Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin-binding proteins by the yeast two-hybrid assay. One of the positive clones contained cDNA encoding a carboxyl-terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix-associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full-length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin-binding site of hnRNP U was located near a carboxyl-terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure.     

Mass Isotopomer Analysis of Metabolically Labeled Nucleotide Sugars and N- and O-Glycans for Tracing Nucleotide Sugar Metabolisms

Nucleotide sugars are the donor substrates of various glycosyltransferases, and an important building block in N- and O-glycan biosynthesis. Their intercellular concentrations are regulated by cellular metabolic states including diseases such as cancer and diabetes. To investigate the fate of UDP-GlcNAc, we developed a tracing method for UDP-GlcNAc synthesis and use, and GlcNAc utilization using ¹³C₆-glucose and ¹³C₂-glucosamine, respectively, followed by the analysis of mass isotopomers using LC-MS.Metabolic labeling of cultured cells with ¹³C₆-glucose and the analysis of isotopomers of UDP-HexNAc (UDP-GlcNAc plus UDP-GalNAc) and CMP-NeuAc revealed the relative contributions of metabolic pathways leading to UDP-GlcNAc synthesis and use. In pancreatic insulinoma cells, the labeling efficiency of a ¹³C₆-glucose motif in CMP-NeuAc was lower compared with that in hepatoma cells.Using ¹³C₂-glucosamine, the diversity of the labeling efficiency was observed in each sugar residue of N- and O-glycans on the basis of isotopomer analysis. In the insulinoma cells, the low labeling efficiencies were found for sialic acids as well as tri- and tetra-sialo N-glycans, whereas asialo N-glycans were found to be abundant. Essentially no significant difference in secreted hyaluronic acids was found among hepatoma and insulinoma cell lines. This indicates that metabolic flows are responsible for the low sialylation in the insulinoma cells. Our strategy should be useful for systematically tracing each stage of cellular GlcNAc metabolism.Protein glycosylation, which is the most abundant post-translational modification, has important roles in many biological processes by modulating conformation and stability, whereas its dysregulation is associated with various diseases such as diabetes and cancer (1, 2). Glycosylation is regulated by various factors including glucose metabolism, the availability and localization of nucleotide sugars, and the expression and localization of glycosyltransferases (3, 4). Thus, ideally all of these components should be considered when detecting changes in a dynamic fashion; namely, it is necessary not only to take a snapshot but also to make movies of the dynamic changes in glycan metabolism.Glucose is used by living cells as an energy source via the glycolytic pathway as well as a carbon source for various metabolites including nucleotide sugars (e.g. UDP-GlcNAc and CMP-NeuAc). These nucleotide sugars are transported into the Golgi apparatus, and added to various glycans on proteins. UDP-GlcNAc is the donor substrate for N-acetylglucosaminyl (GlcNAc)¹ transferases; alternatively, it is used in the cytosol for O-GlcNAc modification (i.e. O-GlcNAcylation) of intracellular proteins (5). The UDP-GlcNAc synthetic pathway is complex as it is a converging point of glucose, nucleotide, fatty acid and amino acid metabolic pathways. Thus, the metabolic flow of glucose modulates the branching patterns of N-glycans via UDP-GlcNAc concentrations because many of the key GlcNAc transferases that determine the branching patterns have widely different K_m values for UDP-GlcNAc ranging from 0.04 mm to 11 mm (6, 7). Indeed, it was demonstrated that the branching formation of N-glycans in T cells is stimulated by the supply from the hexosamine pathway, whereby it regulates autoimmune reactions promoted by T cells (8).UDP-GlcNAc is also used for the synthesis of CMP-NeuAc, the donor substrate for sialyltransferases (9). The CMP-NeuAc concentration is controlled by the feedback inhibition of UDP-GlcNAc epimerase/ManNAc kinase by the final product CMP-NeuAc, and hence a high CMP-NeuAc level reduces metabolic flow in CMP-NeuAc de novo synthesis (10). However, there is still only limited information about how the levels of nucleotide sugars dynamically change in response to the environmental cues, and how such changes are reflected in the glycosylation of proteins.Stable isotope labeling is a promising approach to quantify metabolic changes in response to external cues (11, 12). For example, the use of nuclear magnetic resonance to obtain isotopomer signals of metabolically labeled molecules has been applied to trace the flux in glycolysis and fatty acid metabolism (13). An approach based on the mass isotopomers of labeled metabolites with ¹³C₆-glucose has been developed to monitor the UDP-GlcNAc synthetic pathway (13–15). The method based on the labeling ratio of each metabolite related to UDP-GlcNAc synthesis has clarified the contribution of each metabolic pathway (14). Moseley reported a novel deconvolution method for modeling UDP-GlcNAc mass isotopomers (15).Previous studies into the use of nucleotide sugars in glycosylation have relied on the specific detection of metabolically radiolabeled glycans (16). It is possible not only to deduce the glycan structures but also to trace their relative contributions to glycan synthesis without MS. On the other hand, mass isotopomer analysis of glycans labeled with stable isotope provides the ratios of labeled versus unlabeled molecules from MS spectra and structural details of the glycans. However, there are only a limited number of publications reporting the application of stable isotope labeling of glycans for monitoring the dynamics of glycans (17). To date, there have been no reports describing a systematic method for tracing cellular GlcNAc biosynthesis and use based on mass isotopomer analysis.The aim of this study was to extend our knowledge of the synthesis and metabolism of UDP-GlcNAc as well as its use in the synthesis of CMP-NeuAc, N- and O-glycans. We recently developed a conventional HPLC method for simultaneous determination of nucleotide sugars including unstable CMP-NeuAc (18). We first established an LC-MS method for isotopomer analysis of ¹³C₆-glucose labeled nucleotide sugars for tracing UDP-GlcNAc metabolism from synthesis to use, because previous methods were not suitable for estimating UDP-GlcNAc use in CMP-NeuAc de novo synthesis (15). We also established a method for isotopomer analysis of labeled N- and O-glycan to monitor the metabolic flow of hexosamine into glycans. Using these two methods, we demonstrated the differences in the use of hexosamines between hepatoma and pancreatic insulinoma cell lines. Our approach may be useful for identifying a metabolic “bottleneck” that governs the turnover speed and patterns of cellular glycosylation, which may be relevant for various applications including glycoprotein engineering and discovery of disease biomarkers.     

