Ethoxyformylation of histidine residues in equine growth hormone

Reactivity of histidine residues in equine growth hormone to ethoxyformic anhydride was studied. The existence of two kinetically different sets was demonstrated: one of them including only the slow reacting histidine 169 (k = 0.164 min-1) and the other containing fast reacting histidines 19 and 21 (k = 0.892 min-1). A correlation between the decrease in the capacity to compete with 125I-labeled hormone for rat liver binding sites and the degree of ethoxyformylation of the fast group was found. Circular dichroism studies indicated no significant conformational changes in the protein with all three residues modified. These results fully agree with those obtained for bovine growth hormone which is further evidence supporting the vinculation of histidines 19 and/or 21 with the binding site of these hormones to their specific receptors.     

Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Enhancement of γ-linolenic acid production by Mucor ambiguus with nonionic surfactants

Summary -Linolenic acid (GLA) production by Mucor ambiguus IFO 6742, immobilised in Biomass Support Particles (BSPs), has been investigated in a fluidized-bed fermenter in the presence of nonionic surfactants. In this system, repeated batch cultivation was achieved at higher yield and productivity than by conventional methods, since microbial lipids inlcuding GLA were significantly secreted into the culture broth and/or on the surface of the cell wall.     

Measurement of Serine Acetyltransferase Activity in Crude Plant Extracts by a Coupled Assay System Using Cysteine Synthase

Serine acetyltransferase (SATase) (EC 2.3.1.30 [EC] ) catalyzes theformation of Oacetyl-L-serine (OAS) from L-serine in the presenceof acetyl-CoA. A novel assay method was developed for measuringthis enzyme activity in extracts from plant tissues. The assayconsists of a coupled system in which the OAS formed is convertedto cysteine by the addition of cysteine synthase (CSase) (EC4.2.99.8 [EC] ). Cysteine thus formed is determined colorimetricallyand serves as a measure for SATase activity. This method israpid, simple and sensitive, and can be readily adapted formeasurement of SATase activity in crude tissue extracts or homogenates. (Received January 14, 1987; Accepted April 27, 1987)     

Kyotorphin (tyrosine-arginine) synthetase in rat brain synaptosomes

Kyotorphin (Tyr-Arg) is a unique neuropeptide which produces analgesia by releasing Met-enkephalin from slices of the brain and spinal cord. Recent studies revealed that kyotorphin possesses the properties of neurotransmitter/neuroregulator. In the present study, we identified a kyotorphin synthetase in the soluble fraction of rat brain synaptosomes (synaptosol) and characterized it. The enzyme partially purified with Sephacryl S-300 showed an absolute requirement for ATP, MgCl2, tyrosine, and arginine. The optimal pH was 7.5-9.0 and the pI was determined to be 6.1-6.2 by isoelectric focusing. The Km was 25.6 microM for tyrosine, 926 microM for arginine, 294 microM for ATP, and 442 microM for MgCl2. The Vmax was 34.0 pmol/mg of protein/h. The apparent molecular size of this "kyotorphin synthetase" further purified by the DE52 column was 240,000-245,000 daltons, estimated using TSKgel G4000SW column chromatography. The enzyme reaction is represented by the following equation: Tyr + Arg + ATP + MgCl2 + kyotorphin synthetase----Tyr-Arg (kyotorphin) + AMP + PPi + MgCl2 + kyotorphin synthetase. The regional distribution and subcellular localization of the synthetase showed a close correlation to that of kyotorphin levels in the rat brain. The amounts of kyotorphin formed from amino acids by the synthetase in the dialyzed synaptosol was 3.0-4.0 times higher than that from precursor proteins by processing enzymes within the 30 min incubation.     

A novel proenkephalin processing carboxypeptidase and its activation by cyclic AMP dependent protein kinase

Carboxypeptidase B-like enzymes cleaving Met-enkephalin-Arg from synaptosomes of the rat striatum purified using a DEAE-cellulose column and Met-Arg-CH-Sepharose 4B affinity column proved to be different from enkephalin-convertase, lysosomal carboxypeptidase B-like enzyme, pancreas carboxypeptidase B and carboxypeptidase Y, in effects of inhibitors and activators, pH optimum (7.5-8.5) and molecular size (50,000). This enzyme, named "Processin CP-E" was activated by cAMP dependent protein kinase, and the Vmax was increased from 4.3 to 13.3 microM/min/mg protein, while the Km (28.2 microM) was unchanged.     

Characteristics of secondary flow in steady and pulsatile flows through a symmetrical bifurcation

Steady and pulsatile flow in a glass model simulating an arterial bifurcation was investigated by flow visualization techniques. Secondary flow generated at the bifurcation has a similar pattern to a vortex, called the horseshoe vortex, produced around a wall-based protuberance in a circular tube. The same flow disturbance was clearly observed during the decelerating phase of pulsatile flow. The vortex produces a stagnation point on the top and bottom wall just upstream from the bifurcation apex. When aluminium dust was suspended in the test fluid perfusing the blood vessel model, particles deposited over an area spreading from the stagnation point to the lateral corners of the bifurcation. Comparison between the present results and topographical patterns of atherosclerosis reported in the literature suggests that it is in such low shear regions that lipid deposition tends to occur most.