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Biomedical subjects

M A Barbieri

Publications and source records attributed to M A Barbieri.

At least 19 recordsLinked to original sources
PubMed2003

Lipid kinases play crucial and multiple roles in membrane trafficking and signaling.

Phosphotidylinositols (PIs) are known to play an essential role in membrane trafficking and signaling transduction. PIs serve multiple functions, such as recruitment of cytosolic proteins with PI phosphate (PIP) binding domains and modification of the physical properties of the membranes in which they reside. As substrates for phosphoinositide-specific lipases they function as a switch point in phosphoinositide metabolism. Recent work with epidermal growth factor receptor (EGFR) and colony stimulating factor-1 receptor (CSFR) has identified a possible connection between endocytosis of activated receptors and type-1 phosphatidylinositol-4-phosphate-5-kinase. Furthermore, serine/tyrosine phosphorylation of phosphatidylinositol-4-phosphate-5-kinase seems to be essential for its activities. Indeed, one of the products of the phosphatidylinositol-4-phosphate-5-kinases, PIP2, has been shown to be involved in multiple steps of endocytosis, including the assembly of the clathrin coat, regulation of adaptor proteins, and production of endocytic vesicles via the regulation of dynamin. The discussion in this review focuses primarily on receptors with intrinsic enzymatic activity, specifically on receptor tyrosine kinases (RTKs). We will discuss their structure; mechanism of action and potential role in membrane trafficking and/or signaling through the regulation of phosphatidylinositol phosphate kinases.

Animals↗
PubMed2003

Monitoring of intrathoracic volemia and cardiac output in critically ill children.

AIM: Hemodynamic monitoring is an important step in the management of critically ill children despite the difficulty in measuring preload indices continuously. The aim of the study was to analyze cardiac output parameters and preload indices after acute changes in mean airway pressure and volemia. METHODS: Twenty-three children treated at our unit were enrolled in a prospective non randomized cohort study. Respiration was supported by controlled mechanical ventilation with positive expiratory-end pressure (PEEP), peak inspiratory pressure <20 cm H(2)O and mean airway pressure <10 cm H(2)O, and hemodynamic monitoring using the PiCCO system. Hemodynamic parameters were measured at T0 (base line), T(1) (after an increase in PEEP of 5 cm H(2)O for 10 min), and T(2) (after fluid challenge). The statistical analysis (BMPD New System software package) comprised comparison of changes at T(0) vs T(1), T(1) vs T(2) and T(0) vs T(2), construction of 3 correlation matrices and multiple linear regression analysis. RESULTS: Sixty-nine hemodynamic parameters were measured in the 23 patients. A comparison between T(0) and T(1) showed no significant changes; differences between T(0) and T(2) were found for cardiac index (CI), (p=0.003); between T(0) and T(2) significant differences were found for CI (p=0.0015), intrathoracic blood volume index (ITBVI) (p=0.04) and stroke volume index (SVI) (p=0.06). The analysis of the correlation matrices yielded ITBVI with CI (p=0.0006), ITBVI with SVI (p=1 x 10(-5)), CI with SVI (p=0.002); a significant correlation between CI and extravascular lung water index (EVLWI) was found only at T(1). Multiple linear regression analysis showed that ITBVI and SVI were predictive for variance of CI at each time point. CONCLUSION: ITBVI measured by a volumetric monitoring system such as the PiCCO may be considered a sensitive preload indicator also in critically ill children.

Adolescent↗
PubMed2002

Regulation of Trypanosoma cruzi invasion of nonphagocytic cells by the endocytically active GTPases dynamin, Rab5, and Rab7.

During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.

