Involvement of vesicle-cytoskeleton interaction in AQP5 trafficking in AQP5-gene-transfected HSG cells

We examined the regulatory role of a reduction/oxidation (redox) control protein, thioredoxin (TRX), in tumor necrosis factor-alpha (TNF-alpha)-induced p38 MAP kinase activation and p38 MAP kinase-mediated cytokine expression utilizing TRX-transfected murine L929 cells (TRX14). The results showed that TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production by TRX 14 were less than those by the parental L cells and the control transfected L cells (Neo-1). SB 203580 as the specific inhibitor for p38 MAP kinase activity inhibited TNF-alpha-induced IL-6 production by the parental L cells, indicating that TNF-alpha-activated p38 MAP kinase regulates IL-6 production by the cell lines used in this study. These results showed that overexpression of TRX negatively regulates p38 MAP kinase activation and p38 MAP kinase-mediated IL-6 production by TNF-alpha-stimulated cells, indicating that TRX is critical for p38 MAP kinase activation which regulates cytokine expression.     

Photoperiod- and temperature-dependent regulation of pupal beige/black polymorphism in the small copper butterfly, Lycaena phlaeas daimio Seitz

The small copper butterfly, Lycaena phlaeas daimio, has pupal beige/black polymorphism, the development of which is found to be controlled in an apparent association with the development of adult seasonal polymorphism (spring and summer morphs) by photoperiod and temperature in the larval stages. That is, the pupae of beige and black types developed under long-day and short-day conditions tend to develop into brown-winged and red-winged adults, respectively. In addition, a large proportion of long-day pharate pupae chilled at 4 degrees C for 5 days were observed to develop into pupae whose head-thoracic complexes and abdomens were judged to be of the black and intermediate types, respectively. They developed into adults with redder wings as compared to those obtained from unchilled pupae. The results indicate that the physiological mechanism underlying the photoperiodic control of the development of adult seasonal polymorphism may also play a significant role in the determination of pupal beige/black polymorphism in L. phlaeas daimio. Furthermore, cuticle melanization was found to be induced in the head-thoracic complexes of pupae by chilling of the pharate pupae. Melanization of pupal cuticle seems to occur in a close association with the development of reddish-winged adults.     

Changes of protein profiles during pollen development in<Emphasis Type="Italic"> Lilium longiflorum</Emphasis>

Pollen proteins of Lilium longiflorum were examined at different developmental stages (young, mature and cultured) using two-dimensional differential gel electrophoresis. Quantitative changes of six proteins (MP1–MP6) during pollen development were observed in the acidic and low molecular weight region. After water absorption on the culture medium, the quantities of all six proteins were drastically changed. Mass spectrometric analysis revealed that MP2, MP3, MP4 and MP6 are late embryogenesis abundant (LEA) (D-7) protein, profilin 3, profilin 1 and enolase, respectively. The remaining two proteins (MP1 and MP5) could not be identified by mass spectrometric analysis. Immunogold electron microscopic examination showed the presence of these proteins in different regions: MP1 around lipid bodies, suggesting possible involvement in lipid metabolism, MP4 near actin in the cytoplasm, indicating the possibility of its interaction with actin in the regulatory pathways of pollen, and MP2 and MP6 in the cytoplasm.     

Novel histone deacetylase inhibitors: cyclic tetrapeptide with trifluoromethyl and pentafluoroethyl ketones

Cyclic tetrapeptides containing trifluoromethyl and pentafluoroethyl ketone as zinc binding functional group were synthesized as potent HDAC inhibitors. Evaluation by human HDAC inhibition assay and p21 promoter assay showed that these inhibitors are promising anticancer agents.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Potent and orally active ET(A) selective antagonists with 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acid structures

The synthesis and structure-activity relationships of a series of 5,7-diarylcyclopenteno[1,2-b]pyridine-6-carboxylic acids are described. Our efforts have been focused on modification of the aryl ring at the 5-position and the alkyl substituent at the 2-position of the bottom 4-methoxyphenyl ring in an effort to develop orally available ET(A) selective antagonists with safer profiles in terms of the P-450 enzyme inhibitory activity. Incorporation of a hydroxymethyl group as an alkyl substituent in methylenedioxyphenyl and 6-dihydrobenzofuran derivatives led to the identification of orally bioavailable ET(A) selective antagonists 1f and 7f. These compounds also showed not only excellent binding affinity (IC(50) < 0.10nM, more than 800-fold selectivity for the ET(A) receptor over the ET(B) receptor) but also sufficient oral bioavailability, 48% and 56%, respectively, in rats. Furthermore, these compounds did not exhibit either competitive or mechanism-based inhibition of human cytochrome P450 enzymes.     

