Loss of androgen dependency with preservation of functional androgen receptors in androgen-dependent mouse tumor (Shionogi Carcinoma 115).

Shionogi Carcinoma 115 (SC 115) is an androgen-dependent mouse tumor. Chiba Subline 2 (CS 2) is an androgen-independent subline derived from SC 115. CS 2 contains androgen receptors (AR), but is refractory to androgen and does not exhibit androgen-related responses which are observed in SC 115. In the present study the structure and function of AR in SC 115 and CS 2 are examined using cloned cells. There were no gross rearrangements or deletions in the AR genes of these cell lines when compared by Southern blot analysis with the AR gene in the mouse seminal vesicle. SC 115 and CS 2 expressed AR mRNA of normal size. When the cDNA containing DNA- and androgen-binding domains of the AR genes of both cell lines were amplified by polymerase chain reaction, no mutations were found in these regions. SC 115 and CS 2 were transfected with a plasmid containing a long terminal repeat of mouse mammary tumor virus linked to the chloramphenicol acetyltransferase (CAT) gene. Androgen stimulation of these transfectants resulted in equal elevation of CAT activity. These results indicated that the androgen-independent CS 2 contained functionally normal AR which were identical to those in the androgen-dependent parent tumor.     

Crystallization of water in multilamellar vesicles

The two-step crystallization of water in multilamellar vesicles (MLVs) of phosphatidylcholines has been investigated. The main crystallization occurs near -15 degrees C and involves bulk water. Contrary to unilamellar vesicles, a sub-zero phase transition is observed for MLVs at -40 degrees C that corresponds to the crystallization of interstitial water, as proved by Fourier transform infrared absorption and differential scanning calorimetry (DSC) experiments. Furthermore, by means of the DSC method and, more specifically, using the enthalpy change values Delta H(sub) at the sub-zero transition, the number of water molecules per 1,2-dipalmitoylphosphatidylcholine (DPPC) molecule giving rise to this transition has been estimated for different H(2)O/DPPC molar ratios. The curve of the molecular fraction of water molecules involved in the sub-zero transition versus the H(2)O/DPPC molar ratio exhibits a maximum for H(2)O/DPPC equal to 27 (40% in mass of water) and tends towards zero for H(2)O/DPPC ratio values approaching that of the swelling limit of the membrane. A smaller enthalpy value of the sub-zero transition is found for 1-oleoyl-2-palmitoyl-3-phosphatidylcholine (OPPC) than for DPPC. This may be explained by the decrease of interstitial water's quantity when the lipid contains an unsaturated chain. When troxerutin, a hydrophilic drug, is added to the DPPC multilayers, the decrease of Delta H(sub) and melting enthalpy of bulk water is attributed to a decrease of the entropy of the liquid phase owing to the network of water molecules surrounding troxerutin molecules. In all cases, the experiments revealed that the sub-zero transition occurs only in the presence of excess water with respect to the swelling limit of membranes. This evidence could be, at least qualitatively, related to an increase of membrane pressure on interstitial water subsequent to bulk water crystallization.     

The combined use of oral and topical lipophilic antioxidants increases their levels both in sebum and stratum corneum

The concentration of Vitamin E (vit E) and ubiquinone (CoQ10), which together with squalene (SQ), play a key role against external oxidative insult, has been shown to decrease significantly during ageing. The aim of the present study is to inquire the effect of the combined use of topical bio-cosmetics containing natural active principles (including sebum-like lipid fractions, sebum and epidermal lipophilic and hydrophilic antioxidants), and oral antioxidant supplements on the antioxidant content of sebum and stratum corneum. We therefore treated the face and the back of 50 female volunteers aged 21-40, daily for two months, with a base cream containing 0.05% ubiquinone, 0.1% vit E, and 1% squalene. In addition 50 mg of CoQ10 + 50 mg of d-RRR-alpha-tocopheryl acetate + 50 microg of selenium were administered orally to half of the volunteers (Group A). Group B was represented by 25 volunteers who were treated only topically. Every 15 days during treatment the levels of CoQ10, vit E and SQ were verified in sebum, stratum corneum, and plasma. The daily topical application of the cream led to a significant increase, that peaked after 60 days, of the levels of CoQ10, d-RRR-alpha-tocopherol and SQ in the sebum (Group B), without significantly affecting the stratum corneum or plasma concentrations of the redox couple CoQ10H2/CoQ10 and vit E. The concomitant oral admistration of antioxidants produced in Group A a significant increase of the levels of CoQ10H2/CoQ10 and vit E both in plasma and stratum corneum after 15 and 30 days treatment respectively, compared to Group B. However the sebum levels of lipophilic antioxidants and SQ did not show a significant increase. After the treatments, the levels of CoQ10H2/CoQ10, vit E and SQ went back to basal levels within 6-8 days in sebum, 12-16 days in the stratum corneum, and 3-6 days in plasma. Therefore topical application of the antioxidants was able to increase their level in sebum, while the concomitant oral administration also affected the levels of vit E and CoQ10 in the stratum corneum.     

Mutations in the Plk gene lead to instability of Plk protein in human tumour cell lines

It has been established that mutations in Drosophila Polo cause abnormalities in mitosis. In human cells, maximal Plk activity is reached in the M phase of the cell cycle, and the function of Plk is therefore considered to be required for mitotic cellular events such as spindle formation, chromosome segregation and cytokinesis. Microinjection of anti-Plk antibody into living cells has been found to induce a mitotic abnormality that contributes to the generation of aneuploidy, and this is an important finding in relation to tumour development. Indeed, previous studies have shown that the level of expression of a mitotic checkpoint gene, hsMAD2, is reduced and that another checkpoint gene, BUB1, is mutated in certain human cancer cells.     

Involvement of protein kinase C-regulated ceramide generation in inostamycin-induced apoptosis

Activation of caspases is commonly involved in the apoptosis induced by various anticancer drugs. However, the upstream events leading to the activation of caspases seem to be specific to each anticancer drug. In the present study, we examined the possible involvement of protein kinase C (PKC) and ceramide generation in caspase-3(-like) protease activation induced by inostamycin, a phosphatidylinositol synthesis inhibitor. Treatment of cells with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, suppressed the release of cytochrome c from mitochondria and the activation of caspase-3(-like) proteases in inostamycin-treated cells, but not in other anticancer drug-treated cells. Inostamycin induced the elevation of intracellular ceramide levels, and fumonisin B1, an inhibitor of ceramide synthase, inhibited inostamycin-induced cytochrome c release, caspase-3(-like) protease activation, and apoptosis. Moreover, TPA also inhibited inostamycin-induced ceramide synthesis. Taken together, our results suggest that inostamycin-induced apoptosis is mediated by PKC-regulated ceramide generation, leading to the activation of a caspase cascade.     

Genetic variations in Lycoris radiata var. radiata in Japan

The genetic variations of Lycoris radiata var. radiata, a completely sterile triploid from Japan, were examined by comparing the nucleotide sequences of genomic DNA regions in 11 triploid strains sampled from Japan and four triploid strains sampled from China, and in two diploid strains of Lycoris radiata var. pumila, which is endemic to China and fertile. For this purpose, two genes were analyzed, the lectin gene in the nuclear genome and the maturase gene in the chloroplast genome. A clear genetic constancy was observed in their DNA nucleotide sequences. For both genes, completely identical nucleotide sequences were detected in the 11 Japanese and four Chinese triploid strains and also between the two Chinese diploid strains. However, some genetic variations were observed between the Japanese and Chinese triploid strains, and between the triploid and diploid strains. These results are consistent with the findings obtained from previous chromosome karyotype analyses and allozyme analyses. In addition, in our preliminary FISH analysis of the physical mapping of the rRNA gene family, the 18S-5.8S-26S rRNA and 5S rRNA loci were localized on six and four chromosomes, respectively. Regarding the 18S-5.8S-26S rRNA loci, two were associated with two SAT chromosomes. The remaining four were distinguished by having no secondary constriction. Localization of 5S rRNA loci to chromosome spreads revealed three sites on the proximal part of the long arm of three acrocentric chromosomes and one site on the distal part of the long arm of the SAT chromosome; the latter site was juxtaposed to the 18S-5.8S-26S rRNA loci. These findings indicate that L. radiata var. radiata is not a typical autotriploid. The present paper discusses the possible origin of L. radiata var. radiata from a diploid variety of L. radiata var. pumila, based on the molecular cytogenetic analysis and DNA sequence analysis.     

