The Gene Encoding Flavanone 3-Hydroxylase is Expressed Normally in the Pale Yellow Flowers of the Japanese Morning Glory Carrying the speckled Mutation Which Produce Neither Flavonol nor Anthocyanin but Accumulate Chalcone, Aurone and Flavanone

The Japanese morning glory carrying the recessive mutable speckledallele with the dominant speckled-activator bears colorlessflowers with fine and round colored spots distributed over thecorolla whereas the plant without the speckled-activator producespale yellow flowers. Previous chemical analysis has indicatedthat a mutation in the gene for flavanone 3-hydroxylase (F3H)is a likely candidate for the speckled allele. However, theF3HmRNA without sequence alteration accumulates normally inthe pale yellow flowers, indicating that the speckled alleleis neither the F3H gene nor a regulatory gene acting on theF3H gene expression. (Received April 4, 1997; Accepted June 2, 1997)     

Characterization of tumor-associated fucogangliosides from PC 12 pheochromocytoma cells

PC 12h pheochromocytoma cells were subcutaneously transplanted into rat. We found the transplanted tumors accumulated some fucogangliosides associated with PC 12 cells. These gangliosides were isolated and purified by DEAE-Sephadex A-25 and Iatrobeads column chromatographies. Their structures were determined by fast atom bombardment mass spectrometry, proton nuclear magnetic resonance spectrometry, permethylation study, and sequential degradation using various exoglycosidases and mild acid hydrolysis. Two tumor-associated fucogangliosides were found to possess the blood group B determinant as follows: G6: IV2Fuc alpha, IV3Gal alpha, II3NeuAc, GgOse4Cer; G11: IV2Fuc alpha, IV3Gal alpha, II3 (NeuAc)2, GgOse4Cer. A ganglioside with the similar structure as ganglioside G6 was isolated from rat hepatoma cells (Holmes, E.H., and Hakomori, S-I. (1982) J. Biol. Chem. 257, 7698-7703). However, ganglioside G11 has not previously been reported in the literature. These fucogangliosides reacted with the monoclonal antibody prepared by immunizing mice with PC 12h cells. Other fucogangliosides were also found to accumulate in the transplanted tumor tissues. They were identified as fucosyl-GM1 and fucosyl-GDlb. These fucogangliosides did not react with the monoclonal antibody against PC 12h cells.     

Analyses of azopigments obtained from the delta fraction of bilirubin from mammalian plasma (mammalian biliprotein).

Azopigments were obtained from the delta fraction of bilirubin (mammalian biliprotein) in cholestatic sera of men, rats and guinea pigs by diazo reaction with diazotized p-iodoaniline and analysed by t.l.c. Delta bilirubin of men and rats generated both unconjugated and glucuronide-conjugated azodipyrroles, whereas that of guinea pigs, in which the predominant form of conjugated bilirubin in serum was bilirubin monoglucuronide, generated only unconjugated azodipyrrole. We further analysed the azopigments by reversed-phase h.p.l.c. to distinguish their endovinyl and exovinyl isomers. The results indicated (a) that covalent binding of bilirubin to protein occurs exclusively on the conjugated dipyrrolic (either endovinyl or exovinyl) half of the parent conjugated bilirubin, (b) that both bilirubin monoglucuronide and bilirubin diglucuronide generate delta bilirubin, the latter yielding a 'conjugated' form of delta bilirubin that preserves the glucuronic acid moiety on the dipyrrolic half not bound covalently to protein, and (c) that therefore at least four forms of delta bilirubin exist in jaundiced sera of men and rats.     

Exon-intron organization, expression, and chromosomal localization of the human motilin gene

The human motilin gene has been isolated and characterized. The gene spans about 9 kilobase pairs (kb) and the 0.7 kb motilin mRNA is encoded by five exons. The 22-amino-acid motilin sequence is encoded by exons 2 and 3. The human motilin gene was mapped to the p21.2----p21.3 region of chromosome 6 by hybridization of the cloned cDNA to DNAs from a panel of reduced human-mouse somatic cell hybrids and by in situ hybridization to human prometaphase chromosomes. RNA blotting using RNA prepared from various regions of the human gastrointestinal tract revealed high levels of motilin mRNA in duodenum and lower levels in the antrum of the stomach; motilin mRNA could not be detected by this procedure in the esophagus, cardia of the stomach, descending colon or gallbladder.     

A comparative 1H NMR study of mouse alpha(1-53) and beta(2-53) epidermal growth factors

The three-dimensional structure of the mouse epidermal growth factor (EGF) in solution was studied by comparison of the 1H NMR spectra of alpha EGF (1-53) and beta EGF (2-53, des-asparaginyl 1 form). Using pH dependence of chemical shifts and a two-dimensional difference spectrum, the effect of the N-terminal deletion was investigated based on the complete assignment of the proton resonances. The affected residues were all found to be located exactly in the triple-stranded, beta-sheet core in the N-terminal domain of the EGF molecule.     

