Maricilda Palandi de Mello

Differential gene expression in response to copper in Acidithiobacillus ferrooxidans analyzed by RNA arbitrarily primed polymerase chain reaction

Acidithiobacillus ferrooxidans is a chemoautotrophic bacterium that plays an important role in metal bioleaching processes. Despite the high level of tolerance to heavy metals shown by A. ferrooxidans, the genetic basis of copper resistance in this species remains unknown. We investigated the gene expression in response to copper in A. ferrooxidans LR using RNA arbitrarily primed polymerase chain reaction (RAP‐PCR). One hundred and four differentially expressed genes were identified using eight arbitrary primers. Differential gene expression was confirmed by DNA slot blot hybridization, and approximately 70% of the RAP‐PCR products were positive. The RAP‐PCR products that presented the highest levels of induction or repression were cloned, sequenced and the sequences were compared with those in databases using the BLAST search algorithm. Seventeen sequences were obtained. The RAP‐PCR product with the highest induction ratio showed similarity with the A. ferrooxidans cytochrome c. A high similarity with the thiamin biosynthesis gene thiC from Caulobacter crescentus was observed for another RAP‐PCR product induced by copper. An RAP‐PCR product repressed by copper showed significant similarity with the carboxysome operon that includes the ribulose‐1,5‐bisphosphate carboxylase/oxygenase complex from A. ferrooxidans and another copper‐repressed product was significantly similar to the XyIN outer membrane protein from Pseudomonas putida. Finally, RAP‐PCR products of unknown similarities were also present.

Classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency: a cross-sectional study of factors involved in bone mineral density.

Glucocorticoids are essential in the treatment of patients with congenital adrenal hyperplasia (CAH). The opposite actions of glucocorticoids and androgens in bone mass achievement justify a study of bone mineral density (BMD) in these patients. We evaluated BMD in patients with CAH due to classic 21-hydroxylase (CYP21A2) deficiency and investigated the involvement of clinical and laboratory factors in the BMD. This study assessed the clinical and laboratory factors involved in BMD of 45 patients at the Pediatric Unit of Endocrinology, UNICAMP, who had been diagnosed as having classical CAH due to CYP21A2 deficiency including molecular characterization. The sample consisted of 28 females and 17 males; 23 salt-wasting (SW) and 22 simple virilizing (SV) cases, with average of 9.9 years (ranges, 5.1–16.3 years) when bone densitometry was performed. The DEXA method was used for calculating the areal BMD Z score in L2–L4. The variables were analyzed with reference to the BMD for chronological age (BMD/CA), height age (BMD/HA), and bone age (BMD/BA). The mean Z score for BMD/CA was 0.08 ± 1.21 (−2.55 to 2.64); it was 0.29 ± 1.33 (−2.01 to 4.00) for BMD/HA, and −0.90 ± 1.24 (−3.41 to 1.92) for BMD/BA. The BMD/CA was significantly lower in females and in patients on treatment for a long period and of more advanced chronological age. Weight and body mass index (BMI) Z scores showed a positive correlation with advanced BA. The higher the weight and BMI Z scores, the higher the BMD/HA. The BMD/BA values were significantly higher in the group in which BA was closer to CA. The BMD/BA value was significantly lower when compared to the value obtained with height and chronological ages. Sex, duration of treatment, weight, BMI, and bone age have an effect on areal BMD in patients with CAH due to CYP21A2 deficiency, which may be underestimated when evaluated in relation to CA and HA.

Iron-regulated proteins in Phanerochaete chrysosporium and Lentinula edodes: differential analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis profiles.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and two‐dimensional gel electrophoresis (2‐DE) were used to identify iron‐responsive proteins in the white‐rot species (Phanerochaete chrysosporium and Lentinula edodes), by comparing the differential patterns of cellular and membrane proteins obtained from iron‐sufficient and iron‐deficient mycelia. Six cellular proteins induced by iron restriction have been observed in SDS‐PAGE for P. chrysosporium and twelve for L. edodes. In 2‐DE, the numbers of iron‐restricted induced proteins were 12 and 9, respectively, in a resolution range of 15–60 kDa and pI 4.5–8.1. SDS‐PAGE for the plasma membrane protein did not show differences, whereas the outer‐membrane protein profile showed 6 and 5 proteins induced by iron depletion in P. chrysosporium and L. edodes, respectively. The results presented here are important data to unravel mechanisms of biosynthesis and/or transport of the iron‐complexing agents in ligninolytic fungi and to further correlate them to the ligninolytic processes.

