Stability of fumonisin B<Subscript>1</Subscript>, deoxynivalenol,zearalenone, and T-2 toxin during processing of traditional Nigerian beer and spices

The stability of the Fusarium mycotoxins fumonisin B₁, deoxynivalenol, T-2 toxin, and zearalenone during processing of Nigerian traditional spices (dawadawa, okpehe, and ogiri) and beer (burukutu) using artificially contaminated raw materials was investigated. Results revealed the reduction of these toxins in all the final products. Boiling played a significant role (p?<?0.05) in Fusarium mycotoxin reduction in the traditional spices. The highest percentage reduction of deoxynivalenol (76%) and zearalenone (74%) was observed during okpehe processing (boiled for 12 h). Dehulling and fermentation further demonstrated a positive influence on the reduction of these toxins with a total reduction ranging from 85 to 98% for dawadawa, 86 to 100% for okpehe, and 57 to 81% for ogiri. This trend was also observed during the production of traditional beer (burukutu), with malting and brewing playing a major impact in observed reduction. In addition, other metabolites including deoxynivalenol-3-glucoside, 15-acetyl-deoxynivalenol, α-zearalenol, and β-zearalenol which were initially not present in the raw sorghum were detected in the final beer product at the following concentrations 26?±?11, 16?±?7.7, 22?±?18, and 31?±?16 μg/kg, respectively. HT-2 toxin was also detected at a concentration of 36?±?13 μg/kg along the processing chain (milled malted fraction) of the traditional beer. For the traditional spices, HT-2 toxin was detected (12 μg/kg) in ogiri. Although there was a reduction of mycotoxins during processing, appreciable concentrations of these toxins were still detected in the final products. Thus, the use of good quality raw materials significantly reduces mycotoxin contamination in final products.     

Extreme conservation of non-repetitive non-coding regions near <Emphasis Type="Italic">HoxD</Emphasis>complex of vertebrates

Homeotic gene complexes determine the anterior-posterior body axis in animals. The expression pattern and function of hox genes along this axis is colinear with the order in which they are organized in the complex. This 'chromosomal organization and functional correspondence' is conserved in all bilaterians investigated. Although the molecular basis of this 'colinearity' in not yet understood, it is possible that there are control elements within or in the proximity of these complexes that establish and maintain the expression patterns of hox genes in a coordinated fashion. We report here an unprecedented conservation of non-coding DNA sequences adjacent to the HoxD complex of vertebrates. Stretches of hundreds of base pairs in a 7 kb region, upstream of HoxD complex, show 100% conservation from fish to human. Using primers designed from these sequences of human HoxD complex, we amplified the corresponding regions from different vertebrates, including mammals, aves, reptiles, amphibians and pisces. Such a high degree of conservation, where no variation was allowed during ~500 million years of evolution, suggests critical function for these sequences in the regulation of the HoxD complex. Furthermore, these sequences provide a molecular handle to gain insight into the mechanism of regulation of this complex.     

Effects of paraoxon and ethylparathion on choline oxidase from Alcaligenes species: inhibition and denaturation

The kinetics and thermodynamics of the effects of paraoxon (POX) and ethylparathion (EPA) on choline oxidase (ChOx) were studied. Lineweaver–Burk plots of initial velocity data showed a parallel pattern indicating uncompetitive inhibition versus choline. The inhibition constant (K_I) obtained from the secondary plots for POX and EPA were 0.14 ± 0.01 and 0.48 ± 0.05 mM, respectively, suggesting that POX is a more potent inhibitor of ChOx than EPA. UV absorption was used to monitor the denaturation of ChOx by POX and EPA. A decrease in FAD fluorescence associated with the interaction of POX and EPA with ChOx suggested a tertiary structural change. Interaction of the enzyme molecule with POX or EPA resulted in inhibition and subsequently denaturation of the enzyme. The results indicate that inhibition and denaturation of the enzyme by POX and EPA are linked, but not parallel events, with inhibition occurring at lower concentrations with respect to denaturation. This suggests that the loss of initial velocity of the enzyme is an active site specific effect and not due to global conformational changes induced by the inhibitors.     

