Predicting reliable regions in protein alignments from sequence profiles

For applications such as comparative modelling one major issue is the reliability of sequence alignments. Reliable regions in alignments can be predicted using sub-optimal alignments of the same pair of sequences. Here we show that reliable regions in alignments can also be predicted from multiple sequence profile information alone.Alignments were created for a set of remotely related pairs of proteins using five different test methods. Structural alignments were used to assess the quality of the alignments and the aligned positions were scored using information from the observed frequencies of amino acid residues in sequence profiles pre-generated for each template structure. High-scoring regions of these profile-derived alignment scores were a good predictor of reliably aligned regions.These profile-derived alignment scores are easy to obtain and are applicable to any alignment method. They can be used to detect those regions of alignments that are reliably aligned and to help predict the quality of an alignment. For those residues within secondary structure elements, the regions predicted as reliably aligned agreed with the structural alignments for between 92% and 97.4% of the residues. In loop regions just under 92% of the residues predicted to be reliable agreed with the structural alignments. The percentage of residues predicted as reliable ranged from 32.1% for helix residues to 52.8% for strand residues.This information could also be used to help predict conserved binding sites from sequence alignments. Residues in the template that were identified as binding sites, that aligned to an identical amino acid residue and where the sequence alignment agreed with the structural alignment were in highly conserved, high scoring regions over 80% of the time. This suggests that many binding sites that are present in both target and template sequences are in sequence-conserved regions and that there is the possibility of translating reliability to binding site prediction.     

Cyclin T: three forms for different roles in physiological and pathological functions

Cyclins are members of family of proteins involved in the cell cycle regulation. They are regulatory subunits of complexes with proteins called cyclin-dependent kinases (CDKs). There are three forms of cyclin T: cyclin T1, cyclin T2a, and T2b. All cyclin T contain an N-terminal "cyclin homology box," the most conserved region among different members of the cyclin family that serves to bind CDK9. In addition to the N-terminal cyclin domain, cyclin T contains a putative coiled-coil motif, a His-rich motif, and a C-terminal PEST sequence. The CDK9/cyclin T complex is able to activate gene expression in a catalytic-dependent manner, phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II. In addition, only cyclin T1 supports interactions between Tat and TAR. The interaction of Tat with cyclin T1 alters the conformation of Tat to enhance the affinity and specificity of the Tat:TAR interaction. On the other hand, CDK9/cyclin T2 complexes are involved in the regulation of terminal differentiation in muscle cells.     

cDNA array technology in melanoma: an overview

Genetic aberrations, mostly resulting in changes in gene expression, are critical events in cancer onset and progression. The advent of the cDNA array technology allows the screening and the efficient measurement of expression of thousands genes simultaneously in a wide spectrum of experimental and clinical models. This genomic scale approach is being currently used to obtain global views of human cancer gene expression and to identify genetic markers that might be important for diagnosis, prognosis, and therapy. This review discusses some recent findings obtained by means of cDNA arrays investigating the human melanoma.     

beta 1 Integrin-dependent cell adhesion to EMILIN-1 is mediated by the gC1q domain

EMILIN-1 (Elastin Microfibril Interface Located ProteIN), the prototype of the EMILIN family, consists of a cysteine-rich domain (EMI domain) at the N terminus, an extended region with a high potential coiled-coil structure, a short collagenous stalk, and a self-interacting globular gC1q-l domain. EMILIN-1 is an adhesive extracellular matrix constituent associated with elastic fibers, detected also in the proximity of cell surfaces. To localize the cell attachment site(s), monoclonal antibodies (mAbs) against EMILIN-1 or the gC1q-1 domain were used to inhibit cell attachment to EMILIN-1. Thus, one mAb mapping to the gC1q-1 domain caused complete inhibition of cell attachment. EMILIN-1 and gC1q-1 displayed a comparable dose-dependent ability to promote cell adhesion. Adhesion kinetics was similar to that of fibronectin (FN), reaching the maximum level of attachment at 20 min, but in the absence of cations adhesion was negligible. The relative adhesion strength to detach 50% of the cells was similar for EMILIN-1 and gC1q-1 (250-270 x g) but lower than that for FN (>500). Cell adhesion to EMILIN-1 or gC1q-1 was completely blocked by a function-blocking beta(1) integrin subunit mAb. In contrast, adhesion to the complement C1q component was totally unaffected. Among the various function-blocking mAbs against the alpha integrin subunits only the anti-alpha(4) fully abrogated cell adhesion to gC1q-1 and up to 70% to EMILIN-1. Furthermore, only K562 cells transfected with the alpha(4) integrin chain, but not wild type K562, were able to adhere to EMILIN-1 and were specifically inhibited by anti-alpha(4) function-blocking mAb. Finally, cells attached to EMILIN-1 or gC1q-1, compared with cells plated on FN or vitronectin, which appeared well spread out on the substrate with prominent stress fibers and focal contacts, were much smaller with wide ruffles and a different organization status of the actin cytoskeleton along the cell periphery. This pattern was in accord with the ability of EMILIN-1 to promote cell movement.     

Relationship between monokaryotic growth rate and mating type in the edible basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBalpha and matBbeta) and to correlate the presence of specific matBalpha and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

Rat carotid artery dilation by PTCA balloon catheter induces neointima formation in presence of IEL rupture

The best animal angioplasty model is the porcine model, which is expensive and not available in all laboratories. The aim of this study was to describe a new rat model of angioplasty. An injury was induced with the use of a standard percutaneous transluminal coronary angioplasty (PTCA) 1.5-mm balloon catheter. The neointimal tissue, arterial dimensions, and the injury index were assessed following angioplasty. Ki-67 expression was detected to evaluate cell turnover after balloon angioplasty. In contrast with the standard Clowes model, a significant neointimal formation was detected only in the presence of ruptured internal elastic lamina (IEL). A positive correlation between the percentage of ruptured IEL and the amount of neointimal tissue was also demonstrated. The percentage of IEL fracture correlates with the proliferation index by anti-Ki-67 immunolabeling 7 and 14 days after the angioplasty. Significant arterial negative remodeling was observed following PTCA balloon dilation. In conclusion, our inexpensive animal model of restenosis after angioplasty may have great relevance toward a better understanding of the mechanisms and toward assessment of new therapeutical strategies for this phenomenon.     

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.