New plant growth promoters,repraesentins A,B and C,from Lactarius repraesentaneus

Three new plant growth regulatory sesquiterpenoids were isolated from the Lactarius repraesentaneus fungus. Their structures were elucidated to be a protoilludene sesquiterpene, namely repraesentin A (1), and two related sesquiterpenes, namely repraesentins B (2) and C (3). Compounds 1-3 showed promotion activities toward the radicle elongation of lettuce seedlings by 136%, 118% and 184% at 67 ppm, respectively.     

Fractionation of sugarcane bagasse by hydrothermal treatment

Hydrothermal treatment of sugarcane bagasse was conducted using a semi-batch reactor to develop a new biomass fractionation method that has low impact in the environment. A continuously increasing temperature was used in this treatment. It was found that hemicellulose and lignin could be mainly extracted as a water-soluble fraction at 200-230 degrees C, while the cellulose fraction was hydrolyzed at higher temperatures (230-280 degrees C) or recovered as solid residue from this treatment. Detailed analyses of the solid residue indicated that the crystal structure and the chemical composition of the residue were in good accordance with those of untreated crystalline cellulose. These experimental and analytical findings show that this method is promising for removal of hemicellulose and lignin from woody biomass without any catalyst and organic solvent.     

Experimental prostate carcinogenesis - rodent models

A number of rodent models of prostate carcinoma development have been established to study mechanisms and modifying potential. All except for transgenic mouse models need long experimental periods for generation of a high yield of cancers. Spontaneous prostate tumor models, while not practical in terms of time and tumor incidences, allow the natural course of multistep neoplasia to be followed without a need for chemical exposure. Carcinogens, especially in combination with testosterone, can induce prostate carcinomas in rats, but none are prostate-specific, so that tumor development in other organs is a complicating factor. Induction of invasive prostate carcinomas in the rat frequently requires long-term administration of a pharmacological dose of testosterone with or without application of a chemical carcinogen. While there are several transgenic mouse models, each also has strong and weak points, and it is therefore necessary to select the best model for the purpose of any experimental study.     

Human Dickkopf-1 (huDKK1) protein: characterization of glycosylation and determination of disulfide linkages in the two cysteine-rich domains

Human Dickkopf‐1 (huDKK1), an inhibitor of the canonical Wnt‐signaling pathway that has been implicated in bone metabolism and other diseases, was expressed in engineered Chinese hamster ovary cells and purified. HuDKK1 is biologically active in a TCF/lef‐luciferase reporter gene assay and is able to bind LRP6 coreceptor. In SDS‐PAGE, huDKK1 exhibits molecular weights of 27–28 K and 30 K at ～ 1:9 ratio. By MALDI‐MS analysis, the observed molecular weights of 27.4K and 29.5K indicate that the low molecular weight form may contain O‐linked glycans while the high molecular weight form contains both N‐ and O‐linked glycans. LC‐MS/MS peptide mapping indicates that ～ 92% of huDKK1 is glycosylated at Asn²²⁵ with three N‐linked glycans composed of two biantennary forms with 1 and 2 sialic acid (23% and 60%, respectively), and one triantennary structure with 2 sialic acids (9%). HuDKK1 contains two O‐linked glycans, GalNAc (sialic acid)‐Gal‐sialic acid (65%) and GalNAc‐Gal[sialic acid] (30%), attached at Ser 30 as confirmed by β‐elimination and targeted LC‐MS/MS. The 10 intramolecular disulfide bonds at the N‐ and C‐terminal cysteine‐rich domains were elucidated by analyses including multiple proteolytic digestions, isolation and characterization of disulfide‐containing peptides, and secondary digestion and characterization of selected disulfide‐containing peptides. The five disulfide bonds within the huDKK1 N‐terminal domain are unique to the DKK family proteins; there are no exact matches in disulfide positioning when compared to other known disulfide clusters. The five disulfide bonds assigned in the C‐terminal domain show the expected homology with those found in colipase and other reported disulfide clusters.     

Zymographic demonstration of lactate and malate dehydrogenases isoenzymes in the rodent salivary glands

Summary Analysis of lactate and malate dehydrogenase zymograms of rodent salivary glands showed species and organ specific patterns.Lactate dehydrogenase isoenzyme patterns occupied the middle positions in relation to those of skeletal and heart muscle. Activities of the major salivary glands were in the order submaxillary gland>parotid>sublingual gland. Zymogram of the mouse and rat showed LDH₄ and LDH₅ high activity patterns, while that of the rabbit was the fast moving active one. Hamster salivary gland exhibited a neutral type of the former and the latter.Malate dehydrogenase isoenzyme exhibited very similar patterns for the mouse, rat and hamster. Malate dehydrogenase zymogram of rabbit showed 3 active bands, which was different from the other rodents.     

Seasonal changes in vertical distribution, biomass and faecal production of tubificids in the profundal region of a shallow Japanese lake

Two tubificid species Limnodrilus hoffmeisteri and L. claparedeianus formed more than 93% of the total number of oligochaetes in the profundal. Limnodrilus spp. worms were found down to 33 cm in the sediment but in great numbers in the upper zone in June and October. Worms confined to the top 15 cm of sediment accounted for 53-92% of the total number. There were two annual maxima in population density and biomass, one in late spring (66000 inds m⁻², 17 g wet wt m⁻²) and the other in mid autumn (97000 inds m⁻², 176 g wet wt m⁻²). Two regression lines describing the effect of temperature on faecal production rate were obtained; Log F = 0.0604 T (°C) −0.7660 (below 15°C), Log F = 0.0266 T – 0.2170 (above 15°C). In total 26.8 kg dry wt m⁻² of sediment was defecated annually by Limnodrilus spp. The sediment in the 0–10 cm stratum may pass through the guts of the worms 2.3 times a year. Sedimentation rates in profundal region were very low with respect to the faecal production rates of the tubificids.     

TG1 integrase-based system for site-specific gene integration into bacterial genomes

Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ～2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ～10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ～10-kbp DNA into the genome with an efficiency of ～10⁴ transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.     

Polymorphism of dopamine receptor D4 exon I corresponding region in chicken

In stockbreeding, there are indications that behavioral traits of livestock have an effect on breeding and production. If the variation in individual behavior is related to that in neurotransmitter-related genes such as in humans, it would be possible to breed pedigrees composed of individuals having behavioral traits that are useful to production and breeding using selection based on genotypes. In this study, we investigated the exon I region of dopamine receptor D4 (DRD4), in which variation is related to psychiatric disorder in humans, in major poultry species namely Japanese quail (Coturnix japonica), chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus) and helmeted guinea fowl (Numida meleagris). Furthermore, we investigated Japanese cormorant (Phalacrocorax capillatus) and Japanese jungle crow (Corvus macrorhynchos) as an out-group. In these species of birds, the repeat of proline was identified in the region corresponding to the human polymorphic region. The repeat number was 9 in Japanese quail, ring-necked pheasant and Japanese cormorant; 12 in helmeted guinea fowl; and 3 in Japanese jungle crow. However, no polymorphism was found in these species. In contrast, polymorphism was observed in chicken and two alleles with 8 and 9 repeats were identified. Although 9 repeats (allele 9) were predominant in most chicken breeds, Black Minorca had only 8 repeats (allele 8). Intra-breed polymorphism was found in 6 out of 12 breeds, and two alleles (alleles 8 and 9) were detected in these breeds. This polymorphism, which is the first to be reported on a neurotransmitter-related gene in birds, would contribute significant information for elucidation of differences in behavioral traits in chicken breeds.