Development of improved Sindbis virus-based DNA expression vector

We have constructed an improved DNA expression vector based on the Sindbis virus. Several DNA-based Sindbis virus vectors were constructed to investigate the efficiency of transgene expression. These vectors, when transfected into mammalian cells, have been used to express heterologous genes. A recombinant genome of Sindbis plasmid DNA, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus (SV 40) promoter with a hepatitis delta virus (HDV) antigenomic ribozyme and a polyadenylation signal. Transfection of mammalian cells with this Sindbis-based plasmid vector, pSin-SV40-HDV-SV40pA, resulted in transient high-level expression of the beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSin-SV40-HDV-SV40pA was more than 16-fold higher than that of pSin-Lux originally reported by Herweijer et al. In vivo expression was also detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable after day 14. Furthermore, we demonstrate that the transfection of cells with this Sindbis virus vector results in apoptotic death on glioma cells. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from the pSin-SV40-HDV-SV40pA construct using a Sindbis replication system.     

Spectrometric analysis of degradation of a physiological substrate sigma32 by Escherichia coli AAA protease FtsH

We have established a fluorescence polarization assay system by which degradation of sigma32, a physiological substrate, by FtsH can be monitored spectrometrically. Using the system, it was found that an FtsH hexamer degrades approximately 0.5 molecules of Cy3-sigma32 per min at 42 degrees C and hydrolyzes approximately 140 ATP molecules during the degradation of a single molecule of Cy3-sigma32. Evidence also suggests that degradation of sigma32 proceeds from the N-terminus to the C-terminus. Although FtsH does not have a robust enough unfoldase activity to unfold a tightly folded proteins such as green fluorescent protein, it can unfold proteins with lower [Formula: see text] s such as glutathione S-transferase (Tm = 52 degrees C).     

Role of caveolin-1 and cytoskeletal proteins, actin and vimentin, in adipogenesis of bovine intramuscular preadipocyte cells

We investigated the involvement of caveolin-1 and the cytoskeletal proteins, actin and vimentin, in the adipogenesis of bovine intramuscular preadipocyte (BIP) cells. Immunoblot analysis demonstrated that levels of caveolin-1 and actin gradually increased during adipose conversion in BIP cells, whereas a slight decrease was observed for vimentin. We found that part of the vimentin was clearly distributed to caveolin-1-enriched membrane fractions in BIP cells, but actin was not. During adipogenesis of BIP cells, treatment with the tubulin depolymerizer, nocodazole, significantly increased intracellular triglyceride accumulation compared to non-treated cells. Immunocytochemical analysis showed that actin microfilaments were significantly disrupted in nocodazole-treated cells. Also, a decrease in the localization of vimentin in caveolin-1-enriched fractions and a failure of vimentin to co-immunoisolate with caveolin-1 were observed in nocodazole-treated cells. These results suggest that a rearrangement of cytoskeletal proteins has a role in the intracellular accumulation of lipid droplets during adipogenesis of BIP cells.     

Proliferation of donor mitochondrial DNA in nuclear transfer calves (Bos taurus) derived from cumulus cells

In embryos derived by nuclear-transfer (NT), fusion of donor cell and recipient oocyte caused mitochondrial heteroplasmy. Previous studies from other laboratories have reported either elimination or maintenance of donor-derived mitochondrial DNA (mtDNA) from somatic cells in cloned animals. Here we examined the distribution of donor mtDNA in NT embryos and calves derived from somatic cells. Donor mitochondria were clearly observed by fluorescence labeling in the cytoplasm of NT embryos immediately after fusion; however, fluorescence diminished to undetectable levels at 24 hr after nuclear transfer. By PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, donor mtDNAs were not detected in the NT embryos immediately after fusion (less than 3-4%). In contrast, three of nine NT calves exhibited heteroplasmy with donor cell mtDNA populations ranging from 6 to 40%. These results provide the first evidence of a significant replicative advantage of donor mtDNAs to recipient mtDNAs during the course of embryogenesis in NT calves from somatic cells.