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.     

Hepatitis B core antigen-specific IFN-gamma production of peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

To evaluate the specificity of cellular immune response to hepatitis B virus (HBV) Ag in patients with chronic HBV infection, we have measured IFN-gamma production and proliferation of PBMC of 16 patients with chronic active hepatitis (CAH), 17 asymptomatic carriers of HBV (ASC), 6 anti-hepatitis B surface (HBs)-positive subjects, and 6 control individuals with ELISA procedure and [3H]thymidine incorporation. There was no significant increase in the mean proliferative response to recombinant HB surface and core Ag (rHBsAg and rHBcAg), nor was IFN-gamma production elicited with rHBsAg in any group. In contrast, PBMC of HBeAg-positive and anti-HBe-positive CAH patients, and anti-hepatitis B "e" Ag (HBe)-positive ASC showed significantly enhanced IFN-gamma production in response to HBcAg, whereas those of HBeAg-positive ASC and anti-HBs-positive subjects did not respond to HBcAg. The maximal response was observed in a 5-day culture with 500 ng/ml of rHBcAg when assessed by stimulation index value. Monocytes did not demonstrate an increased suppressor or helper activity for IFN-gamma production in these patients. T cell subset fractionation revealed that CD4+ cells were main population of IFN-gamma production specific for HBcAg and CD8+ cells did not suppress IFN-gamma production of CD4+ cells. Furthermore, CD4+ cells of HBeAg-positive ASC generated lesser amounts of IFN-gamma than HBeAg-positive CAH patients did. These results show that the measurement of IFN-gamma production is useful to determine cellular immune response to HBV Ag and suggest that IFN-gamma production depends on the helper activity of CD4+ T cells sensitized to HBcAg.     

Photoaffinity labeling with dihydropyridine derivatives of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain

The characteristics of photoaffinity labeling with the calcium agonist [3H]Bay K 8644 (Bay) and the calcium antagonists [3H]nitrendipine (Nit) and (+)PN200-110 (PN) of crude membranes from rat skeletal, cardiac, ileal, and uterine muscles and whole brain were investigated. In all these crude membranes, [3H](+)PN (20 nM) was mainly photoincorporated into one protein band with a molecular weight of 30,000 - 41,000 Da. It was also incorporated into some other bands of all these crude membranes. The photoincorporation of [3H](+)PN into these crude membranes was inhibited by the presence of 20 microM unlabeled (+)PN. The photoincorporation of [3H](+)PN into these crude membranes depended on its dose and on the time of UV irradiation. No incorporation of [3H](+)PN was observed in the absence of UV irradiation. The incorporation was not affected by the presence of 1 mM CaCl2 and/or 0.15 M NaCl, but was significantly decreased by 20 microM (+)PN and slightly decreased by 20 microM (-)PN, 20 microM Bay, 1 mM diltiazem, or 1 mM verapamil. Namely, enantiomers of PN caused various extents of stereoselective inhibition of photoaffinity labeling by [3H](+)PN of specific protein bands in these crude membranes. [3H]Nit was photoincorporated into these crude membranes in the same way as [3H](+)PN, but [3H]Bay was not photoincorporated. However, 20 microM unlabeled Nit did not consistently inhibit photoaffinity labeling with [3H]Nit. These findings suggested that measurement of photoaffinity of crude membranes from rat skeletal, cardiac, and uterine muscles and whole brain with [3H](+)PN by UV irradiation is a useful method for investigating the characteristics of the voltage-dependent calcium channels that are affected by 1,4-dihydropyridine derivatives.     

Cetacean relaxin. Isolation and sequence of relaxins from Balaenoptera acutorostrata and Balaenoptera edeni

The tendency toward extremely high variability among relaxins derived from purportedly closely related species has come to an abrupt end with the discovery of quasi-porcine relaxin in the minke whale (Balaenoptera acutorostrata) and the Bryde's whale (Balaenoptera edeni). An aqueous abstract of the corpora lutea of the two baleen whales contained significant amounts of relaxin-like activity as determined by a mouse bioassay and by cross-reactivity with anti-pig relaxin antibodies. The activity could be isolated and purified to homogeneity. Sequence analysis revealed that both whale relaxins differed from each other by about 3 residues, whereas the relaxin of B. edeni differed at only one position from that of pig relaxin. The similarity appears to include even the chain length heterogeneity observed at the C-terminal end of the B chain in porcine relaxin which is produced by a peculiar mode of connecting peptide removal from the pro-hormone. This finding may well represent one of the better documented challenges to the current paradigm of molecular evolution.     

Partial purification and characterization of membrane-associated diacylglycerol kinase of Drosophila heads.

A membrane-associated diacylglycerol kinase of Drosophila heads was purified to near homogeneity from the KCl extract of Drosophila heads. The purification procedure involved chromatography on Q-Sepharose, ammonium sulfate fractionation, Superose 12, hydroxyapatite and ATP-agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fractions after the ATP-agarose column chromatography showed that only a 115 kDa protein correlated well with the enzyme activity. The apparent Km values of partially purified DG kinase were 220 microM for ATP and 540 microM for diolein, respectively. The activity of the DG kinase was inhibited by deoxycholate and was not activated by Ca2+.     

A Drosophila receptor-type tyrosine kinase (DFR1) acts as a fibroblast growth factor receptor in Xenopus embryos

Members of the fibroblast growth factor (FGF) family play important roles in various developmental processes in vertebrates. Since two genes closely related to the vertebrate FGF receptor (FGFR) genes DFR1 and DFR2/breathless have already been reported in Drosophila , the existence of a Drosophila FGF has been predicted. In the present study, we examined whether DFR1 is functionally interchangeable with a vertebrate FGFR in the Xenopus system. First, we found that the expression of DFR1 promoted Ca²⁺ efflux in response to human basic (b)FGF in Xenopus oocytes, whereas the coexpression of a dominant negative form of DFR1 (ΔDFR1) with a chick FGFR1/cek1 inhibited promotion of Ca²⁺ efflux induced by the expression of cek1 in the oocyte. Second, the expression of ΔDFR1 was observed to induce a defect in the posterior structure of the Xenopus embryo at stage 30, as observed with a dominant negative form of cek1 (Δcek1). Third, we found that the expression of ΔDFR1 inhibited the expression of FGF-regulated genes such as Xbra, Xnot , and Xshh in Xenopus embryos at stage 11, while the coexpression of DFR1 with ΔDFR1 could rescue the inhibited expression of FGF-regulated genes. These results indicate that DFR1 acts as an FGFR in Xenopus embryos and that an FGF is likely to exist in Drosophila .