Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.     

Biosynthesis of estradiol-17β in the ovarian follicles of the red seabream Pagrus major during vitellogenesis

ABSTRACT: The red seabream Pagrus major is a useful experimental fish for studying the endocrine control of oogenesis in teleosts. This study investigated the steroidogenic pathway for estradiol-17β (E2) biosynthesis in the ovarian follicles of red seabream. Intact follicles were isolated during vitellogenesis and incubated in vitro with different radiolabeled steroid precursors. When 17-hydroxyprogesterone (17-P), dehydroepiandrosterone (DHEA), or androstenedione (AD) were used as precursors, both testosterone (T) and estrone (E1) were synthesized by follicles, leading to estradiol-17β (E2) production. Serum steroid levels measured by enzyme-linked immunosorbent assay showed that T, E1, and E2 were present in the circulation at levels ranging from 1 ng/mL to 2 ng/mL throughout the day during the spawning season. In vitro conversion of E1 into E2, however, was 15.8-fold greater than T conversion into E2, suggesting that E2 is synthesized mainly via E1 rather than T. The results showed that E2 was synthesized from pregnenolone via 17-hydroxypregnenolone, DHEA, AD, and E1. Thus, the study demonstrated the complete steroidogenic E2 synthesis pathway in the ovarian follicles of red seabream, and revealed that E1 is the major precursor of E2.     

Signature-tagged mutagenesis of Vibrio vulnificus

Vibrio vulnificus is the causative agent of primary septicemia, wound infection and gastroenteritis in immunocompromised people. In this study, signature-tagged mutagenesis (STM) was applied to identify the virulence genes of V. vulnificus. Using STM, 6,480 mutants in total were constructed and divided into 81 sets (INPUT pools); each mutant in a set was assigned a different tag. Each INPUT pool was intraperitoneally injected into iron-overloaded mice, and in vivo surviving mutants were collected from blood samples from the heart (OUTPUT pools). From the genomic DNA of mixed INPUT or OUTPUT pools, digoxigenin-labeled DNA probes against the tagged region were prepared and used for dot hybridization. Thirty tentatively attenuated mutants, which were hybridized clearly with INPUT probes but barely with OUTPUT probes, were negatively selected. Lethal doses of 11 of the 30 mutants were reduced to more than 1/100; of these, the lethal doses of 2 were reduced to as low as 1/100,000. Transposon-inserted genes in the 11 attenuated mutants were those for IMP dehydrogenase, UDP-N-acetylglucosamine-2-epimerase, aspartokinase, phosphoribosylformylglycinamidine cyclo-ligase, malate Na (+) symporter and hypothetical protein. When mice were immunized with an attenuated mutant strain into which IMP dehydrogenase had been inserted with a transposon, they were protected against V. vulnificus infection. In this study, we demonstrated that the STM method can be used to search for the virulence genes of V. vulnificus.     

Effects of grass hay proportion in a corn silage‐based diet on rumen digesta kinetics and digestibility in dairy cows

In this study, we aimed to evaluate the effects of six levels of orchardgrass hay (GH) proportion (0%, 10%, 20%, 30%, 40% or 50% of dry matter) in finely chopped corn silage (CS)‐based diets on digesta kinetics of CS and GH in the rumen. Six non‐lactating, rumen‐cannulated Holstein cows were used in a 6 × 6 Latin square design. Ruminal digesta kinetics was measured by ruminal dosing of feed particle markers (dysprosium for CS, erbium for GH) followed by fecal sampling. The increase of GH proportion had a quadratic effect (P < 0.01) on total tract digestibility of neutral detergent fiber (NDF) and acid detergent fiber. The proportion of GH did not affect the particle size distribution of rumen digesta, total weight of dry matter or NDF in the rumen. The rates of large particle size reduction in the rumen for CS tended to increase linearly with increasing GH proportion (P = 0.077). A quadratic effect (P < 0.05) was found with increasing the GH proportion for the ruminal passage rate of small GH particles, but not for CS particles. The results suggested that associative effects between CS and GH could be generated on rumen digesta kinetics when cows were fed a CS‐based diet with an increased proportion of GH.     

松萝酸的防腐作用研究

以西藏色季拉山长松萝为材料,乙酸乙酯为溶剂回流提取制备松萝酸,并采用滤纸片法对提取物松萝酸进行了抑菌活性和最小抑制浓度测定,同时以苯甲酸为对照,研究了松萝酸的防腐作用。结果表明,松萝酸对细菌、霉菌和酵母菌的生长均有明显的抑制作用;将用松萝酸处理的鲜猪肉、豆腐和牛奶放入37℃的恒温培养箱中1周后,其菌数均不超过5×108／g(mL),而对照不低于15×10-8／g(mL)。细菌菌落数的明显减少表明,松萝酸具有一定的防腐效果;但与相同浓度的苯甲酸相比,松萝酸的抑菌防腐效果稍差。     

Effects of CaCN2 treatment of seeds of Inubie (Echinochloa oryzicola) on synthesis of protein and RNA during germination

