Isolation and characterization of the interspecific fusants from Streptomycetes obtained using a liquid regeneration method

ABSTRACT: Protoplast fusion between different species of Streptomyces was performed using a liquid regeneration method developed for a rapid and simple preparation of the fusants. Consequently, new clones, which could not be obtained using the conventional agar regeneration method, were obtained. In the crosses between S. griseus and S. durhamensis , and between S. californicus and S. catenulae , eight and two recombinants, respectively, were obtained using the liquid regeneration method. Conversely, in the case of crosses between S. ornatus and S. catenulae , and between S. ornatus and S. vendargensis , seven recombinants each were obtained using only the agar method. The physiological characteristics, such as the assimilation of carbohydrate and antibiotic resistance, of these fusants differed considerably from those of their parental strains. Using the proposed liquid regeneration method, a simpler and quicker procedure for protoplast fusion is described.     

The influence of Pueraria mirifica herb containing phytoestrogens on the urinary gonadotropin and estradiol levels in aged menopausal monkeys

We investigated a non‐invasive method of specimen collection for determining the changes of reproductive hormones in aged menopausal monkeys after a long‐term feeding of the Thai herb Pueraria mirifica (PM) containing phytoestrogens. Three groups of aged menopausal monkeys (n = three in each group) were fed daily with 10, 100, or 1000 mg of PM for a 90 day treatment period, and fed with distilled water for 30 and 60 days of the pre‐ and post‐treatment periods, respectively. Urine samples were collected for 14 h daily every 5 days and assayed for follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol levels. The result showed that monkeys fed with PM 10, PM 100, and PM 1000 had a decrease in urinary FSH levels during the treatment period, followed by a rebound increase during the post‐treatment period. Urinary estradiol levels tended to decrease and fluctuated between 4.28 and 266.71, 2.85–42.27, and 6.24–203.50% of the pre‐treatment levels in those three groups, respectively. Decreases in urinary LH levels could not be observed in all the three groups. These results suggest that FSH could be a candidate marker to detect the estrogenic effects of phytoestrogens in aged menopausal monkeys when changes of urinary hormones need to be used as an indicator.     

A Novel Conjugative Plasmid Conferring Multiple-antibiotic Resistance Detected in Epiphytic Strains of Enterobacter cloacae

Several epiphytic strains of Enterobacter cloacae isolated from mulberry leaves were resistant to antibiotics such as streptomycin, kanamycin, ampicillin, tetracycline and chloramphenicol, and harbored a 100-kb plasmid designated pMUL1. Plasmid profile analysis of spontaneous mutants derived from Ent. cloacae MUL1 and MUL1 (RSF1010) suggested that pMUL1 confers resistance to multiple antibiotics. Southern blot analysis using probes of five antibiotic-resistance genes against EcoRI-digested DNA from pMUL1 and defective pMUL1s (mutants) revealed that all these genes were located within a 24-kb region of pMUL1 and that some genes were assigned to the defective plasmids. A similar antibiotic-resistance plasmid was detected in several orchid-pathogenic strains of Ent. cloacae, but not in the type-culture strain (JCM 1232) or strains of Ent. cloacae of insect-origin. Strains MUL1 and MUL1 (RSF1010) were then mated with epiphytic Erwinia spp. and Pseudomonas spp. on filters, respectively. Several recipient strains of epiphytic Er. herbicola simultaneously acquired plasmid pMUL1 and the phenotype of multiple-antibiotic resistance. Thus, pMUL1 was verified to be conjugative and to encode genes for multiple-antibiotic resistance, including genes homologous to the strA-strB of the nonconjugative IncQ plasmid RSF1010. These findings suggest that epiphytic Ent. cloacae may play a role in the dissemination of antibiotic-resistance genes among epiphytic bacteria and plant pathogenic bacteria. Received 10 January 2002/ Accepted in revised form 29 March 2002     

Analysis of the bone morphogenetic protein 6 gene promoter region in young beef cattle affected by enzootic bovine leukosis

Enzootic bovine leukosis (EBL) is typically observed in cattle over 3 years old. However, some cases of EBL onset in young beef cattle have been reported in Japan. The mechanism for early EBL onset is unclear. In Japan, beef cattle are given large amounts of concentrated feed with low vitamin A. Bone morphogenetic proteins (BMPs) are regulators of cell proliferation, differentiation, and apoptosis, and thought to represent one of the key players in tumor malignancy. The purpose of this study was to evaluate the differences in BMP-6 methylation status between EBL beef cattle under 3 years old and other cattle. We investigated the methylation status of the BMP-6 promoter region in 32 EBL beef cattle under 3 years old. We also compared the methylation status of EBL dairy cattle to that of healthy cattle. Median methylation rate of the BMP-6 promoter region in EBL beef cattle under 3 years old was 8.9%, which was significantly higher than that of other groups. Hypermethylation of the BMP-6 promoter region might contribute to early onset of EBL in beef cattle under 3 years old, and animal feeding management practices specific to beef cattle may affect the methylation status of the BMP-6 promoter region.     

