A unique origin for Sicilian (δβ)∘-thalassemia in 33 unrelated families and its rapid diagnostic characterization by PCR analysis

Direct sequencing and restriction enzyme digestion of the polymerase chain reaction (PCR) product encompassing the breakpoint were used to characterize the Sicilian ()-thalassemia deletion in 33 unrelated Italian subjects. All cases showed the same sequencing features at the breakpoint region, suggesting a unique origin for this deletion in Italy. We also describe a one-step PCR assay for the rapid screening of homozygotes and carriers of Sicilian ()-thalassemia by the simultaneous use of three specific oligonucleotides. This procedure could have an impact on genetic counseling of couples at risk for this type of thalassemia, and with respect to compound heterozygotes bearing a Sicilian chromosome.     

Genetic mapping in the presence of genotyping errors

Genetic maps are built using the genotypes of many related individuals. Genotyping errors in these data sets can distort genetic maps, especially by inflating the distances. We have extended the traditional likelihood model used for genetic mapping to include the possibility of genotyping errors. Each individual marker is assigned an error rate, which is inferred from the data, just as the genetic distances are. We have developed a software package, called TMAP, which uses this model to find maximum-likelihood maps for phase-known pedigrees. We have tested our methods using a data set in Vitis and on simulated data and confirmed that our method dramatically reduces the inflationary effect caused by increasing the number of markers and leads to more accurate orders.     

Salicylic acid activates nitric oxide synthesis in Arabidopsis

The relationship between nitric oxide (NO) and salicylic acid (SA) was investigated in Arabidopsis thaliana. Here it is shown that SA is able to induce NO synthesis in a dose-dependent manner in Arabidopsis. NO production was detected by confocal microscopic analysis and spectrofluorometric assay in plant roots and cultured cells. To identify the metabolic pathways involved in SA-induced NO synthesis, genetic and pharmacological approaches were adopted. The analysis of the nia1,nia2 mutant showed that nitrate reductase activity was not required for SA-induced NO production. Experiments performed in the presence of a nitric oxide synthase (NOS) inhibitor suggested the involvement of NOS-like enzyme activity in this metabolic pathway. Moreover, the production of NO by SA treatment of Atnos1 mutant plants was strongly reduced compared with wild-type plants. Components of the SA signalling pathway giving rise to NO production were identified, and both calcium and casein kinase 2 (CK2) were demonstrated to be involved. Taken together, these results suggest that SA induces NO production at least in part through the activity of a NOS-like enzyme and that calcium and CK2 activity are essential components of the signalling cascade.     

Induction of robust diabetes resistance and prevention of recurrent type 1 diabetes following islet transplantation by gene therapy

We have previously shown that the development of type 1 diabetes (T1D) can be prevented in nonobese diabetic (NOD) mice by reconstitution with autologous hemopoietic stem cells retrovirally transduced with viruses encoding MHC class II I-A beta-chain molecules associated with protection from the disease. In this study we examined whether a blockade of the programmed death-1 (PD-1)-programmed death ligand-1 (PD-L1) pathway, a major pathway known to control diabetes occurrence, could precipitate T1D in young NOD mice following reconstitution with autologous bone marrow retrovirally transduced with viruses encoding protective MHC class II I-A beta-chain molecules. In addition, we examined whether the expression of protective MHC class II alleles in hemopoietic cells could be used to prevent the recurrence of diabetes in mice with pre-existing disease following islet transplantation. Protection from the occurrence of T1D diabetes in young NOD mice by the expression of protective MHC class II I-A beta-chain molecules in bone marrow-derived hemopoietic cells was resistant to induction by PD-1-PD-L1 blockade. Moreover, reconstitution of NOD mice with pre-existing T1D autologous hemopoietic stem cells transduced with viruses encoding protective MHC class II I-A beta-chains allowed for the successful transplantation of syngeneic islets, resulting in the long-term reversal of T1D. Reversal of diabetes was resistant to induction by PD-1-PDL-1 blockade and depletion of CD25(+) T cells. These data suggest that expression of protective MHC class II alleles in bone marrow-derived cells establishes robust self-tolerance to islet autoantigens and is sufficient to prevent the recurrence of autoimmune diabetes following islet transplantation.     

Mechanisms involved in the formation of dopamine-induced intracellular bodies within striatal neurons

Recent studies demonstrated that methamphetamine (METH) produces intracellular bodies which are reminiscent of those occurring during degenerative disorders. In vivo studies demonstrate the occurrence of these morphological alterations both in the dopamine (DA) neurons of the substantia nigra and striatal cells. These consist of neuronal bodies staining for a variety of antigens belonging to the ubiquitin-proteasome pathway. The formation of these intracellular bodies both in the substantia nigra and PC12 cells depends on the presence of endogenous DA. In the present study, we analyze the mechanisms which lead to METH-induced intracellular bodies within non-dopaminergic striatal neurons. We found that METH is no longer able to produce inclusions in vivo, in striatal cells, when striatal DA is lost. Similarly, in vitro, in primary striatal cell cultures which do not possess DA, METH administration does not produce inclusions. On the other hand, administration of DA to striatal cell cultures produces neuronal inclusions and cell death, which are both related to the inhibition of the ubiquitin-proteasome system and activation of specific-DA receptors. In line with this, we produced subcellular alterations by administering dopamine agonists.     

PrPSc in salivary glands of scrapie-affected sheep

The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP(Sc)). PrP(Sc) was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP(Sc) concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP(Sc) in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP(Sc) in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

Neovascularization and mast cells with tryptase activity increase simultaneously in human pterygium

Mast cells (MC) have been implicated in both normal and pathological angiogenesis, such as that in chronic inflammatory diseases and tumors. This assumption is partially supported by the close structural association between MC and blood vessels and the recruitment of these cells during tumor growth. MC release a number of angiogenic factors among which tryptase, a serine protease stored in MC granules, is one of the most active. In this study, we correlate the extent of angiogenesis with the number of tryptase-reactive MC in tissue fragments from pterygium and normal bulbar conjunctiva investigated by immunohistochemistry, using two murine monoclonal antibodies against the endothelial cell marker CD31 and the MC marker tryptase. Angiogenesis, measured as microvessel density, was highly correlated with MC tryptase-positive cell count in pterygium tissues. These results suggest that the characteristic neovascularization observed in pterygium may be sustained, at least in part, by MC angiogenic mediators, in particular tryptase.     

A rapid method for the quantification of the enantiomers of Warfarin, Phenprocoumon and Acenocoumarol by two-dimensional-enantioselective liquid chromatography/electrospray tandem mass spectrometry

We describe a new fully validated enantioselective LC-MS/MS method for stereospecific quantification of both the racemic forms of Warfarin (WF), Phenprocoumon and Acenocoumarol in human plasma. Measurement specificity was assessed by using different blank donor plasma samples, where no interfering reagent peak appeared at the retention time (RT) of the targeted analytes. Response was linear for all analytes. Typical linear regression coefficients have >0.99. The recoveries ranged from 98% to 118%. Determinations in 10 normal healthy individuals revealed a high reproducibility of RTs. These findings confer to the method suitability for large population studies.     

The contribution of peroxynitrite generation in HIV replication in human primary macrophages

Background

The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA) derivatives active on the HIV-1 IN strand transfer (ST) step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR) reversing ability.

Results

The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells.

Conclusion

To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.