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

Abstract Background.—Dysgenetic male pseudohermaphroditism (DMP) is a sexual differentiation disorder characterized by bilateral dysgenetic testes, persistent mullerian 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 antimullerian hormone, a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors, and the presence of mullerian 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 micro...

Complete gonadal dysgenesis in clinical practice: the 46,XY karyotype accounts for more than one third of cases

OBJECTIVE To determine the frequency of XY karyotypes among females with complete gonadal dysgenesis (CGD) and to investigate the frequency of both gonadal tumors and SRY mutations. DESIGN Retrospective study based on data from all patients with CGD seen in our service from 1989 to 2010. SETTING Clinic for disorders of sex development, University Hospital, State University of Campinas. PATIENT(S) Thirty-two patients with hypergonadotropic hypogonadism, streak gonads, internal and external female genitalia, and normal karyotype (46,XX or 46,XY); 31 were index cases and 29 did not have a previously determined karyotype. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) None. RESULT(S) The percentage of XY karyotypes among patients with CGD was 34.5% (10/29). Mean age at diagnosis among XY and XX patients was 17.4 years and 19.9 years, respectively. Gonadal tumors were found in 4 of 9 XY girls, and 7 of 10 had SRY gene mutations. CONCLUSION(S) The previously unreported finding of an elevated frequency of 46,XY karyotype among patients with CGD and the high risk of gonadal neoplasia in such cases indicate that this diagnosis must be kept in mind by clinicians and strengthen the importance of karyotype analysis in females with primary hypogonadism. In addition, the frequency of SRY mutations in XY CGD might be higher than previously considered.

Molecular characterization of Acidithiobacillus ferrooxidans and A. thiooxidans strains isolated from mine wastes in Brazil.

Nineteen strains of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, including 12 strains isolated from coal, copper, gold and uranium mines in Brazil, strains isolated from similar sources in other countries and the type strains of the two species were characterized together with the type strain of A. caldus by using a combination of molecular systematic methods, namely ribotyping, BOX- and ERIC-PCR and DNA-DNA hybridization assays. Data derived from the molecular fingerprinting analyses showed that the tested strains encompassed a high degree of genetic variability. Two of the Brazilian A. ferrooxidans organisms (strains SSP and PCE) isolated from acid coal mine waste and uranium mine effluent, respectively, and A. thiooxidans strain DAMS, isolated from uranium mine effluent, were the most genetically divergent organisms. The DNA-DNA hybridization data did not support the allocation of Acidithiobacillus strain SSP to the A. ferrooxidans genomic species, as it shared only just over 40% DNA relatedness with the type strain of the species. Acidithiobacillus strain SSP was not clearly related to A. ferrooxidans in the 16S rDNA tree.

Estudo multicêntrico de pacientes brasileiros com deficiência da 21-hidroxilase: correlação do genótipo com o fenótipo

ABSTRACT Multicentric Study of Brazilian Patients With 21-Hydroxylase Defi-ciency: A Genotype-Phenotype Correlation. We analyzed the clinical and molecular data of 205 patients with thethree different clinical forms of 21-hydroxylase deficiency, in whom theclinical and molecular diagnosis were already defined. The most fre-quent mutations were I2 splice in the salt wasting form, I172N in the sim-ple virilizing and V281L in the nonclassical form, presenting similar fre-quencies as those observed in other populations. We found a lower fre-quency of 21-hydroxylase gene deletion, similar to that previously iden-tified in Argentinean and Mexican populations. Five new mutations weredescribed in our population: G424S, H28+C, Ins 1003^1004 A, R408C andIVS2-2A>G. The genotype was classified in three groups according to theimpairment of enzymatic activity observed in vitro , Group A: 0-2%, GroupB: 3-7% and Group C: >20%. Group A mutations correlated with the saltwasting form, the Group B with simple virilizing form and Group C withthe non classical form. The severity of genotype showed a positive cor-

Association between the MTHFR A1298C polymorphism and increased risk of acute myeloid leukemia in Brazilian children

Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in the metabolism of folate. The presence of polymorphisms that reduce the activity of MTHFR has been linked to the multifactor process of development of acute leukemia. A case control study was conducted on Brazilian children in different regions of the country with the aim of investigating the role of MTHFR C677T and A1298C polymorphisms as risk factors in the development of acute myeloid leukemia (AML). We used the polymerase chain reaction restriction fragment length polymorphism method to genotyping 182 AML and 315 healthy individuals. The genotype 677 CT was associated with decreased risk [odds ratio (OR), 0.37; confidence interval (CI) 95%, 0.14 – 0.92], whereas 1298 AC genotype was linked with an increased risk [OR, 2.90; CI 95%, 1.26 – 6.71] of developing AML in non-white children. Further epidemiological study is needed to unravel the complex multiple gene-environment interactions in the role of the AML leukemogenesis.

The novel p.Cys65Tyr mutation in NR5A1 gene in three 46,XY siblings with normal testosterone levels and their mother with primary ovarian insufficiency

BackgroundDisorders of sex development (DSD) is the term used for congenital conditions in which development of chromosomal, gonadal, or phenotypic sex is atypical. Nuclear receptor subfamily 5, group A, member 1 gene (NR5A1) encodes steroidogenic factor 1 (SF1), a transcription factor that is involved in gonadal development and regulates adrenal steroidogenesis. Mutations in the NR5A1 gene may lead to different 46,XX or 46,XY DSD phenotypes with or without adrenal failure. We report a Brazilian family with a novel NR5A1 mutation causing ambiguous genitalia in 46,XY affected individuals without Müllerian derivatives and apparently normal Leydig function after birth and at puberty, respectively. Their mother, who is also heterozygous for the mutation, presents evidence of primary ovarian insufficiency.Case presentationThree siblings with 46,XY DSD, ambiguous genitalia and normal testosterone production were included in the study. Molecular analyses for AR, SRD5A2 genes did not reveal any mutation. However, NR5A2 sequence analysis indicated that all three siblings were heterozygous for the p.Cys65Tyr mutation which was inherited from their mother. In silico analysis was carried out to elucidate the role of the amino acid change on the protein function. After the mutation was identified, all sibs and the mother had been reevaluated. Basal hormone concentrations were normal except that ACTH levels were slightly elevated. After 1 mcg ACTH stimulation test, only the older sib showed subnormal cortisol response.ConclusionThe p.Cys65Tyr mutation located within the second zinc finger of DNA binding domain was considered deleterious upon analysis with predictive algorithms. The identification of heterozygous individuals with this novel mutation may bring additional knowledge on structural modifications that may influence NR5A1 DNA-binding ability, and may also contribute to genotype-phenotype correlations in DSD. The slightly elevated ACTH basal levels in all three patients with 46,XY DSD and the subnormal cortisol response after 1 mcg ACTH stimulation in the older sib indicate that a long-term follow-up for adrenal function is important for these patients. Our data reinforce that NR5A1 analysis must also be performed in 46,XY DSD patients with normal testosterone levels without AR mutations.

Clinical and Laboratorial Features That May Differentiate 46,XY DSD due to Partial Androgen Insensitivity and 5α-Reductase Type 2 Deficiency

The aim of this study was to search for clinical and laboratorial data in 46,XY patients with ambiguous genitalia (AG) and normal testosterone (T) synthesis that could help to distinguish partial androgen insensitivity syndrome (PAIS) from 5α-reductase type 2 deficiency (5α-RD2) and from cases without molecular defects in the AR and SRD5A2 genes. Fifty-eight patients (51 families) were included. Age at first evaluation, weight and height at birth, consanguinity, familial recurrence, severity of AG, penile length, LH, FSH, T, dihydrotestosterone (DHT), Δ4-androstenedione (Δ4), and T/DHT and T/Δ4 ratios were evaluated. The AR and SRD5A2 genes were sequenced in all cases. There were 9 cases (7 families) of 5α-RD2, 10 cases (5 families) of PAIS, and 39 patients had normal molecular analysis of SRD5A2 and AR genes. Age at first evaluation, birth weight and height, and T/DHT ratio were lower in the undetermined group, while penile length was higher in this group. Consanguinity was more frequent and severity of AG was higher in 5α-RD2 patients. Familial recurrence was more frequent in PAIS patients. Birth weight and height, consanguinity, familial recurrence, severity of AG, penile length, and T/DHT ratio may help the investigation of 46,XY patients with AG and normal T synthesis.