Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4'-imidazo-lidine+ ++)-2',5'-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.     

Microsomal monooxygenase system in Morris hepatoma: purification and characterization of cytochromes P-450 from Morris hepatoma 5123D of 3-methylcholanthrene-treated rats

Two forms of cytochrome P-450 (hepatoma P-450MCI and P-450MCII) were purified from hepatoma 5123D microsomes of tumor-bearing rats treated with 3-methylcholanthrene. Hepatoma P-450MCI had a specific content of 18.4 nmol/mg protein and showed a main protein band with a minimum molecular weight of 56,000 on sodium dodecyl sulfate-polyacrylamide gel. Hepatoma P-450MCII had a specific content of 7.38 nmol/mg protein and a minimum molecular weight of 50,000. The carbon monoxide-reduced difference spectral peak of hepatoma P-450MCI was at 446.5 nm, whereas the peak of hepatoma P-450MCII was at 451 nm. In the reconstituted system, hepatoma P-450MCI catalyzed 3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin, but showed low activities for N-demethylation of benzphetamine and aminopyrine, O-demethylation of p-nitroanisole, and p-hydroxylation of aniline. On the other hand, hepatoma P-450MCII did not catalyze hydroxylation of any of the substrates tested. By Ouchterlony double-diffusion analysis, hepatoma P-450MCI was immunologically indistinguishable from rat liver cytochrome P-450c, but hepatoma P-450MCII was distinct from hepatoma P-450MCI and rat liver cytochrome P-450c. Peptide maps of hepatoma P-450MCI and rat liver cytochrome P-450c after proteolysis with Staphylococcus aureus V8 protease demonstrated the similarity of the two cytochromes P-450.     

Fibroblast growth factor in the human placenta

Fibroblast growth factor (FGF) has been purified 333,000-fold from human placenta by a combination of salt precipitation, cation-exchange chromatography, and Heparin-Sepharose affinity chromatography. Molecular weight (15-16 kDaltons), amino acid composition, bioactivity and immunological crossreactivity with bovine pituitary FGF indicate that the mitogens from the two species are closely related molecules.     

Promotion of Rooting in Azukia Cuttings by Possible Glycoproteins Extracted from Lentinus edodes Culture

Macromolecules purified from Lentinus edodes mycelia cultureenhanced adventitious root formation in Azukia epicotyl cuttings.Partial purification by sequential column chromatographies gavematerial composed of 71% polysaccharides and 29% proteins. Thesugar moiety consisted of mainly xylose, glucose and arabinose,the sum of their contents being more than 76% of the total carbohydrates.The protein moiety consisted of mainly glycine and serine, whichaccounted for more than 40% of the total amino acid residues. (Received June 9, 1984; Accepted November 12, 1984)     

Genome type analysis of adenovirus type 4 isolates, recently obtained from clinically different syndromes in some areas in Japan

DNA cleavage analyses with EcoRI, HindIII and BamHI were carried out to investigate genome types of recent Ad 4 isolates obtained from acute respiratory disease (8 strains), and ocular disease (11 strains) in Japan. DNA cleavage patterns of all 19 isolates studied were identical regardless of whether they were recovered from respiratory tract or conjunctiva, but were distinct from that of the prototype strain.     

Characterization of antisera to 2-hydroxyestradiol and 4-hydroxyestradiol using 6-(O-carboxymethyl)oxime- and 17-hemisuccinate-bovine serum albumin conjugates and radioimmunoassay

For radioimmunoassay of the catechol estrogens, four hapten-bovine serum albumin (BSA) conjugates were prepared from 6-oxo-2-hydroxyestradiol 6-(O-carboxymethyl)oxime, 2-hydroxyestradiol 17-hemisuccinate, 6-oxo-4-hydroxyestradiol 6-(O-carboxymethyl)oxime and 4-hydroxyestradiol 17-hemisuccinate by coupling with BSA, employing the mixed anhydride method. The antisera elicited in rabbits by immunization with these antigens showed high affinity and specificity for 2-hydroxyestradiol or 4-hydroxyestradiol with cross-reactivities to a few structurally related estrogens. The specificity of antisera obtained is discussed in relation to the site of attachment of the hapten to BSA.     

Control of chromatophore movements in dermal chromatic units of blue damselfish--I. The melanophore

Mechanisms controlling pigment movements in the melanophore of the blue damselfish, Chrysiptera cyanea, were studied. Histological observations revealed that the melanophore had three-dimensionally developed processes to envelop overlying small iridophores, and thus participated in the construction of a simple dermal chromatophore unit. Nervous stimulation, catecholamines and melatonin brought about melanosome aggregation in the melanophore. The actions of the nervous stimulation and catecholamines were antagonized by alpha adrenolytic agents. A beta adrenergic agonist, metaproterenol, adenosine and adenine nucleotides, and alpha-MSH acted as pigment-dispersing agents. These results indicate that the melanophore of the present material is controlled quite orthodoxly by adrenergic nerves and endocrines, notwithstanding the fact that it has quite a unique morphology among fish species, and that its motile rate is remarkably high.     

Molecular epidemiology of adenovirus type 8 (Ad 8) in Taiwan: four subtypes recovered during the period of 1980-1981 from patients with epidemic keratoconjunctivitis

Adenovirus type 8 (Ad 8) has been the major and important causative agent of epidemic keratoconjunctivitis (EKC). By enzymatic cleavage analysis with five endonucleases, PstI, HindIII, BamHI, SalI and SstI, 27 out of 149 Ad 8 isolates recovered from patients with EKC during the period from July, 1980 to July, 1981 in Kaohsiung, Taiwan, were studied. By cleavage patterns, 27 Ad 8 isolates in Kaohsiung were classified into four subtypes which were found to be different from the subtypes (Ad 8A, Ad 8B) prevalent in Sapporo (1).