Inhibition of surgical trauma-enhanced peritoneal dissemination of tumor cells by human catalase derivatives in mice

Surgical trauma, which is inevitably associated with the surgical removal of cancer, has been reported to accelerate tumor metastasis. The close association of reactive oxygen species with the trauma and tumor metastasis supports the possibility of using antioxidants for the inhibition of metastasis. To inhibit surgical trauma-enhanced peritoneal dissemination, human catalase (hCAT) derivatives, i.e., hCAT-nona-arginine peptide (hCAT-R9) and hCAT-albumin-binding peptide (hCAT-ABP), were designed to increase the retention time of the antioxidant enzyme in the abdominal cavity after intraperitoneal administration. Both ¹²⁵I-labeled derivatives showed significantly prolonged retention in the cavity compared to ¹²⁵I-hCAT. Cauterization of the cecum of mice with a hot iron, an experimental model of surgical trauma, induced abdominal adhesions. In addition, cauterization followed by colon26 tumor cell inoculation increased lipid peroxidation in the cecum and mRNA expression of molecules associated with tissue repair/adhesion and inflammation in the peritoneum. hCAT derivatives significantly suppressed the increased mRNA expression. The cauterization also increased the number of tumor cells in the abdominal organs, and the number was significantly reduced by hCAT-R9 or hCAT-ABP. These results indicate that hCAT-R9 and hCAT-ABP, both of which have a long retention time in the peritoneal cavity, can be effective at inhibiting surgery-induced peritoneal metastasis.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Will krill fare well under Southern Ocean acidification?

Antarctic krill embryos and larvae were experimentally exposed to 380 (control), 1000 and 2000 µatm pCO₂ in order to assess the possible impact of ocean acidification on early development of krill. No significant effects were detected on embryonic development or larval behaviour at 1000 µatm pCO₂; however, at 2000 µatm pCO₂ development was disrupted before gastrulation in 90 per cent of embryos, and no larvae hatched successfully. Our model projections demonstrated that Southern Ocean sea water pCO₂ could rise up to 1400 µatm in krill''s depth range under the IPCC IS92a scenario by the year 2100 (atmospheric pCO₂ 788 µatm). These results point out the urgent need for understanding the pCO₂-response relationship for krill developmental and later stages, in order to predict the possible fate of this key species in the Southern Ocean.     

PSF1 is essential for early embryogenesis in mice

Psf1 (partner of sld five 1) forms a novel heterotetramer complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three, respectively, in Japanese), with Sld5, Psf2, and Psf3. The formation of this complex is essential for the initiation of DNA replication in yeast and Xenopus laevis egg extracts. Although all of the components are well conserved in higher eukaryotes, the biological function in vivo is largely unknown. We originally cloned the mouse ortholog of PSF1 from a hematopoietic stem cell cDNA library and found that PSF1 is expressed in blastocysts, adult bone marrow, and testis, in which the stem cell system is active. Here we used the gene-targeting technique to determine the physiological function of PSF1 in vivo. Mice homozygous for a nonfunctional mutant of PSF1 died in utero around the time of implantation. PSF1-/- blastocysts failed to show outgrowth in culture and exhibited a cell proliferation defect. Our data clearly indicate that PSF1 is required for early embryogenesis.     

