In silico analysis of mutations occurring in the protein N-acetylgalactosamine-6-sulfatase (GALNS) and causing mucopolysaccharidosis IVA.

The goals were to analyze and characterize the secondary structure, regions of intrinsic disorder and physicochemical characteristics of three classes of mutations described in the enzyme N-acetylgalactosamine-6-sulfatase that cause mucopolysaccharidosis IVA: missense mutations, insertions and deletions. All mutations were compared to wild-type enzyme, and the results showed that with 25 of 129 missense mutations secondary structure was maintained and that 104 mutations showed minor changes, such as an increase or decrease in the size of the elements. The secondary structure of all insertions and deletions introduced important changes, such as increase in the number and size of elements. The results obtained from intrinsic disorder analysis revealed that missense mutations caused no alterations. However, the insertions and deletions led to major regions of intrinsic disorder. The physicochemical characteristics of the amino acids found in missense mutations revealed unchanged characteristics in 32 of the 129 mutations. However, the remainder had changes that could lead to a modification of tertiary structure. The results proved that it was feasible and necessary to obtain the three-dimensional structure of the enzyme with its mutants to better understand the change in function.

Search for methylation-sensitive amplification polymorphisms in mutant figs.

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that originated from gamma-irradiated buds. Therefore, we conducted methylation-sensitive amplified polymorphism analysis to verify the existence of variability due to epigenetic DNA methylation among these mutant selections compared to the main cultivar 'Roxo-de-Valinhos'. Samples of genomic DNA were double-digested with either HpaII (methylation sensitive) or MspI (methylation insensitive) and with EcoRI. Fourteen primer combinations were tested, and on an average, non-methylated CCGG, symmetrically methylated CmCGG, and hemimethylated hmCCGG sites accounted for 87.9, 10.1, and 2.0%, respectively. MSAP analysis was effective in detecting differentially methylated sites in the genomic DNA of fig mutants, and methylation may be responsible for the phenotypic variation between treatments. Further analyses such as polymorphic DNA sequencing are necessary to validate these differences, standardize the regions of methylation, and analyze reads using bioinformatic tools.

miRNApath: a database of miRNAs, target genes and metabolic pathways.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate target gene expression and hence play important roles in metabolic pathways. Recent studies have evidenced the interrelation of miRNAs with cell proliferation, differentiation, development, and diseases. Since they are involved in gene regulation, they are intrinsically related to metabolic pathways. This leads to questions that are particularly interesting for investigating medical and laboratorial applications. We developed an miRNApath online database that uses miRNA target genes to link miRNAs to metabolic pathways. Currently, databases about miRNA target genes (DIANA miRGen), genomic maps (miRNAMap) and sequences (miRBase) do not provide such correlations. Additionally, miRNApath offers five search services and a download area. For each search, there is a specific type of input, which can be a list of target genes, miRNAs, or metabolic pathways, which results in different views, depending upon the input data, concerning relationships between the target genes, miRNAs and metabolic pathways. There are also internal links that lead to a deeper analysis and cross-links to other databases with more detailed information. miRNApath is being continually updated and is available at http://lgmb.fmrp.usp.br/mirnapath.

miRNApath: a database of miRNAs, target genes and metabolic pathways

MicroRNAs (miRNAs) are small non-coding RNAs that regulate target gene expression and hence play important roles in metabolic pathways. Recent studies have evidenced the interrelation of miRNAs with cell proliferation, differentiation, development, and diseases. Since they are involved in gene regulation, they are intrinsically related to metabolic pathways. This leads to questions that are particularly interesting for investigating medical and laboratorial applications. We developed an miRNApath online database that uses miRNA target genes to link miRNAs to metabolic pathways. Currently, databases about miRNA target genes (DIANA miRGen), genomic maps (miRNAMap) and sequences (miRBase) do not provide such correlations. Additionally, miRNApath offers five search services and a download area. For each search, there is a specific type of input, which can be a list of target genes, miRNAs, or metabolic pathways, which results in different views, depending upon the input data, concerning relationships between the target genes, miRNAs and metabolic pathways. There are also internal links that lead to a deeper analysis and cross-links to other databases with more detailed information. miRNApath is being continually updated and is available at http://lgmb.fmrp.usp.br/mirnapath.

Gene Class expression: analysis tool of Gene Ontology terms with gene expression data

Serial analysis of gene expression (SAGE) technology produces large sets of interesting genes that are difficult to analyze directly. Bioinformatics tools are needed to interpret the functional information in these gene sets. We present an interactive web-based tool, called Gene Class, which allows functional annotation of SAGE data using the Gene Ontology (GO) database. This tool performs searches in the GO database for each SAGE tag, making associations in the selected GO category for a level selected in the hierarchy. This system provides user-friendly data navigation and visualization for mapping SAGE data onto the gene ontology structure. This tool also provides graphical visualization of the percentage of SAGE tags in each GO category, along with confidence intervals and hypothesis testing.

