Synthetic Route to Glycosyl ß-1C-(phosphino)-phosphonates as Unprecedented Stable Glycosyl Diphosphate Analogs and Their Preliminary Biological Evaluation.

The synthesis of glycosyl-ß-1C-(phosphino)-phosphonates is a challenge since it has not yet been described. In this paper, we report an innovative synthetic method for their preparation from Glc-, Man-, and GlcNAc- lactone derivatives. The proposed original strategy involves the addition of the corresponding δ-hexonolactones onto the dianion of (methylphosphino) phosphonate as a key step, followed by dehydration and stereoselective addition of dihydrogen on the resulting double bond. Final deprotection provides the new glycosyl diphosphate analogs in 35%, 36%, and 10% yield over 6 steps from the corresponding δ-hexonolactones. The synthetized compounds were evaluated as inhibitors of phosphatase and diphosphatase activities and found to have complex concentration-dependent activatory and inhibitory properties on alkaline phosphatase. The synthetized tools should be useful to study other enzymes such as transferases.

NUDT7 Loss Promotes KrasG12D CRC Development.

Studies have suggested that dysregulation of peroxisomal lipid metabolism might play an important role in colorectal cancer (CRC) development. Here, we found that KrasG12D-driven CRC tumors demonstrate dysfunctional peroxisomal b-oxidation and identified Nudt7 (peroxisomal coenzyme A diphosphatase NUDT7) as one of responsible peroxisomal genes. In KrasG12D-driven CRC tumors, the expression level of Nudt7 was significantly decreased. Treatment of azoxymethane/dextran sulfate sodium (AOM/DSS) into Nudt7 knockout (Nudt7-/-) mice significantly induced lipid accumulation and the expression levels of CRC-related genes whereas xenografting of Nudt7-overexpressed LS-174T cells into mice significantly reduced lipid accumulation and the expression levels of CRC-related genes. Ingenuity pathway analysis of microarray using the colon of Nudt7-/- and Nudt7+/+ mice treated with AOM/DSS suggested Wnt signaling as one of activated signaling pathways in Nudt7-/- colons. Upregulated levels of ß-catenin were observed in the colons of KrasG12D and AOM/DSS-treated Nudt7-/- mice and downstream targets of ß-catenin such as Myc, Ccdn1, and Nos2, were also significantly increased in the colon of Nudt7-/- mice. We observed an increased level of palmitic acid in the colon of Nudt7-/- mice and attachment of palmitic acid-conjugated chitosan patch into the colon of mice induced the expression levels of b-catenin and CRC-related genes. Overall, our data reveal a novel role for peroxisomal NUDT7 in KrasG12D-driven CRC development.

Fluorescent probe displacement assays reveal unique nucleic acid binding properties of human nudix enzymes.

Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds. The substrates for a subset of Nudix enzymes are all nucleotides linked to RNA, like the m7G mRNA caps and the more recently discovered NAD(H) RNA caps. However, the RNA affinity and nucleic acid specificity of Nudix proteins has not yet been explored in depth. In this study we designed new fluorescence-based assays to examine the interaction of purified recombinant E. coli NudC and human Nudt1 (aka MTH1) Nudt3, Nudt12, Nudt16, and Nudt20 (aka Dcp2). All Nudix proteins except Nudt1 and Nudt12 bound both RNA and DNA stoichiometrically with high affinity (dissociation constants in the nanomolar range) and no clear sequence specificity. In stark contrast, Nudt12 binds RNA but not similar DNA oligonucleotides. Nudt12 also bound RNAs with 5' NAD+ caps more tightly than those with NADH or m7G cap. NudC was similarly selective against m7G caps but did not differentiate between NAD+ and NADH capped RNA. Nudt3, Nudt16, and Nudt20 bound m7G capped RNA more tightly than RNA with NADH caps.

Overexpression of SSH1 in gastric adenocarcinoma and its correlation with clinicopathological features.

BACKGROUND: Gastric adenocarcinoma is known to be the fourth most common cancer type and the second cause of cancer-related deaths. Movement and invasion of cancer cells is one of the major characteristics of the cancer phenotype that various types of network regulate this. Expression levels of slingshot diphosphatase 1 (SSH1) gene has been modulated in this pathway. SSH1 acts as a dephosphorylation and activator of cofilin that this regulating and activating by SSH1 can play a major role in the mobility and migration of the cell. The aim of this study was to compare the expression level of SSH1 genes between tumor and corresponding adjacent non tumor gastric tissues and healthy tissue of gastric adenocarcinoma. METHODS: In this study, mRNA of 40 gastric adenocarcinoma and corresponding adjacent non tumor gastric tissues and 15 healthy biopsy samples was extracted, then after cDNA synthesis, real-time polymerase chain reaction was performed to measure gene expression. RESULTS: According to REST analysis, the relative expression of SSH1 was significantly increased in gastric cancer tissues compared to the corresponding adjacent non tumor gastric tissue samples and normal tissue. Nevertheless, the result revealed no substantial correlation between the expression levels of SSH1 with clinical features. The biomarker index for SSH1 was obtained as 0.89. CONCLUSIONS: The results obtained from investigating SSH1 expression are indicative of significant changes in the expression of this gene in gastric adenocarcinoma. This gene can also be used as a biomarker for gastric cancer.

Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings.

The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.

Synthesis and biological evaluation of chemical tools for the study of Dolichol Linked Oligosaccharide Diphosphatase (DLODP).

Citronellyl- and solanesyl-based dolichol linked oligosaccharide (DLO) analogs were synthesized and tested along with undecaprenyl compounds for their ability to inhibit the release of [3H]OSP from [3H]DLO by mammalian liver DLO diphosphatase activity. Solanesyl (C45) and undecaprenyl (C55) compounds were 50-500 fold more potent than their citronellyl (C10)-based counterparts, indicating that the alkyl chain length is important for activity. The relative potency of the compounds within the citronellyl series was different to that of the solanesyl series with citronellyl diphosphate being 2 and 3 fold more potent than citronellyl-PP-GlcNAc2 and citronellyl-PP-GlcNAc, respectively; whereas solanesyl-PP-GlcNAc and solanesyl-PP-GlcNAc2 were 4 and 8 fold more potent, respectively, than solanesyl diphosphate. Undecaprenyl-PP-GlcNAc and bacterial Lipid II were 8 fold more potent than undecaprenyl diphosphate at inhibiting the DLODP assay. Therefore, at least for the more hydrophobic compounds, diphosphodiesters are more potent inhibitors of the DLODP assay than diphosphomonoesters. These results suggest that DLO rather than dolichyl diphosphate might be a preferred substrate for the DLODP activity.

A putative adenosine kinase family protein possesses adenosine diphosphatase activity.

Adenosine kinase is a potential target for development of new types of drugs. The COG1839 family has been defined as "adenosine-specific kinase" family based on structural analysis and the adenosine-binding ability of a family member, PAE2307. However, there has been no experimental evidence with regard to the enzymatic function of this protein family. Here we measured the enzymatic activity of TTHA1091, a COG1839 family protein from Thermus thermophilus HB8. The phosphorylation of adenosine by TTHA1091 was undetectable when ATP or ADP were used as phosphate donor. However, the degradation of ADP to AMP was detected, indicating that this protein possessed adenosine diphosphatase (ADPase) activity. The (ADPase) activity was inhibited by divalent cations and was specific to ADP and CDP. Thus, this study provides the first experimental evidence for the enzymatic function of the "adenosine-specific kinase" family and suggests a need to reexamine its functional annotation.

Brefeldin A promotes the appearance of oligosaccharyl phosphates derived from Glc3Man9GlcNAc2-PP-dolichol within the endomembrane system of HepG2 cells.

We reported an oligosaccharide diphosphodolichol (DLO) diphosphatase (DLODP) that generates dolichyl-phosphate and oligosaccharyl phosphates (OSPs) from DLO in vitro. This enzyme could underlie cytoplasmic OSP generation and promote dolichyl-phosphate recycling from truncated endoplasmic reticulum (ER)-generated DLO intermediates. However, during subcellular fractionation, DLODP distribution is closer to that of a Golgi apparatus (GA) marker than those of ER markers. Here, we examined the effect of brefeldin A (BFA), which fuses the GA with the ER on OSP metabolism. In order to increase the steady state level of truncated DLO while allowing formation of mature DLO (Glc3Man9GlcNAc2-PP-dolichol), dolichyl-P-mannose Man7GlcNAc2-PP-dolichol mannosyltransferase was partially downregulated in HepG2 cells. We show that BFA provokes GA endomannosidase trimming of Glc3Man9GlcNAc2-PP-dolichol to yield a Man8GlcNAc2-PP-dolichol structure that does not give rise to cytoplasmic Man8GlcNAc2-P. BFA also strikingly increased OSP derived from mature DLO within the endomembrane system without affecting levels of Man7GlcNAc2-PP-dolichol or cytoplasmic Man7GlcNAc2-P. The BFA-provoked increase in endomembrane-situated OSP is sensitive to nocodazole, and BFA causes partial redistribution of DLODP activity from GA- to ER-containing regions of density gradients. These findings are consistent with BFA-provoked microtubule-dependent GA-to-ER transport of a previously reported DLODP that acts to generate a novel endomembrane-situated OSP population.

Demonstration of an oligosaccharide-diphosphodolichol diphosphatase activity whose subcellular localization is different than those of dolichyl-phosphate-dependent enzymes of the dolichol cycle.