Protein and RNA induction during the germination of Inubie (Echinochloa oryzicola) seeds pretreated with CaCN₂ was studied. It was observed that the germination of Echinochloa oryzicola seeds was delayed after pretreatment with a high concentration of CaCN₂ for 18 h. A considerable difference in the protein patterns was detected in the SDS-PAGE gel between the control (H₂O) and CaCN₂ treatment, particularly a 48 kilodalton (kD) protein band disappeared in CaCN₂ treatment. At the tested three levels of CaCN₂, the induction of this 48 kD protein was apparently inhibited during the germination process even when the duration of the pretreatment was as short as 2 h. When imbibed with water, the amount of the 48 kD protein increased rapidly within 1 h from the trace level in the dry seeds, and reached the maximum level after about 1–2 d. The results showed that the CaCN₂ treatment also decreased the total RNA level in the germinating seeds. The relation of this protein induction and RNA decline with the beginning of Echinochloa oryzicola germination was discussed.     

Factors regulating acetylene reduction assay for measuring heterotrophic nitrogen fixation in water-logged soils

In the C₂H₂-C₂H₄ assay for measurement of heterotrophic N₂ fixation in water-logged soils, the diffusion of C₂H₂ into the soil and the recovery of C₂H₄ from it are critical factors regulating the assay result. To establish an C₂H₂-C₂H₄ assay technique suitable for waterlogged soils, the C₂H₂-reducing activities (ARA), assayed by varying the method of assay gas filling, the pC₂H₂ of the assay gas, the duration of assay incubation and of soil vibration before the gas sampling, were compared.

A maximum ARA was measured when the following set of procedures were applied to the soil sample in assay flasks: 1) a 4-fold repetition of I-min evacuation under 0.01 atmospheric pressure and the subsequent I-min filling under 1 atmospheric pressure with assay gas at pC₂H₂ of 0.1 atm, 2) an assay incubation for 3 hr, and 3) a sampling of an aliquot of the headspace gas after strongly vibrating the flask for 1 min.

The ARA measured by this technique was several times larger than those measured by the techniques hitherto applied, and corresponded to an almost 80% of the V _max of the sample. This technique was, therefore, proposed for the assay of heterotrophic N₂ fixation in waterlogged soils.

A striking depression of ARA in the soil sample prepared with agitation indicated that a microbial ecosystem established in the soil should be kept as undisturbed as possible throughout the C₂H₂-C₂H₄ assay.     

Determination of periods of sensitivity to low-salinity and low-temperature conditions during the early development of cultured Japanese eel Anguilla japonica larvae with respect to the rate of morphological deformity at completion of yolk resorption

The present study analyzed the rate of occurrence of deformities at completion of yolk resorption after Japanese eel larvae were exposed to low-salinity (29 psu) or low-water-temperature (20 °C) conditions (after they had been reared under suitable conditions: 34 psu, 25 °C) at various points between 0 and 8 days post-fertilization (dpf) in order to determine the period of sensitivity of the larvae to low-salinity or low-temperature conditions. No significant differences were observed between the rate of deformed larvae obtained in the groups reared under suitable conditions and the corresponding rates in groups that were exposed to low-salinity or low-temperature conditions at 4 dpf or later, but the frequency of normal larvae showed a downward trend in experimental groups that were subjected to low-salinity or low-temperature conditions at 3 dpf or earlier. Occurrence rates of pericardial edema were significantly higher in experimental groups that were exposed to low-salinity or low-temperature conditions at 2 dpf or earlier. Therefore, it was concluded that the rearing conditions for Japanese eel larvae should be maintained at 34 psu salinity and 25 °C until at least 4 dpf in order to reduce the occurrence of larval deformity at completion of yolk resorption.     

Total Synthesis and Absolute Configuration of the Marine Norditerpenoid Xestenone

Xestenone is a marine norditerpenoid found in the northeastern Pacific sponge Xestospongia vanilla. The relative configuration of C-3 and C-7 in xestenone was determined by NOESY spectral analysis. However the relative configuration of C-12 and the absolute configuration of this compound were not determined. The authors have now achieved the total synthesis of xestenone using their developed one-pot synthesis of cyclopentane derivatives employing allyl phenyl sulfone and an epoxy iodide as a key step. The relative and absolute configurations of xestenone were thus successfully determined by this synthesis.     

Exploring an East Asian melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) collection for parthenocarpic ability

In melon (Cucumis melo L.), the amount of fertile pollen decreases at low temperature during winter and early spring and thereby restricts fertilization. A promising way to overcome this problem is to develop parthenocarpic cultivars. Parthenocarpic cucumber and squash, but not melon cultivars are available. In this study, we explored 172 accessions from an East Asian melon collection and identified accessions that exhibited strong parthenocarpic ability throughout the year. Crosses between parthenocarpic accessions and a non-parthenocarpic cultivar, and among parthenocarpic accessions indicated that parthenocarpy may be inherited in a recessive manner and is likely controlled by the same gene or genes in these accessions. The parthenocarpic indices, such as the size and number of parthenocarpic fruits, differed among the cultivation periods, indicating the importance of environmental factors for parthenocarpic fruit development. We conclude that it is possible to breed new cultivars with stable parthenocarpic ability throughout the year by using the identified accessions.