Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Arginine, the common substrate for production of nitric oxide (NO) and polyamines in mammals, increases in the uterine lumen during the peri-implantation period of pregnancy. However, functional roles of arginine within the uterine lumen for conceptus (embryo and extraembryonic membranes) development have not been elucidated in vivo. To assess roles of arginine in reproductive tissue for survival and development of the conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown of SLC7A1 mRNA, the arginine transporter in ovine conceptus trophectoderm (Tr). Translational knockdown of SLC7A1 mRNA resulted in retarded conceptus development and abnormal function compared to MAO control. Use of MAO-SLC7A1 knockdown in conceptuses decreased arginine transport (73%, P<0.01), the abundance of ornithine decarboxylase, and nitric oxide synthase (NOS3) proteins, arginine-related amino acids [citrulline (76%, P<0.05) and ornithine (40%, P<0.05)], and polyamines, which likely accounts for their retarded development. Also, no alternative arginine precursors (glutamine and glutamate), isoforms of nitric oxide synthase (NOS1 and NOS2), or alternative pathways for polyamine biosynthesis via arginine decarboxylase and agmatinase were activated to rescue conceptus development. Collectively, SLC7A1 is the key transporter of arginine by conceptus Tr, and arginine is essential for conceptus survival and development.-Wang, X., Frank, J. W., Little, D. R., Dunlap, K. A., Satterfield, M. C., Burghardt, R. C., Hansen, T. R., Wu, G., and Bazer, F. W. Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Mycobacterium indicus pranii downregulates MMP-9 and iNOS through COX-2 dependent and TNF-α independent pathway in mouse peritoneal macrophages in vitro.

Despite the popular belief that granulomas are innate immune mechanism to restrict mycobacterial growth, evidences suggest that granulomas facilitate growth of Mycobacterium by recruiting large numbers of uninfected macrophages to the site of infection. Matrix metalloproteinase-9 (MMP-9) has been shown to be directly involved in recruitment of macrophages at the site of infection, contributing to nascent granuloma maturation and bacterial growth. In this manuscript it is reported that heat-killed Mycobacterium indicus pranii (MIP) leads to a significant downregulation of MMP-9 in murine peritoneal macrophages in vitro. The downregulation of MMP-9 is mediated through cyclooxygenase-2 (COX-2), but independent of tumor necrosis factor-α (TNF-α). By limiting nuclear to cytoplasmic export of COX-2 and iNOS transcripts, MIP inhibits excessively-high levels of nitric oxide which can be damaging to the host during acute phases of infection. MIP has been shown to provide clinical improvement in all phases of leprosy and used for treatment of leprosy and tuberculosis.

Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site.

Plasminogen activation by the urokinase-type plasminogen activator (uPA) is facilitated in the presence of cells expressing the glycolipid-anchored high-affinity receptor for uPA (denoted uPAR). Structures involved in the interaction between human uPAR and a decamer peptide antagonist of uPA binding (SLNFSQYLWS) were previously tagged by specific site-directed photoaffinity labeling [Ploug, M., Ostergaard, S., Hansen, L. B. L., Holm, A., and Dano, K. (1998) Biochemistry 37, 3612-3622]. Replacement of the key functional residues Phe4 and Trp9 with either benzophenone or (trifluoromethyl)aryldiazirine rendered this peptide antagonist photoactivatable, and as a consequence, it incorporated covalently upon photolysis into either uPAR domain I or domain III depending on the actual position of the photophore in the sequence. The residues of uPAR specifically targeted by photoaffinity labeling were identified by matrix-assisted laser desorption mass spectrometry, NH2-terminal sequence analysis, and amino acid composition analysis after enzymatic fragmentation and HPLC purification. According to these data, the formation of the receptor-ligand complex positions Phe4 of the peptide antagonist very close to Arg53 and Leu66 in uPAR domain I and Trp9 of the antagonist in the vicinity of His251 in uPAR domain III. The gross molecular arrangement of the deduced receptor-ligand interface provides a rational structural basis for the observed requirement for the intact multidomain state of uPAR for achieving high-affinity ligand binding, since according to this model ligand binding must rely on a close spatial proximity of uPAR domains I and III. In addition, these data suggest that the assembly of the composite ligand binding site in uPAR may resemble the homophilic interdomain dimerization of kappa-bungarotoxin, a structural homologue of the Ly-6/uPAR domain family.