Structural investigation of a heteropolysaccharide isolated from the pods (fruits) of Moringa oleifera (Sajina)

A water-soluble polysaccharide was isolated from the aqueous extract of pods of Moringa oleifera. The polysaccharide contains d-galactose, 6-O-Me-D-galactose, D-galacturonic acid, l-arabinose, and l-rhamnose in a molar ratio of 1:1:1:1:1. On the basis of total hydrolysis, methylation analysis, periodate oxidation, and NMR ((1)H, (13)C, TOCSY, DQF-COSY, NOESY, ROESY, HSQC, and HMBC) studies, the repeating unit of the polysaccharide is established as [structure: see text].     

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using ¹³C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO₂ via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

Fluctuation in recoverable nickel and zinc resistant copiotrophic bacteria explained by the varying zinc ion content of Torsa River in different months

Heavy metal content analysis of River Torsa in India did not indicate any alarming level of toxicity for human consumption but revealed variation at the ppb level in different months. The variation in recoverable nickel and zinc resistant copiotrophic (or eutrophic) bacterial counts was explained by the variation of the zinc content (34.0–691.3 ppb) of the river water in different sampling months. Growth studies conducted with some purified nickel and/or zinc resistant strains revealed that pre-exposure of the cells to ppb levels of Zn²⁺, comparable to the indigenous zinc ion concentration of the river, could induce the nickel or zinc resistance. A minimum concentration of 5–10 μM Zn²⁺ (325–650 ppb) was found effective in inducing the Nickel resistance of the isolates. Zinc resistance of the isolates was tested by pre-exposing the cells to 4 μM Zn²⁺ (260 ppb). The lag phase was reduced by 6–8 h in all the cases. Biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicated that some of the Torsa River isolates, having inducible nickel and zinc resistance, are members of the genus Pseudomonas, Acinetobacter, Bacillus, Enterobacter, Serratia and Moraxella.     

Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-nitrilotriacetic acid

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.     

Genetic admixture and population structure analysis of Indian water buffaloes (Bubalus bubalis) using STR markers

Molecular Biology Reports - India has a vast riverine and swamp buffalo diversity adapted to various agro-ecological conditions. In the present study, genetic diversity data for 10 different...     

A Novel Role for Protein Kinase Kin2 in Regulating HAC1 mRNA Translocation,Splicing, and Translation

A signaling network called the unfolded protein response (UPR) resolves the protein-folding defects in the endoplasmic reticulum (ER) from yeasts to humans. In the yeast Saccharomyces cerevisiae, the UPR activation involves (i) aggregation of the ER-resident kinase/RNase Ire1 to form an Ire1 focus, (ii) targeting HAC1 pre-mRNA toward the Ire1 focus that cleaves out an inhibitory intron from the mRNA, and (iii) translation of Hac1 protein from the spliced mRNA. Targeting HAC1 mRNA to the Ire1 focus requires a cis-acting bipartite element (3′BE) located at the 3′ untranslated leader. Here, we report that the 3′BE plays an additional role in promoting translation from the spliced mRNA. We also report that a high dose of either of two paralogue kinases, Kin1 and Kin2, overcomes the defective UPR caused by a mutation in the 3′BE. These results define a novel role for Kin kinases in the UPR beyond their role in cell polarity and exocytosis. Consistently, targeting, splicing, and translation of HAC1 mRNA are substantially reduced in the kin1Δ kin2Δ strain. Furthermore, we show that Kin2 kinase domain itself is sufficient to activate the UPR, suggesting that Kin2 initiates a signaling cascade to ensure an optimum UPR.     

Regulation of CTP Synthase Filament Formation During DNA Endoreplication in Drosophila

CTP synthase (CTPsyn) plays an essential role in DNA, RNA, and lipid synthesis. Recent studies in bacteria, yeast, and Drosophila all reveal a polymeric CTPsyn structure, which dynamically regulates its enzymatic activity. However, the molecular mechanism underlying the formation of CTPsyn polymers is not completely understood. In this study, we found that reversible ubiquitination regulates the dynamic assembly of the filamentous structures of Drosophila CTPsyn. We further determined that the proto-oncogene Cbl, an E3 ubiquitin ligase, controls CTPsyn filament formation in endocycles. While the E3 ligase activity of Cbl is required for CTPsyn filament formation, Cbl does not affect the protein levels of CTPsyn. It remains unclear whether the regulation of CTPsyn filaments by Cbl is through direct ubiquitination of CTPsyn. In the absence of Cbl or with knockdown of CTPsyn, the progression of the endocycle-associated S phase was impaired. Furthermore, overexpression of wild-type, but not enzymatically inactive CTPsyn, rescued the endocycle defect in Cbl mutant cells. Together, these results suggest that Cbl influences the nucleotide pool balance and controls CTPsyn filament formation in endocycles. This study links Cbl-mediated ubiquitination to the polymerization of a metabolic enzyme and reveals a role for Cbl in endocycles during Drosophila development.     

