Inhibitory NANC nerves in human tracheal smooth muscle: a quest for the neurotransmitter.

Inhibitory nonadrenergic noncholinergic (i-NANC) nerves are the only neural bronchodilator pathway in human airways. Possible candidates for the neurotransmitter include vasoactive intestinal peptide (VIP) and nitric oxide (NO) and purines such as ATP. We have investigated the potential role of these neurotransmitters. Phosphoramidon (10(-5) M) significantly potentiated relaxations to low doses of VIP with no effect on i-NANC responses. Relaxations induced by VIp were abolished with alpha-chymotrypsin (2 U/ml), but i-NANC responses were unaffected. Reactive blue 2 had no effect on i-NANC neural responses, indicating that endogenous ATP was not involved. The NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 10(-4) M) produced a concentration-dependent inhibition of the i-NANC response, producing almost complete inhibition at every frequency studied (0.5-40 Hz), whereas L-NG-monomethyl arginine was effective only at low stimulation frequencies. The inhibitory effect of L-NAME was partially reversed by L- but not D-arginine, and D-NAME was without effect. These results suggest that in human tracheal segments the neural bronchodilator response is mediated by NO, and there is no functional evidence for implicating VIP in this response.     

Seroepizootiological survey on bluetongue virus infection in cattle in Japan

Bovine sera collected in various parts of Japan were subjected to seroepizootiological tests with bluetongue virus type 1 (BTV1), type 12 (BTV12), and type 20 (BTV20). All these viruses have been widely disseminated among cattle in the southern part of Japan in 1974. Relatively high incidences of neutralizing (NT) antibody against the three viruses were shown among cattle in the Kyushu district, including Okinawa Prefecture, or the southern part of Japan, but extremely low or incidences in Hokkaido, or the northern part of Japan. The incidence of reactors was higher in old animals. Cattle in Okinawa Prefecture showed a high rate of seroconversion for all the viruses during the summer of 1979. None of the animals seroconverted, however, manifested any sign of disease. Seroepizootiological investigation made it clear that BTV1, BTV12 and BTV20 had existed in Japan and that the epizootic of bluetongue virus infection started during a period from summer through early autumn.     

Absence of pantothenic acid in gramicidin S synthetase 2 obtained from some mutants of Bacillus brevis

The pantothenic acid content of gramicidin S synthetase 2(GS 2) was estimated microbiologically with enzymes obtained from the wild strain and gramicidin S-lacking mutant strains of Bacillus brevis. Four mutant enzymes from BI-4, C-3, E-1, and E-2 lacked pantothenic acid. Other mutant enzymes from BII-3, BI-3, BI-9, and BI-2 contained the same amount of pantothenic acid as the wild-type enzyme. Pantothenic acid-lacking GS 2 belonged to group V of mutant enzymes, which could activate all amino acids related to gramicidin S; their complementary enzyme, gramicidin S synthetase 1(GS 1), lacked racemizing activity. To ascertain whether 4'-phosphopantetheine is involved in the formation of D-phenylalanyl-L-prolyl diketopiperazine (DKP) and gramicidin S, combinations were tested of intact GS 1 from the wild strain with various mutant GS 2 either containing or lacking pantothenic acid. Only the combinations of wild-type GS 1 with mutant GS 2 containing pantothenic acid could synthesize DKP. Combinations with pantothenic acid-lacking GS 2 also failed to elongate peptide chains. Pantothenic acid-lacking GS 2 could bind the four amino acids which constitute gramicidin S as acyladenylates and thioesters, but the binding abilities were lower than those of the wild-type enzyme and other mutant enzymes containing the pantothenic group.     

Identification of a protease inhibitor from rat peritoneal macrophages as poly(ADP-ribose)

Rat peritoneal macrophages contain a chymotrypsin-like protease and its specific inhibitor, both being associated with chromatin of the cells. The inhibitor was separated from the protease by gel filtration through a Sephadex G-75 column, further treated with trypsin, DNase and RNase, and then purified successively on Sephadex G-75, Sephadex G-25, and dihydroxyboryl Bio-Gel P-60 columns. The purified inhibitor had a molecular weight in the range from 2,000 to 3,500 and an absorption maximum at 260 nm at pH 7.0. When the inhibitor was digested by snake venom phosphodiesterase, the inhibitory potency was lost, yielding 5'-AMP and 2'-(5'-phosphoribosyl)-5'-AMP as the digestion products which were identified by high pressure liquid chromatography. The inhibitory potency was neutralized specifically by anti-poly(ADP-ribose) antiserum. The profile of inhibition by the isolated inhibitor was nearly identical with that produced by authentic poly(ADP-ribose). It was therefore concluded that the inhibitor isolated was identical with poly(ADP-ribose), whose chain length ranged from 4 to 7 ADP-ribosyl units. This is the first demonstration that a intracellular protease inhibitor can be endogenous poly(ADP-ribose).     

Performance of affinity chromatography columns

Performance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B-soybean trypsin inhibitor column and a Sepharose 4B-arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break-through curves of trypsin, and elution profiles bed height of Sepharose 4B-STI column was estimated based on these results.     

Normalization of phospholipids concentration of the gastric mucosa was observed in patients with peptic ulcer after eradication of Helicobacter pylori

Background. Phospholipids concentration in the gastric mucosa decreased in patients with Helicobacter pylori infection. The aim of this study is to examine the effects of eradication of H. pylori on decreasing the phospholipids concentration in the gastric mucosa in patients with gastric or duodenal ulcer. Materials and Methods. Phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingonomyeline) were measured in biopsy specimens from the antrum and corpus using thin‐layer chromatography. In H. pylori positive patients with gastric ulcer (n = 26) and duodenal ulcer (n = 13), and H. pylori negative controls (n = 20), the biopsy specimens were obtained before and 3 months after eradication. Eradication was performed using lansoprazole, amoxycillin, and clarithromycin. Results. Compared with the H. pylori negative control group, the concentrations of phosphatidylcholine and phosphatidylethanolamine decreased significantly in the gastric ulcer group in both antrum and corpus mucosa, and in the duodenal ulcer group in antrum mucosa. This decrease returned to the control level after eradication. Conclusions. This study demonstrates that the eradication of H. pylori in patients with peptic ulcer normalized the decrease of phosphatidylcholine and phosphatidylethanolamine in the gastric mucosa.     

Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.