Alkane Oxidation by a Particulate Preparation from Candida

The oxidation of decane by a cell-free particulate preparation from Candida intermedia was studied. Decane is oxidized to decanoate via decanol and decanaldehyde. Oxidation of decane to decanol requires molecular oxygen. Decanol is oxidized to decanaldehyde by a nicotinamide adenine dinucleotide-linked dehydrogenase differing greatly in specificity from ordinary yeast alcohol dehydrogenase. Decanaldehyde is oxidized to decanoate by a nicotinamide adenine dinucleotide-linked dehydrogenase that oxidizes long-chain aldehydes but not short-chain aldehydes. The enzymes that oxidize decane, decanol, and decanaldehyde are all induced when decane is present in the medium. These enzymes are apparently located in the cell membrane.     

The Organic Acid Metabolism of Swede 'Root' Tissue

Data are recorded on the quantitative changes in the contentof succinic, malic and citric acids of discs of washed ‘root’tissue of ‘swede’ (Brassica napus), subjected toexperimental treatments involving the addition of succinate,malate, citrate, -ketoglutarate, pyruvate and malonate. Theresults are interpreted as showing the acids to be associated,in part at least, with the operation of an active Krebs cycle.It is suggested that they are also involved, by providing carbonskeleton material, in the enhanced protein synthesis resultingon washing discs of swede tissue and treating washed discs withphosphate buffer.     

Enzymatic Oxidation of Tetramethyl-p-Phenylenediamine and p-Phenylenediamine by the Electron Transport Particulate Fraction of Azotobacter vinelandii

The ability of the electron transport particulate fraction of Azotobacter vinelandii strain O to oxidize tetramethyl-p-phenylenediamine (TMPD) and p-phenylenediamine (PPD) was examined in detail. The highest specific activity for TMPD and PPD oxidation concentrated in the A. vinelandii O R(3) fraction. The A. vinelandii O R(3) fraction was used to develop a standard manometric assay which gave optimal oxidation rates for both of these dyes. The conditions of the assay and all essential related enzymatic kinetic parameters are presented. Other para derivatives of phenylenediamines also were oxidized readily, whereas ortho and meta derivatives were not. Hydroquinone, p-hydroxybenzoic acid, p-cresol, tyrosine, pyrogallol, pyrocatechol, and diphenylamine were not able to serve as electron donors for the A. vinelandii O R(3) system. The probable involvement of a particle-bound cytochrome oxidase is indicated by the marked sensitivity of both TMPD and PPD oxidation to cyanide, axide, phenylhydrazine, hydroxylamine, and, to a lesser degree, carbon monoxide.     

Constituent Proteins of Globulin Fraction Obtained from Egg White

A globulin fraction which was salted out from egg white by ammonium sulfate was constituted of five kinds of proteins. One of them was macroglobulin and two were G₂ and G₃ of Longsworth et al. [J. Am. Chem. Soc., 62, 2580 (1940)]. Remaining two proteins were assumed to be ovoinhibitors. G₂ and G₃ were separated from each other by CM-cellulose chromatography. The molecular weights of G₂ and G₃ were almost the same, being about 49,000. An euglobulin-like protein which was precipitated during the dialysis of the globulin fraction consisted mainly of macroglobulin and aggregates of some other egg white proteins.     

Decomposition of Urea by the Larger Particulate Fraction and the Free Bacteria Fraction in a Pond Water

An aliquot of a water sample taken from an artificial pond on the campus of the Faculty of Science, Tokyo Metropolitan University, was filtered with Whatman GF/C glass fiber filters. Urea was added to the filtered and unfiltered waters, respectively to a final concentration of around 10 μg-at-N/l. These samples were kept in 10 liter glass bottles, which were kept on the surface of the pond with rope. Changes in the concentrations of urea, ammonium, nitrate, nitrite, and the number of bacteria were traced from 15 June through 13 July, 1973. The urea added to the unfiltered water decreased to about 1 μg-at-N/l within first three days. The decrement of the urea seemed to proceed as a first-order reaction with rate constant of 0.73 day⁻¹. On the other hand, the urea added in the filtered water kept nearly the initial concentration for 18 days. As the number of bacteria in the filtered and unfiltered water were not significantly different, decrease of the urea in the unfiltered water may be ascribable to the participate fraction removed by the filtration. The urea was not decomposed by free bacteria.     

Deoxyribonucleic Acid Polymerase Activity Associated with a Plasma Particulate Fraction from Patients with Chronic Lymphocytic Leukemia

Plasma from two cases of chronic lymphocytic leukemia have been examined for deoxyribonucleic acid (DNA) polymerase activity. In both cases, detectable enzyme activity was present. In the plasma from a patient known to have chronic lymphocytic leukemia for 10 years, the enzyme activity was sufficiently high to study product formation, buoyant density of the enzyme activity, deoxyribonucleoside triphosphate requirements, and kinetics of DNA synthesis. These studies are presented in this report.     

Sections from Double Mesas Obtained at the Same Tissue Plane

A method for obtaining sections from two areas in the face plane of a tissue block is described. It facilitates ultrathin sectioning where virtually identical planes of section are essential but where areas of interest are too far apart to be included in a single section. Two horizontally separated mesas are prepared; sections are cut from the first with the knife rotated around its vertical axis by 2-3° to provide clearance for the other. The second mesa is then sectioned with the knife rotated 4-6° in the opposite direction. Similarly, by changing the vertical inclination of the block, two additional vertically separated mesas can be cut. This procedure is of great value for comparative morphometric studies of material from opposite sides of individual specimens.     