Animals↗
PubMed2001

Phosphatidylinositol-4-phosphate 5-kinase-1beta is essential for epidermal growth factor receptor-mediated endocytosis.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is known to play an important role in signal transduction and membrane trafficking. We show that one enzyme responsible for PIP(2) production, phosphatidylinositol-4-phosphate 5-kinase type 1beta (PIPKbeta), is essential for epidermal growth factor receptor (EGFR)-mediated endocytosis. Expression of murine PIPKbeta in NR6 cells expressing EGFR strikingly increased receptor internalization. Moreover, the kinase was shown to form an immunoprecipitable complex with EGFR. Expression of either a truncated kinase or a kinase dead mutant inhibited EGFR endocytosis and also blocked the membrane recruitment of PIPKbeta and both clathrin light chain and dynamin. Our results delineate a novel mechanism by which PIPKbeta regulates receptor-mediated endocytosis and receptor tyrosine kinase membrane traffic.

Animals↗
PubMed2001

Trypanosoma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is associated with parasite invasion.

Multiple signal transduction events are triggered in the host cell during invasion by the protozoan parasite Trypanosoma cruzi. Here, we report the regulation of host cell phosphatydilinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt) activities by T. cruzi during parasite-host cell interaction. Treatment of nonphagocytic cells (Vero, L(6)E(9), and NIH 3T3) and phagocytic cells (human and J774 murine macrophages) with the selective PI3K inhibitors Wortmannin and LY294002 significantly impaired parasite invasion in a dose-dependent fashion. A strong activation of PI3K and PKB/Akt activities in Vero cells was detected when these cells were incubated with trypomastigotes or their isolated membranes. Consistently, we were unable to detect activation of PI3K or PKB/Akt activities in host cells during epimastigote (noninfective) membrane-host cell interaction. Infection of transiently transfected cells containing an inactive mutant PKB showed a significant inhibition of invasion compared with the active mutant-transfected cells. T. cruzi PI3K-like activity was also required in host cell invasion since treatment of trypomastigotes with PI3K inhibitors prior to infection reduced parasite entry. Taken together, these results indicate that PI3K and PKB/Akt activation in parasites, as in host cells induced by T. cruzi, is an early invasion signal required for successful trypomastigote internalization.

3T3 Cells↗
PubMed2001

Drosophila rab GDI mutants disrupt development but have normal Rab membrane extraction.

Rab GTPases are essential for vesicular transport. Rab GDP dissociation inhibitor (GDI) binds to GDP-bound rabs, removes rabs from acceptor membranes and delivers rabs to donor membranes. We isolated lethal GDI mutations in Drosophila and analyzed their developmental phenotypes. To learn how these mutations affect GDI structure, the crystal structure of Drosophila GDI was determined by molecular replacement to a resolution of 3.0 A. Two hypomorphic, missense mutations are located in domain II of GDI at highly conserved positions, but not in previously identified sequence conserved regions. The mutant GDIs were tested for ability to extract rabs from membranes and showed wild-type levels of rab membrane extraction. The two missense alleles showed intragenic complementation, indicating that domain II of GDI may have two separable functions. This study indicates that GDI function is essential for development of a complex, multicellular organism and that puparium formation and pole cell formation are especially dependent on GDI function.

Alleles↗
PubMed2001

Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.

Animals↗
PubMed2001

Risk factors for low birthweight in north-east Brazil: the role of caesarean section.

Caesarean section (CS) delivery is associated with low birthweight (LBW) in south-east Brazil. A hospital-based study was conducted on singleton infants from mothers residing in São Luís, to assess if an association between CS and LBW was found in the northern part of the country, where the CS rate is lower than in the south-east. A standardised questionnaire was administered to a sample of 2541 mothers in 10 hospitals, representing 94% of all deliveries, from March 1997 to February 1998. In a logistic model, type of delivery was the independent variable, the other variables were treated as confounders, and interaction terms were added between type of delivery and all other factors. LBW was associated with low maternal height, maternal smoking, primiparity, previous LBW, public insurance, preterm birth and CS. The CS rate was 33.7%. The risk of CS was higher for primiparous and married mothers, those with high level of schooling and attended by the same physician during prenatal and delivery care, deliveries held in private hospitals, daylight hours or evenings, and for those mothers who had adequate prenatal care. Because it appears unlikely that only medical reasons are operative, it is a possibility that CS could cause LBW, reflecting abusive indications for elective CS.

