Inhibition of human immunodeficiency virus type 1 and type 2 Tat function by transdominant Tat protein localized to both the nucleus and cytoplasm.

We introduced various mutations into the activation and RNA binding domains of human immunodeficiency virus type 1 (HIV-1) Tat in order to develop a novel and potent transdominant Tat protein and to characterize its mechanism of action. The different mutant Tat proteins were characterized for their abilities to activate the HIV LTR and inhibit the function of wild-type Tat in trans. A Tat protein containing a deletion of the basic domain (Tat(delta)49-57) localized exclusively to the cytoplasm of transfected human cells was nonfunctional and inhibited both HIV-1 and HIV-2 Tat function in a transdominant manner. Tat proteins containing mutations in the cysteine-rich and core domains were nonfunctional but failed to inhibit Tat function in trans. When Tat nuclear or nucleolar localization signals were fused to the carboxy terminus of Tat(delta)49-57, the chimeric proteins localized to the nucleus or nucleolus, respectively, and remained capable of acting in a transdominant manner. Introduction of secondary mutations in the cysteine-rich and core domains of the various transdominant Tat proteins completely eliminated their abilities to act in a transdominant fashion. Our data best support a mechanism in which these transdominant Tat proteins squelch a cellular factor or factors that interact with the Tat activation domain and are required for Tat to function.     

Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.     

Genetic polymorphisms in juvenile-onset diabetes

Nine genetic polymorphic systems (ACP1, PGM1, ADA, AK, G-6-PD, Hp, ABO, Rh, MN), were studied in a series of 138 subjects affected by JOD. Differences between diabetic patients and controls were observed in the distribution of phenotypes of the red cell acid phosphatase (ACP1), and the ABO and MN blood groups.     

Dentin Matrix Protein 1 and Dentin Sialophosphoprotein in Human Sound and Carious Teeth: An Immunohistochemical and Colorimetric Assay

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are extracellular matrix proteins produced by odontoblasts involved in the dentin mineralization. The aim this study was to compare the distribution of DMP1 and DSPP in human sound dentin vs human sclerotic dentin. Sixteen sound and sixteen carious human molars were selected, fixed in paraformaldehyde and processed for immunohistochemical detection of DMP1 and DSPP by means of light microscopy, transmission electron microscopy (TEM) and high-resolution field emission in-lens scanning electron microscopy (FEI-SEM). Specimens were submitted to a pre-embedding or a post-embedding immunolabeling technique using primary antibodies anti DMP1 and anti-DSPP and gold-conjugated secondary antibodies. Other samples were processed for the detection of DMP1 and DSPP levels. Dentin from these samples was mechanically fractured to powder, then a protein extraction and a protein level detection assay were performed. DMP1 and DSPP were more abundant in carious than in sound samples. Immunohistochemical analyses in sclerotic dentin disclosed a high expression of DMP1 and DSPP inside the tubules, suggesting an active biomineralization of dentin by odontoblasts. Furthermore, the detection of small amounts of these proteins inside the tubules far from the carious lesion, as shown in the present study, is consistent with the hypothesis of a preventive defense of all dentin after a noxious stimulus has undermined the tooth.Key words: sclerotic dentin, dentin matrix protein, dentin sialophosphoprotein, immunohistochemistry     

BlaSTorage: a fast package to parse, manage and store BLAST results

Background

Large-scale sequence studies requiring BLAST-based analysis produce huge amounts of data to be parsed. BLAST parsers are available, but they are often missing some important features, such as keeping all information from the raw BLAST output, allowing direct access to single results, and performing logical operations over them.

Findings

We implemented BlaSTorage, a Python package that parses multi BLAST results and returns them in a purpose-built object-database format. Unlike other BLAST parsers, BlaSTorage retains and stores all parts of BLAST results, including alignments, without loss of information; a complete API allows access to all the data components.

Conclusions

BlaSTorage shows comparable speed of more basic parser written in compiled languages as C++ and can be easily integrated into web applications or software pipelines.     

Effect of nitric oxide on the transport and release of oxygen in fetal blood

It is well known that nitric oxide (NO), the most important vasodilator agent, plays an important role in lowering vascular resistance in the human umbilical-placental circulation and that its deficiency is related to the pathogenesis of pre-eclamptic disorder. Besides it has recently been demonstrated that human hemoglobin (HbA) is able to transport nitric oxide, as S-nitrosohemoglobin (SNO-Hb), from the arterial to the venous blood. In the present study we examine the functional properties of the adult and fetal nitrosated hemoglobins to see if the double transport of oxygen and NO may influence the fetal oxygenation and the relation between maternal and fetal blood. Our results show that S-nitrosation significantly increases the oxygen affinity of the adult Hb (HbA) with respect to native protein (no-nitrosated) while the functional properties of HbF are less influenced. The oxygen affinity modification, found for SNO-HbA, was ascribed to the nitrosation of cysteine beta 93: really, the same residue is also present in the gamma chains of fetal hemoglobin, while the increase of affinity is less evidenced; hence, it is probable that the 39 aminoacidic substitutions between beta and gamma chains allay the effects of S-nitrosation. As regards the physiological modulators (protons, chloride ions, 2,3-diphosphoglyceric acid, and temperature), they influence the oxygen affinity of the two hemoglobins S-nitrosated, in equal mode with respect to the native forms determining the same variation on the oxygen affinity. Hence, our results evidence the fact that the NO release by SNO-HbA "in vivo" would be limited to regions of extremely low oxygen tension (such as hypoxic regions), while in fetus, SNO-HbF would unload nitric oxide and oxygen at pressure values close to normal.