Oligosaccharyl phosphates (OSPs) are hydrolyzed from oligosaccharide-diphosphodolichol (DLO) during protein N-glycosylation by an uncharacterized process. An OSP-generating activity has been reported in vitro, and here we asked if its biochemical characteristics are compatible with a role in endoplasmic reticulum (ER)-situated DLO regulation. We demonstrate a Co(2+)-dependent DLO diphosphatase (DLODP) activity that splits DLO into dolichyl phosphate and OSP. DLODP has a pH optimum of 5.5 and is inhibited by vanadate but not by NaF. Polyprenyl diphosphates inhibit [(3)H]OSP release from [(3)H]DLO, the length of their alkyl chains correlating positively with inhibition potency. The diphosphodiester GlcNAc2-PP-solanesol is hydrolyzed to yield GlcNAc2-P and inhibits [(3)H]OSP release from [(3)H]DLO more effectively than the diphosphomonoester solanesyl diphosphate. During subcellular fractionation of liver homogenates, DLODP codistributes with microsomal markers, and density gradient centrifugation revealed that the distribution of DLODP is closer to that of Golgi apparatus-situated UDP-galactose glycoprotein galactosyltransferase than those of dolichyl-P-dependent glycosyltransferases required for DLO biosynthesis in the ER. Therefore, a DLODP activity showing selectivity toward lipophilic diphosphodiesters such as DLO, and possessing properties distinct from other lipid phosphatases, is identified. Separate subcellular locations for DLODP action and DLO biosynthesis may be required to prevent uncontrolled DLO destruction.

Cod liver oil ameliorates sodium nitrite-induced insulin resistance and degradation of rat hepatic glycogen through inhibition of cAMP/PKA pathway.

AIMS: Sodium nitrite is used to inhibit the growth of microorganisms and is responsible for the desirable red color of meat; however, it can be toxic in high quantities for humans and other animals. Moreover, glycogen, a branched polysaccharide, efficiently stores and releases glucose monosaccharides to be accessible for metabolic and synthetic requirements of the cell. Therefore, we examined the impact of dietary sodium nitrite and cod liver oil on liver glycogen. MAIN METHODS: Thirty-two Sprague-Dawley rats were treated daily with sodium nitrite (80 mg/kg) in the presence/absence of cod liver oil (5 ml/kg). Liver sections were stained with Periodic acid-Schiff. Hepatic homogenates were used for measurements of glycogen, cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), glycogen synthase, glycogen synthase kinase, pyruvate carboxylase, fructose 1,6-diphosphatase, glucose 6-phosphatase, phosphodiesterase and glycogen phosphorylase. Glucose, pyruvate tolerances and HOMA insulin resistance were also determined. KEY FINDINGS: Sodium nitrite significantly increased plasma glucose and insulin resistance. Moreover, sodium nitrite significantly reduced hepatic glycogen content as well as activities of glycogen synthase, glycogen synthase kinase-3, and phosphodiesterase. Sodium nitrite elevated hepatic cAMP, PKA, pyruvate carboxylase, fructose 1,6-diphosphatase, glucose 6-phosphatase and phosphorylase. Cod liver oil significantly blocked all of these except pyruvate carboxylase, fructose 1,6-diphosphatase and glucose 6-phosphatase. SIGNIFICANCE: Sodium nitrite inhibited liver glycogenesis and enhanced liver glycogenolysis and gluconeogenesis, which is accompanied by hyperglycemia and insulin resistance through the activation of cAMP/PKA and the inhibition of phosphodiesterase. Cod liver oil blocked the sodium nitrite effects on glycogenesis and glycogenolysis without affecting gluconeogenesis.

Decoding Alzheimer's disease from perturbed cerebral glucose metabolism: implications for diagnostic and therapeutic strategies.

Alzheimer's disease (AD) is an age-related devastating neurodegenerative disorder, which severely impacts on the global economic development and healthcare system. Though AD has been studied for more than 100 years since 1906, the exact cause(s) and pathogenic mechanism(s) remain to be clarified. Also, the efficient disease-modifying treatment and ideal diagnostic method for AD are unavailable. Perturbed cerebral glucose metabolism, an invariant pathophysiological feature of AD, may be a critical contributor to the pathogenesis of this disease. In this review, we firstly discussed the features of cerebral glucose metabolism in physiological and pathological conditions. Then, we further reviewed the contribution of glucose transportation abnormality and intracellular glucose catabolism dysfunction in AD pathophysiology, and proposed a hypothesis that multiple pathogenic cascades induced by impaired cerebral glucose metabolism could result in neuronal degeneration and consequently cognitive deficits in AD patients. Among these pathogenic processes, altered functional status of thiamine metabolism and brain insulin resistance are highly emphasized and characterized as major pathogenic mechanisms. Finally, considering the fact that AD patients exhibit cerebral glucose hypometabolism possibly due to impairments of insulin signaling and altered thiamine metabolism, we also discuss some potential possibilities to uncover diagnostic biomarkers for AD from abnormal glucose metabolism and to develop drugs targeting at repairing insulin signaling impairment and correcting thiamine metabolism abnormality. We conclude that glucose metabolism abnormality plays a critical role in AD pathophysiological alterations through the induction of multiple pathogenic factors such as oxidative stress, mitochondrial dysfunction, and so forth. To clarify the causes, pathogeneses and consequences of cerebral hypometabolism in AD will help break the bottleneck of current AD study in finding ideal diagnostic biomarker and disease-modifying therapy.