Adult↗
PubMed2001

Use of purified protein derivative to assess the risk of infection in children in close contact with adults with tuberculosis in a population with high Calmette-Guérin bacillus coverage.

SETTING: Household contacts <15 years of age of adults with tuberculosis (TB) attending a reference center in Aracaju, Sergipe, Northeast Brazil. OBJECTIVE: To assess the use of purified protein derivative (PPD) and frequency of infection in children with high Calmette-Guérin bacillus (BCG) coverage who were recently exposed to TB. DESIGN: Cross-sectional study of 141 exposed household contacts <15 years of age and 506 nonexposed neighborhood controls. Children were examined and assessed for degree of exposure to index cases, BCG vaccination and scar and were tested with PPD. RESULTS: Exposed children were younger and less up to date in their vaccination schedule than controls (P < 0.05). BCG had been given to 95.6% of exposed children and 97.4% of controls, but only 80.9% of exposed vs. 88.5% of controls had a scar (P < 0.05). Scar sizes of exposed children were smaller (medians, 4.5 and 7 mm, respectively; P < 0.05). Children had lower weight for age z scores and height than the National Center for Health Statistics standards. Exposed children had lower weight for age z scores than controls (P < 0.05). Sixty-seven (47.5%) exposed children and 18 (3.6%) controls had PPD readings of >10 mm. Positivity and induration sizes increased with age, although this was significant only in the controls. The presence of a BCG scar was not associated with having a positive PPD. The degree of exposure was an important factor for PPD positivity; 66 (60.6%) of the 109 children with close exposure were positive compared with 1 (3.1%) of 32 with less intimate exposure. PPD indurations among close contacts were also larger than those with restricted exposure and controls (16.3, 11 and 9.4 mm, respectively; P < 0.05). PPD positivity was associated with the quantification of acid-fast bacilli in the adult; 38.5% of contacts with adults with sputum with (+) were positive, compared with 42.3% of those with (++) and 58.4% with (+++) (P < 0.05). CONCLUSION: PPD is a useful test to identify children infected with TB independently of whether they had received BCG or not. Children exposed to adults with TB are at high risk of infection. The risk of infection is associated with the intimacy of contact and the number of bacilli expectorated in sputum.

Adolescent↗
PubMed2001

Infant mortality rates according to socioeconomic status in a Brazilian city.

OBJECTIVE: Data from municipal databases can be used to plan interventions aimed at reducing inequities in health care. The objective of the study was to determine the distribution of infant mortality according to an urban geoeconomic classification using routinely collected municipal data. METHODS: All live births (total of 42,381) and infant deaths (total of 731) that occurred between 1994 and 1998 in Ribeirão Preto, Brazil, were considered. Four different geoeconomic areas were defined according to the family head's income in each administrative urban zone. RESULTS: The trends for infant mortality rate and its different components, neonatal mortality rate and post-neonatal mortality rate, decreased in Ribeirão Preto from 1994 to 1998 (chi-square for trend, p<0.05). These rates were inversely correlated with the distribution of lower salaries in the geoeconomic areas (less than 5 minimum wages per family head), in particular the post-neonatal mortality rate (chi-square for trend, p<0.05). Finally, the poor area showed a steady increase in excess infant mortality. CONCLUSIONS: The results indicate that infant mortality rates are associated with social inequality and can be monitored using municipal databases. The findings also suggest an increase in the impact of social inequality on infant health in Ribeirão Preto, especially in the poor area. The monitoring of health inequalities using municipal databases may be an increasingly more useful tool given the continuous decentralization of health management at the municipal level in Brazil.

Brazil↗
PubMed2001

Clinical experience with infants with Robin sequence: a prospective study.

