Identification of a Site in Sar1 Involved in the Interaction with the Cytoplasmic Tail of Glycolipid Glycosyltransferases

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1^D198A) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1^D189A also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.     

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.     

Sugarcane (Saccharum X officinarum): A Reference Study for the Regulation of Genetically Modified Cultivars in Brazil

Global interest in sugarcane has increased significantly in recent years due to its economic impact on sustainable energy production. Sugarcane breeding and better agronomic practices have contributed to a huge increase in sugarcane yield in the last 30?years. Additional increases in sugarcane yield are expected to result from the use of biotechnology tools in the near future. Genetically modified (GM) sugarcane that incorporates genes to increase resistance to biotic and abiotic stresses could play a major role in achieving this goal. However, to bring GM sugarcane to the market, it is necessary to follow a regulatory process that will evaluate the environmental and health impacts of this crop. The regulatory review process is usually accomplished through a comparison of the biology and composition of the GM cultivar and a non-GM counterpart. This review intends to provide information on non-GM sugarcane biology, genetics, breeding, agronomic management, processing, products and byproducts, as well as the current technologies used to develop GM sugarcane, with the aim of assisting regulators in the decision-making process regarding the commercial release of GM sugarcane cultivars.     

DNA vaccines against dengue virus type 2 based on truncate envelope protein or its domain III

Two DNA vaccines were constructed encoding the ectodomain (domains I, II and III) of the DENV2 envelope protein (pE1D2) or only its domain III (pE2D2), fused to the human tissue plasminogen activator signal peptide (t-PA). The expression and secretion of recombinant proteins was confirmed in vitro in BHK cells transfected with the two plasmids, detected by immunofluorescence or immunoprecipitation of metabolically labeled gene products, using polyclonal and monoclonal antibodies against DENV2. Besides, results reveal that the ectodomain of the E protein can be efficiently expressed in vivo, in a mammalian system, without the prM protein that is hypothesized to act as a chaperonin during dengue infection. Balb/c mice were immunized with the DNA vaccines and challenged with a lethal dose of DENV2. All pE1D2-vaccinated mice survived challenge, while 45% of animals immunized with the pE2D2 died after infection. Furthermore, only 10% of pE1D2-immunized mice presented some clinical signs of infection after challenge, whereas most of animals inoculated with the pE2D2 showed effects of the disease with high morbidity degrees. Levels of neutralizing antibodies were significantly higher in pE1D2-vaccinated mice than in pE2D2-immunized animals, also suggesting that the pE1D2 vaccine was more protective than the pE2D2.     

Biological and conformational evaluation of angiotensin II lactam bridge containing analogues

Angiotensin II (AII) is the active octapeptide product of the renin enzymatic cascade, which is responsible for sustaining blood pressure. In an attempt to establish the AII-receptor-bound conformation of this octapeptide, we designed conformationally constrained analogues by scanning the entire AII sequence with an i-(i+2) and i-(i+3) lactam bridge consisting of an Asp-(Xaa)(n)-Lys scaffold. Most analogues presented low agonistic activity when compared to AII in the different bioassays tested. The exceptions are cyclo(0-1a) [Asp(0), endo-(Lys(1a))]-AII (1) and [Asp(0), endo-(Lys(1a))]-AII (2), both of which showed activity similar to AII. Based on peptide 1 and the analogue cyclo(3-5)[Sar(1), Asp(3), Lys(5)]-AII characterized by Matsoukas et al., we analyzed the agonistic and antagonistic activities, respectively, through a new monocyclic peptide series synthesized by using the following combinations of residues as bridgehead elements for the lactam bond formation: D- or L-Asp combined with D- or L-Lys or L-Glu combined with L-Orn. Six analogues showed an approximately 20% increase in biological activity when compared with peptide (1) and were equipotent to AII. In contrast, six analogues presented antagonistic activity. These results suggest that the position of the lactam bridge is more important than the bridge length or chirality for recognition of and binding to the angiotensin II AT1-receptor.     

