NaPi-IIc induces vacuole formation in OK cells

NaPi-IIc/SLC34A3 is a sodium-dependent inorganic phosphate (Pi) transporter in the renal proximal tubules and its mutations cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). In the present study, we created a specific antibody for opossum SLC34A3, NaPi-IIc (oNaPi-IIc), and analyzed its localization and regulation in opossum kidney cells (a tissue culture model of proximal tubular cells). Immunoreactive oNaPi-IIc protein levels increased during the proliferative phase and decreased during differentiation. Moreover, stimulating cell growth upregulated oNaPi-IIc protein levels, whereas suppressing cell proliferation downregulated oNaPi-IIc protein levels. Immunocytochemistry revealed that endogenous and exogenous oNaPi-IIc proteins localized at the protrusion of the plasma membrane, which is a phosphatidylinositol 4,5-bisphosphate (PIP2) rich-membrane, and at the intracellular vacuolar membrane. Exogenous NaPi-IIc also induced cellular vacuoles and localized in the plasma membrane. The ability to form vacuoles is specific to electroneutral NaPi-IIc, and not electrogenic NaPi-IIa or NaPi-IIb. In addition, mutations of NaPi-IIc (S138F and R468W) in HHRH did not cause cellular PIP2-rich vacuoles. In conclusion, our data anticipate that NaPi-IIc may regulate PIP2 production at the plasma membrane and cellular vesicle formation

Identification and functional analysis of a splice variant of mouse sodium-dependent phosphate transporter Npt2c

Mutations in the SLC34A3 gene, a sodium-dependent inorganic phosphate (Pi) cotransporter, also referred to as NaPi IIc, causes hereditary hypophosphatemic rickets with hypercalciuria (HHRH), an autosomal recessive disorder. In human and rodent, NaPi IIc is mainly localized in the apical membrane of renal proximal tubular cells. In this study, we identified mouse NaPi IIc variant (Npt2c-v1) that lacks the part of the exon 3 sequence that includes the assumed translation initiation site of Npt2c. Microinjection of mouse Npt2c-v1 cRNA into Xenopus oocytes demonstrated that Npt2c-v1 showed sodium-dependent Pi cotransport activity. The characterization of pH dependency showed activation at extracellular alkaline-pH. Furthermore, Npt2c-v1 mediated Pi transport activity was significantly higher at any pH value than those of Npt2c. In an in vitro study, the localization of the Npt2c-v1 protein was detected in the apical membrane in opossum kidney cells. The expression of Npt2c-v1 mRNA was detected in the heart, spleen, testis, uterus, placenta, femur, cerebellum, hippocampus, diencephalon and brain stem of mouse. Using mouse bone primary cultured cells, we showed the expression of Npt2c-v1 mRNA. In addition, the Npt2c protein was detected in the spermatozoa head. Thus, Npt2c-v1 was expressed in extra-renal tissues such as epididymal spermatozoa and may function as a sodium-dependent phosphate transporter

Characterization of Hexenuronosyl Xylan-degrading Enzymes Produced by Paenibacillus sp. 07

The enzyme involved in hexenuronic acid (HexA) removal from kraft pulp was identified in Paenibacillus sp. strain 07. Extracellular and intracellular enzymes of Paenibacillus sp. were assessed for their hexenuronosyl-xylotriose (∆X3) degradation activity. First, ∆X3 was obtained from hardwood kraft pulp by enzymatic hydrolysis using three commercial enzymes. Crude extracellular and intracellular enzyme fractions were obtained from Paenibacillus cultures cultivated in 0.5% (w/v) birch wood xylan as the sole carbon source. The ∆X3-degrading activities of the enzyme fractions were measured by hydrolysis assays in sodium acetate buffer containing ∆X3 substrate (pH 6) at 50 °C. The reaction products were analyzed by high-performance anion-exchange chromatography with pulsed amperometric detection. The enzyme fractions displayed different chromatogram patterns. After treatment with the intracellular enzyme fraction, the chromatograms displayed xylose and hexenuronosyl xylobiose (∆X2) peaks. The chromatogram patterns of the extracellular fraction assays indicated xylose, xylotriose, and ∆X2 production. Thus, the intracellular enzymes of Paenibacillus can hydrolyze the xylosidic linkages at the reducing ends of ∆X3, whereas a specific extracellular enzyme can hydrolyze HexA. This enzyme is potentially applicable to HexA removal during bio-bleaching

Toxicity and Feeding Deterrent Effect of 2-Methylanthraquinone from the Wood Extractives of Tectona grandis on the Subterranean Termites Coptotermes formosanus and Reticulitermes speratus

