Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance

In eukaryotes, the flap endonuclease of Rad27/Fen-1 is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments, and in base excision repair by cleaving a 5' flap structure that may result during base excision repair. Saccharomyces cerevisiae rad27Delta mutants further display a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result from duplications of DNA sequence, indicating a role in mutation avoidance. Two conserved motifs in Rad27/Fen-1 show homology to the 5' --> 3' exonuclease domain of Escherichia coli DNA polymerase I. The strain defective in the 5' --> 3' exonuclease domain in DNA polymerase I shows essentially the same phenotype as the yeast rad27Delta strain. In this study, we expressed the yeast RAD27 gene in an E. coli strain lacking the 5' --> 3' exonuclease domain in DNA polymerase I in order to test whether eukaryotic RAD27/FEN-1 can complement the defect of its bacterial homolog. We found that the yeast Rad27 protein complements sensitivity to methyl methanesulfonate in an E. coli mutant. On the other hand, Rad27 protein did not reduce the high rate of spontaneous mutagenesis in the E. coli tonB gene which results from duplication of DNA. These results indicate that the yeast Rad27 and E. coli 5' --> 3' exonuclease act on the same substrate. We argue that the lack of mutation avoidance of yeast RAD27 in E. coli results from a lack of interaction between the yeast Rad27 protein and the E. coli replication clamp (beta-clamp).     

Mass‐rearing conditions do not affect responsiveness to sex pheromone and flight activity in sweetpotato weevils

For ensuring the effectiveness of sterile insect technique (SIT) programmes, maintaining the reproductive competitiveness and dispersal ability of mass‐reared sterile males is essential. Inadvertent selection is an important genetic process that frequently occurs during mass rearing to produce sterile males. We investigated the effect of mass‐rearing conditions on the responsiveness to sex pheromones and spontaneous flight activity of males of the sweetpotato weevil Cylas formicarius (Coleoptera: Brentidae). There were no significant differences in the responsiveness to sex pheromones and spontaneous flight activity between wild and mass‐reared strains. These results indicate that mass‐reared strains of C. formicarius might not cause serious problems for implementing SIT programmes.     

The Number of Larval Molts Is Controlled by Hox in Caterpillars

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Metalloproteinase 1 downregulation in neurofibromatosis 1: Therapeutic potential of antimalarial hydroxychloroquine and chloroquine

Neurofibromatosis type 1 is an autosomal dominant genetic disorder caused by mutation in the neurofibromin 1 (NF1) gene. Its hallmarks are cutaneous findings including neurofibromas, benign peripheral nerve sheath tumors. We analyzed the collagen and matrix metalloproteinase 1 (MMP1) expression in Neurofibromatosis 1 cutaneous neurofibroma and found excessive expression of collagen and reduced expression of MMP1. To identify new therapeutic drugs for neurofibroma, we analyzed phosphorylation of components of the Ras pathway, which underlies NF1 regulation, and applied treatments to block this pathway (PD184352, U0126, and rapamycin) and lysosomal processes (chloroquine (CQ), hydroxychloroquine (HCQ), and bafilomycin A (BafA)) in cultured Neurofibromatosis 1 fibroblasts. We found that downregulation of the MMP1 protein was a key abnormal feature in the neurofibromatosis 1 fibroblasts and that the decreased MMP1 was restored by the lysosomal blockers CQ and HCQ, but not by the blockers of the Ras pathway. Moreover, the MMP1-upregulating activity of those lysosomal blockers was dependent on aryl hydrocarbon receptor (AHR) activation and ERK phosphorylation. Our findings suggest that lysosomal blockers are potential candidates for the treatment of Neurofibromatosis 1 neurofibroma.Subject terms: Drug development, Mechanisms of disease     

Ubenimex (Bestatin), an aminopeptidase inhibitor, modulates protein kinase C in K562 cells.

