Effects of diesel exhaust particles on the male reproductive system in strains of mice with different aryl hydrocarbon receptor responsiveness

Diesel exhaust particles (DEPs) contain polycyclic aromatic hydrocarbons (PAH) that bind to aryl hydrocarbon receptors (AhRs) and decrease sperm production. Since it is not clear if AhR mediates DEP toxicity, we investigated the effect of DEPs in four strains of mice that have different AhR responsiveness. We treated BALB/c, C57BL/6, ICR and DBA/2 mice with DEP suspensions and compared their toxicity in each strain. In both the vehicle- and DEP-treated groups, ethoxyresorufin-O-deethylase (EROD) activity, as an indirect index of AhR activity, was increased in the order of BALB/c > C57BL/6 > ICR > DBA/2. Only BALB/c and C57BL/6 mice had significantly lower daily sperm production (DSP) than vehicle-treated mice. All strains exhibited increased sperm abnormalities. In particular, the C57BL/6, ICR and DBA/2 mice exhibited significantly increased abnormalities. A significant correlation was found between EROD activity and DSP or incidence of morphologically abnormal sperm. These data suggest that DEP toxicity may affect the male reproductive system in an AhR-dependent manner.     

Vitellogenin in tilapia (Oreochromis mossambicus): Induction of two forms by estradiol,quantification in plasma and characterization in oocyte extract

Two forms of vitellogenin were isolated by DEAE agarose ion-exchange chromatography from plasma of the tilapia, Oreochromis mossambicus. The monomers have apparent molecular masses of 200 and 130 kDa, as indicated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and a total amount of phosphorus of 1.7 and 0.1%, respectively. Antibodies specific to the two forms, designated tVTG-200 and tVTG-130, were generated in rabbits and used to develop enzyme-linked immunosorbent assays (ELISAs) and in Western blot analyses of plasma and oocyte extract. SDS-PAGE of the oocyte extract showed a major protein band at 106.6, minor bands at 26.6, 24.2, and 23.7 kDa, and very faint bands at 83.4 and 17.5 kDa. Western blots of the oocyte extract revealed that the antiserum to tVTG-200 recognized strongly the protein bands at 24.2 and 23.7 kDa, and less strongly the bands at 25.1 and 22.6 kDa, whereas the antiserum to tVTG-130 recognized mainly the protein band at 106.6 kDa. The presence of both VTGs in untreated male tilapia was detected with the ELISAs using relatively high plasma volumes. Their presence in males was confirmed by VTG-like immunoreactive materials eluting from the ion-exchange column at the same positions as tVTG-200 and tVTG-130. The concentrations of the VTGs in males were several orders of magnitude lower than in vitellogenic females. Treatment of male tilapia with estradiol-17β (E₂) induced both VTGs within 24h. After 7 days, tVTG-130 reached a maximum concentration in plasma, whereas tVTG-200 continued to increase. Our findings demonstrate that the two vitellogenins are biochemically distinct, possibly differentially regulated, and made by both sexes.     

The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants

We report the draft genome sequence of the model moss Physcomitrella patens and compare its features with those of flowering plants, from which it is separated by more than 400 million years, and unicellular aquatic algae. This comparison reveals genomic changes concomitant with the evolutionary movement to land, including a general increase in gene family complexity; loss of genes associated with aquatic environments (e.g., flagellar arms); acquisition of genes for tolerating terrestrial stresses (e.g., variation in temperature and water availability); and the development of the auxin and abscisic acid signaling pathways for coordinating multicellular growth and dehydration response. The Physcomitrella genome provides a resource for phylogenetic inferences about gene function and for experimental analysis of plant processes through this plant's unique facility for reverse genetics.     

