Coprinus cinereus DNA ligase I during meiotic development

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.     

The significance of N-linked glycosylation in pig endogenous retrovirus infectivity

The significance of the envelope glycoprotein in the transmission of pig endogenous retrovirus (PERV) to human cells was investigated. Pig endothelial cells (PEC) were transduced with the LacZ gene by a pseudotype infection and then infected with PERV subtype B. Culture supernatants of the infected PEC previously incubated with several types of drugs were inoculated into HEK293 cells. The inoculated cells were then stained and the number of LacZ-positive foci was counted. PERV from tunicamycin treated PEC was not transmitted to human cells, indicating the importance of N-linked sugars in this process. Moreover, while inhibition of the terminal alpha-glucose residues from the precursor N-glycan by castanospermine and 1-deoxynojirimycin attenuated PERV infectivity, the mannosidase inhibitors, 1-deoxymannojirimycin and swainsonine, upregulated the infectivity. In addition, treatment with alpha-mannosidase and incubation with concanavalin A completely abrogated the transmission of PERV to HEK293. These data imply that the high-mannose type of N-glycan plays a key role in PERV infectivity.     

Neurite outgrowth-stimulating activities of beta-casomorphins in Neuro-2a mouse neuroblastoma cells

Endogenous opioid peptides and opiate drugs are known to affect the development of the nervous system. beta-Casomorphins (beta-CMs) belong to a family of exogenous opioid peptides derived from the milk protein beta-casein by proteolytic fragmentation. We investigated the effects of various fragments and analogues of beta-CM on neurite outgrowth in Neuro-2a mouse neuroblastoma cells. The fragments beta-CM-5 to -9 and beta-CM-5 amide stimulated neurite outgrowth. Fragments shorter than beta-CM-5 (beta-CM-3, -4, and beta-CM-4 amide) and longer than beta-CM-9 (beta-CM-13 and -21) had no effects. The activity of beta-CMs to promote neurite outgrowth does not correlate with their opioid activity in guinea-pig ileum. The effect of the most potent fragment, beta-CM-5, was prevented by the micro-opioid receptor-selective antagonist D-Phe-Cys(2)-Tyr(3)-D-Trp-Orn(5)-Thr(6)-Pen(7)-Thr(8)-NH(2) (CTOP), or by pretreatment with pertussis toxin. These results suggest that the stimulatory effects of beta-CMs on neurite outgrowth were mediated through G protein-coupled micro-opioid receptors.     

Deletion of N-terminal residues 23-88 from prion protein (PrP) abrogates the potential to rescue PrP-deficient mice from PrP-like protein/doppel-induced Neurodegeneration

Accumulating evidence has suggested that prion protein (PrP) is neuroprotective and that a PrP-like protein/Doppel (PrPLP/Dpl) is neurotoxic. A line of PrP-deficient mice, Ngsk Prnp0/0, ectopically expressing PrPLP/Dpl in neurons, exhibits late-onset ataxia because of Purkinje cell death that is prevented by a transgene encoding wild-type mouse PrP. To elucidate the mechanisms of neurodegeneration in these mice, we introduced five types of PrP transgene, namely one heterologous hamster, two mouse/hamster chimeric genes, and two mutants, each of which encoded PrP lacking residues 23-88 (MHM2.del23-88) or with E199K substitution (Mo.E199K), into Ngsk Prnp0/0 mice. Only MHM2.del23-88 failed to rescue the mice from the Purkinje cell death. The transgenic mice, MHM2.del23-88/Ngsk Prnp0/0, expressed several times more PrP than did wild-type (Prnp+/+) mice and PrPLP/Dpl at an equivalent level to Ngsk Prnp0/0 mice. Little difference was observed in the pathology and onset of ataxia between Ngsk Prnp0/0 and MHM2.del23-88/Ngsk Prnp0/0. No detergent-insoluble PrPLP/Dpl was detectable in the central nervous system of Ngsk Prnp0/0 mice even after the onset of ataxia. Our findings provide evidence that the N-terminal residues 23-88 of PrP containing the unique octapeptide-repeat region is crucial for preventing Purkinje cell death in Prnp0/0 mice expressing PrPLP/Dpl in the neuron.     

