A putative met-enkephalin releaser, kyotorphin enhances intracellular Ca2+ in the synaptosomes

Kyotorphin (Tyr-Arg) at 1 to 100 microM increased the intracellular [Ca2+]i, determined with Quin-II in the slice and the entry of 45Ca2+ entry into synaptosomes of the lower brain stem of the rat. These effects were not antagonized by nifedipine nor verapamil. However, since this dipeptide caused no changes on the membrane potentials of the synaptosomes, measured with Rhodamine 6G, it is suggested that the kyotorphin-induced increase in the [Ca2+]i may be due not to effects on the voltage dependent Ca2+ channels and Na+-Ca2+ exchange mechanisms caused by the changes of the membrane potentials, but to the specific receptor (kyotorphin receptor)-mediated mechanisms.     

Unique cell surface phenotypes of proliferating lymphocytes in mice homozygous for lpr and gld mutations, defined by monoclonal antibodies to MRL/Mp-lpr/lpr T cells

In mice bearing the autosomal recessive gene of either lpr or gld, generalized T-cell proliferation and autoimmunity occurs. The surface antigen profiles of these proliferating cells were analyzed using two-color flow cytometry analysis with two newly established rat monoclonal antibodies (ALP-1, ALP-2) directed to lpr cells. The Lp-1 antigen, defined by ALP-1, is expressed exclusively on approximately one-half of proliferating lpr and gld lymph node cells. The Lp-2 antigen, like B 220, is expressed on 80-90% of lpr and gld lymph node cells, the cells in B-cell lineage and a small population of Ly-2+ T cells from normal mice. Thus, the lpr and gld lymph node cells were classified into three subsets, Lp-1+/Lp-2+, Lp-1-/Lp-2+ and Lp-1-/Lp-2-. After stimulation with Con A or a combination of IL-2 and phorbol ester, a small population of T cells from normal mice became Lp-1+. The same treatment increased Lp-2+/Ly-2+ and induced Lp-2+/L3T4+ T-cell populations. Therefore, it seems likely that these phenotypically unique T cells are generated at some stage during the proliferation and differentiation of certain normal T-cell subpopulations. The aberrant T cells in mice with lpr and gld mutations may even be normal regulatory T cells, if they are not proliferating abnormally.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Establishment of ex vivo systems to identify compounds acting on innate immune responses and to determine their target molecules using transgenic Drosophila

Innate immunity is an evolutionarily conserved self-defense mechanism against microbial infections. In Drosophila, induction of antimicrobial peptides is a major immune response that is regulated by two distinct signaling pathways called the IMD pathway and the Toll pathway, similar to the tumor necrosis factor-alpha signaling and Toll-like receptor/interleukin-1 signaling pathways, respectively, in mammals. In mammals, innate immunity interacts with adaptive immunity and has a key role in the regulated immune response. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Previously, based on the striking conservation between the mechanisms that regulate Drosophila immunity and human innate immunity, we established an ex vivo culture in which compounds acting on innate immunity can be evaluated using a reporter gene that reflects activation of the IMD pathway [Yajima et al. [Yajima, M., Takada, M., Takahashi, N., Kikuchi, H., Natori, S., Oshima, Y., Kurata, S., 2003. A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity. The Biochemical Journal 371(Pt 1), 205-210] Biochem J 371, 205-210]. Here, we combined the ex vivo culture with a reporter gene that reflects the heat shock response and demonstrated that the resulting systems are useful for screening compounds that act specifically on innate immunity, including mammalian innate immune responses. Identification of target molecules is essential for the development of more potent medicines with fewer side effects. In this study, we also established ex vivo systems capable of identifying target molecules of the identified compounds using targeted activation of the IMD pathway.     

