Determination of enzymes from Colletotrichum sp. AHU9748 essential for lepidimoide production from okra polysaccharide

The allelopathic substance lepidimoide (Lp), which exhibits multiple functions in the growth and development of plants, was produced by Colletotrichum sp. AHU9748 from okra polysaccharide. Okra polysaccharide has the repeating structure (1-->4)-O-alpha-(d-galactopyranosyluronic acid)-(1-->2)-O-alpha-l-rhamnopyranose in its hexasaccharide repeating unit of its main chain. To determine the enzymes essential for Lp production, the supernatant of a culture broth was fractionated by repeated column chromatographies to identify two serial fractions responsible for Lp production and non-Lp production by measuring Lp production together with beta-galactosidase (beta-gal), rhamnogalacturonan lyase (RG-lyase) and acetylesterase (AE) activities, which we hypothesized to be necessary for Lp production from the structure of Lp. We confirmed the presence of these three enzymatic activities in the highest-Lp-producing fraction. The addition of purified RG-lyase to fractions producing no or a small amount of Lp demonstrated that beta-gal and RG-lyase activities are necessary for Lp production. The N-terminal amino acid sequences of the three separated proteins on SDS-PAGE confirmed the presence of enzymes identical to beta-gal, RG-lyase and AE in the Lp-producing fractions.     

Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes

In pigs, failure of sperm nuclear decondensation has been reported after injection into oocytes. We examined the effects of pretreating sperm heads with Triton X-100 (TX-100) and dithiothreitol (DTT) and of electrical stimulation of oocytes after sperm head injection on time-dependent morphologic changes in sperm nuclei and in vitro development to the blastocyst stage. In experiment 1, spermatozoa were pretreated with 1% TX-100 and 5 mM DTT (T + D) or not treated, and then injected into in vitro matured oocytes. Electrical stimulation (1.5 kV/cm, 20 mus DC pulse) was applied to the oocytes 1 h after injection (stimulated group) or was not applied (unstimulated group). Some of the oocytes in each group were evaluated at hourly intervals until 10 h after injection for morphologic changes in the sperm nuclei. Unstimulated oocytes injected with untreated spermatozoa showed a delayed peak in the rate of nuclear decondensation (39.4-44.1%, 3-6 h after injection) compared with oocytes injected with T + D-treated spermatozoa (57.0% and 52.6%, 1 and 2 h, respectively). The rate of male pronucleus formation peaked 6 h after stimulation (by 40-60%) after injected oocytes had been stimulated with an electrical pulse, irrespective of whether or not the spermatozoa had been pretreated. In unstimulated oocytes, the rate of male pronucleus formation did not increase and stayed at the basal level (less than 20%) throughout the culture period, regardless of the sperm treatment. Thus, T + D treatment of spermatozoa did not affect completion of fertilization. In experiment 2, we evaluated the effects of electrical stimulation and sperm treatment with T + D on the rate of blastocyst formation and the mean number of cells per blastocyst. Oocytes stimulated after injection with either T + D-treated or untreated spermatozoa showed significantly higher percentages of blastocyst formation (24.8% and 27.1% respectively) than did unstimulated oocytes (1.1% and 4.1% for T + D-treated and untreated respectively; P < 0.01 by Duncan's multiple-range test). The rate of blastocyst formation did not differ between the T + D-treated and untreated groups. The mean number of cells per blastocyst did not differ among any of the groups (14.0-29.4 cells). These results suggest that pretreatment of sperm with TX-100 and DTT shifted the timing of sperm nuclear decondensation forward. However, pronucleus formation and development to the blastocyst stage in vitro were not improved by sperm treatment. Thus, electrical stimulation of injected oocytes enhances in vitro development to the blastocyst stage in pigs.     

Sanger Gap Sequencing for Genetic Alphabet Expansion of DNA

Genetic alphabet expansion technology, creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four-letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing) for DNAs containing our UBP, Ds-Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds-Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds-Px pairs in various sequence contexts. This method is useful for UBP applications such as high-affinity DNA aptamer generation and semisynthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, thus suggesting further development of UBPs.     