Tissue Slice and Particulate beta-Glucan Synthetase Activities from Pisum Epicotyls

β-Glucan synthetase activity in growing regions of pea (Pisum sativum L.) epicotyls was assayed by supplying UDP-glucose to particulate fractions of tissue homogenates or to thin tissue slices. Particulate fractions are less active in forming alkali-insoluble glucan than slices from the same tissue, although many kinetic characteristics (pH and Mg²⁺ optimum, apparent K_m) are similar for the two systems. Synthesis by tissue slices progresses linearly without lag period for at least an hour and is proportional to cut surface area. It is much more rapid from UDP-glucose than from glucose, glucose-1-P, or sucrose. Tests with plasmolyzing agents and trypsin support the conclusion that synthesis from UDP-glucose by slices occurs at accessible surfaces of cut cells. Analyses of glucan products by GLC of partially methylated and acetylated derivatives and by hydrolysis with various β-glucanases all show that both β-1,3 and β-1,4 linkages are formed by particulate fractions and slices at substrate concentrations ranging from micro- to millimolar. β-1,4 Linkages predominate at low substrate (5 μm) concentration. Kinetic data indicate that the capacity to synthesize β-1,3-glucan is substrate-activated, and this product predominates in preparations supplied with high (5 mm) substrate.     

Phosphoryl Group Transfer by a Fraction of the Soluble Proteins of Catecholamine Storage Vesicles

Abstract: The terminal phosphate group of ATP was transferred to ADP by an enzyme present in the soluble core proteins of adrenal medulla catecholamine storage vesicles. It was purified 10–30-fold by DEAE Sephadex chromatography (Fraction I). The enzyme required divalent metal ions for activation; Mn²⁺ was almost as effective as Mg²⁺, but Ca²⁺ was only a weak activator. Activation by Mg²⁺ took place over a very narrow concentration range (0.5–3 m m ). The specificity of the enzyme activity to nucleoside triphosphates was broad, to the nucleoside diphosphates narrow, favouring adenosine diphosphate. In dependence on the pH the activity increased from pH 4 to pH 7 and remained constantly high between pH 7 and 9. The Arrhenius plot was linear between 5 and 70°C, with an activation energy of 11.1 kcal/mol. The phosphoryl group transfer reaction depended on the function of thiol groups; p -hydroxymercuribenzoate inhibited 50% of the enzyme activity; dithioerythritol reactivated it completely. Gel electrophoresis revealed that in Fraction I, a protein of molecular weight about 45,000, was enriched compared with the total soluble proteins. The enzyme-enriched Fraction I differed significantly in its relative amino acid composition from that of the total soluble proteins; in general, the acidic amino acids were reduced and the more basic acids enhanced.     

Purification and Characterization of DNA Polymerase Obtained from the Membrane Fraction of E. coli

Purification studies were conducted on DNA polymerase bound to the membrane fraction of E. coli HF 4704. Purified enzyme (Fraction V) required Mg²⁺ and showed an optimun pH of 7.2. Various kinds of salt indicated a stimulative effect at concentrations lower than 0.1 m. Fraction V was unstable at an acidic condition (pH 5.0) but was rather stable at an alkaline condition (pH 9.0). The enzyme activity was lost by incubation at 45°C for 30min but was stabilized by the addition of DNA. The enzyme contained exonuclease activity but no endonuclease activity. The enzyme produced only light density DNA of various sizes. The function of this enzyme as considered to fill single stranded region of the double stranded primer DNA.     

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 μg of mitochondria indicate that 1.0 μg is sufficient to measure respiration and that 5.0 μg provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.     

Morphological Variants of Sindbis Virus Obtained from Infected Mosquito Tissue Culture Cells

Tissue-cultured Aedes albopictus cells infected with morphologically homogeneous Sindbis virus were found to produce progeny virions which could be divided into three classes based on size. The thickness of the envelope was constant on all three sizes of progeny virions suggesting that the variability in size rested with the viral nucleocapsid. It is suggested that the three classes of virions have icosahedral nucleocapsids composed of common subunits organized in decreasing triangulation numbers.     

Gene Expression Profiles of Beta-Cell Enriched Tissue Obtained by Laser Capture Microdissection from Subjects with Type 2 Diabetes

Background

Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover.

Methodology/Principal Findings

Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated.

Conclusions/Significance

This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.     

Effects of Anoxia on Solute Loss from Beetroot Storage Tissue

Beetroot storage tissue that had been aged in an aerated solutionwas particularly suited for studies of solute losses duringanoxia;retention of betacyanin being a good indicator of tonoplastintegrity. During anoxia, loss of K⁺ was nearly always greater than thatof Na⁺ while Cl^– loss was intermediate. Supply of glucoseduringageing increased the tolerance of beetroot tissue to anoxia.In these tolerant tissues, there were three phases of soluteloss.During the first phase, losses of K⁺ and amino acids wererapid, presumably due to membrane depolarization from –156to –95 mV. In contrast, losses of Na⁺ and Cl^– wereslow. During the second phase, K⁺ loss had decreased to a lowrate, while losses of Na⁺ and Cl⁺ remained slow. Furthermore,the membrane potential remained at –95 to –90mV,which was consistent with the diffusion potential estimatedfrom the modified Goldman equation. In the third and final phase,loss of K⁺ Na⁺ Cl⁺,sugars, and amino acids began to increase,soon followed by loss of betacyanin. Tissues that had lost their betacyanin during anoxia were irreversiblyinjured, as shown by rapid uptake of Evans Blue and afailureto take up K⁺ , Na⁺ and Cl⁺ during re–aeration. In contrast,tissues which had retained their betacyanin did not take upEvansBlue, but took up substantial amounts of K⁺ , Na⁺ , and Cl^–after re–aeration. After return to air for 1.5 h, tissuethat hadretained its betacyanin had a membrane potential of– 154 mV. Key words: Anoxia, beetroot, solute, membrane potential