Hypocretin/orexin is produced exclusively in the dorsal and lateral hypothalamus but its projection is widespread within the brain and plays important roles. In this paper, we review the independent discoveries of the hypocretin/orexin peptides, the neuroanatomy of this system, and the link to the sleep disorder narcolepsy that has led to the idea that this system plays a crucial role in the regulation of sleep and wakefulness.

Ixodes persulcatus Schulze (I. persulcatus) is distributed in Russia and Far East Asia including Japan, and has been implicated as the vector of several human pathogens. In particular, I. persulcatus acts as the only tick vector for human lyme borreliosis in Japan. In order to elucidate the mechanism of transmission of I. persulcatus-borne pathogens, we developed a laboratory colony of I. persulcatus. Ticks were fed on Syrian hamster and engorged ticks that had dropped off the animals were collected and maintained to allow them to molt. Tick rearing was performed in incubator at 20degC with 95% relative humidity and 12-hour light/dark photo-period regimen. We found out that adult females fed for 8+-2 days and had a pre-oviposition period lasting for 7+-2 days. The minimum egg incubation period was 1 month with the hatched larvae feeding for 3+-1 days and molting to nymphs 3-4 months thereafter. Meanwhile, the nymphs fed for 4+-1 days and molted to adult 2-3 months thereafter. For future analysis of gene expression profiles in I. persulcatus, we cloned and sequenced the actin gene (a housekeeping gene), and found that it is 92.7% to 98.6% homologous to the published sequences of related ixodid ticks. This laboratory colony of I. persulcatus will facilitate investigations on the role of tick-derived molecules on the transmission of I. persulcatus-borne pathogens and will be important for identification of potential anti-tick vaccine and acaricide target molecules.

alpha-Hemoglobin stabilizing protein (AHSP) functions as the erythroid-specific molecular chaperon for alpha-globin. AHSP gene expression has been reported to be downregulated in hematopoietic tissues of animals suffering from prion diseases though the mechanism remains to be clarified. Herein, we demonstrate that MELhipod8 cells, a subclone of murine erythroleukemia (MEL) cells, have prion protein (PrPsup(C)) on the cell surface and have highly inducible expression of the AHSP and alpha- and beta-globin genes, resembling the expression pattern of the PrP and AHSP genes in bipotential erythroid- and megakaryocyte-lineage cells followed by erythroid differentiation in normal erythropoiesis. Moreover, MELhipod8 cells exhibit greater effective erythroid differentiation with a population of hemoglobinized normoblast-like cells than that observed for the parental MEL cells. These findings suggest that MELhipod8 cells could provide a mechanism for downregulation of the AHSP gene in prion diseases.

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokhaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 virus as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.

Rhipicephalus appendiculatus serpin-3 (RAS-3), R. appendiculatus serpin-4 (RAS-4) and a 36kDa immuno-dominant protein of R. appendiculatus (RIM36) were reported as candidate antigens for the anti-tick vaccine to control ixodid ticks. In the present study, we generated recombinant proteins of RAS-3 (rRAS-3), RAS-4 (rRAS-4) and RIM36 (rRIM36), and assessed their potency as an anti-tick cocktail vaccine in cattle model. RT-PCR analysis showed that RAS-3, RAS-4 and RIM136 transcripts were detected in both adult male and female ticks during feeding. Immunization of cattle with the combination of rRAS-3, rRAS-4 and rRIM36 had raised antibodies against all recombinants and anti-sera had reacted with the molecules from the tick salivary gland extract. Tick infestation challenge demonstrated protective immunity against female ticks, resulting in mortality rates of 39.5 and 12.8 % for the vaccinated and control groups, respectively. Moreover, the mortality rate of Theileria parva-infected female ticks was 48.5 and 10.8 % in the vaccinated and control group, respectively. In order to evaluate the levels of pathogen transmission capacity by T. parva-infected ticks fed on immunized cattle, the occurrence of T. parva in the bovine parotid lymph node and peripheral blood was also determined and quantified by real-time PCR. Although the infection with T. parva could not be protected by the vaccine, the occurrence of pathogen in peripheral blood was delayed 1 to 2 days after the infestation challenge in vaccinated group. These results suggest that this cocktail vaccine plays a role in the prevention of tick infestation.

This study was performed to examine whether the brain activities induced by noxious algesic chemical substances in anesthetized animals could be detected by blood oxygen-level-dependent functional magnetic resonance imaging (BOLD-fMRI). Multislice gradient echo images of the primary somatosensory cortex were obtained using a 7.05 T superconducting system and a one-turned surface coil centered over the primary somatosensory cortex of the 1.0%-isoflurane-anesthetized rat. The Z-score t-map of BOLD signals and its time-course analysis revealed that subcutaneous injection of formalin into the left forepaw immediately induced an early response in the contralateral primary sensory cortex lasting for a few minutes, followed by a late response until 20 min after stimulation. In contrast, injection of capsaicin into the left forepaw evoked only the early response. Furthermore, pretreatment with morphine completely abolished these responses induced by the chemical algesic substances. Thus BOLD-fMRI is a useful method to analyze the brain activities of painful stimulation in anesthetized animals.

