Potentiating effects of methylxanthines on teratogenicity of mitomycin C in mice

A previous study demonstrated that caffeine strongly potentiated the teratogenic action of mitomycin C in mice. In the present study the effect of methylxanthines including caffeine, theophylline, theobromine (theobromine sodium salicylate), paraxanthine, and 1-methylxanthine was compared in order to analyze the structure-activity relationship. Jcl:ICR mice were injected IP with 3 mg/kg of mitomycin C, immediately followed by SC injection of each methylxanthine on day 11 of gestation. The doses of methylxanthines were calculated so that the mice received 50 mg/kg of caffeine or the equimolecular amount of the other methylxanthines. Fetuses were examined for external malformations on day 18 of gestation. Mitomycin C at 3 mg/kg and the methylxanthines at the doses used were not teratogenic. Combined administration of caffeine or theophylline with mitomycin C produced more than 80% of malformed fetuses. Although less effective than caffeine or theophylline, paraxanthine also significantly increased the incidence of malformed fetuses. Theobromine and 1-methylxanthine were virtually ineffective. From these findings, it is suggested that the methyl group at N-1 position of the xanthines is important for the enhancement but the N-1 methylation alone is ineffective unless accompanied with the substitution of the methyl moiety at the other position(s).     

Manifestation of cerebral malaria-like symptoms in the WM/Ms rat infected with Plasmodium berghei strain NK65

Conditions required for the induction of cerebral malaria (CM)-like symptoms were investigated using 12 strains of rats and 5 murine malaria strains. Among various combinations, only inbred WM/Ms rats infected with P. berghei (NK65) developed neuropathological complications that closely resembled human CM cases. When young WM/Ms rats were infected with the parasites, neurologic signs were induced followed by death in 5-10 days with almost 100% incidence, whereas aged hosts revealed strong resistance. Histologically, edematous changes, occlusion of vessels, and petechial hemorrhages were found in the brain. There was an optimum dose of parasites to induce the manifestations, and a low incidence was obtained by increased or decreased inoculum size. No correlation was found between the level of parasitemia and incidence of the disease. The other 11 rat strains inoculated with this parasite showed high levels of parasitemia, but most of their infections were self-limiting or malarial death occurred without CM-like signs. Inoculation into WM/Ms rats with other murine malaria parasites, including P. chabaudi, P. vinckei, P. yoelii (17X), and P. yoelii (nigeriensis) failed to induce CM-like manifestations irrespective of the inoculation size and the degree of parasitemia. These results indicated that P. berghei (NK65)-infected WM/Ms rats represent an experimental model for CM and certain appropriate conditions are needed for its development in both parasite and host sides.     

Calcium/calmodulin-dependent inhibition of microtubule assembly by brain synaptic junction

The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37°C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca²⁺/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0°C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca²⁺ concentration level required for the inhibition of microtubule assembly was 10^–6 M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca²⁺/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca²⁺ concentration (10^–6M) The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca²⁺/CaM-dependent protein kinase associated with SJ; i. e., major postsynaptic density protein.Abbreviations used CaM calmodulin - DTT dithiothreitol - MAPs microtubule-associated proteins - MES 2-(N-morphorino)ethanesulfonic acid - mPSDp major postsynaptic density protein - PSD postsynaptic density - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride     

Inhibition of microtubule assembly by poly(L-glutamic acid) and the site of its action

Poly(L-glutamic acid) (PGA) suppresses the polymerization of porcine brain microtubule proteins and induces the depolymerization in vitro in a concentration-dependent manner. The extent of inhibition increases with increasing molecular weight of the PGA tested. A 50% inhibition of the protein polymerization was observed at a PGA (molecular weight = 60,000) to microtubule protein ratio of 0.04 (w/w), and complete inhibition was obtained at a ratio of 0.07. Such an inhibition on the polymerization by PGA is greatly decreased when Mg2+ is present at a higher concentration. The addition of PGA raises the critical concentration of microtubule proteins necessary for assembly. During incubation with PGA, microtubule proteins retain the ability to assemble, i.e., substoichiometric amounts of taxol considerably relieve the inhibition of assembly by PGA. PGA interacts with microtubule-associated proteins (MAPs) preferentially, because the amount of MAPs binding to PGA-Sepharose 4B is much larger than that of tubulin. Tau proteins were observed only in adsorbed fractions, while MAP-2 was present in both unbound and adsorbed fractions.     

A molecular orbital study on the oxidation of hydrogen donor molecules by peroxidase compound II

The electronic characteristics of some hydrogen donor substrate (phenol and aniline derivatives) for peroxidase reaction were calculated with the aid of the CNDO/2 and other methods. These results were compared with the experimental data concerning the rate of oxidation of these compounds by peroxidase and hydrogen peroxide. No simple relationship between the total or frontier electron densities on the oxygen or nitrogen atoms, or the lowest unoccupied orbital energies, and the rate of oxidation was found. It was, however, found that the logarithm of the rate of oxidation for the compounds studied correlates linearly with the highest occupied orbital energies. Based on these results, the mechanism of electron transfer from the substrate to compound II is discussed.     

