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Biomedical subjects

G Guerra

Publications and source records attributed to G Guerra.

At least 19 recordsLinked to original sources
PubMed2002

Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46,XY pure gonadal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes.

The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians. In humans mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases. We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites. One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis. The Escherichia coli expressed SRY(N65H) protein did not present DNA-binding activity in vitro. The other mutation, a G to T transversion, causes the R30I substitution. This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings. The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR. The R30I mutation is located upstream to the HMG box, within the (29)RRSSS(33) phosphorylation site. The E. coli expressed SRY(R30I) protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro.

Blotting, Western↗
PubMed2001

Family-based association of HLA class II alleles and haplotypes with type I diabetes in Brazilians reveals some characteristics of a highly diversified population.

The association of HLA class II haplotypes with type I diabetes was analyzed in 56 Southeastern Brazilian families using affected family-based controls (AFBAC) method. DRB1-DQA1-DQB1 alleles were determined by polymerase chain reaction/sequence-specific primer genotyping. This study first revealed the great haplotype diversity of Brazilians (65 different haplotypes even with incomplete DRB1 subtyping), probably due to the admixture of Africans genes with European and Amerindian genes in this population. The results revealed increased frequencies of the DRB1*03-DQA1*0501-DQB1*02 and DRB1*0401-DQA1*03-DQB1*0302 haplotypes in the patient group The highest risk for type I diabetes was associated with the heterozygote DRB1*03/*04 genotype as largely reported, and DRB1*03/X and DRB1*04/Y genotypes conferred a significant, but much lower disease risk. Protection from type I diabetes revealed some peculiarities in Southeastern Brazilians: a lack of significant protecting effect of the DRB1*1501-DQA1*0102-DQB1*0602 haplotype, and an apparent protection conferred by the DRB1*13-DQB1*0301, DRB1*11-DQB1*0301, and DRB1*01-DQB1*0501 two-locus haplotypes. The risk to type I diabetes in the highly diversified Southeastern Brazilians evidenced specific information to the prediction of the disease in this region of the country.

Adolescent↗
PubMed2001

H28+C insertion in the CYP21 gene: a novel frameshift mutation in a Brazilian patient with the classical form of 21-hydroxylase deficiency.

In the classical form of 21-hydroxylase deficiency, CYP21- affected genes either carry mutations present in the CYP21P pseudogene (microconversions) or bear a chimeric gene that replaces the active gene as a result of large conversion or deletion mutational events. Previous genotyping of 41 Brazilian patients revealed 64% microconversion, whereas deletions and large gene conversions accounted for up to 21% of the molecular defect. The present paper describes a new mutation disclosed by sequencing an entire gene in which no pseudogene-originated mutation had been found. The patient with the classical form of 21-hydroxylase deficiency is the daughter of a consanguineous marriage, and she is homozygous for a novel frameshift H28+C within exon 1. The mutation causes a stop codon at amino acid 78. Both parents are heterozygous for the mutation as confirmed by allele-specific oligonucleotide PCR. The H28+C is not present in the published CYP21P sequences and is likely to result in an enzyme with no activity.

Adrenal Hyperplasia, Congenital↗
PubMed2001

Morphometry and histology of gonads from 13 children with dysgenetic male pseudohermaphroditism.

BACKGROUND: Dysgenetic male pseudohermaphroditism (DMP) is a sexual differentiation disorder characterized by bilateral dysgenetic testes, persistent müllerian structures, and cryptorchidism in individuals with a 46,XY karyotype. However, the histologic criteria for the diagnosis of DMP are poorly established. OBJECTIVE: To determine gonadal histology in children with DMP. PATIENTS AND METHODS: Between 1996 and 1998, 13 patients with DMP were evaluated on our service. The clinical diagnosis of DMP was based on a 46,XY karyotype, sex ambiguity, high levels of follicle-stimulating hormone and low levels of antimüllerian hormone, a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors, and the presence of müllerian structures. Molecular sequencing the HMGbox region of the SRY gene did not reveal any mutations. Biopsies were performed for 22 of 26 gonads (patient age at the time of biopsy, 16 months to 10 years). Conventional microscopy was used to evaluate mean tubular diameter, tubular fertility index, and number of Sertoli cells per tubular profile. RESULTS: All 26 gonads were located outside of the labioscrotal folds. Their histologic features varied from only a reduction in tubular size to features of a streak gonad. Five of the 22 gonads grossly resembled a streak gonad. The mean tubular diameter was severely reduced (>30% reduction relative to the normal tubular diameter for the patient's age) in 4 gonads, markedly reduced (10%-30%) in 11 gonads, slightly reduced (<10%) in one gonad, and normal in one gonad. The tubular fertililty index, expressed as the percentage of tubular profiles containing germ cells, was severely reduced (<30% of normal values) in 9 gonads, markedly reduced (50%-30%) in 2 gonads, and normal in 6 gonads. The number of Sertoli cells per tubular profile was elevated in 16 gonads and normal in one gonad. Thin tubules surrounded by fibrous tissue were occasionally observed. CONCLUSION: The histologic findings confirmed the clinical diagnosis of DMP in every patient in the present series. However, gonadal histology was variable, and careful morphometric evaluation may be necessary to establish the diagnosis.

