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     

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.     

Evidence of a local spawning site of marine eels along northeastern Japan, based on the distribution of small leptocephali

Evidence of recent spawning by several species of marine eels of the families Congridae, Ophichthidae, and Nettastomatidae was found over the continental shelf along the coast of northeastern Japan in October 2003. Sixty‐three leptocephali of at least three taxa that ranged from 3.8 to 12.3 mm total length were collected in a relatively small area, just to the south of Cape Shioya, primarily over the 100‐and 200‐m‐depth contours. The leptocephali of Gnathophis nystromi nystromi were the most abundant, followed by those of G. nystromi ginanago. This spawning area is located in an area that appeared to include water originating from the Tsugaru Warm Current and is further south than the typical latitude of the cold water of the Oyashio First Intrusion. These findings suggest that some species of marine eels may not migrate past the slope to spawn, and that the oceanographic features of the region may provide favorable conditions for the growth and retention of leptocephali in the area to the south of Cape Shioya.     

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.     

Naupliar copepod concentrations in the spawning grounds of Japanese sardine, Sardinops melanostictus, along the Kuroshio Current

Naupliar copepods were distributed at similar concentrations over the waters inshore and offshore of the Kuroshio Current off central Japan in early spring 1993 and 1994, overlapping with the distribution of early feeding larvae of Sardinops melanostictus . Although N, P, Si and chlorophyll a concentrations were higher in the waters inshore of the Kuroshio axis than in the offshore waters, the mean concentrations of nauplii were not statistically different between the two waters. Food availability for larval S. melanostictus did not seem to be different between the two waters in terms of the mean food concentrations. Using the critical food concentration (>9 nauplii L^–1) for 25% survival during 3 days after first feeding derived from a rearing experiment, percentages in number of stations or water samples with nauplii concentrations >9 nauplii L^–1 were higher in the inshore waters than in the offshore waters in both years examined. Considering that fish larvae may depend on small-scale patchiness of food for their survival, the inshore waters seemed to be more favourable for first-feeding larvae than the offshore waters.     

Morphological analysis of cell distribution and network structure via gap junctions in swine corneal stroma

Because of corneal transplantation limitations, there is a need for cornea-specific regenerative medicine. The development of such regenerative medicine has been delayed because of the complex and unique structure of the corneal stroma. Few studies have explored the corneal stroma cell distribution and cell types in vivo. This study investigated regional differences in morphological characteristics and distributions of corneal keratocytes and immunocompetent cells in the corneal stroma to clarify their functions and structural characteristics. The porcine eyeballs were subjected to light microscopy, transmission electron microscopy, scanning electron microscopy, and immunofluorescence staining analyses. Corneal cells were primarily located in the limbus, rather than the center of the cornea; the long keratocyte diameter was largest on the epithelial side of the corneal limbus, while the short diameter was largest on the endothelial side of the central cornea. Moreover, there were significantly more corneal cells on the epithelial side than on the endothelial side in both the central and limbus areas. Gap junctions between cells in the corneal stroma were present on the surfaces of cytoplasmic processes. Many cytoplasmic processes were scattered throughout the corneal stroma; they were connected both vertically and horizontally, forming an intercellular network. Additionally, immunocompetent cells on the epithelial side suggested to participate in this network via gap junctions. The morphology of keratocytes and immunocompetent cells on the epithelial side suggests that they play important roles in corneal homeostasis.     

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.     

Taste-active components in a Vietnamese fish sauce

Taste-active components were determined for a typical first-grade Vietnamese fish sauce Nuoc mam , of which total content of extractive compounds reached as high as 20 g/ 100 mL. A complete synthetic extract prepared from 35 compounds according to the analytical data reproduced almost satisfactorily the taste of the original fish sauce. From the synthetic extract, 11 compounds were identified to be the taste-active components by a series of omission and addition tests. The components consisted of glutamic and aspartic acids, threonine, alanine, valine, histidine, proline, tyrosine, cystine, methionine, and pyroglutamic acid. The most effective compound for recreating the characteristic flavor of fish sauce was glutamic acid, followed by pyroglutamic acid and alanine. Many of these components contribute to umami, sweetness, and overall taste of fish sauce. Even though the simplified 11-component extract reproduced the taste of fish sauce, the taste strength was weaker than that of the complete synthetic extract or fish sauce itself.