The N Terminus of Pro-endothelial Monocyte-activating Polypeptide II (EMAP II) Regulates Its Binding with the C Terminus,Arginyl-tRNA Synthetase,and Neurofilament Light Protein

Pro-endothelial monocyte-activating polypeptide II (EMAP II), one component of the multi-aminoacyl tRNA synthetase complex, plays multiple roles in physiological and pathological processes of protein translation, signal transduction, immunity, lung development, and tumor growth. Recent studies have determined that pro-EMAP II has an essential role in maintaining axon integrity in central and peripheral neural systems where deletion of the C terminus of pro-EMAP II has been reported in a consanguineous Israeli Bedouin kindred suffering from Pelizaeus-Merzbacher-like disease. We hypothesized that the N terminus of pro-EMAP II has an important role in the regulation of protein-protein interactions. Using a GFP reporter system, we defined a putative leucine zipper in the N terminus of human pro-EMAP II protein (amino acid residues 1–70) that can form specific strip-like punctate structures. Through GFP punctum analysis, we uncovered that the pro-EMAP II C terminus (amino acids 147–312) can repress GFP punctum formation. Pulldown assays confirmed that the binding between the pro-EMAP II N terminus and its C terminus is mediated by a putative leucine zipper. Furthermore, the pro-EMAP II 1–70 amino acid region was identified as the binding partner of arginyl-tRNA synthetase, a polypeptide of the multi-aminoacyl tRNA synthetase complex. We also determined that the punctate GFP pro-EMAP II 1–70 amino acid aggregate colocalizes and binds to the neurofilament light subunit protein that is associated with pathologic neurofilament network disorganization and degeneration of motor neurons. These findings indicate the structure and binding interaction of pro-EMAP II protein and suggest a role of this protein in pathological neurodegenerative diseases.     

Radiological and Pathological Features Associated with IDH1-R132H Mutation Status and Early Mortality in Newly Diagnosed Anaplastic Astrocytic Tumours

Background

Glioblastoma can occur either de novo or by the transformation of a low grade tumour; the majority of which harbor a mutation in isocitrate dehydrogenase (IDH1). Anaplastic tumours are high-grade gliomas that may represent the final step in the evolution of a secondary glioblastoma or the initial presentation of an early primary glioblastoma. We sought to determine whether pathological and/or radiological variables exist that can reliably distinguish IDH1-R132H-positive from IDH1-R132H-negative tumours and to identify variables associated with early mortality.

Methods

Patients diagnosed with anaplastic astrocytic tumours were included. Magnetic resonance imaging was performed and immunohistochemistry was used to identify tumours with the IDH1-R132H mutation. Survival was assessed 12 months after diagnosis. Variables associated with IDH1-R132H status were identified by univariate and ROC analysis.

Results

37 gliomas were studied; 18 were positive for the IDH1-R132H mutation. No tumours demonstrated a combined loss of chromosomes 1p/19q. Patients with IDH1-R132H-positive tumours were less likely to die within 12 months of diagnosis (17% vs. 47%; p=0.046), more likely to have tumours located in the frontal lobe (55% vs. 16%; p=0.015), and have a higher minimum apparent diffusion coefficient (1.115 x 10^-3 mm²/sec vs. 0.838 x 10^-3 mm²/sec; p=0.016), however, these variables demonstrated only moderate strength for predicting the IDH1-R132H mutation status (AUC=0.735 and 0.711, respectively). The Ki-67 index was significantly lower in IDH1-R132H-positive tumours (0.13 vs. 0.21; p=0.034). An increased risk of death was associated with contrast-enhancement ≥ 5 cm³ in patients with IDH1-R132H-positive tumours while edema ≥ 1 cm beyond the tumour margin and < 5 mitoses/mm² were associated with an increased risk of death in patients with IDH1-R132H-negative tumours.

Conclusions

IDH1-R132H-positive and -negative anaplastic tumours demonstrate unique features. Factors associated with early mortality are also dependent on IDH1-R132H status and can be used to identify patients at high risk for death.