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Biomedical subjects

G Casari

Publications and source records attributed to G Casari.

At least 19 recordsLinked to original sources
PubMed1997

Mutational analyses support a model for the HRV2 2A proteinase.

The proteinase 2A of human rhinovirus 2 is a cysteine proteinase which contains a tightly bound Zn ion thought to be required for structural integrity. A three-dimensional model for human rhinovirus type 2 proteinase 2A (HRV2 2A) was established using sequence alignments with small trypsin-like Ser-proteinases and, for certain regions, elastase. The model was tested by expressing selected proteinase 2A mutants in bacteria and examining the effect on both intramolecular ("cis") and intermolecular ("trans") activities. The HRV2 proteinase 2A is proposed to have a two domain structure, with the catalytic site and substrate binding region on one face of the molecule and a Zn-binding motif on the opposite face. Residues Gly 123, Gly 124, Thr 121, and Cys 101 are proposed to be involved in the architecture of the substrate binding pocket and to provide the correct environment for the catalytic triad of His 18, Asp 35, and Cys 106. Residues Tyr 85 and Tyr 86 are thought to participate in substrate recognition. The presence of an extensive C-terminal helix, in which Asp 132, Arg 134, Phe 130, and Phe 136 play important roles, explains why mutations in this region are generally detrimental to proteinase activity. The proposed Zn-binding motif comprises Cys 52, Cys 54, Cys 112, and His 114. Exchange of these residues inactivates the enzyme. Furthermore, as measured by atom emission spectroscopy, Zn was absent from purified preparations of proteinase 2A in which His 114 had been replaced by Asn. The absence of disulphide bridges was confirmed by subjecting highly purified HRV2 proteinase 2A to one- and two-step alkylation procedures.

Amino Acid Sequence↗
PubMed1997

Polymorphisms of alpha-adducin and salt sensitivity in patients with essential hypertension.

BACKGROUND: Abnormalities in renal sodium transport may be involved in hypertension. Adducin, an alpha/beta heterodimeric protein found in the renal tubule is thought to regulate ion transport through changes in the actin cytoskeleton. We investigated whether an alpha-adducin polymorphism (Gly 460 Trp) is involved in essential hypertension in two separate populations. METHODS: Linkage analysis of three DNA markers at different distances from the alpha-adducin locus (20-2500 kb) was done in 137 hypertensive sibling-pairs. 477 hypertensive and 322 normotensive individuals were genotyped for the alpha-adducin polymorphism. The blood-pressure response to acute and chronic changes in sodium balance was studied in hypertensive individuals with and without the 460 Trp alpha-adducin allele. FINDINGS: Significant linkage was found for all three markers in the sibling-pair study. The extra shared alleles (9.1%, 6.5%, and 4.7%) and the significance level for linkage (p = 0.0006, p = 0.0119, and p = 0.0211) both decreased with increasing distance from the alpha-adducin locus. There was a significant association between the 460 Trp mutation and hypertension (p = 0.0003). In the salt-sensitivity test, to assess the acute blood-pressure response to changes in body sodium in 86 hypertensive patients, the decrease in mean arterial pressure was greater in 65 patients who were heterozygous for the mutant allele (Gly/Trp) than in 21 wild-type homozygotes (Gly/Gly) (mean decrease 15.9 [SE 2.0] vs 7.4 [1.3] mm Hg; p = 0.001). Similarly, 21 heterozygous hypertensive patients showed a greater fall in mean arterial pressure in response to 2 months' treatment with hydrochlorothiazide than did 37 wild-type homozygous hypertensive patients (mean decrease 14.7 [2.2] vs 6.8 [1.4] mm Hg; p = 0.002). INTERPRETATION: Our findings of significant linkage of the alpha-adducin locus to essential hypertension and greater sensitivity to changes in sodium balance among patients with the mutant allele suggest that alpha-adducin is associated with a salt-sensitive form of essential hypertension. We suggest the alpha-adducin polymorphism may identify hypertensive patients who will benefit from diuretic treatment or manoeuvres to reduce total body sodium.

Aged↗
PubMed1997

Conserved clusters of functionally related genes in two bacterial genomes.

An approach for genome comparison, combining function classification of gene products and sequence comparison, is presented. The genomes of Haemophilus influenzae and Escherichia coli are analyzed, and all genes are classified into nine major functional classes, corresponding to important cellular processes. To study gene order relationships and genome organization in the two bacteria, we performed statistics on neighboring pairs of genes. To estimate the significance of the observations, a statistical model based on binomial distributions has been developed. Significant patterns of gene order are observed within, as well as between, the two bacterial genomes: Functionally related genes tend to be neighbors more often than do unrelated genes. Some of these groups represent well-known operons, but additional gene clusters are identified. These clusters correspond to genomic elements that have been conserved during bacterial evolution. In addition to nearest-neighbor relationships, the method is also useful to study the relative direction of transcription in genomes, which is also highly conserved between homologous gene pairs. This new approach combines the high-level description of molecular function with pair statistics that express genome organization. It is expected to complement traditional methods of sequence analysis in the study of genomic structure, function, and evolution.

