Very-long-chain fatty acid-containing phospholipids accumulate in fatty acid synthase temperature-sensitive mutant strains of the fission yeast Schizosaccharomyces pombe fas2/lsd1

Fission yeast lsd1 strains show aberrant mitosis with a lsd phenotype, large and small daughter nuclei, and a very thick septum, the phenotypic expression being temperature-sensitive. The lsd1(+) gene is the homologue of the budding yeast FAS2 gene encoding the fatty acid synthase alpha-subunit as reported previously (S. Saitoh, K. Takahashi, K. Nabeshima, Y. Yamashita, Y. Nakaseko, A. Hirata, M. Yanagida, J. Cell Biol. 134 (1996) 949--961). In this paper, lsd1 is considered to represent fas2. Here, three fas2 strains were investigated and found to have missense point mutations at different sites in the gene encoding the alpha-subunit of fatty acid synthase. The mutation affected only slightly the enzymatic activities monitored in vitro. Unexpectedly, abnormal phospholipids, phosphatidylcholine and phosphatidylethanolamine, both of which contain a very-long-chain fatty acyl residue (1-melissoyl-2-oleolyl-sn-glycero-3-phosphocholine and 1-melissoyl-2-oleolyl-sn-glycero-3-phosphoethanolamine), accumulated in fas2 strains in a temperature-sensitive manner. Rescue of the fas2 strains by addition of palmitate to the medium at restrictive temperature was accompanied by disappearance of these abnormal phospholipids. Accumulation of these lipids in membranes may cause alteration of various cellular functions.     

Molecular cloning of Bombyx cerebral opsin (Boceropsin) and cellular localization of its expression in the silkworm brain

We have cloned a cDNA for a novel opsin from the larval brain of the silkworm Bombyx mori in which the photoperiodic photoreceptor had been supposed to reside in the cephalic central nervous system (CNS). Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins and showed the greatest degree of homology (84%) with the green visual photoreceptor in the sphingid moth. We have designated this Bombyx cerebral opsin as Boceropsin. Southern blotting experiments indicated that the Boceropsin gene is present in a single copy, and RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain but not in the subesophageal ganglion (Sg) or thoracic ganglion (Tg). Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of Bombyx larvae. This is the first report of expression of an opsin-based protein in CNS of an insect. The possibility that the Boceropsin functions as the photoperiodic receptive pigment in the silkworm is also discussed.     

Absorption of acylated anthocyanins in rats and humans after ingesting an extract of Ipomoea batatas purple sweet potato tuber

We evaluated the absorbability of anthocyanins in humans and rats administered with a beverage prepared from an extract of the tuber of purple sweet potato (Ipomoea batatas Cultivar Ayamurasaki), or with an anthocyanin concentrate. Two major anthocyanin components, cyanidin 3-O-(2-O-(6-O-(E)-caffeoyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside) and peonidin 3-O-(2-O-(6-O-(E)-caffeoyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside)-5-O-beta-D-glucopyranoside), were detected in the plasma and urine of both rats and humans by HPLC or liquid chromatography/mass spectrometry (LC/MS). The plasma concentration of anthocyanins in humans reached a maximum 90 minutes after ingestion, and the recovery of anthocyanins in the urine was estimated as 0.01-0.03%. These results indicate that acylated anthocyanins could be selectively absorbed after ingesting food.     

Characterization of diabetes-related traits in MSM and JF1 mice on high-fat diet

We examined the effect of a high-fat diet on the diabetes-related traits of the Japanese Fancy mouse 1 (JF1), MSM, and C57BL/6J (B6J) mice. MSM and JF1 mice were derived from Mus musculus molossinus. B6J is a commonly used laboratory strain, with the vast majority of genome segments derived from Mus musculus domesticus and Mus musculus musculus, and is susceptible to high-fat diet-induced type 2 diabetes. None of the strains showed symptoms of diabetes or obesity when fed a laboratory chow diet. Under a high-fat diet, JF1 mice developed impaired glucose tolerance, hyperglycemia, hyperinsulinemia, and obesity. B6J mice fed a high-fat diet mildly developed these diabetes-related traits compared to JF1 mice fed a high-fat diet. JF1 mice fed a high-fat diet were classified as having type 2 diabetes and were susceptible to high-fat diet-induced diabetes and obesity. On the other hand, MSM mice were resistant to high-fat diet-induced diabetes. These results indicate that the JF1 strain, with its unique genetic origin, is a useful new animal model of high-fat diet-induced diabetes and obesity. Further investigations using JF1 mice will help to clarify the role of the high-fat diet on human diabetes and obesity.     

