Analysis of electrophoretic mobility histograms of mouse splenocytes and thymocytes during tumor growth and after combined chemotherapy and immunotherapy

Changes in electrophoretic mobility histograms of splenocytes and thymocytes were studied in plasmacytoma X5563-bearing mice as an indicator of response to treatment with mitomycin C (MMC) alone or combined with the immunomodulator Krestin (PSK). Tumor growth was inhibited by 80-90% in the MMC-treated and was further inhibited in the MMC and PSK-treated group. Electrophoretic mobility histograms of splenocytes were used to determine the fraction of cells having intermediate mobility between high mobility (T cells) and low mobility (B cells). This fraction of intermediate-mobility cells increased in tumor-bearing mice, but a normal electrophoretic mobility pattern was obtained following successful antitumor treatment. In the electrophoretic mobility histogram of thymocytes, on the other hand, the low-mobility cells (cortical thymocytes) decreased in number during tumor growth and were further reduced in the MMC-treated group. This reduction was less in the MMC and PSK-treated group. These results suggest that combined therapy with MMC and PSK prevents damage of the host defence mechanism and allows more efficient antitumor treatment. Analysis of electrophoretic mobility histograms of splenocytes and thymocytes using a fully automated cell electrophoretic instrument makes possible the rapid evaluation of the immunological effects of drug therapy of tumor-bearing mice.     

The role of peroxisomal fatty acyl-CoA beta-oxidation in bile acid biosynthesis

The physiological role of the peroxisomal fatty acyl-CoA beta-oxidizing system (FAOS) is not yet established. We speculated that there might be a relationship between peroxisomal degradation of long-chain fatty acids in the liver and the biosynthesis of bile acids. This was investigated using [1-14C]butyric acid and [1-14C]lignoceric acid as substrates of FAOS in mitochondria and peroxisomes, respectively. The incorporation of [14C]lignoceric acid into primary bile acids was approximately four times higher than that of [14C]butyric acid (in terms of C-2 units). The pools of these two fatty acids in the liver were exceedingly small. The incorporations of radioactivity into the primary bile acids were strongly inhibited by administration of aminotriazole, which is a specific inhibitor of peroxisomal FAOS in vivo [F. Hashimoto and H. Hayashi (1987) Biochim. Biophys. Acta 921, 142-150]. Aminotriazole inhibited preferentially the formation of cholate, the major primary bile acid, from both [14C]lignoceric acid and [14C]butyric acid, rather than the formation of chenodeoxycholate. The former inhibition was about 70% and the latter was approximately 40-50%. In view of reports that cholate is biosynthesized from endogenous cholesterol, the above results indicate that peroxisomal FAOS may have an anabolic function, supplying acetyl CoA for bile acid biosynthesis.     

Dual loading of the fluorescent indicator fura-2 and 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) in isolated myocytes

Isolated rat heart myocytes were loaded with both the Ca2+ sensitive fluorescent probe fura-2/AM and the fluorescent pH indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM). Changes in [Ca2+]i and pHi were measured simultaneously using digitized video fluorescence microscopy. In measurement of [Ca2+]i and pHi, the ratios of dual-loaded cells were not different from single-loaded cells. Using this method, [Ca2+]i and pHi in myocytes were 48 +/- 7 nM and 7.17 +/- 0.05. It is concluded that [Ca2+]i and pHi could be measured simultaneously in isolated myocyte using dual-loading of fura-2 and BCECF.     

Monoclonal antibodies specific to a Ca2(+)-bound form of lipocortin I distinguish its Ca2(+)-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2.

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Protection of mice against Sendai virus pneumonia by non-neutralizing anti-F monoclonal antibodies

Nine monoclonal antibodies (MAbs) directed to F protein of Sendai virus were obtained and characterized for their protective ability against Sendai virus infection in mice. None of the MAbs showed hemagglutination-inhibition (HI), hemolysis-inhibition (HLI), or neutralization (NT) activities in vitro when assayed by standard methods. Some of the MAbs, however, showed complement-requiring NT (C-NT) and complement-requiring hemolysis (C-HL) activities when assayed in the presence of complement. Passive immunization experiments revealed that the MAbs with higher C-NT and C-HL activities showed protective activity against Sendai virus pneumonia in mice, and that some MAbs with IgG1 isotype having neither C-NT nor C-HL activity also showed the protective activity. Digestion of the MAbs with pepsin which split immunoglobulin molecules into F(ab')2 and Fc fragments greatly suppressed the protective activity. These results suggest that not only complement-mediated immunological responses such as immune virolysis but also antibody-dependent cellular cytotoxicity (ADCC) and/or immune phagocytosis, in which complement system is not necessarily involved, play an important role in the protection of mice from Sendai virus infection.     

Effects of hydrocortisone and aminophylline on plasma leukotriene C4 levels in patients during an asthmatic attack

To study the role of leukotriene C4(LTC4) and the effect of hydrocortisone and aminophylline on plasma LTC4 levels in patients with asthmatic attacks, we measured LTC4 in plasma of 18 asthmatics during a wheezing attack and of 7 normal subjects. Blood samples were obtained before and after treatment with aminophylline and/or hydrocortisone injections. We extracted LTC4 using a Sep-Pak C18 cartridge for the measurement of LTC4 by radioimmunoassay. The plasma levels of immunoreactive LTC4 (i-LTC4) of the normal subjects were 142 +/- 25 pg/ml (n = 7), while those of nonatopic type asthmatic patients with wheezing attacks were 208 +/- 68 pg/ml (n = 15) (p less than 0.01). Before and after treatment with both hydrocortisone succinate (100 mg) and aminophylline (250 mg), 6 asthmatic patients with wheezing attacks had a mean plasma level of i-LTC4 181 +/- 24 and 132 +/- 18 pg/ml (p less than 0.01), respectively. On the other hand, the treatment with aminophylline 250 mg alone increased the i-LTC4 levels from 178 +/- 19 pg/mg to 213 +/- 16 pg/mg (n = 6)(p less than 0.05), while treatment with hydrocortisone succinate 100 mg decreased the i-LTC4 level 0.05 from 284 +/- 99 pg/ml to 249 +/- 85 pg/ml (n = 4)(p less than 0.05). In conclusion, the present study shows that the i-LTC4 level in venous blood of patients with asthmatic attacks is decreased significantly by treatment with hydrocortisone succinate.     

Biochemical switching device realizing McCulloch-Pitts type equation

We analyzed cyclic enzyme systems, one of the best candidates for biochemical switching devices, especially focusing on their control mode against external perturbations. Since these systems have the reliability of ON-OFF types of operation (McCulloch-Pitts' neuronic equation), we shall present here the mechanical difference between these systems and electronic switching circuit, especially on the mnemonic mechanism of biochemical switching devices.     

A comparative 1H NMR study of mouse alpha(1-53) and beta(2-53) epidermal growth factors

The three-dimensional structure of the mouse epidermal growth factor (EGF) in solution was studied by comparison of the 1H NMR spectra of alpha EGF (1-53) and beta EGF (2-53, des-asparaginyl 1 form). Using pH dependence of chemical shifts and a two-dimensional difference spectrum, the effect of the N-terminal deletion was investigated based on the complete assignment of the proton resonances. The affected residues were all found to be located exactly in the triple-stranded, beta-sheet core in the N-terminal domain of the EGF molecule.