Mycotoxin contamination of home-grown maize in rural northern South Africa (Limpopo and Mpumalanga Provinces).

The aim of this study was to assess mycotoxin contamination of crops grown by rural subsistence farmers over two seasons (2011 and 2012) in two districts, Vhembe District Municipality (VDM, Limpopo Province) and Gert Sibande District Municiality (GSDM, Mpumalanga Province), in northern South Africa and to evaluate its impact on farmers' productivity and human and animal health. A total of 114 maize samples were collected from 39 households over the two seasons and were analysed using a validated liquid chromatography-tandem mass spectrometry mycotoxins method. Aflatoxin B1 (AFB1) occurrence ranged from 1 to 133 µg kg(-1) in VDM while AFB1 levels in GSDM were less than 1.0 µg kg(-1) in all maize samples. Fumonisin B1 levels ranged from 12 to 8514 µg kg(-1) (VDM) and 11-18924 µg kg(-1) (GSDM) in 92% and 47% positive samples, respectively, over both seasons. Natural occurrence and contamination with both fumonisins and aflatoxins in stored home-grown maize from VDM was significantly (p < 0.0001) higher than from GSDM over both seasons.

A Dutch field survey on fungal infection and mycotoxin concentrations in maize.

Mycotoxins are secondary metabolites produced by fungi that can cause adverse health effects. Due to climate change, temperatures are expected to rise and changes in rainfall patterns are foreseen. These developments may increase fungal occurrence and mycotoxin concentrations in maize. It is therefore useful to monitor mycotoxin levels in maize and record the accompanying agronomic factors and weather parameters. This paper describes a field survey in the Netherlands in which information on soil, cultivar, green manure, tillage as well as sowing, emergence, flowering and harvest dates of silage maize were collected from 148 growers. A small number of these growers (42 in total) were visited to collect maize samples revealing that 50% of the samples were contaminated with Fusarium species and mycotoxins were detected in 25% of the samples. The Fusarium species that was most commonly found was F. crookwellense followed by F. graminearum, F. culmorum, F. sporotrichiodes and F. equiseti. In total 31 mycotoxins were analysed. The predominant mycotoxins present were (sum of 3 and 15)-acetyl-DON and nivalenol; other mycotoxins found were alternariol, beauvericin, deoxynivalenol, diacetoxyscirpenol, moniliformin and zearalenone. Nivalenol was present in concentrations up to 1670 µg kg⁻¹ and acetylated DON was usually present at higher concentrations than DON. Statistical analysis of the current data showed no correlation between mycotoxins present and agronomic factors recorded. Field studies as described in this paper are useful and need to be continued in the future in order to observe trends in mycotoxin occurrence.

Occurrence of Fusarium Head Blight species and Fusarium mycotoxins in winter wheat in the Netherlands in 2009.

Most recent information on the occurrence of Fusarium Head Blight species and related mycotoxins in wheat grown in the Netherlands dates from 2001. This aim of this study was to investigate the incidence and levels of Fusarium Head Blight species and Fusarium mycotoxins, as well as their possible relationships, in winter wheat cultivated in the Netherlands in 2009. Samples were collected from individual fields of 88 commercial wheat growers. Samples were collected at harvest from 86 fields, and 2 weeks before the expected harvest date from 21 fields. In all, 128 samples, the levels of each of seven Fusarium Head Blight species and of 12 related mycotoxins were quantified. The results showed that F. graminearum was the most frequently observed species at harvest, followed by F. avenaceum and M. nivale. In the pre-harvest samples, only F. graminearum and M. nivale were relevant. The highest incidence and concentrations of mycotoxins were found for deoxynivalenol, followed by zearalenone and beauvericin, both pre-harvest and at harvest. Other toxins frequently found--for the first time in the Netherlands--included T-2 toxin, HT-2 toxin, and moniliformin. The levels of deoxynivalenol were positively related to F. graminearum levels, as well as to zearalenone levels. Other relationships could not be established. The current approach taken in collecting wheat samples and quantifying the presence of Fusarium Head Blight species and related mycotoxins is an efficient method to obtain insight into the occurrence of these species and toxins in wheat grown under natural environmental conditions. It is recommended that this survey be repeated for several years to establish inter-annual variability in both species composition and mycotoxin occurrence.

Determination of fumonisins B1 and B2 in corn by LC/MS with immunoaffinity column cleanup: interlaboratory study.

An interlaboratory validation study was conducted to establish the method performance characteristics of an immunoaffinity column (IAC) cleanup procedure followed by LC/MS for the determination of fumonisins B1 (FB1) and B2 (FB2) and combined FB1 + FB2 in corn. The test portion is extracted with acetonitrile-methanol-water (25 + 25 + 50). The extract is filtered, diluted with phosphate-buffered saline solution, and applied to an IAC. FB1 and FB2 are removed with methanol, followed by water, then directly determined by RPLC with MS detection using selected-ion monitoring of two characteristic ions in each case. Naturally contaminated corn samples were milled to a fine powder and mixed to produce three samples with target levels of combined FB1 + FB2 ranging from 350 to 4000 microg/kg. Of 15 initially participating laboratories, two failed to report results and another did not follow the prescribed method. Thus, valid results were obtained from 12 participants located in 11 countries. Statistical analysis of the results produced RSDr values of 4.6-11.9, 1.9-12.6, and 1.4-11.5% for FB1, FB2, and combined FB1 + FB2, respectively; the corresponding RSDR values were 19.8-23.8, 18.2-25.5, and 18.8-23.2%. The three concentration levels of combined FB1 + FB2 were 534, 1194, and 1954 microg/kg. HorRat values for r and R were all < 2.0, indicating that the method is suitable as a regulatory method for the enforcement of European Union limits for fumonisins in corn.

