A new clinical and molecular form of Unverricht-Lundborg disease localized by homozygosity mapping

Progressive myoclonus epilepsy (PME) has a number of causes, of which Unverricht-Lundborg disease (ULD) is the most common. ULD has previously been mapped to a locus on chromosome 21 (EPM1). Subsequently, mutations in the cystatin B gene have been found in most cases. In the present work we identified an inbred Arab family with a clinical pattern compatible with ULD, but mutations in the cystatin B gene were absent. We sought to characterize the clinical and molecular features of the disorder. The family was studied by multiple field trips to their town to clarify details of the complex consanguineous relationships and to personally examine the family. DNA was collected for subsequent molecular analyses from 21 individuals. A genome-wide screen was performed using 811 microsatellite markers. Homozygosity mapping was used to identify loci of interest. There were eight affected individuals. Clinical onset was at 7.3 +/- 1.5 years with myoclonic or tonic-clonic seizures. All had myoclonus that progressed in severity over time and seven had tonic-clonic seizures. Ataxia, in addition to myoclonus, occurred in all. Detailed cognitive assessment was not possible, but there was no significant progressive dementia. There was intrafamily variation in severity; three required wheelchairs in adult life; the others could walk unaided. MRI, muscle and skin biopsies on one individual were unremarkable. We mapped the family to a 15-megabase region at the pericentromeric region of chromosome 12 with a maximum lod score of 6.32. Although the phenotype of individual subjects was typical of ULD, the mean age of onset (7.3 years versus 11 years for ULD) was younger. The locus on chromosome 12 does not contain genes for any other form of PME, nor does it have genes known to be related to cystatin B. This represents a new form of PME and we have designated the locus as EPM1B.

Characterization of temperature dependent and substrate-binding cleft movements in Bacillus circulans family 11 xylanase: A molecular dynamics investigation

Background: Xylanases (EC 3.2.1.8) hydrolyze xylan, one of the most abundant plant polysaccharides found in nature, and have many potential applications in biotechnology. Methods: Molecular dynamics simulations were used to investigate the effects of temperature between 298 to 338 K and xylobiose binding on residues located in the substrate-binding cleft of the family 11 xylanase from Bacillus circulans (BcX). Results: In the absence of xylobiose the BcX exhibits temperature dependent movement of the thumb region which adopts an open conformation exposing the active site at the optimum catalytic temperature (328 K). In the presence of substrate, the thumb region restricts access to the active site at all temperatures, and this conformation is maintained by substrate/protein hydrogen bonds involving active site residues, including hydrogen bonds between Tyr69 and the 2` hydroxyl group of the substrate. Substrate access to the active site is regulated by temperature dependent motions that are restricted to the thumb region, and the BcX/substrate complex is stabilized by extensive intermolecular hydrogen bonding with residues in the active site. General significance: These results call for a revision of both the ""hinge-bending"" model for the activity of group 11 xylanases, and the role of Tyr69 in the catalytic mechanism. (C) 2009 Elsevier B.V. All rights reserved.

Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon

Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon. A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation. Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae, The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented. Other, less commonly observed types were pink or orange pigmented, A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization. 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria, Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group, Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives. Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species, A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P. brasiliensis, and exhibited distinctive morphology.

Molecular profiling of isolated histological components of Wilms tumor implicates a common role for the Wnt signaling pathway in kidney and tumor development