A pulsatile flow model for in vitro quantitative evaluation of prosthetic valve regurgitation.

A pulsatile pressure-flow model was developed for in vitro quantitative color Doppler flow mapping studies of valvular regurgitation. The flow through the system was generated by a piston which was driven by stepper motors controlled by a computer. The piston was connected to acrylic chambers designed to simulate "ventricular" and "atrial" heart chambers. Inside the "ventricular" chamber, a prosthetic heart valve was placed at the inflow connection with the "atrial" chamber while another prosthetic valve was positioned at the outflow connection with flexible tubes, elastic balloons and a reservoir arranged to mimic the peripheral circulation. The flow model was filled with a 0.25% corn starch/water suspension to improve Doppler imaging. A continuous flow pump transferred the liquid from the peripheral reservoir to another one connected to the "atrial" chamber. The dimensions of the flow model were designed to permit adequate imaging by Doppler echocardiography. Acoustic windows allowed placement of transducers distal and perpendicular to the valves, so that the ultrasound beam could be positioned parallel to the valvular flow. Strain-gauge and electromagnetic transducers were used for measurements of pressure and flow in different segments of the system. The flow model was also designed to fit different sizes and types of prosthetic valves. This pulsatile flow model was able to generate pressure and flow in the physiological human range, with independent adjustment of pulse duration and rate as well as of stroke volume. This model mimics flow profiles observed in patients with regurgitant prosthetic valves.

A pulsatile flow model for in vitro quantitative evaluation of prosthetic valve regurgitation

A pulsatile pressure-flow model was developed for in vitro quantitative color Doppler flow mapping studies of valvular regurgitation. The flow through the system was generated by a piston which was driven by stepper motors controlled by a computer. The piston was connected to acrylic chambers designed to simulate "ventricular" and "atrial" heart chambers. Inside the "ventricular" chamber, a prosthetic heart valve was placed at the inflow connection with the "atrial" chamber while another prosthetic valve was positioned at the outflow connection with flexible tubes, elastic balloons and a reservoir arranged to mimic the peripheral circulation. The flow model was filled with a 0.25 per cent corn starch/water suspension to improve Doppler imaging. A continuous flow pump transferred the liquid from the peripheral reservoir to another one connected to the "atrial" chamber. The dimensions of the flow model were designed to permit adequate imaging by Doppler echocardiography. Acoustic windows allowed placement of transducers distal and perpendicular to the valves, so that the ultrasound beam could be positioned parallel to the valvular flow. Strain-gauge and electromagnetic transducers were used for measurements of pressure and flow in different segments of the system. The flow model was also designed to fit different sizes and types of prosthetic valves. This pulsatile flow model was able to generate pressure and flow in the physiological human range, with independent adjustment of pulse duration and rate as well as of stroke volume. This model mimics flow profiles observed in patients with regurgitant prosthetic valves.

Investigation of distal aortic compliance and vasodilator responsiveness in heart failure due to proximal aortic stenosis in the guinea pig.

Hypotension and syncope are recognized features of chronic aortic stenosis. This study examined vasomotor responses and dynamic compliance in isolated abdominal aortae after chronic constriction of the ascending aorta. Guinea pigs underwent constriction of the ascending aorta or sham operation. Sections of descending aorta were removed for studies of contractile performance and compliance. Dynamic compliance was measured using a feedback-controlled pulsatile pressure system at frequencies of 0.5, 1.5 and 2.5 Hz and mean pressures from 40 to 100 mmHg. Chronic (149+/-6 days) aortic constriction resulted in significant increases in organ weight/body weight ratios for left ventricle (58%), right ventricle (100%) and lung (61%). The presence of heart failure was indicated by increased lung weights, left ventricular end-diastolic pressure and systemic vascular resistance, reduced cardiac output and increased levels of plasma atrial natriuretic peptide (166%), adrenaline (x20), noradrenaline (106%) and dopamine (x3). Aortic rings showed similar constrictor responses to phenylephrine and angiotensin II, but maximal vasodilator responses to acetylcholine and isoprenaline were significantly increased (144% and 48% respectively). Dilator responses to sodium nitroprusside, forskolin and cromokalim were unchanged. Compliance of all vessels decreased with increasing pulsatile frequency and to a lesser extent with increased mean pressure, but were similar in aortic-constricted and control groups. Chronic constriction of the ascending aorta resulted in heart failure and increased vasodilator responses to acetylcholine and isoprenaline in the distal aorta while dynamic compliance was unchanged. We hypothesize that increased endothelium-mediated vasodilatation may contribute to hypotension and syncope in patients with left ventricular outflow obstruction.