v-myb Transformation of Xeroderma pigmentosum human fibroblasts: Overexpression of the c-Ha-ras oncogene in the transformed cells

Human Xeroderma pigmentosum “normal” fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental “normal” AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotipically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.     

Evidence of doubly uniparental inheritance of the mitochondrial DNA in Polititapes rhomboides (Bivalvia,Veneridae): Evolutionary and population genetic analysis of F and M mitotypes

Doubly uniparental inheritance (DUI) is a particular mitochondrial DNA inheritance mode reported in a number of bivalves. DUI species show two types of mtDNA, one transmitted from females to daughters and sons (F mitotype) and another one from males to sons (M mitotype). In Veneridae, the existence of DUI has been investigated in several species but it was found in only two of them. In this study, we obtained partial sequences of rrnL, cytb and cox1 genes of males and females of Polititapes rhomboides from NW Spain and we demonstrated the existence of heteroplasmy in males, as expected under DUI. F and M mitotypes showed a taxon-specific phylogenetic pattern and similar evolutionary rates. We focused on cox1 for population genetic analysis, examining separately F and M mitotypes, but also F mitotypes from females (F_♀) and males (F_♂). In all cases, cox1 bears signs of strong purifying selection, with no apparent evidence of relaxed selection in the M genome, while the divergence between F and M genomes is in agreement with the neutral model of evolution. The cox1 polymorphism, higher at the M than at the F genome, also shows clear footprints of genetic hitchhiking with favourable mutations at other mtDNA loci, except for F_♂. In terms of population structure, results suggest that the pattern depends on the examined mitotype (F, F_♀, F_♂ or M).     

Synthesis and Antiviral Activity of 5′-O-Sulfamoyluridine Derivatives

Abstract

A series of 5′-O-[[[[(alkyl)oxy]carbonyl] amino] sulfonyl] uridines have been synthesized by reaction of cyclohexanol, palmityl alcohol, 1,2-di-O-benzoylpropanetriol and 2,3,4,6-tetra-O-benzoyl-L-glucopyranose with chlorosulfonyl isocyanate and 2,3′-O-isopropylidene-uridine. Another series of 5′-O-(N-ethyl and N-isopropylsulfamoyl) uridines have been prepared by reaction of 2′,3′-O-isopropylidene and 2′,3′-di-O-acetyluridine with N-ethylsulfamoyl and N-isopropylsulfamoyl chlorides. All compounds were tested against HSV-2, VV, SV and ASFV viruses. 2′,3′-Di-O-acetyl-5′-O-(N-ethyl and N-isopropylsulfamoyl) uridine showed significant activities against HSV-2. 5′-O-[[[[(2,3,4,6-Tetra-O-benzoyl-β-L-glucopyranosyl)oxy]carbonyl]amino] sulfonyl]-2′,3′-O-isopropylideneuridine was very active against ASFV.     

Generalized Baum-Welch Algorithm Based on the Similarity between Sequences

The profile hidden Markov model (PHMM) is widely used to assign the protein sequences to their respective families. A major limitation of a PHMM is the assumption that given states the observations (amino acids) are independent. To overcome this limitation, the dependency between amino acids in a multiple sequence alignment (MSA) which is the representative of a PHMM can be appended to the PHMM. Due to the fact that with a MSA, the sequences of amino acids are biologically related, the one-by-one dependency between two amino acids can be considered. In other words, based on the MSA, the dependency between an amino acid and its corresponding amino acid located above can be combined with the PHMM. For this purpose, the new emission probability matrix which considers the one-by-one dependencies between amino acids is constructed. The parameters of a PHMM are of two types; transition and emission probabilities which are usually estimated using an EM algorithm called the Baum-Welch algorithm. We have generalized the Baum-Welch algorithm using similarity emission matrix constructed by integrating the new emission probability matrix with the common emission probability matrix. Then, the performance of similarity emission is discussed by applying it to the top twenty protein families in the Pfam database. We show that using the similarity emission in the Baum-Welch algorithm significantly outperforms the common Baum-Welch algorithm in the task of assigning protein sequences to protein families.     

Porcine reproductive and respiratory syndrome (PRRS) virus in wild boar and Iberian pigs in south-central Spain