No-choice feeding tests using ethanol, chloroform, and acetone extractives of teak (Tectona grandis) heartwood clearly showed feeding deterrent activity and toxicity to the subterranean termite Reticulitermes speratus. The amount of 2-methylanthraquinone (MAQ) in teak wood extractives was not related to the feeding deterrents or toxicity, as shown by the no-choice feeding tests conducted using crude extractives containing various amounts of MAQ, MAQ alone, and fractions of crude extractives. As a native pest, the subterranean termite Coptotermes formosanus was more tolerant to the fractions of crude extractives than Reticulitermes speratus, and the mortality observed in C. formosanus was not due to the presence of MAQ

Mutagenicity of <i>Tectona grandis</i> Wood Extracts and Their Ability to Improve Carbohydrate Yield for Kraft Cooking Eucalyptus Wood

Considering the toxicity of the impurities of synthesized anthraquinone, this study clarified new catalytic compounds for kraft cooking with improved carbohydrate yield and delignification and less mutagenicity, which are important for ensuring the safety of paper products in contact with food. The 2-methylanthraquinone contents of teak (Tectona grandis) woods were 0.18–0.21%. Acetone extracts containing 2-methylanthraquinone from Myanmar and Indonesia teak woods as additives improved lignin removal during kraft cooking of eucalyptus wood, which resulted in kappa numbers that were 2.2–6.0 points lower than the absence of additive. Myanmar extracts and 2-methylanthraquinone improved carbohydrate yield in pulps with 1.7–2.2% yield gains. Indonesia extracts contained more deoxylapachol and its isomer than 2-methylanthraquinone. The residual content of 2-methylanthraquinone in the kraft pulp was trace. Although Ames tests showed that the Indonesia and Myanmar extracts were mutagenic to Salmonella typhimurium, 2-methylanthraquinone was not. The kraft pulp obtained with the additives should be safe for food-packaging applications, and the addition of 0.03% 2-methylanthraquinone to kraft cooking saves forest resources and fossil energy in industries requiring increased pulp yield

Characterization of Residual Lignin Obtained by the Enzymatic Hydrolysis of Oil Palm Empty Fruit Bunch Pulps

Residual lignin present in alkali pulps prepared from oil palm (Elaeis guineensis) empty fruit bunch was isolated using an enzymatic method and characterized successfully by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). These pulps were prepared by soda-anthraquinone (AQ) and prehydrolysis (PH) soda-AQ cooking methods (pulp yields were 45.3 and 33.9%, respectively). Py-GC/MS of the residual lignin showed that two pyrolysis products which were indole and methylindole originating from the enzyme (contents 12 to 44%), in addition to eight pyrolysis products originating from syringyl (S) and guaiacyl (G) structure of lignin. Furthermore, palmitic acid was also detected in the residual lignin (contents 0.11 to 0.28%). The residual lignin was subjected to further purification by a cellobiose treatment method, and the quantity of enzyme present in the residual lignin was found to decrease after the treatment. Residual lignin in PH-soda-AQ pulp exhibited a higher S/G ratio (0.86 to 0.98) than that in soda-AQ pulp (0.76 to 0.97). This study showed that the contents of lignin (19 to 44%) and enzyme in enzymatically isolated lignin can be estimated by the Py-GC/MS method

Thermal Melting of Lignin Derivatives Prepared from Dried Black Liquor Powder of Softwood Soda-AQ Cooking and Polyethylene Glycol

Softwood lignin prepared by soda-anthraquinone (AQ) cooking does not have thermal melting characteristics. To improve the properties of softwood soda-AQ lignin, we have invented a new method of lignin modification using dried black liquor powder by a spray dryer system and polyethylene glycol (PEG). In this process, black liquor powder was directly treated with PEG under alkaline conditions to produce PEG-modified lignin (alkaline PEG treatment). Dried black liquor powder prepared by a spray dryer was dissolved into PEG and heated at either 120 or 160 °C at atmospheric pressure. The modified lignin (alkaline PEG-treated lignin) was precipitated with acid and recovered by filtration. The alkaline PEG-treated lignin showed adequate thermal melting characteristics. The treatment temperature and the molecular weights of PEG considerably affected the thermal properties of the alkaline PEG-treated lignin. There was an addition reaction of the PEG to the lignin hydroxyl group at the alpha- (-) carbon. However, in the acid precipitation step, if the mixture was allowed to set unfiltered for a long time, the PEG bonded with the lignin was hydrolyzed, which yielded the original soda-AQ lignin and PEG polymer