Human peroxisomal L-alanine: glyoxylate aminotransferase. Evolutionary loss of a mitochondrial targeting signal by point mutation of the initiation codon.

The amino acid sequence of human hepatic peroxisomal L-alanine: glyoxylate aminotransferase 1 (AGTI) deduced from cDNA shows 78% sequence identity with that of rat mitochondrial AGTI, but lacks the N-terminal 22 amino acids (the putative mitochondrial targeting signal). In humans this signal appears to have been deleted during evolution by a point mutation of the initiation codon ATG to ATA. These data suggest that the targeting defect in primary hyperoxaluria type 1, in which AGT1 is diverted from the peroxisomes to the mitochondria, could be due to a point mutation that reintroduces all or part of the mitochondrial signal sequence.     

Transformation of cucumber (Cucumis sativus L.) plants using Agrobacterium tumefaciens and regeneration from hypocotyl explants

Summary Transgenic cucumber (Cucumis sativus L.) plants were successfully obtained from hypocotyl explants inoculated with Agrobacterium tumefaciens, which harbored a binary vector plasmid with NOS-nptII, CaMV 35S-I-gus and CaMV 35S-hph genes. Acetosyringone enhanced the efficiency of transformation at the cut surface cells of hypocotyl explants during five days of co-cultivation. Transformed cells were more effectively selected using 20–30 mg/l hygromycin B than using 50–100 mg/l kanamycin. Shoot regeneration occurred within 4–6 wks, and 12 of 21 regenerated plantlets displayed strong GUS expression in the very young leaves. All of 8 GUS-positive R0 plants examined showed single or a few positive bands by Southern blot analysis. The expression of the CaMV 35S-I-gus gene was observed in various tissues and organs of R0 and R1 transgenic cucumber plants.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds

Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds.     

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B^*5101 binding self-peptides, 27 paptides bound to HLA-B^*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B^*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B^*5102 and B^*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B^*5102) and that of Gly for Trp at residue 167 (B^*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B^*5102 or HLA-B^*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.     

Genomic imprinting of the human serotonin-receptor (HTR2) gene involved in development of retinoblastoma.

Epidemiological and genetic studies of retinoblastoma (RB) suggested that imprinted genes might be genetically linked to the RB gene. In this study, we found that the human serotonin-receptor, HTR2, gene, which had been mapped nearby the RB gene on chromosome 13, was expressed only in human fibroblasts with a maternal allele and not in cells without a maternal allele. The 5' genomic region of the human HTR2 gene was cloned by PCR-mediated method. Only the 5' region of the gene was methylated in cells with the maternal gene, and it was not methylated in cells without the maternal gene. A polymorphism of PvuII site of the gene was also found and useful for the segregation analysis in a family of a RB patient and for analysis of loss of heterozygosity on chromosome 13 in tumor and its parental origin. These results suggest that the human HTR2 gene might be affected by genomic imprinting and that exclusive expression of the maternal HTR2 gene may be associated with the delayed occurrence of RB, which had lost the maternal chromosome 13.     

Seasonality and vertical structure of light-attracted insect communities in a dipterocarp forest in Sarawak

Nocturnal flying insects were collected monthly for 13 months using ultra violet light-traps set at various vertical levels in a weakly-seasonal, tropical lowland dipterocarp forest in Sarawak, Malaysia. Abundance, faunal composition, size distribution and guild structure of these samples were analyzed with respect to temperal and vertical distributions. The nocturnal flying insect community in the canopy level was highly dominated by fig wasps (84%) in individual number, and by scarabaeid beetles (28%) in weight. A principal component analysis on monthly catches detected non-random, seasonal trends of insect abundance. The first two principal trends were an alternation of wetter (September to January) and less wet seasons (February to August) and an alternation between the least wet (January to March) and the other seasons. Many insect groups were less abundant in the least wet season than the other seasons, whilst inverse patterns were found in Scarabaeidae and Tenebrionidae. Significantly positive and negative correlations between monthly catch and rainfall were detected only in ovule-feeders and in phloem-feeders, respectively. Delayed, significant negative correlations between monthly catch and 1–3 month preceding rainfall were more frequently detected in phytophages, phloem-feeders, seed-feeders, wood-borers and scavengers. The peak in abundance along vertical levels were found at the canopy level (35 m) for phloem-, ovule-, seed-, root-, fungal-feeders and nectar collectors, at an upper subcanopy level (25 m) for scavengers and aquatic predators, and at a middle subcanopy level (17 m) for ants. Catches at the emergent level (45 m) did not exceed those at the canopy level.     

Comparative biochemical studies of carotenoids in sea urchins—III. Relationship between developmental mode and carotenoids in the Australian echinoids Heliocidaris erythrogramma and H. tuberculata and a comparison with Japanese species

The sea urchin Heliocidaris tuberculata is typical of most echinoids in having a small egg and a feeding larva, while H. erythrogramma has a large egg and modified development through a non-feeding larvae. The carotenoids in the gonads of these two species were investigated from the comparative biochemical points of view. The carotenoid content of the buoyant eggs of H. erythrogramma was approximately 60 times that of the negatively-buoyant eggs of H. tuberculata. With respect to cytoplasmic volume, however, the carotenoid concentration in the eggs of H. tuberculata was approximately twice that in the eggs of H. erythrogramma. In both species β-echinenone was the principal carotenoid found and their carotenoid patterns were similar. It is very interesting from a functional point of view that carotenoid levels per cytoplasmic volume are conserved across most of the species we have examined irrespective of phylogeny and egg size. In light of this result we suggest that carotenoids may play an important role in developing stage in all echinoids including indirect and direct developers.     

Fungal succession in the early decomposition process of pine cones on the floor ofPinus densiflora forests

The fungal succession on pine cones on the floor ofPinus densiflora forest was investigated in the early decomposition process (within ca. 30% decrease in dry weight). The fungal flora was examined by both washing and surface-sterilization methods on artificially placed cones and naturally fallen cones. The decomposition rates of artificially placed cones were 0.081–0.082 yr^–1. On withered cones still attached to the tree,Pestalotiopsis spp. were dominant. These fungi also occurred with higher frequencies after cones had lain on the floor and on cones in the L and FH horizons.Xylaria sp. andPhomopsis sp., which seem to colonize the interior of the tissue, occurred with higher frequencies on the cones on the tree, but their occurrence frequencies decreased after cones had lain on the forest floor. Conversely,Mortierella spp. andTrichoderma spp. newly occurred or their occurrence frequencies increased on lying cones. Of these,Trichoderma koningii increased rapidly and showed high occurrence frequencies.Thysanophora penicillioides, which prefers coniferous substrates, showed higher occurrence frequencies in the early stages of lying on the forest floor. On cones lying on the floor, the fungal flora did not significantly change during the investigation period.