Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives.

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-PheP(OPh)2]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-ß-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-PheP(OPh)2 (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman-Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin.

Development of longitudinal smooth muscle in the posterior mesenteric artery and purinergic regulation of its contractile responses in chickens.

This study was designed to clarify development and the neural regulation of longitudinal smooth muscle in the chicken posterior mesenteric artery to generate new hypotheses for the roles of arterial longitudinal muscles. The existence of longitudinal muscles was examined with hematoxylin-eosin staining. A well-developed longitudinal muscle layer exists in the posterior mesenteric artery of adult female chickens but not adult male chickens. The muscle layer is poorly developed in chickens aged < 15 weeks, even in female chickens. Mechanical responses of muscles were recorded and perivascular nerves were stimulated by electrical field stimulation (EFS). EFS induced monophasic contractions in longitudinal muscle of the posterior mesenteric artery segment, and those responses were inhibited by pretreatment with tetrodotoxin. Blockers for cholinoceptors and adrenoceptors did not affect EFS-evoked contractions but an antagonist for P2X purinoceptors blocked them. The present study demonstrated that the longitudinal muscle in the posterior mesenteric artery of the domestic fowl develops between the 5th and 15th week of life, suggesting that its development is involved in oviposition. The longitudinal muscle might have a role in resisting extensional stress from the oviduct containing eggs. Moreover, the arterial longitudinal muscle is regulated by purinergic neurons via P2X purinoceptors.

Matrix metalloproteinases (MMPs) activity in cultured canine bone marrow stromal cells (BMSCs).

Autologous bone marrow stromal cells (BMSCs) infusion therapy improves the hepatic fibrosis. To investigate the mechanism of remission, we evaluated the matrix metalloproteinase (MMP)-2 and -9 activity in canine BMSCs and the effect of pro-inflammatory cytokines on their expression. The activity and the gene expression of MMPs were analyzed by gelatin zymography and quantitative RT-PCR, respectively. The specific gelatinase bands were indicative effect of MMP-2 and -9 in canine BMSCs. MMP-2 expression seemed to be increased by TNF-α and IL-1ß while MMP-9 was enhanced by TNF-α and IL-6. These results suggested that remissive effect on liver fibrosis might be partly attributable to the MMP-2 and -9 activity in BMSCs under the inflammatory condition.