Inactivation of D-glucosyltransferases from oral Streptococcus mutans and Streptococcus sanguis by photochemical oxidation

Cell-free D-glucosyltransferase of D-glucose-grown Streptococcus mutans AHT was completely inactivated in the presence of 0.002% of Methylene Blue at 25 degrees and pH 7.0 after illumination with a 150-W incandescent lamp. The rate of inactivation was decreased at pH values less than 7.0. Histidine was the only amino acid residue modified to a significant extent, and the rates of oxidation of histidine residues and loss of enzyme activity closely agreed. Production of both water-insoluble and -soluble D-glucan fractions from sucrose by the oxidized D-glucosyltransferase preparations was significantly inhibited. Photooxidation with 0.002% of Rose Bengal at pH 7.0 or higher also induced complete inactivation of the D-glucosyltransferase. These results strongly suggest that the imidazole portion of histidine may function as part of the active sites of both D-glucosyltransferase isozymes of S. mutans AHT, which are responsible for the synthesis of (1 goes to 3)- and (1 goes to 6)-alpha-D-glucosidic linkages. The D-glucosyltransferases from S. mutans 6715 and AHT-mutant M1, and Streptococcus sanguis ATCC 10558 were also almost completely inactivated by Methylene Blue-sensitized photooxidation.     

Identification of nucleobase chemical modifications that reduce the hepatotoxicity of gapmer antisense oligonucleotides

Currently, gapmer antisense oligonucleotide (ASO) therapeutics are under clinical development for the treatment of various diseases, including previously intractable human disorders; however, they have the potential to induce hepatotoxicity. Although several groups have reported the reduced hepatotoxicity of gapmer ASOs following chemical modifications of sugar residues or internucleotide linkages, only few studies have described nucleobase modifications to reduce hepatotoxicity. In this study, we introduced single or multiple combinations of 17 nucleobase derivatives, including four novel derivatives, into hepatotoxic locked nucleic acid gapmer ASOs and examined their effects on hepatotoxicity. The results demonstrated successful identification of chemical modifications that strongly reduced the hepatotoxicity of gapmer ASOs. This approach expands the ability to design gapmer ASOs with optimal therapeutic profiles.     

Plant reproductive intervals and pollinators in the aseasonal tropics: a new model

What factors determine reproductive intervals and modes of pollination in plants of the aseasonal tropics? To answer this general question, we present a new explanation for some community patterns of plant reproductive intervals and pollinators observed in a lowland dipterocarp forest in Sarawak, Malaysia, using a mathematical model featuring different display effects for different types of pollinators. Predictions from the model matched with the following observed patterns: (i) flowering intervals were different among forest strata (forest floor < understorey < canopy < subcanopy and emergent), and not in the exact order of stratum height; (ii) among generalist pollinators, the proportion of social foragers was maximum in intermediate forest stratum; and (iii) plants pollinated by specialist pollinators were found on the forest floor and in gaps.     

Purification and characterization of the messenger RNA coding for bovine corticotropin/beta-lipotropin precursor.

The mRNA coding for the common precursor of corticotropin and beta-lipotropin has been purified to homogeneity from neurointermediate lobes of bovine pituitaries. The homogeneity of the mRNA preparation is evidenced by analysis of its translation product, electrophoresis on polyacrylamide gel in the presence of formamide and analysis of the kinetics of hybridization with its cDNA. The purification procedure involves the isolation of RNA from membrane-bound polysomes, chromatography on oligo(dT)-cellulose and on poly(U)-Sepharose and sucrose density gradient centrifugation. The mRNA has a molecular weight of approximately 450000, equivalent to approximately 1360 nucleotides in length, and contains a polyadenylate sequence with an average length of 68 nucleotides. The size of the mRNA is sufficiently large to encode the corticotropin/beta-lipotropin precursor.     

Plasmic degradation of bovine fibrinogen and non-crosslinked fibrins in solution and in gel form.

