Immunization with recombinant adenovirus synthesizing the secretory form of Japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis

Replication-defective recombinant adenoviruses (RAds) were constructed that synthesized the pre-membrane and envelope (E) proteins of Japanese encephalitis virus (JEV). Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa replicated poorly in HEK 293A cells and synthesized lower amounts of E protein. Oral immunization of mice with RAds generated low titers of anti-JEV antibodies that had little JEV neutralizing activity. Intra-muscular (IM) immunization of mice with either RAd generated high titers of anti-JEV antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing antibodies. Titers were significantly higher in case of RAdEs immunization. Splenocytes from mice immunized IM with RAds secreted large amounts of interferon-γ and moderate amounts of interleukin-5 in the presence of JEV and showed cytotoxic activity against JEV-infected cells. Naïve mice immunized IM with RAdEs showed complete protection against a lethal dose of JEV given intra-cerebrally. In order to study the effect of the pre-existing adenovirus 5 (Ad5) immunity on the outcome of the RAdEs immunization, mice were exposed to Ad5 through IM or intra-nasal (IN) routes before immunization with RAdEs. Mice exposed to Ad5 through the IN route, when immunized with RAdEs given IM, or those exposed to Ad5 through the IM route, when immunized with RAdEs given IN, were completely protected against lethal JEV challenge.     

Removing the threat of diclofenac to critically endangered Asian vultures

Veterinary use of the nonsteroidal anti-inflammatory (NSAID) drug diclofenac in South Asia has resulted in the collapse of populations of three vulture species of the genusGyps to the most severe category of global extinction risk. Vultures are exposed to diclofenac when scavenging on livestock treated with the drug shortly before death. Diclofenac causes kidney damage, increased serum uric acid concentrations, visceral gout, and death. Concern about this issue led the Indian Government to announce its intention to ban the veterinary use of diclofenac by September 2005. Implementation of a ban is still in progress late in 2005, and to facilitate this we sought potential alternative NSAIDs by obtaining information from captive bird collections worldwide. We found that the NSAID meloxicam had been administered to 35 captiveGyps vultures with no apparent ill effects. We then undertook a phased programme of safety testing of meloxicam on the African white-backed vultureGyps africanus, which we had previously established to be as susceptible to diclofenac poisoning as the endangered AsianGyps vultures. We estimated the likely maximum level of exposure (MLE) of wild vultures and dosed birds by gavage (oral administration) with increasing quantities of the drug until the likely MLE was exceeded in a sample of 40G. africanus. Subsequently, sixG. africanus were fed tissues from cattle which had been treated with a higher than standard veterinary course of meloxicam prior to death. In the final phase, ten Asian vultures of two of the endangered species(Gyps bengalensis,Gyps indicus) were dosed with meloxicam by gavage; five of them at more than the likely MLE dosage. All meloxicam-treated birds survived all treatments, and none suffered any obvious clinical effects. Serum uric acid concentrations remained within the normal limits throughout, and were significantly lower than those from birds treated with diclofenac in other studies. We conclude that meloxicam is of low toxicity toGyps vultures and that its use in place of diclofenac would reduce vulture mortality substantially in the Indian subcontinent. Meloxicam is already available for veterinary use in India.     

A biophotonic approach to measure pH in small volumes in vitro: Quantifiable differences in metabolic flux around the cumulus‐oocyte‐complex (COC)

Unfertilised eggs (oocytes) release chemical biomarkers into the medium surrounding them. This provides an opportunity to monitor cell health and development during assisted reproductive processes if detected in a non‐invasive manner. Here we report the measurement of pH using an optical fibre probe, OFP1, in 5 μL drops of culture medium containing single mouse cumulus oocyte complexes (COCs). This allowed for the detection of statistically significant differences in pH between COCs in culture medium with no additives and those incubated with either a chemical (cobalt chloride) or hormonal treatment (follicle stimulating hormone); both of which serve to induce the release of lactic acid into the medium immediately surrounding the COC. Importantly, OFP1 was shown to be cell‐safe with no inherent cell toxicity or light‐induced phototoxicity indicated by negative DNA damage staining. Pre‐measurement photobleaching of the probe reduced fluorescence signal variability, providing improved measurement precision (0.01‐0.05 pH units) compared to previous studies. This optical technology presents a promising platform for the measurement of pH and the detection of other extracellular biomarkers to assess cell health during assisted reproduction.     

Pheromone perception in the tsetse fly Glossina morsitans morsitans

The perception of cuticular female sex pheromone (15, 19, 23-trimethylheptatriacontane=morsilure) in Glossina m. morsitans was studied electrophysiologically and behaviourally. Electrophysiological studies indicated no sensitivity of the tibial sensilla or the contact chemoreceptive hairs on the legs of males to the pheromone. However, electroantennograms were recorded during stimulation of antennae from male flies with the odour of morsilure, which indicated that the pheromone is detected by the insect via olfactory receptors on the antennae. This was further confirmed by behavioural experiments in which the antennal movements of intact males stimulated with the odour of both synthetic pheromone and female decoys were studied. Behavioural responsiveness to morsilure increased with increasing starvation and was not due to a general sensitivity to paraffins. These studies indicate that tsetse flies may be able to recognize conspecifics at close range using their sense of olfaction.