Antioxidant activity of new ruthenium compounds

Many biological properties have been attributed to ruthenium complexes including anti-tumor activity and the attenuation of reperfusion damage and infarct size. In this work, we characterize the antioxidant activity of trans-[RuCl2(nic)4] where nic is 3-pyridinecarboxylic acid and trans-[RuCl2(i-nic)4] where i-nic is 4-pyridinecarboxylic acid by (i) evaluation of total antioxidant potential (TRAP); (ii) prevention of DNA damage induced by hydrogen peroxide using the alkaline comet assay; and (iii) the prevention of lipid peroxidation and cell death induced by iron in liver slices. Our results suggest that nic has stronger antioxidant potential when compared to the i-nic. Higher doses (above 200 microM) of these compounds gave genotoxic effects, but the antioxidant potential could be obtained with the use lower doses (0.1-10 microM).     

Crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A(2) complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors at 1.9- and 1.45-A resolution

An acidic phospholipase A(2) (PLA(2)) isolated from Bothrops jararacussu snake venom was crystallized with two inhibitors: alpha-tocopherol (vitamin E) and p-bromophenacyl bromide (BPB). The crystals diffracted at 1.45- and 1.85-A resolution, respectively, for the complexes with alpha-tocopherol and p-bromophenacyl bromide. The crystals are not isomorphous with those of the native protein, suggesting the inhibitors binding was successful and changes in the quaternary structure may have occurred.     

Expression of Pathogenicity-Related Genes of Xylella fastidiosa In Vitro and In Planta

Xylella fastidiosa is responsible for several economically important plant diseases. It is currently assumed that the symptoms are caused by vascular occlusion due to biofilm formation. Microarray technology was previously used to examine the global gene expression profile of X. fastidiosa freshly isolated from symptomatic plants or after several passages by axenic culture medium, and different pathogenicity profiles have been obtained. In the present study the expression of some pathogenicity-related genes was evaluated in vitro and in planta by RT-PCR. The results suggest that adhesion is important at the beginning of biofilm formation, while the genes related to adaptation are essential for the organisms maintenance in planta. Similar results were observed in vitro mainly for the adhesion genes. The pattern of expression observed suggests that adhesion modulates biofilm formation whereas the expression of some adaptation genes may be related to the environment in which the organism is living.     

Chrysanthemum chlorotic mottle viroid RNA: dissection of the pathogenicity determinant and comparative fitness of symptomatic and non-symptomatic variants

Chrysanthemum chlorotic mottle viroid (CChMVd) is a small RNA (398-401nt) with hammerhead ribozymes in both polarity strands that mediate self-cleavage of the oligomeric RNA intermediates generated in a rolling-circle mechanism of replication. Within the in vivo branched RNA conformation of CChMVd, a tetraloop has been identified as a major determinant of pathogenicity. Here we present a detailed study of this tetraloop by site-directed mutagenesis, bioassay of the CChMV-cDNA clones and analysis of the resulting progenies. None of the changes introduced in the tetraloop, including its substitution by a triloop or a pentaloop, abolished infectivity. In contrast to observations for other RNAs, the thermodynamically stable GAAA tetraloop characteristic of non-symptomatic CChMVd-NS strains was not functionally interchangeable for other stable tetraloops of the UNCG family, suggesting that the sequence, rather than the structure, is the major factor governing conservation of this motif. In most cases, the changes introduced initially led to symptomless infections, which eventually evolved to be symptomatic concurrently with the prevalence in the progeny of the UUUC tetraloop characteristic of symptomatic CChMVd-S strains. Only in one case did the GAAA tetraloop emerge and eventually dominate the progeny in infected plants that were non-symptomatic. These results revealed two major fitness peaks in the tetraloop (UUUC and GAAA), whose adjacent stem was also under strong selection pressure. Co-inoculations with CChMVd-S and -NS variants showed that only when the latter was in a 100- or 1000-fold excess did the infected plants remain symptomless, confirming the higher biological fitness of the S variant and explaining the lack of symptom expression previously observed in cross-protection experiments.