L-Proline methyl amide hydrochloride

Arginase and nitric oxide synthase (NOS) share a common substrate, l-arginine, and have opposing effects on vascular remodeling. Arginase is the first step in polyamine and proline synthesis necessary for cellular proliferation, while NO produced from NOS promotes apoptosis. Previously, we identified a single nucleotide polymorphism (SNP) in the arginase-1 (ARG1) gene, rs2781666 (T-allele) that was associated with a decreased risk for developing pulmonary hypertension (PH) in a cohort of infants with bronchopulmonary dysplasia (BPD). In this study, we utilized lymphocytes from neonates (the only readily available cells from these patients expressing the two genotypes of interest) with either the rs2781666 SNP (TT) or wild type (GG) to test the hypothesis that the protection of the ARG1 SNP against the development of PH in BPD would involve augmented NO production leading to more apoptosis. Lymphocytes were stimulated with IL-4, IL-13, and phorbol myristate acetate (PMA). We found that TT lymphocytes had similar levels of arginase I and arginase II expression, but there was a tendency for lower urea production (a surrogate marker of arginase activity), than in the GG lymphocytes. The TT lymphocytes also had significantly greater NO production than did GG lymphocytes despite no differences in iNOS expression between genotypes. Furthermore, the TT lymphocytes had lower numbers of viable cells, and higher levels of cleaved caspase-3 than did GG lymphocytes. Inhibiting NOS activity using Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME) significantly decreased cleaved caspase-3 levels in the TT lymphocytes. These data demonstrate that the TT genotype results in greater levels of NO production leading to more apoptosis, which is consistent with the concept that BPD patients with the TT genotype are protected against the development of PH by producing greater basal levels of endogenous NO.

A human pituitary-derived serine protease, immunologically identical to human lung tryptase (Smith, T. J., Hougland, M.W., and Johnson, D.A. (1984) J. Biol. Chem. 259, 11046-11051), was found immunohistochemically to be associated with mast cells present in pituitary connective tissue. Western blotting combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of multiple forms: a major Mr 36,300 form and three minor forms with Mr 32,400, 33,400, and 34,600. Two major forms with Mr 35,600 and 34,100 were detected by affinity labeling with 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl. Treatment of the pituitary tryptase preparation with N-glycosidase F indicated that some of the molecular weight heterogeneity results from N-linked glycosylation. The multiple molecular weight forms appear to have the same NH2-terminal sequence: Ile-Val-Gly-Gly-Gln-Glu-Ala-Pro. Pituitary tryptase has an apparent Mr = 110,000 by gel filtration on Sephadex G-200 in the presence of 0.3 M NaCl, indicating that the enzyme may be a tetramer of Mr = 32,400-36,300 subunits. However, this quaternary structure was not stable to gradient polyacrylamide gel electrophoresis. Human pituitary tryptase was so reactive toward synthetic tripeptide coumarin-containing substrates containing a pair of basic amino acids at the site of cleavage such as benzyloxylcarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide (k cat/Km = 2.38 X 10(8) M-1 s-1) that Briggs-Haldane kinetics may apply. The reversible inhibitor NaCl at a concentration of 1 M decreased the k cat/Km for benzyloxylcarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide to 6.53 X 10(6) M-1 s-1, which reflected a 100-fold increase in apparent Km. Based on active site titration with fluorescein mono-p-guanidinobenzoate hydrochloride, NaCl had no effect on the number of accessible active sites. Substrate specificity studies with prohormones indicated that pituitary tryptase has a preference for cleaving COOH-terminal to arginine or lysine residues which are preceded by a proline residue 4 or 6 residues NH2-terminal to the site of cleavage.

A lactam analog of actinomycin D (AMD) has been synthesized as a potential antitumor chemotherapeutic agent. Both L-threonine residues were replaced by L-alpha,beta-diaminopropionic acid. Starting with Nalpha-benzyloxycarbonyl-Nbeta-tert-butyloxycarbonyl-L-alpha,beta-diaminopropionic acid methyl ester hydrochloride the linear intermediate Nalpha-benzyloxycarbonyl-Nbeta-(tert-butyloxycarbonylsarcosyl-L-N-methylvalyl)-L-alpha,beta-diaminopropionyl-D-valyl-L-proline p-nitrophenyl ester was prepared by conventional methods of peptide synthesis in solution. Selective cleavage of the Nbeta-tert-butyloxycarbonyl group and lactam formation afforded the desired cyclic pentapeptide derivative. The chromophore precursor, Nalpha-(2-nitro-3-benzyloxy-4-methylbenzoyl) substituent, was introduced via its symmetric anhydride. Catalytic reduction followed by ferricyanide-mediated phenoxazinone formation provided the lactam analog, [di(1'-L-alpha,beta-diaminopionic acid)]actinomycin D ([Dpr1]2-AMD). Its binding to natural and synthetic DNA and that of an analogous L-threo-alpha,beta-diaminobutyric acid containing lactam ([Dbu1]2-AMD) compared with the binding of AMD (in which the peptides are in lactone form) was studied by circular dichroic (CD) spectroscopy. The visible and uv CD spectra of free AMD differed from those of the free lactam analogs, indicating that the asymmetric environment of the pentapeptide rings in the region of the chromophore differs in free actinomycin lactone and lactams. In the presence of calf thymus DNA, PM2 DNA, and the synthetic d(A-T)-like copolymers containing 2,6-diaminopurine (DAP), poly[d(DAP-T)], and poly[d(DAP-A-T)], the rotational strengths of the optically active transitions in the visible region of the actinomycins increased, and the CD spectra in the presence of the various DNA duplexes were qualitatively similar. The CD spectra of bound actinomycin lactams resembled the spectrum of bound AMD. This suggests that the lactone and lactam actinomycins acquire a similar environment when bound to DNA. [Dpr1]2-AMD was less cytotoxic than AMD in antibacterial assays but exhibited somewhat higher toxicity in mice than AMD. At optimal dose levels the lactam analog had little or no antitumor activity in three murine tumor systems.