Seven new mutations in the nicotinamide adenine dinucleotide reduced-cytochrome b(5) reductase gene leading to methemoglobinemia type I.

Cytochrome b(5) reductase (b5R) deficiency manifests itself in 2 distinct ways. In methemoglobinemia type I, the patients only suffer from cyanosis, whereas in type II, the patients suffer in addition from severe mental retardation and neurologic impairment. Biochemical data indicate that this may be due to a difference in mutations, causing enzyme instability in type I and complete enzyme deficiency or enzyme inactivation in type II. We have investigated 7 families with methemoglobulinemia type I and found 7 novel mutations in the b5R gene. Six of these mutations predicted amino acid substitutions at sites not involved in reduced nicotinamide adenine dinucleotide (NADH) or flavin adenine dinucleotide (FAD) binding, as deduced from a 3-dimensional model of human b5R. This model was constructed from comparison with the known 3-dimensional structure of pig b5R. The seventh mutation was a splice site mutation leading to skipping of exon 5 in messenger RNA, present in heterozygous form in a patient together with a missense mutation on the other allele. Eight other amino acid substitutions, previously described to cause methemoglobinemia type I, were also situated in nonessential regions of the enzyme. In contrast, 2 other substitutions, known to cause the type II form of the disease, were found to directly affect the consensus FAD-binding site or indirectly influence NADH binding. Thus, these data support the idea that enzyme inactivation is a cause of the type II disease, whereas enzyme instability may lead to the type I form.

Characterisation of extracellular polysaccharides produced by Crypthecodinium cohnii.

The valuable polyunsaturated fatty acid, docosahexaenoic acid, can be produced by cultivation of the heterotrophic microalga, Crypthecodinium cohnii. During batch growth of C. cohnii on glucose, sea salt and yeast extract for 5 days, so far unreported extracellular polysaccharides were produced. These caused an increased viscosity and a strong drop in the maximum oxygen transfer. The viscosity increased most markedly as cells entered the stationary phase. The polysaccharides varied in size (from 6 kDa to >1,660 kDa) and monomer distribution. A high molecular mass fraction (from 100 kDa to >1,660 kDa) and a medium molecular mass fraction (6-48 kDa) were prepared. The high molecular mass fraction contained (on a molar basis) 71.7% glucose, 13.1% galactose and 3.8% mannose, whereas the medium molecular mass fraction contained 37.7% glucose, 19.8% galactose and 28.1% mannose. Other monomers present in both fractions were fucose, uronic acid and xylose. Monomers were coupled mainly via alpha-(1-3) links. Increased viscosity due to polysaccharide production complicates the development of commercial, high cell-density processes for the production of docosahexaenoic acid.

A missense mutation in the heavy subunit of gamma-glutamylcysteine synthetase gene causes hemolytic anemia.

gamma-Glutamylcysteine synthetase (GCS) catalyzes the initial and rate-limiting step in the biosynthesis of glutathione. gamma-GCS consists of a heavy and a light subunit encoded by separate genes. Hereditary deficiency of GCS has been reported in 6 patients with hemolytic anemia and low erythrocyte levels of glutathione and gamma-glutamylcysteine. In addition, 2 patients also had generalized aminoaciduria and developed neurologic symptoms. We have examined a Dutch kindred with 1 suspected case of GCS deficiency. The proband was a 68-year-old woman with a history of transient jaundice and compensated hemolytic anemia. One of her grandchildren was also GCS deficient; he was 11 years old and had a history of neonatal jaundice. The enzyme defect was confirmed and GCS activity was found to be less than 2% of normal in the erythrocytes of both patients. The complementary DNA (cDNA) for the heavy subunit of GCS was sequenced in these patients and in several members of the family. The proband and her GCS- deficient grandson were identified as homozygous for a 473C-->T substitution, changing codon 158 from CCC for proline into CTC for leucine. Several family members with half-normal GCS activity in their erythrocytes were heterozygous for the mutation.

Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections.

We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability.

Expression of GABAA receptor subunit mRNAs in hippocampal pyramidal and granular neurons in the kindling model of epileptogenesis: an in situ hybridization study.