Genetic variations in the chloroplast genome and phylogenetic clustering of Lycoris species

The genus Lycoris of Amaryllidaceae comprises approximately 20 species that are distributed only in the moist warm temperate woodlands of eastern Asia. The objectives of this study were: (1) to clarify the phylogeny of the Lycoris species by using the definitive DNA sequencing method and (2) to examine the possible maternal donor of the hybrid origin Lycoris species and the Japanese triploid strains of Lycoris radiata var. radiata. The nucleotide sequence of the maturase K (matK) gene and the noncoding intergenic spacer (IGS) between the atpB and rbcL genes in the chloroplast genome were determined in a total of 27 strains of 11 species of the genus Lycoris. Variation among taxa was mainly due to nucleotide substitution, although deletions and an insertion were found in the IGS. For two chloroplast regions, the phylogenetic trees showed essentially similar topology, indicating the existence of four clades, I, II, III, and IV. For all the species except L. radiata, intraspecific variation was smaller than interspecific variation. For L. radiata, triploid strains were divided into clades I and II, and diploid strains were divided into clades I and IV. This implies that the diploid species of L. radiata var. pumila is a probable ancestral species. The clustering indicated that the chloroplast genome has not evolved in parallel with the karyotype in genus Lycoris. Regarding the hybrid origin species, the maternal parents of L. squamigara, L. albiflora and L. rosea were revealed to be L. longituba, L. radiata and L. radiata var pumila, respectively. We also suggest that a diploid strain of L. radiata var. pumila in clade I might be a candidate of the maternal donor of the Japanese triploid strains. A possible model of the maternal donor of Lycoris species is proposed.     

Suppression of cellular invasion by glybenclamide through inhibited secretion of platelet-derived growth factor in ovarian clear cell carcinoma ES-2 cells

It has been demonstrated that potassium channels (K(+) channels) play significant roles in some malignant phenotypes. Here, we provide the first evidence that treatment with glybenclamide, an ATP-sensitive K(+) channel blocker, inhibited cell migration in an ovarian clear cell carcinoma cell line, ES-2. Treatment with glybenclamide or knockdown by siRNA targeted against K(+) channel subunits demonstrated the suppression of ovarian cancer cell invasion, which occurred via inhibition of PDGF-AA secretion. Therefore, our findings suggest that K(+) channel blockers may be useful chemotherapeutic drugs for blocking the invasiveness of ovarian cancers.     

Role of the SEL1L:LC3-I complex as an ERAD tuning receptor in the mammalian ER

Several regulators of endoplasmic reticulum (ER)-associated degradation (ERAD) have a shorter half-life compared to conventional ER chaperones. At steady state, they are selectively removed from the ER by poorly defined events collectively referred to as ERAD tuning. Here we identify the complex comprising the type-I transmembrane protein SEL1L and the cytosolic protein LC3-I as an ERAD tuning receptor regulating the COPII-independent, vesicle-mediated removal of the lumenal ERAD regulators EDEM1 and OS-9 from the ER. Expression of?folding-defective polypeptides enhances the lumenal content of EDEM1 and OS-9 by inhibiting their SEL1L:LC3-I-mediated segregation. This raises ERAD activity in the absence of UPR-induction. The mouse hepatitis virus (MHV) subverts ERAD tuning for replication. Consistently, SEL1L or LC3 silencing impair the MHV life cycle. Collectively, our data provide new molecular information about the ERAD tuning mechanisms that regulate ERAD in mammalian cells at the post translational level and how these mechanisms are hijacked by a pathogen.