Wilms tumor (WT), a tumor composed of three histological components - blastema (BL), epithelia and stroma - is considered an appropriate model system to study the biological relationship between differentiation and tumorigenesis. To investigate molecular associations between nephrogenesis and WT, the gene expression pattern of individual cellular components was analyzed, using a customized platform containing 4,608 genes. WT gene expression patterns were compared to genes regulated during kidney differentiation. BL had a closer gene expression pattern to the earliest stage of normal renal development. The BL gene expression pattern was compared to that of fetal kidney (FK) and also between FK and mature kidney, identifying 25 common de-regulated genes supposedly involved in the earliest events of WT onset. Quantitative RT-PCR was performed, confirming the difference in expression levels for 13 of 16 genes (81.2%) in the initial set and 8 of 13 (61.5%) in an independent set of samples. An overrepresentation of genes belonging to the Wnt signaling pathway was identified, namely PLCG2, ROCK2 and adenomatous polyposis coli (APC). Activation of the Wnt pathway was confirmed in WT, using APC at protein level and PLCG2 at mRNA and protein level. APC showed positive nuclear immunostaining for an independent set of WT samples, similarly to the FK in week 11. Lack of PLCG2 expression was confirmed in WT and in FK until week 18. Taken together, these results provided molecular evidence of the recapitulation of the embryonic kidney by WT as well as involvement of the Wnt pathway in the earliest events of WT onset. Copyright (C) 2008 S. Karger AG, Basel.

Molecular evidence of human T-cell lymphotropic virus types 1 and 2 (HTLV-1 and HTLV-2) infections in HTLV seroindeterminate individuals from Sao Paulo, Brazil

Background: Using enzyme immunoassays and Western blot (Wb) tests, HTLV serodiagnosis yields indeterminate results in a significant number of cases. Objective: To determine the prevalence of HTLV infection among HTLV-seroindeterminate individuals. Study design: We studied peripheral blood mononuclear cells from 65 anti-HTLV Wb-seroindeterminate individuals by attempting to amplify proviral DNA sequences (tax and pot) to identify HTLV-I and HTLV-2 infections. Results: These 65 specimens exhibited predominantly (43%) anti-HTLV antibodies to gag-coded antigens in the absence of anti-p24 on Wb analysis. Tax proviral sequences were detected in 6 (9.2%) samples. According to restricted fragment polymorphism analysis (RFLP), we identified HTLV-1 proviral DNA in 4 samples. HTLV-2 in one and sequences from both in another. Nested PCR for the pot region was positive in 3 (4.6%) specimens, which were also positive for tax sequences. After hybridization HTLV-1 infection was confirmed in 2 samples (3.1%) and HTLV-2 in another (1.5%). Detection of a single HTLV DNA sequence may be due to infection by defective provirus, but its significance remains undefined. In this cohort, no Wb reactivity pattern was predictive of proviral detection. HTLV-I infection was demonstrated in an individual who had Wb reactivity to gag-coded antigens only. Conclusions: This emphasizes the importance of clinical and laboratory follow-up of HTLV-seroindeterminate individuals from endemic areas. (c) 2009 Elsevier B.V. All rights reserved.

Endomyocardial fibrosis: pathological and molecular findings of surgically resected ventricular endomyocardium

Endomyocardial fibrosis (EMF) is a restrictive cardiomyopathy of unknown etiology prevalent in tropical regions affecting the inflow tract and apex of one or both ventricles, which show fibrous thickening of the endocardium and adjacent myocardium. Surgical treatment is recommended for patients in functional classes III or IV (New York Heart Association). The gross and histological features of the heart have been comprehensively studied in autopsies, but studies in surgical samples are still lacking. Histological and immunohistochemical features of EMF in surgical samples collected from 32 patients were described and correlated with clinical data. Polymerase chain reaction (PCR) and reverse transcription-PCR, performed on formalin fixed endomyocardial samples, were used retrospectively to detect genomes of certain cardiotropic viruses and Toxoplasma gondii. Ventricular endocardium was thickened by superficial acellular hyaline collagen fibers type I and III, with predominance of the former type. Besides fibrosis, a chronic inflammatory process and an anomalous lymphatic rich vascular pattern were observed in the deep endocardium, connected to the terminal coronary circulation of the myocardium, which might be an important pathological finding concerning EMF pathogenesis. Molecular analysis of the endomyocardium revealed high incidence of cardiotropic infective agents (6/12, 50%); however, their role in the disease pathogenesis is still controversial.

