Alpha-hydroxylation of lignoceroyl-CoA by a cyanide-sensitive oxygenase in rat brain microsomes

The enzymatic mechanism of alpha-hydroxylation of lignoceroyl-CoA, an intermediate in the synthesis of hydroxyceramide, was studied. In the presence of NADPH, sphingosine and microsomes from 20-day-old rat brain, 14C from [1-14C]lignoceroyl-CoA was incorporated into hydroxyceramide. Activity was linear with time (up to 40 min) and with protein (up to 0.8 mg). The apparent Km for lignoceroyl-CoA was about 10 microM. NADPH was a more efficient electron donor than NADH. Oxygen was required for activity, which increased linearly up to 20% O2. In 5 and 10% oxygen, the reaction was inhibited by 0.1 mM cyanide and by electron transfer chain inhibitors, cytochrome c, ferricyanide, menadione, and p-chloromercuriphenyl sulphonate; CO and SKF-525A had no effect. Moreover none of the inhibitors affected the formation of hydroxyceramide. Lignoceroyl-CoA alpha-hydroxylase appears to be an oxygenase requiring NADPH and oxygen, which involves cyanide-sensitive enzyme.     

Selection of an atrazine-resistant tobacco cell line having a mutant psbA gene

Summary A mutant cell line that shows high resistance to the photosynthesis-inhibiting herbicide atrazine was selected from cultured photomixotrophic Nicotiana tabacum cv. Samsun NN cells by repeated exposure to toxic levels of the herbicide. This resistance was confirmed by measurements of Hill reaction activity in isolated thylakoid membranes. Nucleotide sequencing revealed that the resistant cell line had a point mutation in its chloroplast psbA gene. The 264th codon, AGT (serine) was changed to ACT (threonine) in this mutant. This new type of mutation also conferred moderate cross-resistance to diuron and subsequently was stable in the absence of continued selection pressure.     

Surface Charge Density Estimation of Vigna mungo Protoplasts Using a Fluorescent Dye, 9-Aminoacridine

We showed that the surface charge density of protoplasts canbe estimated by the 9-aminoacridine method. The estimated surfacecharge density of the protoplasts isolated from elongating regionsof Vigna mungo root was – 39 ? 8 mC/m². The negative surfacecharge density increased when protoplasts were treated withglutaraldehyde or when EDTA was added to the protoplast suspensionmedium. These results support the validity of our estimationof the surface charge density of protoplasts by the 9-aminoacridinemethod. The concentration of amino groups at the surface ofthe protoplasts was estimated to be 34 mC/m². (Received June 19, 1987; Accepted April 11, 1988)     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(β-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH₄. Reaction of the modified enzyme with [-³²P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with M_r 140000 by covalently linked ApU. Labelling was inhibited by 1μg/ml -amanitin.     

Reverse effect of gram-positive bacteria vs. gram-negative bacteria on adjuvant-induced arthritis in germfree rats

Germfree (GF) F344 rats developed severe adjuvant-induced arthritis with a 100% incidence after a single intradermal injection of heat-killed Mycobacterium bovis (BCG). Specific pathogene-free (SPF) rats developed less severe arthritis with a lower incidence. The rats colonized with Escherichia coli or Bacteroides developed mild disease comparable to that in SPF rats. The rats colonized with Bifidobacterium, Propionibacterium acnes, Lactobacillus casei, L. fermentum, L. murini, and L. acidophilus developed more severe disease than that in GF rats. Furthermore, the rats colonized with a mixture of E. coli and the above lactobacilli developed very mild disease similar to that in SPF rats. These results suggest that gram-negative bacteria, such as E. coli and Bacteroides, may suppress the disease, possibly through their lipopolysaccharides, and may be responsible for the lower susceptibility of SPF rats; gram-positive bacteria, such as Bifidobacterium, P. acnes, and lactobacilli, may enhance the disease, possibly through their peptidoglycans; and E. coli may play a dominant role in modulating the development of adjuvant-induced arthritis.     