The usefulness of CHAPS as a non-cytotoxic stabilizing agent in purification of growth factors

Among several detergents, a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), was found to be least cytotoxic for cultured mammalian cells. CHAPS improved the activity recovery and elution profile of crude and purified fibroblast growth factors (FGFs) during chromatographies. Diluted preparations of FGFs were stabilized by CHAPS against the loss during storage. Amino acid sequence analysis was not disturbed by CHAPS. CHAPS was removable by reversed-phase high-performance liquid chromatography. These results indicate that CHAPS is useful as a non-cytotoxic stabilizing agent in purification of various kinds of bioactive polypeptides.Abbreviations -MEM Alpha Modification of Eagle's Minimal essential medium - CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate - CHAPSO 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate - CS Calf Serum - EGF Epidermal Growth Factor - FGF Fibroblast Growth Factor - HPLC High-Performance Liquid Chromatography - NGF Nerve Growth Factor - NOG 1-O-n-octyl--D-glucopyranoside - NP-40 Nonidet P-40 - PBS Phosphate-Buffered Saline - SB 12 3-(dodecylmethylammonio)-1-propane sulfonate - SDS Sodium Dodecyl Sulfate - TGF- and Transforming Growth Factor type and      

Thermostable alanine racemase from Bacillus stearothermophilus: DNA and protein sequence determination and secondary structure prediction

The nucleotide sequence of the alanine racemase (EC 5.1.1.1) gene from a thermophile, Bacillus stearothermophilus, was determined by the dideoxy chain termination method with universal and synthetic site-specific primers. The amino acid sequence of the enzyme predicted from the nucleotide sequence was confirmed by peptide sequence information derived from the N-terminal amino acid residues and several tryptic fragments. The alanine racemase gene consists of 1158 base pairs encoding a protein of 386 amino acid residues; the molecular weight of the apoenzyme is estimated as 43,341. The racemase gene of B. stearothermophilus has a closely similar size (1158 vs 1167 base pairs) to that of the gene of a mesophile, B. subtilis, but shows a higher preference for codons ending in G or C. A comparison of the amino acid sequence with those of Bacillus subtilis and Salmonella typhimurium dadB and alr enzymes revealed overall sequence homologies of 31-54%, including an identical octapeptide bearing the pyridoxal 5'-phosphate binding site. Although the residues common in the four racemases are not continuously arrayed, these constitute distinct domains and their hydropathy profiles are very similar. The secondary structure of B. stearothermophilus alanine racemase was predicted from the results obtained by theoretical analysis and circular dichroism measurement.     

Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization

The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B. stearothermophilus. A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones. Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment. The overproduced enzyme was confirmed to originate from B. stearothermophilus by an immunochemical cross-reaction with the enzyme of B. stearothermophilus. The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000. At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine). Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B. stearothermophilus is a pyridoxal 5'-phosphate enzyme.     

N-(2-Aminoethyl)-5-isoquinolinesulfonamide, a newly synthesized protein kinase inhibitor, functions as a ligand in affinity chromatography. Purification of Ca2+-activated, phospholipid-dependent and other protein kinases

We designed a simple procedure for the purification of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) from rabbit brain, using affinity chromatography with a new affinity adsorbent. The adsorbent was synthesized by attaching the amino residue of N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) to cyanogen bromide-activated Sepharose. H-9 is a potent competitive inhibitor of protein kinase C, cGMP-, and cAMP-dependent protein kinase with respect to ATP and exhibits inhibition constants of 18, 0.87, and 1.9 microM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, 23, 5036). A 960-fold purification was achieved in the two-step procedure, which entailed DEAE-cellulose and the affinity chromatography. The resultant preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis under conditions of denaturation with sodium dodecyl sulfate. The affinity of protein kinase C for the H-9-Sepharose was high, and the enzyme could not be eluted either by a high concentration of sodium chloride or by 40% glycerol. The protein kinase C could be eluted from H-9-Sepharose by the buffer containing both 0.2 M NaCl and 20% glycerol, thereby suggesting that the binding between protein kinase C and H-9-Sepharose was due to both hydrophobic and electrostatic interactions. H-9 coupled to Sepharose retained both cyclic nucleotide-dependent protein kinases and protein kinase C, and these enzymes could be eluted separately by the buffer containing L-arginine, a potent inhibitor of these three kinases. The novel aspects of these three multifunctional protein kinases can thus be investigated using isoquinolinesulfonamide derivatives.     

Differences in hair density of Japanese monkeys (Macaca fuscata fuscata) with locality and age

The hair density of free-ranging Japanese monkeys (Macaca fuscata fuscata) living in three different areas was investigated. The Japanese monkeys had thicker hair than other macaques. The hair density in the Japanese monkeys varied with locality: the northern monkeys had thicker hair than the southern ones. The density did not vary markedly with age up to 3 years of age, but then decreased gradually up to adult age (≧7 years old). The remarkable growth of the trunk suggested that the total number of hairs increased with age, especially during the period as a juvenile.