A sensitive colorimetric assay for various proteases using naphthyl ester derivatives as substrates

A convenient and highly sensitive colorimetric assay for various proteases, such as trypsin, chymotrypsin, plasmin, thrombin, and urokinase is described. The substrates used are alpha-naphthyl ester derivatives of N alpha-tosyl-L-lysine, N alpha-acetylglycyl-L-ination of alpha-naphthol released from them. Use of these alpha-naphthyl ester derivatives made the method more sensitive than the use of the corresponding methyl or ethyl ester derivatives. The minimum detectable concentrations of trypsin, chymotrypsin, plasmin, thrombin and urokinase in this method were about 0.002 micrograms, 0.01 microgram, 0.002 CU, 0.01 IU, and 2 IU, respectively. The Km values of trypsin and thrombin for TLNE were 0.11 mM and 0.15 mM while those for TLME were 2.5 mM and 6.7 mM, respectively; the Km values of chymotrypsin for ATNE and ATEE were 0.18 mM and 0.7 mM, respectively; and the Km values of urokinase for AGLNE and AGLME were 0.17 mM and 4 mM, respectively. Zymograms of various proteases were easy to prepare using these alpha-naphthyl ester substrates, and zymograms of trypsin and chymotrypsin were made with TLNE and ATNE, respectively, as substrates.     

Exposure of aromatic residues of Streptomyces subtilisin inhibitor. A photo-CIDNP study

Exposure of aromatic residues, Tyr 7, Tyr 75, Tyr 93, His 43, His 106, and Trp 8, was studied by laser-induced photo-CIDNP in the 1H NMR spectrum of Streptomyces subtilisin inhibitor at 360 MHz. Only Tyr 7 and Tyr 75 gave strong CIDNP signals, whereas the rest of the aromatic residues gave no detectable signals in the temperature range 25-55 degrees C. From the temperature dependence data, it is concluded that Tyr 7 is well exposed at all temperatures, whereas the exposure of Tyr 75 increases with temperature, in agreement with the conclusion obtained by other methods. Agreements and discrepancies between the conclusions derived from the CIDNP data and the results so far obtained by other methods are compared for all the aforementioned aromatic residues.     

Maternal Exogastrula-Inducing Peptides (EGIPs) and Their Changes during Development in the Sea Urchin Anthocidaris crassispina

Exogastrula-inducing activity was examined in eggs and embryos of the sea urchin Anthocidaris crassispina at various stages. During fractionation on a column of DEAE-cellulose, the exogastrula-inducing activity was found in the flow-through fraction at all developmental stages. In particular, the activity present in the flow-through fraction of unfertilized eggs represents the presence of maternal exogastrula-inducing peptides (EGIPs). The flow-through fractions from the column of DEAE-cellulose were applied to a column of Sephadex G-100 and the activities in the eluate were assayed. The active low-molecular-weight fraction was obtained in all cases with the exception of pluteus larvae, extracts of which contained another active fraction. Immunoblots of protein samples from eggs and embryos probed with antiserum against EGIP-D indicated that there is a major immunoreactive protein that migrates with an apparent molecular weight of about 6 kDa in all cases with the exception of pluteus larvae, and that there are two major immunoreactive proteins that migrate with apparent molecular weights of 6 kDa and 35 kDa, respectively, in pluteus larvae.     

Kinetics of the hydrolysis of monodispersed dihexanoylphosphatidylcholine catalyzed by bovine pancreatic phospholipase A2: roles of Ca2+ binding and ionizations of amino acid residues in the active site.

Phospholipases A2 (PLA2s) are classified into two groups, I and II, according to differences in the polypeptide-chain length and intramolecular disulfide bondings. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by bovine pancreas PLA2 (Group I) was studied at 25 degrees C and ionic strength 0.2, and the initial velocity data were analyzed by means of the Michaelis-Menten equation. The Michaelis constant, Km, was found to be practically independent of Ca2+ concentration and also of pH value. The latter result indicates no participation of the ionizable groups in the active site in the substrate binding. The pH-dependence curve of the logarithm of the catalytic center activity, kcat, obtained in the presence of a practically saturating amount of Ca2+, showed three transitions ascribable to the participation of three ionizable groups with pK values of 5.00, 8.40, and 9.50. The respective groups were tentatively assigned to the catalytic group His 48, the N-terminal alpha-amino group, and invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48. Deprotonation of His 48 and protonation of Tyr 52 were shown to be essential to the catalysis. The importance of the ionization state of the alpha-amino group was also indicated.(ABSTRACT TRUNCATED AT 250 WORDS)