Localization of atrial natriuretic peptide, ANP-(99-126) binding sites in the rat thymus and spleen with quantitative autoradiography

Binding sites for atrial natriuretic peptide, ANP-(99-126) were studied in lymphoid organs of the rat with quantitative autoradiography. Tissue sections were incubated in the presence of 0.13 nM 125I-ANP-(99-126) followed by autoradiography using [3H]-Ultrofilm, and the results were analyzed by computerized densitometry and comparison to 125I-standards. Specific ANP binding sites were localized in the medulla and the cortex of the rat thymus and in the white pulp of the rat spleen, with apparent binding sites concentrations of 93, 65, and 126 fmol/mg protein, respectively. The presence of ANP binding sites in areas related to the maturation and function of lymphocytes, and to the production of thymic hormones, suggests the possibility of a role of circulating ANP in the modulation of the immune response.     

Quantitative distribution of angiotensin II binding sites in rat brain by autoradiography

Angiotensin II binding sites were localized and quantified in individual brain nuclei from single rats by incubation of tissue sections with 1 nM 125I-[Sar1]-angiotensin II, [3H]-Ultrofilm autoradiography, computerized microdensitometry and comparison with 125I-standards. High angiotensin II binding was present in the circumventricular organs (organon vasculosum laminae terminalis, organon subfornicalis and area postrema), in selected hypothalamic nuclei (nuclei suprachiasmatis, periventricularis and paraventricularis) and in the nucleus tractus olfactorii lateralis, the nucleus preopticus medianus, the dorsal motor nucleus of the vagus and the nucleus tractus solitarii. High affinity (KA from 0.3 to 1.5 X 10(9) M-1) angiotensin II binding sites were demonstrated in the organon subfornicalis, the nucleus tractus solitarii and the area postrema after incubation of consecutive sections from single rat brains with 125I-[Sar1]-angiotensin II in concentrations from 100 pM to 5 nM. These results demonstrate and characterize brain binding sites for angiotensin II of variable high affinity binding both inside and outside the blood-brain barrier.     

Molecular and enzymatic properties of 1,2-α-d-mannosidase fromAspergillus saitoi

The molecular properties, such as molecular weight, N-and C-terminal amino acids, amino acid composition, and circular dichroism, of 1,2--mannosidase isolated from the culture filtrate ofAspergillus saitoi were determined.The enzyme had aK _m of 0.67 mM andk _cat of 1.27/s with mannobiose at pH 50.0 and 30°C. The anomeric configuration of the reaction products of the enzyme was examined by studying the -anomer. A single Manl2Man linkage in intact Taka-amylase A fromAspergillus oryzae was hydrolyzed, producing free mannose.     

The Eiken Latex test for detection of a cryptococcal antigen in cryptococcosis

A latex agglutination test for cryptococcal antigen, the Eiken Latex test (Eiken, Tokyo, Japan), was compared with a monoclonal antibody-based agglutination assay, Pastorex^® Cryptococcus (Diagnostics Pasteur, Marneur-la-Coquette, France). In a murine model of disseminated cryptococcosis, the kinetics of the antigen titers by the Eiken Latex were similar to those by the Pastorex^® Cryptococcus, but sensitivity was much higher. In HIV-negative patients with pulmonary cryptococcosis, a cryptococcal antigen was detected in 6 of 10 patients by the Eiken Latex test and in only 3 of those patients by the Pastorex^® Cryptococcus. The results indicate that the Eiken Latex is more sensitive for the detection of the cryptococcal antigen, even in non-disseminated cryptococcosis. The sensitivity and specificity of the Eiken Latex were examined using 195 sera from 25 patients with pulmonary cryptococcosis and 170 patients with non-cryptococcosis. The cutoff value of 1:8 showed a sensitivity of 76% (19/25) and a specificity of 98.9% (168/170).