Conformational studies of the pentapeptides with weak adrenocorticotropin (ACTH) activities by means of nuclear magnetic resonance spectroscopy

The conformations of a pentapeptide L -His-L -Arg-L -Trp-Gly with weak adrenocorticotropin (ACTH) activity and its analogs, where each L -amino acid residue is substituted by D -residue, were investigated by means of proton and carbon-13 nmr spectroscopy on their DMSO-d₆ solutions. The spectra indicated the presence of slowly exchangeable conformation isomers for D -Phe and D -Arg analogs, due to steric hindrance around the arginine residue. The activation energy of the hindered rotation of the arginine side chain was estimated to be more than 19 ～ 20 kcal/mol. Spin-lattice relaxation times of carbon-13 nuclei also indicated slow segmental motion of the arginine side chain of the D analogs. An effect on proton chemical shifts by intermolecular electrostatic interaction between the arginine side chain and the C terminal carboxylic residue was observed. We did not observe, however, a direct correlation between pentapeptide activity and molecular conformation at this stage of the experiments.     

Multiple proteolytic action of rat liver cathepsin B: specificities and pH-dependences of the endo- and exopeptidase activities.

Dipeptidylcarboxypeptidase, endopeptidase, and carboxypeptidase activities of rat liver cathepsin B were investigated using soluble denatured protein substrates, reduced and S-(3-trimethylammonio)propylated proteins and their derivatives. It was found that the soluble denatured proteins were degraded mainly by the dipeptidylcarboxypeptidase activity and in a few cases by the endopeptidase and carboxypeptidase activities. The eipeptidylcarboxypeptidase activity showed broad substrate specificity with broad pH optimum at 4-6. A peptide having the alpha-carboxyl group amidated with methylamine could also be a good substrate for this activity. These results suggest that this activity is dependent not upon the dissociated alpha-carboxyl group at the P2' site but upon the hydrogen-bonding abilities of the alpha-imino moiety and the protonated or amidated alpha-carboxyl moiety at P2'. On the other hand, the endopeptidase and carboxypeptidase activities were observed in a few cases, suggesting that special amino acid sequences in the substrates are responsible for these activities. These activities showed sharp pH optima at 6 and seemed to prefer basic amino acid residues at P1 site. Therefore, we suppose that cathepsin B has a carboxyl group with a pKa of about 5.5 at the S1 subsite which more effectively interacts with a positive charge at the P1 site of the substrate at pH 6 than at pH 5. Based on these results, a model of the binding subsites of this enzyme is proposed.     

Isolation of a cardiotonic glycoprotein,striatoxin, from the venom of the marine snail Conus striatus

Striatoxin, a powerful cardiotonic glycoprotein has been isolated from the venom of the marine snail Conus striatus, monitored by the inotropic action on the guinea-pig left atria. The molecular weight was estimated to be 25,000 by gel filtration. The purified glycoprotein is electrophoretically homogeneous. The toxin at concentrations above 10^?7 g/ml has a long-lasting inotropic action, which was abolished by tetrodotoxin (10^?6 M). The minimum lethal dose in the fish Rhodeus ocellatus smithi was 1 μg/g body weight. A possible biological role of striatoxin in C. striatus is briefly discussed.     

The effect of sound location in a song-discrimination task by Bengalese finches (<Emphasis Type="Italic">Lonchura striata</Emphasis> var. <Emphasis Type="Italic">domestica</Emphasis>)

To understand the relationship between song perception and stimulus location, Bengalese finches were trained to discriminate between a normally played conspecific song and the reversed song. Loudspeakers on the opposite side of the response key (experimental condition) and above the subject (control condition) were used to present stimuli. All control birds learned the task, whereas some experimental subjects did not. In general, controls learned the task more quickly than experimental animals. These results suggest that sound location may be closely associated with behavioral responses in this species. Bioacoustical investigations should be performed while taking these results into consideration.     

