High performance liquid chromatographic separation of steroids from ovarian follicles of fresh water perch Anabas testudineus: identification and characterization of the maturation-inducing hormone

Accurate separation and identification of steroids from the postvitellogenic ovarian follicles of Indian climbing perch Anabas testudineus was performed by high-performance liquid chromatography (HPLC) and specific radioimmunoassay (RIA). The steroids from such follicles, incubated in Cortland's saline with or without homologous fish pituitary extract (FPE), were extracted with dichloromethane and separated on a micro Bondapak C(18) column. Identification of the HPLC fractions was further confirmed by thin layer chromatography. As HPLC peaks for 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) and testosterone (T) were close, clear separation of these steroids and accurate measurement of their quantities was achieved by RIA of HPLC fractions using specific antibodies. Altogether, nine eluted fractions in the FPE-untreated and ten in FPE-treated samples were obtained. Of these, six were identified as: 5 beta-pregnan-3 alpha,17 alpha,20 beta-triol (5 beta-3 alpha,17 alpha,20 beta-P); DHP; T; 17 alpha-hydroxyprogesterone (17 alpha-P(4)); progesterone (P(4)); and androstenedione (AD). Three fractions from untreated and four from FPE-treated samples, however, remained unidentified. Of all the HPLC fractions examined for their relative maturational inducing (MI) potency on full grown (postvitellogenic) ovarian follicles of perch, the fraction identified as DHP was found to be the most effective inducer of germinal vesicle breakdown (GVBD) both at low and high concentrations. Fractions identified as 5 beta-3 alpha, 17 alpha, 20 beta-P and 17alpha-P(4) could induce only 32% and 20% GVBD at their highest concentration, while none of the unidentified fractions showed any MI activity. FPE caused increased production of DHP, testosterone, and 5 beta-3 alpha, 17 alpha, 20 beta-P. The qualitative differences between the fractions obtained from FPE-treated samples and those from FPE-untreated samples were only the appearance of a new polar metabolite of unknown function. The present study showed that, as a single steroid, DHP was the most potent MIH for A. testudineus.     

Neuronal interactions between galanin-like-peptide- and orexin- or melanin-concentrating hormone-containing neurons

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain alpha-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.     

Characterization of the water quality of dam lakes on Shikoku Island, Japan

This study clarifies the distribution and variation of bioelements in dam lakes of the Shikoku region, Japan. The survey was carried out from 2002 to 2003 on three dam lakes in the Shikoku Mountains with high precipitation levels and on three dam lakes in the Sanuki mountain range with low precipitation levels. By the conventional classification for eutrophication, the dam lakes in the Shikoku Mountains were classified as oligotrophic to mesotrophic, whereas those in the Sanuki mountain range were classified as mesotrophic to eutrophic. Furthermore, for the dam lakes in the Sanuki mountain range, it was indicated that the amount of precipitation directly and indirectly affected lake water quality, such as high nitrogen concentration, low dissolved oxygen concentration, and increase in primary production. Especially, in a hypertrophic dam lake, during the period of low precipitation the surface water flowed out and the water under the thermocline, with no dissolved oxygen, appeared on the lake surface. As a result, the anaerobic environment extended across all the layers. Because a compulsory water-level operation was carried out in the dam lake, it was considered that the effect of the hydrological environment in the catchment area on the dam lake was relatively large in comparison with that in natural lakes.     

Extracts of Larix Leptolepis effectively augments the generation of tumor antigen-specific cytotoxic T lymphocytes via activation of dendritic cells in TLR-2 and TLR-4-dependent manner

Type-1 immunity plays a crucial role in host defense against various tumors and infectious diseases. Here, we first demonstrated that extract of Larix Leptolepis (ELL), one of the most popular timbers at Hokkaido area in Japan, strongly activated Type-1 immunity. ELL induced production of Type-1 cytokines such as IL-12 and TNF-α from bone marrow-derived dendritic cells (BMDCs) in TLR2- and TLR4-dependent manner and remarkably up-regulated the expression of MHC and co-stimulatory molecules. In addition, antigen-specific CTLs were significantly augmented by the combined administration of ELL, antigen and BMDCs. Finally, we revealed that combination therapy using ELL, antigen and BMDCs significantly inhibited the growth of established tumor in mouse model. Thus, these findings suggested that ELL would be a novel adjuvant for inducing an activation of Type-1-dependent immunity including activation of BMDCs and induction of tumor-specific CTLs, which is applicable to the therapy of cancer and infectious diseases.     

Assignment of RAPD marker probes designed from 12 linkage groups of Flammulina velutipes to CHEF-separated chromosomal DNAs

Electrophoretic karyotype analyses of Flammulina velutipes FSB and its monokaryotic progeny, omFSB1 and omFSB2, obtained from oidia were performed by contour-clamped homogeneous electric field (CHEF) gel electrophoresis. At least 11 chromosome-sized DNA bands (CB 1 through CB 11) for FSB, 6 bands for omFSB1, and 7 bands for omFSB2, respectively, were resolved on a CHEF gel. Southern hybridization analysis on CHEF-separated chromosomal DNA of FSB was carried out using RAPD marker probes prepared from each of the 12 linkage groups. The bands CB 1, 2, and 4 each hybridized to two or three probes for different linkage groups. The bands CB 5 and 6 both hybridized to a common probe. The bands CB 3, 7, 8, and 9 each hybridized to a single specific probe for different linkage groups. The two smallest bands (CB 10 and 11) did not hybridize with any probes.     

Identification of nobiletin,a polymethoxyflavonoid,as an enhancer of adiponectin secretion

Adiponectin, an adipocyte-derived protein with insulin-sensitizing, anti-diabetic and anti-atherogenic activities, is known to be induced during adipocyte differentiation. Nobiletin, a citrus polymethoxy flavonoid, was found to induce the differentiation of ST-13 preadipocytes into mature adipocytes and enhance the production of adiponectin protein at a concentration of 10 μM.     

CTL-mediated selective pressure influences dynamic evolution and pathogenic functions of HIV-1 Nef

HIV-1 Nef plays multiple roles in modulating immune responses, even though it is a dominant CTL target itself. How Nef accomplishes the balance between such conflicting selective pressures remains elusive. By genetic and functional studies, we found that Arg75Thr and Tyr85Phe mutations, located in a well-conserved proline-rich region in Nef, were differently associated with escape from CTL responses specific for two overlapping HLA-B35-restricted epitopes. CTLs specific for an epitope, that selected Tyr85Phe, were elicited earlier and had more potent functional avidities than did those that selected Arg75Thr. Although the double mutant could escape from both CTLs, the mutations are rarely observed in combination naturally. Introduction of both mutations reduced Nef's HLA class I down-regulation activity and increased the susceptibility of virus-infected cells to recognition by CTLs targeting other epitopes. Moreover, the mutant Nef was impaired in the association with activated cellular kinases and in the enhancement of viral replication. These results highlight CTL immunosurveillance as important modulators of Nef's biological activity in the infected host.