Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

Presence and localization of proteins immunologically related to erythrocyte protein 4.1 in human skin

Analogues of human erythrocyte protein 4.1 have been examined in the human skin by immunochemical techniques using anti-human erythrocyte protein 4.1 antibodies. Immunoblot analysis revealed that human epidermis contains 4.1-like proteins of 80 kDa and 78 kDa that cross react with anti-protein 4.1 antibodies. Analysis with immunofluorescence microscopy revealed that the plasma membrane of the human epidermal keratinocyte was stained intensively in the basal cells, whereas spinous cells were moderately stained. It is noted that eccrine sweat gland cells and ductal cells were also stained in the peripheral cytoplasma. Taken together, these results demonstrate that 4.1-like proteins are present in human epidermal keratinocytes, eccrine sweat gland cells and ductal cells. The present findings enable us to suggest that a membrane skeletal protein lattice might exist in these cells.     

Seasonal changes in the biomass of floating leaved plant,Trapa spp., and its relation with a leaf beetle,Galerucella nipponensis,in Lake Inba,Japan

Limnology - Trapa spp. dominate many shallow eutrophic lakes in Japan, which must affect the nutrient dynamics in lakes. Trapa spp. are utilized by several animals, in particular the leaf beetle,...     

A water channel of the nematode C.elegans and its implications for channel selectivity of MIP proteins

A genome project focusingon the nematode Caenorhabditis elegans has demonstrated thepresence of eight cDNAs belonging to the major intrinsic proteinsuperfamily. We functionally characterized one of these cDNAs namedC01G6.1. Injection of C01G6.1 cRNA increased the osmotic waterpermeability (P_f) of Xenopusoocytes 11-fold and the urea permeability 4.5-fold but failed toincrease the glycerol permeability. It has been speculated that the MIPfamily may be separated into two large subfamilies based on thepresence or absence of two segments of extra amino acid residues (~15amino acids) at the second and third extracellular loops. BecauseC01G6.1 (designated AQP-CE1), AQP3, and glycerol facilitator (GlpF) all have these two segments, we replaced the segments of AQP-CE1 with thoseof AQP3 and GlpF to identify their roles. The functional characteristics of these mutants were principally similar to that ofwild-type AQP-CE1, although the values of P_f andurea permeability were decreased by 39-74% and 28-65%,respectively. These results suggest that the two segments of extraamino acid residues may not contribute to channel selectivity orformation of the route for small solutes.

     

Coevolution and Hierarchical Interactions of Tomato mosaic virus and the Resistance Gene Tm-1

During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.     

Anti‐Tumor‐Initiating Effects of Spiro‐biflavonoids from Abies sachalinensis

Cancer chemoprevention, the prevention of cancer by ingestion of chemical agents that reduce the risk of carcinogenesis, is one of the potent ways to reduce morbidity and mortality. We have been searching for cancer chemopreventive agents from the leaves and barks of coniferous trees that have been treated as waste in the forestry industry. We have previously reported the isolation of spiro‐biflavonoids, named as abiesinols, and a neolignan from the MeOH extract of the bark of Abies sachalinensis. These compounds were tested for their inhibitory effects on the activation of (±)‐(E)‐methyl‐2‐[(E)‐hydroxyimino]‐5‐nitro‐6‐methoxyhex‐3‐enamide (NOR 1), a nitric oxide (NO) donor, as a primary screening test for anti‐tumor initiators. All compounds tested exhibited potent inhibitory effects on NOR 1 activation. Furthermore, abiesinol A, bearing a spiro‐biflavonoid skeleton, showed remarkable anti‐tumor‐initiating activity in the in vivo two‐stage mouse skin carcinogenesis test using peroxynitrite (ONOO^?; PN) as the initiator and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) as the promoter.     

Involvement of protein kinase C in the phosphorylation of 46 kDa proteins which are phosphorylated in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes

Two proteins (Mr 46,000, pI 6.4 and 7.0), the phosphorylation of which was increased by any of the membrane-perturbing agents in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes in our previous study (Okamura, N., Ohashi, S., Nagahisa, N. and Ishibashi, S. (1984) Arch. Biochem. Biophys. 228, 270-277), were also phosphorylated in a cell-free system prepared from the leukocytes. The in vitro phosphorylation of these two proteins was stimulated by the addition of phosphatidylserine in the presence of higher concentrations of Ca2+ (300-500 microM). The phosphorylation was further increased when protein kinase C partially purified from guinea-pig brain was added to the system. At a low concentration of Ca2+ (about 10 microM), stimulation of the phosphorylation was not attained by phosphatidylserine alone but required the addition of diacylglycerol or phorbol myristate acetate. On the other hand, the increase in the phosphorylation was inhibited by H-7, an inhibitor for protein kinase C. These results indicate that protein kinase C is involved in the phosphorylation of the two proteins, which may be related to the superoxide anion production stimulated by various membrane-perturbing agents.     

Possible involvement of cathepsin L in processing of rat liver hexokinase to eliminate mitochondria-binding ability.

A previously found proteinase possibly involved in the modification of hexokinase to eliminate the mitochondria-binding ability without appreciable change in the catalytic activity (called hexokinase-processing enzyme hereafter), was purified by sequential chromatographies from rat liver and its properties were examined. The hexokinase-processing enzyme had carbohydrate moieties as evidenced by adsorption on immobilized concanavalin A, and had a molecular weight of about 23,000 as estimated by SDS-PAGE and gel filtration chromatography. Benzyloxycarbonyl-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA)-hydrolyzing activity was co-purified with this processing activity throughout the purification, while the hydrolyzing activity for benzyloxycarbonyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide (Z-Arg-Arg-MCA) was not. The processing activity, as well as Z-Phe-Arg-MCA hydrolyzing activity, was highly sensitive to cysteine proteinase inhibition, for example, by leupeptin and N-[N-3-(trans-carboxirane-2-carbonyl)-L-leucyl]agmatine (E-64). Furthermore, the enzyme preparation reacted with an antibody against cathepsin L purified from rat kidney. These results indicated that cathepsin L may be involved in the above-mentioned processing of hexokinase.