Synthesis of Isoprenyl Chalcone “Sophoradin”

Nicotine has been found an effective photosensitizer for DDT. At DDT: nicotine (1:5), DDT along with its formed degradation products DDD, DDE and DBP disappeared within 18 and 60 days under UV and sunlight respectively. Because of persistence, nicotine proved a superior photosensitizer to N,N′-diethylaniline. In DDT emulsifiable concentrates it led to high alkalinity but no DDT degradation up to 60 days at 20~25°C. 0.1 and 0.5% DDT emulsions from these formulations showed no adverse effect on Daucus carrota, Vicia faba, Brassica oleracea var. botrytis, and Dahlia sp., but showed mild to severe phytotoxicity against Pisum sativum and Cicer arietinum; caused by high concentration of nicotine. On Clerodendrum sp. in sunlight, these formulations showed over 20% faster DDT loss between 3~15 days of application. DDT-nicotine mixtures showed no synergism against Tribolium castaneum Herbst.     

l-Sorbose Oxidase from Trametes sanguinea

A crude inhibitor for pancreatic lipase was extracted from soybean seeds. The lipase activity decreased curvilinearly with an increase in inhibitor concentration. At a low inhibitor concentration, enhanced inhibition was observed by the co-existence of protein such as bovine serum albumin in the reaction mixture. The lipase activity was inhibited immediately after the addition of inhibitor which did not cause the significant destraction of substrate emulsion. The lipase activities of Aspergillus niger, Rhizopus delemar and castor bean seeds were also inhibited. The inhibition was observed when various oil substrates such as soybean oil, linseed oil, olive oil emulsions and Ediol were used, and the extent of inhibition varied among them. Column chromatography of inhibitor on Sephadex G–100 showed that the molecular weight of a main peak of inhibitor was estimated as about 80,000.     

