Molecular biology in tropical dermatoses

Initially, basic concepts are presented concerning the cell, genetic code and protein synthesis, and some techniques of molecular biology, such as PCR, PCR-RFLP, DNA sequencing, RT-PCR and immunoblotting. Protocols of nucleotides and of proteins extraction are supplied, such as salting out in peripheral blood allied to phenol-chloroform and trizol methods in skin samples. To proceed, commented examples of application of those techniques of molecular biology for the etiologic diagnosis and for research in tropical dermatoses, with emphasis to American tegumentary leishmaniasis and leprosy are presented.

Mucoepidermoid Carcinoma of the Lung Arising at the Primary Site of A Bronchogenic Cyst: Clinical, Cytogenetic, and Molecular Findings

Primary lung tumors are rare in children, and mucoepidermoid carcinoma (MEC) represents less than 10% of them. Additionally, MEC arising from bronchogenic cysts (BC) is particularly unusual. We describe the clinical and genetic findings on a MEC occurring within a previous location of a BC in an adolescent. This particular association has not been previously reported. The lesion revealed normal karyotype without the typical t(11;19)(q21;p13) translocation. Cyclin D1 overexpression (165-fold increase) was demonstrated by real-time PCR although FISH assessment showed normal hybridization at 11q13. Information on these unusual clinical presentations may present relevant insight on tumorigenesis of infrequent pediatric pulmonary tumors. Pediatr Blood Cancer 2011;56:311-313. (C) 2010 Wiley-Liss, Inc.

Molecular Sieve Silica Membranes

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Blood and Salivary Oxidative Stress Biomarkers Following an Acute Session of Resistance Exercise in Humans

The aim of the present study was to compare oxidative stress biomarkers determined in blood and saliva before and after acute resistance exercise. 1 week after 1 maximum repetition (1RM) test 11 healthy well-trained males completed a hypertrophy acute session of resistance training including 3 sets of 10 repetitions at 75% of the 1RM, with 90s rest periods between sets. Venous blood and saliva samples were collected before (pre) and 10 min after (post) the resistance training session. A significant (p < 0.05) rise in blood lactate accumulation (pre: 1.6 +/- 0.4 vs. post: 9.5 +/- 2.4) was found post-acute resistance training compared with baseline values. Significant increases (p < 0.05) in TBARS (42%), AOPP (28%), uric acid (27%) and GSH (14%) were detected post-acute resistance training in relation to pre in blood samples. A significant increase (p < 0.05) in uric acid (36%) was found in saliva post-acute resistance training as well as a significant correlation (p < 0.05) between uric acid determined in blood and saliva. Statistical analysis did not reveal any other change in the salivary oxidative stress biomarkers. In conclusion, an acute session of resistance exercise induces oxidative stress in plasma of trained men after acute resistance training, which was not found in saliva samples except for uric acid.

The Association of Lipodystrophy and Oxidative Stress Biomarkers in HIV-Infected Men

The aim of this study was to describe the status of oxidative stress and antioxidant biomarkers and their association with metabolic and body composition components of HIV-lipodystrophy syndrome. In a cross-sectional study of blood samples from HIV-infected men with lipodystrophy syndrome (HIV+LIPO+ = 10), HIV-infected men without lipodystrophy syndrome (HIV+LIPO- = 22), and healthy subjects (control = 12), the following oxidative stress biomarkers were analyzed: total hydroperoxide, thiobarbituric acid reactive substances (TBARS), and advanced oxidation protein products (AOPP). In addition, antioxidant biomarkers, including total glutathione, uric acid, alpha-tocopherol, and metabolic components were tested. Dual-energy x-ray absorciometry (DXA) was used to measure the fat mass. The duration of HIV infection and the duration and type of highly active antiretroviral therapy were similar between the two HIV-infected groups. Higher levels of total hydroperoxide were observed in the HIV+LIPO+ (50 +/- 33 H(2)O(2)/L) group compared to the HIV+LIPO-(19 +/- 13 H(2)O(2)/L) and control (5 +/- 5 H(2)O(2)/L) groups (p < 0.05). Similarly, higher levels of AOPP were observed in the HIV+LIPO+ (326 +/- 173 mu mol/L) group compared to the HIV+LIPO- (105 +/- 92 mu mol/L) and control groups (80 +/- 20 mu mol/L) (p < 0.05). Total hydroperoxide significantly correlated with insulin serum levels in the HIV+LIPO+ (r = 0.47, p < 0.05) and HIV+LIPO- groups (r = 0.29, p < 0.05), while AOPP significantly correlated with insulin serum levels in the HIV+LIPO+ (r = 0.73, p < 0.05) and HIV+LIPO- (r = 0.54, p < 0.05) groups. Therefore, higher lipid and protein oxidation were found in HIV-infected patients with lipodystrophy syndrome, and both were associated with insulin levels.

