Ability of CD8+ T cell anti-feline immunodeficiency virus (FIV) activity and FIV proviral DNA load in mononuclear cells in FIV-infected cats

We investigated the relationship between CD8+ T cell anti-feline immunodeficiency virus (FIV) activity and FIV proviral DNA load integrated in mononuclear cells. The anti-FIV activity and the proviral DNA load were correlated, and the number of proviral DNA copies was high in cats with decreased anti-FIV activity. Particularly, no anti-FIV activity was detected in the cats staged as having an acquired immunodeficiency syndrome (AIDS)-related complex or AIDS, and the number of proviral DNA copies was obviously increased compared to those in the cats in the asymptomatic stage. These results suggest that decreased anti-FIV activity destroys the control of in vivo FIV replication, which leads to an increased proviral DNA load with the progression of the clinical stage of disease.     

Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.     

Molecular study on chicken tumor necrosis factor receptor-II and tumor necrosis factor receptor-associated factor-5

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were identified as signal transducers for the tumor necrosis factor receptor (TNFR) superfamily. In this study, we cloned and characterized two genes that encode chicken TNFR-II and TRAF5. The initial cDNA fragments were obtained by suppressive subtractive hybridization (SSH) of chicken spleen cells with or without lipopolysaccharide stimulation (Salmonella typhimurium SL1181 (RE-mutant)). The results showed that chicken TNFR-II is 1518 bp in length with an open reading frame (ORF) of 1386 bp having 31% homology with human TNFR-II. Expression analysis of chicken TNFR-II revealed that it is highly expressed in the spleen and bursa of Fabricius. The chicken cell lines IN24, MSB1 and 1104B express TNFR-II abundantly. The time course analysis of expression in spleen, bursa of Fabricius and IN24 cell line showed that TNFR-II is maximally expressed at 6 h after stimulation in bursa of Fabricius and after 8 h stimulation in the IN24 cell line. With regard to TRAF5, the complete sequence was 1936 bp in length with an ORF of 1671 bp that showed 71.3% homology with human TRAF5. Expression analysis showed that, among the tissues examined, TRAF5 was strongly expressed in spleen and bursa of Fabricius, while among the cell lines examined, it was maximally expressed in IN24. Thus, both genes were expressed in the same tissues and cell line among examined materials. These results suggest that chicken TNFR-II may interact with TRAF5 adaptor protein to complete its signal transduction pathway.     

Evaluation of fetal growth and estimation of fetal age based on skeletal growth in Hokkaido sika deer (Cervus nippon yesoensis Heude, 1884)

We investigated fetal development and the estimation of fetal age of 127 Hokkaido sika deer fetuses, categorizing them into three groups according to the nutritional condition of populations. The order and time of the appearance of ossification centers were clarified, and fetal age was determined based on bone length and the appearance of ossification centers. Then we observed the differences in fetal growth among three populations, and discussed the effect of poor nutrition on the fetal growth. The results suggest that fetal diaphysial length of the femur was affected very little by nutritional conditions, whereas conception dates were delayed and fetal weight was restricted as the nutritional condition became poorer. Although it is impossible to know the exact accurate fetal age in wild populations, it was possible to create a standard to estimate fetal age more precisely by the method described in this study. Both the bone length and the appearance of ossification centers are reliable indices to estimate fetal age precisely in measurements available from fetuses of unknown age, and can be applied to estimate the fetal age of other populations of sika deer, whereas estimation of fetal age based on weight is prone to great errors.     

Relationship between serum sex hormone concentrations and histology of seminiferous tubules of captured baleen whales in the Western North Pacific during the feeding season

The present study was conducted to obtain new information on relationships among serum testosterone (T), estradiol-17 beta (E(2)), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) concentrations and histology of seminiferous tubules in captured common minke and Bryde's whales during the feeding season. Blood samples and testes were collected from common minke (n=39 for blood samples, n=15 for testes) and Bryde's (n=14 for blood samples, n=7 for testes) whales captured from May 2001 to August 2001 in the Western North Pacific. Serum T concentrations, in 35.9% of the common minke and 57.1% of Bryde's whales, were below the detection limit (< 2.5 pg/ml). There were no significant differences in the serum concentrations of E(2), FSH, and LH among immature, mature common minke and Bryde's whales except that LH levels of immature Bryde's whales was higher than those of common minke whales. In most seminiferous tubules of mature whales, only a single-layer of spermatogonia was observed. However, spermatozoa were observed in seminiferous tubules in 2/13 of mature common minke and 4/4 of mature Bryde's whales with the low or undetectable T levels. These results indicate that the low serum T concentrations reflect the inactivity of spermatogenesis in both baleen whales, and that it is not possible to assess gonadal activity in either common minke or Bryde's whales using serum sex hormone concentrations during the feeding season.     

Tyrosinase inhibitory activity of citrus essential oils

Thirteen kinds of citrus essential oils and their volatile flavor constituents were investigated for tyrosinase inhibitory activity. Eureka lemon, Lisbon lemon, Keraji, and Kiyookadaidai significantly inhibited the oxidation of L-dihydroxy phenylalanine (L-DOPA) by mushroom tyrosinase. Citral and myrcene among volatile flavor constituents of citrus essential oils exhibited tyrosinase inhibitory activities with Ki values of 0.318 and 2.38 mM, respectively. The inhibition kinetics analyzed by a Lineweaver-Burk plot indicated that citral is a noncompetitive inhibitor and myrcene is a competitive inhibitor. These results indicated that citral and myrcene are responsible for the tyrosinase inhibitory activity of citrus essential oils.     