On‐Line Estimation and Control of Apparent Extract Concentration in Low‐Malt Beer Fermentation

Apparent extract concentration, which is defined as the apparent total dry matter concentration in the media calculated by measurement of the specific gravity, is a very important variable in the process of beer and low‐malt beer fermentation. In the present study, ethanol formation and the residual apparent extract concentration were estimated using the CO₂ concentration in the exhaust gas. A simple mathematical model involving the CO₂ production rate, temperature and cell activity was constructed and a novel system for on‐line estimation and control of apparent extract concentration was developed. In the control system for the residual apparent extract concentration, cell activity was estimated by monitoring the differences in actual CO₂ production rate. In addition, the future trajectory of the apparent extract concentration was predicted. The degree of fermentation, i.e., the residual apparent extract concentration trajectory, was controlled by raising or lowering the temperature, taking into account the estimated cell activity at that time. The residual apparent extract concentration reached the target value at a given time, using the on‐line estimation of apparent extract concentration and temperature control.     

Determination of isothiocyanates and related compounds in mustard extract and horseradish extract used as natural food additives

Amounts of isothiocyanates and related compounds in a mustard extract and a horseradish extract for food additive use were determined by GC, after confirmation of the identity of GC peaks by GC/MS. Amounts of allyl isothiocyanate, which included that of allyl thiocyanate, because most of the allyl thiocyanate detected in the sample was assumed to have been formed from allyl isothiocyanate during GC analysis, were 97.6% and 85.4%, in the mustard extract and the horseradish extract, respectively. Total amounts of the identified isothiocyanates in the mustard extract and the horseradish extract were 98.5% and 95.4%, respectively. Allyl cyanide, a degradation product of allyl isothiocyanate, was found in the mustard extract and the horseradish extract at the levels of 0.57% and 1.73%, respectively. beta-Phenylethyl cyanide, a possible degradation product of beta-phenylethyl isothiocyanate, and allyl sulfides were found in the horseradish extract, at the levels of 0.13% and 0.46%, respectively. Allylamine, which is another degradation product of allyl isothiocyanate, was determined after acetylation, and was found in the mustard extract and the horseradish extract at the levels of 8 micrograms/g and 67 micrograms/g, respectively.     

Calculation of metabolic flow of xylose in Lactococcus lactis

A circuit diagram is proposed on the basis of an analysis of metabolic pathways of lactic acid bacteria, namely, a phosphoketolase pathway and a pentose phosphate/glycolic pathway. An augmented matrix was derived from carbon balances and stoichiometries from the circuit diagram, and solved by Gaussian elimination. Results indicated that 70% of the amount of xylose was metabolized through the pentose phosphate/glycolic pathway and that the yield of lactic acid from xylose ranged from 0.34 to 0.52 (mol/mol), whereas that of pyruvic acid was more than 1.4 (mol/mol).     

Eleven Novel Polymorphic Microsatellite Loci for Oval Squid Sepioteuthis Lessoniana (Shiro-Ika Type)

The oval squid Sepioteuthis lessoniana is one of the most economically important squid species in Japan; however, its population structure is poorly understood due to the lack of hypervariable markers. Such information is critical for managing sustainable fisheries, as well as for ensuring the existence of wild S. lessoniana stocks. Eleven candidate microsatellite loci were isolated from a small insert genomic DNA library. Polymorphisms in these 11 loci were screened in 24 wild individuals. The number of alleles per locus was found to range from 5 to 19 alleles, and the observed heterozygosity ranged from 0.292 to 0.958. No evidence for linkage disequilibrium was detected among all the loci. The genotypic proportions conformed to Hardy-Weinberg equilibrium, except at one locus. In conclusion, these polymorphic microsatellite loci may be used to develop a genetic framework to manage S. lessoniana in the future.