Response and Specificity of Antibodies for Candida albicans

Rabbit antibodies for Candida albicans, reacting in agglutination and fluorescent-antibody reactions, were present in both IgM and IgG protein fractions. The two types of immune globulins were separated from ammonium sulfate-precipitated gamma-globulin either by filtration through a column of Sephadex G-200 or by diethylaminoethyl column chromatography performed by stepwise elution with various concentrations of sodium chloride. In the fluorescent-antibody test, initial separation of the IgG fraction, prior to its conjugation with dye, proved to be essential for the high specificity of this reaction. Investigation of the specificities of the two types of antibodies revealed that the IgG was highly specific, whereas the IgM was not very specific. Each antigen fraction, extracted by various methods, demonstrated its own characteristic antibody response. Only the IgG fraction yielded serotype-specific antibody useful for detection of a serotype of C. albicans in agglutination and fluorescent-antibody tests. The results indicate the importance of IgG for specific serological reactions with the Candida species.     

THE FINE STRUCTURE AND IMMUNOLOGICAL LABELING OF THE ACHROMATIC MITOTIC APPARATUS AFTER DISRUPTION OF CELL MEMBRANES

After treatment of HeLa and L cells with vinblastine sulfate the material of microtubules (tubulin) was reorganized into (a) large paracrystals (PC) of tightly packed tubules; (b) smaller aggregates of tubules with greater diameter whose walls are constituted from well defined, helically arranged morphological subunits; and (c) microtubules associated with helices of polyribosomes of uniform size. All of these structures survived disruption of cellular membranes by means of a nonionic detergent. Following a thorough stripping of membranes there remained a subcellular fraction sedimenting at 1,500 g for 15 min, in which were contained nuclei, centrioles, and the above mentioned microtubular elements, maintained as a complex of organelles by an interconnecting network of 80 Å microfibrils. As a result of membrane disruption it was possible to localize precisely in the electron microscope the binding of ferritin antibody conjugates. Specific labeling at the surface of PC and microtubule aggregates could be demonstrated. This result was substantiated by means of the immunoperoxidase method of labeling the PC. A concentrated deposit of ferritin was also found in the vicinity of centrioles and related structures, the annuli of the nuclear pore complex and the annulate lamellae. However, the specificity of the label on these organelles remains questionable because ferritin, albeit in lower concentration, was also present on them in control preparations reacted with preimmune sera.     

Identification of N‐octylnortadalafil and its Stereoisomers in Dietary Supplements with Chiral Liquid Chromatography–Circular Dichroism

A direct chiral liquid chromatography–circular dichroism (LC‐CD) method was developed for the simple and rapid identification of N‐octylnortadalafil [(6R, 12aR)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione; RR‐OTDF] and its stereoisomers in dietary supplements. Samples were extracted with methanol. Compounds were then separated by chiral LC‐CD using Chiralcel OD‐RH (4.6 × 1 50 mm, 5 µm) with 5 mM ammonium formate (pH 3)/0.1% formic acid in acetonitrile (95:5, v/v) mixture solution (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B). The isocratic elution used was mobile phase A / mobile phase B (3:7, v/v) at a flow rate of 1 ml/min. The column temperature was held at 30°C. RR‐OTDF and its stereoisomers were separated within 20 min with the resolution factors being over 2.0. Using this method, RR‐OTDF and (6R, 12aS)‐6‐(1,3‐benzodioxol‐5‐yl)‐2‐octyl‐2,3,6,7,12,12a‐hexahydropyrazino[1’,2’:1,6]pyrido[3,4‐b]indole‐1,4‐dione were detected in a dietary supplement. Chirality 28:204–208, 2016. © 2016 Wiley Periodicals, Inc.     

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4‐like viruses isolated in Japan

Bacteriophages (phages) belonging to the family Podoviridae genus N4‐like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4‐like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4‐like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.     

Serological survey of viral diseases relating to reproductive failure among Artiodactyla in Ethiopian Camelus dromedarius

Serological surveys were performed on Ethiopian camels with a history of abortion to investigate the presence of antibodies against viruses that infect animals classified in the order Artiodactyla. In 2013, 120 serum samples were collected from camels in various parts of Ethiopia. Several viruses related to abortion in ruminants were prevalent. In particular, antibodies against bluetongue virus, were detected at a high rate (76.7% of samples). Additionally, antibodies against Akabane virus and Japanese encephalitis virus were also detected in samples from more than 40% of the camels; however, their antibody titers were relatively low.     

