal function.

Animals↗
PubMed1995

Organ-specific binding system for beta-galactosidase in the male reproductive tract.

This study reports on the binding of beta-galactosidase obtained from different organs of the rat urogenital tract to membranes of these organs. Homologous and cross binding saturation assays indicated that: (1) high-affinity sites that recognize fructose-6-phosphate derivates (FPR) are present in spermatozoa from the rete testis, epididymal membranes and testes, although the latter may reflect binding to testicular spermatozoa; (2) the membranes of the other organs studied do not have FPR; (3) the FPR of the epididymis does not recognize enzymes purified from other organs of the reproductive tract. These results suggest that the FPR-binding system belongs to a peculiar transport route that permits maturing spermatozoa to acquire hydrolytic enzymes secreted by the epididymal epithelium. In the epididymis and seminal vesicles more than 50% of the enzymatic activity of beta-galactosidase was recovered in cytosol, suggesting that the enzyme is located mainly in the secretory fluid of these organs.

Animals↗
PubMed1994

Rab5, an early acting endosomal GTPase, supports in vitro endosome fusion without GTP hydrolysis.

Endocytosis is regulated by several GTPases including Rab5 and one or more heterotrimeric G proteins. We show here that Rab5, in the GTP gamma S (guanosine 5'-O-(thiotriphosphate))-bound form, fully supports in vitro endosome fusion, indicating that GTP hydrolysis is not required, whereas Rab5:S34N and Rab5:N133I, mutants unable to bind GTP, are potent inhibitors of endosome fusion. Double mutants (Rab5:S34N/delta C4 and Rab5:N133I/delta C4) lacking the C-terminal prenylation site were inactive, indicating that prenylation is required. Endosomes became resistant to the inhibitory effects of Rab5:S34N by preincubating the vesicles with cytosol prior to the addition of the inhibitor. The acquisition of resistance to Rab5:S34N was more rapid than to N-ethylmaleimide, indicating that Rab5 mutants are early acting. G beta gamma subunits of heterotrimeric G proteins block endosome fusion. However the effect of G beta gamma was abrogated by Rab5-GTP gamma S, indicating that a heterotrimeric G protein may operate upstream of Rab5.

Endocytosis↗
PubMed1994

Structural features of the GTP-binding defective Rab5 mutants required for their inhibitory activity on endocytosis.

Rab5 is a Ras-like small GTPase that regulates early events of endocytosis. Previous work indicates that two GTP-binding defective Rab5 mutants (Rab5:S34N and Rab5:N133I) are dominant inhibitors of endocytosis. In this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two Rab5 mutants. Second-site mutations were introduced into Rab5:S34N and Rab5:N133I, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector. Endocytic activity of the cells was monitored by following the uptake of a fluid-phase endocytic marker (horseradish peroxidase). The effects of the Rab5 mutants on endosome fusion in vitro were also examined. Truncation of the C-terminal isoprenylation motif CCSN abolished the inhibitory activity of both Rab5:S34N and Rab5:N133I. The same held true when the secondary mutation was a substitution mutation (F57S) in the effector domain. Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:N133I. The final mutation (R81A) was created immediately downstream of the second GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on the structural model of Ras. This mutation greatly decreased the isoprenylation of Rab5:N133I and its inhibitory activity on endocytosis. It is believed that Rab5 function requires protein-protein interactions with Rab5-specific regulators and effectors. Some of these interactions are disrupted by Rab5:S34N and Rab5:N133I. By analogy to Ras, both Rab5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor. This interaction requires the effector domain Phe57 residue and C-terminal isoprenylation of Rab5.

Amino Acid Sequence↗
PubMed1994

Affinity sites for N-acetyl-beta-D-glucosaminidase on the surface of rat epididymal spermatozoa.

The binding of N-acetyl-beta-D-glucosaminidase from rat epididymal fluid to the surface of spermatozoa from the cauda epididymis was measured in the presence of sugars, its phosphorylated derivatives, or after treatment of the cells or the enzyme with agents that alter the integrity of proteins or carbohydrates. The binding was saturable, with a Kd in the nanomolar range, was inhibited with phosphorylated derivates of fructose, and did not depend on Ca2+, showing that it is different from the mannose 6-P-recognizing system existing in other tissues for this and other acid hydrolases. Treatment of the cells with sodium periodate or trypsin inhibited the binding, showing that a glycoprotein of the plasmalemma is involved in the affinity site. Fructose or phosphorylated derivates were not detected in the proteins of the epididymal fluid with HPLC. However, with the method used, the presence of these compounds cannot be ruled out, if among the proteins of the fluid there are only a small number of acid hydrolases containing this sugar.

