109 research outputs found
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A Fluorescent Antibody Test for Detection of the Rickettsia Causing Disease in Chilean Salmonids
An indirect fluorescent antibody test (IFAT) was developed for detection of the rickettsia that was causing epizootics among salmonids cultured in seawater net-pens in southern Chile. Antiserum against the rickettsial agent was produced in New Zealand white rabbits with a preparation grown in antibiotic-free chinook salmon embryo (CHSE-214) cell cultures and partially purified by a combination of filtration and centrifugation steps. The IFAT was effectively used on blood films, tissue sections, and smears. Two gram-negative and two gram-positive bacterial pathogens of salmonids did not react in this test. Detection of the rickettsial agent has previously been restricted to examination by light microscopy or isolation in salmonid cells. The IFAT provides a simple, rapid, sensitive method for detection of the agent and diagnosis of the disease. The rickettsia is thought to be a member of the tribe Ehrlichieae and was tested by IFAT with sera from animals infected with other rickettsial agents.Keywords: Fish diseases, Indirect fluorescent antibody test, Salmonids, Rickettsia, ChileKeywords: Fish diseases, Indirect fluorescent antibody test, Salmonids, Rickettsia, Chil
Heat Shock Protein 70 Expression in Juvenile Eastern Oysters, Crassostrea virginica (Gmelin, 1791), Exposed to Anoxic Conditions
Anoxic water events in conjunction with summer high temperatures are thought to be one of the causes of declines in natural oyster reefs on the eastern shore of Mobile Bay. Work is underway to determine whether tolerance to low oxygen can be selected for in hatchery-produced oysters. As a component of this work, the expression of heat shock protein 70 (HSP 70) was examined in control (normoxia) and anoxia-challenged juvenile oysters. Parental Eastern oysters, Crassostrea virginica were collected from 2 sites, Cedar Point Reef (CP), an area considered to have normoxic conditions, and White House Reef (WH), an area suspected to experience periodic anoxia. F1 generation oysters were produced from CP and WH parents that survived an anoxic exposure of 96 h. Control F1 generation oysters from both parental stocks not exposed to anoxia were also produced. The F1 generation oysters were subsequently exposed to anoxia or control normoxic conditions, and differences in expression of HSP 70 were examined. Nitrogen was used to create the anoxic conditions for both the parental and F1 generations. Three HSP 70 isoforms—2 constitutive forms (HSC 77 and HSC 72) and 1 inducible form (HSP 69)—were expressed in both anoxia- and normoxia-exposed oysters from all groups. Although there were differences among groups of oysters from the 2 sites, there were no differences in the expression of HSC 77 and HSC 72 between the control and anoxia-treated oysters within a group. Interestingly, the expression of HSP 69 was higher in oysters exposed to normoxia than the ones from anoxia treatments. These differences are thought to reflect a combination of responses to nutritional stress in the controls and facultative anaerobiosis and metabolic arrest in the anoxia groups
Open Sequence Initiative: a part submission standard to complement modern DNA assembly techniques
The discipline of synthetic biology emphasizes the application of engineering principles such as standardization, abstraction, modularity, and rational design to complex biological systems. The archetypical example of such standardization is BioBrick RFC[10], introduced in 2003 by Tom Knight at MIT. BioBricks are stored on a standard plasmid, pSB1C3, which contains prefix and suffix sequences flanking the DNA sequence specifying a biological part. The prefix and suffix sequences contain two pairs of 6 base-pair (bp) restriction enzyme sites (EcoRI+XbaI and SpeI+PstI), which can be used for both part assembly and quality control. BioBricks are intended to be well- characterized biological parts, such as genes or promoters, that function in a predictable fashion and can be readily combined to make complex systems. The rules of the RFC[10] BioBrick assembly method require that none of the restriction sites used in the prefix and suffix be present in the parts themselves. This requirement can be an onerous imposition for iGEM teams developing large, novel parts, such as genes or entire operons that are obtained by amplifying DNA sequences from environmental samples or microorganisms.
While iGEM teams may use methods such as site-directed mutagenesis to remove illegal restriction sites from a part's sequence, it is certainly possible that this mutation will alter the functionality of the part – a very undesirable outcome. In addition, the mutagenesis of illegal restriction sites is an unnecessary burden on teams, given the limited time and resources available to teams during each year’s iGEM competition. Efforts spent mutagenizing sites would be better spent characterizing and improving parts. This RFC proposes an alternative submission standard to eliminate these problems
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Infectivity of a rickettsia isolated from coho salmon Oncorhynchus kisutch
Two species of salmonids were tested for their susceptibility to infection by a rickettsia isolated in cell culture from diseased coho salmon Oncorhynchus kisutch in Chile. Mortality approached 100% in coho and Atlantic salmon Salmo salar injected with 10-fold dilutions of cell culture medium containing the rickettsia. Typical disease signs were present in the coho salmon but were not observed in inoculated Atlantic salmon. However, the rickettsia was recovered in pure culture from moribund fish in each of the injected groups of either species. The rickettsia was thereby demonstrated to be pathogenic and the cause of the ongoing epizootic affecting salmonids cultured in Chile. Horizontal transmission was not demonstrated in a group of uninoculated coho salmon held in the same tank with experimentally infected fish.Keywords: Salmo salar, Rickettsia, Oncorhynchus kisutch, pathogensKeywords: Salmo salar, Rickettsia, Oncorhynchus kisutch, pathogen
An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either Piscirickettsia salmonis or IPNV
