Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.     

Influence of aflatoxin B1 on seed germination, seedling growth, chlorophyll and carotenoid contents of mustard (brassica juncea L. var. pusa bold) seeds

Five different concentrations (100, 250, 500, 1000 and 2000 μg/L of aflatoxin B₁ were found to be inhibitory to seed germination and seedling growth (root and shoot lengths) of mustard seeds (variety Pusa bold). These also lowered the levels of chlorophyll and carotenoids in the emerging leaves during seedling growth. The inhibitory effect was correlated with the concentration of applied toxin.     

Effect of dietary supplements on lean mass and strength gains with resistance exercise: a meta-analysis.

The purpose of this study was to quantify which dietary supplements augment lean mass and strength gains during resistance training. Peer-reviewed studies between the years 1967 and 2001 were included in the analysis if they met a predetermined set of experimental criteria, among which were at least 3-wk duration and resistance-training 2 or more times a week. Lean mass and strength were normalized for meta-analysis by conversion to percent change per week and by calculating the effect size for each variable. Of the 250 supplements examined, only 6 had more than 2 studies that met the criteria for inclusion in the meta-analysis. Creatine and beta-hydroxy-beta-methylbutyrate (HMB) were found to significantly increase net lean mass gains of 0.36 and 0.28%/wk and strength gains of 1.09 and 1.40%/wk (P < 0.05), respectively. Chromium, dehydroepiandrosterone, androstenedione, and protein did not significantly affect lean gain or strength. In conclusion, two supplements, creatine and HMB, have data supporting their use to augment lean mass and strength gains with resistance training.     

Phenotypic and molecular characterization of inducible human neuroblastoma cell lines

Two new neuroblastoma (NB) cell lines, NUB-6 and NUB-7, were established from recurrent and primary NB tumours respectively and identified conclusively as NB by their phenotypic characteristics, catecholamine production and N-myc amplification. The cell lines could be distinguished on the bases of distinctive growth patterns in monolayer culture and semi-solid media (collagen gel and agarose), neurite formation and their response to four classes of growth and differentiation modulators. The NUB-6 cell line consisted of two distinct cell subtypes, small typical neuroblasts and larger spheroid-forming cells, while NUB-7 was homogeneously neuroblastic. Class-I agents (dibutyrl cyclic AMP [dbcAMP], butyrate, and papaverine) inhibited growth of both cell lines, while only dbcAMP stimulated the formation of short neurites by NUB-6 neuroblast cells in monolayer culture and collagen. Of the class-II agents (vitamins), retinoic acid inhibited growth of both cell lines and stimulated formation of long neurites by NUB-6 cells and NUB-7 cells in later passages. In contrast, vitamin E inhibited growth of NUB-6 and late-passage NUB-7, but stimulated early passage NUB-7. The class III agent (nerve growth factor) resembled vitamin E. The class-IV agents (interferons; rIFN-alpha 2a and rIFN-gamma 1) inhibited growth of both cell lines in monolayer culture and agarose, but stimulated NUB-6 neuroblasts and early passage NUB-7 cells to form long neurites. Thus phenotypically distinct NB cell lines were established in vitro and shown to be differentially influenced by various growth and differentiation modulators. The potent effect of IFN suggests a role for these modulators in NB behaviour in vivo.     

Validity of the multiphasic concept of ion absorption in plants

Borstlap (Naturwissenschaften 68: 41–43, 1981; Plant Cell Environ. 4: 189–195, 1981) has alleged that the concept of multiphasic uptake of solutes in plants is invalid. This claim is considered in detail and shown not to be justified. Contrary to Borstlap, the concentration-dependence of uptake cannot be adequately represented by the sum of two Michaelis-Menten terms and a linear term, or by similar continuous functions, if sufficiently detailed and precise data are used. Published evidence for the multiphasic concept, including the existence of discontinuities and separate uptake and transition sites, remains valid.     

Multiphasic uptake of potassium by corn roots: no linear component

Concentration-dependence data (LV Kochian, WJ Lucas Plant Physiol 1982 70: 1723-1731; [1983] 73: 208-215; [1985] 429-436; LV Kochian, J Xin-zhi, WJ Lucas [1985], 79: 771-776) for potassium uptake by corn roots have been claimed to be resolvable into a saturable and a linear component representing two separate uptake mechanisms. A comprehensive analysis shows, however, that the data are not compatible with this concept. They can, in contrast, be precisely represented by multiphasic isotherms, in agreement with many findings for uptake of potassium and other solutes. Solute uptake in plants is proposed to be mediated by multistate entities having carrier-like properties at low external solute concentrations and channel-like properties at high concentrations.     

Relationship between Indole-3-Acetic Acid Levels in Apple (Malus pumila Mill) Rootstocks Cultured in Vitro and Adventitious Root Formation in the Presence of Indole-3-Butyric Acid

In vitro rooting response and indole-3-acetic acid (IAA) levels were examined in two genetically related dwarfing apple (Malus pumila Mill) rootstocks. M.26 and M.9 were cultured in vitro using Linsmaier-Skoog medium supplemented with benzyladenine (BA), indole-3-butyric acid (IBA), and 1,3,5-trihydroxybenzoic acid (PG). Rooting response was tested in Lepoivre medium supplemented with IBA and PG. IBA concentrations of 12.0 and 4.0 micromolar induced the maximum rooting percentages for M.9 and M.26, respectively. At these concentrations rooting response was 100% for M.26 and 80% for M.9. Free and conjugated IAA levels were determined in M.26 and M.9 shoots prior to root inducing treatment by high performance liquid chromatography with fluorescence detection and validated by gas chromatography-mass spectrometry using ¹³[C₆]IAA as internal standard. Basal sections of M.26 shoots contained 2.8 times more free IAA than similar tissue in M.9 (477.1 ± 6.5 versus 166.6 ± 6.7 nanograms per gram fresh weight), while free IAA levels in apical sections of M.26 and M.9 shoots were comparable (298.0 ± 4.4 versus 263.7 ± 9.3 nanograms per gram fresh weight). Conjugated IAA levels were significantly higher in M.9 than in M.26 indicating that a greater proportion of total IAA was present as a conjugate in M.9. These data suggest that differences between M.26 and M.9 rooting responses may be related to differences in free IAA levels in the shoot base.     

Uptake of Benzyladenine by Tuber Slices of Jerusalem Artichoke (Helianthus tuberosus L.) over a Wide Concentration Range

Uptake of the cytokinin benzyladenine by tuber slices of Jerusalem artichoke (Helianthus tuberosus L.) increased with the concentration of benzyladenine in the medium in the range 10⁻⁸ to 10⁻⁴ molar. The kinetics of uptake could be precisely represented by four phases of a single, multiphasic isotherm. K_m and V_max increased in a regular manner upon transition to higher phases. The fit to multiphasic kinetics was significantly better than the fit to cooperative kinetics or to the sum of four independent Michaells-Menten terms.