Creatinine measurements in 24 h urine by liquid chromatography--tandem Mass Spectrometry

A simple, sensitive, and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determining urinary creatinine was developed and used to evaluate 24 h urine samples collected during an exposure study. Urine (1 microL) was diluted with methanol and then directly applied to LC-MS/MS. Under electrospray ionization (ESI) conditions, the transition molecules of creatinine and creatinine- d3 were observed at m/ z 114 > 44 and m/ z 117 > 47, respectively. The retention time of creatinine was 0.59 min. The linear range was 1-2000 ng/mL, with a detection limit in urine of 1 ng/mL. LC-MS/MS and colorimetric end-point methods were significantly associated ( R2 = 0.8785, p < 0.0001). The LC-MS/MS method to determine creatinine in 24 h urine samples had shorter retention times, was more sensitive, reliable, reproducible, simple, selective, and used a smaller sample size than other LC-MS/MS or commercial methods.     

Molecular and Gelatinization Properties of Rice Starches from IR24 and Sinandomeng Cultivars

The differences in pasting properties involving gelatinization and retrogradation of rice starches from IR24 and Sinandomeng cultivars during heating‐cooling processes were investigated using a Rapid Visco Analyser (RVA)and a dynamic rheometer. The results were discussed in relation to the molecular structure, actual amylose content (AC), and concentration of the starches. Generally, both starches possessed a comparable AC (≈11 wt%), amylose average chain length (CL), iodine absorption properties, and dynamic rheological parameters on heating to 95°C at 10 wt% and on cooling to 10°C at higher concentrations. In contrast to Sinandomeng, IR24 amylose had a greater proportion of high molecular weight species and number‐average degree of polymerization (DP_n). IR24 amylopectin possessed a lower DP_n and greater CL, exterior CL (ECL), and interior CL (ICL). Comparing the results of RVA analysis and dynamic rheology, the gelatinization properties and higher retrogradation tendencies of IR24 starch can be related to the structural properties and depend on starch concentration. In addition, the exponent n of starch concentration for storage moduli at 25°C (G′₂₅ ∝ Cⁿ) increased linearly with increasing AC.     

Isolation and antifungal and antioomycete activities of staurosporine from Streptomyces roseoflavus strain LS-A24

The actinomycete strain LS-A24 active against some plant fungal and oomycete pathogens was isolated from a soil sample of the Sunghwan Lake in Korea. The cell wall composition and spore shape of strain LS-A24 were LL-diaminopimelic acid and spiral type, respectively. On the basis of the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, strain LS-A24 was identical to Streptomyces roseoflavus. An antifungal and antioomycete antibiotic was isolated from LS-A24 using various chromatographic procedures. The molecular formular of the antibiotic was determined to be C(28)H(26)N(4)O(3), and on the basis of the NMR data, the antibiotic was confirmed to be staurosporine, 2,3,10,11,12,13-hexahydro-10R-methoxy-9S-methyl-11R-methylamino-9S,13R-epoxy-1H,9H-diindolo[1,2,3-gh:3',2',1'-lm]pyrrolo[3,4-j][1,7]benzodiazonin-1-one. Staurosporine completely inhibited the mycelial growth of Colletotrichum orbiculare, Phytophthora capsici, Rhizoctonia solani, Botrytis cinerea, and Cladosporium cucumerinum with minimum inhibitory concentration (MIC) values of 1-50 microg/mL for MICs. Staurosporine also was active against Saccharomyces cerevisiae, Bacillus subtilis ssp. subtilis, and Xanthomonas vesicatoria. Staurosporine and the commercial fungicide metalaxyl inhibited the development of Phytophthora blight on pepper plants. However, the control efficacy of staurosporine against the Phytophthora disease was somewhat less than that of metalaxyl. This is the first study to isolate staurosporine from S. roseoflavus and demonstrate its in vitro and in vivo antioomycete activity against P. capsici.     

