Characterization by restriction fragment length polymorphism and sequence analysis of field and vaccine strains of infectious laryngotracheitis virus involved in severe outbreaks

At the end of 2002 and throughout 2003, there was a severe outbreak of infectious laryngotracheitis (ILT) in an intensive production area of commercial hens in the Sao Paulo State of Brazil. ILT virus was isolated from 28 flocks, and 21 isolates were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) using four genes and eight restriction enzymes, and by partial sequencing of the infected cell protein 4 (ICP4) and thymidine kinase (TK) genes. Three groups resulted from the combinations of PCR-RFLP patterns: 19 field isolates formed Group I, and the remaining two isolates together with the chicken embryo origin (CEO) vaccine strains formed Group II. Group III comprised the tissue-culture origin (TCO) vaccine strain by itself. The PCR-RFLP results agreed with the sequencing results of two ICP4 gene fragments. The ICP4 gene sequence analysis showed that the 19 field isolates classified into Group I by RFLP-PCR were identical among themselves, but were different to the TCO and CEO vaccines. The two Group II isolates could not be distinguished from one of the CEO vaccines. The nucleotide and amino acid sequence analyses discriminated between the Brazilian and non-Brazilian isolates, as well as between the TCO and CEO vaccines. Sequence analysis of the TK gene enabled classification of the field isolates (Group I) as virulent and non-vaccine. This work shows that the severe ILT outbreak was caused by a highly virulent, non-vaccine strain.

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha-lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position-1689 (g. AG) and+15 (g. AG) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide-46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A-1689, A-46, and A+15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5`-flanking and the 5`-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

Physical therapy assessment tools to evaluate disease progression and phenotype variability in Golden Retriever muscular dystrophy

Dogs suffering from Golden Retriever muscular dystrophy (GRMD) present symptoms that are similar to human patients with Duchenne muscular dystrophy (DMD). Phenotypic variability is common in both cases and correlates with disease progression and response to therapy. Physical therapy assessment tools were used to study disease progression and assess phenotypic variability in dogs with GRMD. At 5 (TO), 9 (T1), 13 (T2) and 17 (T3) months of age, the physical features, joint ranges of motion (ROM), limb and thorax circumferences, weight and creatine kinase (CK) levels were assessed in 11 dogs with GRMD. Alterations of physical features were higher at 13 months, and different disease progression rates were observed. Passive ROM decreased until 1 year old, which was followed by a decline of elbow and tarsal ROM. Limb and thorax circumferences, which were corrected for body weight, decreased significantly between TO and T3. These measurements can be used to evaluate disease progression in dogs with GRMD and to help discover new therapies for DMD patients. (C) 2011 Elsevier Ltd. All rights reserved.

Fossil trees in ancient fluvial channel deposits: evidence of seasonal and longer-term climatic variability

It has been established that large numbers of certain trees can survive in the beds of rivers of northeastern Australia where a strongly seasonal distribution of precipitation causes extreme variations in flow on both a yearly and longer-term basis. In these rivers, minimal flow occurs throughout much of any year and for periods of up to several years, allowing the trees to become established and to adapt their form in order to facilitate their survival in environments that experience periodic inundation by fast-flowing, debris-laden water. Such trees (notably paperbark trees of the angiosperm genus Melaleuca) adopt a reclined to prostrate, downstream-trailing habit, have a multiple-stemmed form, modified crown with weeping foliage, development of thick, spongy bark, anchoring of roots into firm to lithified substrates beneath the channel floor, root regeneration, and develop in flow-parallel, linear groves. Individuals from within flow-parallel, linear groves are preserved in situ within the alluvial deposit of the river following burial and death. Four examples of in situ tree fossils within alluvial channel deposits in the Permian of eastern Australia demonstrate that specialised riverbed plant communities also existed at times in the geological past. These examples, from the Lower Permian Carmila Beds, Upper Permian Moranbah Coal Measures and Baralaba Coal Measures of central Queensland and the Upper Permian Newcastle Coal Measures of central New South Wales, show several of the characteristics of trees described from modern rivers in northeastern Australia, including preservation in closely-spaced groups. These properties, together with independent sedimentological evidence, suggest that the Permian trees were adapted to an environment affected by highly variable runoff, albeit in a more temperate climatic situation than the modem Australian examples. It is proposed that occurrences of fossil trees preserved in situ within alluvial channel deposits may be diagnostic of environments controlled by seasonal and longer-term variability in fluvial runoff, and hence may have value in interpreting aspects of palaeoclimate from ancient alluvial successions. (C) 2001 Elsevier Science B.V. All rights reserved.

