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.     

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.     

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.     

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.     

Origin of mitotic cells of the chorionic villi in direct chromosome analysis

Summary The origin of mitotic cells was investigated histologically in chorionic tissue, metaphase plates of which were used for direct cytogenetic study. Mitotic figures were often observed in the Langhans' cell layers, but absolutely none were seen in the syncytium and stromal cells.     

Conditionally Expressed Missense Mutations: The Basis for the Unusual Phenotype of an Apparent trpD Nonsense Mutant of SALMONELLA TYPHIMURIUM

Tryptophan auxotroph trp-28 is anomalous since preliminary mapping and suppression studies indicate the presence of a single amber nonsense mutation either late in trpE or early in trpD, but enzymological tests indicate the complete inactivation of both genes in this strain. Since the trpE and trpD genes are contiguous and encode the two subunits of a multifunctional enzyme complex, it was of interest to learn the mechanism of action of this apparent pleiotropic nonsense mutation. Our study has revealed that the phenotype of this strain derives not from a single mutation, but from the presence and interaction of multiple mutations. Besides the recognized amber mutation (designated trpD28), this strain carries two additional, conditionally expressed missense mutations (designated trpE1651 and trpD1652). The trpD28 amber codon maps in the promoter-proximal region 1 of trpD and eliminates the glutamine amidotransferase activity of the bifunctional trpD polypeptide. The trpD1652 mutation maps in the promoter-distal region 2 of trpD and severely reduces (but does not eliminate) the phosphoribosyl transferase activity of the trpD polypeptide. The trpE1651 mutation maps in the anterior part of trpE and causes a rapid loss of activity of the trpE polypeptide, but only when it exists as an uncomplexed subunit. The existence of the two missense mutations escaped prior notice in standard recombinational tests since the nature of each mutation is such that neither is detectable by the nutritional screens normally used in such tests unless an unsuppressed chainterminating mutation, such as trpD28, is also present.     

Internal reinitiation of translation in polar mutants of the trpB gene of Salmonella typhimurium.

Summary The trpB gene of S. typhimurium codes for the bifunctional component II subunit of the AS-PRT complex which catalyzes the first two steps of tryptophan biosynthesis. It has previously been shown that the amino-terminal 40% of the component II molecule possesses the catalytic sites determining glutamine amidotransferase (GAT) activity, demonstrable indirectly by complementation with component I, the product of trpA, in the synthesis of anthranilic acid from chorismic acid and glutamine (AS activity), while its carboxy-terminal 60% possesses the catalytic sites determining anthranilate-PRPP phosphoribosyl transferase (PRT) activity, demonstrable by direct enzymatic assay. Here we further demonstrate the functional independence of the two regions of the component II subunit by providing evidence for the existence of monofunctional (GAT^-, PRT⁺) carboxy-terminal restart fragments of component II in certain chain terminating trpB mutants. Nonsense and frameshift mutants of the operator-proximal portion (region 1) of trpB have been found to grow well in media supplemented with anthranilic acid, implying the presence of PRT activity in the cell. Analysis of extracts of these strains has demonstrated the presence of low, but variable levels of PRT activity, but no GAT activity. Correlation of the map location of these mutations with the intensity of their polar effects on the expression of operator-distal genes suggests the existence of at least two gradients or units of polarity within region 1. Furthermore, in double mutant polarity tests, multiplicatiove polar effects were found in certain region 1 trpB-trpB double mutants strains. Taken together, these results lead us to conclude that at least two sites for reinitiation of translation exist within region 1 of trpB which can be activated by the presence of a nearby chain terminating codon. Such reinitation leads to the synthesis of labile carboxy-terminal restart fragments of component II which possess PRT function, but lack GAT function.