Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Combined use of COSY and double quantum two-dimensional NMR spectroscopy for elucidation of spin systems in polymyxin B

Two-dimensional single quantum correlation NMR spectroscopy (COSY) and two-dimensional double quantum NMR spectroscopy (2QT) are used to study spin systems in the 1H NMR spectrum of polymyxin B. Because of different frequency relationships, the two types of two-dimensional NMR experiments are found to be highly complementary. This is demonstrated by combined use of COSY and 2QT spectroscopy to obtain a complete analysis of the complicated spectral overlap which occurs in the 1H NMR spectrum of polymyxin B.     

Bacterial flora of the root surface of wheat grown in nutrient solutions deficient in nitrogen and phosphorus

Изучался состав бактериальной флоры поверхности корней пшеницы, выращиваемой в питательном растворе Кнопа—как с полным составом питательных веществ, так и при отсутствии азота и фосфора. Недостаток азота заметно отражался на росте растений и на составе бактериальной флоры: отмечено понижение встречаемости менее требовательных к питанию бактерий и повышение числа бактерий, требующих питательных веществ, содержащихся в дрожжевом и почвенном экстрактах —при понижении встречаемости быстро растущих типов. Не было отмечено разницы в составе бактериальной флоры поверхности корней пшеницы, выращиваемой в питательном растворе полного состава—или же в среде, лишенной фосфора.      

Biological decomposition of fulvic acid preparations

Decomposition of preparations of various fractions of fulvio acids in pure cultures of bacteria and in the soil was investigated. In the soils enriched with fulvic acids the amount of bacteria increased, oxygen consumption and formation of carbon dioxide followed a typical sigmoid curve. The above measurements indicated that mineralization occurred after a very short or negligible lag phase. During the decomposition of fulvic acids the ability of microorganisms to oxidize aromatic compounds,e.g. vanillio andphydroxybenzoic acid, increased. The presence of aromatic structures in the used preparations of fulvic acids was demonstrated on the basis of their IR spectra and according to the results of Chromatographic analyses of their hydrolysates. The results indicated a relationship between metabolism offulvio acids and aromatic oompounds. In samples of the soil preincubated with glucose the fulvic acids decomposed more rapidly than in untreated samples.     

Effect of glucose on the decomposition of organic materials added to soil

The effect of glucose on the decomposition of plant material in soil was studied. Soil samples were enriched with different fractions of labelled alfalfa: the soluble dialyzable fraction, the soluble nondialyzable fraction, cellulose-lignin, lucerne meal. The added substances were decomposed for 42 days. One soil sample in every experimental variant was then percolated with water and another with 200 ml. 0.5% glucose for another 21 days. It was found that the effect of glucose on decomposition of the plant material depended on the latter’s composition and degree of decomposition. It was small in soils pre-enriched with soluble alfalfa fractions, decomposition of which was largely completed within the given time. Glucose markedly inhibited the release of radioactive carbon from the celluloselignin fraction and stimulated the formation of radioactive carbon dioxide from alfalfa meal.     

The development ofAzotobacter in the oat rhizosphere and its effect on the yield

ИccлeДOBAлocь paэBиTиe paэлиЧHьix Πitammb aэotoбaкtepa b pиэocфepe obca и иx bлияhиe ha ypoжa и. ΠepbohaЧaлЧhЧie Πitammьi cpabhиbaлиcь c t. h. iiaccaж иpobahhьimh шtammamи (te жe шtammьi, Aotopьie длиte иьho пobtopho bьipaщиba лиcь B pиэOCфepe OBca, pocшe гo B CTepилиэoBaHHoй Π). БaкTepиэaция ceMяB OBca BceMи шTaMMaMи aэOTOбaкTepa Bлиялa лa Ha кOлиЧecTBO aэOToбaкTepa B ΠOЧBe PиэOCъepьI, HO He Ha кOPHяX. Πaccaжи aэOTOбaкTepa Чepeэ pиэocфepy OBca B HaшиX ycлoBияX OΠьITa He yBeлиЧиBaли CΠOCOбHOCTи эTиX шTaMMOB paэBиBaTьCя бOлee yCилeHHO Ha кOPHяX или B ΠOЧBe PиэOCфePяI, ЧeM пePBOHaЧaлпHпIe шTaMMьI, — B OTлиЧиe OT дaHHьIX OпьITOB CO шTaMMaMи aэOTOбaкTepa, кOTOPьIe BьIPaщиBaлиCь C AOPHяMи PaCTeHий B CPeдe C aг;apoM (эиHOBьeBa 1950‐1954, BaHЧypa C COTP. 1959). БaкTepиэaпия BCeMи шTaMMaMи aэOTOбaкTepa цOBяIшaлa ypoжaй B CPaBHeHии C кOHTpOлeM.     

