Involvement of polyamines in gibberellin-induced development of seedless grape berries

The roles of polyamines (PAs) in the development of seedless grape berries induced by gibberellin (GA₃) was investigated. The development of seedless grape berries was stimulated by the application of putrescine (Put), but not by that of spermidine (Spd) and spermine (Spm), regardless of the presence of GA₃. At harvest, the fresh weight of seedless grape berries treated with 500 ppm Put + 25 ppm GA₃ and 500 ppm Put increased to 111 and 112%, respectively, of the control. Treatment with methylglyoxal-bis (guanyl hydrazone), a potent inhibitor of S-adenosylmethionine decarboxylase that plays a role in Spd and Spm synthesis, did not affect the development of seedless grape berries induced by 100 ppm GA₃. The application of 100 ppm GA₃ significantly increased endogenous free Put levels. Levels of free Spd and Spm were not affected by GA₃. Although the levels of endogenous perchloric acid insoluble bound PAs were higher than those of free PAs, obvious changes in the levels of bound PAs were not observed. These results indicate that free Put is implicated in the development of seedless grape berries induced by GA₃.     

On-line measurement of cell mass and specific growth rate by a laser turbidity meter coupled with data smoothing using a one-pass method

Summary A data smoothing algorithm by a one-pass method, employing spline function was applied to a laser turbidity meter for on-line measurement of cell mass and specific growth rate in the culture using recombinant E.coli The outputs from the laser turbidity meter containing noise and errors were successfully filtered by the method, leading to more reliable estimation of cell mass and specific growth rate in real time.     

ARPP-16/ARPP-19: a highly conserved family of cAMP-regulated phosphoproteins

ARPP-16 and ARPP-19 are closely related cAMP-regulated phosphoproteins that were initially discovered in mammalian brain as in vitro substrates for protein kinase A (PKA). ARPP-16 is enriched in dopamine-responsive medium spiny neurons in the striatum, while ARPP-19 is ubiquitously expressed. ARPP-19 is highly homologous to alpha-endosulfine and database searches allowed the identification of novel related proteins in D. melanogaster, C. elegans, S. mansoni and yeast genomes. Using isoform-specific antibodies, we now show that ARPP-19 is composed of at least two differentially expressed isoforms (termed ARPP-19 and ARPP-19e/endosulfine). All ARPP-16/19 family members contain a conserved consensus site for phosphorylation by PKA (RKPSLVA in mammalian ARPP-16 and ARPP-19), and this site was shown to be efficiently phosphorylated in vitro by PKA. An antibody that specifically recognized the phosphorylated form of ARPP-16/19/19e was used to examine the phosphorylation of ARPP-16/19 family members in intact cells. In striatal slices, the phosphorylation of ARPP-16 was increased in response to activation of D(1)-type dopamine receptors, and decreased in response to activation of D(2)-type dopamine receptors. In non-neuronal cells, ARPP-19 was highly phosphorylated in response to activation of PKA. These results establish that ARPP-16/19 proteins constitute a family of PKA-dependent intracellular messengers that function in all cells. The high levels of ARPP-16 in striatal neurons and its bi-directional regulation by dopamine suggest a specific role in dopamine-dependent signal transduction. The conservation of this protein family through evolution suggests that it subserves an important cellular function that is regulated by PKA.     

5'-upstream structure of the gene coding for chicken riboflavin-binding protein and its relation to estrogen induction

To elucidate the mechanism of the estrogen-dependent induction of chicken riboflavin-binding protein (RfBP), we analyzed the 5'-upstream structure of its gene. A noncoding exon exists there, and around this sequence, 9 widely spaced half-palindromic estrogen-response element (ERE) motifs (5'-GGTCA or 5'-TGACC) were found. Furthermore, an imperfect ERE-like palindromic sequence (5'-ATGTCANNNTGACAT-3') was also found at the 2.25 kb upstream region. No consensus palindromic ERE was observed. By luciferase reporter assay, the regions containing the half ERE motifs and the imperfect ERE showed estrogen-dependent enhancer activities, suggesting that these two characteristic sequences might confer estrogen-inducibility upon the chicken RfBP gene. However the activities were lower than that of a consensus ERE. It remains uncertain whether these sequences act cooperatively.     

Filamentous phage replication initiator protein gpII forms a covalent complex with the 5' end of the nick it introduced

Rolling circle type DNA replication is initiated by introduction of a nick in the leading strand of the origin by the initiator protein, which in most cases binds covalently to the 5' end of the nick. In filamentous phage, however, such a covalent complex has not been detected. Using a suitable substrate and short reaction time, we show that filamentous phage initiator gpII forms a covalent complex with nicked DNA, which rapidly dissociates unless gpII is inactivated. A peptide-DNA complex was isolated from trypsin digest of the complex by ion-exchange column chromatography and gel filtration, and its peptide sequence was determined. The result indicated that gpII was linked to DNA by the tyrosine residue at position 197 from the N-terminus. The mutant protein in which this tyrosine was replaced by phenylalanine did not show any detectable activity to complement gene II amber mutant phage in vivo. In vitro, the mutant protein recognized the origin and bent DNA as well as the wild-type does, but failed to introduce a nick and to relax the superhelicity of cognate DNA.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria

The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.     

Effects of different beta73 amino acids on formation of 14-stranded fibers of Hb S versus double-stranded crystals of Hb C-Harlem

Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.     

CD36-mediated endocytic uptake of advanced glycation end products (AGE) in mouse 3T3-L1 and human subcutaneous adipocytes

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. We here show that AGE-modified bovine serum albumin (BSA) is endocytosed by adipocytes via CD36. Upon differentiation, 3T3-L1 and human subcutaneous adipose cells showed marked increases in endocytic uptake and subsequent degradation of [(125)I]AGE-BSA, which were inhibited effectively by the anti-CD36 antibody. Ligand specificity of CD36 for modified BSAs was compared with that of LOX-1 and scavenger receptor class A. Effect of fucoidan on [(125)I]AGE-BSA binding showed a sharp contrast to that on [(125)I]-oxidized low density lipoprotein. These results implicate that CD36-mediated interaction of AGE-modified proteins with adipocytes might play a pathological role in obesity or insulin-resistance.     

Kinetic analysis of microbial desulfurization of model and light gas oils containing multiple alkyl dibenzothiophenes

The reaction mechanism of biodesulfurization was investigated using whole cells of Rhodococcus erythropolis KA2-5-1, which have the ability to convert dibenzothiophene (DBT) into 2-hydroxybiphenyl. The desulfurization patterns of alkyl DBTs were represented by the Michaeis-Menten equation. The values of rate constants, the limiting maximal velocity (Vmax) and Michaelis constant (Km), for desulfurization of alkyl DBTs were calculated. The relative desulfurization activities of various alkyl DBTs were reduced in proportion to the total carbon numbers of alkyl substituent groups. Alkyl DBTs that had a total of six carbons of alkyl substituent groups were not desulfurized. The type or position of alkyl substituent groups had little effect on desulfurization activity. The desulfurization activity of each alkyl DBT, when mixed together, was reduced. This phenomenon was caused by apparent competitive inhibition of substrates. Using the apparent competitive inhibition model, the desulfurization pattern of a multiple components system containing alkyl DBTs was elucidated. This model was also applicable for biodesulfurization of light gas oil.