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An outbreak of type A foodborne botulism in Taiwan due to commercially preserved peanuts.

Until recently, botulism was not recognized as an important public health problem in Taiwan. In 1986, an outbreak of type A foodborne botulism resulted in nine cases, two of them fatal. The vehicle in this outbreak was commercially preserved peanuts processed by an improperly equipped, unlicensed cannery. A single batch of peanuts was implicated; however, we could not determine why this particular batch was contaminated. Efforts to recall the product were hampered by a lack of distribution records. Mass media announcements were used to warn the public about the outbreak, and preliminary data suggest the ensuing publicity improved botulism surveillance. The local preference for low-acid preserved foods, increasing consumerism, the shortage of adequately trained inspectors are factors which probably contributed to this outbreak. Stricter enforcement of food sanitation policies are needed to meet the changing situation in Taiwan.

Adult↗

Management of spotted wilt vectored by Frankliniella fusca (Thysanoptera: Thripidae) in Virginia market-type peanut.

Field tests were conducted during 2001 and 2002 in northeastern North Carolina to evaluate the impact of cultural practices and in-furrow insecticides on the incidence of Tomato spotted wilt virus (genus Tospovirus, family Bunyaviridae, TSWV), which is transmitted to peanut, Arachis hypogaea L., primarily by tobacco thrips, Frankliniella fusca Hinds (Thysanoptera: Thripidae). Treatments included in row plant populations of 7, 13, and 17 plants per meter; the virginia market-type 'NC V-11' and 'Perry'; planting dates of early and late May; and phorate and aldicarb insecticide applied in-furrow. The incidence of plants expressing visual symptoms of spotted wilt was recorded from mid-June through mid-September. Treatment factors that reduced the incidence of symptoms of plants expressing spotted wilt symptoms included establishing higher plant densities, delaying planting from early May until late May, and applying the in-furrow insecticide phorate. Peanut cultivar did not have a consistent, significant effect on the incidence of symptomatic plants in this experiment.

Agriculture↗

Conversion of peanut trypsin-chymotrypsin inhibitor B-III to a chymotrypsin inhibitor by deimination of the P1 arginine residues in two reactive sites.

The deimination of the arginine residues in peanut trypsin-chymotrypsin inhibitor B-III caused the disappearance of its trypsin-inhibitory activity. Peanut protease inhibitor B-III was incubated with peptidylarginine deiminase, resulting in the conversion of 2.5 mol of arginine to citrulline and in the loss of its trypsin-inhibitory activity. However, the ability of the deiminated inhibitor to inhibit chymotrypsin was as strong as before. Structural analysis of the deiminated B-III indicated that the P1 arginine residues at both reactive sites, Arg(10) and Arg(38), were completely modified to citrulline by the action of peptidylarginine deiminase, and that the Arg(60) in the C-terminal region of B-III was partially deiminated. These residues seem to be exposed on the surface of the molecule. The P1' arginine residue at the first reactive site, Arg(11), was not deiminated at all.

Amino Acid Sequence↗

Amino acid sequences of trypsin-chymotrypsin inhibitors (A-I, A-II, B-I, and B-II) from peanut (Arachis hypogaea): a discussion on the molecular evolution of legume Bowman-Birk type inhibitors.

The amino acid sequences of four peanut protease inhibitors (A-I, A-II, B-I, and B-II) were determined by conventional methods and by comparison of peptide maps of their tryptic digests with that of B-III on HPLC. A-I, A-II, B-I, and B-III had the same amino acid sequence except for differences in their N-terminal regions. This suggests that the four inhibitors would be derived from an original inhibitor with a longer N-terminal amino acid sequence by proteolysis of its N-terminal region. But B-II possessed an extremely different amino acid sequence from those of the other peanut inhibitors and was thought to be biosynthesized from a gene different from that of the other inhibitors. A phylogenetic tree of legume double-headed inhibitors was constructed on the basis of the matrix of amino acid differences among their sequences. The double-headed inhibitors whose sequences have been determined were classified into four groups.

Amino Acid Sequence↗

Peanut hulls as a litter source for broiler breeder replacement pullets.