Dietary Zn deficiency does not influence systemic blood pressure and vascular nitric oxide signaling in normotensive rats

Because zinc (Zn) is an important component for cell protection against certain oxygen species, it has been suggested that Zn deficiency impairs the potent oxidant defense capacity, which is constitutively provided in the vascular system. However, the influence of dietary Zn deficiency on systemic blood pressure and vascular system is controversial and unclear. We therefore examine the effect of dietary Zn deficiency on systemic blood pressure, a potent superoxide scavenger, aortic Cu/Zn superoxide dismutase (SOD) activity, a most representative synthase of the endothelium-derived relaxing factor, and aortic endothelial nitric oxide synthase (eNOS) expression. Furthermore, the direct effects of intravenous administration of NOS inhibitor, N ^ω-nitro-l-arginine methyl ester (l-NAME), and a SOD mimetic compound, tempol, in normotensives were tested in Wistar-Kyoto (WKY) rats. A Zn-deficient diet (4 wk) contributed to growth retardation, the decrease in thymus weight, and the lower levels of serum Zn compared with the standard diet group. However, no significant difference in conscious systolic and diastolic blood pressure was found in the Zn-deficiency group. The administration of l-NAME caused an increase in the mean arterial pressure (MAP) levels in the two groups of rats and the involvement of the vasodilator nitric oxide (NO) in the regulation of systemic BP in the normotensive state. On the other hand, administration of the superoxide scavenger, tempol, led to a decrease in MAP levels in the two groups of rats, indicating the participation of the oxygen free radical, superoxide, in the maintenance of the systemic BP in a normotensive state. There were no significant differences between the Zn-deficient diet group and the standard diet group in the normotensive state. eNOS expression and Cu/Zn SOD activity in the aorta were also intact in Zn-deficient normotensive rats. These findings suggest that the 4 wk of Zn deficiency was inadequate to alter systemic blood pressure and focal NO signaling in the normotensive state. Long-term Zn deficiency affects the neuronal, immune, and hematopoietic systems, which contribute to systemic and/or local circulation. However, Zn deficiency alone does not cause hypertension and local vascular dysfunction in the normotensive state.     

Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.     

Development and characterization of CATS markers for genetic linkage mapping in the house musk shrew, Suncus murinus.

To serve as an initial step in developing an ideal genetic marker map for the house musk shrew, Suncus murinus, 318 comparative anchor tagged sequence (CATS) primer pairs were assessed for polymorphism ascertainment and linkage mapping. Of the 112 (35.2%) CATS primer pairs that were successfully amplified by PCR in the shrew, 18 (16.1%) showed polymorphism between two mutant strains, BAN-kc, oeb and WZ. Linkage analysis of the polymorphic CATS markers and three visible mutant genes, kc, oeb and wz, genotyped in a 77 F2 mapping panel from a cross of the two mutant strains, assigned wz and five CATS markers into three linkage groups. Sequence analysis revealed that two (ADA and TXN) out of nine CATS amplified sequences had a total of six deletions of varying sizes and 17 single nucleotide polymorphisms (SNPs). BLAST search identified three CATS (ADA, CYP1A2, and TXN) products matching the genes from which they were originally designed, while the remaining six markers could not be identified. Together with the use of the detected SNPs as genetic markers, the five CATS markers linkage mapped in this species will serve as anchors in establishing the first framework map for locating loci affecting all heritable qualitative and quantitative traits in the musk shrew.