Application of highly differentiated SNPs between Japanese Black and Holstein to a breed assignment test between Japanese Black and F1 (Japanese Black x Holstein) and Holstein

Two taurine breeds, Japanese Black and Holstein, established from geographically distant origins and selected for different uses, beef and dairy, were extensively genotyped using a genome‐wide single nucleotide polymorphism (SNP) chip with more than 1000 animals of each breed. The genetic structure was examined by principal component analysis, in which the first principal component clearly separated the two breeds and explained more than 15% of the variance. Highly differentiated SNPs were detected throughout the genome, some of which were clustered within small regions on BTA4 (79.2–79.7 Mb, Btau4.0) and BTA26 (22.2–23.6 Mb). A breed assignment test was developed using 18 highly differentiated SNPs to distinguish Japanese Black from F₁ (Japanese Black × Holstein) and Holstein. The error rate that an F₁ or Holstein animal is misjudged as Japanese Black was expected to be < 0.8%, while the error rate that a Japanese Black animal is misjudged as F₁ or Holstein was expected to be < 0.001%. This test provides a reliable and powerful method to detect breed label falsification in retail beef.     

Biological nitrification inhibition by Brachiaria humidicola roots varies with soil type and inhibits nitrifying bacteria, but not other major soil microorganisms

The tropical pasture grass Brachiaria humidiola (Rendle) Schweick releases nitrification inhibitory compounds from its roots, a phenomenon termed 'biological nitrification inhibition' (BNI). We investigated the influence of root exudates of B. humidicola on nitrification, major soil microorganisms and plant growth promoting microorganisms using two contrasting soil types, Andosol and Cambisol. The addition of root exudates (containing BNI activity that is expressed in Allylthiourea unit (ATU) was standardized in a bioassay against a synthetic inhibitor of nitrification, allylthiourea, and their function in soil was compared to inhibition caused by the synthetic nitrification inhibitor dicyandiamide. At 30 and 40 ATU g⁻¹soil, root exudates inhibited nitrification by 95% in fresh Cambisol after 60 days. Nitrification was also similarly inhibited in rhizosphere soils of Cambisol where B. humidicola was grown for 6 months. Root exudates did not inhibit other soil microorganisms, including gram-negative bacteria, total cultivable bacteria and fluorescent pseudomonads. Root exudates, when added to pure cultures of Nitrosomonas europaea , inhibited their growth, but did not inhibit the growth of several plant growth promoting microorganisms, Azospirillum lipoferum , Rhizobium leguminosarum and Azotobacter chroococcum. Our results indicate that the nitrification inhibitors released by B. humidicola roots inhibited nitrifying bacteria, but did not negatively affect other major soil microorganisms and the effectiveness of the inhibitory effect varied with soil type.     

Methanogenic archaeal communities developed in paddy fields in the Kojima Bay polder, estimated by denaturing gradient gel electrophoresis, real-time PCR and sequencing analyses

Methanogenic archaeal communities inhabiting the paddy field soils in the Kojima Bay polder were investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), real-time PCR and sequencing analyses. Soil samples of the plow and subsoil layers were collected in 2006 from four paddy fields that were reclaimed between 1692 and 1954. The DGGE band patterns of the targeted 16S rRNA genes amplified from the extracted DNA from the samples were different from the patterns from the paddy field soils in diluvial and alluvial areas. The numbers of targeted 16S rRNA genes, which were involved with methanogenic archaeal and other archaeal sequences, were approximately 10⁷–10⁸ and 10⁶ g⁻¹ dry soil in the plow and subsoil layers, respectively. Sequences of methanogenic archaeal 16S rRNA genes belonging to Methanocellales (Rice cluster I), Methanosarcinales and Methanobacteriales were obtained from the major DGGE bands. Whereas sequences in Methanomicrobiales, which were predominant methanogens in the diluvial and alluvial paddy fields, were not recovered. Known halophilic and methylotrophic methanogens, which are characteristic of saline and marine environments, were not detected. These results indicate that distinctive methanogenic archaeal communities have developed in the paddy field soils in the Kojima Bay polder.     

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis)

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.     

Modelling the dispersal of larval Japanese sardine, Sardinops melanostictus, by the Kuroshio Current in 1993 and 1994

Acoustic Doppler current profiler (ADCP) data collected during routine monitoring surveys of the distribution and abundance of Japanese sardine larvae ( Sardinops melanostictus ) off the Pacific coast of Japan in February 1993 and 1994 were used to construct stationary average flowfields for three levels in the upper 100 m in each year. No large-scale meanders in the path of the Kuroshio Current were present in either year, but the axis of the current was closer to the coast in 1993 than in 1994. The flowfields were used to drive a particle-tracking model representing the dispersal of sardine eggs and larvae. Particles were released in accordance with the observed distribution of eggs, and their positions tracked for up to 40 days. In 1993, the model indicated that ≈ 50% of the egg production was carried north-eastwards out of the survey area into the area of the NW Pacific referred to as the Kuroshio Extension Zone. In contrast, only 5% of the egg production was exported to the Extension Zone in 1994, the remainder being retained in Japanese coastal waters. The consequences of the different dispersal patterns are discussed in relation to subsequent recruitment to the sardine stock. Based on commercial catch data, survival of the 1993 year class was 15% of that for the 1994 class. Hence, the results indicate that export of larvae to the Kuroshio Extension cannot in itself lead to successful recruitment.