Detection and differentiation of yellow head complex viruses using monoclonal antibodies

Three monoclonal antibodies (MAbs) raised against pathogenic yellow head virus (YHV) from Thailand were tested against tissues of shrimp from Thailand, Australia, Ecuador and India that were purported to be infected with yellow head complex viruses. MAbs V-3-2B and Y-18 were specific to gp116 and gp64 envelope proteins, respectively, while Y-19 was specific to a 20 kDa putative nucleoprotein p20. As a preliminary step, the site of reactivity of the 3 MAbs in YHV was determined by immuno-electron microscopy using ultra-thin sections of YHV-infected shrimp tissue and negatively stained, semi-purified YHV particles. As expected, MAb Y-19 reacted with viral nucleocapsids in ultra-thin sections but not with negatively stained, whole virions; MAb V-3-2B did react with negatively stained, whole virions, but not with virions or nucleocapsids in ultra-thin sections. Unexpectedly, MAb Y-18 did not react with whole or sectioned virions. By immunohistochemistry, MAbs Y-19 and Y-18 reacted with Penaeus monodon tissues infected with either YHV or with gill-associated virus (GAV) from Australia, while MAb V-3-2B reacted with YHV only. In addition, all the YHV and GAV tissue samples gave positive in situ hybridization reactions with a cDNA probe specific to the ORF1b gene of YHV. They also gave expected differential RT-PCR results for YHV and GAV. By contrast, 2 natural Thai shrimp specimens with no gross signs of disease gave similar immunohistochemical reactions and RT-PCR reactions to GAV. However, sequencing of their RT-PCR products showed that they shared 92.7% identity with GAV, but only 79.0% identity with YHV. Although specimens from Ecuador and India displayed histopathology suggestive of YHV infection, they gave negative immunohistochemical reactions with all 3 Mabs, and negative in situ hybridization results. Additional work is required to determine whether a virus from the yellow head complex was responsible for their observed histopathology. These data show that the 3 YHV MAbs could be used in diagnostic situations to differentiate some viruses in the yellow head virus complex.     

First isolation of an aquatic birnavirus from farmed and wild fish species in Australia

During routine sampling and testing, as part of a systematic surveillance program (the Tasmanian Salmonid Health Surveillance Program), an aquatic birnavirus was isolated from 'pin-head' (fish exhibiting deficient acclimatisation on transfer to saltwater) Atlantic salmon Salmo salar, approximately 18 mo old, farmed in net-pens located in Macquarie Harbour on the west coast of Tasmania, Australia. The isolate grows readily in a range of fish cell lines including CHSE-214, RTG-2 and BF-2 and is neutralised by a pan-specific rabbit antiserum raised against infectious pancreatic necrosis virus (IPNV) Ab strain and by a commercial pan-specific IPNV-neutralising monoclonal antibody. Presence of the virus was not associated with gross clinical signs. Histopathological examination revealed a range of lesions particularly in pancreatic tissue. The virus was localised in pancreas sections by immunoperoxidase staining using the polyclonal antiserum and by electron microscopy. Examination by electron microscopy demonstrated that the virus isolated in cell culture (1) belongs to the family Birnaviridae, genus Aquabirnaviridae; (2) was ultrastructurally and antigenically similar to virus identified in the index fish; (3) is related to IPNV. Western blot analysis using the polyclonal rabbit antiserum confirmed the cross-reactions between various aquatic birnavirus isolates. In addition, PCR analysis of isolated viral nucleic acid from the index case indicated that the virus is more closely related to IPNV fr21 and N1 isolates than to other birnavirus isolates available for comparison. Sampling of other fish species within Macquarie Harbour has demonstrated that the virus is present in several other species of fish including farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes.     