<p>Abstract</p> <p>Background</p> <p>Due to the limited number of species specific antibodies against fish proteins, differential gene expression analyses are vital for the study of host immune responses. Quantitative real-time reverse transcription PCR (qRT-PCR) is one of the most powerful tools for this purpose. Nevertheless, the accuracy of the method will depend on the careful selection of genes whose expression are stable and can be used as internal controls for a particular experimental setting.</p> <p>Findings</p> <p>The expression stability of five commonly used housekeeping genes [beta-actin (<it>ACTB</it>), elongation factor 1-alpha (<it>EF1A</it>), ubiquitin (<it>UBQ</it>), glyceraldehyd-3-phosphate dehydrogenase (<it>GAPDH</it>) and tubulin alpha (<it>TUBA</it>)] were monitored in salmonid cell lines CHSE-214 and RTS11 after infection with two of the most fastidious fish pathogens, the facultative bacterium <it>Piscirickettsia salmonis </it>and the aquabirnavirus IPNV (Infectious Pancreatic Necrosis Virus). After geNorm analysis, <it>UBQ </it>and <it>EF1A </it>appeared as the most stable, although <it>EF1A </it>was slightly upregulated at late stages of <it>P. salmonis </it>infection in RTS11. <it>ACTB </it>instead, showed a good performance in each case, being always considered within the three most stable genes of the panel. In contrast, infection-dependent differential regulation of <it>GAPDH </it>and <it>TUBA </it>was also demonstrated.</p> <p>Conclusion</p> <p>Based on the data presented here with the cell culture models CHSE-214 and RTS11, we suggest the initial choice of <it>UBQ</it>, <it>ACTB </it>and <it>EF1A </it>as reference genes in qRT-PCR assays for studying the effect of <it>P. salmonis </it>and IPNV on the host immune response.</p
Differential glucocorticoid metabolism in patients with persistent versus resolving inflammatory arthritis
Introduction: Impairment in the ability of the inflamed synovium to generate cortisol has been proposed to be a factor in the persistence and severity of inflammatory arthritis. In the inflamed synovium, cortisol is generated from cortisone by the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme. The objective of this study was to determine the role of endogenous glucocorticoid metabolism in the development of persistent inflammatory arthritis. Methods: Urine samples were collected from patients with early arthritis (symptoms ≤12 weeks duration) whose final diagnostic outcomes were established after clinical follow-up and from patients with established rheumatoid arthritis (RA). All patients were free of disease-modifying anti-rheumatic drugs at the time of sample collection. Systemic measures of glucocorticoid metabolism were assessed in the urine samples by gas chromatography/mass spectrometry. Clinical data including CRP and ESR were also collected at baseline. Results: Systemic measures of 11β-HSD1 activity were significantly higher in patients with early arthritis whose disease went on to persist, and also in the subgroup of patients with persistent disease who developed RA, when compared with patients whose synovitis resolved over time. We observed a significant positive correlation between systemic 11β-HSD1 activity and ESR/CRP in patients with established RA but not in any of the early arthritis patients group. Conclusions: The present study demonstrates that patients with a new onset of synovitis whose disease subsequently resolved had significantly lower levels of systemic 11β-HSD1 activity when compared with patients whose synovitis developed into RA or other forms of persistent arthritis. Low absolute levels of 11β-HSD1 activity do not therefore appear to be a major contributor to the development of RA and it is possible that a high total body 11β-HSD1 activity during early arthritis may reduce the probability of disease resolution
Effects of cigarette smoke condensate on proliferation and wound closure of bronchial epithelial cells in vitro: role of glutathione
BACKGROUND: Increased airway epithelial proliferation is frequently observed in smokers. To elucidate the molecular mechanisms leading to these epithelial changes, we studied the effect of cigarette smoke condensate (CSC) on cell proliferation, wound closure and mitogen activated protein kinase (MAPK) activation. We also studied whether modulation of intracellular glutathione/thiol levels could attenuate CSC-induced cell proliferation. METHODS: Cells of the bronchial epithelial cell line NCI-H292 and subcultures of primary bronchial epithelial cells were used for the present study. The effect of CSC on epithelial proliferation was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Modulation of epithelial wound repair was studied by analysis of closure of 3 mm circular scrape wounds during 72 hours of culture. Wound closure was calculated from digital images obtained at 24 h intervals. Activation of mitogen-activated protein kinases was assessed by Western blotting using phospho-specific antibodies. RESULTS: At low concentrations CSC increased proliferation of NCI-H292 cells, whereas high concentrations were inhibitory as a result of cytotoxicity. Low concentrations of CSC also increased epithelial wound closure of both NCI-H292 and PBEC, whereas at high concentrations closure was inhibited. At low, mitogenic concentrations, CSC caused persistent activation of ERK1/2, a MAPK involved in cell proliferation. Inhibition of cell proliferation by high concentrations of CSC was associated with activation of the pro-apoptotic MAP kinases p38 and JNK. Modulation of intracellular glutathione (GSH)/thiol levels using N-acetyl-L-cysteine, GSH or buthionine sulphoximine (BSO), demonstrated that both the stimulatory and the inhibitory effects of CSC were regulated in part by intracellular GSH levels. CONCLUSION: These results indicate that CSC may increase cell proliferation and wound closure dependent on the local concentration of cigarette smoke and the anti-oxidant status. These findings are consistent with increased epithelial proliferation in smokers, and may provide further insight in the development of lung cancer
Caspase Inhibitors of the P35 Family Are More Active When Purified from Yeast than Bacteria
Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a “reactive site loop” within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity
Characterizing the anti-inflammatory and tissue protective actions of a novel Annexin A1 peptide
This work was supported by a
collaborative project between Unigene Corp. and
Queen Mary University of London and by the
William Harvey Research Foundation. JD is
supported by a Sir Henry Dale Fellowship jointly
funded by the Wellcome Trust and the Royal
Society (grant no: 107613/Z/15/Z). MP was
supported by the Wellcome Trust (grant no:
086867/Z/08)
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