Cholesterol accumulation caused by low density lipoprotein receptor deficiency or a cholesterol-rich diet results in ectopic bone formation during experimental osteoarthritis

Introduction

Osteoarthritis (OA) is associated with the metabolic syndrome, however the underlying mechanisms remain unclear. We investigated whether low density lipoprotein (LDL) accumulation leads to increased LDL uptake by synovial macrophages and affects synovial activation, cartilage destruction and enthesophyte/osteophyte formation during experimental OA in mice.

Methods

LDL receptor deficient (LDLr^−/−) mice and wild type (WT) controls received a cholesterol-rich or control diet for 120 days. Experimental OA was induced by intra-articular injection of collagenase twelve weeks after start of the diet. OA knee joints and synovial wash-outs were analyzed for OA-related changes. Murine bone marrow derived macrophages were stimulated with oxidized LDL (oxLDL), whereupon growth factor presence and gene expression were analyzed.

Results

A cholesterol-rich diet increased apolipoprotein B (ApoB) accumulation in synovial macrophages. Although increased LDL levels did not enhance thickening of the synovial lining, S100A8 expression within macrophages was increased in WT mice after receiving a cholesterol-rich diet, reflecting an elevated activation status. Both a cholesterol-rich diet and LDLr deficiency had no effect on cartilage damage; in contrast, ectopic bone formation was increased within joint ligaments (fold increase 6.7 and 6.1, respectively). Moreover, increased osteophyte size was found at the margins of the tibial plateau (4.4 fold increase after a cholesterol-rich diet and 5.3 fold increase in LDLr^−/− mice). Synovial wash-outs of LDLr^−/− mice and supernatants of macrophages stimulated with oxLDL led to increased transforming growth factor-beta (TGF-β) signaling compared to controls.

Conclusions

LDL accumulation within synovial lining cells leads to increased activation of synovium and osteophyte formation in experimental OA. OxLDL uptake by macrophages activates growth factors of the TGF-superfamily.     

Mapping hypoxia-induced bioenergetic rearrangements and metabolic signaling by 18O-assisted 31P NMR and 1H NMR spectroscopy

Brief hypoxia or ischemia perturbs energy metabolism inducing paradoxically a stress-tolerant state, yet metabolic signals that trigger cytoprotection remain poorly understood. To evaluate bioenergetic rearrangements, control and hypoxic hearts were analyzed with 18O-assisted 31P NMR and 1H NMR spectroscopy. The 18O-induced isotope shift in the 31P NMR spectrum of CrP, betaADP and betaATP was used to quantify phosphotransfer fluxes through creatine kinase and adenylate kinase. This analysis was supplemented with determination of energetically relevant metabolites in the phosphomonoester (PME) region of 31P NMR spectra, and in both aromatic and aliphatic regions of 1H NMR spectra. In control conditions, creatine kinase was the major phosphotransfer pathway processing high-energy phosphoryls between sites of ATP consumption and ATP production. In hypoxia, creatine kinase flux was dramatically reduced with a compensatory increase in adenylate kinase flux, which supported heart energetics by regenerating and transferring beta- and gamma-phosphoryls of ATP. Activation of adenylate kinase led to a build-up of AMP, IMP and adenosine, molecules involved in cardioprotective signaling. 31P and 1H NMR spectral analysis further revealed NADH and H+ scavenging by alpha-glycerophosphate dehydrogenase (alphaGPDH) and lactate dehydrogenase contributing to maintained glycolysis under hypoxia. Hypoxia-induced accumulation of alpha-glycerophosphate and nucleoside 5'-monophosphates, through alphaGPDH and adenylate kinase reactions, respectively, was mapped within the increased PME signal in the 31P NMR spectrum. Thus, 18O-assisted 31P NMR combined with 1H NMR provide a powerful approach in capturing rearrangements in cardiac bioenergetics, and associated metabolic signaling that underlie the cardiac adaptive response to stress.