Hormonal variation of rat uterine contractile responsiveness to selective neurokinin receptor agonists

Regulated uterine contractions are important in many reproductive functions such as sperm transport and embryo positioning during implantation. The role of classical neurotransmitters including acetylcholine and norepinephrine in regulating myometrial contractility has been well studied; however, the peripheral role of sensory neurotransmitters such as the neurokinins is less clear. The major neurokinins are substance P, neurokinin A, and neurokinin B, which predominantly activate neurokinin receptors (NK-Rs) 1, 2, and 3, respectively. This study utilized selective receptor agonists to examine the role of NK-Rs in uterine contractility. Uterine tissues, obtained from the major stages of the rat estrous cycle, were stimulated with selective NK-R agonists. Addition of each agonist resulted in a significant contractile response. However, the magnitude and nature of the response were dependent upon the stage of the estrous cycle, with responses to all agonists being significantly decreased in tissue from proestrus and estrus. Furthermore, the nature of NK3-R-mediated contraction was different in tissue from proestrus and estrus compared to metestrus and diestrus. The hormonal dependence of NK-R-mediated contractility was then examined in the ovariectomized estrogen-supplemented rat model. These studies confirmed that the magnitude and nature of uterine contractility in response to NK-R activation depend upon the hormonal environment.     

CXCR4 receptor expression on human retinal pigment epithelial cells from the blood-retina barrier leads to chemokine secretion and migration in response to stromal cell-derived factor 1 alpha

Retinal pigment epithelial (RPE) cells form part of the blood-retina barrier and have recently been shown to produce various chemokines in response to proinflammatory cytokines. As the scope of chemokine action has been shown to extend beyond the regulation of leukocyte migration, we have investigated the expression of chemokine receptors on RPE cells to determine whether they could be a target for chemokine signaling. RT-PCR analysis indicated that the predominant receptor expressed on RPE cells was CXCR4. The level of CXCR4 mRNA expression, but not cell surface expression, increased on stimulation with IL-1beta or TNF-alpha. CXCR4 protein could be detected on the surface of 16% of the RPE cells using flow cytometry. Calcium mobilization in response to the CXCR4 ligand stromal cell-derived factor 1alpha (SDF-1alpha) indicated that the CXCR4 receptors were functional. Incubation with SDF-1alpha resulted in secretion of monocyte chemoattractant protein-1, IL-8, and growth-related oncogene alpha. RPE cells also migrated in response to SDF-1alpha. As SDF-1alpha expression by RPE cells was detected constitutively, we postulate that SDF-1-CXCR4 interactions may modulate the affects of chronic inflammation and subretinal neovascularization at the RPE site of the blood-retina barrier.     

Redistribution of cyclic GMP in response to sodium butyrate in colon cells

The effect of butyrate on the response to guanylin and Escherichia coli heat-stable enterotoxin, STa, was assessed in T84 cells and Caco-2 cells, cultured colon cell lines possessing the guanylyl cyclase C which is the receptor for these peptides. Butyrate treatment of these cells resulted in an apparent increase in cyclic GMP (cGMP) accumulation when the cGMP content of cells and the supernatant medium was measured. Butyrate treatment did not change the guanylyl cyclase activity or (125)I-STa binding parameters in T84 cells, but the butyrate effect was completely blocked by cycloheximide. Butyrate did not have any effect on STa-stimulated cGMP accumulation in COS cells transfected with the human or porcine GC-C. Further experiments showed that butyrate treatment caused a large increase in the cGMP released into the culture medium, and in cells grown in polarized fashion in Transwell inserts, cGMP efflux was predominantly from the basolateral surface of the cell; intracellular cGMP was actually lowered by butyrate treatment. Exposure of T84 cells to butyrate had no effect on the disposition of cyclic AMP generated in response to forskolin. The effects of butyrate on cGMP were reversible within 24 h of butyrate withdrawal. In colon cells, butyrate treatment induced a previously undescribed, cGMP-specific efflux mechanism which lowered intracellular cGMP and elevated extracellular cGMP in response to peptide agonists such as guanylin and STa.     