Cross-talk between the estrogen receptor-, protein kinase A-, and mitogen-activated protein kinase-mediated signaling pathways in the regulation of lactotroph proliferation in primary culture

Using pharmacological means we investigated the functional interactions between the estrogen receptor (ER)-, protein kinase A (PKA)-, and mitogen-activating protein kinase (MAPK)-mediated pathways in the regulation of lactotroph proliferation in primary culture. Treatment of lactotrophs for 28 h with the PKA inhibitor H89 or KT5720, an effective inhibitor of forskolin-induced proliferation, inhibited both insulin- and estradiol-induced proliferation. Inhibition of the MAPK activity by PD98059 or U0126 suppressed not only insulin-induced proliferation but also forskolin- and estradiol-induced proliferation. However, treatment for 28 h with the antiestrogens 4-hydroxy tamoxifen and ICI182780 failed to antagonize estradiol-induced lactotroph proliferation but instead enhanced it. Prolonging the antiestrogen treatment time from 28 to 88 h was effective in antagonizing estradiol-induced proliferation with this long-term treatment also inhibiting insulin- and forskolin-induced proliferation. There was no decrease in these mitogen-induced proliferations following treatment with a progesterone antagonist or protein kinase C inhibitor. These results suggest that cross-talk occurs between the ER-, PKA-, and MAPK-mediated signaling pathways in the regulation of lactotroph proliferation, and that antiestrogens stimulate and inhibit estradiol-induced proliferation in a time-dependent manner.     

Catheter-mediated delivery of adenoviral vectors expressing beta-adrenergic receptor kinase C-terminus inhibits intimal hyperplasia and luminal stenosis in rabbit iliac arteries

BACKGROUND: Previous studies have shown that incubation of balloon-injured rat carotid arteries with adenoviral vectors encoding the carboxyl terminus of the beta-adrenergic receptor kinase (Ad2/betaARKct) for 30 min reduces neointima formation. However, it is unclear whether this beneficial effect of betaARKct could be achieved using a catheter-based vector delivery system and whether the observed inhibition of neointima formation translated into a reduction of vessel stenosis. METHODS: In this study, Ad2/betaARKct was infused into the balloon-injured site of rabbit iliac arteries using a porous infusion catheter over 2 min. Twenty-eight days after gene transfer, angiographic and histological assessments were performed. RESULTS: Angiographic and histological assessments indicate significant (p < 0.05) inhibition of iliac artery neointima formation and lumen stenosis by Ad2/betaARKct. Our studies demonstrate that an inhibitory effect of Ad2/betaARKct on neointima formation is achievable using a catheter-based vector delivery system and that the inhibition of neointima formation translates into a gain in the vessel minimal luminal diameter. The extent of inhibition (35%) was comparable to that observed with adenoviral-mediated expression of thymidine kinase plus ganciclovir treatment, a cytotoxic gene therapy approach for restenosis. CONCLUSIONS: These results suggest that adenoviral-mediated gene transfer of betaARKct is a clinically viable cytostatic gene therapy strategy for the treatment of restenosis.     

Physiological and subjective responses in the elderly when using floor heating and air conditioning systems

The purpose of this study was to investigate the effects of a floor heating and air conditioning system on thermal responses of the elderly. Eight elderly men and eight university students sat for 90 minutes in a chair under the following 3 conditions: air conditioning system (A), floor heating system (F) and no heating system (C). The air temperature of sitting head height for condition A was 25 degrees C, and the maximum difference in vertical air temperature was 4 degrees C. The air and floor temperature for condition F were 21 and 29 degrees C, respectively. The air temperature for condition C was 15 degrees C. There were no significant differences in rectal temperature and mean skin temperature between condition A and F. Systolic blood pressure of the elderly men in condition C significantly increased compared to those in condition A and F. No significant differences in systolic blood pressure between condition A and F were found. The percentage of subjects who felt comfortable under condition F was higher than that of those under condition A in both age groups, though the differences between condition F and A was not significant. Relationships between thermal comfort and peripheral (e.g., instep, calf, hand) skin temperature, and the relationship between thermal comfort and leg thermal sensation were significant for both age groups. However, the back and chest skin temperature and back thermal sensation for the elderly, in contrast to that for the young, was not significantly related to thermal comfort. These findings suggested that thermal responses and physiological strain using the floor heating system did not significantly differ from that using the air conditioning system, regardless of the subject age and despite the fact that the air temperature with the floor heating system was lower. An increase in BP for elderly was observed under the condition in which the air temperature was 15 degrees C, and it was suggested that it was necessary for the elderly people to heat the room somehow in winter. Moreover, it is particularly important for elderly people to avoid a decrease in peripheral skin temperature, and maintain awareness of the warmth of peripheral areas, such as the leg, in order to ensure thermal comfort.     

Structural basis for new pattern of conserved amino acid residues related to chitin-binding in the antifungal peptide from the coconut rhinoceros beetle Oryctes rhinoceros

Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4-35 is 0.728 +/- 0.217 A from the mean structure. The solution structure consists of a two-stranded antiparallel beta-sheet connected by a type-I beta-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18-36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process.