Cancer chemotherapy and somatic cell mutation

The occurrence of a second neoplasm is one of the major obstacles in cancer chemotherapy. The elucidation of the genotoxic effects induced by anti-cancer drugs is considered to be helpful in identifying the degree of cancer risk. Numerous investigations on cancer patients after chemotherapy have demonstrated: (i) an increase in the in vivo somatic cell mutant frequency (Mf) at three genetic loci, including hypoxanthine–guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), and (ii) alterations in the mutational spectra of hprt mutants. However, the time required for and the degree of such changes are quite variable among patients even if they have received the same chemotherapy, suggesting the existence of underlying genetic factor(s). Accordingly, some cancer patients prior to chemotherapy as well as patients with cancer-prone syndrome have been found to show an elevated Mf. Based on the information obtained from somatic cell mutation assays, an individualized chemotherapy should be considered in order to minimize the risk of a second neoplasm.     

Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

Biochemical properties of a penicillinase from Escherichia coli carrying Rms 298.

We obtained two R plasmids, i.e., Rms195 and Rms298, from a clinical isolate, E. coli GN5503. Penicillin beta-lactamase (PCase) was extracted from ML1410 Rms195+ and Rms298+, and was purified by chromatography. Rms195 PCase was identical to the type I PCase mediated by R-TEM, RI and Rms212. The isoelectric point of Rms298 PCase was 5.9 and its molecular weight was 21,000 +/- 1,000. The substrate profile and physiochemical properties indicate that Rms298 PCase belongs to the type IV PCase mediated by Rms139 isolated from Pseudomonas aeruginosa.     

Effect of various reagents on the susceptibility of target cells to the cytotoxicity of guinea pig lymphotoxin (GLT).

The cytotoxic activity of guinea pig lymphotoxin (GLT) was increased when the target cells were treated with the following metabolic inhibitors: puromycin, actinomycin D, NaN3, NaF, and 2,4-dinitrophenol, whereas calf serum, N-acetyl-D-galactosamine, colchicine, and vinblastin inhibited GLT-cytotoxicity. Puromycin, an inhibitor of protein synthesis, exhibited the most potent enhancing effect on GLT-cytotoxicity. It seems likely that there are certain cellular metabolic processes depending on cellular protein synthesis which antagonize GLT-cytotoxicity. Taking advantage of this effect of puromycin, a time-saving assay method for GLT-cytotoxicity was developed.     

Immunoprotective human monoclonal antibodies against five major serotypes of Pseudomonas aeruginosa

Human monoclonal antibodies (Mabs) against the O antigens of Pseudomonas aeruginosa lipopolysaccharides (LPS) were produced by cell fusion between human tonsillar lymphocytes and P3-X63-Ag8-U1 (P3U1) mouse myeloma cells. To obtain human Mabs efficiently, 6 d culture supernatants of pokeweed-mitogen-stimulated lymphocytes (21 cultures from peripheral blood and 76 from tonsils) were assayed by ELISA. Five tonsillar lymphocytes which produced IgG antibody specific for P. aeruginosa LPS were preselected for fusion. The human Mabs, named P1-1 (IgG2, kappa), P5-1 (IgG2, lambda), P7-1 (IgG2, lambda), P8-1 (IgG2, lambda) and P10-1 (IgG2, kappa), bound with high specificity to Homma standard serotype strains A, E, B, G and I, respectively, and recognized O antigens. Each Mab showed opsonophagocytic killing activity of the corresponding serotype strain. Four of the Mabs caused agglutination at a very low concentration; a rather higher concentration of P7-1 was required for this effect. Although all the Mabs conferred type-specific protection against peritoneal infection, the strongly agglutinating Mabs provided better protection than the moderately agglutinating P7-1. The protective activity of P8-1 was estimated in compromised mice. A low dose (PD50 0.5-0.6 microgram per mouse) of P8-1 prevented subcutaneous infection in burned mice and peritoneal infection in leucopenic mice. All the hybridomas described here could be cultured in serum-free medium, and they have continued to secrete human Mabs for more than 14 months at rates of 10-20 micrograms per 10(6) cells in 24 h. These results suggested that these five human Mabs specific for O antigens might be useful in the prophylaxis and treatment of P. aeruginosa infections.     

N-type Calcium Channel in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca²⁺ channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca²⁺ channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca²⁺ channel α_1B-deficient (α_1B^−/−) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35–55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α_1B^−/− mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α_1B^−/− mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α_1B^−/− mice and was significantly inhibited by a selective N-type Ca²⁺ channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca²⁺. These results suggest that the N-type Ca²⁺ channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.     

Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis.

The specificity for 11-deoxycortisol (11-DOC) of a monoclonal antibody (mAb), designated SCET, was changed to specificity for cortisol (CS) by site-specific mutagenesis followed by random mutagenesis. The Fab form of SCET was expressed on the surface of a phage. During the first step, mutations were introduced at 14 amino acid positions in three complementarity-determining regions (CDRs) of the VH domain that seemed likely to form the steroid-binding pocket. A clone, DcC16, was isolated from the resultant library with multiple mutations and this clone was shown to have CS-binding activity but also to retain high 11-DOC-binding activity. During the second step, mutations were introduced randomly into the entire VH-coding region of the DcC16 clone by an error-prone polymerase chain reaction, and CS-specific mutant antibodies were selected in the presence of 11-DOC as a competitor. Three representative clones were analyzed with the BIAcore instrument, and each revealed a large increase in the binding constant for CS and a decrease in that for 11-DOC. Structural models, constructed by computer simulation, indicated the probable molecular basis for these changes in specificity.