Porcine reproductive and respiratory syndrome (PRRS) is a swine infectious disease causing major economic problems on the intensive pig industry. This virus has been reported worldwide in domestic pigs and there is evidence of PRRS virus (PRRSV) infection in wild boar (Sus scrofa). Nonetheless, the epidemiological role of wild boar and extensively kept domestic pigs remains unclear. The aim of this study was to determine the occurrence of PRRS in wild boar and Iberian pigs in the dehesa ecosystem of the Castile-La Mancha region of Spain, which boasts one of the most important free-roaming porcine livestock and hunting industries in the country. Using geo-spatial analysis of literature data, we first explored the relationship between domestic pig density and PRRS occurrence in wild boar in Europe. Results revealed that PRRS occurrence in wild boar may be influenced, albeit not significantly, by domestic pig density. Next, we analyzed sera from 294 wild boar and 80 Iberian pigs by indirect enzyme-linked immunosorbent assay for PRRSV antibodies. The sera and 27 wild boar tissue samples were analyzed by two real-time RT-PCR assays, targeting the most conserved genes of the PRRSV genome, ORF1 and ORF7. Seven wild boar (2.4 %) and one Iberian pig (1.3 %) were seropositive, while none of the animals tested positive for PRRSV by RT-PCR. Our results confirm the limited spread of PRRSV in free-roaming Iberian pigs and wild boar living in mutual contact. Further studies would be necessary to address whether this low seroprevalence found in these animals reflects transmission from intensively kept pigs or the independent circulation of specific strains in free-roaming pigs.     

Loss of the SV2-like Protein SVOP Produces No Apparent Deficits in Laboratory Mice

Neurons express two families of transporter-like proteins − Synaptic Vesicle protein 2 (SV2A, B, and C) and SV2-related proteins (SVOP and SVOPL). Both families share structural similarity with the Major Facilitator (MF) family of transporters. SV2 is present in all neurons and endocrine cells, consistent with it playing a key role in regulated exocytosis. Like SV2, SVOP is expressed in all brain regions, with highest levels in cerebellum, hindbrain and pineal gland. Furthermore, SVOP is expressed earlier in development than SV2 and is one of the neuronal proteins whose expression declines most during aging. Although SV2 is essential for survival, it is not required for development. Because significant levels of neurotransmission remain in the absence of SV2 it has been proposed that SVOP performs a function similar to that of SV2 that mitigates the phenotype of SV2 knockout mice. To test this, we generated SVOP knockout mice and SVOP/SV2A/SV2B triple knockout mice. Mice lacking SVOP are viable, fertile and phenotypically normal. Measures of neurotransmission and behaviors dependent on the cerebellum and pineal gland revealed no measurable phenotype. SVOP/SV2A/SV2B triple knockout mice did not display a phenotype more severe than mice harboring the SV2A/SV2B gene deletions. These findings support the interpretation that SVOP performs a unique, though subtle, function that is not necessary for survival under normal conditions.     

Apolipoprotein A-I Helsinki promotes intracellular acyl-CoA cholesterol acyltransferase (ACAT) protein accumulation

Reverse cholesterol transport is a process of high antiatherogenic relevance in which apolipoprotein AI (apoA-I) plays an important role. The interaction of apoA-I with peripheral cells produces through mechanisms that are still poorly understood the mobilization of intracellular cholesterol depots toward plasma membrane. In macrophages, these mechanisms seem to be related to the modulation of the activity of acyl-CoA cholesterol acyltransferase (ACAT), the enzyme responsible for the intracellular cholesterol ester biosynthesis that is stored in lipid droplets. The activation of ACAT and the accumulation of lipid droplets play a key role in the transformation of macrophages into foam cells, leading to the formation of atheroma or atherosclerotic plaque. ApoA-I Helsinki (or ?K107) is a natural apoA-I variant with a lysine deletion in the central protein region, carriers of which have increased atherosclerosis risk. We herein show that treatment of cultured RAW macrophages or CHOK1 cells with ?K107, but not with wild-type apoA-I or a variant containing a similar deletion at the C-terminal region (?K226), lead to a marked increase (more than 10 times) in the intracellular ACAT1 protein level as detected by western blot analysis. However, we could only detect a slight increase in cholesteryl ester produced by ?K107 mainly when Chol loading was supplied by low-density lipoprotein (LDL). Although a similar choline-phospholipid efflux is evoked by these apoA-I variants, the change in phosphatidylcholine/sphyngomyelin distribution produced by wild-type apoA-I is not observed with either ?K107 or ?K226.     

Willow seedlings from photooxidized seeds accelerate cotyledon death and anticipate first leaf emergence: a histological and biochemical study following germination

In willow seeds, photooxidative damage is mainly restricted to the outer cotyledonary tissues, significantly reducing normal germination. Here we analyzed the damage generated in cotyledonary tissues and investigated whether the increase in reactive oxygen species (ROS) generation in seedlings from photooxidized seeds can affect the morphogenetic capacity of the shoot apical meristem. Seeds were photooxidized under different light intensities and the evolution of the damage during seedling growth was studied by light and transmission electron microscopies. The level of lipid peroxidation and changes in antioxidant capacity were measured following the time course of superoxide dismutase, catalase, ascorbate peroxidase and guaiacol peroxidase enzyme activities, and the effect of photooxidative stress on the genesis of new leaf primordia and lateral roots was examined. Early and active endocytosis and autophagy, changes in chloroplast morphology, as well as the accumulation and diffusion of ROS all play important roles in the early cell death observed in cotyledonary tissues. Following germination, seedlings from photooxidized seeds anticipated the emergence of first leaves, which complemented the altered functionality of the damaged cotyledons.