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.     

Effect of IL-2 in vitro on CTL generation in spleen cells of young and old mice: asialo GM1+ cells are required for the apparent restoration of the CTL response

The role of asialo GM1+ (ASGM1+) cells and exogenous IL-2 in the age-related decline in allospecific CTL activity was evaluated. Primary CTL were generated in mixed leukocyte culture (MLC) [BALB/cANN (H-2d) anti C57BL/6N (H-2b)] and tested for allospecific lytic activity against the EL-4 (H-2b) cell culture line, and for non-MHC-restricted activity against WEHI-3 (H-2d) and YAC-1 (H-2a). Cultures included responder cell populations which had been treated with antibody to ASGM1 plus complement or complement alone, and irradiated stimulator cells, in the presence or absence of rIL-2 or crude IL-2-containing supernatants. The amount of rIL-2 used to accommodate the age-related decline in IL-2 production was determined empirically to be 500 U by assessing IL-2 production in MLCs containing responder cells from young versus old animals. rIL-2 appeared to restore the allospecific CTL activity generated by spleen cells of old mice to the level of that of young. However, treatment with anti-ASGM1 antibody revealed that this restoration was due to an effect of the IL-2 on ASGM1+ cells. The allospecific target cells, EL-4, were not sensitive to lymphokine-activated killer (LAK) cells induced by IL-2 alone under the conditions used. It is suggested that the apparent restoration was due to increased LAK-like (or MHC-nonrestricted) activity mediated by an ASGM1+ cell in the CTL precursor population.     

Three types of vitronectin in human blood

Vitronectin is a cell-adhesive glycoprotein in serum and plasma, also termed serum spreading factor and complement S-protein. It consists of a mixture of a polypeptide of molecular weight 75 kilodalton (kDa) and its nicked product of 65 kDa plus 10 kDa. By a quantitative immunoblotting assay, human blood samples could be classified into three distinct vitronectin types; type I (58% of the population) was 75 kDa rich and 65 kDa poor, type II (35% of the population) contained approximately equal amounts of 75 kDa and 65 kDa, and type III (5% of the population) was 75 kDa poor and 65 kDa rich. The vitronectin type did not correlate with age, sex, or ABO blood type.     

Chromatin diminution and chromosome elimination in four Japanese hagfish species

Cytogenetic examination of four Japanese hagfish species belonging to the order Myxinida (Eptatretus okinoseanus, E. burgeri. Paramyxine atami, and Myxine garmani) revealed differences in chromosome number between germ cells (spermatocytes and spermatogonia) and somatic cells (liver, blood, gill, and kidney). The differences in chromosome number between spermatogonia (54, 52, 48, and 16) and somatic cells (34, 36, 34, and 14) were 20, 16, 14, and 2 in E. okinoseanus, E. burgeri, P. atami, and M. garmani, respectively. The amount of DNA in a somatic cell (2C) relative to that in a germ cell (2C) averaged 54.6% (E. okinoseanus type A), 44.9% (E. okinoseanus type B), 79.1% (E. burgeri), 60.0% (P. atami), and 70.2% (M. garmani). These results clearly indicate that chromosome elimination takes place during early cleavage in the four hagfish species of Myxinida living in Japanese waters, except in the ancestral germline cells. C-banding of metaphase chromosome preparations of germline and somatic cells from each hagfish species revealed that the C-band-positive chromatin in the ancestral somatic cells had been almost completely eliminated. Three patterns of elimination of this chromatin are discussed.     

Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers

A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.     

Purification and characterization of an antibacterial factor from snail mucus

The antibacterial factor from the body surface of the African giant snail, Achatina fulica Férussac, was isolated by DEAE-Toyopearl 650M ion exchange chromatography. The isolated preparation exhibited highly positive antibacterial activity both for the Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus and for the Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, but it lost such activity when heated at 75 degrees C for 5 min. The antibacterial factor of the snail mucus was a glycoprotein whose molecular weight (MW) was about 160,000. It was composed of two subunits of MW 70,000-80,000.     

