Selective inhibition of thrombin by (2R,4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl++ +) sulfonyl]-l-arginyl)]-2-piperidinecarboxylic acid

The potency of thrombin inhibition by 4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfony l]- L-arginyl]-2-piperidinecarboxylic acid (MQPA) depended on the stereoconformation of the 2-piperidinecarboxylic acid moiety. Ki values for bovine alpha-thrombin were 0.019 microM with (2R,4R)-MQPA, 0.24 microM with (2R,4S)-MQPA, 1.9 microM with (2S,4R)-MQPA, and 280 microM with (2S,4S)-MQPA. (2R,4R)-MQPA of the four stereoisomers of MQPA was also the most potent inhibitor for other trypsin-like serine proteases with Ki values of 5.0 microM for trypsin, 210 microM for factor Xa, 800 microM for plasmin, and 1500 microM for plasma kallikrein. Examination of the potency of thrombin inhibition by arginine derivatives related to MQPA in structure suggested the presence of a specific binding site for the carboxamide portion (C-terminal side). The relative inhibitory potency of the four stereoisomers of MQPA for trypsin was nearly identical with that for thrombin, suggesting that the specific binding site for the carboxamide portion is present in both enzymes. Modification of thrombin by phosphopyridoxylation or the presence of heparin did not significantly alter the binding of MQPA.     

Structural changes of alpha2- and ovomacroglobulins

The plasma α₂-macroglobulin and its egg white homologue ovomacroglobulin were purified from several different species and their structure before and after the reaction with proteinases studied by electron microscopy. The negatively stained specimens showed either a ringlike structure or a flowerlike one before the reaction with proteinses, but their structures changed into open rectangular ones after the reaction. The translational frictional ratio f/f ₀ of human α2-macroglobulin and crocodilian ovomacroglobulin given in the literature is between 1.5 and 1.6 before and after the reaction with proteinases. The value reflects asymmetry due not to a high axial ratio, but rather to an openness of the structure resulting in a partially free draining character of the molecules. The computational method developed by Bloomfield and his co-workers based on the formalism of Kirkwood is used to calculate the frictional ratio of several models constructed from small spheres. The overall shape of the models is derived from electron micrographs. Although the degree of hydration is an unknown parameter in the calculation, reasonable agreement is obtained between the experimental values of f/f ₀ and the calculated ones. Combination of electron microscopic and hydrodynamic methods would be fruitful in the structural study of giant proteins such as α₂-macroglobulin.     

Molecular characterization of mutant actin genes which induce heat-shock proteins in Drosophila flight muscles

Heat-shock proteins (hsps) are constitutively induced by the mutant actins in the Drosophila indirect flight muscles (IFM). We compared primary structures of the mutant actin genes (KM75 and HH5) which induce hsps and of the non-inducing alleles (KM129 and KM88). The KM75 actin has lost 20 amino acids at the C-terminus. The HH5 actin has only one amino acid substitution, from Gly-336 to Ser. In KM129, the C-terminal part of actin is replaced by novel amino acids. KM88 is a null allele, with an amber mutation early in the coding region of the mutated actin gene. Although all of the KM75, HH5 and KM129 actins have defects near the C-terminus, only hsp-inducing mutant actins cause enlargement of the IFM nuclei as well as a disruption of myofibrils even in the presence of two copies of the normal genes. We further consider the underlying mechanisms linking these features of the hsp-inducing alleles.     

Continuous requirement of cAMP for pre-spore differentiation in Dictyostelium discoideum

Abstract Using a shaking culture system, we have previously shown that both cell contact and cAMP are required for pre-spore differentiation in Dictyostelium discoideum [2]. In the present study, cAMP was removed from the medium by the use of a hydrolysing enzyme after cells had formed agglomerates. This treatment left the agglomerates unchanged, but caused a rapid decrease in the activity of UDP galactose transferase, a pre-spore-specific enzyme. This result indicates that cAMP is required even after agglomerate formation to maintain pre-spore differentiation.     

Detection of carcinogen-induced DNA breaks by nick translation in permeable cells

A nick-translation reaction with $\text{E. coli}$ DNA polymerase I (pol. I) was used to detect $\text{in situ}$ DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [³H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.     

Changes in cerebral norepinephrine induced by vibration or noise stress

To investigate the effects of whole body vibration on the central nervous system, rats were exposed to various whole body vibrations and examined for changes in the levels of norepinephrine (NE) in whole brain or regions of the brain. Whole brain NE had decreased significantly (P less than 0.05) after an acceleration of 5.0G with a frequency of 20 Hz; and the decrease was also observed in the hypothalamus (P less than 0.01) and the hippocampus (P less than 0.10). Exposure to noise [100 dB (A)] caused a significant decrease in NE. This decrease related particularly to a significant decrease in midbrain NE (P less than 0.05) and a non-significant decrease of NE in the hypothalamus.     

Optimal control mode of a biochemical feedback system

An optimal feedback system for constant-value control of biochemical reaction system was investigated by computer simulations. A feedback system containing a cyclic enzyme system where two enzyme types share a substrate in a cyclic manner, was found to be the most reliable one. This feedback system has a capability to keep the stationary value of the end product at a desired level against not only exogenous substrate supply but also endogenous parametric disturbances. The cyclic enzyme system installed as a control element of this feedback system played the role of comparator in this feedback system. The control mode of this feedback system was in good agreement with that of a system established by means of optimization technique based on the maximum principle. Also bang - bang control could be performed in this biochemical feedback system as well as in electrical one.