Broiler breeder pullets were reared on either peanut hulls or pine shavings to determine effects of litter type on growth performance and litter characteristics. Pullets were reared to 20 wk of age in rooms initially bedded with 8 cm of clean shavings or hulls. Heating and ventilation were standardized in all rooms. Restricted skip-a-day feeding was used to attain recommended growth curves. Water was continuously provided for ad libitum consumption. Litter and environmental variables were measured throughout rearing and 2 wk after pullets were removed from the litter materials. Feed consumption, BW, mortality, and uniformity at 20 wk were not affected by litter type; however, gizzard weights were decreased in pullets reared on hulls. Litter bulk density increased with use and was greater for shavings through 11 wk, but not thereafter. Particle size decreased with use in both litter types. Through 11 wk, there were more particles in the > 4 mm range and less in the < 1.7 mm range with hulls. Litter moisture increased with use but was not affected by litter type. Litter pH was greater in unused shavings, but during and after use was generally greater in hulls. With both litter types, litter and environmental ammonia levels increased to 11 wk then decreased; however, this effect was more pronounced for hulls. Bacteria populations were not affected by litter type; however, greater fungal populations were observed in shavings at 7 and 15 wk. Aflatoxins were detected in unused hulls but not shavings. Because aflatoxin levels decreased during use and Aspergillus flavus and Aspergillus parasiticus populations were not detected in samples collected during use, aflatoxins observed were presumed to have been formed prior to use. Peanut hulls performed similarly to pine shavings as a litter source for breeder pullets; however, the specific influence of the aflatoxins contained in this litter source on bird performance deserves further study.

Aflatoxins↗

Comparison of peanut meal and soybean meal as protein supplements for laying hens.

Peanut protein is severely limiting in threonine and has been used to create threonine deficiency in animals. The availability of purified threonine at low cost raises the possibility of economically using peanut meal (PNM) and threonine combinations in poultry diets. An experiment was conducted to compare corn and PNM based diets to corn and soybean meal (SBM) based diets at three protein levels (16, 18.5, and 21%) in diets for 22-to-34-wk-old commercial Leghorns. Birds were housed two per cage with four cages per replicate and six replicates per treatment. Feed consumption, egg production, and feed per dozen eggs were almost identical for PNM (93.8 g/hen per d, 92.2 eggs per 100 hens/d, and 1.22 kg/dozen) and SBM (93.7 g/hen per d, 92.2 eggs per 100 hens/d, and 1.22 kg/dozen). Dietary protein level had no consistent effect on any of these parameters but did significantly improve body weight gains and egg weights (1.2 to 2.5 g/egg). PNM-fed hens laid slightly smaller eggs during the first 6 wk (P<0.05), but there were no egg size differences during the last 6 wk of the experiment (P>0.14). PNM-fed hens laid eggs with better interior quality at 26 and 30 wk of age. After 2 wk of storage, Haugh units remained better for eggs from hens fed PNM than SBM when kept refrigerated (4 degrees C; P<0.05) or at room temperature (20 degrees C; P<0.10). Egg specific gravity was slightly lower for hens fed PNM. It is concluded that PNM is an excellent ingredient for laying hen diets.

Animal Feed↗

Circumstantial evidence for phytoalexin involvement in the resistance of peanuts to Aspergillus flavus.

Three stilbene phytoalexins, elicited by slicing and incubating imbibed peanut kernels under aerobic conditions, inhibited spore germination and hyphal extension of Aspergillus flavus with ED50 values in the range 4.9-12.8 micrograms ml-1. Phytoalexin yield was dependent on cultivar, conditions and duration of incubation after slicing, and crop history. The yield of phytoalexin from ten cultivars studied, after slicing and incubating at 25 degrees C for 24 h, ranged from 28 to 935 micrograms per g fresh weight and was negatively correlated with dry kernel colonization by A. flavus [r = -0.868 when plotted as 1n (phytoalexin concn) against 1n (percentage peanut colonization)]. When the incubation period was extended to 96 h there was no such correlation. Reduced phytoalexin yields were obtained when sliced kernels of one cultivar studied were incubated in water or at 37 degrees C, and no phytoalexin was obtained when the slices were incubated under nitrogen gas or frozen before aerobic incubation. Drought stress during pod development in four cultivars studied reduced phytoalexin yields of sliced kernels incubated at 25 degrees C for 24 h by 17-65% compared with non-stressed controls.

Arachis↗

Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons.

The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [(3)H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min(-1) mg(-1). The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 +/- 1.5 kD. The K(m) values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 microM, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, or p-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants.

Arachis↗

Effects of protein inhibitors and auxin on nucleic Acid metabolism in peanut cotyledons.