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Résumé La perception de la phéromone sexuelle des femelles de Glossina m.morsitans, la morsilure (15, 19, 23-triméthylheptatriacontane) a été examinée par électrophysiologie et par le comportement. L'électrophysiologie n'a pas révélé de sensibilité des sensilles tibiales, ni des poils chimioréceptives de contact sur les pattes des mâles à la phéromone. Cependant, les électroantennogrammes recueillis pendant la stimulation des antennes des mâles par l'odeur de morsilure, ont montré que la phéromone est décelée par l'insecte grâce aux récepteurs olfactifs des antennes. Ceci a été confirmé ultérieurement par des expériences de comportement au cours desquelles ont été examinés les mouvements antennaires de mâles intacts stimulés par l'odeur de phéromone synthétique et de femelles.La réponse comportementale à la morsilure a augmenté avec le jeûne, elle n'était pas due à une sensibilité générale aux paraffines.Ces études montrent que les mouches tsé-tsé peuvent être capables de reconnaître leurs congénères à faible distance grâce à l'olfaction.

     

Seed Germination in Chenopodium album L: Relationships between Nitrate and the Effects of Plant Hormones

Effects of ethylene, gibberellins, and kinetin on the germination of two lots of Chenopodium album L. seeds, collected from the field in 1982 and 1983, were studied in relation to the availability of nitrate. The experiments were conducted in darkness and at temperatures ranging from 12 to 32°C. Ethylene induced over 75% germination in the 1983 seed but had little effect on the 1982 seed. Nitrate was only slightly promotive in either of the two seed lots. A combination of ethylene and nitrate, however, acted synergistically on 1982 seed, resulting in as much germination as that induced in 1983 seed by ethylene alone. In 1983 seed, a combination of ethylene and nitrate was only marginally more effective than ethylene. A similar relationship was observed in the effects of gibberellic acid₄₊₇ (GA₄₊₇) and nitrate on seeds from the two lots. The 1982 seed, which responded synergistically to combinations of nitrate with ethylene or GA₄₊₇ was found to contain an extremely low endogenous level of nitrate as compared to 1983 seed. Thus, high levels of either endogenous or applied nitrate appeared to enhance the germination response to ethylene or GA₄₊₇.

Kinetin had no effect on 1982 seed and only a small promotive effect on 1983 seed. There was no synergism between kinetin and nitrate in either of the seed lots.

     

Mechanism of action of aflatoxin B1 in Bacillus megaterium

Bacillus megaterium cells from various growth phases were equally susceptible to the lethal effects of aflatoxin B1. Known surfactants (EDTA and Tween-80) accentuated the effects of aflatoxin B1. Viability and inulin uptake in aflatoxin B1-exposed cells decreased considerably. The effect was concentration dependent. A straight-line relationship observed in the death curve indicated a single target for aflatoxin B1 action in B. megaterium. Leakage of intracellular constituents in B. megaterium was also concentration dependent, and this can be related to the extent of cell membrane damage.     

Circadian features of carbohydrate metabolism in domestic fowls exposed to weekly 120 degree-shifts of synchronizer schedule.

Effects of weekly 8 hr advance- or delay-shifts on the circadian rhythm of plasma glucose, liver glycogen and muscle glycogen in male domestic fowls, beginning at about 3 days of age, were examined. Circadian rhythm in the aforesaid indices of carbohydrate metabolism in control birds was also studied. Blood and tissue samples were collected from birds in all the three groups at 4 hr intervals over a single 24 hr time scale both at 6th and 12th week of age. Plasma glucose and glycogen content in the tissues were determined by employing standard techniques. Cosinor rhythmometry was used for analyzing time series data. In general, a statistically significant circadian rhythm was documented for all the three indices in control and advance-schedule birds, irrespective of age. In contrast, in delay-schedule birds, statistically significant circadian rhythm could not be detected, excluding in muscle glycogen at 12th week of age. The poor growth rate in the delay-schedule birds could be imputed to the disappearance of circadian rhythm in the indices of carbohydrate metabolism.     

Bioemulsifier production by an oleaginous yeastRhodotorula glutinis IIP-30

Summary A locally isolated oleaginous strain ofRhodotorula glutinis strain IIP-30 produced a growth associated extracellular emulsifying agent while utilizing glucose during fed batch fermentation under nitrogen limitation at 30°C and pH 4. 0. Similar optimum conditions were also noted for intracellular lipid accumulation.     

Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over,ovarian follicles and egg activation

Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. The impact of alterations in MMR family of proteins (bacterial MutS and MutL homologues) on mammalian fertility is well documented. However, an insight to the role of MMR in reproduction of non-mammalian organisms is limited. Hence, in the present study, we analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1^e00130 hypomorphic allele, we report female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1^e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1^e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1^e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility.