Biotin derivatives of endothelin: utilization for affinity purification of endothelin receptor.

Three different types of biotinylated endothelin 1 (ET-1) derivatives, [Cys1]-biotinylated ET-1, [Lys9]-biotinylated ET-1, and [Cys1][Lys9]-dibiotinylated ET-1, were obtained when the biotinylation reaction was carried out with sulfosuccinimidyl-6-(biotinamido)hexanoate in an aqueous solvent. The binding of [Lys9]-biotinylated ET-1 to the ET receptor was as efficient as that of natural ET-1, whereas the binding of either [Cys1]-biotinylated ET-1 or [Cys1][Lys9]-dibiotinylated ET-1 was significantly reduced. When ET-1 was reacted with succinimidyl-6-(biotinamido)hexanoate in an organic solvent, ET-1 was exclusively modified at lysine 9. The ET receptor was then isolated from human placenta by affinity chromatography with [Lys9]-biotinylated ET-1 and avidin-agarose. The purified ET receptor was active in ET binding and was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two polypeptides with apparent molecular masses of 45 and 35 kDa. The NH2-terminal amino acid sequence indicated that the two polypeptides were from an identical subtype of the ET receptor (ETB, the ligand-nonselective type). A signal peptide from Met1 to Gly26 was missing from the 45-kDa ETB, whereas 64 amino acids at the NH2 terminus were missing from the 35-kDa ETB due to proteolytic cleavage which occurred between Arg64 and Ser65. Indeed, incubation of purified ETB with endopeptidase Arg-C resulted in degradation of the 45-kDa ETB, giving rise to the 35-kDa species by a specific cleavage at Arg64. The 35-kDa ETB was active in binding to ET-1, indicating that the NH2-terminal 64-amino-acid residues are not essential for ligand binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absence of correlations between radiosensitivities of human T-lymphocytes in G0 and skin fibroblasts in log phase

Dose-survival curves were obtained for matched samples of peripheral T-lymphocytes and skin fibroblasts from a total of 22 patients who underwent various surgical procedures using loss of colony-forming ability as the end point. The results showed that the mean D10 (dose required to kill 90% of cells) +/- SD was 3.58 +/- 0.21 Gy for T-lymphocytes irradiated in G0 and 3.19 +/- 0.37 Gy for skin fibroblasts irradiated in log phase. The coefficients of variation were found to be 6 and 11%, respectively. Contrary to the expectation, regression analysis of D10 values for the two types of cells revealed no significant correlations. The absence of correlation most probably derives from the fact that the apparent interindividual variability of dose-survival curves is caused primarily by random experimental fluctuations at least in the case of lymphocytes. Possible reasons for the greater variability observed in the fibroblast assay are discussed.     

Role of the autonomic nervous system in cyclophosphamide-induced heart mitochondrial dysfunction in rats

This study was designed to clarify mechanisms responsible for cyclophosphamide-induced cardiotoxicity. Rats were divided into 2 groups: the cyclophosphamide group, which received cyclophosphamide (100 mg/kg) intraperitoneally once a day for 4 consecutive days; and the control group, which remained untreated. In each group, myocardial mitochondrial respiratory function, enzymic activities in the respiratory chain, and ventricular acetylcholine and norepinephrine concentrations were measured. In the cyclophosphamide group, decreases in mitochondrial respiratory function and in enzymic activities in the respiratory chain were observed compared with those of the control group. Administration with cyclophosphamide caused increases in acetylcholine and norepinephrine in the myocardium. As an increase in tissue acetylcholine level is reported to be linked with the genesis of myocardial damage, we conclude that cyclophosphamide-induced cardiotoxicity is closely related to mitochondrial dysfunction and that alterations in the autonomic nervous system might be related to this dysfunction.     

Gene frequencies of S-adenosylhomocysteine hydrolase (SAHH) in a Japanese population

Summary Genetic polymorphism of S-adenosylhomocysteine hydrolase (SAHH) was investigated in a total of 214 red blood cell samples from unrelated Japanese using the starch gel electrophoresis and the enzyme-specific staining procedures. Three common phenotypes were observed which corresponded to SAHH 1, SAHH 2-1, and SAHH 2, controlled by two alleles, SAHH^*1 and SAHH^*2. The estimated gene frequencies of SAHH^*1 and SAHH^*2 in Japanese were 0.953 and 0.047, respectively. This result was not different from European samples reported by Bissbort et al. (1983).     

Direct observation of the properties of cholesterol in membranes by deuterium NMR

The properties of cholesterol in bilayers of egg phosphatidylcholine (PC) were investigated directly by means of ²H-NMR of specifically-deuterated species (C3, C7, C26, C27). Quadrupole splittings were a measure of molecular ordering, and relaxation times T₁ and T_2e were indicators of rates of motion. The importance of the use of echoes for spectral acquisition is emphasised, particularly to obtain accurate values of the quadrupole splitting. In the case of overlapping powder patterns from two labelled positions, the use of the absolute value mode of spectral presentation is shown to yield reasonable estimates of the individual quadrupole splittings. Spectral properties were monitored as a function of cholesterol concentration and temperature. Increasing cholesterol concentration led to a high degree of ordering for the rigid ring system of cholesterol, approaching a molecular order parameter of 0.8 at 50 mol% cholesterol. The isopropyl methyl groups were in all cases less ordered anmore mobile than the ring system, but responded in a similar fashion to variable cholesterol concentration and temperature. The observation of a minimum in the temperature dependence of T₁ for cholesterol-7,7-d₂ led to a direct estimate of its correlation time for molecular motion, 3.5 × 10^?9 s rad^?1. This indicates that the overall rate of motion of cholesterol is considerably slower than that of the lipids in which it is located. The short T_2e values suggest that the motional spectrum of cholesterol is rich in low frequencies. The parallel temperature and cholesterol dependences of quadrupole splittings for different positions on the rigid ring system of cholesterol indicate that the position of the axis of motional averaging of the molecule is not changing, and is the same as that determined in an earlier study. It is emphasised that the steep temperature dependence and small quadrupole splittings for the chain isopropyl methyl groups of cholesterol do not necessarily indicate a high degree of disorder, but may be due to their axes of motional averaging lying at angles close to 54° with respect to the director of the ordered lipids.     

The structure of fibronectin and its role in cellular adhesion

Fibronectin is a large, adhesive glycoprotein which is found in a number of locations, most notably on cell surfaces, in extracellular matrixes, and in blood. Fibronectin has been detected in all vertebrates tested and in many invertebrates. Its presence in sponges is significant because this suggests that fibronectin may have appeared very early in evolution, possibly with the most primitive multicellular organisms. Cellular and plasma fibronectins have many striking similarities. However, the locations of the polypcptide chain differences between these two proteins indicate that plasma fibronectin cannot be derived from cellular fibronectin by means of simple post-translational proteolysis. Instead, these different types of fibronectin may be products of different genes or of differentially spliced messenger RNA molecules. Amniotic fluid fibronectin is possibly a third form of the protein. Cellular and plasma fibronectins are composed of at least six protcaseresistant domains which contain specific binding sites for actin, gelatin, heparin, Staphylococcus aureus, transglutarninase, fibrin, DNA, and a cell surface receptor. The relative locations of these domains have been mapped in the primary structure of fibronectin. The cell surface receptor for fibronectin has not been positively identified, but may be a glycoprotein, a glycolipid, or a complex of the two. Although cell-substratum adhesion is mediated by fibronectin, the locations of the areas of closest approach of the cell to the substratum (the adhesion plaques) and fibronectin are not coincident under conditions of active cell growth. Under conditions of cell growth arrest in low scrum concentrations, some fibronectin may become localized at the adhesion plaques. Models describing the domain structure of fibronectin and the molecular organization of the adhesion plaque area are presented.     

Stimulated rapid expression in vitro for early detection of in vivo T-cell receptor mutations induced by radiation exposure

The T-cell receptor (TCR) mutation assay for in vivo somatic mutations is a sensitive indicator of exposure to ionizing radiation. However, this assay cannot be immediately applied after radiation exposure because expression of a mutant phenotype may require as long as several months. In the present study, we eliminate this time lag by stimulating lymphocytes with a mitogen that can accelerate the turnover of TCR protein expression in T-cells. When lymphocytes obtained from healthy donors were irradiated with various doses of X-rays and cultured with human interleukin-2 after phytohemagglutinin (PHA) pulse stimulation, the mutant frequency (MF) of CD4⁺ T-cells increased dose dependently during the first 7 days, then decreased rapidly due to the growth disadvantage of mutant cells. This suggests that PHA stimulation can shorten the expression time of a mutant phenotype to within a week after radiation exposure. The relationship between radiation dose and TCR MF on the seventh day was best fitted by a linear-quadratic dose–response model. We applied this improved TCR mutation assay to gynecological cancer patients who received 5 days of localized radiotherapy, totaling about 10 Gy. The in vivo TCR MF in the patients did not change within a week after radiotherapy, whereas the in vitro TCR MF of PHA-stimulated lymphocytes from the same patients significantly increased 7 days after initiating culture. The estimated mean radiation dose to the peripheral blood lymphocytes of the cancer patients was about 0.9 Gy, based on the in vitro linear-quadratic dose–response curve. This estimated dose was close to that described in a previous report on unstable-type chromosome aberrations from cervical cancer patients after receiving the same course of radiotherapy. On the basis of these findings, we propose that the improved TCR mutation assay is a useful biological dosimeter for recent radiation exposure.     

High-performance liquid chromatography of methamphetamine and its related compounds in human urine following derivatization with fluorescein isothiocyanate

A high-performance liquid chromatographic method with fluorescence detection for the determination of methamphetamine and its related compounds is reported. Methamphetamine, amphetamine, norephedrine, p-hydroxymethamphetamine and 1-phenylethylamine as an internal standard were extracted from human urine, derivatized with fluorescein-4-isothiocyanate, and then separated on a reversed-phase column within 36 min. The fluorescence intensity of the effluent was monitored at excitation and emission wavelengths of 496 and 518 nm, respectively. Calibration curves were confirmed to be linear up to at least 100 pmol on the column with a correlation coefficient (r) of 0.994–0.999 for the target compounds. The detection limits (S/N=3) were 55–105 fmol per 20-μl injection. The method was successfully applied to urine samples taken from methamphetamine addicts.     

In Vivo Electrochemical Measurement of the Long-Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10⁻⁵ M ) and high (5 × 10⁻³ M ) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10⁻⁵ KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA-induced changes were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA-induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA-mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.