Thioredoxin h System and Wheat Seed Quality

Thioredoxin is one of the key systems controlling cellular redox balance in all living organisms. Plant thioredoxins are a diverse multigene family divided into two systems, the chloroplastic and the cytoplasmic systems, which are distinguishable by the electron donor and by the enzyme that catalyses thioredoxin reduction. In cereal seed, the thioredoxin (Trx) h system acts in the developing phase, controlling the delivery of compounds during seed filling. Early in the development of the imbibed seed, it promotes the mobilization of storage nitrogen and carbon in the endosperm by inhibiting the inactivators of amylolytic enzyme and by activating a specific serine protease, thiocalsin. During seed maturation and germination, the Trx h system controls oxidative stress in the living tissues, specifically in the scutellum and the aleurone layer, where it accumulates in the nucleus. The overexpression and the suppression of Trx confirm these features and constitute a powerful tool to manage seed quality, due to the large number of Trx h isoforms and to the specific nature of some of them.     

Quality Evaluation of Sediments from 24 Tributaries of the Po River, Italy

Sediment samples from 24 tributaries of the Po River (Italy) were screened for selected trace elements (Cd, Cu, Hg, Pb, and Zn) and extractable organic compounds; a proxy for contamination by organic microcontaminants. The toxicity of sediment extracts was evaluated using a battery of biotests (Dugesia gonocephala, Paracentrotus lividus, and Tamnocephalus platyurus). Contamination by trace elements (including very high Hg pollution – 4 to 16ppm total Hg – in one sub-basin) reached potentially harmful levels only in the sediments of four tributaries; while contamination by organic microcontaminants was present in most sub-basins. Sediments from most study sites did actually show signs of anthropogenic stress and were able to elicit a toxic response. A more detailed evaluation of sediment quality in the Po River tributaries seems to be urgently needed for developing the necessary remediation strategies. Research priorities should include more thorough testing of sediment toxicity, determination of metal background levels in the various sub-basins and a more detailed identification of the organic micropollutants of possible concern.     

Determination of pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated peanut allergen Ara h 1

This study was aimed at the determination of the pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated Ara h 1, a major allergen from peanuts. Both the oligomeric structure and the trimeric structure of the allergen were investigated. Under the in vitro conditions applied, oligomeric Ara h 1, either unheated or preheated, was hydrolyzed by pepsin at a lower rate than trimeric Ara h 1. Peptides with relatively high molecular masses were shown to be able to bind IgE, whereas peptides with lower molecular masses (<2 kDa) did not. In these latter fractions, fragments of 15 previously published epitopes of mature Ara h 1 were identified. As a result, these epitopes are not likely responsible for the induction of systemic food allergic reactions to peanuts. Using sequential chymotrypsin digestion, the pepsin-resistant IgE-binding peptides were deduced to contain the previously identified intact epitopes EDWRRPSHQQ (amino acids 50-59) and PRKIRPEG (amino acids 60-67). The presence of four additional earlier published intact epitopes (covering amino acids 6-13, 14-21, 24-31, and 40-47) on the pepsin-resistant peptides could be neither deduced nor ruled out. The two deduced and four possible pepsin-resistant epitopes are all situated in the N-terminal part of Ara h 1, which does not show homology with other vicilin proteins. Consequently, this unique N-terminal part of Ara h 1 is proposed to be responsible for the allergen's ability to induce systemic allergic reactions.     

cDNA clone of a putative peanut (Arachis hypogaea L.) trypsin inhibitor has homology with peanut allergens Ara h 3 and Ara h 4

Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.     

Effect of ammonium and phosphorus supply on h+ production in gel by two tropical forage grasses