Child↗
PubMed2000

Gonadotropin-independent precocious puberty due to luteinizing hormone receptor mutations in Brazilian boys: a novel constitutively activating mutation in the first transmembrane helix.

Naturally occurring activating mutations in the human LH receptor (hLHR) gene are the cause of sporadic or familial male gonadotropin-independent precocious puberty. We have previously reported three different activating mutations of the hLHR gene in four unrelated Brazilian boys with male-limited precocious puberty. In the current study, we examined three other Brazilian boys, two brothers and one unrelated boy, with gonadotropin-independent precocious puberty. Direct sequencing of the entire exon 11 of the hLHR gene in the two brothers revealed a heterozygous substitution of T for C at nucleotide 1103, resulting in the substitution of leucine at position 368 by proline in the first transmembrane helix. Their mother carried the same mutation, establishing the familial nature of this mutation. Human embryonic 293 cells expressing hLHR(L368P) bound hCG with the same high affinity as cells expressing the wild-type hLHR. Cells expressing the novel L368P mutation displayed up to a 12-fold increase in basal cAMP production compared with cells expressing the same number of cell surface wild-type hLHR, indicating constitutive activation of the mutant receptor. In addition, the cAMP levels in cells expressing the hLHR mutant were further augmented by hCG. Molecular dynamics simulations suggest that substitution of L368 of the hLHR by proline results in lack of a salt bridge interaction between D405 and R464 (distance 9. 0 A vs. 4.7 A in wild-type hLHR) as well as by the opening of a crevice between the second and third intracellular loops, which may allow G proteins greater accessibility. These structural features were shared by other activating mutants of the hLHR. Sequencing of exon 11 of the hLHR gene of the unrelated boy revealed that he carried a homozygous nucleotide substitution causing an A568V mutation in the third cytoplasmic loop of the receptor. This mutation was previously found in two unrelated Brazilian boys, but in heterozygous state. Clinical and hormonal data of the patient with the homozygous A568V were not different from those individuals with the Ala568Val mutation in a heterozygous state. Furthermore, the phenotype caused by dominant activating mutations of the hLHR gene are not altered when both alleles carry a mutant sequence. Our studies show that the A568V is the most frequent cause of male-limited precocious puberty in Brazilian boys. Lastly, the identification of a novel activating L368P mutation in the first transmembrane helix of two Brazilian boys with familial male-limited precocious puberty provides further insights into the mechanism of activation of the hLHR.

Amino Acid Substitution↗
PubMed2000

Turner's syndrome and thyroid disease: a transverse study of pediatric patients in Brazil.

An increased prevalence of autoimmune thyroid disease (AITD) has been described in Turner's syndrome (TS), but the extent of this association is still controversial. Some studies also suggest that AITD is more frequent among patients with X-isochromosome. In order to determine the prevalence of AITD among girls with TS, and to look for an association with age and karyotype, we evaluated 71 patients with a mean age of 11.4 years (range 0-19.9). 15.5% (11/71) were hypothyroid, 17 (23.9%) were positive for thyroid peroxidase (TPO) and/or thyroglobulin (Tg) antibodies, and 24 (33.8%) had thyromegaly. No abnormality was observed before 4 years, and the highest frequencies were observed after 16 years. There were no significant differences concerning thyroid findings among patients with a 45,X karyotype, mosaics, and structural rearrangements. Half of the patients (35/71) exhibited one or more abnormalities, which demonstrates the importance of careful evaluation of thyroid function in all girls with TS.