OBJECTIVE: To study the clinical course of patients with Robin sequence (RS) during the first 6 months of life. DESIGN: A longitudinal prospective study of children with RS. SETTING: Hospital de Reabilitação de Anomalias Craniofaciais, Universidade de São Paulo, Bauru-SP, Brazil, 1997 and 1998. PATIENTS: Sixty-two children were studied from hospital admission to 6 months of age. Thirty-three (53.2%) presented with probable isolated RS (PIRS), 25 (40.3%) presented with syndromes or other malformations associated with RS, and 4 (6.5%) presented with RS with neurological involvement. INTERVENTIONS: The type of respiratory tract obstruction was defined by nasopharyngoscopy. The patients with type 1 and type 2 obstruction underwent nasopharyngeal intubation (NPI), and glossopexy was indicated in patients with type 1 obstruction who did not show clinical improvement with this procedure. Tracheostomy was indicated in patients with type 2 obstruction who did not show a good course after NPI, in patients with type 1 obstruction who did not show good course after glossopexy, and in patients with type 3 and type 4 obstruction. RESULTS: Prone position treatment (PPT) or NPI was the definitive treatment in 25 cases (75.8%) of PIRS and in 13 cases (52%) of syndromes or other malformations. Among the children with type 1 obstruction, 24 (51.1%) were submitted exclusively to PPT and 12 (25.5%) to NPI. With the type 2 groups, only one (12.5%) received PPT, and three (37.5%) were treated exclusively with NPI. All 15 infants treated exclusively with NPI (24.4%) presented with good weight, length, and neuromotor development. CONCLUSIONS: Most patients with PIRS and type 1 obstruction improved without surgical intervention. NPI should be the initial treatment in all patients with RS with type 1 and type 2 obstruction who present with important respiratory and feeding difficulties.

Abnormalities, Multiple↗
PubMed2000

Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a.

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH(2) and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A "kinase-dead" EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.

Animals↗
PubMed2000

A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion.

The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.

Amino Acid Sequence↗
PubMed2000

Factors associated with preterm births in southeast Brazil: a comparison of two birth cohorts born 15 years apart.

An increase in preterm deliveries in Ribeirão Preto stimulated an analysis of possible explanatory factors. Two cohorts of singleton livebirths were studied, the first based on 6746 births in 1978-9 and the second based on 2846 births in 1994. A logistic regression was carried out to assess the association of preterm birth with several sociodemographic, behavioural and clinical variables, including year of survey. Delivery in private settings compared with a public setting, maternal age of < or = 17 compared with any other age group, and mothers who had had previous abortions and previous stillbirths were associated with greater rates of preterm birth. Although there was an increase in preterm birth rates regardless of mode of delivery, the increase was greater in the caesarean section group than in the vaginal delivery group. Over the study period, deliveries in private hospitals and caesarean section operations increased markedly (from 4% to 36% and from 30% to 51% respectively). Caesarean section may be the main contributor to the increase of preterm birth rate in this study. It is essential to ensure that health-care staff, especially those in private facilities, are properly educated and audited.

Adolescent↗
PubMed2000

[Comparison between two cohorts of adolescent mothers in municipality of the Southeastern, Brazil].

OBJECTIVE: To compare the prevalence of pregnancy among adolescents and analyze trends in some social and biological variables among two cohorts of adolescent mothers in Ribeirão Preto, Brazil, during 1978/79 and 1994. METHODS: Two cohorts of adolescent mothers were interviewed shortly after delivery, comprising 943 women in the first survey (1978/79) and 499 in the second (1994). Both surveys covered more than 98% of births in all childbirth clinics. Only singleton livebirths from adolescent mothers living in the municipality were included in the analysis. The Chi-square test was used for statistical analysis, with a 0.05 significance level. RESULTS: The percentage of adolescent mothers increased from 14.1% in 1978/79 to 17.5% in 1994 (p<0.05), mainly due to an increase in deliveries in the 13-17 years age group. There was a reduction in the number of smokers and mothers with lower schooling. The number of prenatal care visits increased. There was also an increase in the proportion of single mothers, of those who were employed and got private delivery care. Low birth weight and preterm birth rates remained unchanged for all of them. Cesarean section and forceps delivery rates increased substantially. Mothers in the 13-17 years age group however showed twice as much premature and low birth weight children, a higher proportion of single mothers with lower schooling, and higher rate of forceps delivery. CONCLUSIONS: Some characteristics of the adolescent mothers in the 13-17 years age group were more unfavorable when compared to the 18-19 years age group. The characteristics of adolescent age groups need to be better understood and taken into account in the prenatal and delivery care planning.