To investigate the molecular changes underlying kindling epileptogenesis in the rat hippocampus, the expression levels of the genes encoding for 13 different gamma-aminobutyric acid type-A receptor (GABAAR) subunits were measured in hippocampal principal neurons using in situ hybridization techniques and semi-quantitative analysis of the autoradiograms. Schaffer collateral-commissural pathway kindled rats were investigated at three different stages of kindling acquisition, at 24 h after the last seizure and at long-term (28 days) after termination of kindling stimulations. Changes were distinct for the different subunits in the three analyzed regions (CA1, CA3, fascia dentata) and also different for the various kindling stages. In all hippocampal areas at the early phases of kindling epileptogenesis, before the appearance of generalized seizures, an increase was found of those transcripts that constituted the majority of the expressed variants in control animals (alpha 1, alpha 2, alpha 4, beta 1, beta 2, beta 3, gamma 2/gamma 2L mRNA). In these stages, the increased levels of different variants in the granular neurons of the fascia dentata were more pronounced when compared to the pattern of changes in pyramidal cells of CA1 and CA3. In fully kindled animals, the expression levels of several subunits returned to control levels, whereas beta 3 and gamma 2/gamma 2L mRNA expression was still significantly enhanced in all areas. At long-term, few changes were encountered. The long-splice variant of gamma 2 was decreased within pyramidal and granular neurons while the total level of gamma 2 mRNA was not different from controls. The increased GABAAR subunit expression in the fascia dentata may underly the reported increased GABAAR ligand binding and the increased GABA mediated inhibition. However, the decreased GABAAR binding and the attenuation of GABAergic inhibition in CA1, could not be explained by a decrement of receptor subunit expression.

Opposite changes in GABAA receptor function in the CA1-3 area and fascia dentata of kindled rat hippocampus.

Muscimol-stimulated radiotracer 36CI- uptake in synaptoneurosomes was used to investigate the function of the GABAA receptor complex in the CA1-3 area and fascia dentata (granular and molecular layers and hilus) of rats kindled by stimulation, twice a day, of the Schaffer collateral fibers. Two kindled groups were studied: (a) 24 h after the last generalized tonic-clonic seizure [fully kindled (FK) stage] and (b) 28 days after the last generalized seizure (long-term stage). Synaptoneurosomes were prepared in parallel from subslices of the CA1-3 area and fascia dentata. In FK animals, the muscimol-stimulated 36CI- uptake was significantly reduced by 21% in the CA1-3 area in comparison with nonstimulated controls, whereas a significant increase of 29% was found in the fascia dentata. Significant changes were no longer present at 4 weeks after the last generalized seizure. The observed changes in muscimol-stimulated 36CI- uptake at the FK stage closely parallel the recently observed changes in [3H]muscimol binding in the CA1 area and fascia dentata. These results indicate that kindling causes a transiently decreased GABAA receptor-mediated function in the CA1-3, in contrast to an increased GABAA receptor-mediated function in the fascia dentata.

Rat hippocampal kindling induces changes in the glutamate receptor mRNA expression patterns in dentate granule neurons.

The expression level of the mRNAs encoding the Flip and Flop versions of the AMPA-selective glutamate receptor subunits A, B, C and D was studied using in situ hybridization in the hippocampus of rats kindled by Schaffer collateral/commissural fibre stimulation. The expression levels of the Flip variant of GluR-A, B and C mRNAs were bilaterally enhanced in the dentate granule neurons of fully kindled animals 24 h after the last seizure. These changes were already observed after the sixth kindling stimulation (preconvulsive-stage), but not after a single afterdischarge. Four weeks after the last seizure, when the animals were still hypersensitive to kindling stimulations, only GluR-A Flip expression was enhanced. These results suggest that kindling epileptogenesis is accompanied by an increased number and enhanced sensitivity of the expressed AMPA type glutamate receptors in the fascia dentata, leading to an enhanced excitatory synaptic transmission which may contribute to the process of kindling epileptogenesis.

GABAA receptor beta 1-3 subunit gene expression in the hippocampus of kindled rats.

The effect of Schaffer collateral/commissural fiber kindling on the expression levels of GABAA receptor beta 1, beta 2 and beta 3 subunit mRNA in the pyramidal and granular neurons of the rat dorsal hippocampus was studied, using semi-quantitative in situ hybridization. In pyramidal neurons of CA1 and CA3, only small changes (10-15%) were found. In dentate granule neurons, the expression level of GABAA R-beta 3 mRNA was significantly, enhanced, bilaterally, in animals that were partial or fully kindled. At long-term, 4 weeks after the last convulsion no significant changes were found in pyramidal or granular neurons.

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.

Hippocampal kindling increases the expression of glutamate receptor-A Flip and -B Flip mRNA in dentate granule cells.

The level of the mRNAs encoding the AMPA-selective glutamate receptors-A and -B, alternatively splice variants, Flip and Flop, was studied by in situ hybridization in the brains of rats kindled by Schaffer collateral/commissural-fiber stimulation. In comparison to control animals, the expression level of the Flip variant of both GluR-A and GluR-B mRNAs was bilaterally enhanced in the dentate granule neurons of kindled animals 24 h after last-generalized seizure, whereas no obvious alterations were observed in the GluR-A Flop and GluR-B Flop mRNA variants. In kindled animals, studied 1 month after the last seizure, GluR-A Flip and GluR-B Flip mRNA had returned to control levels. We suggest that these changes may result in an enhanced glutamate receptor sensitivity in the fascia dentata during kindling.