Molecular Evolution of the Pathogenicity Island of Enterotoxigenic Bacteroides fragilis Strains

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.     

Gill infection of Leporinus macrocephalus Garavello & Britski, 1988 (Osteichthyes: Anostomidae) by Henneguya leporinicola n. sp. (Myxozoa: Myxobolidae). Description, histopathology and treatment

Piau?us (Leporinus macrocephalus), were raised in 300 m2 ponds (density of 10 fish/m2) presenting asphyxia signals and daily mortality of 27 fishes. Specimens with 8-cm total body length, were collected for necropsy. Mucus of body surface and pieces of organs were collected and examined microscopically, in wet mounts, stained or in histological sections. The smears examination showed the presence of several spores in the secondary lamellae of the gill filaments, identified as Henneguya leporinicola n.sp (Myxozoa: Myxobolidae). Histopathological study showed epithelial hyperplasia and fulfilling of the spaces between the secondary lamellae, congestion and telangiectasia sinusoidal. It was also observed hyperplasia of the goblet cells and several cysts of parasite with 70.3 microns diameter. Such cysts were situated among the secondary lamellae, covered or not by the hyperplasic epithelium. With this diagnostic, three applications of formalin solution 10 ml/m3 were carried out. Fifteen days after that, fish were examined again to ascertain whether the treatment was efficient on disease caused by the protozoa. The tissue alterations present in the gills after the treatment were just a moderate sinusoidal congestion and a slight epithelial hyperplasia on the base of the secondary lamellae.     

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.     

Biophysical properties and functional significance of stem water storage tissues in Neotropical savanna trees

Biophysical characteristics of sapwood and outer parenchyma water storage compartments were studied in stems of eight dominant Brazilian Cerrado tree species to assess the impact of differences in tissue capacitance on whole-plant water relations. The rate of decline in tissue water potential with relative water content (RWC) was greater in the outer parenchyma than in the sapwood for most of the species, resulting in tissue-and species-specific differences in capacitance. Sapwood capacitance on a tissue volume basis ranged from 40 to 160 kg m-3 MPa-1, whereas outer parenchyma capacitance ranged from 25 to only 60 kg m-3 MPa-1. In addition, osmotic potentials at full turgor and at the turgor loss point were more negative for the outer parenchyma compared with the sapwood, and the maximum bulk elastic modulus was higher for the outer parenchyma than for the sapwood. Sapwood capacitance decreased linearly with increasing sapwood density across species, but there was no significant correlation between outer parenchyma capacitance and tissue density. Midday leaf water potential, the total hydraulic conductance of the soil/leaf pathway and stomatal conductance to water vapour (gs) all increased with stem volumetric capacitance, or with the relative contribution of stored water to total daily transpiration. However, the difference between the pre-dawn water potential of non-transpiring leaves and the weighted average soil water potential, a measure of the water potential disequilibrium between the plant and soil, increased asymptotically with total stem capacitance across species, implying that overnight recharge of water storage compartments was incomplete in species with greater capacitance. Overall, stem capacitance contributes to homeostasis in the diurnal and seasonal water balance of Cerrado trees.     

Haematological and biochemical reference intervals for farmed channel catfish

The reference intervals for biochemical variables and red blood cell indices of healthy intensively bred channel catfish Ictalurus punctatus were determined. The blood variables were determined using standardized clinical methods. The reference intervals (25th and 75th percentiles) were established using a non-parametric method. Reference intervals for plasma glucose, serum total protein, sodium, potassium, calcium, magnesium, chloride concentration, primary and secondary red blood cell indices were established. The haematological and biochemical reference intervals established may allow important clinical decisions about channel catfish.     

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.     

Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria

Heat and cold are environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of insects. The thermotolerance and cold activity of 60 entomopathogenic fungal isolates, including five species of Beauveria and one isolate of Engyodontium albus (=Beauveria alba) were examined as to tolerance of temperatures that might be encountered during field use. In addition, cold activity of eight Metarhizium spp. isolates was evaluated. The isolates were from various geographic regions, arthropod hosts or substrates. High variability in conidial thermotolerance was found among the Beauveria spp. isolates after exposure to 45 °C for 2 h, as evidenced by low (0-20%), medium (20-60%), or high germination (60-80%). The thermal death point (0% germination) for three rather thermotolerant B. bassiana isolates (CG 138, GHA and ARSEF 252) was 46 °C for 6 h. At low temperatures (5 °C), with few exceptions (e.g. CG 66, UFPE 479, CG 227, CG 02), most of the B. bassiana isolates germinated well (ca. 100%). On the other hand, only one isolate of Metarhizium sp. was cold-active (i.e. ARSEF 4343 from Macquarie Island, 54.4°S, Australia). This probably is a M. frigidum isolate. The E. albus isolate (UFPE 3138) was the most susceptible isolate to both heat and cold stress. Isolates ARSEF 252 and GHA of B. bassiana, on the other hand, presented exceptionally high thermotolerance and cold activity. Some isolates with high cold activity, however, were thermosensitive (e.g. ARSEF 1682) and others with high thermotolerance had low cold activity (e.g. CG 227). An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that B. bassiana isolates from higher latitudes were more cold-active than isolates from nearer the equator, but there was not a similar correlation for heat.