Histopathological characteristics of Ito cells and Kupffer cells in the feline liver

The histopathological characteristics of Ito cells and Kupffer cells were investigated in the liver of 21 cats (age range: 6 months -18 years) autopsied in our laboratory during 2003. Immunohistochemical examinations were performed using antibodies against lysozyme, desmin and alpha-smooth muscle actin. No Kupffer cells reacted with the antibody against lysozyme. However, macrophages in the lung and spleen showed a positive reaction with the antibody. This finding suggests a possibility that the amount of lysozyme in the Kupffer cells of feline liver is comparatively small. On the other hand, large vacuole-laden cells were observed in the hepatic perisinusoid of some feline cases, and these cells showed a positive reaction with antibodies against desmin and alpha-smooth muscle actin. These cells could be Ito cells with large lipid vacuoles. This conclusion was supported by electron microscopic observation and oil red O staining. However, no such large vacuole-laden perisinusoidal cells were detected in the liver of young cats less than 2 years old. The present study revealed the histopathological features of Kupffer cells and Ito cells in the feline liver.     

Comparison of cell populations derived from canine olfactory bulb and olfactory mucosal cultures

OBJECTIVE: To evaluate the numbers and proportions of olfactory ensheathing cells (OECs) in cell cultures derived from the olfactory bulb (OB) and olfactory mucosa of dogs. ANIMALS: 7 dogs. PROCEDURES: OB tissue and olfactory mucosa from the nasal cavity and frontal sinus were obtained from euthanatized dogs and prepared for cell culture. At 7, 14, and 21 days of culture in vitro, numbers and proportions of OECs, astrocytes, and fibroblasts were determined via immunocytochemistry. Antibody against the low-affinity nerve growth factor receptor p75 was used to identify OECs, antibody against glial fibrillary acidic protein was used to identify astrocytes, and antibody against fibronectin was used to identify fibroblasts. RESULTS: Cultured OECs derived from the olfactory mucosa of the nasal cavity and frontal sinus had similar characteristics. However, whereas OECs in the OB cell cultures constituted approximately 50% of the cells at 7 days and approximately 75% at 21 days the proportion of OECs in cultures derived from both mucosal types was much lower, with approximately 40% OECs at 7 days and approximately 25% at 21 days. Analysis of OEC numbers revealed that these changes were accompanied by corresponding decreases and increases in the population of cells with fibronectin receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Although olfactory mucosal cell cultures yielded a sufficient number of OECs for spinal cord transplantation procedures in dogs, modification of culture conditions would be required to ensure that the derived cell population contained a sufficient proportion of OECs.     

Green-odour compounds have antifungal activity against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Four green-odour compounds—trans-2-hexenal, cis-3-hexenol, n-hexanal, and cis-3-hexenal—were applied (0.85 μg ml⁻¹ as vapour) to rice plants in laboratory conditions to observe their biological activity against the phytopathogenic fungus Maganporthe oryzae, which causes rice blast disease worldwide. Two compounds, trans-2-hexenal and cis-3-hexenal, showed remarkable disease suppression efficacy (99.7% and 100% suppression, respectively), while n-hexanal had moderate (86.5%) and cis-3-hexenol had weak (20.8%) disease-suppressing effects. Pre-application and post-application of trans-2-hexenal or cis-3-hexenal had slight effects on blast incidence, suggesting that these compounds had direct effects to suppress M. oryzae infection. In fact, trans-2-hexenal and cis-3-hexenal exhibited a growth suppression effect on M. oryzae. Interestingly, these two compounds inhibited appressorium formation at lower concentrations than the growth suppression. Studies on the hypersensitive response (HR)-like reaction and plant β-1,3-glucanase activity in rice plant confirmed that induced resistance was not the major factor involved in the disease suppression mechanism. Results of this study conclusively showed that trans-2-hexenal and cis-3-hexenal possess potent inhibitory activities against the growth and the appressorium formation of M. oryzae and could be used as antifungal agents to significantly reduce M. oryzae infections in rice.     

Development of EST-SSR markers of Ipomoea nil

Although Japanese morning glory (Ipomoea nil (L.) Roth.) has been used intensively for genetic studies, DNA markers have not been developed in Ipomoea nil sufficient to cover all chromosomes. Therefore, we conducted microsatellite (simple sequence repeats, SSR) marker development in I. nil for future genetic studies. From 92,662 expressed sequence tag (EST) sequences, 514 unique microsatellite-containing ESTs were identified. Primer pairs were designed automatically in 326 SSRs. Of 150 SSRs examined, 75 showed polymorphisms among strains. A phenogram based on the SSR genotypes revealed the genetic relation among seven Japanese morning glories from five different regions of the world and an ivyleaf morning glory (I. hederacea Jacq.). The developed SSR markers might be applicable for genetic studies of morning glories and their relatives.