Conditioning of karyoplasts for producing somatic nuclear transferred gonadal germ cells in domestic chickens

The objective of this study was to establish a protocol for generating karyoplasts that can be used to produce somatic nuclear transferred gonadal germ cells (snt-GGCs) in domestic chickens. Karyoplasts were produced by centrifuging cultured fibroblasts from 10-day-old chick embryos at 10,000 x g in the presence of 1.0 microg/ml cytochalasin B. The number of karyoplasts was significantly (P<0.05) higher and the diameters of the karyoplasts were significantly (P<0.05) smaller when fibroblasts were centrifuged for 60 min than for 10 or 30 min. It was possible to generate snt-GGCs by electrofusion of GGCs with karyoplasts produced from cryopreserved or serum-starved fibroblasts. These results indicate that karyoplasts generated from 10-day-old chick embryos can be used to produce snt-GGCs even after cryopreservation and serum starvation of the fibroblasts.     

Comparative aspects on the role of polypyrimidine tract-binding protein in internal initiation of hepatitis C virus and picornavirus RNAs

We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.     

Evaluation of a recombinant measles virus expressing hepatitis C virus envelope proteins by infection of human PBL-NOD/Scid/Jak3null mouse

In this study, we infected NOD/Scid/Jak3null mice engrafted human peripheral blood leukocytes (hu-PBL-NOJ) with measles virus Edmonston B strain (MV-Edm) expressing hepatitis C virus (HCV) envelope proteins (rMV-E1E2) to evaluate the immunogenicity as a vaccine candidate. Although human leukocytes could be isolated from the spleen of mock-infected mice during the 2-weeks experiment, the proportion of engrafted human leukocytes in mice infected with MV (10³–10⁵ pfu) or rMV-E1E2 (10⁴ pfu) was decreased. Viral infection of the splenocytes was confirmed by the development of cytopathic effects (CPEs) in co-cultures of splenocytes and B95a cells and verified using RT-PCR. Finally, human antibodies against MV were more frequently observed than E2-specific antibodies in serum from mice infected with a low dose of virus (MV, 10⁰–10¹ pfu, and rMV-E1E2, 10¹–10² pfu). These results showed the possibility of hu-PBL-NOJ mice for the evaluation of the immunogenicity of viral proteins.     

Experimental transmission of Bovine leukemia virus in cattle via rectal palpation

We examined whether Bovine leukemia virus (BLV) was transmitted by rectal palpation using a common sleeve between a BLV-infected cow and BLV-negative steers. Three of four steers developed antibodies against BLV as determined by agar-gel immunodiffusion (AGID) test between 7 to 10 weeks after the first rectal palpation using common sleeves from BLV-infected cow. In the steers, BLV proviral DNA were detected by PCR 1 to 5 weeks earlier than detection of the antibodies by the AGID test. Our experiments demonstrated that rectal palpation is a potential cause of BLV spread in herds and that detection of BLV proviral DNA in cattle by PCR is useful screening test for early diagnosis of BLV infection.     

Development of novel clubroot resistant rapeseed lines (Brassica napus L.) effective against Japanese field isolates by marker assisted selection

Clubroot is an important disease infectible to cruciferous plants and a major threat to rapeseed production in Japan. However, no clubroot resistant rapeseed cultivars have been released. We surveyed pathotype variation of six isolates collected from rapeseed fields and found they were classified as pathotype groups 2 and 4 using Japanese F₁ Chinese cabbage cultivars. We produced the resynthesized clubroot resistant Brassica napus harboring two resistant loci, Crr1 and Crr2, by interspecific crossing and developed resistant rapeseed lines for southern and northern regions by marker-assisted selection and backcrossing. We improved the DNA marker for erucic acid content to remove linkage drag between Crr1 and high erucic acid content and successfully selected lines with clubroot resistance and zero erucic acid for northern regions. A novel line, ‘Tohoku No. 106’, suitable for southern regions showed stable resistance against all six isolates and high performance in infested fields. We conclude that Crr1 and Crr2 are important genes for CR rapeseed breeding and marker-assisted selection is effective in improving clubroot resistance.