Stimulatory effect of glycine betaine on l-lysine fermentation

Summary The growth rate, sugar consumption rate, and production rate of an l-lysine producing Brevibacterium lactofermentum mutant were stimulated by addition of exogenous glycine betaine. Glycine betaine stimulated the growth rate especially in media of inhibitory osmotic stress, and the stimulation was independent of any specific solute. Therefore growth stimulation by glycine betaine was considered to be an osmoprotective effect. A strong enhancement of the sugar consumption rate and the l-lysine production rate was observed even with resting cells under osmotic stress as well as in a fermentation with growing cells. These data indicated that the osmoprotective effects of glycine betaine on l-lysine production can be independent of protein synthesis. Offprint requests to: Yoshio Kawahara     

Mechanism of the stimulatory effect of cyclodextrins on lankacidin-producing Streptomyces

Summary Gel-filtration analysis of a mixture of cyclodextrin (CyD) and lankacidin C showed that -CyD had strong, -CyD weak and -CyD no affinity for lankacidin C. Lankacidin C production activity, which was assayed by measuring the incorporation of l-[methyl-¹⁴C-]methionine into the lankacidin molecule, was the greatest with cells grown in the presence of -CyD, less with -CyD and the least with -CyD. Lankamycin and T-2636M, which are by-products in lankacidin C fermentation, were not included by -CyD and their production was not stimulated by -CyD. It was apparent that the stimulatory effect of CyD was closely related to the formation of an inclusion complex between CyD and the antibiotic. Lankacidin C biosynthesis was repressed by preincubating cells with lankacidin C, while the repressive effect of lankacidin C was abrogated by the inclusion by -CyD. Thus, abrogation of feed-back repression seems to be a main mechanism of the effect of CyD. However, -CyD, which had no affinity for lankacidin C, stimulated the production to the least extent and exhibited a complementary effect on the stimulation by -CyD or -CyD. -CyD also caused a change in cell morphology and cell-surface hydrophobicity. It was assumed that the modification of the cell surface is a secondary mechanism of the effect of CyD.The second report of the stimulatory effect of cyclodextrins on lankacidin fermentationOffprint requests to: H. Sawada     

Chinese hamster ovary cells continuously secrete a cysteine endopeptidase

Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide, was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium. In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases. This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization).     

Comparison of fish assemblages among an artificial reef, a natural reef and a sandy-mud bottom site on the shelf off Iwate, northern Japan

Synopsis Fish assemblages at an artificial reef site, a natural reef site and a sandy-mud bottom site, on the shelf (depth 130 m) off Iwate Prefecture, northern Japan, were surveyed by using a bottom trammel net from May 1987 to March 1993. A total of 12 173 fishes of 48 species were recorded. Physiculus maximowiczi was dominant and comprised 69% of the total numerical abundance. Total fish number was lowest in March at all the 3 sites when P. maximowiczi migrated to deeper and warmer waters. Assemblage equitability and species diversity also varied seasonally in accordance with the abundance fluctuation of P. maximowiczi. P. maximowiczi, Alcichthys alcicornis and Hexagrammos otakii were more abundant at the artificial reef and natural reef sites, while Dexistes rikuzenius and Hemitripterus villosus were more abundant at the sandy-mud bottom site; total fish abundance was largest at the artificial reef site mainly due to the large number of P. maximowiczi. Species richness was similar among sites, but equitability, and consequently species diversity, was lowest at the artificial reef site. The main effect of the artificial reef seemed the attraction of P. maximowiczi from nearby bottoms, especially from natural rocky reefs; its large abundance determined the structure of the artificial reef fish community.     

Proliferation and Teratogenicity of Aino Virus in Chick Embryos

Aino virus (AIV; JaNAr 28 strain) 10³ TCID₅₀/0.2 ml was inoculated in the yolk sac of 8-day-old chick embryos. Recovery and titration of the virus from various organs including the central nervous system (CNS) and skeletal muscle were performed at 2, 4, 7, 10 and 13 days after inoculation (PI). AIV was systemically disseminated and proliferated even 2 days PI. The titers of the recovered virus from the CNS and from skeletal muscle was the highest at 4 days PI and declined with time, whereas hydranencephaly, arthrogryposis and cerebellar hypoplasia developed at 7 days PI and gradually progressed until 13 days PI.     