Ubenimex (Bestatin) is a potent inhibitor of aminopeptidases (APase) including APase N (EC 3.4.11.2), a widely distributed membrane-bound metalloprotease. Binding of Ubenimex (UBX) to cells has been implicated in a variety of its biological activities, while little evidence has yet been provided as to any subsequent mechanisms of intracellular signal transduction. We now examined the possible involvement of protein kinase C (PKC), a key regulator in transmembrane signaling. Human leukemia K562 cells were cultured in the presence or absence of UBX (1 to 50 micrograms.ml-1, 1 to 72 h), and the subcellular distribution as well as phorbol-12, 13-dibutyrate (PDBu)-induced redistribution of PKC activities were assessed. The membrane-bound enzymatic activity tended to increase in the presence of UBX, while a significant loss of the activity was demonstrable upon subsequent exposure to PDBu (100 nM, 10 min) in both the cytosolic and membrane fractions. Specific binding of [3H]PDBu to intact K562 cells was also down-modulated with UBX concentration- and time-dependently, suggesting loss of PKC enzyme protein on the cell surface. Western blot analysis of the total cell extracts disclosed no appreciable alteration in the amount of PKC protein. APase inhibition with UBX was observable independently of PKC modulation. The present findings were discussed with reference to the possible differential mechanisms of PKC-mediated regulation of cellular responses depending on cell types.     

Differentiated implication of Lactobacillus GG and L. gasseri TMC0356 to immune responses of murine Peyer's patch

Lactobacillus GG and L. gasseri TMC0356 were examined for their potential to alter the immune responses of murine PP cells in vitro and in vivo. Lactobacillus GG and L. gasseri TMC0356 characteristically stimulated the production of IL-12, IL-6, IFN-γ and IgA from isolated PP cells in vitro . Anatomical analysis indicated uptake of these bacteria by the PP tissue after giving orally in mice. Isolated PP cells exposed to Lactobacillus GG in vivo secreted more IFN-γ, IL-6 and total IgA, whereas those exposed to L. gasseri TMC0356 in vivo did not exhibit altered immune responses in terms of cytokine and IgA production. Therefore, these two bacteria might exhibit different immunodulatory effects in host animals by strain-dependent interaction with gut-associated lymphoid tissues in vivo .     

Use of high pressure liquid chromatography for analysis of deoxyribonucleotides in the epidermis

An analytical method for free deoxyribonucleotide (nucleoside monophosphate) in the epidermis is presented. Free nucleotides were extracted from tissue using a methylal ethanol mixture. The deoxyribonucleotides were separated using DEAE-cellulose and DBAE-cellulose. The analysis of free deoxyribonucleotides was carried out by high pressure liquid chromatography on a column of Lichrosorb-NH₂ with a single buffer of potassium phosphate. The elution order of nucleotides was CMP, AMP, UMP, IMP, GMP, and XMP. This procedure employing high pressure liquid chromatography for the detection of deoxyribonucleotides in the epidermis makes it possible to elucidate the biological characteristics and significance of DNA metabolism in normal epidermis and changes that occur in pathological conditions.     

Performance and phenology of alpine herbs along a snow-melting gradient

Responses of plants to the length of the growing season were studied in an alpine snow-bed by setting five plots along a snow-melting gradient. Performance and phenology were compared between the plots for five herbaceous species (Peucedanum multivittatum, Primula cuneifolia, Veronica stelleri var.longistyla, Solidago virga-aurea var.leiocarpa andPotentilla matsumurae). Performance characteristics measured were flower height, leaf height, leaf number, flower number and fruit number. In the late exposed plots with short snow-free periods, fruit-set was reduced in many species due to the decrease in flower number and/or the short growing season for fruit development.Veronica stelleri var.longistyla andSolidago virga-aurea var.leiocarpa, which decreased in flower and leaf numbers due to the short snow-free period, were sensitive to the short growing season.Peucedanum multivittatum was vigorous in the late exposed plots, but its slow flowering and fruiting prevented the fruit-set from developing in the last exposed plot.Primula cumeifolia andPotentilla matsumurae, quick flowering species that maintained their flower number throught the snow-melting gradient, were considered the most successful species in late exposed habitats.