Isao Chinen

A male and hermaphrodite specific RAPD marker for papaya (Carica papaya L.)

Abstract The random amplified polymorphic DNA (RAPD) technique was used to determine the sex of a dioecious species, Carica papaya L., with three sex types, male, female and hermaphrodite. A 450 bp marker fragment, named PSDM(Papaya Sex Determination Marker), exists in all male and hermaphrodite plants but not in the female plants so far analyzed. The DNA sequence of PSDM exhibited no significant similarity to previously reported sequences. A sequence-characterized amplified region (SCAR) marker, SCARps, was developed from PSDM to determine the sex of papaya. Southern hybridization, using PSDM as a probe, showed that PSDM exists in the male and hermaphrodite genomes, but not in the female genome. This result strongly suggests that PSDM is located on the chromosome region that is specific to the male and the hermaphrodite. SCARps is a suitable marker for the precise and rapid diagnosis of sex in papaya.

Composition of lipids, fatty acids and sterols in Okinawan corals

A survey of lipid composition was made for 15 cnidarians from Okinawa, Japan. Eleven zooxanthellate scleractinian corals, an azooxanthellate scleractinian coral Tubastrea sp., a soft coral Lobophytum crassum, a hydroid coral Millepora murrayi and a sea anemone Boloceroides sp. were examined to elucidate the total lipid content, fatty acid composition for each lipid class and sterol composition. All specimens contained monoalkyldiacylglycerol which migrated between the triacylglycerols and esters on thin layer chromatography (TLC). Analysis by high performance thin layer chromatography (HPTLC) and Gas chromatography-mass spectrometry (GC-MS) revealed that these cnidarians were rich in wax ester and triacylglycerol, and that palmitic acid (16:0) was the most abundant fatty acid component of these lipid classes, followed by stearic (18:0) and oleic (18:1, n-9) acid in order of concentration. Of 11 sterols separated, four sterols were identified. It is suggested that sterol composition may be more useful for the biochemical classification of these cnidarians than fatty acid composition.

Inhibition of Osteoporosis Due to Restricted Food Intake by the Fish Oils DHA and EPA and Perilla Oil in the Rat

Seven-week old female rats fed restricted foods including the fish oils Docosahesaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) and perilla oil with food intake decreased by 50%, had increases of fracture force and bone mineral density (BMD) and decreases in levels of Deoxypiridinoline (Dpd) and Calcium (Ca) in the urine, compared with those of rats with osteoporosis due to restricted soy bean oil food intake. Therefore, the fish oils DHA and EPA and perilla oil depressed excretion of urinary Ca and inhibited osteoporosis due to restricted food intake.

New Phenolic Compounds from Kokuto, Non-centrifuged Cane Sugar

Five new phenolic compounds, 4-(β-D-glucopyranosyloxy)-3,5-dimethoxyphenyl-propanone (8), 3-[5-[(threo) 2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxybenzofurany]]-propanoic acid (12), 2-[4-(3-hydroxy-1-propenyl)-2,6-dimethoxyphenoxy]-3-hydroxy-3-(4-hydroxy-3,5-dimethoxyphenyl)propyl-β-D-glucopyranoside (13), 4-[(erythro) 2,3-dihydro-3(hydroxymethyl)-5-(3-hydropropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenyl-β-D-glucopyranoside (14), 9-O-β-D-xylopyranoside of icariol A2 (15), and known phenolic compounds were isolated from Kokuto, non-centrifuged cane sugar (Saccharum officinarum L.). Their structures were determined by a spectral investigation.

Biased distribution of the branched-chain fatty acids in ceramides of vernix caseosa

The compositions of ester- and amide-linked fatty acids from ceramides of human vernix caseosa were described with emphasis on the distribution of the branched-chain fatty acid (BCFA). Two novel ceramides were isolated from vernix caseosa in the course of this study: the acylated type of esterified α-OH-hydroxyacid/sphingosine ceramide (Cer[EAS]) and nonacylated type of non-OH fatty acid/hydroxysphingosine ceramide (Cer[NH]). Their chemical structures were identified by nuclear magnetic resonance and chemical procedure. The Cer[EAS] was an acylceramide and consisted of the highest concentrations of ester- and amide-linked BCFA (62 and 67%, respectively). The iso- or anteiso-branching structures of the aliphatic chains were confirmed by the mass spectra of their picolinyl or pyrrolidide derivatives. As a whole, amide-linked fatty acids of ceramides 1–7 and Cer[NH] were normal types of straight-chain fatty acids with or without α- or ω-hydroxylation. The BCFA concentrations of amide-linked fatty acids in these ceramides (ceramides 1–7 and Cer[NH]) were low and less than 10%. The BCFA thus occurred exclusively in a novel acylceramide of Cer[EAS] in the vernix caseosa.

