Heat-inducible translational coupling in Bacillus subtilis.

Bacillus subtilis plasmid pGR71 is a promoter-probe shuttle vector derived from pUB110. The expression of the cat gene on pGR71 in B. subtilis requires the insertion of a Bacillus promoter and a ribosomal binding site (RBS) into the HindIII cloning site immediately upstream from the cat gene. A recombinant plasmid of pGR71, named pGR71-369, was obtained by a spontaneous deletion of a fragment containing most of the inserted HindIII fragment and the replication origin necessary for multiplication in Escherichia coli. The expression of the cat gene in B. subtilis cells carrying this plasmid was inducible by heat. Nucleotide sequence analysis of the upstream region of the cat gene, deletion analysis, and dot blot hybridization analysis of mRNA in various conditions revealed that the cat gene was expressed by heat-inducible translational coupling and that the regulatory region of heat inducibility was present in the upstream region of the cat gene.     

Mass-spectrometric identification of trehalose 6-monomycolate synthesized by the cell-free system of Bacterionema matruchotii

The fluffy layer fraction prepared from Bacterionema matruchotii was found to possess high activity for the biosynthesis of mycolic acids which were bound to an unknown compound by an alkali-labile linkage [T. Shimakata, M. Iwaki, and T. Kusaka (1984) Arch. Biochem. Biophys. 229, 329-339]. To determine the structure of the mycolate-containing compound, it was purified and analyzed by field desorption (FD) and secondary ion mass spectrometry (SI-MS). When non-labelled palmitic acid was used as a precursor in the in vitro biosynthetic system, the underivatized product had a cationized molecular ion, [M + Na]+, at m/z 843 in FD-MS and a protonated ion, [M + H]+, at m/z 821 in SI-MS, corresponding to the quasimolecular ion of trehalose monomycolate (C32:0). In SI-MS, characteristic fragment ions due to cleavage of glycosidic linkages were clearly detected in addition to the molecular ion. If [1-13C]palmitic acid was the precursor, 2 mass unit increases in both the quasimolecular and fragment ions were observed, indicating that two molecules of palmitate were incorporated into the product. alpha-Trehalose was found in the aqueous phase after saponification of the product. By the electron impact mass spectrometry of the trimethylsilylated product, the mycolate was found to be esterified with an hydroxyl group at position 6 of the trehalose molecule. These results clearly demonstrated that the predominant product synthesized by the fluffy layer fraction with palmitate as substrate was 6-monomycolate (C32:0) of alpha-D-trehalose. Because newly synthesized mycolic acid was mainly in the form of trehalose monomycolate instead of free mycolate or trehalose dimycolate, the role of trehalose in the biosynthesis of mycolic acid is discussed.     

Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Effects of serine protease inhibitors on oxyradical burst from phagocytizing neutrophils—analysis by chemiluminescence counting and its microscopic imaging

Reaction difference of oxyradical generation and luminol-dependent photoemission of zymosan- and phorbol ester-treated neutrophils were investigated using a conventional photomultiplier and ultrasensitive photonic imaging technique. Zymosan-treated cells released a concentrated photonic burst corresponding to the cellular distribution. In contrast, phorbol ester-treated cells produced a negligible level of photoemission, and the additional application of Ca²⁺ ionophore enhanced the photonic burst, which was gradually spread out into extracellular space. Serine protease inhibitors did not attenuate PMA-induced chemiluminescence but did attenuate zymosan-induced chemiluminescence. This suggests the involvement of serine protease in the respiratory burst of phagocytizing neutrophils.     

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with ¹²⁵ I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K_d values of 1 × 10^?10 M and 5 × 10^?9 M. The numbers of high- and low- affinity receptors were 1 × 10⁴ and 2 × 10⁵ per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.     

Characterization and expression of a P-450-like mycinamicin biosynthesis gene using a novel Micromonospora-Escherichia coli shuttle cosmid vector

A 29 kb shuttle cosmid vector, pTYS507, was constructed from a cryptic Micromonospora griseorubida plasmid and the Escherichia coli cosmid pJB8. Subcloning of mycinamicin II biosysnthesis genes in pTYS507 led to the identification of a DNA region that could complement a mutant of M. griseorubida that lacked both hydroxylase and epoxidase activities. Nucleotide sequence and mutational analysis suggested that a single P-450-like protein catalyzes both reactions.     

Chromosomes of Temnocephala minor,an ectosymbiotic turbellarian on Australian crayfish found in Kagoshima Prefecture,with karyological notes on exotic turbellarians found in Japan

Records of exotic turbellarian species found in Japan are reviewed from taxonomic and karyological viewpoints. Temnocephala minor Haswell, 1888, an ectocommensal on a freshwater crayfish of Australia, was found from culture ponds of Cherax tenuimanus (introduced from W. Australia) in Kagoshima Prefecture. T. minor had the chromosome number of 2x = 18 (2sm + 2m + 2m + 2sm + 2m + 2m + 2m + 2sm + 2m). The following 3 species of exotic freshwater triclads were recorded from tanks and ponds used for tropical fish culture: Dugesia austroasiatica Kawakatsu, 1985 (2x = 16), Dugesia tigrina (Girard, 1850) (2x = 16) and Rhodax? sp. (3x = 24; 3x = 24 &; 3x + 1LB + 1SB = 25 + 1SB). The following 3 species of exotic terrestrial triclads were recorded: Bipalium nobile Kawakatsu et Makino, 1982 (2x = 10), Bipalium kewense Moseley, 1878 (2x = 18), and Platydemus manokwari de Beauchamp, 1962 (n = 6, 2x = 12). An extensive occurrence of P. manokwari in the Southwest Islands of Japan may be due to an unexpected introduction of the animal in very recent years.     

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

 An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration. Accepted: 18 March 1997     

Over-production of Thermus protease in dense culture of Escherichia coli using two carbon sources

Escherichia coli JM109(DE3) harboring expression plasmid pkAQNÆC30, which carries the Thermus protease aqualysin I (AQI) gene, was cultivated with glucose as a sole carbon source. The final cell concentration was over 15 g dry weight/l and the amount of AQI produced reached approximately 130 kU/ml broth. Moreover, by using two carbon sources, glucose and glycerol, the production yield was increased to over 200 kU AQI/ml, while suppressing the formation of inhibitory acetic acid.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.