Conformation of sequential polypeptides of (Lysi-Leuj), (Lysi-Serj), and (Lys-Gly) in sodium dodecyl sulfate solution

A series of sequential polypeptides (Lys_iR_j)_n (R is Leu, Ser, or Gly) and random copolypeptides, (Lys^x, Leu^y)_n, were synthesized. Their conformation in NaDodSO₄ solution was determined by CD. Only (Lys-Leu)_n, (Lys-Ser)_n, and (Lys₃-Ser)_n adopt a stable β-form in the surfactant solution; (Lys-Ser₂)_n, (Lys-Ser₃)_n, (Lys₂-Ser₂)_n, and (Lys₂-Ser)_n have an unstable β-form, which reverts to an unordered form in high NaDodSO₄ concentrations, even though both Ser and DodSO-bound Lys⁺ are β-formers. In contrast, (Lys-Gly)_n remains unordered in NaDodSO₄ solution. On the other hand, Lys-rich (Lys₂-Leu)_n forms an unstable helix and (Lys₂-Leu₂)_n a stable helix in NaDodSO₄ solution. In 25 mM NaDodSO₄ (Lys^x, Leu^y)_n also forms a helix up to x = 75 and reverts to the β-form at x = 90. This compares with the helical conformation of (Lys^x, Ala^y)_n up to x = 65 and its β-form at x = 90, suggesting that Leu is an even stronger helix-former than Ala. Our results may provide a plausible explanation for the increase in helicity and disruption of the β-form for many proteins in NaDodSO₄ solution, that is, the polypeptide chain of a protein usually favors a helical conformation over a β-form in the presence of excess surfactant.     

Antithrombotic effect of topically applied 3-Oxa-Methano-prostaglandin I1 in the microcirculation and antiplatelet functions of its active form

The antithrombotic effect of topical application of the 3-oxamethano-prostaglandin (PG) I₁ analog, SM-10902 in the microcirculation and in vitro antiplatelet functions of its active form SM-10906 were estimated in comparison with PGI₂ and PGE₁. In rat platelets, SM-10906 evoked accumulation of intracellular cyclic adenosine 3′,5′-monophosphate, and exhibited antiaggregatory and disaggregatory activities, which were all enhanced by the phosphodiesterase inhibitor theophylline. Additionally, SM-10906 was shown to inhibit platelet adhesion to collagen in human platelet-rich plasma. PGI₂ and PGE₁ also showed in vitro antiplatelet effects in the order of PGI₂ > SM-10906 ≥ PGE₁. SM-10902 exhibited a dose-dependent antithrombotic effect in the guinea pig mesenteric arteriole by a topical application, and this activity might be exerted by the antiplatelet functions of SM-10906. Although SM-10906, PGI₂ and PGE₁ also showed the antithrombotic effects, SM-10902 was the most potent. In conclusion, the present studies indicate that an external topical preparation of SM-10902 may be useful for the therapy of peripheral circulatory insufficiency.     

Alkaline phosphatase reactivity in rabbit airway epithelium: a potentially useful marker for airway basal cells

The purpose of this study was to investigate alkaline phosphatase (ALPase) reactivity in rabbit airway epithelial cells. Acetone-fixed, methyl benzoate and xylene-cleared (AMeX-treated) paraffin sections of trachea, bronchus, and lung tissue were stained by an azo dye coupling method for ALPase and examined by light microscopy. Electron histochemical staining was also performed in order to study the sensitivity and specificity of reactivity in each cell type. ALPase reactivity at the light microscopic level was observed exclusively in trachco-bronchial basal cells, and not in bronchiolar basal cells. By electron microscopy, ALPase reactivity was noted in 97.9% of basal cells in the trachea, 97.0% of basal cells in the bronchus, and 94.5% of basal cells and 15.4% of Clara cells in the bronchiole. This was also true for dispersed tracheal epithelial cells. Reactivity was rarely observed in ciliated cells, non-goblet-type secretory cells, and undetermined cells. The reactivity was heatlabile, levamisole-sensitive, and of a non-specific type. These findings indicate that basal cells of rabbit trachea and bonchus have fairly high specificity for ALPase of a non-specific isozyme (92.2% and 95.6%, respectively). Therefore, ALPase is considered to be a useful marker for these cells.