Recording of blood pressure,heart rate and aortic nerve activity during parabolic flight in the rat via radio-telemetry

Exposure to microgravity induces cardiovascular deconditioning characterized by orthostatic hypotension when astronauts return to the earth. In order to understand the mechanism of cardiovascular deconditioning, it is necessary to clarify the changes in hemodynamics and the cardiovascular regulation system over the period of space flight. The telemetry system applied to freely moving animals will be a useful and appropriate technique for this kind of long term study of the cardiovascular system in the conscious animal during space flight. The purpose of the present study is twofold: firstly, to observe the detailed changes of arterial pressure and heart rate (HR) during microgravity elicited by the parabolic flight in order to study the acute effect of microgravity exposure on the cardiovascular system; and secondly, to test the feasibility of the telemetry system for recording blood pressure, HR and autonomic nervous activities continuously during space flight.     

Identification and expression of the kosena radish (Raphanus sativus cv. Kosena) homologue of the ogura radish CMS-associated gene,orf138

A CMS-associated gene, orf125, present in the Japanese radish cultivar Kosena, has a sequence homologous to that of the ogura CMS-associated gene, orf138, except for two amino acid substitutions and a 39 bp deletion in the orf138 coding region. In Kosena radish, orf125 is linked with orfB, whereas the orf125 locus differs in a Brassica napus CMS cybrid derived from protoplast fusion between Kosena radish and B. napus. A novel mtDNA sequence is present in the 3-flanking region of orf125 in the B. napus kosena CMS cybrid. The orf125 is expressed both in the radish and the B. napus kosena CMS cybrid. Its accumulation is strongly associated with the CMS phenotype in B. napus. Fertility restoration was accompanied by a decrease in the amount of ORF125 in B. napus.     

Characterization and Molecular Cloning of a Novel Enzyme, Inorganic Polyphosphate/ATP-Glucomannokinase, of Arthrobacter sp. Strain KM

A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The K_m values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.     

Oxidation of chlorinated olefins by Escherichia coli transformed with dimethyl sulfide monooxygenase genes or cumene dioxygenase genes

In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes. The specific activities of these oxygenases were determined for C(2) to C(4) chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol.min(-1).mg(-1) of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol.min(-1).mg(-1) of dry cells by the cumene dioxygenase. The oxidation products were identified by gas chromatography-mass spectrometry. Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides. In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.     

Extracellular signal-regulated kinase, stress-activated protein kinase/c-Jun N-terminal kinase, and p38mapk are involved in IL-10-mediated selective repression of TNF-alpha-induced activation and maturation of human peripheral blood monocyte-derived dendritic cells

TNF-alpha or IL-10 has been implicated to reversibly regulate physiological states of dendritic cells (DCs). However, little is known about dual stimulations of these cytokines on DC properties and the intracellular signaling events that are responsible for the regulation of these states. Here, we show that a family of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 2 (ERK2), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and p38mapk, are potentially involved in IL-10-mediated selective suppression of TNF-alpha-induced changes of the monocyte-derived DC properties. TNF-alpha induced the cluster formation of the cells and the enhancement of cell surface expression levels of CD83, CD86, and HLA-DR, and T cell stimulatory capacity, whereas the capacities for the endocytosis and the chemotactic migration were suppressed in these cells. Treatment of monocyte-derived DCs with IL-10 resulted in the reduction of the cell surface expression levels of CD86, HLA-DR, and T cell stimulatory capacity, whereas both endocytic and chemotactic migratory capacities were increased by IL-10. Dual stimulations of monocyte-derived DCs with TNF-alpha and IL-10 selectively antagonized their respective effects on these DC properties. TNF-alpha induced tyrosine phosphorylation and enzymatic activation of ERK2, SAPK/JNK, and p38mapk, whereas IL-10 did not induce these events. Dual stimulations of TNF-alpha plus IL-10 abolished TNF-alpha-induced changes of these MAPKs in DCs. These results suggest that the blockage in the MAPKs cascades contributes to IL-10-mediated repression of TNF-alpha-induced changes of DC properties.