The accumulation of labeled phosphorus into newly synthesized nucleic acids or peanut cotyledon slices incubated with chloramphenicol, puromycin, or 2,4-dichlorophenoxyacetic acid (2,4-D) was reduced. Promotion of nucleic acid synthesis was not noted by any of these chemicals. Chloramphenicol completely inhibited the synthesis of the DNA-RNA fraction at 1.25 x 10(-3)m while soluble and ribosomal RNA was inhibited by 70% and 80%, respectively. At the same concentration messenger RNA was inhibited by only 40%. These effects suggest that chloramphenicol inhibit nucleic acid synthesis in peanut cotyledons in a differential manner. Similar results were noted for DNA at low concentrations of 2,4-D. However, at high concentrations of 2,4-D, DNA as well as RNA fractions were inhibited in a similar manner at a given concentration. Puromycin did not differentially inhibit nucleic acid synthesis except at 2 x 10(-3)m where DNA was least inhibited.Nondifferential inhibition suggests that a common site or precursor pool essential for the synthesis of all nucleic acid fractions is altered. Differential inhibition may be due to the interference with a specific rate-limiting step, directly or indirectly, in the formation of a particular nucleic acid.

Journal Article↗

Evidence for de novo synthesis of isocitratase and malate synthesis in germinating peanut cotyledons.

Evidence for de novo synthesis of isocitratase and malate synthetase in cotyledons of germinating peanut (Arachis hypogaea L.) was obtained by the density labeling method. When dry peanut cotyledons were cultured in H(2) (18)O, a 2.4% increase in the buoyant density of malate synthetase in a cesium chloride gradient was observed. In 100% D(2)O the buoyant density shift was 5.5% for isocitratase and 3.5% for malate synthetase in comparison to the water controls. These data suggest that isocitratase and malate synthetase do not pre-exist in some inactive form in the cotyledons, but are completely synthesized after onset of germination from a pool of amino acids which do not derive directly from hydrolysis of storage proteins.

Journal Article↗

Physiology of Oil Seeds: II. Dormancy Release in Virginia-type Peanut Seeds by Plant Growth Regulators.

Germination, ethylene production, and carbon dioxide production by dormant Virginia-type peanuts were determined during treatments with plant growth regulators. Kinetin, benzylaminopurine, and 2-chloroethylphosphonic acid induced extensive germination above the water controls. Benzylaminopurine and 2-chloroethylphosphonic acid increased the germination of the more dormant basal seeds to a larger extent above the controls than the less dormant apical seeds. Coumarin induced a slight stimulation of germination while abscisic acid, 2,4-dichlorophenoxyacetic acid, and succinic acid 2,2-dimethylhydrazide did not stimulate germination above the controls. In addition to stimulating germination, the cytokinins also stimulated ethylene production by the seeds. In the case of benzylaminopurine, where the more dormant basal seeds were stimulated to germinate above the control to a larger extent than the less dormant apical seeds, correspondingly more ethylene production was induced in the basal seeds. However, the opposite was true of kinetin for both germination and ethylene production. When germination was extensively stimulated by the cytokinins, maximal ethylene and carbon dioxide evolution occurred at 24 and 72 hours, respectively. Abscisic acid inhibited ethylene production and germinaton of the seeds while carbon dioxide evolution was comparatively high. The crucial physiological event for germination of dormant peanut seeds was enhancement of ethylene production by the seeds.

Journal Article↗

Identification of Cultivar Differences in Seed Polypeptide Composition of Peanuts (Arachis hypogaea L.) by Two-Dimensional Polyacrylamide Gel Electrophoresis.

Seed polypeptides from several cultivars of peanut (Arachis hypogaea L.) have been compared by means of a two-dimensional polyacrylamide gel electrophoresis. Protein was extracted from the defatted peanut meal by homogenizing in 5 millimolar K(2)CO(3)-9.5 molar urea. After addition of Nonidet P-40 (2%, v/v) and dithiothreitol (0.5%, w/v) the solution was centrifuged at 25,000 g. This procedure led to solubilization of more than 95% of the total protein. The clear supernatant fraction was then subjected to two-dimensional polyacrylamide gel electrophoresis, employing isoelectric focusing in the first dimension and electrophoresis in presence of sodium dodecyl sulfate in the second. After examining several cultivars, it was possible to construct a composite map to include all of the polypeptide species found among all of the cultivars examined. At least 74 major and between 100 and 125 minor components were detectable by Coomassie blue staining. The majority of these had isoelectric points between pH 4.4 and 8.0, and molecular weights between 16,000 and 75,000. Several different cultivars have been compared using this method and it has been shown that considerable variation exists among the major polypeptides present. The method should prove valuable for analyzing different genotypes and selecting varieties with a particular storage protein make-up, as well as for following compositional changes that occur during seed development and germination.