Binomial Distribution↗
PubMed1997

Classification of protein families and detection of the determinant residues with an improved self-organizing map.

Using a SOM (self-organizing map) we can classify sequences within a protein family into subgroups that generally correspond to biological subcategories. These maps tend to show sequence similarity as proximity in the map. Combining maps generated at different levels of resolution, the structure of relations in protein families can be captured that could not otherwise be represented in a single map. The underlying representation of maps enables us to retrieve characteristic sequence patterns for individual subgroups of sequences. Such patterns tend to correspond to functionally important regions. We present a modified SOM algorithm that includes a convergence test that dynamically controls the learning parameters to adapt them to the learning set instead of being fixed and externally optimized by trial and error. Given the variability of protein family size and distribution, the addition of this features is necessary. The method is successfully tested with a number of families. The rab family of small GTPases is used to illustrate the performance of the method.

Algorithms↗
PubMed1997

Probing the structure of the HIV-1 Rev trans-activator protein by functional analysis.

Human immunodeficiency virus type 1 (HIV-1) encodes a trans-acting regulatory protein, termed Rev, which is critically required for virus replication. Rev is a sequence-specific RNA binding protein which mediates the nuclear export of unspliced and incompletely spliced viral mRNAs encoding the viral structural proteins. While CD and fluorescence measurements have provided several possible structural models of Rev, all attempts employing X-ray crystallography and NMR techniques have so far failed to provide more accurate data. We present a new approach to validate alternative structural models of the N-terminal region of Rev which contains the nuclear localization/RNA binding domain. Points of contact between structural elements in a protein were determined by introduction of targeted amino acid substitutions and subsequent scoring of the biological activities. Our data resulted in the suggestion of a new and more refined model of HIV-1 Rev structure which to date has been impossible to obtain by other means.

Animals↗
PubMed1996

Molecular cloning, expression pattern, and chromosomal localization of the human Na-Cl thiazide-sensitive cotransporter (SLC12A3).

Electrolyte homeostasis is maintained by several ion transport systems. Na-(K)-Cl cotransporters promote the electrically silent movement of chloride across the membrane in absorptive and secretory epithelia. Two kidney-specific Na-(K)-Cl cotransporter isoforms are known, so far, according to their sensitivity to specific inhibitors. We have cloned the human cDNA coding for the renal Na-Cl cotransporter selectively inhibited by the thiazide class of diuretic agents. The predicted protein sequence of 1021 amino acids (112 kDa) shows a structure common to the other members of the Na-(K)-Cl cotransporter family: a central region harboring 12 transmembrane domains and the 2 intracellular hydrophilic amino and carboxyl termini. The expression pattern of the human Na-Cl thiazide-sensitive cotransporter (hTSC, HGMW-approved symbol SLC12A3) confirms the kidney specificity. hTSC has been mapped to human chromosome 16q13 by fluorescence in situ hybridization. The cloning and characterization of hTSC now render it possible to study the involvement of this cotransport system in the pathogenesis of tubulopathies such as Gitelman syndrome.

Adult↗
PubMed1996

Computational comparisons of model genomes.

Complete genomes from model organisms provide new challenges for computational molecular biology. Novel questions emerge from the genome data obtained from the functional prediction of thousands of gene products. In this review, we present some approaches to the computational comparison of genomes, based on sequence and text analysis, and comparisons of genome composition and gene order.

Biotechnology↗
PubMed1996

Endothelin-B receptor mutations in patients with isolated Hirschsprung disease from a non-inbred population.

Hirschsprung disease (HSCR), or aganglionic megacolon, is the most common cause of congenital intestinal obstruction. Two different loci have been found to be tightly linked to HSCR on chromosomes 10 and 13, respectively. Recently, mutations in the RET protooncogene on chromosome 10q11.2 were identified in several HSCR patients. In addition, a missense mutation in the endothelin-B receptor (EDNRB) gene on chromosome 13q22 was found in an inbred Mennonite kindred affected by HSCR and associated abnormalities, demonstrating the involvement of EDNRB in HSCR pathogenesis. To test whether mutations in the EDNRB gene could account for Hirschsprung in patients from non-inbred populations, we analysed DNA samples from 17 probands of Italian origin with HSCR. We have identified two novel EDNRB mutations: a missense mutation in a sporadic case, S305N, which leads to a change of a serine to an asparagine, disrupting a putative phosphorylation site; and a single nucleotide deletion in a familial case, N378I, resulting in a truncated protein. Both mutations were found in one of the healthy parents, and neither of these mutations were found in any of the normal individuals tested. These data confirm the involvement of EDNRB in HSCR pathogenesis and demonstrate that EDNRB mutations could contribute to HSCR disease in non-inbred populations.

Adolescent↗
PubMed1996

The prediction of protein contacts from multiple sequence alignments.