Adolescent↗
PubMed1999

An alternative model for the transmembrane segments of the yeast H+-ATPase.

An alternative topological model for the yeast plasma membrane H(+)-ATPase from K. lactis was deduced by joint prediction, using 11 algorithms for the prediction of transmembrane segments complemented with hydrophobic moment analysis. Similarly to the model currently used in the literature, this alternative model contains 10 transmembrane segments, four in the N-half and six in the C-half of the protein. However, the distribution of the membrane-associated segments on the C-half of the enzyme differs in both models. Nine of the 10 transmembrane segments are highly hydrophobic with low hydrophobic moments, and are probably involved in structural roles. The fifth transmembrane segment is, on the other hand, less hydrophobic, with the highest hydrophobic moment, suggesting that this segment might have a dynamic role in the coupling of the hydrolysis of ATP with the translocation of protons across the membrane. The alignment of the Ca(2+)-ATPase, the Na(+)/K(+)-ATPase and the H(+)-ATPase sequences showed that these proteins have the same topology in the N-half, but important differences were found at the C-half of the enzymes. In contrast with the mammalian ATPases, the fifth transmembrane segment in the H(+)-ATPase appears early in the sequence, giving rise to a shorter cytoplasmic central loop. This alternative model will be useful in the designing of site-directed mutagenesis experiments and contains information for the fitting of the amino acid sequence into the transmembrane region of the three-dimensional model of the ATPase.

Algorithms↗
PubMed1999

Mutation distribution and CYP21/C4 locus variability in Brazilian families with the classical form of the 21-hydroxylase deficiency.

Deficiency of adrenal steroid 21-hydroxylase is the most common form of congenital adrenal hyperplasia and it is considered to be responsible for 90% of the disease. This paper describes for the first time the CYP21B mutation profile in Brazilian patients. We genotyped 41 families with at least one individual affected with the classical form of the 21-hydroxylase deficiency, representing 74 unrelated alleles. In order to characterize different disease-causing alleles, genotyping was performed by Southern blot analysis with three restriction enzymes, allele-specific oligonucleotide hybridization, and allele-specific PCR. Different alleles were distinguished by TaqI C4B RFLP, gene duplications or deletions of either CYP21A + C4B or CYP21B + C4B, large gene conversions and eight mutations that might have been introduced into CYP21B from CYP21A by microconversion events. At least one mutation was detected in 24 different disease-causing alleles, which represents about 85% of the affected alleles in those families. The frequency of the 30 kb deletion of CYP21B was lower than that described for Caucasians. The mutation Sp2 showed the highest frequency (24.65%) and was present mainly in salt-wasting patients, although it was also detected in some patients with the simple virilizing form of the disease. Conversely, I172N showed a frequency of 18.91% and was found mostly in patients affected with the simple virilizing form of the disease. Five other mutations were determined at low frequency, but CL6 was not found in any of the tested alleles.

Adrenal Hyperplasia, Congenital↗
PubMed1999

Adaptation of corticosterone-but not beta-endorphin-secretion to repeated blood sampling in rats.

Effects of short-term repeated blood sampling on the secretion of corticosterone (CORT) and beta-endorphin (beta-END) were evaluated in male Wistar rats. Blood was drawn from the tail vein of conscious rats four times within 2 h both at the peak and trough period of the diurnal corticosterone secretion cycle. All rats were well accustomed to the procedure. The main findings were: (1) At both sampling intervals, CORT increased significantly in response to the first sampling and declined to baseline values in successive samples. (2) beta-END also increased significantly in response to the first sampling but remained elevated in successive samples. (3) Intensities of initial CORT and beta-END responses correlated positively with each other and with the baseline beta-END values. Feedback inhibition of CORT secretion with sustained elevation of beta-END titres suggests a moderate stress intensity of the repeated blood sampling procedures. In general, due to lack of short-term feedback inhibition, beta-END seems to reflect the effects of repeated administration of moderate intense stressors more closely than CORT.

Animal Welfare↗
PubMed1998

Trehalose-mediated protection of the plasma membrane H+-ATPase from Kluyveromyces lactis during freeze-drying and rehydration.