The effect of the supply of ammonium (NH₄ ⁺) and phosphorus (P) in gel on the amounts of hydrogen ion (H⁺) excreted from plant roots was studied with Brachiaria humidicola (a highly acid‐soil tolerant tropical grass) and B. brizantha (less acid‐soil tolerant) grown in soil in a glasshouse. The H⁺ production was measured over 24 h in agar gel containing full nutrient solution with a range of NH/‐N levels (0, 0.25, 0.5, and 5.0 mM NH₄ ⁺‐N). Highly soluble P, K₂HPO₄, or relatively insoluble P, rock P, was supplied at four concentrations (0, 11.5, 34.5, or 115 μM p) in the gel. Increasing NH₄ ⁺ concentration in the gel increased H⁺ production for both grasses, but there was some inhibition of growth for B. brizantha at the highest N concentration. For B. humidicola, but not B. brizantha H⁺ production was greater with 34.5 μM K₂HPO₄ than 11.5 μM K₂HPO₄. At 34.5 μM P for both grasses there was no difference in H⁺ production when P was supplied as rock P or K₂HPO₄. With 11.5 μM P both grasses produced less acid in the gel with the rock P compared with K₂HPO₄. The reduced H⁺ production is probably due to a lower availability of P in the rock P compared with K₂HPO₄. This effect was greater with B. brizantha than B. humidicola, implying that 11.5 μM rock P was not able to supply sufficient P for the growth of B. brizantha. Brachiaria humidicola was able to dissolve more rock P than B. brizantha or alternatively, the growth of B. humidicola was less adversely affected by the low P supply from rock P than B. brizantha. Plant‐induced acidity does not seem to occur as a response to a lack of available P, but rather these grasses only produce acid if there are enough nutrients for growth, i.e., both NH₄ ⁺ and P. If either N or P is limiting, growth is limited as is NH₄ ⁺ uptake, so that H⁺ production is curtailed.     

Ovine metabolism of lithogenic sapogenins. Synthesis of [2,2,4,4-(2)h(4)]sarsasapogenone, [2,2,4,4-(2)h(4)]sarsasapogenin,and [2,2,4,4-(2)h(4)]episarsasapogenin and evaluation of deuterium retention in a sheep-dosing trial

The suitability of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), [2,2,4,4-(2)H(4)]sarsasapogenin (2b), and [2,2,4,4-(2)H(4)]episarsasapogenin (3b) as isotopically labeled dosing substrates to determine the levels of free and conjugated sapogenins present in feces from sheep grazing saponin-containing plants implicated in the development of ovine heptagenous photosentization diseases was investigated. A 1:4 mixture of [2,2,4,4-(2)H(4)]sarsasapogenin (2b) and [2,2,4,4-(2)H(4)]episarsasapogenin (3b), obtained by reduction of [2,2,4,4-(2)H(4)]sarsasapogenone (1b), was found to retain 94% of incorporated deuterium, when dosed to one sheep. The recovery of the dosed mixture of genins 2b and 3b was calculated to be 85%. Considerable loss of deuterium and a lower recovery of genin material were observed when [2,2,4,4-(2)H(4)]sarsasapogenone (1b) was dosed.     

Flavor authenticity studies by (2)h/(1)h ratio determination using on-line gas chromatography pyrolysis isotope ratio mass spectrometry

Based on (2)H/(1)H ratio measurements of commercial synthetic and "natural" references, the recently developed on-line gas chromatography pyrolysis isotope ratio mass spectrometry (HRGC-P-IRMS) technique was used to determine the delta(2)H(SMOW) values of the flavor compounds decanal, linalool, and linalyl acetate, as well as those of E-2-hexenal and E-2-hexenol in foods and essential oils. In preceding model studies, the influence of sample preparation steps (simultaneous distillation extraction, SDE; solvent extraction, SE; liquid liquid extraction, LLE) on the delta(2)H values was found to be negligible. For decanal, the typical (2)H abundance, with higher content of (2)H for synthetic material (delta(2)H(SMOW) from -90 to -156 per thousand) and lower (2)H content for natural references (delta(2)H(SMOW) from -138 to -262 per thousand) was observed. Although the delta(2)H data recorded for linalool did not allow one to distinguish between synthetic (delta(2)H(SMOW) from -207 to -301 per thousand) and natural (delta(2)H(SMOW) from -234 to -333 per thousand) materials, the situation was somewhat more encouraging for linalyl acetate; delta(2)H(SMOW) values from -199 to -239 per thousand and from -213 to -333 per thousand were found for synthetic and natural samples, respectively. E-2-Hexenal and E-2-hexenol showed clear-cut origin-dependent differences in their (2)H/(1)H ratios; that is, delta(2)H(SMOW) values from -14 to -109 per thousand and from -263 to -415 per thousand as well as from -41 to -131 per thousand and from -238 to -348 per thousand were recorded for products from synthetic and natural origins, respectively.     