Serum-free culture of rat keratinocytes

SummaryProcedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 µg/ml), hydrocortisone (0.1 µg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.

1-O-alkyl-2,3-diacylglycerols in the skin surface lipids of the hairless mouse

A neutral lipid class was isolated by thin-layer chromatography from the skin surface lipids of the hairless mouse. The fraction migrated faster than triglycerides and had a migration rate similar to that of diacyl alkanediols (diester wax). Upon deacylation, however, the long-chain diols were identified as 1-alkylglycerol ethers based on their chromatographic properties and on the mass spectra of their nicotinylidene derivatives. Thus, the skin lipid fraction was identified as 1-O-alkyl-diacylglycerol. The alkyl moieties were all saturated and even-numbered and ranged in chainlength from C16 to C22 with 1-O-hexadecylglycerol amounting to 34% of the total glycerol ether moieties. The fatty acids derived from this lipid fraction were mostly monoenoic with chainlengths ranging from C16 to C24. The major acyl component was eicosenoic acid (20∶1) representing 61% of the total fatty acids.

Lipoprotein and apoprotein profile of Japanese quail.

The present study delineated the lipoprotein and apoprotein distribution in Japanese quail. Quail lipoprotein was composed of three fractions: VLDL, d < 1.020; LDL, 1.020 < d < 1.081; HDL, 1.081 < d < 1.210. When animals were fed the cholesterol-free diet, HDL was the predominant form, LDL intermediate, VLDL and chylomicron were smallest in amount. Feeding of cholesterol induced a marked change in the lipoprotein profile: VLDL or chylomicron predominated over HDL and LDL. An apoprotein of 26 kD (molecular weight) was the major protein moiety comprising more than 50% of total apoprotein in the entire density range of lipoprotein class. Amino acid composition of 26 kD protein was similar to hen, rat and human apo A-I. N-Terminal 36 amino acid sequence of 26 kD protein showed 92% homology to chicken apo A-I and 11% homology to human apo A-1. The 26 kD protein did not react with the antibody raised against human apo A-I. These observation showed that the 26 kD protein was partially identical to apo A-I.

Inhibition of Osteoporosis in Rats Fed with Sugar Cane Wax

Rats fed on a restricted, semi-purified diet containing a 50%-reduced level of carbohydrate and oil, but normal levels of protein, minerals and vitamins, exhibited osteoporosis. However, rats fed on this restricted diet, but containing sugar cane wax, did not exhibit this bone disease. Sugar cane wax, containing a long-chain carbohydrate with an OH radical, prevented the development of osteoporosis via a non-estrogenic mechanism.

Accumulation of 1-o-alkyl-2,3-diacylglycerols in cultured rat keratinocytes.

The present study was undertaken to identify the chemical structure of neutral lipid accumulated in cultured rat keratinocytes and to address their metabolism. Neutral lipid of similar mobility with alkyldiacylglycerol was isolated from cultured rat keratinocytes by thin layer chromatography. The long-chain diols derived from the neutral lipids were identified as 1-alkylglycerol based on the mass spectra of their nicotinylidene derivatives. Thus these neutral lipids were identified as 1-o-alkyl-2,3-diacylglycerols (ADAG). Addition of rat serum elevated the level of ADAG with increasing trend of linoleic acid concentration in this fraction. [14C]Acetate added to the confluent plates was incorporated into alkyl- and acyl-chains of ADAG with incubation in 24 h, and remained un-metabolized up to 72 h. This, however, is not the case for the label incorporation into phospholipid and triacylglycerol. Radioactivities of these two lipid fractions appeared to reach the maximum in 24 h, and thereafter decreased to 72 h with a similar decay curve. Incorporation of [14C]acetate into phospholipid and ADAG was significantly depressed, and that into triacylglycerol and free cholesterol was increased by the supplementation of the medium with rat serum. In concomitance with the accumulation of ADAG, the concentration of ethanolamine-plasmalogen increased in the cultured keratinocytes. The results of the present study first showed the elevated level of ether lipid synthesis in the proliferating primary culture of rat keratinocytes.