Journal Article↗

Reassociation Kinetics and Cytophotometric Characterization of Peanut (Arachis hypogaea L.) DNA.

The base composition of peanut (var. NC-17) DNA determined from thermal denaturation profiles showed an average guanine plus cystosine content of 34% which was in close approximation to 36% guanine plus cytosine calculated from the buoyant density. Buoyant density also indicated the absence of satellite DNA. The genome size, 2.0 x 10(9) base pairs, as determined by reassociation kinetics of the single copy DNA was close to the genome size determined by cytophotometry, 2.1 x 10(9) base pairs. Peanut DNA averaging 450 to 600 base pairs long, reassociated in phosphate buffer and fractionated by hydroxylapatite, indicated a DNA genome composition of 36% nonrepetitive or single copy DNA; reassociation in formamide and followed by optical methods indicated the repetitive DNA possesses highly repeated, intermediately repeated and rarely repeated components of DNA with DNA sequences repeated on the average about 38,000, 6,700, and 200 times each. Different criteria of reassociation in formamide revealed further subdivisions of these four separate components of DNA. The DNA of above mentioned NC-17 variety compared to Florigiant variety showed no differences in thermal denaturation profiles, buoyant density, or in genome size.

Journal Article↗

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons : II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA.

THERE ARE TWO TYPES OF MITOCHONDRIA PRESENT IN IMBIBED PEANUT COTYLEDONS: a light type (density 1.182 grams per cubic centimeter) and a heavy type (density 1.205 grams per cubic centimeter). The membrane fractions from these two types can be distinguished using sucrose density gradient analysis, and differences in membrane density between the light and heavy types are reflected in differences in their protein N and phospholipid P composition. With increasing time after imbibition, there is a substantial increase in the amount and activity of the light type of mitochondria due to their de novo synthesis. The membrane density of the light mitochondrial fraction declines over 5 days after the start of imbibition as the phospholipid P to protein N ratio increases. The heavy mitochondrial fraction declines during the first 3 days after the start of imbibition, and then it remains at a low, but constant, level thereafter. Even during the decline, however, there is synthesis of proteins comparable to that into light mitochondria. The mitochondrial biogenesis that has been observed in peanut cotyledons is of the light type, the function and physiological importance of the minor heavy type is not known.

Journal Article↗

Glutamine Synthetase of Germinating Peanuts : PROPERTIES OF TWO CHROMATOGRAPHICALLY DISTINCT FORMS AND THEIR ACTIVITY TOWARD 4-METHYLENEGLUTAMIC ACID.

Glutamine synthetase activity, extracted from an acetone powder of 7-day germinated peanuts (Arachis hypogaea L.), was precipitated by ammonium sulfate (40-60% saturation) and further purified by gel filtration and calcium phosphate gel treatment. When it was adsorbed to and subsequently eluted from a column of diethylaminoethyl-cellulose, two peaks of activity (designated glutamine synthetase 1 and 2) were obtained which were enriched 150- and 20-fold, respectively, over the initial extract. Glutamine synthetase 1 was present in ungerminated seeds and in the cotyledons during germination; glutamine synthetase 2 appeared during germination and was found largely in the developing plant. Compared with glutamine synthetase 2, glutamine synthetase 1 appeared to have a slightly smaller molecular weight and was more stable to heat and storage. The catalytic properties of the two forms were essentially the same. Whereas neither form catalyzed gamma-glutamyltransferase activity with 4-methyleneglutamine, both glutamine synthetases 1 and 2 catalyzed an ATP- and NH(4) (+)-dependent conversion of [(14)C]-4-methyleneglutamic acid to [(14)C]-4-methyleneglutamine, but the K(m) value for 4-methyleneglutamic acid was 10-fold greater and the V(max) only one-fourth that measured with l-glutamic acid. This is the first report of glutamine synthetase activity with 4-methyleneglutamic acid as substrate, although the level of this activity does not appear adequate to account for the rapid synthesis of 4-methyleneglutamine observed in germinating peanuts.

Journal Article↗

Photocontrol of Peanut (Arachis hypogaea L.) Embryo and Ovule Development in Vitro.