Thermal dissociation and conformational lock of superoxide dismutase

The kinetics of thermal dissociation of superoxide dismutase (SOD) was studied in 0.05 M Tris-HCl buffer at pH 7.4 containing 10(-4) M EDTA. The number of conformational locks and contact areas and amino acid residues of dimers of SOD were obtained by kinetic analysis and biochemical calculation. The cleavage bonds between dimers of SOD during thermal dissociation and type of interactions between specific amino acid residues were also simulated. Two identical contact areas between two subunits were identified. Cleavage of these contact areas resulted in dissociation of the subunits, with destruction of the active centers, and thus, lost of activity. It is suggested that the contact areas interact with active centers by conformational changes involving secondary structural elements.     

Cation Modulation of Hemoglobin Interaction with Sodium <Emphasis Type="Italic">n</Emphasis>-Dodecyl Sulfate (SDS). II: Calcium Modulation at pH 5.0

We investigate the conformational differences between HbA and HbS in the presence and absence of Ca(2+) concentrations (0-40 μM) akin to those within the erythrocyte cytoplasm and the membrane mimetic and native structure disrupting environments of the Plasmodium parasite food vacuole at pH 5.0. The experiments were monitored by UV-Vis spectrophotometery in the range of 250-650 nm. Our results suggest that the HbS, on interacting with both the membrane mimic and 40 μM Ca(2+), undergoes an "expansion" akin to the burst phase of proteins accompanied by tyrosine exposure while that of the HbA occurred with tryptophan exposure. Our results suggest conformational flexibility in the HbS unlike in the HbA. Besides, the spectral results also suggest that the HbS complexes with the Ca(2+) in its immediate environment without strain (due to its inherent conformational flexibility), unlike the HbA, thus appropriating the cation from its vicinity. The implications of these results are discussed in the light of possible mechanisms employed by the HbS to resist protease digestion or at least slow down the kinetics of the protease activities and on how these same factors can predispose the homozygous HbS individuals to sickling and consequent vaso-occlusive crisis.     

Fungal and bacterial metabolites of stored maize (Zea mays,L.) from five agro-ecological zones of Nigeria

Seventy composite samples of maize grains stored in five agro-ecological zones (AEZs) of Nigeria where maize is predominantly produced were evaluated for the presence of microbial metabolites with the LC-MS/MS technique. The possible relationships between the storage structures and levels of mycotoxin contamination were also evaluated. Sixty-two fungal and four bacterial metabolites were extracted from the grains, 54 of which have not been documented for maize in Nigeria. Aflatoxin B₁ and fumonisin B₁ were quantified in 67.1 and 92.9 % of the grains, while 64.1 and 57.1 % exceeded the European Union Commission maximum acceptable limit (MAL) for aflatoxin B₁ and fumonisins, respectively. The concentration of deoxynivalenol was, however, below the MAL with occurrence levels of 100 and 10 % for its masked metabolite, deoxynivalenol glucoside. The bacterial metabolites had low concentrations and were not a source of concern. The storage structures significantly correlated positively or negatively (p?<?0.01 and p?<?0.05), respectively with the levels of grain contamination. Consumption of maize grains, a staple Nigerian diet, may therefore expose the population to mycotoxin contamination. There is need for an immediate action plan for mycotoxin mitigation in Nigeria, especially in the Derived Savannah zone, in view of the economic and public health importance of the toxins.     

Kinetic analysis of urea-inactivation of beta-galactosidase in the presence of galactose

The effect of galactose on the inactivation of purified beta-galactosidase from the black bean, Kestingiella geocarpa, in 5 M urea at 50 degrees C and at pH 4.5, was determined. Lineweaver-Burk plots of initial velocity data in the presence and absence of urea and galactose were used to determine the relevant K(m) and V(max) values, with p-nitrophenyl beta-D-galactopyranoside (PNPG) as substrate, S. The inactivation data were analysed using the Tsou equation and plots. Plots of ln([P](infinity) - [P](t) ) against time in the presence of urea yielded the inactivation rate constant, A. Plots of A vs [S] at different galactose concentrations were zero order showing that A was independent of [S]. Plots of [P](infinity) vs [S] were used to determine the mode of inhibition of the enzyme by galactose, and slopes and intercepts of the 1/[P](infinity) vs. 1/[S] yielded k(+0) and k '(+0), the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k(+0) and k '(+0) vs. galactose concentrations showed that galactose protected the free enzyme and not the enzyme-substrate complex against urea inactivation via a noncompetitive mechanism at low galactose concentrations and a competitive pattern of inhibition at high galactose concentrations. The implication of the different modes of inhibition in protecting the free enzyme was discussed.     

