NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Biochemical characteristics and fatty acid compositions of some armadillo-derived mycobacteria and their relation to Mycobacterium gordonae

The long-chain components of 75 strains of mycobacteria, cultivated from Mycobacterium leprae-infected or non-infected armadillos, and of eight clinical and 15 environmental isolates of M. gordonae, were compared. Four major groups could be distinguished based on the presence of 10-methyloctadecanoic (tuberculostearic) and 2-methyl 3-hydroxyeicosanoic acids and secondary alcohols (2-octadecanol and 2-eicosanol). Some heterogeneity was found in strains assigned to M. gordonae: the characteristic absence of tuberculostearic acid and secondary alcohols and the presence of the branched C14 and the hydroxylated C20 acids were seen in only 34 of the 49 strains studied. Three strains were identified as M. malmoense, one as M. kansasii, ten as belonging to the M. avium-M. intracellulare-M. scrofulaceum complex and eight as belonging to new groups of armadillo-derived mycobacteria (ADM 1, ADM 2 and ADM 3) by conventional bacteriological tests and fatty acid compositions, though M. malmoense was heterogeneous in its fatty acids composition. Four strains, identified as M. avium by conventional tests, differed from this species by their fatty acid compositions. Thirteen strains showed some similarity to M. simiae and ten strains differed from all other known mycobacteria.     

Effect of auranofin treatment on aberrant splenic interleukin production in adjuvant arthritic rats

Adjuvant-induced arthritis (AA) in rats is associated with a number of immunologic abnormalities which include a marked decrease in spleen cell mitogenic responses. In this study we investigated the altered production of interleukins in arthritic rats and evaluated the effects of auranofin treatment on disease progression and aberrant interleukin production. The capacity of the AA rat spleen cells to produce interleukin (IL) 2 and IL-3 was found to decrease during the development of the arthritic lesion, with maximum suppression occurring 16 to 17 days after adjuvant injection. In contrast, the production of IL-1 by splenic adherent cells from arthritic rats was markedly increased. Prophylactic treatment of AA rats with auranofin resulted in a slight reduction in paw edema, a complete normalization of the depressed IL-2 production, and a reduction of the elevated IL-1 production, but had no effect on the depressed IL-3 production. In contrast, auranofin administered to normal rats, in the same dosing regimen, did not affect interleukin production. Therapeutic administration of auranofin to AA rats with established disease resulted in normalization of IL-1 production without affecting the suppressed IL-2 and IL-3 levels. In contrast, while indomethacin treatment effectively decreased paw edema, it did not appreciably affect the systemic aberrant interleukin production. Taken together, these results suggest that disease-associated abnormalities in interleukin production may be mediated by different mechanisms with differential sensitivity to the effects of the disease-modifying drug auranofin. Furthermore, defining the relationship between drug-mediated normalization of aberrant immune parameters and clinical improvement will provide a basis for the elucidation of the mechanism of action of disease-modifying antiarthritic drugs as well as for assessment of clinical efficacy of drug treatment.     

Synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives of ribavirin

The synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives (13 and 15, respectively) of the antiviral agent ribavirin are described. Direct glycosylation of 2',3'-O-isopropylideneribavirin with either tetra-O-acetyl-alpha-D-glucopyranosyl bromide (4) or tetra-O-acetyl-alpha-D-galactopyranosyl bromide (8) under Koenigs-Knorr conditions (i.e., silver carbonate, silver perchlorate, and Drierite in dichloromethane) followed by O-deacetylation of the reaction product gave the corresponding ortho esters. However, treatment of 2',3'-di-O-acetyl-5'-O-tritylribavirin (11) with 4 under the Bredereck modification of the Koenigs-Knorr reaction (i.e., silver perchlorate and Drierite in nitromethane) and subsequent deacetylation furnished the desired 1-(5-O-beta-D-glucopyranosyl-beta-D-ribofuranosyl)-1,2,4-triazole-3-carb oxamide (13). Similarly, reaction of 11 with 8 in the presence of AgClO4, and deprotection of the condensation product, gave 5'-O-beta-D-galactopyranosylribavirin (15). The beta-anomeric configuration of the D-glucosyl and D-galactosyl groups of 13 and 15 was assigned by 1H-n.m.r. studies.     

