VAL-GLN

The 3C protease of poliovirus is distinguished from that of all other picornaviruses in that it only cleaves at Gln-Gly amino acid pairs within the viral polyprotein. To determine whether this strict cleavage specificity is an intrinsic property of the poliovirus 3C protease, amino acid substitutions were introduced at one of the Gln-Gly cleavage sites. Oligonucleotide-directed site-specific mutagenesis of an infectious poliovirus type 1 (Mahoney strain) cDNA was used to change the Gln-Gly site at the 3C/3D junction of the polyprotein into Gln-Val, Gln-Ala, Gln-Ser or Gln-Pro. The effects of these substitutions were studied in vivo after transfection of primate cells by the mutated cDNAs. The Gln-Gly to Gln-Pro substitution was lethal for virus growth, and the corresponding altered 3CD polypeptide expressed in insect cells using a recombinant baculovirus vector did not appear to undergo autocleavage. The Gln-Gly to Gln-Val change was also lethal, although production of virus was occasionally observed as a result of reverse mutations. Mutants with Gln-Ala and Gln-Ser sequences were viable, indicating that these dipeptides can be cleaved by the poliovirus protease in vivo. However, processing at the 3C/3D junction occurred relatively inefficiently in the case of the Gln-Ser virus. Furthermore, the Gln-Gly to Gln-Ala substitution seemed to result in an additional cleavage event within the N-terminal part of polypeptide 3D.

We earlier screened overlapping synthetic peptides from the globular domain of the laminin alpha1 chain to identify active sites for cell attachment. We report here that one of the active cell-adhesion peptides, AG-73 (Arg-Lys-Arg-Leu-Gln-Val-Gln-Leu-Ser-Ile-Arg-Thr; RKRLQVQLSIRT) causes B16F10 murine melanoma cells to metastasize to the liver, a site not normally colonized by these cells. Increases in liver metastases and in lung colonization are observed in immune-deficient beige/nude/xid and in C57Bl/6 mice with this peptide. This metastatic activity was observed with i.v. and with i.p. peptide injections, regardless of tumor cell or of peptide-injection times. In vitro, the AG-73 peptide enhances tumor cell adhesion, migration, invasion, and gelatinase production, and blocks laminin-1-mediated cell migration. AG-73 was found to significantly inhibit cell adhesion to a proteolytic laminin-1 fragment, E3, containing the AG-73 sequence. Cell attachment to AG-73, the E3 fragment, and laminin-1 involved cation-dependent receptors. We report that a laminin peptide has the novel and unexpected activity of causing B16F10 melanoma cells, a lung selected cell line, to metastasize to the liver. The minimal active sequence of AG-73, LQVQLSIR, could be one of the most important biologically active sites of laminin-1, especially in promotion of the malignant phenotype. Activation of the malignant phenotype by this peptide provides a significant new model for understanding metastatic mechanisms.

Red deer (Cervus elaphus) blood is widely used as a health product. Mixed culture fermentation improves the flavor and bioavailability of deer blood (DB), and both DB and its enzymatic hydrolysates exhibit anti-fatigue activities in vivo. To elucidate the bioactive ingredients, enzymatic hydrolysates were fractioned into different peptide groups using reversed phase resin chromatography, and then evaluated using an exhaustive swimming mice model to assess swimming time and biochemical parameters. The structures of the bioactive peptides were elucidated by high performance liquid chromatography with tandem mass detection. Thirty-one compounds were identified as glutamine or branched-chain amino acids containing short peptides, of which Val-Ala-Asn, Val-Val-Ser-Ala, Leu(Ile)-Leu(Ile)-Val-Thr, Pro-His-Pro-Thr-Thr, Glu-Val-Ala-Phe and Val-Leu(Ile)-Asp-Ala-Phe are new peptides. The fractions containing glutamine or valine short peptides, Ala-Gln, Val-Gln, Val-Val-Ser-Ala, Val-Leu(Ile)-Ser improved exercise endurance by increasing hepatic glycogen (HG) storage. The peptides group containing Leu(Ile)-Leu(Ile), Asp-Gln, Phe- Leu(Ile), Val-Val-Tyr-Pro contributed to decreased muscle lactic acid (MLA)accumulation and to an increase in HG. The anti-fatigue activities of DB hydrolysates were attributed to the synergistic effects of different types of peptides.