During freeze-drying and rehydration, the activity of the H+-ATPase from the plasma membrane of Kluyveromyces lactis was preserved by increasing concentrations of carbohydrates. When the H+-ATPase was freeze-dried in the absence of carbohydrates the activity was lost. The protective efficiency of carbohydrates was as follows: trehalose > maltose > sucrose > glucose > galactose. Each carbohydrate exhibited the maximal protection at a concentration of 20 mg carbohydrate per milligram of protein or above. No structural changes of the rehydrated H+-ATPase were detected by intrinsic fluorescence measurements. Trehalose, at 20 mg/mg protein, protected the enzyme activity completely during freeze-drying and rehydration. Rehydration temperature was critical; at 20 degrees C or below, activity was fully retained, while at 30, 40, or 50 degrees C activity decreased in proportion with temperature. The trehalose-protected freeze-dried H+-ATPase was stored at different temperatures for up to 60 days. Storage at 4 degrees C resulted in retention of most of the enzymatic activity, while storage at 20 or 30 degrees C resulted in loss of activity. The protection of the H+-ATPase by trehalose suggests that this carbohydrate might protect other membrane enzymes from inactivation during handling.

Cell Membrane↗
PubMed1997

Bile salt-induced cytotoxicity and ursodeoxycholate cytoprotection: in-vitro study in perifused rat hepatocytes.

OBJECTIVES: Membrane toxicity induced by hydrophobic bile salts may be important in liver diseases. Administration of ursodeoxycholate reduces serum liver enzymes in chronic liver diseases, but the nature of this effect is still unclear. We aimed at establishing a convenient in-vitro system for investigating the hepatotoxic properties of hydrophobic bile salts and the putative hepatoprotective effect of ursodeoxycholate. METHODS: About 100 mg of freshly isolated rat hepatocytes were suspended on a resin column (Bio-Gel P4 fine) and perifused with different concentrations of bile salts. The effluent was collected at 5-min intervals and assayed for lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. Enzyme leakage induced by bile salts was compared with that induced by Triton X-100 (Union Carbide, Danbury, CT, USA) at different concentrations. After perifusion, hepatocytes were collected for electron microscopic observation. RESULTS: Cytotoxicity of individual bile salts, assessed by enzyme release, was time and concentration dependent and corresponded to their hydrophilic-hydrophobic balance. Perifusion with hydrophilic bile salts, cholate and ursodeoxycholate, did not result in a significant enzyme release in concentrations up to 5 mmol/l, whereas hydrophobic bile salts, chenodeoxycholate and deoxycholate, induced significant enzyme leakage even in low concentrations, 0.5 and 0.1 mmol/l, respectively. Addition of ursodeoxycholate significantly reduced the hepatotoxic effect of deoxycholate. This protective effect was evident within minutes. The ultrastructural appearance of hepatocytes exposed to hydrophobic bile salts was very similar to the non-specific cellular lysis observed after exposition to Triton X-100, suggesting that they act mainly in a detergent-like fashion. CONCLUSION: Perifused rat hepatocytes seem a convenient in-vitro system for investigating the hepatotoxic properties of bile salts and hepatoprotective effect of ursodeoxycholate, offering the opportunity to investigate the effects of bile salts under dynamic conditions, mimicking the in-vivo situation, and allowing continuous enzyme release monitoring. Hydrophobic bile salts seem to act mainly in a detergent-like fashion; ursodeoxycholate-related hepatoprotection could be due not only to a dilution effect of toxic bile salts, but also to a direct cytoprotective effect.

Animals↗
PubMed1996

Changes in human plasma nerve growth factor level after chronic alcohol consumption and withdrawal.

Numerous studies reported in recent years have shown that withdrawal from chronic consumption of drugs induces high levels of anxiety, both in humans and in animal models. In the present study, we demonstrated that withdrawal from chronic consumption of either ethanol or heroin causes a significant increase in plasma nerve growth factor, suggesting that the resulting anxiety condition triggers the release of this molecule. Although the functional significance of this phenomenon needs to be better defined, it is hypothesized that the increased levels of circulating nerve growth factor might be involved in homeostatic adaptive and/or reparative mechanisms.

Adult↗
PubMed1996

The determination of plasma transferrin receptor as good index of erythropoietic activity in renal anemia and after renal transplantation.

