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Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.

Sixteen species of Thai traditional herbs were selected for this study. They were Andrographis paniculata, Cassia alata, Clinacanthus nutans, Eclipta alba, Momordica charantia, Phyllanthus acidus, P. amarus, P. debilis, P. pulcher, P. reticulatus, P. urinaria, Psidium guajava, Tinospora cordifolia, T. crispa and white and red strains or Ocimum sanctum. Using an agar plate dilution method, they were tested for antibacterial activity against the pathogenic bacteria: Aeomonas hydrophila, a Streptococcus species and 10 strains of Vibrio. Eleven of the tested herbs showed antibacterial activity. Among them, P. guajava and M. charantia displayed the highest activity against Vibrio harveyi and V. parahaemolyticus. The minimal inhibitory concentration (MIC) of P. guajava against the tested bacteria was found to be 0.625mg/ml, while the MIC of M. charantia was 1.25 mg/ml.

Six alginolytic, facultatively anaerobic, non-motile marine bacteria were isolated from the gut of abalone Haliotis discus hannai. DNA-DNA hybridization data showed that the six strains constituted a single genospecies. Phylogenetic analyses of 16S rDNA sequences indicated that the isolates should be assigned to the genus Vibrio. The phenotypic features of the isolates were closely related to Vibrio fischeri and Vibrio pelagius biovar I, but 13 traits (motility, luminescence, alginase production, lipase production, lysine decarboxylase, indole production, growth in 1 and 6% NaCl and assimilation of five carbon compounds) distinguished these strains from V. fischeri, and 17 traits (motility, growth at 37 degrees C, lipase production, indole production, growth in 1 and 6% NaCl, acid from sucrose and D-sorbitol, and assimilation of nine carbon compounds) distinguished these strains from V. pelagius. The G + C content of the isolates was 41.6-43.1 mol%. According to DNA-DNA hybridization data and 16S rDNA phylogenetic analyses, it was concluded that the six isolates constitute a new species different from any other Vibrio species. The name Vibrio halioticoli sp. nov. (type strain IAM 14596T) is proposed. A set of phenotypic features which enables differentiation of the new species from other species of the Vibrionaceae family is described.

An aerobic, polarly flagellated marine bacterium that produces a prodigiosin-like pigment was isolated from the red-spotted culture beds of Laminaria japonica. Five isolates had unique bacteriolytic activity for both Gram-positive and -negative bacteria, which had never been observed among Alteromonas or related species. The isolates were identified as the causative agent of red spot disease of L. japonica seeds. The phenotypic features of the isolates were similar to these of Pseudoalteromonas rubra ATCC 29570T, but they could be differentiated using 10 traits (growth at 37 degrees C, requirement for organic growth factors, bacteriolytic activity, utilization of sucrose, N- acetylglucosamine, fumarate, succinate, D-galactose, L-proline and acetate). The G+C content of DNAs from the isolates was 44-46 mol%. The isolates constitute a new species, distinct from the other Alteromonas and Pseudoalteromonas species, as shown by DNA-DNA hybridization experiments and phylogenetic clustering of 16S rRNA gene sequences, for which the name Pseudoalteromonas bacteriolytica sp. nov. (type strain = IAM 14595T) is proposed. A set of phenotypic features which differentiate this new species from closely related Pseudoalteromonas and Alteromonas species is provided.

Three continuous cell lines were established: JSKG from gonads of Japanese striped knife jaw Oplegnathus fasciatus, KRE from embryos of a hybrid of kelp Epinephelus moara and red spotted grouper E. akaara, and PAS from the skin of greater amberjack (also called purplish amberjack) Seriola dumerili; these cell lines were passed 60, 89, 120 times, respectively. Although initially cultured in Leibovitz's L-15 medium, two of the cell lines, JSKG and PAS, exhibited optimal growth response in Eagle's minimum essential medium buffered with a combination of tris and sodium bicarbonate. These cell lines were initiated at a higher NaCl concentration of 0.206 M but gradually adapted to the low NaCl concentration of 0.116 M after several subcultures. Optimum growth temperature was 25℃ for JSKG and PAS cells, and 30℃ for KRE cells. The modal chromosome number is 83 for the JSKG cell line, 92 for the KRE cell line, and 96 for the PAS cell line. Results for efficiency of plating indicate that all three cell lines are composed of transformed cells. Cell lines JSKG and PAS are susceptible to nine fish viruses, including channel catfish virus (CCV) and chum salmon virus (CSV). The KRE cell line is susceptible to CCV and fish rhabdoviruses of the vesiculovirus group. None of the cells showed cytopathic effect for Oncorhynchus masou virus (OMV) or Herpesvirus salmonis. Yields of infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), hirame rhabdovirus (HRV), and CSV were relatively low in these cell lines.

Seven continuous cell lines were established from salmonid and nonsalmonid fishes. Salmonid cell lines derived from rainbow trout Oncorhynchus mykiss and chum salmon O. keta were designated RTE and RTE-2 (rainbow trout embryo), RTT (rainbow trout tail), and SEH ("sake" or chum salmon embryo head). Nonsalmonid cell lines derived from pond smelt Hypomesus olidus, chevron snakehead Channa striata, and goldfish Carassius auratus were designated WF-1 ("wakasagi" fin), SHH (snakehead heart), and EPG (epithelioma papulosum of goldfish), respectively. Optimum growth for most of the cell lines was observed in Eagle's minimum essential medium buffered with sodium bicarbonate (26 mM) or a combination of sodium bicarbonate (8.9 mM) and tris (16 mM). Likewise, most of the cell lines showed optimum growth at the lowest NaCl concentration tested (0.116 M). Optimum growth temperatures ranged from 15 to 20℃ for the salmonid cell lines and from 15 to 30℃ for nonsalmonid cell lines. Except for RTT, the cell lines were heteroploid. Eleven fish viruses were used to test the susceptibility of these cell lines. Cell lines derived from salmonids developed cytopathic effects (CPE) when infected with 10 of the 11 fish viruses tested, except for RTT, which produced CPE with only 8 of the fish viruses. Six fish rhabdoviruses used in this study elicited a pronounced CPE when inoculated into nonsalmonid cell lines EPG, WF-1, and SHH. Among the new cell lines, RTE-2 showed the best potential for the isolation of fish viruses.

Oji International Symposium on Salmonid Diseases. 22-25 October 1991. Sapporo, Japan