The health hazards of individual organophosphorus insecticides have been characterized by their acute toxicity, mainly by investigating their cholinesterase inhibition. However, the chronic effects of most of these toxicants on the drug-metabolizing enzymes have not been investigated. Profenofos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate) is an organophosphorus pesticide widely used in cotton cultivation. In the present study, we investigated the effect of profenofos on male-specific cytochrome P450 (CYP) enzymes in adult Wistar rats. We orally administered 17.8 mg/kg body weight, twice weekly for 65 days. Profenofos downregulated levels of hepatic and testicular CYP2C11 and CYP3A2 mRNA and protein expression. Testicular aromatase (CYP19A) mRNA was decreased in the profenofos-treated rats compared to controls. Overall, the present study suggests that profenofos acts as an endocrine disruptor of male-specific CYP enzymes and affects testosterone concentration, which implicates its deleterious effects on animal or human males chronically exposed to organophosphorus pesticide.

Nigella sativa (family Ranunculaceae ) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNFalpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflamnatory IL-10.

Puumala virus (PUUV), a causative agent of hemorrhagic fever with renal syndrome (HFRS), is prevalent in Europe and European Russia. No vaccine has been developed for PUUV-associated HFRS, primarily because of the low viral yield in cultured cells. A PUUV strain known as DTK/Ufa-97 was isolated in Russia and adapted for growth in Vero E6 cells maintained in serum-free medium. The DTK/Ufa-97 strain produced a higher viral titer in serum-free medium, suggesting that it may prove useful in the development of an HFRS vaccine. When PUUV-infected Vero E6 cells were grown in serum-free medium, the DTK/Ufa-97 strain yielded more copies of intracellular viral RNA and a higher viral titer in the culture fluid than did the Sotkamo strain. Phylogenetic analysis revealed that PUUVs can be classified into multiple lineages according to geographical origin, and that the DTK/Ufa-97 strain is a member of the Bashkiria-Saratov lineage. The deduced amino acid sequences of the small, medium, and large segments of the DTK/Ufa-97 strain were 99.2% to 100%, 99.3% to 99.8%, and 99.8% identical, respectively, to those of the Bashkirian PUUV strains and 96.9%, 92.6%, and 97.4% identical, respectively, to those of the Sotkamo strain, indicating that the PUUVs are genetically diverse. However, DTK/Ufa-97 and other strains of PUUV exhibited similar patterns of binding to a panel of monoclonal antibodies against Hantaan virus. In addition, diluted antisera (i.e., ranging from 1:160 to 1:640) specific to three strains of PUUV neutralized both homologous and heterologous viruses. These results suggest that the DTK/Ufa-97 strain is capable of extensive growth and is antigenically similar to genetically distant strains of PUUV.

With the aim to evaluate the risk of citrus pulp to induce acute rumen lactic acidosis (ARLA), 15 rumen cannulated cattle didn't adapted to concentrates were randomly allocated in 3 groups: G1- CONTROL - group fed only the basal diet; G2 - SUCROSE animals with ARLA induced by rumen administration of sucrose; G3 CITRUS PULP animals received citrus pulp into rumen (1.65% of BW). Blood and rumen samples were drawn throughout the next 24 h to determine pH, lactic acid concentration and the packet cell volume, blood base excess. Clinical signs were also recorded and food intake followed by the next 7 days. Sucrose caused a systemic and ruminal acidosis and characteristic ARLA signs such as, apathy, dehydration, diarrhea and tachycardia, while citrus pulp gave rise to mild and brief rumen acidosis, reaching the lowest pH (5.35) at the 6th h, without any changing in the blood pH and any typical clinical sign, but temporary reduction in the rumination and excretion of semi liquid feces. Appetite was fully recovered after two and seven days in the pulp and sucrose group, respectively. These results showed that citrus pulp may be used as a feedstuff for cattle with low risk to cause ARLA. | Com o objetivo de avaliar o potencial da polpa cítrica (PC) em provocar acidose láctica ruminal aguda (ALRA), 15 bovinos com peso médio de 160 kg providos de cânula ruminal não-adaptados à dieta contendo concentrado foram alocados aleatoriamente em três grupos: CONTROLE animais receberam apenas a dieta basal; SACAROSE animais receberam sacarose diretamente no rumem a fim de provocar ALRA; POLPA - grupo que recebeu subitamente alta quantidade de PC no rúmen (equivalente a 1,65% do peso corporal). Em vários tempos no decorrer de 24 horas, após administração dos substratos, foram determinados o volume globular, pH, excesso de bases (BE) e lactato total no sangue e pH e concentração de ácido láctico total no conteúdo ruminal. Exame clínico foi realizado no decorrer do 1º dia e o consumo de alimento acompanhado nos próximos sete dias. A administração de sacarose provocou um característico quadro de ALRA com o desenvolvimento de acidose ruminal e sistêmica, apatia, desidratação, diarréia e taquicardia. Por outro lado, a polpa cítrica produziu discreta e temporária acidose ruminal, atingindo na 6ª hora o pH ruminal mais baixo (5,35), sem provocar acidose sistêmica e quadro clínico mais evidente de ALRA, com exceção de uma diminuição temporária na ruminação e eliminação de fezes semilíquidas. A regularização do apetite ocorreu após dois dias no grupo com PC e sete dias no grupo com sacarose. Tais resultados indicam que a polpa cítrica pode ser utilizada na alimentação de bovinos com baixo risco de provocar ALRA.