Heterogeneity in the molecular basis of ACTH resistance syndrome

Objective: ACTH resistance syndromes are rare, autosomal, and genetically heterogeneous diseases that include familial glucocorticoid deficiency (FGD) and triple A syndrome. FGD has been shown to segregate with mutations in the gene coding for ACTH receptor (MC2R) or melanocortin 2 receptor accessory protein (MRAP), whereas mutations in the triple A syndrome (AAAS, Allgrove syndrome) gene have been found in segregation with triple A syndrome. We describe the clinical findings and molecular analysis of MC2R, MRAR and AAAS genes in five Brazilian patients with ACTH resistance syndrome. Design and methods: Genomic DNA from patients and their unaffected relatives was extracted from peripheral blood leucocytes and amplified by PCR, followed by automated sequencing. Functional analysis was carried out using Y6 cells expressing wild-type and mutant MC2R. Results: All five patients showed low cortisol and elevated plasma ACTH levels. One patient had achalasia and alacrima, besides the symptoms of adrenal insufficiency. The molecular analysis of FGD patients revealed a novel p.Gly116Val mutation in the MC2R gene in one patient and p.Met1Ile mutation in the MRAP gene in another patient. Expression of p.Glyll.6Val MC2R mutant in Y6 cells revealed that this variant failed to stimulate cAMP production. The analysis of the AAAS gene in the patient with triple A syndrome showed a novel g.782_783deITG deletion. The molecular analysis of DNA from other two patients showed no mutation in MC2R, MRAP or AAAS gene. Conclusions: In conclusion, the molecular basis of ACTH resistance syndrome is heterogeneous, segregating with genes coding for proteins involved with ACTH receptor signaling/expression or adrenal gland development and other unknown genes.

Meningiomas and schwannomas: Molecular subgroup classification found by expression arrays

Microarray gene expression profiling is a high-throughput system used to identify differentially expressed genes and regulation patterns, and to discover new tumor markers. As the molecular pathogenesis of meningiomas and schwannomas, characterized by NF2 gene alterations, remains unclear and suitable molecular targets need to be identified, we used low density cDNA microarrays to establish expression patterns of 96 cancer-related genes on 23 schwannomas, 42 meningiomas and 3 normal cerebral meninges. We also performed a mutational analysis of the NF2 gene (PCR, dHPLC, Sequencing and MLPA), a search for 22q LOH and an analysis of gene silencing by promoter hypermethylation (MS-MLPA). Results showed a high frequency of NF2 gene mutations (40%), increased 22q LOH as aggressiveness increased, frequent losses and gains by MLPA in benign meningiomas, and gene expression silencing by hypermethylation. Array analysis showed decreased expression of 7 genes in meningiomas. Unsupervised analyses identified 2 molecular subgroups for both meningiomas and schwannomas showing 38 and 20 differentially expressed genes, respectively, and 19 genes differentially expressed between the two tumor types. These findings provide a molecular subgroup classification for meningiomas and schwannomas with possible implications for clinical practice.