Atrium contributes to osmoregulation in eels acclimated to sea water

Since highly concentrated NaCl is suspected to enter into the heart of the seawater eels, effects of high NaCl concentration on the atrial beating was examined, and plasma ion concentrations and osmolality were measured simultaneously in the blood collected from the bulbus arteriosus and from the caudal vessels. When 100 mmole l(-1) NaCl was added to the incubation medium, atrial contraction was enhanced significantly. Similar enhancement in the atrial contractility was also observed after addition of NaCH3SO4 (100 mmole l(-1)) or Tris HCl (100 mmole l(-1)), indicating that Na(+) and Cl(-) are not indispensable for the positive inotropic effect. Furthermore, an addition of sucrose (200 mmole l(-1)) also enhanced the contraction. Inversely, hypoosmotic solution reduced the atrial contraction. These results indicate that the eel atrium is sensitive to environmental osmolarity. The eel atrium responses even at 20 mmole l(-1) sucrose. Such an inotropic effect of sucrose was not depressed after blocking adrenoceptor with betaxolol, a beta1-adrenoceptor antagonist, indicating that the effect is not due to adrenaline release from nerve endings. Plasma osmolality and Na(+) concentration were higher in bulbus arteriosus than in caudal vessels, indicating that the eel heart is really exposed to hyperosmotic blood in sea water. The osmotically enhanced atrial contraction may increase the cardiac outflow into the gill. Such property of the atrium would have clear advantages for seawater teleosts, since the concentrated NaCl from the esophagus can be excreted immediately through the gill, without circulating their body, and blood homeostasis can be maintained efficiently.     

Cloning and expression of gene encoding a novel endoglycoceramidase of Rhodococcus sp. strain C9

Endoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active site of EGCase of Rhodococcus sp. strain M-777. This paper describes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly distinguishable from M-777 by 16S rDNA analysis. C9 EGCase possessed an open reading frame of 1,446 bp encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respectively. Interestingly, C9 EGCase showed the different specificity to the M-777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly than the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent.     

Identification of mammalian TOM22 as a subunit of the preprotein translocase of the mitochondrial outer membrane

A mitochondrial outer membrane protein of approximately 22 kDa (1C9-2) was purified from Vero cells assessing immunoreactivity with a monoclonal antibody, and the cDNA was cloned based on the partial amino acid sequence of the trypsin-digested fragments. 1C9-2 had 19-20% sequence identity to fungal Tom22, a component of the preprotein translocase of the outer membrane (the TOM complex) with receptor and organizer functions. Despite such a low sequence identity, both shared a remarkable structural similarity in the hydrophobicity profile, membrane topology in the Ncyt-Cin orientation through a transmembrane domain in the middle of the molecule, and the abundant acidic amino acid residues in the N-terminal domain. The antibodies against 1C9-2 inhibited the import of a matrix-targeted preprotein into isolated mitochondria. Blue native polyacrylamide gel electrophoresis of digitonin-solubilized outer membranes revealed that 1C9-2 is firmly associated with TOM40 in the approximately 400-kDa complex, with a size and composition similar to those of the fungal TOM core complex. Furthermore, 1C9-2 complemented the defects of growth and mitochondrial protein import in Deltatom22 yeast cells. Taken together, these results demonstrate that 1C9-2 is a functional homologue of fungal Tom22 and functions as a component of the TOM complex.     

Relationship between contact inhibition and intranuclear S100C of normal human fibroblasts

Many lines of evidence indicate that neoplastic transformation of cells occurs by a multistep process. For neoplastic transformation of normal human cells, they must be first immortalized and then be converted into neoplastic cells. It is well known that the immortalization is a critical step for the neoplastic transformation of cells and that the immortal phenotype is recessive. Thus, we investigated proteins downregulated in immortalized cells by two-dimensional gel electrophoresis. As a result, S100C, a Ca(2+)-binding protein, was dramatically downregulated in immortalized human fibroblasts compared with their normal counterparts. When the cells reached confluence, S100C was phosphorylated on threonine 10. Then the phosphorylated S100C moved to and accumulated in the nuclei of normal cells, whereas in immortalized cells it was not phosphorylated and remained in the cytoplasm. Microinjection of the anti-S100C antibody into normal confluent quiescent cells induced DNA synthesis. Furthermore, when exogenous S100C was compelled to localize in the nuclei of HeLa cells, their DNA synthesis was remarkably inhibited with increase in cyclin-dependent kinase inhibitors such as p16(Ink4a) and p21(Waf1). These data indicate the possible involvement of nuclear S100C in the contact inhibition of cell growth.     

Antioxidant activities of phycocyanobilin prepared from Spirulina platensis

The antioxidative activity of phycocyanobilin fromSpirulina platensis was evaluated againstoxidation of methyl linoleate in a hydrophobic systemor with phosphatidylcholine liposomes. Phycocyanobilin as well as phytochemicals including-tocopherol, caffeic acid and zeaxanthin,effectively inhibited the peroxidation of methyllinoleate and produced a prolonged induction period.Oxidation of phosphatidylcholine liposomes was alsocontrolled markedly by adding phycocyanobilin or-tocopherol. Phycocyanobilin was distributedoutside in the liposomes to scavenge radicals fromAAPH and to prevent initiation of radical chainreactions. When the concentrations of phycocyanin andphycocyanobilin in the reaction mixture were adjustedequally on a phycocyanobilin basis, the activity ofphycocyanobilin was almost the same as that ofphycocyanin in the AAPH-containing reaction mixture.The antioxidizing action of phycocyanin prepared fromspray-dried Spirulina almost agreed with thatfrom fresh Spirulina in the AAPH-containingreaction mixture. These results suggest thatphycocyanobilin is responsible for the majority of theantioxidative activity of phycocyanin and may act asan effective antioxidant in a living human body.