Identification of a new natural Ara h 6 isoform and of its proteolytic product as major allergens in peanut

Numerous food allergens of plant origin belong to the 2S albumin family, including peanut Ara h 2. In addition to Ara h 2, several other conglutins related to 2S albumins are present in peanut seeds. We evaluated the allergenicity of different peanut conglutins as compared with Ara h 2. Several conglutins were isolated from the kernel, i.e. Ara h 2, a new isoform of Ara h 6 and its derived product, which is likely to be naturally formed during seed processing. Enzyme allergosorbent tests performed on sera of peanut allergic patients showed that more than 94% of 47 analyzed patients had positive IgE responses to Ara h 6 isoform and to its degradation product. Skin prick tests with the new isoform of Ara h 6 led to a positive response in seven out of the eight tested patients. Both enzyme allergosorbent tests and skin prick tests showed that the reactivity of Ara h 6 was similar to, or even higher than, that of Ara h 2, suggesting that the present isoform of Ara h 6 is as allergenic as Ara h 2. In addition the IgE response to the plant processed (i.e., hydrolyzed) Ara h 6 new isoform is equivalent to the IgE response to the native isoform. The IgE immunoreactivity is mostly abrogated by chemical reduction and denaturation of Ara h 6 isoforms, which underlined the importance of tertiary structure in Ara h 6 immunoreactivity. These results, and particularly the high correlation between anti-Ara h 2 and anti-Ara h 6 IgE responses, emphasise the major role of 2S albumins in peanut allergenicity.     

Art- und Gattungsgrenzen bei höheren Discomyceten,IV

Zusammenfassung Vergleichende Discomyceten-Studien der letzten Jahre haben gezeigt, daß strittige Arten oft nur durch Heranziehung lebender Exemplare geklärt werden können, da Exsikkate einen Teil ihrer makroskopischen Merkmale verloren haben und dadurch leicht Fehlschlüsse entstehen. Einige Beispiele der GattungHelvella L. ex. Fr. ss. str. werden kritisch erörtert,H. pithyophila Boud. undH. sulcata Afz. ex. Fr. als wohlbegründete Arten bestätigt undH. platycephala Bx. nov. spec. erstmalig beschrieben.Ein Nachtrag zur Gliederung der Discinaceen (cf. Kulturpflanze 17) unterscheidet in der GattungParadiscina Bx. zwei neue Sektionen (Leucoxanthae Bx. nov. sect. —Melaleucae Bx. nov. sect.) und vervollständigt sie durch 3 Umkombinationen. Für die europäischen Discinaceen ist ein Artenschlüssel hinzugefügt.

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Specific and generic limits within higherDiscomycetes, IV

Summary For the last years comparative studies on higherDiscomycetes pointed out, that uncertain species may only be proved by employment of fresh specimens, because the dried ones mostly lost some of their macroscopic criteria and easily produce errors. Some examples of the genusHelvella L. ex Fr. ss. str. are critically discussed, wherebyH. pithyophila Boud. andH. sulcata Afz. ex Fr. are confirmed as well-founded species, andH. platycephala Bx. nov. spec. is described for the first time.A supplement to theDiscinaceae (cf. Kulturpflanze17) divides the genusParadiscina Bx. into the new sectionsLeucoxanthae Bx. nov. sect. andMelaleucae Bx. nov. sect. and completes this genus by 3 new combinations. A key to European species ofDiscinaceae is added.

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, IV

, . , . Helvella L. ex Fr. s. str.;H. pithyophila Boud. H. sulcata Afz. ex Fr. ; H. platycephala Bx. nov. spec. Discinaceae (. . 17) Paradiscina Bx. (Leucoxanthae Bx. nov. sect. Melaleucae Bx. nov. sect.) . .

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Teil I in Kulturpflanze10, S. 359–371; Teil II in Kulturpflanze14, S. 359 bis 379; Teil III in Kulturpflanze17, S. 253–284.