The purpose of the present study was to determine the nature of the photoreceptor that controls the light-dependent development of the peanut ovule and embryo, and to determine whether this photoreceptor activity is located in the ovular or the embryonic tissue. Peanut ovules were aseptically excised from surface sterilized gynophores (pegs) and cultured in darkness, continuous far red (FR), or given 15 minutes daily irradiations with white (W), red (R), blue (B); FR, R/FR, FR/R light for 21 days. Increases in the volume of ovules cultured in dark, continuous FR, FR, or R/FR were significantly greater than for ovules cultured in W, R, FR/R, or B radiation. Halving the ovule or removing the micropyle did not affect the relative change in ovule volume in response to different light treatments. When the light sources were altered after the first 10 days of culture, the ovules exposed first to darkness or FR radiation began to increase in ovule volume which then ceased after subsequent exposure to W, R, or B radiation. Likewise, if W, R, or B radiation was given during the first 10 days, ovule development was inhibited, and could be stimulated by exposure to darkness or FR light. Growth of embryos, when removed from the influence exerted by ovular tissue, was unaffected by the light. However, when embryos remained attached to the ovular tissue, their growth was stimulated in darkness or by FR light and inhibited by W, R, or B light. The R/FR reversibility indicates that the photoreceptor that regulates ovular growth is phytochrome and that the maternal ovular tissue appears to be the site of photoreception, which may then transmit some developmental signal to the embryo.

Journal Article↗

Nitrogen Nutrition and Xylem Sap Composition of Peanut (Arachis hypogaea L. cv Virginia Bunch).

The principal forms of amino nitrogen transported in xylem were studied in nodulated and non-nodulated peanut (Arachis hypogaea L.). In symbiotic plants, asparagine and the nonprotein amino acid, 4-methyleneglutamine, were identified as the major components of xylem exudate collected from root systems decapitated below the lowest nodule or above the nodulated zone. Sap bleeding from detached nodules carried 80% of its nitrogen as asparagine and less than 1% as 4-methyleneglutamine. Pulse-feeding nodulated roots with (15)N(2) gas showed asparagine to be the principal nitrogen product exported from N(2)-fixing nodules. Maintaining root systems in an N(2)-deficient (argon:oxygen, 80:20, v/v) atmosphere for 3 days greatly depleted asparagine levels in nodules. 4-Methyleneglutamine represented 73% of the total amino nitrogen in the xylem sap of non-nodulated plants grown on nitrogen-free nutrients, but relative levels of this compound decreased and asparagine increased when nitrate was supplied. The presence of 4-methyleneglutamine in xylem exudate did not appear to be associated with either N(2) fixation or nitrate assimilation, and an origin from cotyledon nitrogen was suggested from study of changes in amount of the compound in tissue amino acid pools and in root bleeding xylem sap following germination. Changes in xylem sap composition were studied in nodulated plants receiving a range of levels of (15)N-nitrate, and a (15)N dilution technique was used to determine the proportions of accumulated plant nitrogen derived from N(2) or fed nitrate. The abundance of asparagine in xylem sap and the ratio of asparagine:nitrate fell, while the ratio of nitrate:total amino acid rose as plants derived less of their organic nitrogen from N(2). Assays based on xylem sap composition are suggested as a means of determining the relative extents to which N(2) and nitrate are being used in peanuts.

Journal Article↗

Production and Preliminary Characterization of Monoclonal Antibodies against Cationic Peanut Peroxidase.

Ten monoclonal antibodies (McAbs) have been produced against the cationic peroxidase from peanut suspension cell culture. Eight of these antibodies were found to be of the immunoglobulin (Ig)G(1) subclass and two were of IgA subclass. A combination of competitive enzyme-linked immunosorbent assay, Western blotting analysis, and direct antigen-binding assay revealed that the antibodies are directed against four different epitopes on the cationic peroxidase and the McAbs can be subdivided into four groups. Only group A inhibits peroxidase activity. Group B and D bind equally well to the native and the denatured form of cationic peroxidase, whereas the remaining McAbs react with more or less reduced affinity to the denatured antigen. Group C probably recognizes a conformation-dependent epitope. All the McAbs cross react weakly with the anionic peanut peroxidase, suggesting a structural nonidentity as well as some similarity between these two peroxidase isozymes. Cross reactivities of these McAbs with peroxidases of various plant species were also demonstrated.

Journal Article↗