Variability in reproductive traits in Jatropha curcas L. accessions during early developmental stages under warm subtropical conditions

Variability in floral, fruit, and seed characteristics, and oil content of 15 accession of Jatropha curcas during early development were assessed during two flowering periods in south Florida subtropical climate. The two flowering periods had leaf flushing in March. Field evaluation using 18 quantitative traits showed significant variation among accessions. The number of female flowers and female : male flower ratio ranged from 1 to 15 and 1 : 8.8 to 1 : 67.8, respectively. Fruit set by natural pollination was 89 and 66% during the first (1st) and second (2nd) flowering periods, respectively. A higher number of female‐type inflorescences were observed during summer. There were significant differences in seed traits, except for number of seeds per fruit. Accession TREC 31 had the highest individual seed dry weight and 100‐seed weight (0.83 g and 79.7 g, respectively). The oil content varied from 19.30% to 35.62%. Seed dry weight had positive correlation with seed fresh weight, seed length, seed thickness, seed width, and 100‐seed weight, but negative correlation with oil content. Based on the cluster analysis using 15 morphological traits, jatropha accessions were grouped into five main clusters and accessions from different geographic regions grouped together in a cluster. Principal component analyses (PCA) revealed morphological variation. The first three components explained 73.5% of the total variation and seed dry weight, 100‐seed weight, total flowers per inflorescence, male flowers per inflorescence and fruit set can be used to distinguish accessions. The PCA also indicated that flowering traits were more influenced by seed origin while seed traits were affected by flowering spans. Although evaluations were performed in plants during the juvenile phase, accessions TREC 31 and TREC 55 had superior averages for almost all characters evaluated. These results provide a preliminary assessment of the high variability in jatropha accessions evaluated and their potential for use in breeding and genetic improvement programs.     

Microbial processes of the carbon and sulfur cycles in the Chukchi Sea

The research performed in August 2004 within the framework of the Russian-American Long-term Census of the Arctic (RUSALCA) resulted in the first data concerning the rates of the key microbial processes in the water column and bottom sediments of the Bering strait and the Chukchi Sea. The total bacterial counts in the water column varied from 30 × 10³ cells ml^?1 in the northern and eastern parts to 245 × 10³ cells ml^?1 in the southern part. The methane content in the water column of the Chukchi sea varied from 8 nmol CH₄l^?1 in the eastern part of the sea to 31 nmol CH₄l^?1 in the northern part of the Herald Canyon. Microbial activity occurred in the upper 0–3 cm of the bottom sediments; the methane formation rate varied from 0.25 to 16 nmol CH₄dm^?3 day^?1. The rates of methane oxidation varied from 1.61 to 14.7 nmol CH₄dm^?3 day^?1. The rates of sulfate reduction varied from 1.35 to 16.2 μmol SO ₄ ^2? dm^?1 day^?1. The rate of methane formation in the sediments increased with depth, while sulfate reduction rates decreased (less than 1 μmol SO ₄ ^2? dm^?3 day^?1). These high concentrations of biogenic elements and high rates of microbial processes in the upper sediment layers suggest a specific type of trophic chain in the Chukchi Sea. The approximate calculated balance of methane emission from the water column into the atmosphere is from 5.4 to 57.3 μmol CH₄m^?2 day^?1.     

Rapid measurement of deuterium-labeled long-chain fatty acids in plasma by HPLC-ESI-MS

Imbalanced fatty acid metabolism contributes significantly to the increased incidence of metabolic disorders. Isotope-labeled fatty acids (2H, 13C) provide efficient means to trace fatty acid metabolism in vivo. This study reports a new and rapid method for the quantification of deuterium-labeled fatty acids in plasma by HPLC-MS. The sample preparation protocol developed required only hydrolysis, neutralization, and quenching steps followed by high-performance liquid chromatography-electrospray ionization-mass spectrometry analysis in negative ion mode using single ion monitoring. Deuterium-labeled stearic acid (d7-C18:0) was synthesized to reduce matrix interference observed with d5 analog, which improved the limit of detection (LOD) significantly, depending on the products analyzed. Linearity > 0.999 between the LOD (100 nM) and 30 microM, accuracy > 90%, precision > 88%, and adequate recovery in the dynamic range were obtained for d7-C18:0 and d7-oleic acid (C18:1). Upon oral dosing of d7-C18:0 in rats, the parent compound and its desaturation and beta-oxidation products, d7-C18:1 and d7-C16:0, were circulating with a maximal concentration ranging from 0.6 to 2.2 microM, with significant levels of d7-fatty acids detected for up to 72 h.     

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.