Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Characterization of a diphosphonopentaosylceramide containing 3-O-methylgalactose from the skin of Aplysia kurodai (sea hare)

The complete structure is proposed for a ceramide (Cer), bis(2-aminoethylphosphono)-pentaoside, isolated from the skin of Aplysia kurodai. This new phosphonoglycosphingolipid was purified using two systems of column chromatography on silicic acid. The purity of the glycolipid was confirmed by thin-layer chromatography, analysis of its composition, and proton magnetic resonance spectrometry. The component carbohydrates were glucose, galactose, N-acetylgalactosamine, and 3-O-methylgalactose. Most (90%) of the fatty acid was palmitic acid and the major sphingosine bases were octadeca-4-sphingenine (51%) and anteisononadeca-4-sphingenine (38%). 2-Aminoethylphosphonyl-6-galactose was identified after its partial hydrolysis. From studies by methanolysis, permethylation, mild acid hydrolysis, hydrogen fluoride treatment, chromium trioxide oxidation combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry, the structure of the glycolipid was concluded to be 3-OMeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)-Gal alpha 1----2](2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Cer.     

Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450reductase, was purified by chromatography through DEAE-cellulose,2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilizationfrom microsomes from Ceratocystis fimbriata-infected sweet potatoroot tissue with Emulgen 913. The enzyme existed in three formsafter solubilization which migrated to positions correspondingto molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamidegel. Trypsin treatment of the enzyme species with the largestpolypeptide yielded the species with the smallest one. Aftersucrose density gradient centrifugation of the pellet fractionobtained by centrifugation at 100,000?g of the crude extract,the enzyme species with the largest polypeptide was presentin the particulate fractions, whereas that with the smallestone was only found at the top of the gradient. We conclude thatthe enzyme species with the largest polypeptide is in an intact,amphipathic form, whereas that with the smallest one, and probablyalso the other species, is its hydrophilic domain produced byan endogenous protease(s). The K_m values of the enzyme in theintact form for NADPH and cytochrome c were 7.7 and 2.3 µM,respectively. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)     

Two positional isomers of sialylheptasaccharides isolated from the urine of a patient with sialidosis

We describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of beta-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows: AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----3(Man alpha 1----6)Man beta 1----4GlcNac; AcNeu alpha 2----6Gal beta 1----4GlcNac beta 1----2Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNac. Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, alpha-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.     

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Occurrence of anthocyanoplasts in cell suspension cultures of sweet potato

Intensely pigmented and spherical vesicles (anthocyanoplasts) were found in anthocyanin-containing cells of sweet potato (Ipomoea batatas) suspension cultures. Anthocyanin synthesis began to first occur 24–48 h after exposure to light, and then numerous small red vesicles were detected under a microscope. The frequency of anthocyanoplast-containing cells rapidly increased to finally about 80% of the total cultured cells after 5 days of irradiation. Fully developed anthocyanoplasts reached 10–15 m in diameter. On the other hand, neither anthocyanin synthesis nor development of anthocyanoplasts was induced in the dark-cultured cells. 2,4-D also inhibited anthocyanin synthesis and development of these vesicles. The results suggest that anthocyanoplasts might be a site of anthocyanin synthesis and/or accumulation.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Receptive field mechanisms of ganglion cells in the cat retina

On the basis of anatomical and physiological results of the vertebrate retina, a method is proposed for analysing the respective fields of ganglion cells in the cat retina. In the model, we assume the following: (a) Ganglion cells receive their input from bipolar and/or amacrine cells. (b) The nonlinearity of ganglion cell responses is due to the activities of transient type amacrine cells. The method has been proved to be effective. According to the results of this investigation, the receptive field properties of X type and Y type ganglion cells are heterogeneous. Thus, it may be considered that their receptive fields consist of center and surround mechanisms. The receptive field properties of X-cells are almost linear and the X-cells seem to receive most of their input from bipolar cells. On the other hand, the ones of Y-cells are highly nonlinear. Consequently, it is conceivable that the Y-cells receive their input mainly from transient type amacrine cells.