Cation Modulation of Hemoglobin Interaction with Sodium <Emphasis Type="Italic">n</Emphasis>-Dodecyl Sulfate (SDS). I: Calcium Modulation at pH 7.20

A comparative denaturation of HbA and HbS in the R states using sodium n-dodecyl sulfate (SDS) was carried out at pH 7.20 in the presence and absence of Calcium (0–40 μM) and monitored by UV–Vis spectrophotometry in the range of 250–650 nm. In the HbS spectra, the calcium alone caused little or no perturbation of the aromatic region but caused a decrease in oxygen affinity when compared to the HbA. The combinations of [SDS] and [Ca] perturbed the HbS the most, relative to the individual spectra of the [SDS] and [Ca]. However, the presence of Ca appeared to diminish the adverse effects of the SDS on HbA. The denaturation pathway of the HbA involved mainly the formation of heme dimers and some ferryl heme species. For the HbS, heme monomers and a large amount of ferryl species were formed. It is suggested that the greater monomer species formed by the HbS denaturation pathway would result in both Fenton and enhanced enzymatic reactions, compared to the dimer. This could lead ultimately to the formation of ferryl radicals. Thus, at physiological pH for the HbS, the Ca–SDS interaction increases the tendency for protein denaturation in comparison to the HbA.     

Proteome‐wide systems analysis of a cellulosic biofuel‐producing microbe

Fermentation of plant biomass by microbes like Clostridium phytofermentans recycles carbon globally and can make biofuels from inedible feedstocks. We analyzed C. phytofermentans fermenting cellulosic substrates by integrating quantitative mass spectrometry of more than 2500 proteins with measurements of growth, enzyme activities, fermentation products, and electron microscopy. Absolute protein concentrations were estimated using Absolute Protein EXpression (APEX); relative changes between treatments were quantified with chemical stable isotope labeling by reductive dimethylation (ReDi). We identified the different combinations of carbohydratases used to degrade cellulose and hemicellulose, many of which were secreted based on quantification of supernatant proteins, as well as the repertoires of glycolytic enzymes and alcohol dehydrogenases (ADHs) enabling ethanol production at near maximal yields. Growth on cellulose also resulted in diverse changes such as increased expression of tryptophan synthesis proteins and repression of proteins for fatty acid metabolism and cell motility. This study gives a systems‐level understanding of how this microbe ferments biomass and provides a rational, empirical basis to identify engineering targets for industrial cellulosic fermentation.     

Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367

Microbial cellulose degradation is a central part of the global carbon cycle and has great potential for the development of inexpensive, carbon‐neutral biofuels from non‐food crops. Clostridium phytofermentans has a repertoire of 108 putative glycoside hydrolases to break down cellulose and hemicellulose into sugars, which this organism then ferments primarily to ethanol. An understanding of cellulose degradation at the molecular level requires learning the different roles of these hydrolases. In this study, we show that interspecific conjugation with Escherichia coli can be used to transfer a plasmid into C. phytofermentans that has a resistance marker, an origin of replication that can be selectively lost, and a designed group II intron for efficient, targeted chromosomal insertions without selection. We applied these methods to disrupt the cphy3367 gene, which encodes the sole family 9 glycoside hydrolase (GH9) in the C. phytofermentans genome. The GH9‐deficient strain grew normally on some carbon sources such as glucose, but had lost the ability to degrade cellulose. Although C. phytofermentans upregulates the expression of numerous enzymes to break down cellulose, this process thus relies upon a single, key hydrolase, Cphy3367.