ALDH2 polymorphism for the risk of cervical carcinogenesis

To investigate the clinical significance of ALDH2 genetic polymorphisms in cervical carcinogenesis. ALDH2 polymorphisms together with human papillomavirus (HPV) types were examined in a total of 195 cervical smear in exfoliated cervical cell samples using Real-Time polymerase chain reaction (PCR) System. The frequency for the AG+AA genotype was seven in the normal group (70.0 %), 16 in the LSIL group (57.1 %), and 27 in the HSIL group (90.0 %). A significant difference was found between the LSIL and HSIL groups (P = 0.0064). Patients with HSIL lesions frequently had high-risk HPV infections and concurrently belonged to the AG+AA group. ALDH2 genotype in cervical cell samples may be associated with more severe precancerous lesions of the cervix in a Japanese population.     

Colorimetric dimethyl sulfide sensor using Rhodovulum sulfidophilum cells based on intrinsic pigment conversion by CrtA

A colorimetric whole-cell sensor for dimethyl sulfide (DMS) was constructed based on the in vivo conversion of intrinsic pigments in response to the analyte. In a marine bacterium, Rhodovulum sulfidophilum, carotenoids are synthesized via the spheroidene pathway. In this pathway, demethylspheroidene, a yellow carotenoid, is converted to spheroidene under catalysis of O-methyltransferase. Spheroidene monooxygenase (CrtA) catalyzes the terminal step of the pathway and converts spheroidene to spheroidenone, a red carotenoid. Here, the CrtA gene in R. sulfidophilum was removed and then reintroduced downstream of the DMS dehydrogenase gene promoter. Using this whole-cell sensor, 3 μM DMS or dimethyl sulfoxide can be detected without adding any color-forming reagent. The ratio of the red spheroidenone to total carotenoids increased, as the DMS concentration was raised to 0.3 mM. Comparison of the signal to the background color indicated a shift in the color coordinate from a yellow to a red hue. An intense signal was obtained with 1-day incubation at a high cell density when sensor cells at the exponential growth phase were used. These results show that the genetically engineered R. sulfidophilum cells can be used to monitor the quality of marine aquacultural environments by the naked eye.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

Identification of a glycoprotein, gp21, of adult T cell leukemia virus by monoclonal antibody

A mouse hybridoma cell line producing monoclonal antibody, F10, was established from mice hyperimmunized with cells bearing adult T cell leukemia (ATL) virus (ATLV). F10 antibody reacted with an ATLV structural polypeptide ( gp21 ) with a m.w. of 21,000 that was glycosylated on cell surfaces. The gp21 was expressed on cell surfaces of all ATL-associated antigen (ATLA)-positive human cell lines but not on ATLA-negative cell lines nor peripheral blood leukocytes stimulated with mitogens. The gp21 was also detected with anti-ATLA-positive human serum, and the binding of F10 antibody to ATLA-positive cell surfaces was significantly blocked by pretreatment with anti-ATLA-positive human sera. Double immunofluorescence staining with F10 antibody and anti-ATLA-positive human serum caused co-capping on cell surfaces, which suggests that gp21 co-exists with other ATLV antigens expressed on cell surfaces. Immunoprecipitation studies also suggested that the gp21 is a minor component of the ATLV envelope.     

Salmonid olfactory system-specific protein (N24) exhibits glutathione S-transferase class pi-like structure

A salmonid olfactory system-specific protein (N24) that has been identified in lacustrine sockeye salmon (Oncorhynchus nerka) was characterized by biochemical and molecular biological techniques. N24 is a homodimer, and the intact molecular mass is estimated as approximately 43.3 kDa by gel filtration. Furthermore, N24 was located only in the cytosolic fraction of the olfactory tissues as determined by subcellular fractionation. cDNA encoding the lacustrine sockeye salmon N24 was isolated and sequenced. This cDNA contained a coding region encoding 216 amino acid residues and the molecular mass of this protein is calculated to be 242,224.77. The protein and nucleotide sequencing demonstrates the existence of a remarkable homology between N24 and glutathione S-transferase (GST; EC 2.5.1.18) class pi enzymes. Northern analysis showed that N24 mRNA with a length of 950 bases is expressed in lacustrine sockeye salmon olfactory epithelium. Olfactory receptor cells showed strong hybridization signals for N24 mRNA in the olfactory epithelium. N24 demonstrated glutathione binding activity in affinity-purified GST column experiments. The present study describes for the first time cDNA cloning of GST in fish olfactory epithelium.