Adolescent↗
PubMed2000

Risk factors for the increasing trend in low birth weight among live births born by vaginal delivery, Brazil.

OBJECTIVE: To identify risk factors for low birth weight (LBW) among live births by vaginal delivery and to determine if the disappearance of the association between LBW and socioeconomic factors was due to confounding by cesarean section. METHODS: Data were obtained from two population-based cohorts of singleton live births in Ribeirão Preto, Southeastern Brazil. The first one comprised 4,698 newborns from June 1978 to May 1979 and the second included 1,399 infants born from May to August 1994. The risks for LBW were tested in a logistic model, including the interaction of the year of survey and all independent variables under analysis. RESULTS: The incidence of LBW among vaginal deliveries increased from 7.8% in 1978--79 to 10% in 1994. The risk was higher for: female or preterm infants; newborns of non-cohabiting mothers; newborns whose mothers had fewer prenatal visits or few years of education; first-born infants; and those who had smoking mothers. The interaction of the year of survey with gestational age indicated that the risk of LBW among preterm infants fell from 17.75 to 8.71 in 15 years. The mean birth weight decreased more significantly among newborns from qualified families, who also had the highest increase in preterm birth and non-cohabitation. CONCLUSIONS: LBW among vaginal deliveries increased mainly due to a rise in the proportion of preterm births and non-cohabiting mothers. The association between cesarean section and LBW tended to cover up socioeconomic differences in the likelihood of LBW. When vaginal deliveries were analyzed independently, these socioeconomic differences come up again.

Adult↗
PubMed2000

Dynamics of rab5 activation in endocytosis and phagocytosis.

Fluid-phase endocytosis is stimulated by H-ras-linked growth factor receptors and this stimulation requires activation of rab5. We utilized a GFP-rab5a:wt fusion protein to monitor GFP-rab5a:wt activation in living fibroblasts and in J774 macrophages. Control CHO cells that expressed GFP-rab5a:wt were cultured in serum-free conditions and showed GFP-rab5a:wt localized to endosomal vesicles with a mean diameter of 0.3 +/- 0.1 microm. Endosome fusion, membrane ruffling, and pinosome formation were rarely detected in these cells. Coexpression of H-ras:G12V, a constitutively active H-ras mutant that activates rab5a, in cells resulted in marked enlargement of labeled endosomes (mean diameter 0.7 +/- 0.2 microm) and large numbers of giant GFP-rab5a:wt-positive endosomes were present. Time-lapse recordings showed abundant fusion among giant labeled endosomes, and membrane ruffling and pinosome formation were commonly observed. Alterations in GFP-rab5a:wt endosome structure and activity in cells expressing H-ras:G12V were linked to rab5a activation because these changes were identical to those found in cells expressing GFP-rab5a:Q79L, a constitutively activated rab5a mutant. Furthermore, cells co-expressing H-ras:G12V and GFP-rab5a:S34N, an inactive rab5a mutant, exhibited no evidence of H-ras:G12V-induced endosome enlargement. To observe changes in endosome structure and activity that directly followed activation of GFP-rab5a:wt, we performed time-lapse recordings of cells cultured overnight in serum-free media after addition of EGF. EGF caused a rapid increase in endosome fusion and in membrane ruffling activity. Membrane ruffling was often associated with GFP-rab5a:wt-positive vesicle (pinosome) formation at the base of membrane ruffles. Endosome and pinosome fusion were common in EGF-stimulated cells. Phagocytosis is also regulated by GFP-rab5a:wt. J774 macrophages that expressed GFP-rab5a:wt showed transiently activation and recruitment of GFP-rab5a:wt to newly formed phagosomes that contained rhodamine-labeled Escherichia coli. These studies show that GFP-rab5a:wt activation results in dynamic alterations in the structure and activity of the early endosomal and early phagosomal elements.

Animals↗