Vitamin K2 Biosynthetic Enzyme,UBIAD1 Is Essential for Embryonic Development of Mice

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K₂ biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1 ^−/−) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1 ^−/− embryonic stem (ES) cells failed to synthesize vitamin K₂ but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1 ^+/− mice developed normally, exhibiting normal growth and fertility. Vitamin K₂ tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K₂ synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1 ^+/− E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1 ^−/− mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1 ^+/− mice. These results suggest that UBIAD1 is responsible for vitamin K₂ synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K₂, but may have additional functions beyond the biosynthesis of vitamin K₂.     

Gene expression profile analysis of the mouse liver during bacteria-induced fulminant hepatitis by a cDNA microarray system

Fulminant hepatic failure (FHF) is a disease characterized by sudden and severe impairment of liver function. To elucidate the mechanism involved in FHF, we adopted a murine model of FHF by administrating mice with heat-killed Propionibacterium acnes (P. acnes), followed by a low dose of lipopolysaccharide (LPS), and analyzed the dynamic change of gene expression profile of the murine liver using an in-house cDNA microarray system which contained most of the cDNAs encoding chemokines/cytokines and their receptors (33 chemokines/21 chemokine receptors, 28 cytokines/35 cytokine receptors) as well as 230 liver related proteins mostly selected by serial analysis of gene expression (SAGE). Among them, 335 genes were found to differ by more than 2-fold in at least one time point comparing with normal liver. Hierarchical cluster analysis revealed that except for a few genes, such as heme oxygenase (HO)-1 and nicotinamide N-methyltransferase (NNMT) of which expression increased, the expression of most of the genes encoding drug metabolizing enzymes decreased with the progress of the disease. The expression of the genes encoding chemokines/cytokines was dramatically changed, such as Mig, IP-10, RANTES, TNF-alpha, and IFN-gamma. In addition, the expression of those that were not previously linked to this murine model was also identified to be changed. These include endogenous IL-18 binding protein (IL-18BP), CXCL16 (the ligand of Bonzo, CXCR6) as well as ESTs. Taken together this study has shown the systemic and comprehensive gene expression profile during FHF and may contribute to better understanding of the mechanism of FHF.     

Design, synthesis, and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors

Aiming to develop selective anticancer drugs, we designed and synthesized three disulfides bearing a folic acid moiety as candidate folate receptor (FR)-targeted prodrugs of thiolate histone deacetylase inhibitors. Among them, compound 1 displayed growth-inhibitory activity toward folate receptor-positive MCF-7 breast cancer cells. The activity of 1 was significantly reduced by free folic acid, suggesting that cellular uptake of 1 is mediated by FR.     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Expression and localization of aquaporins in rat gastrointestinal tract

A family of water-selective channels, aquaporins (AQP), has beendemonstrated in various organs and tissues. However, the localizationand expression of the AQP family members in the gastrointestinal tracthave not been entirely elucidated. This study aimed to demonstrate theexpression and distribution of several types of the AQP family and tospeculate on their role in water transport in the rat gastrointestinal tract. By RNase protection assay, expression of AQP1-5 and AQP8 was examined in various portions through the gastrointestinal tract.AQP1 and AQP3 mRNAs were diffusely expressed from esophagus to colon,and their expression was relatively intense in the small intestine andcolon. In contrast, AQP4 mRNA was selectively expressed in the stomachand small intestine and AQP8 mRNA in the jejunum and colon.Immunohistochemistry and in situ hybridization demonstrated cellularlocalization of these AQP in these portions. AQP1 was localized onendothelial cells of lymphatic vessels in the submucosa and laminapropria throughout the gastrointestinal tract. AQP3 was detected on thecircumferential plasma membranes of stratified squamous epithelialcells in the esophagus and basolateral membranes of cardiac glandepithelia in the lower stomach and of surface columnar epithelia in thecolon. However, AQP3 was not apparently detected in the smallintestine. AQP4 was present on the basolateral membrane of the parietalcells in the lower stomach and selectively in the basolateral membranesof deep intestinal gland cells in the small intestine. AQP8 mRNAexpression was demonstrated in the absorptive columnar epithelial cellsof the jejunum and colon by in situ hybridization. These findings mayindicate that water crosses the epithelial layer through these waterchannels, suggesting a possible role of the transcellular route forwater intake or outlet in the gastrointestinal tract.     