Diagnostic model based on Raman spectra of normal, hyperplasia and prostate adenocarcinoma tissues in vitro

This study evaluated the use of Raman spectroscopy to identify the spectral differences between normal (N), benign hyperplasia (BPH) and adenocarcinoma (CaP) in fragments of prostate biopsies in vitro with the aim of developing a spectral diagnostic model for tissue classification. A dispersive Raman spectrometer was used with 830 nm wavelength and 80 mW excitation. Following Raman data collection and tissue histopathology (48 fragments diagnosed as N, 43 as BPH and 14 as CaP), two diagnostic models were developed in order to extract diagnostic information: the first using PCA and Mahalanobis analysis techniques and the second one a simplified biochemical model based on spectral features of cholesterol, collagen, smooth muscle cell and adipocyte. Spectral differences between N, BPH and CaP tissues, were observed mainly in the Raman bands associated with proteins, lipids, nucleic and amino acids. The PCA diagnostic model showed a sensitivity and specificity of 100%, which indicates the ability of PCA and Mahalanobis distance techniques to classify tissue changes in vitro. Also, it was found that the relative amount of collagen decreased while the amount of cholesterol and adipocyte increased with severity of the disease. Smooth muscle cell increased in BPH tissue. These characteristics were used for diagnostic purposes.

Gastrointestinal stromal tumor in Brazil: Clinicopathology, immunohistochemistry, and molecular genetics of 513 cases

The aim of the present study was to evaluate the clinicopathological, immunohistochemical, and molecular genetic features of gastrointestinal stromal tumors in Brazil and compare them with cases from other countries. Five hundred and thirteen cases were retrospectively analyzed. HE-stained sections and clinical information were reviewed and the immunohistochemical expression of CD117, CD34, smooth-muscle actin, S-100 protein, desmin, CD44v3 adhesion molecule, p53 protein, epidermal growth factor receptor, and Ki-67 antigen was studied using tissue microarrays. Mutation analysis of KIT and platelet-derived growth factor receptor-alpha genes was also performed. There was a slight female predominance (50.3%) and the median age at diagnosis was 59 years. The tumors were mainly located in the stomach (38.4%). Immunohistochemistry showed that CD117 was expressed in 95.7% of cases. Epidermal growth factor receptor expression was observed in 84.4% of tumors. p53 protein expression was found only in 2.6% of cases but all belonged to the high-risk group for aggressive behavior according to the National Institutes of Health consensus approach. No CD44v3 adhesion molecule expression was detected. KIT exon 11 mutations were the most frequent (62.2%). The present data confirm that gastrointestinal stromal tumors in Brazilian patients do not differ from tumors occurring in other countries.

Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.

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.

Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocysts derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules

In vitro culture conditions affect both the maternal and embryonic expression of genes and is likely to alter both oocyte and embryo developmental competence. The search for better and less variable culture conditions simulating those in vivo has led to the development of defined culture media, with lower impact on the molecular reprogramming of oocytes and embryos. We evaluated embryo development and relative abundance (RA) of Hsp-70 and Bax transcripts in bovine blastocysts produced from oocytes matured in a chemically defined IVM system with synthetic polymers. Immature cumulus oocyte complexes (COCs) were matured for 22-24 h in alpha-MEM supplemented with IGF-1, insulin, 0.1% polyvinyl alcohol (PVA), or 0.1% polyvinylpyrrolidone (PVP), but without FSH or LH. The control group consisted of COCs matured it, TCM plus FSH and 10% estrous cow serum. After fertilization. presumptive zygotes were co-cultured with cumulus cells until 224 h post-insemination. Total RNA was isolated from embryo pools, reverse transcribed into cDNA, and subjected to transcript analysis by real-time PCR. Cleavage rate was higher (P < 0.05) for the control group (68.3%) than for the PVA (54.4%) and PVP-40 (58.3%) groups. Nevertheless. there was no difference among the PVA, PVP-40 and control groups in blastocyst or hatching rates. similarly, no difference in relative abundance of Hsp-70 and Bax transcripts was detected in comparison to the control group. We inferred that bovine oocytes can be matured in serum- and gonadotrophin-free medium supplemented with PVA or PVP, enriched with IGF-I and insulin, without altering post-cleavage development and relative abundance of some genes associated with stress and apoptosis. (C) 2009 Elsevier Inc. All rights reserved.

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)