Suppression of gravitropic response of primary roots by submergence

Primary roots of six plant species were placed horizontally either in humid air or under water, and their growth and gravitropic responses were examined. In air, all the roots showed a normal gravitropic curvature. Under water without aeration, roots of rice (Oryza sativa L.), oat (Avena sativa L.), azuki bean (Vigna angularis Ohwi et Ohashi), and cress (Lepidium sativum L.) curved downward at almost same rate as in air, whereas the curvature of roots of maize (Zea mays L.) and pea (Pisum sativum L.) was strongly suppressed. Submergence did not cause a decrease in growth rate of these roots. When roots of maize and pea were placed horizontally under water without aeration and then rotated in three dimensions on a clinostat in air, they showed a significant curvature, suggesting that the step suppressed by submergence is not graviperception but the subsequent signal transmission or differential growth process. Constant bubbling of air through the water partly restored the gravitropic curvature of maize roots and completely restored that of pea roots. The curvature of pea roots was also partly restored by the addition of an inhibitor of ethylene biosynthesis, aminooxyacetic acid. In air, ethylene suppressed the gravitropic curvature of roots of maize and pea. Furthermore, the level of ethylene in the intercellular space of the roots was increased by submergence. These results suggest that the accumulation of ethylene in the tissue is at least partly involved in suppression of transmission of the gravity signal or of differential growth in maize and pea roots under conditions of submergence.Abbreviations AOA aminooxyacetic acid - 3-D three-dimensional Dedicated to Professor Andreas Sievers on the occasion of his retirementWe thank Professor H. Suge and Drs. H. Takahashi and H. Kataoka, Tohoku University and Dr. T. Suzuki, Yamagata University, for helpful suggestions. The present study was supported in part by a Grant for Basic Research in Space Station Utilization from the Institute of Space and Astronautical Science, Japan.     

A new yeast gene, HTR1, required for growth at high temperature, is needed for recovery from mating pheromone-induced G1 arrest

A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).     

Flower-Inducing Activity of Exogenous Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Rich Medium

Flower-inducing activity of lysine was examined in Lemna paucicostata151, a weakly responsive short-day plant, cultured on nitrogen-richmedium under long-day conditions (continuous light). Lemna paucicostata151 was homogenized in a solution of lysine and the homogenatewas centrifuged. The supernatant (lysine-containing extract)was added to nitrogen-rich medium after passage through a membranefilter to give various concentrations of lysine in the medium.Flowering was induced in plants grown for six days on mediumthat contained lysine at concentrations above 0.25 µM.In plants grown on medium that contained 1 µM lysine,a significant flowering response was observed on the fourthday of culture. However, the flower-inducing activity of lysinedisappeared when the lysine-containing extract was added tothe medium and the medium was then autoclaved, suggesting thatthe active principle is unstable to autoclaving. Among derivativesof lysine tested, lysine hydroxamate had the highest flower-inducingactivity and lysyl lysine had almost same activity as that oflysine. When added to the medium without homogenization withplant material, lysine and lysyl lysine had flower-inducingactivity but lysine hydroxamate did not induce flowering. (Received April 26, 1993; Accepted November 8, 1993)     

Computer-aided image analysis applied to immunogold-silver staining: evaluation of proliferating cell nuclear antigen (PCNA)-reactive sites in paraffin sections

Feasibility of the combination of the immunogold-silver staining method (IGSS) and computer-aided image analysis was assessed for the detection of antigen in an immunostained, paraffin-embedded section. Using low-temperature IGSS, we stained a specimen of human oral squamous cell carcinoma with a monoclonal antibody, PC 10, against a proliferating cell nuclear antigen (PCNA/cyclin), and the section was analyzed by ACAS 570 interactive laser cytometry. The PCNA-positive cells, exhibiting a heteromorphic texture, were contrasted by the dark staining of their nuclei, but showed heterogeneity in staining intensity from cell to cell. Using a conventional microscope light source rather than a laser, and by employing the COMPLEMENT DATA program (which permits inversion of the data values) installed in the ACAS 570 software system, we were able to obtain a complemental image which replicated the real immunohisto-morphology. Approximately 30–35 cells from three different areas in the same section were selected by DEFINE CELL and MARK AREA programs, and quantitative image analysis was performed in terms of cell integrated value, area, perimeter, and shape factor indicated in histogram form. The combined utilization of IGSS with computer-aided image analysis was demonstrated to offer a crucial advantage for the quantitative assessment of immunostained sections.