We have studied the question of how much extra predictive power the correlated mutational behaviour of pairs of amino acid residues separated along a sequence has concerning the likelihood of those residues being in contact in the folded protein. The mutational behaviour is deduced from multiple sequence alignments. Our findings are that there is, indeed, some valuable information available from this source and that it is sufficient to make a significant improvement in our ability to predict contacts, when compared with earlier methods that do not take into account the correlations between the mutations. This improvement is approximately twice as large as can be obtained by the more economical method of simply averaging pair preferences over the same sequence alignment. Even when using a method based on pair preferences, a further significant improvement can be made by penalizing more variable regions (on the reasonable assumption that invariant residues are relatively more likely to be in contact), though we have found no way of improving the pair preference method to the extent that it matches the method based on correlated behaviour. Our new method is thought to be the best data-based method of contact prediction developed so far, achieving, on average, an improvement over a random (i.e. information-free) prediction of a factor of five when the number of contacts predicted is chosen to match the number that actually occur.

Algorithms↗
PubMed1996

The locus for a novel syndromic form of neuronal intestinal pseudoobstruction maps to Xq28.

The neuronal type of primary chronic idiopathic intestinal pseudoobstruction (CIIP) results from the developmental failure of enteric neurons to migrate or differentiate correctly. This leads to intestinal motility disorders, which are characterized by symptoms and signs of bowel obstruction in the absence of a mechanical obstacle. Most of these conditions are congenital, and among them some are inherited. One syndromic condition characterized by intestinal pseudoobstruction with morphological abnormalities of the argyrophil neurons in the myenteric plexus, associated with short small bowel, malrotation, and pyloric hypertrophy, has been previously described. We have studied a family affected by this disorder, in which the disease appeared to segregate as an X-linked recessive trait. In order to map the CIIP locus in this family, we performed linkage analysis in 26 family members by use of highly polymorphic microsatellite markers from the X chromosome. One of these markers, DXYS154, located in the distal part of Xq28, shows no recombination with a maximum lod score of 2.32. Multipoint analysis excluded linkage with markers spanning other regions of the X chromosome. Our results, integrated with the current genetic and physical map of Xq28, determine the order of loci as cen-DXS15-(CIIPX)-DXS1108/DXYS154-tel. This study establishes, for the first time, the mapping assignment of a neuropathic form of CIIP other than Hirschsprung disease.

Chromosome Mapping↗
PubMed1996

Novel molecular variants of the Na-Cl cotransporter gene are responsible for Gitelman syndrome.

A hereditary defect of the distal tubule accounts for the clinical features of Gitelman syndrome (GS), an autosomal recessive disease characterized by hypokalemia, hypomagnesemia, metabolic alkalosis, and hypocalciuria. Recently, we cloned the cDNA coding for the human Na-Cl thiazide-sensitive cotransporter (TSC; also known as ¿NCCT¿ or ¿SLC12A3¿) as a possible candidate for GS, and Simon et al., independently, described mutations in patients with GS. Now, we show 12 additional mutations consistent with a loss of function of the Na-Cl cotransporter in GS. Two missense replacements, R209W and P349L, are common to both studies and could represent ancient mutations. The other mutations include three deletions, two insertions, and six missense mutations. When all mutations from both studies are considered, missense mutations seem to be more frequently localized within the intracellular domains of the molecule, rather than in transmembrane or extracellular domains. One family, previously reported as a GS form with dominant inheritance, has proved to be recessive, with the affected child being a compound heterozygote. A highly informative intragenic tetranucleotide marker, useful for molecular diagnostic studies, has been identified at the acceptor splice site of exon 9.

Adult↗
PubMed1995

Characterisation and chromosomal localisation of the rat alpha- and beta-adducin-encoding genes.

A polymorphism in the genes encoding alpha- and beta-adducin (ADD) was described as being associated with blood-pressure variation in a genetically hypertensive strain of rats (MHS). ADD is a cytoskeletal heterodimeric protein which may be involved in cellular signal transduction and interacts with other membrane skeleton proteins which affect ion transport across the cell membrane. The cDNA encoding the alpha subunit of rat ADD was isolated using PCR methods. The cDNA consists of about 3900 bp and encodes a protein of 735 amino acids (aa) which shows 91% aa identity with the human counterpart. In spleen and kidney, three alternative spliced exons were found by PCR amplification and confirmed by RNase protection analysis. 17 inbred rat strains were genotyped for the polymorphism in the alpha- and beta-ADD genes. Chromosomal localisation mapped rat alpha-ADD on chromosome 14 and rat beta-ADD on chromosome 4.

Alleles↗
PubMed1995

New protein functions in yeast chromosome VIII.

The analysis of the 269 open reading frames of yeast chromosome VIII by computational methods has yielded 24 new significant sequence similarities to proteins of known function. The resulting predicted functions include three particularly interesting cases of translation-associated proteins: peptidyl-tRNA hydrolase, a ribosome recycling factor homologue, and a protein similar to cytochrome b translational activator CBS2. The methodological limits of the meaningful transfer of functional information between distant homologues are discussed.

Alcohol Oxidoreductases↗