Quantitative analysis of hemin-induced neutrophil extracellular trap formation and effects of hydrogen peroxide on this phenomenon

Formation of neutrophil extracellular traps (NETs) can perpetuate sterile inflammation; thus, it is important to clarify their pathophysiological characteristics. Free heme, derived via hemolysis, is a major contributor to organ damage, and reportedly induces neutrophil activation as well as reactive oxygen species (ROS) production and NET formation. For this study, we examined hemin (Fe³⁺ -protoporphyrin IX)-induced NET formation quantitatively in vitro as well as the effects of oxidative stress.NETs formed in vitro from cultured neutrophils were quantitatively detected by using nuclease treatment and Sytox Green, a nucleic acid stain. Hemin-induced NET production was found to be in a dose-dependent manner, NADPH oxidase-dependent and toll-like receptor (TLR)-4 independent. Additionally, the iron molecule in the porphyrin ring was considered essential for the formation of NETs. In the presence of low concentrations of hydrogen peroxide, low concentrations of hemin-induced NETs were enhanced, unlike those of phorbol myristate acetate (PMA)-induced NETs.Quantitative analysis of NET formation may prove to be a useful tool for investigating NET physiology, and hemin could function as a possible therapeutic target for hemolysis-related events.     

Purification and characterization of formaldehyde dismutases of Methylobacterium sp. FD1

ABSTRACT

In the present study, we purified and characterized three formaldehyde dismutases (Fdms) (EC 1.2.98.1) (Fdm1, Fdm2, and Fdm3) of Methylobacterium sp. FD1. These Fdms (with His-tag) were produced in the recombinant E. coli and purified by immobilized metal affinity chromatography from the E. coli extracts. In each of the three Fdms, the enzyme-bound coenzyme was nicotinamide adenine dinucleotide (NAD(H)) and the enzyme-bound metal was zinc. The quaternary structures of these Fdms were estimated as homotetrameric. The optimal pHs and temperatures of Fdm1, Fdm2, and Fdm3 were approximately 6.5, 6.0, and 6.0, and 35°C, 25°C, and 30°C, respectively. The K_m values of Fdm1, Fdm2, and Fdm3 were 621, 865, and 414 mM, respectively. These results were similar to the properties of already-known Fdms. However, each of the Fdms of FD1 had methanol:p-nitroso-N,N-dimethylaniline oxidoreductase activity that is not found in already-known Fdms.     

Identification of cellular proteins interacting with octaarginine (R8) cell-penetrating peptide by photo-crosslinking

Octaarginine (R8) is a representative cell-penetrating peptide. Lanthionine synthetase component C-like protein 1 (LanCL1) was identified as a potential intracellular target of R8 by using a photo-crosslinking assay that utilized a phenyl-trifluoromethyl diazirine moiety and peptide mass fingerprinting. Increased cellular uptake of R8 by LanCL1-overexpressing cells was observed.     

Flexibacter japonensis sp. nov., a New Species That Produces a Novel Inhibitor of Human Leukocyte Elastase Isolated from Soil

This strain 758 of a new member of Flexibacter species isolated from a soil is a Gram-negative, nonflagellated, gliding, long rod or filamentous. The GC contents of the deoxyribonucleic acid of this strain is 49.8 mol %GC. The isolate can be distinguished from other Flexibacter species on the basis of physiological and biochemical properties and DNA relatedness data. Therefore, we propose a new species, Flexibacter japonensis, for this strain. The strain 758 is deposited to the Japan Collection of Microorganisms as JCM 9735. Received: 27 November 1995 / Accepted: 30 January 1996     

New O and H antigens ofPlesiomonas shigelloides and their O antigenic relationships toShigella boydii

Twenty six new O (O51-O76) and 24 new H (H18-H41) antigens forPlesiomonas shigelloides serotyping scheme were defined. The O antigens 54 and 57 were closely related to those ofShigella boydii 2 and 9, respectively, in an a, b-a, c type of relationship.