Both the plasma determinations of erythropoietic (EPO) and transferrin receptor (TfR) would provide a good characterization of anemia especially when mixed erythron disorders underlie, such as in renal failure. Immunologic assays of EPO and TfR, as well as standard hematologic determinations (hematocrit, reticulocyte count, serum iron, transferrin, ferritin) were performed in patients with chronic renal failure (CRF), in regular dialysis treatment (RDT) and in transplanted (TX) patients. In nonanemic TX patients both EPO and TfR ranged normally, whereas in anemic TX ones (Hct < 40%) both values were increased suggesting the physiologic response both of the kidney and of the erythron to decreased red cell mass. In transitory posttransplant erythrocytosis the increased values of TfR, with normal EPO values, would hypothesize a defective feedback to EPO release. Both EPO and TfR values were found increased in TX patients with adult polycystic kidney disease with persistent erythrocytosis (Hct > 50%), thus confirming previous observations. In CRF and RDT patients, all anemic, both EPO and TfR were normal, even though significantly low with respect to the degree of anemia. In RDT seriously anemic patients, the administration of recombinant human EPO induced different patterns of bone marrow response. We conclude that the determination of TfR would provide further information on renal anemia since the receptor increase mostly preceded the rise of Hct, evidencing those patients who will not have an effective bone marrow response to the therapy.

Adult↗
PubMed1996

Red blood cells expressing fetal antigens: their presence in adults with certain forms of anemia.

Using the IgG fraction of an antiserum against cord red blood cell (RBC) membranes (F-IgG), antigenic properties of RBC of newborns (n = 24) and patients suffering from anemia (n = 46) [either due to beta-thalassemia intermedia (n = 37) or hemorrhage (n = 9)] as compared to those of normal adults (n = 18) were examined with fluorescence microscopy, flow cytometry and radioimmunoassays (RIA). With fluorescence microscopy and flow cytometry 1.01 +/- 0.31 and 0.82 +/- 0.28% (mean +/- SD), respectively, of cord RBC and 0.79 +/- 0.31 and 0.53 +/- 0.28% of RBC from anemic patients reacted with F-IgG. RBC of normal adults showed virtually no F-IgG reactivity. In anemic patients there was a good correlation between the percent of F-IgG-reactive cells and the percent of reticulocytes, although the former were only two thirds of the latter; the ratio of F-IgG-reactive cells to reticulocytes was higher in posthemorrhagic anemia than in thalassemia. Moreover, double stainings revealed that the majority of F-IgG-reactive RBC were at the reticulocyte stage (80%), and coexpressed transferrin receptor (96%). Furthermore, the F-IgG-positive RBC correlated inversely with Hb levels. When RIA was employed, F-IgG binding to RBC of anemic patients and newborns was similar and considerably and significantly higher than that to RBC from healthy adults. The results demonstrate the reappearance in certain forms of anemia of F-IgG-reactive RBC, which are likely to represent a subpopulation of reticulocytes.

Adult↗
PubMed1996

Molecular analysis of CYP21 and C4 genes in Brazilian families with the classical form of steroid 21-hydroxylase deficiency.

The most common enzymatic defect of steroid synthesis is deficiency of the adrenal steroid 21-hydroxylase. Inhibition of the formation of cortisol results in an increased pituitary release of ACTH which in turn drives the adrenal cortex to overproduce androgens. This hormonal setting affects the development of genetic females by misdirecting the differentiation of external genitalia towards the male type. Since the isolation of the gene encoding 21-hydroxylase enzyme in 1984, gene deletions, large gene conversions, and microconversions have been reported to be responsible for the disease. In this paper, we report a study of this genetic defect in 22 families with one or more affected offspring diagnosed as having the classical form of congenital adrenal hyperplasia. The DNA from 30 patients was analyzed with three restriction enzymes. Hybridization with a 21-hydroxylase cDNA probe and the 5' end of a C4 genomic probe disclosed gene deletion in 7.3% (3/41) of the disease-related chromosomes. The rate of large gene conversion was 17.1% (7/41), and no abnormality in the hybridization pattern was observed in 75.6% (31/41) of the disease alleles. Densitometry of the autoradiographs was used to determine the ratio of the copy-number of the 21-hydroxylase gene (CYP21B) to the copy-number of its pseudogene (CYP21A). Differences in phenotype, the low frequency of gene deletion, and the high frequency of gene conversion compared with other studies in different populations indicated that 21-hydroxylase deficiency in the Brazilian population may involve different molecular mutations.

Adrenal Hyperplasia, Congenital↗