MHM assay: molecular sexing based on the sex-specific methylation pattern of the MHM region in chickens

Bird sex determination using molecular methods has proved to be a valuable tool in different studies. Although it is possible to sex most birds by coupling the CHD assay with others available methods, no sex-determining gene like SRY in mammalians has been identified in birds. The male hypermethylated (MHM) region on the Z chromosome has been found to be hypermethylated in males and hypomethylated in females in birds of the order Galliformes. We analyzed the DNA from feathers of 50 adult chickens to verify the methylation pattern of the MHM region by PCR and the restriction enzyme HpaII (a method named MHM assay). The results, visualized in agarose gel, were compared with PCR amplification of the CHD-Z and CHD-W genes (polyacrylamide gel) and with the birds` phenotype. All males (25) showed hypermethylation of the MHM region, and all females (25) showed hypomethylation. The sexing by MHM assay was in according with phenotype and CHD sexing. To our knowledge, this is the first study that uses the MHM region for sexing birds. Although the real role of the MHM region in the sex determination is still unclear, this could be a universal marker for sexing birds and may be involved in sex determination by its influence on transcriptional processes. The MHM assay could be a good alternative for CHD assay in developmental studies.

Clinical and molecular characterization of Brazilian families with von Hippel-Lindau disease: a need for delineating genotype-phenotype correlation

von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumours, especially cerebellar haemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). The etiology and manifestations are due to germline and somatic mutations in the VHL tumour suppressor gene. VHL disease is classified into type 1 and type 2, showing a clear genotype-phenotype correlation, as type 2 is associated with phaeochromocytoma and essentially caused by missense mutations. The aim of this study is to characterize the phenotype and genotype of families with VHL disease. Eighteen of twenty patients from ten unrelated families underwent genetic testing, nine of them fulfilled VHL disease criteria and one had an apparently sporadic cerebellar haemangioblastoma. Four different germline mutations in the VHL gene were identified: c.226_228delTTC (p.Phe76del); c.217C > T (p.Gln73X); IVS1-1 G > A and IVS2-1 G > C. The first three mutations were associated with type 1 disease and the last one with type 2B, which had never been identified in the germline. The transcriptional processing of a novel splice-site mutation was characterised. Three type 1 VHL families showed large deletions of the VHL gene, two of them encompassed the FANCD2/C3orf10 genes and were not associated with renal lesions. We also suggest that such families should be subclassified according to the risk of RCC and the extent of the VHL gene deletions. This study highlights the need for a through clinical and molecular characterisation of families with VHL disease to better delineate its genotype-phenotype correlation.

Imbalanced circulating matrix metalloproteinases in polycystic ovary syndrome

Altered levels of matrix metalloproteinases (MMPs) may reflect relevant pathogenetic mechanisms of disease conditions. The objective of this study was to compare the plasma levels of MMPs and tissue inhibitors of MMPs (TIMPs) in polycystic ovary syndrome (PCOS) patients with those found in healthy ovulatory controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical features of this syndrome. Sixty-five healthy ovulatory subjects (controls) and 80 patients with PCOS were include in this study. MMP-2, MMP-8, MMP-9, TIMP-1, TIMP-2 concentrations were measured in plasma samples by gelatin zymography or enzyme-linked immunoassays. MMP-2, MMP-8, MMP-9, and TIMP-1 levels were similar in PCOS patients and in healthy controls (P > 0.05). PCOS patients had lower plasma TIMP-2 levels than healthy controls (P < 0.05). We found higher MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios in PCOS patients than in healthy controls (all P < 0.05). Testosterone levels correlated positively with the MMP-9/TIMP-1 ratio and negatively with TIMP-2 levels (r = 0.26, P < 0.01 and r = -0.21, P = 0.02, respectively). In addition, only testosterone was an independent predictor of TIMP-2 levels (estimate = -0.35, P = 0.04) and the MMP-9/TIMP-1 ratio (estimate = 0.01, P = 0.04). We found evidence indicating that the balance between MMPs and TIMPs in women with PCOS is altered, probably due to androgen excess found in these women.