Acetylglucosaminidase↗
PubMed1993

Accuracy of clinical diagnoses in a teaching hospital: a review of 997 autopsies.

OBJECTIVES: To analyse the concordance between clinical and autopsy diagnoses. DESIGN: Nine-hundred-and-ninety-seven autopsies were studied comparing the diagnoses of the autopsy requests with those of the death certificates and autopsy reports. The cases were grouped according to the 17 categories of diseases of the International Classification of Diseases (ICD) and the concordance was analysed with the kappa (kappa) coefficient of concordance. SETTING: The Hospital da Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRPUSP), Ribeirão Preto, SP, Brazil. SUBJECTS: The patients autopsied at HCFMRPUSP during the period between 1978 and 1980. MAIN OUTCOME MEASURES: kappa statistics offer an alternative approach to measuring the concordance between clinical and autopsy diagnoses. RESULTS: The kappa-value obtained was equal to 0.601 with a variance of 1.545 x 10(-4) when comparing the clinical diagnoses and the autopsy diagnoses, and equal to 0.661 with a variance of 1.531 x 10(-4) comparing the clinical diagnoses with those obtained after the gross examination. These values are significant at the level of 5%, i.e. there is an overall statistical concordance between clinical and autopsy diagnoses although the value is not absolute (kappa = 1.00). CONCLUSIONS: If autopsies are heeded without bias, they will continue to give important feedback concerning medical diagnosis.

Autopsy↗
PubMed1992

Trypanosoma cruzi: inhibition of metacyclogenesis by mannose.

Metacyclogenesis of Trypanosoma cruzi epimastigotes was evaluated in a medium supplemented with Triatoma infestans intestinal homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N-acetylglucosamine, mannose 6-P, and fructose 1,6-P at a concentration of 25 mM. Only mannose significantly inhibited metacyclogenesis. Sodium metaperiodate and trypsin treatment of the intestinal homogenate also inhibited differentiation. In our opinion there exists a proteinic factor in the intestine of the vector that promotes metacyclogenesis and is incorporated by the parasite. Treatment of the intestinal homogenate with alkaline phosphatase had no effect. Instead, high ionic strength in the medium (0.4 M NaCl) strongly inhibited metacyclogenesis indicating that, in these conditions, the possible binding of the differentiation factor to the parasite surface was inhibited.

Animals↗
PubMed1992

[Correlation between maternal age, social class and smoking, and birth weight].

An epidemiological survey was carried out in Ribeirão Preto, Brazil, from June 1978 to May 1979. Interviews were held with mother of singleton live borne children, delivered in hospitals, which accounted for 98% of all births in the area. The higher percentages of low birthweight children related to the offspring of smokers, young mother and women belonging to the working class. The majority of young women were found in the working class and the prevalence of smoking was higher in the group of women below 20 years of age. There was no statistical difference in the smoking habit as between different social classes. A larger number of low birthweight children were observed in nonsmoking women of the working class than among women smokers of the middle class. A log model was adjusted to the data in order to study the possible multiple association of smoking, maternal age and social class with birthweight. The results indicated that maternal smoking, maternal age and social class had independent effects on birthweight. The was no interaction between them. These findings suggest that the higher prevalence of low birthweight in nonsmoking mothers of the working class in relation to smoking mothers of the middle class probably reflects clustering of other risk factors-such as poor education inadequate prenatal care, high parity and differences in reproductive behavior in women of the working class.

Adult↗
PubMed1991

Binding of beta-galactosidase from rat epididymal fluid to the sperm surface by high-affinity sites different from phosphomannosyl receptors.

beta-Galactosidase, known to be secreted by epithelial cells lining the rat epididymal duct, binds to the surface of spermatozoa from the caudal region with high affinity and in a saturable form. The binding was not inhibited by mannose-6-phosphate, but was inhibited by fructose phosphate derivatives, a peculiarity previously demonstrated for the membranes of epididymal tissue. Fructose phosphate derivatives released 55% of beta-galactosidase activity from the spermatozoa. These results suggest that in the epididymis there is a special transport system for hydrolases, which could be involved in the secretion of enzymes destined for spermatozoa. This transport would require receptors that recognize sugar ligands other than mannose-6-phosphate. These receptors were present in the epididymal tissue and on the sperm surface.

Animals↗