1. Further studies on the three-step-increase in activity due to the aromatic amino acids B24-26 (-Phe-Phe-Tyr-)
G Weitzel, K Eisele, F U Bauer Hoppe Seylers Z Physiol Chem . 1976 Feb;357(2):187-200. doi: 10.1515/bchm2.1976.357.1.187.
Using a reaction suite which was suggested by Ruttenberg  for the semisynthesis of insulin variants, insulin hexamethyl ester was digested by trypsin, then the N-terminal amino groups of the resulting desoctapeptide insulin pentamethyl ester were protected with the Boc residue. The free carboxyl group of the arginyl residue (B22) of this product was coupled to two different series of synthetic peptide methyl esters: I) Gly-OMe, Gly-Phe-OMe, Gly-Phe-Phe-OMe, Gly-Phe-Phe-Tyr-OMe and II) Gly-Ala-OMe, Gly-Phe-Ala-OMe, Gly-Phe-Phe-Ala-OMe, Gly-Phe-Phe-Tyr-Ala-OMe. Removal of all protecting groups yielded the corresponding insulin variants. The syntheses of these peptide methyl esters are described. Following the original prescription of Ruttenberg, we were not able to prepare the desired variants. That is why we were forced to change some important details of the Ruttenberg recipe. The activity determinations by the mouse fall test showed the weak activity (ca. 4%) of the desoctapeptide insulin (C-terminus Arg B22). This activity increases drastically in three steps, when the amino acids Phe, Phe, Tyr (B24-26) are added successively to the insulin trunk. Coupling of Gly-Phe yields 14%, -Gly-Phe-Phe 36%, and -Gly-Phe-Phe-Tyr 61% of the biological activity (cryst. insulin=100%). The same peptides, elongated at their C-terminis with an alanyl residues (see above, series II) yield higher activities. Coupling these peptides to the arginyl residue B22 increases the activity as follows: -Gly-Phe-Ala, 36%, -Gly-Phe-Phe-Ala, 59%, and -Gly-Phe-Phe-Tyr-Ala, 91%. Comparing the activities of the variants with the C-termini-Gly-Phe-Phe (36%) and -Gly-Phe-Ala (36%) or -Gly-Phe-Phe-Tyr (61%) and -Gly-Phe-Phe-Ala (59%), it becomes clear that the aromatic amino acids Phe (B25) and Tyr (B26) can be substituted by Ala without loss of activity. In our preceding work (published 1969-1973 [3, 6-8]), we synthesized successively shortened insulin B-chains which yielded, after combination with natural A-chain, practically the same activity values as we have now obtained with the Ruttenberg semisynthesis. As we have already mentioned l.c.[1-4], it is obvious that the activity of insulin proceeds from the arginyl residue (B22) and is only intensified by the aromatic amino acids (B24-26). We[2,3] observed the same three-step increase in activity in the case of our synthetic oligopeptides Arg-Gly-Phe, Arg-Gly-Phe-Phe and Arg-Gly-Phe-Phe-Tyr (B22-26), which we assume to be the active region of insulin (1971).
2. Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence
A E Chung, L J Dong, N A Edgeworth, F D Yelian, D R Armant J Cell Biol . 1993 May;121(4):923-9. doi: 10.1083/jcb.121.4.923.
In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose-dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.
3. Pseudosymmetry in the structure of luteinizing hormone-releasing hormone. Studies on a series of novel analogs
L A Lowe, P J Goodford, C R Beddell, P J Fraser, D Gilbert, S Wilkinson J Med Chem . 1975 Apr;18(4):417-23. doi: 10.1021/jm00238a019.
Pseudosymmetry in the LH-RH structure is described. Eleven analogs of LH-RH (SMALLER THAN Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) have been synthesized by the fragment condensation method and the repetitive excess mixed anhydride method. Multiple substitutions have been made in the LH-RH sequence, which retain the pseudosymmetry of the LH-RH molecule, while presenting fewer problems of synthesis than the corresponding residues in the natural decapeptide. Thus Trp3, Ser4, Tyr5, Gly6, Leu7, and Arg8 residues were replaced by amino acids having similar properties to the residues that they replace. In all but one of the peptides the Gly10-NH2 residue was replaced by ethylamide, while in the remaining peptide, 1-methyl-5-aminomethyltetrazole (AMT-Me) was substituted at position 10. The compounds were assayed in vitro and in vivo. The following analogs had in vivo and in vitro activities in the range 1-28 percent relative to LH-RH: I, smaller than Glu-His-Phe-Ala-Tyr-Gly-Leu-Arg-Pro-NHEt; II, smaller than Glu-His-Phe-Gly-Tyr-Gly-Leu-Arg-Pro-NHEt; VII, smaller than Glu-His-Phe-Ala-Tyr-Gly-Phe-Arg-Pro-NHEt; IX, smaller than Glu-His-Phe-Ala-Tyr-D-Ala-Leu-Arg-Pro-NHEt; XI, smaller than Glu-His-Phe-Gly-Tyr-Gly-Leu-Arg-Pro-AMT-Me.