Appearance of retrogradely labeled neurons in the rat superior cervical ganglion after injection of wheat-germ agglutinin-horseradish peroxidase conjugate into the contralateral ganglion

Summary Injection of wheat-germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) into the superior cervical ganglion (SCG) of the rat results in accumulation of WGA-HRP in sympathetic postganglionic neurons in the contralateral SCG. The sympathetic pathways involved and the mechanism underlying the labeling were investigated. The labeling in neurons in the contralateral SCG was apparent 6 h after injection and increased in intensity with longer survival times. The number of labeled neurons reached 1300 at 72 h after the injection. Transection of the external (ECN) or internal carotid nerves (ICN) resulted in considerable reduction in the number of labeled neurons. Combined transection of both ECN and ICN virtually eliminated labeling in the contralateral SCG. This provides strong evidence that these two nerves are the major pathways for WGA-HRP transport out of the SCG. No labeling was observed in the contralateral SCG following injection of horseradish peroxidase (HRP). Therefore, it seems unlikely that a direct nerve connection exists between the bilateral ganglia. Instead, the labeling of contralateral SCG neurons appears to depend on the transneuronal transport capacity of WGA-HRP, which conveys the marker in an anterograde direction along the postganglionic fibers to terminals in sympathetic target organs, and then delivers it transneuronally to contralateral SCG neurons. We suggest that the sympathetic nerve fibers originating in the bilateral SCGs run intermingled and are in close contact in their peripheral target organs.     

Characterization of the {alpha}-helix region in domain 3 of the haemolytic lectin CEL-III: implications for self-oligomerization and haemolytic processes

CEL-III is a haemolytic lectin, which has two beta-trefoil domains (domains 1 and 2) and a beta-sheet-rich domain (domain 3). In domain 3 (residues 284-432), there is a hydrophobic region containing two alpha-helices (H8 and H9, residues 317-357) and a loop between them, in which alternate hydrophobic residues, especially Val residues, are present. To elucidate the role of the alpha-helix region in the haemolytic process, peptides corresponding to different parts of this region were synthesized and characterized. The peptides containing the sequence that corresponded to the loop and second alpha-helix (H9) showed the strongest antibacterial activity for Staphylococcus aureus and Bacillus subtilis through a marked permeabilization of the bacterial cell membrane. The recombinant glutathione S-transferase (GST)-fusion proteins containing domain 3 or the alpha-helix region peptide formed self-oligomers, whereas mutations in the alternate Val residues in the alpha-helix region lead to decreased oligomerization ability of the fusion proteins. These results suggest that the alpha-helix region, particularly its alternate Val residues are important for oligomerization of CEL-III in target cell membranes, which is also required for a subsequent haemolytic action.     

Amphotericin B covalent dimers bearing a tartarate linkage

Amphotericin B (AmB, 1) is known to assemble together and form an ion channel across biomembranes, by which the drug presumably exerts its antimicrobial activity. To access the whole architecture of this channel assemblage, the understanding of binary interaction between AmB molecules is of prime importance because the dimeric interaction is the basis of the assemblage. In this context, we have recently reported covalently conjugated AmB dimers such as 2 and 3 with a long linker, which show prominent hemolytic potency and ion-channel activity. To evaluate the effect of the length and hydrophilicity of linker parts on the activity, we prepared new dimers bearing tartarate linkages (4 and 5). Especially, 5 exhibited potent hemolytic activity (EC50, 0.03 microM) surpassing those of AmB, 2, and 3. Measurements of UV and CD spectra of 5 in liposomes indicated that AmB portions of 5 could adopt appropriate arrangements in molecular assemblage in spite of the short linkage, and also indicated that the assemblage formed by 5 appeared more stable than AmB. These short-tethered dimers are expected to be a promising tool to reveal the mechanism of dimeric interaction in the ion channel formed by AmB.     

Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo

The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimer’s disease (AD) and Down’s syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. Furthermore, cerebral ischemia also induced the degradation of drebrin in vivo. These findings suggest that calpain-mediated degradation of drebrin is a fundamental pathology of neurodegenerative diseases mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin regulates the synaptic clustering of NMDARs; therefore, degradation of drebrin under excitotoxic conditions may modulate NMDAR-mediated signal transductions, including pro-survival signaling. Overall, the results presented here provide novel insights into the molecular basis of cellular responses to excitotoxicity in vitro and in vivo.