TEI-9063, a stable and highly specific prostacyclin analogue for the prostacyclin receptor in mastocytoma P-815 cells.

The prostacyclin (PGI2) analogues, TEI-9063 and its methyl ester, TEI-1324, have been compared with another stable analogue, iloprost, with respect to binding to the PGI2 receptor, stimulation of adenylate cyclase activity and inhibition of thrombin-induced Ca2+ mobilization in mastocytoma P-815 cells. TEI-9063 displaced the [3H]iloprost binding to the membrane fraction, the IC50 value being 3 nM, but showed very low affinity for the PGE receptor. TEI-9063 dose dependently stimulated cAMP formation in the cells and GTP-dependent adenylate cyclase activity in the membrane fraction, the EC50 value being 50 and 10 nM, respectively. Furthermore, TEI-9063 prevented the thrombin-induced increase in the intracellular Ca2+ concentration, the IC50 value being 50 nM. These IC50 and EC50 values are lower than those obtained for iloprost. On the other hand, those of TEI-1324 were about two-orders higher. Although PGI2 lost its ability to stimulate cAMP formation by preincubation for 20 min at 37 degrees C, TEI-9063 completely retained its ability after 60-min preincubation. These results demonstrate that TEI-9063 is a stable and stronger agonist for the PGI2 receptor than iloprost, and that it prevents thrombin-induced Ca2+ mobilization through stimulation of the adenylate cyclase system in mastocytoma cells.     

Changes of carp myosin subfragment-1 induced by temperature acclimation

Summary Heavy meromyosin subfragment-1 (S1) was prepared by -chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca²⁺- and K⁺ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 mol P_i·min^-1·mg^-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (K_D) of S1 from cold-acclimated carp was 32.1x10^-4· s^-1, compared to 13.2x10^-4·s^-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg²⁺-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s^-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - K _m apparent dissociation constant - P _i inorganic -phosphate - PMSF phenylmethane-sulfonyl fluoride - S ₁ heavy meromyosin subfragment-1 - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TPCK N-tosyl-l-phenylalanyl chloromethyl ketone - V _max maximum initial velocity     

Detection of tetrodotoxin by thin-layer chromatography/fast atom bombardment mass spectrometry

A new method for detection of tetrodotoxin (TTX) by thin-layer chromatography/fast atom bombardment (FAB) mass spectrometry was developed. TTX and/or related substances were separated by TLC on LHP-K high-performance precoated plates, with a solvent system of pyridine:ethyl acetate:acetic acid:water (15:5:3:4). The plates were subjected to positive FAB mass spectrometry, under scanning within a mass range from m/z 100 to 500. TTX was identified by selected ion-monitored chromatograms at m/z 320 (M + H)+ and 302 (M + H - H2O)+, along with full scan positive ion FAB mass spectrometry. The limit of detection for TTX was about 0.1 micrograms. TTX was also detected by cellulose acetate membrane electrophoresis/FAB mass spectrometry.     

The amino acid sequence of ribonuclease U1, a guanine-specific ribonuclease from the fungus Ustilago sphaerogena

The complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11,235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.     

Changes in aerobic and anaerobic ATP-synthesizing activities in hypoxic mouse brain

The changes in cerebral metabolism in mice in severe hypoxia were investigated by analyses of changes in the levels of energy metabolites and near-infrared spectrophotometric assessment of the states of hemoglobin and cytochrome oxidase. Under 4.4% O2, the contribution of anaerobic ATP production was at most about 20% of the demand. However, the cerebral ATP level was kept at the control level until about 1 min before death. Pentobarbital anesthesia, which reduced the cerebral rate of metabolism, prolonged the survival time, although anaerobic ATP production still did not support ATP demand. Under these conditions, deoxygenation of hemoglobin and reduction of cytochrome oxidase proceeded rapidly within 1 min. Hemoglobin reached the maximum state of deoxygenation in the middle phase of hypoxia, with no further change until death. However, cytochrome oxidase was reduced slowly with one phase of partial reoxidation due to increase of cerebral blood volume, and reached the completely reduced state at death. From these results it was concluded that the aerobic ATP synthesis, which supplied more than 80% of the cerebral demand, decreased gradually because of limitation of oxygen supply, and that the failure of oxidative phosphorylation to meet demand triggered the decrease in the cellular ATP level that led to death.     

A male-specific renal cytochrome P-450 isozyme(s) responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene in mice

Renal microsomes from male mice (BALB/c, DBA/2 and BALB/c x DBA/2 F1) showed about 10-fold greater activity for mediating mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) toward Salmonella typhimurium TA98 than did the corresponding hepatic microsomes, as compared on the basis of nmol of microsomal cytochrome P-450. On the other hand, female renal microsomes and other extrahepatic microsomes (lung, small intestine and colon) in both sexes of mice showed little or no activity for converting 3-MeO-AAB to mutagen(s). The mutagenic activation of 3-MeO-AAB with the male renal enzyme(s) was definitely inhibited by cytochrome P-450 inhibitors, 7,8-benzoflavone and SKF 525A. All these findings suggest that in mice, there is a male-specific renal 3-MeO-AAB activation enzyme(s), a cytochrome P-450 isozyme(s), which is different, at least in proportion and/or in nature, from hepatic cytochrome P-450 isozymes.     

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.     

Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA:cholesterol acyltransferase

The influence of chronic ethanol ingestion on hepatic acyl-CoA: cholesterol acyltransferase activity was investigated to determine the relationship between alcohol intake and cholesterol ester accumulation. Rats were given nutritionally complete liquid diets supplemented with 6.3% ethanol or an isocaloric equivalent of dextrin-maltose for 5 weeks. During this period, the hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed male rats remained constant, whereas the same activity in pair-fed controls as well as chow-fed rats exhibited a 30% decrease in activity. Unlike alcohol-fed male rats, the hepatic acyl-CoA: cholesterol acyltransferase activity of female rats decreased by approximately 30% by the fifth week of ethanol ingestion. Despite the fact that the gender of the animals led to disparate levels of acyl-CoA: cholesterol acyltransferase activity in response to ethanol ingestion, similar levels of cholesteryl ester accumulation were observed. The altered levels of acyl-CoA: cholesterol acyltransferase activity caused no significant change in the cholesterol concentration, cholesterol/phospholipid ratio, phospholipid fatty acid composition, or the membrane fluidity of the hepatic microsomes. We conclude that the altered hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed female rats cannot be directly responsible for ethanol-induced accumulation of cholesteryl esters.