Essential role of the N-terminal autoregulatory sequence in the regulation of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine and inhibited by its cofactor tetrahydrobiopterin (BH4). The crystal structure of PAH revealed that the N-terminal sequence of the enzyme (residues 19-29) partially covered the enzyme active site, and suggested its involvement in regulation. We show that the protein lacking this N-terminal sequence does not require activation by phenylalanine, shows an altered structural response to phenylalanine, and is not inhibited by BH4. Our data support the model where the N-terminal sequence of PAH acts as an intrasteric autoregulatory sequence, responsible for transmitting the effect of phenylalanine activation to the active site, (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia

Henneguya lesteri n. sp, (Myxosporea) is described from sand whiting, Sillago analis, from the southern Queensland coast of Australia. H. lesteri displays a preference for the pseudobranchs and is typically positioned along the afferent blood vessels, displacing the adjoining lamellae and disrupting their normal array, The plasmodia appeared as whitish-hyaline, elliptical cysts (mean dimensions 230 x 410 mum) attached to the oral mucosa lining of the hyoid arch on the inner surface of the operculum. Infections of the gills were also found, in which the plasmodia were spherical, averaged 240 x 240 mum in size and were located on the inner hemibranch margin. The parasites lodged in the gill filament crypts and generated a mild hyperplastic response of the branchial epithelium, In histological sections, the plasmodium wall and adjoining ectoplasm appeared as a finely granulated, weakly eosinophilic layer, Ultrastructurally, this section of the host-parasite interface contained an intricate complex of pinocytotic channels. H. lesteri is polysporic, disporoblastic and pansporoblast forming. Sporogenesis is asynchronous, with the earliest developmental stages aligned predominantly along the plasmodium periphery, and maturing sporoblasts and spores toward the center. Ultrastructural details of sporoblast and spore development are in agreement with previously described myxosporeans. The mature spore is drop-shaped, length (mean) 9.1 mum, width 4.7 mum, thickness 2.5 mum, and comprises 2 polar capsules positioned closely together, a binucleated sporoplasm and a caudal process of 12.6 mum. The polar capsules are elongated, 3.2 x 1.6 mum, with 4 turns of the polar filament. Mean length of the everted filament is 23.2 mum, Few studies have analyzed the 18S gene-of marine Myxosporea. In fact, H. lesteri is the first marine species of Henneguya to be characterized at the molecular level: we determined 1966 bp of the small-subunit (18S) rDNA, The results indicated that differences between this and the hitherto studied freshwater Henneguya species are greater than differences among the freshwater Henneguya species.

Laser-derived particle size data from CRP-3, Victoria Land Basin, Antarctica: Implications for sequence and seismic stratigraphy

Seven hundred and nineteen samples from throughout the Cainozoic section in CRP-3 were analysed by a Malvern Mastersizer laser particle analyser, in order to derive a stratigraphic distribution of grain-size parameters downhole. Entropy analysis of these data (using the method of Woolfe and Michibayashi, 1995) allowed recognition of four groups of samples, each group characterised by a distinctive grain-size distribution. Group 1, which shows a multi-modal distribution, corresponds to mudrocks, interbedded mudrock/sandstone facies, muddy sandstones and diamictites. Group 2, with a sand-grade mode but showing wide dispersion of particle size, corresponds to muddy sandstones, a few cleaner sandstones and some conglomerates. Group 3 and Group 4 are also sand-dominated, with better grain-size sorting, and correspond to clean, well-washed sandstones of varying mean grain-size (medium and fine modes, respectively). The downhole disappearance of Group 1, and dominance of Groups 3 and 4 reflect a concomitant change from mudrock- and diamictite-rich lithology to a section dominated by clean, well-washed sandstones with minor conglomerates. Progressive downhole increases in percentage sand and principal mode also reflect these changes. Significant shifts in grain-size parameters and entropy group membership were noted across sequence boundaries and seismic reflectors, as recognised in others studies.

Immunization with tumor-associated epitopes fused to an endoplasmic reticulum translocation signal sequence affords protection against tumors with down-regulated expression of MHC and peptide transporters