Osteopontin expression according to molecular profile of invasive breast cancer: a clinicopathological and immunohistochemical study

Osteopontin (OPN) is a secreted, calcium-binding phosphorylated glycoprotein involved in several physiological and pathological events such as angiogenesis, apoptosis, inflammation, wound healing, vascular remodeling, calcification of mineralized tissues, and induction of cell proteases. There is growing interest in the role of OPN in breast cancer. In an attempt to obtain new insight into the pathogenesis of OPN-associated breast carcinomas, an immunohistochemical panel with 17 primary antibodies including cytokeratins and key regulators of the cell cycle was performed in 100 formalin-fixed paraffin-embedded samples of invasive breast carcinomas. OPN was expressed in 65% of tumors and was negatively correlated with estrogen (p=0.0350) and progesterone (p=0.0069) receptors, but not with the other markers and clinicopathological features evaluated including age, menstrual status, pathological grading, tumor size, and metastasis. There was no correlation between OPN expression and carcinomas of the basal-like phenotype (p=0.1615); however, OPN correlated positively with c-erbB-2 status (p=0.0286) and negatively with carcinomas of the luminal subtype (p=0.0353). It is well known that carcinomas overexpressing c-erbB-2 protein have a worse prognosis than luminal tumors. Here, we hypothesize that the differential expression of OPN in the first subtype of carcinomas may contribute to their more aggressive behavior. (Int J Biol Markers 2008; 23: 154-60)

Proteomic analysis of low- to high-grade astrocytomas reveals an alteration of the expression level of raf kinase inhibitor protein and nucleophosmin

Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p < 0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p < 0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.

Pompe disease in a Brazilian series: clinical and molecular analyses with identification of nine new mutations

Pompe disease (glycogen storage disease type II or acid maltase deficiency) is an inherited autosomal recessive deficiency of acid alpha-glucosidase (GAA), with predominant manifestations of skeletal muscle weakness. A broad range of studies have been published focusing on Pompe patients from different countries, but none from Brazil. We investigated 41 patients with either infantile-onset (21 cases) or late-onset (20 cases) disease by muscle pathology, enzyme activity and GAA gene mutation screening. Molecular analyses identified 71 mutant alleles from the probands, nine of which are novel (five missense mutations c.136T > G, c.650C > T, c.1456G > C, c.1834C > T, and c.1905C > A, a splice-site mutation c.1195-2A > G, two deletions c.18_25del and c.2185delC, and one nonsense mutation c.643G > T). Interestingly, the c.1905C > A variant was detected in four unrelated patients and may represent a common Brazilian Pompe mutation. The c.2560C > T severe mutation was frequent in our population suggesting a high prevalence in Brazil. Also, eight out of the 21 infantile-onset patients have two truncating mutations predicted to abrogate protein expression. Of the ten late-onset patients who do not carry the common late-onset intronic mutation c.-32-13T > G, five (from three separate families) carry the recently described intronic mutation, c.-32-3C > A, and one sibpair carries the novel missense mutation c.1781G > C in combination with known severe mutation c.1941C > G. The association of these variants (c.1781G > C and c.-32-3C > A) with late-onset disease suggests that they allow for some residual activity in these patients. Our findings help to characterize Pompe disease in Brazil and support the need for additional studies to define the wide clinical and pathological spectrum observed in this disease.

Molecular characteristics of extended-spectrum beta-lactamase-producing Escherichia coli from Rio de Janeiro, Brazil

Twenty-five extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli clinical isolates from Rio de Janeiro, Brazil were characterized by isoelectric focusing, PCR and sequencing of bla(ESBL) genes, plasmid-mediated quinolone resistance determinants, phylogenetic groups, replicon typing, pulsed-field electrophoresis, and multilocus sequencing typing. Twenty-three (92%) ESBL-producing E. coli isolates were positive for bla(CTX-M) genes, aac(6`)-lb-cr, and qnrB. Genetic relatedness of ESBL producers clustered seven (28%) CTX-M-15-producing isolates as sequence type (ST) 410, clonal complex (CC) 23, and two (8%) as clone O25-ST131. Our results illustrate the predominance of phylo-group A (52%), ST410 (CC 23) and CTX-M-15 among ESBL-producing E. coli isolates from hospitals in Rio de Janeiro.