1. Analysis of degradation processes of bovine fibrinogen by bovine plasmin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a study on the mode of changes of the properties related to clotting of digestion products as a function of time were performed. Gross features and patterns very similar to those which had been reported with human fibrinogen-plasmin systems were obtained. 2. Based on the molecular size of the degradation products and the mode of appearance and disappearance of the degradation products, the processes could tentatively be divided into three stages: stage 1, where fibrinogen (mol. wt 370 000) was degraded to produce fragments X1 (330 000) and X2 (290 000); stage 2, fragment X2 was degraded with appearance of Y (210 000) and D1 (140 000); stage 3, appearance of fragments D1, D2 (110 000), and D3 (100 000) sequentially and E (68 000) with concomitant disappearance of Y. 3. A microseparation method, which is a combination of dansylation and sodium dodecylsulfate-polyacrylamide gel electrophoresis, was devised to analyze the events of stage 1 in detail, and a molecular model for the process was proposed. 4. The plasmic degradation processes of bovine non-cross-linked fibrins in solution and in gel form were compared with that of fibrinogen and it was found that the state of the substrates, fibrins, could cause differences in the degradation patterns. With the former substrate, essentially the same sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns as those with fibrinogen were obtained. With the latter substrate, however, a distinct difference in the mode of degradation of beta chains was observed.     

Transesterification catalyzed by polystyrene-supported chymotrypsin in toluene: the effect of neutralization of basic or acidic groups attaching to polystyrene resins

Crosslinked polystyrene resins containing a low level of either basic or acidic groups were used for supports of alpha-chymotrypsin (CT), which catalyzed the transesterification of N-acetyl-L-phenylalanine ethyl ester (AcPheOEt) with propanol in toluene. With a minimal amount of water, CT was sorbed to the resins, basic or acidic groups of which were partly or fully neutralized by several soluble acids or bases. With an increasing degree of neutralization of basic resins by free acids, the rate of disappearance of AcPheOEt was decreased, whereas the by-product formation of AcPheOH, due to hydrolysis, was considerably suppressed, compared with the ester-exchange product, AcPheOPr. The pK(a) value of the neutralizing acid was also important for both CT activity and reaction selectivity. AcPheOPr was selectively produced at a certain range of pK(a) values. On the other hand, the neutralization of acidic resins with free amines enhanced the CT activity but a strong base promoted the formation of hydrolysis product. (c) 1995 John Wiley & Sons, Inc.     

Mutations affecting early distribution of primordial germ cells in Medaka (Oryzias latipes) embryo

The development of germ cells has been intensively studied in Medaka (Oryzias latipes). We have undertaken a large-scale screen to identify mutations affecting the development of primordial germ cells (PGCs) in Medaka. Embryos derived from mutagenized founder fish were screened for an abnormal distribution or number of PGCs at embryonic stage 27 by RNA in situ hybridization for the Medaka vasa homologue (olvas). At this stage, PGCs coalesce into two bilateral vasa-expressing foci in the ventrolateral regions of the trunk after their migration and group organization. Nineteen mutations were identified from a screen corresponding to 450 mutagenized haploid genomes. Eleven of the mutations caused altered PGC distribution. Most of these alterations were associated with morphological abnormalities and could be grouped into four phenotypic classes: Class 1, PGCs dispersed into bilateral lines; Class 2, PGCs dispersed in a region more medial than that in Class 1; Class 3, PGCs scattered laterally and over the yolk sac area; and Class 4, PGCs clustered in a single median focus. Eight mutations caused a decrease in the number of PGCs. This decrease was observed in the offspring of heterozygous mothers, indicating the contribution of a maternal factor in determining PGC abundance. Taken together, these mutations should prove useful in identifying molecular mechanisms underlying the early PGC development and migration.     

Essential role of the PSI–LHCII supercomplex in photosystem acclimation to light and/or heat conditions by state transitions

Light and temperature affect state transitions through changes in the plastoquinone (PQ) redox state in photosynthetic organisms. We demonstrated that light and/or heat treatment induced preferential photosystem (PS) I excitation by binding light-harvesting complex II (LHCII) proteins. The photosystem of wheat was in state 1 after dark overnight treatment, wherein PQ was oxidized and most of LHCII was not bound to PSI. At the onset of the light treatment [25 °C in the light (100 µmol photons m^?2 s^?1)], two major LHCIIs, Lhcb1 and Lhcb2 were phosphorylated, and the PSI–LHCII supercomplex formed within 5 min, which coincided with an increase in the PQ oxidation rate. Heat treatment at 40 °C of light-adapted wheat led to further LHCII protein phosphorylation of, resultant cyclic electron flow promotion, which was accompanied by ultrafast excitation of PSI and structural changes of thylakoid membranes, thereby protecting PSII from heat damage. These results suggest that LHCIIs are required for the functionality of wheat plant PSI, as it keeps PQ oxidized by regulating photochemical electron flow, thereby helping acclimation to environmental changes.