Treatment of human cancers with an inherent antigen-processing defect due to a loss of peptide transporters (TAP-1 and TAP-2) and/or MHC class I antigen expression remains a considerable challenge. There is now an increasing realization that tumor cells with down-regulated expression of TAP and/or MHC class I antigens display strong resistance to cytotoxic T lymphocyte (CTL)mediated immune control, and often fail to respond to the conventional immunotherapeutic protocols based on active immunization with tumor-associated epitopes (TAE) or adoptive transfer of tumor-specific T cells, In the present study, we describe a novel approach based on immunization with either genetically modified tumor cells or naked DNA vectors encoding TAE fused to an endoplasmic reticulum (ER) signal sequence (ER-TAE) which affords protection against challenge by melanoma cells with down-regulated expression of TAP-1/2 and MHC class I antigens. In contrast, animals immunized with a vaccine based on TAE alone showed no protection against tumor challenge. Although MHC-peptide tetramer analysis showed a similar frequency of antigen-specific CTL in both ER-TAE- and TAE-immunized mice, functional analysis revealed that CTL activated following immunization with ER-TAE displayed significantly higher avidity for TAE when compared to animals immunized with the TAE alone, These observations provide a new strategy in anti-cancer vaccine design that allows activation of a highly effective and well-defined CTL response against tumors with down-regulated expression of TAP and MHC class I antigens.

Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF(35)-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine,tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.

Valproic acid has temporal variability in urinary clearance of metabolites

The reasons for the intra- and interindividual variability in the clearance of valproic acid (VPA) have not been completely characterized. The aim of this study was to examine day-night changes in the clearance of 3-oxo-valproate (3-oxo-VPA), 4-hydroxy-valproate (4-OH-VPA), and valproic acid glucuronides under steady state. Six diurnally active healthy male volunteers ingested 200 mg sodium valproate 12 hourly, at 0800 and 2000, for 28 days. On the last study day, two sequential 12-h urine samples were collected commencing at 2000 the evening before. Plasma samples were obtained at the end of each collection. Following alkaline hydrolysis, urine was analyzed for concentrations of VPA, 3-oxo-VPA, and 4-OH-VPA. A separate aliquot was assayed for creatinine (CR). The plasma concentrations of VPA, 3-oxo-VPA, 2-en-VPA, and CR were determined. The analysis of VPA and its metabolites was performed by CC-MS. There was an increase in plasma 3-oxo-VPA concentration at 0800, sampling as compared to 2000 sampling (p < .05). The urinary excretion of 3-oxo-VPA and VPA glucuronides were decreased between 2000 and 0800, compared to between 0800, and 2000, by 30% and 50% respectively (p < .05). These results indicate a nocturnal decrease in renal clearance of 3-oxo-VPA rather than a decrease in the beta -oxidation of VPA at night. These differences were not explained by differences between the sampling periods in CR excretion. These results indicate the importance of collecting samples of 24-h duration when studying metabolic profiles of VPA.

The sequence of a 200 kb portion of a Plasmodium vivax chromosome reveals a high degree of conservation with Plasmodium falciparum chromosome 3

Within a 199 866 base pair (bp) portion of a Plasmodium vivax chromosome we identified a conserved linkage group consisting of at least 41 genes homologous to Plasmodium falciparum genes located on chromosome 3. There were no P. vivax homologues of the P. falciparum cytoadherence-linked asexual genes clag 3.2, clag 3.1 and a var C pseudogene found on the P. vivax chromosome. Within the conserved linkage group, the gene order and structure are identical to those of P. falciparum chromosome 3. This conserved linkage group may extend to as many as 190 genes. The subtelomeric regions are different in size and the P. vivax segment contains genes for which no P. falciparum homologues have been identified to date. The size difference of at least 900 kb between the homologous P. vivax chromosome and P. falciparum chromosome 3 is presumably due to a translocation. There is substantial sequence divergence with a much higher guanine + cytosine (G + C) content in the DNA and a preference for amino acids using GC-rich codons in the deduced proteins of P. vivax. This structural conservation of homologous genes and their products combined with sequence divergence at the nucleotide level makes the P. vivax genome a powerful tool for comparative analyses of Plasmodium genomes. (C) 2001 Elsevier Science B.V. All rights reserved.

Genetic variability in Australian isolates of Puccinia coronata f. sp avenae assessed with molecular and pathogenicity markers

In a preliminary survey of genetic variability among 12 Australian isolates of Puccinia coronata f. sp. avenae Fraser and Led (Pca) collected from 1966 to 1993, two relatively diverse (

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled-coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi. membranes. By immunoelectron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)-p230(GRIP) fusion protein, we show binding specifically to a subset of membranes of the trans-Golgi network (TGN). Real-time imaging of live HeLa cells revealed that the GFP-p230(GRIP) was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP-p230(GRIP) transfected cells. Analysis of Golgi-derived vesicles by sucrose gradient fractionation demonstrated that GFP-p230(GRIP) binds to a specific population of vesicles distinct from those labelled for beta -COP or gamma -adaptin. The GFP-p230(GRIP) fusion protein is recruited to the same vesicle population as full-length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans-Golgi network tubulovesicular carriers.