Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells.

Whole cells of Brevibacterium ammoniagenes IAM 1645 having the polyphosphate NAD-kinase were successfully immobilized in a polyacrylamide gel lattice. The immobilized cells were activated by treatment with organic solvents or detergents. The pH optimum of the immobilized cells for the production of NADP was 7.0, and divalent metal ions were required to maintain the elevated activity of polyphosphate NAD-kinase. Highly pure NADP was continuously produced in high yield by the immobilized cell column. The half-life of this column was about eight days.     

Continuous production of glucose-6-phosphate by immobilized Achromobacter butyri cells

Summary Whole cells of Achromobacter butyri OUT 8004 having polyphosphate glucokinase activity were immobilized in polyacrylamide gel. The immobilized cells were activated by organic solvents, especially acetone. The immobilization resulted in increased stability of polyphosphate glucokinase. Continuous high yield production of G-6-P from glucose and metaphosphate was performed with an immobilized cell column, which had a half-life of approximately 20 days.Abbreviations G-6-P glucose-6-phosphate - G-1-P glucose-1-phosphate - Cation-S stearyl trimethyl ammonium chloride - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)-aminomethane; p-NPP, p-nitrophenyl phosphate - S.V. space velocity     

Entrapment of microbial cells and organelles with hydrophilic urethane prepolymers

Summary Microbial cells and cellular organelles were immobilized by mixing aqueous suspensions of the biocatalysts with water-miscible urethane prepolymers. Thus immobilized preparations of acetone-dried cells of Arthrobacter simplex and thawed cells of Nocardia rhodocrous showed appreciable {ie351-1} activities in the transformation of hydrocortisone into prednisolone and 4-androstene-3,17-dione to androst-1,4-diene-3,17-dione, respectively. The activities of catalase and alcohol oxidase were observed in the immobilized peroxisomes (microbodies) of a methanol-grown yeast Kloeckera sp. No. 2201. Yeast mitochondria entrapped with the prepolymer showed adenylate kinase activity. These results indicate the usefulness of the urethane prepolymers as convenient materials for entrapment of not only enzymes, but also organelles and microbial cells.     

Characterization of O-trimethylsilyl derivatives of d-glucose,d-galactose,and d-mannose by gas-liquid chromatography-chemical-ionization mass spectrometry with ammonia as reagent gas

The per(trimethylsilyl) ethers of d-glucose, d-galactose, and d-mannose were analyzed by g.l.c.-c.i.m.s. with ammonia as the reagent gas. C.i.m.s. gave simple fragmentation and fragment ions of high intensity in the high-mass range where the QM⁺ ion is also detected. The β-d anomers gave ions at m/e 558 showing intensities 3–12 times those of the α-d anomers. The epimers could be distinguished by differences in the intensities of the ions and by the observation that d-glucose gave a base peak at m/e 198, d-galactose at m/e 468, and d-mannose at m/e 204. The pyranose and furanose structures could be distinguished by comparing the ion intensities at m/e 198, m/e 271, m/e 361, m/e 396, and m/e 451. A similar analysis was also performed with 2-methylpropane as the reagent gas.     

Studies on the phosphomannose isomerase of Amorphophallus konjac C. Koch I. Its isolation and some enzymic properties

A method is described for isolating phosphomannose isomerasefrom young konjak corms. The enzyme is believed to catalyzethe mannose forming reaction in growing corm tissues. The purifiedenzyme preparation was free from phosphoglucose isomerase activity,and was stable at pH 6–9. Maximum enzyme activity wasobserved at pH 6.5–7.0. The molecular weight of the enzymewas estimated as 45,000 using Sephadex gel filtration. The followingkinetic parameters were obtained: Km (mannose-6-P), 0.73 mM,K_eq (fructose-6-P/mannose-6-P), 1.06 at pH 6.5, and activationenergy, 11,600 cal/mole. The enzyme was inhibited by the metalbinding agents EDTA, o-phenanthroline, and a,a'-bipyridyl. Theinhibitory effect of these agents was markedly influenced bythe pH level of the incubation mixture, being more pronouncedat pH 6 than at pH 8. ¹ This paper constitutes part 3 of studies on konjak mannanbiosynthesis. (Received March 3, 1975; )     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

Nontraditional translation is the key to UFMylation and beyond

The Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein that can also be conjugated to protein substrates and subsequently alter their fates. Both UFMylation and de-UFMylation are mediated by Ufm1-specific proteases (UFSPs). In humans, it is widely believed that UFSP2 is the only active Ufm1 protease involved in Ufm1 maturation and de-UFMylation, whereas UFSP1 is thought to be inactive. Here, Liang et al. provide strong evidence showing that human UFSP1 is also an active Ufm1 protease. These results solve an age-old mystery in the human Ufm1 conjugation system and could have a greater impact not only on Ufm1 biology but also on the translation of genes employing nontraditional start codons.