A case of freemartin with atresia recti and ani in Japanese Black calf

A female Japanese Black calf was born on 25 March 2003 at Hiroshima University Farm as a co-twin to a male Japanese Black calf. The male calf showed no external urogenital abnormalities. The absence of anal opening and external features of freemartinism were observed in the female. A small opening to the vulva (about 1.5 cm in length) with fused lips and a prominent clitoris were seen. The hair around the vulva was 3.5 cm in length and was heavy and dense. The distance from the vulva to the atretic anus was 9.0 cm. There were no other detectable abnormalities on physical examination. The PCR-based DNA test showed male-specific sequences confirming the calf to be freemartin. At autopsy 1 day after the calf birth, the gonads were found to be small and hard and the left uterine horn showed segmental aplasia near its proximal end. Two seminal glands (remnants of mesonephric duct) were located on both sides of the uterine body. A cervix was absent. The vagina was underdeveloped and looked like a tubual structure. The rectal end was closed, while the distance from the end of the atretic rectum to the absent anal opening was about 4.0 cm. On histological examination, the gonads exhibited extensive morphologic alteration; there was no cortex with the absence of ovarian structures. The seminal glands consisted of hypoplastic glandular tissue surrounded by extensive fibrous connective tissue. In conclusion, this is a case report of a freemartin with atresia recti and ani.     

Cyanidioschyzon merolae genome. A tool for facilitating comparable studies on organelle biogenesis in photosynthetic eukaryotes

The ultrasmall unicellular red alga Cyanidioschyzon merolae lives in the extreme environment of acidic hot springs and is thought to retain primitive features of cellular and genome organization. We determined the 16.5-Mb nuclear genome sequence of C. merolae 10D as the first complete algal genome. BLASTs and annotation results showed that C. merolae has a mixed gene repertoire of plants and animals, also implying a relationship with prokaryotes, although its photosynthetic components were comparable to other phototrophs. The unicellular green alga Chlamydomonas reinhardtii has been used as a model system for molecular biology research on, for example, photosynthesis, motility, and sexual reproduction. Though both algae are unicellular, the genome size, number of organelles, and surface structures are remarkably different. Here, we report the characteristics of double membrane- and single membrane-bound organelles and their related genes in C. merolae and conduct comparative analyses of predicted protein sequences encoded by the genomes of C. merolae and C. reinhardtii. We examine the predicted proteins of both algae by reciprocal BLASTP analysis, KOG assignment, and gene annotation. The results suggest that most core biological functions are carried out by orthologous proteins that occur in comparable numbers. Although the fundamental gene organizations resembled each other, the genes for organization of chromatin, cytoskeletal components, and flagellar movement remarkably increased in C. reinhardtii. Molecular phylogenetic analyses suggested that the tubulin is close to plant tubulin rather than that of animals and fungi. These results reflect the increase in genome size, the acquisition of complicated cellular structures, and kinematic devices in C. reinhardtii.     

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) > D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).     

Cloning and characterization of a cDNA encoding beta-amyrin synthase from petroleum plant Euphorbia tirucalli L

Euphorbia tirucalli L., known as the petroleum plant, produces a large amount of triterpenes, such as beta-amyrin. Degenerate RT-PCR based on the sequences conserved among known beta-amyrin synthases led to cloning of a putative triterpene synthase cDNA, EtAS, from leaves of E. tirucalli. The deduced amino acid sequence of the EtAS cDNA showed the highest identity of 82% to the Panax ginseng beta-amyrin synthase. Heterologous expression of the EtAS ORF in the methylotrophic yeast, Pichia pastoris, resulted in production of beta-amyrin, revealing that the EtAS cDNA codes for a beta-amyrin synthase. This is the first report of a gene involved in the triterpene synthetic pathway from Euphorbiaceae plants.     

A sorting nexin PpAtg24 regulates vacuolar membrane dynamics during pexophagy via binding to phosphatidylinositol-3-phosphate

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.     

Discovery of diphenylcarbamate derivatives as highly potent and selective IP receptor agonists: orally active prostacyclin mimetics. Part 3

The new classes of diphenylcarbamate derivatives with a tetrahydronaphthalene skeleton as highly potent and selective IP agonists have been discovered. The optimized diphenylcarbamate type compound FK-788: (R)-4 exhibited potent antiaggregative potency with an IC50 of 18 nM and high binding affinity for the human recombinant IP receptor with K(i) values of 20 nM and selectivity for human IP over all other members of the human prostanoid receptor family. Compound (R)-4 was shown to exhibit good pharmacokinetic properties in rats and dogs, and also good bioavailability in healthy volunteers.