Simultaneous demonstration of two antigens in ultrathin cryosections by a novel application of an immunogold staining method using primary antibodies from the same species

A recently developed immunocytochemical double-staining method for ultrathin Epon and Lowicryl K4M sections has been adopted for use on ultrathin cryosections. The essential features of the method include: staining for the first antigen by the indirect method using sufficient concentrations of second antibodies conjugated to colloidal gold particles to saturate available epitopes on the primary antibodies; supporting the cryosections by methyl cellulose followed by paraformaldehyde vapour treatment (30-60 min at 80 degrees C); removal of the methyl cellulose followed by staining for the second antigen using primary antiserum from the same species and another size class of colloidal gold particles conjugated to second antibodies. Contaminating staining does not occur if the paraformaldehyde vapour treatment exceeds 30 min, as this treatment destroys the combining sites on the second antibodies applied in the first staining cycle. Successful double-staining was documented using primary rabbit antibodies to growth hormone and corticotropin and anti-rabbit IgG conjugated to 5 and 15 nm colloidal gold particles. Following double-staining, the ultrathin cryosections may be silver-enhanced to improve detectability of the markers at low magnification.     

Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress

Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.     

Base pair opening dynamics of a 2-aminopurine substituted Eco RI restriction sequence and its unsubstituted counterpart in oligonucleotides.

Studies of 1H NMR selective saturation recovery were performed to determine the imino proton exchange with solvent water of the base pairs in the Eco RI endonuclease recognition sequence GAATTC, placed at the center of self-complementary decamer and dodecamer oligonucleotides. In one oligonucleotide the innermost adenine was replaced by the fluorescent base analogue 2-aminopurine (2AP). From the measurements at different concentrations of TRIS buffer acting as proton exchange catalyst, base pair lifetimes were evaluated. The results at 25 degrees show that the AT base pairs have lifetimes of the order of a few ms, whereas the surrounding GC base pairs in a dodecamer have lifetimes of about 100 ms. The (2AP)T base pair has a shorter lifetime than the corresponding AT base pair. The temperature dependent optical absorption, and for the 2AP containing oligonucleotide fluorescence, were used to study the single strand-duplex equilibrium of the decamers. The results indicate that NMR and the optical techniques, although applied at very different concentrations, monitor the same conformational transition of the oligonucleotide.     

Species relationship in the Pennisetum gene pool: enzyme polymorphism

Summary Variation in leaf esterases (EST), 6-phosphogluconate dehydrogenase (PGD), shikimate dehydrogenase (SKDH), leucine aminopeptidase (AMP), phosphoglucomutase (PGM) and malate dehydrogenase (MDH) is reported in the Pennisetum gene pool. In the primary gene pool, polymorphism for EST, AMP, SKDH was very high, as compared to the near-monomorphic isozymes of PGD. Two loci controlling leaf esterases Est-1 and Est-2, were identified in the primary gene pool. Differences in allelic frequency distribution of the polymorphic Est-1 locus occur between the cultivated and wild pearl millet. The prevalent alleles of Est-1 are absent in P. purpureum Schumach (secondary gene pool). A monomorphic band of the -esterase-specific Est-2 locus was identified in most of the secondary gene pool accessions, P. squamulatum Fresen and an accession of P. pedicellatum. SKDH and EST revealed differences between most of the tertiary gene pool species. By contrast, a PGD zymogram was prevalent in several species of different sectional taxa. Gene duplication for PGD isozymes occurs in the diploid species, P. ramosum, of the tertiary gene pool. Heterodimers of PGD and EST were observed in the hybrid between pearl millet and P. squamulatum, whereas a monomeric structure characterized SKDH and AMP.     

Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.