Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors

In the presence of complementing adeno-associated virus type 2 (AAV-2) Rep proteins, AAV-2 genomes can be pseudotyped with the AAV-5 capsid to assemble infectious virions. Using this pseudotyping strategy, the involvement of the ubiquitin-proteasome system in AAV-5 and AAV-2 capsid-mediated infections was compared. A recombinant AAV-2 (rAAV-2) proviral luciferase construct was packaged into both AAV-2 and AAV-5 capsid particles, and transduction efficiencies in a number of cell lines were compared. Using luciferase expression as the end point, we demonstrated that coadministration of the viruses with proteasome inhibitors not only increased the transduction efficiency of rAAV-2, as previously reported, but also augmented rAAV-5-mediated gene transfer. Increased transgene expression was independent of viral genome stability, since there was no significant difference in the amounts of internalized viral DNA in the presence or absence of proteasome inhibitors. Western blot assays of immunoprecipitated viral capsid proteins from infected HeLa cell lysates and in vitro reconstitution experiments revealed evidence for ubiquitin conjugation of both AAV-2 and AAV-5 capsids. Interestingly, heat-denatured virus particles were preferential substrates for in vitro ubiquitination, suggesting that endosomal processing of the viral capsid proteins is a prelude to ubiquitination. Furthermore, ubiquitination may be a signal for processing of the capsid at the time of virion disassembly. These studies suggest that the previously reported influences of the ubiquitin-proteasome system on rAAV-2 transduction are also active for rAAV-5 and provide a clearer mechanistic framework for understanding the functional significance of ubiquitination.     

Selective excitation of GABAergic neurons in the substantia nigra of the rat by orexin/hypocretin in vitro

Dysfunction of the orexin/hypocretin neurotransmitter system leads to the sleep disorder narcolepsy. Narcolepsy is characterized by excessive daytime sleepiness and the occurrence of cataplexy--a sudden loss of muscle tone triggered by emotionally arousing events. Both symptoms can be treated with drugs that act on dopaminergic systems. Here we have investigated the effect of orexins on the firing of dopaminergic and GABAergic neurons of the substantia nigra (SN) in brain slices. Surprisingly, dopaminergic neurons in pars compacta were unaffected by orexins. In contrast, bath application of orexin A (100 nM) or orexin B (5-300 nM) greatly increased the firing rate of GABAergic neurons in pars reticulata. The orexin B-mediated excitation was unaffected by blocking synaptic transmission (using low-Ca2+/high-Mg2+ solution). However, the effect of orexin B was reduced significantly by thapsigargin (1 microM) and inhibitors of protein kinase A. The presence of orexinergic fibres in the SN pars reticulata was demonstrated by immunohistochemical methods with the fibre density increasing in the rostrocaudal direction. The orexin excitation of SN reticulata cells may help to maintain their high firing rate during waking. Furthermore, the absence of orexin effects in narcolepsy may predispose affected individuals to attacks of cataplexy.     

Clinical efficacy of teflubenzuron (Calicide) for the treatment of Lepeophtheirus salmonis infestations of farmed Atlantic salmon Salmo salar at low water temperatures

The efficacy of teflubenzuron (Calicide) for the treatment of farmed Atlantic salmon Salmo salar L. infested with sea lice Lepeophtheirus salmonis (Kr?yer, 1838), was investigated at low water temperatures in 2 commercial salmon farms. Calicide, coated on commercial feed pellets, was administered orally at 10 mg kg(-1) d(-1) for 7 consecutive days. Fish were randomly sampled and lice numbers recorded from both treated and control groups on 3 or 4 sampling occasions post-medication. Statistically significant reductions in the number of L. salmonis per fish were recorded. Maximum efficacy was observed toward chalimus and preadult stages of L. salmonis, and was achieved approximately 26 d post-medication. No adverse drug reactions or palatability problems were associated with the treatments.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Multifactor dimensionality reduction software for detecting gene-gene and gene-environment interactions

MOTIVATION: Polymorphisms in human genes are being described in remarkable numbers. Determining which polymorphisms and which environmental factors are associated with common, complex diseases has become a daunting task. This is partly because the effect of any single genetic variation will likely be dependent on other genetic variations (gene-gene interaction or epistasis) and environmental factors (gene-environment interaction). Detecting and characterizing interactions among multiple factors is both a statistical and a computational challenge. To address this problem, we have developed a multifactor dimensionality reduction (MDR) method for collapsing high-dimensional genetic data into a single dimension thus permitting interactions to be detected in relatively small sample sizes. In this paper, we describe the MDR approach and an MDR software package. RESULTS: We developed a program that integrates MDR with a cross-validation strategy for estimating the classification and prediction error of multifactor models. The software can be used to analyze interactions among 2-15 genetic and/or environmental factors. The dataset may contain up to 500 total variables and a maximum of 4000 study subjects. AVAILABILITY: Information on obtaining the executable code, example data, example analysis, and documentation is available upon request. SUPPLEMENTARY INFORMATION: All supplementary information can be found at http://phg.mc.vanderbilt.edu/Software/MDR.     

Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

We analyzed protein expression in preparations from whole testis in adult male Sprague-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8 (JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m(3) +/- 10%), simulating a range of possible human occupational exposures. Whole body inhalation exposures were carefully controlled to eliminate aerosol phase, and subjects were sacrificed within 48 h postexposure. Organ fractions were solubilized and separated via large-scale, high resolution two-dimensional electrophoresis, and gel patterns scanned, digitized and processed for statistical analysis. Seventy-six different testis proteins were significantly increased or decreased in abundance in vapor-exposed groups, compared to controls, and dose-response profiles were often nonlinear. A number of the proteins were identified by peptide mass fingerprinting and related to histopathological or physiological deficits shown in previously published studies to occur with repeated exposure to hydrocarbon fuels or solvents. These results demonstrate a significant effect of JP-8 exposure on protein expression, particularly in protein expression in the rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changes of equal or greater magnitude to those reported previously for shorter duration JP-8 aerosol exposures.     

While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.     

Collection of wind-borne haematophagous insects in the Torres Strait,Australia

Circumstantial evidence has implicated wind-borne mosquitoes (Diptera: Culicidae) in the introduction of Japanese encephalitis (JE) virus into Australia from the New Guinea mainland. A study was initiated on Saibai Island in the northern Torres Strait, during January and February 2000, to identify the potential source of insects collected in aerial (kytoon) and surface-level traps. Wind speed and direction were recorded to determine wind profiles during insect sampling. Northerly winds capable of carrying insects from New Guinea to Saibai Island were only present on three out of 18 nights sampled. Only three male mosquitoes, comprising two Verrallina funerea (Theobald) and one Ochlerotatus vigilax (Skuse), were collected in aerial samples, and were most likely of local origin. Culicoides midges were also collected in aerial nets and included gravid/parous C. bundyensis Lee and Reye, and one parous C. histrio Johannsen. Highest densities of arthropods (up to 1562/million m3) were on 30 January 2000 when NW winds, sustained for six hours, probably introduced midges from the New Guinea mainland. Adult mosquitoes (including three female Ve. funerea and a single female Ficalbia) and Culicoides (including two gravid C. bundyensis and one parous C. cordiger Macfie) were also collected in 2 m high mast nets during northerly surface winds. Although the results do not provide evidence that wind-blown mosquitoes introduced JE from New Guinea into Australia, they do not preclude that strong N winds associated with low pressure systems SW of the Torres Strait could have done so. However, results suggest that Culicoides were more likely than mosquitoes to reach high altitude and travel long distances during the light N winds experienced during the study.