Tachykinins (Substance P, Neurokinin A and Neuropeptide γ) and Neurotensin from the Intestine of the Burmese Python, Python molurus

Conlon, J. M., T. E. Adrian and S. M. Secor. Tachykinins (substance P, neurokinin A and neuropeptide γ,) and neurotensin from the intestine of the burmese python, Python molurus. Peptides 18(10) 1505–1510, 1997.—Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH₂) shows one amino acid substitution (Phe⁸ → Tyr) compared with chicken/alligator substance P and an additional substitution (Lys³ → Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide γ (Asp-Ala-Gly-Tyr-Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH₂ shows three substitutions (Gly⁵ → Ser, Gln⁶ → Pro and Ile⁷ → Leu) compared with alligator neuropeptide γ and an additional substitution (His⁴ → Tyr) compared with mammalian neuropeptide γ. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met.NH₂) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.     

Loss of CDK5RAP2 affects neural but not non-neural mESC differentiation into cardiomyocytes

Biallelic mutations in the gene encoding centrosomal CDK5RAP2 lead to autosomal recessive primary microcephaly (MCPH), a disorder characterized by pronounced reduction in volume of otherwise architectonical normal brains and intellectual deficit. The current model for the microcephaly phenotype in MCPH invokes a premature shift from symmetric to asymmetric neural progenitor-cell divisions with a subsequent depletion of the progenitor pool. The isolated neural phenotype, despite the ubiquitous expression of CDK5RAP2, and reports of progressive microcephaly in individual MCPH cases prompted us to investigate neural and non-neural differentiation of Cdk5rap2-depleted and control murine embryonic stem cells (mESC). We demonstrate an accumulating proliferation defect of neurally differentiating Cdk5rap2-depleted mESC and cell death of proliferative and early postmitotic cells. A similar effect does not occur in non-neural differentiation into beating cardiomyocytes, which is in line with the lack of non-central nervous system features in MCPH patients. Our data suggest that MCPH is not only caused by premature differentiation of progenitors, but also by reduced propagation and survival of neural progenitors.     

Synthesis of alkyne derivatives of a novel triazolopyrazine as A(2A) adenosine receptor antagonists

A novel [1,2,4]triazolo[1,5-a]pyrazine core was synthesized and coupled with terminal acetylenes. The structure-activity relationship of the alkynes from this novel template was studied for their in vitro and in vivo adenosine A(2A) receptor antagonism. Selected compounds from this series were shown to have potent in vitro and in vivo activities against adenosine A(2A) receptor. Compound 12, in particular, was found to be orally active at 3mg/kg in both a mouse catalepsy model and a 6-hydroxydopamine-lesioned rat model.     

Isolation and primary structure of urotensin II from the brain of a tetrapod, the frog Rana ridibunda.

A peptide related to urotensin II has been isolated in pure form from an extract of the brain of the European green frog, Rana ridibunda. The primary structure of the peptide was established as Ala-Gly-Asn-Leu-Ser-Glu-Cys-Phe-Trp-Lys-Tyr-Cys-Val and this sequence was confirmed by chemical synthesis. Frog urotensin II contains an additional amino acid residue compared with fish urotensin II peptides but the structure of the cyclic region of the molecule has been fully conserved. The data show that urotensin II is not confined to the caudal neurosecretory system of fish but is present in the central nervous system of a tetrapod.     

Novel 2-imidazoles as potent and selective alpha1A adrenoceptor partial agonists

Novel 2-imidazoles have been identified as potent partial agonists of the α_1A adrenergic receptor, with good selectivity over the α_1B, α_1D and α_2A receptor sub-types. Sulfonamide 23 possessed attractive drug-like properties with respect to physicochemical and ADME properties and wide ligand selectivity.     

Novel 2-imidazoles as potent, selective and CNS penetrant alpha1A adrenoceptor partial agonists

A novel series of central nervous system (CNS) penetrant indane 2-imidazoles have been identified as potent, partial agonists of the alpha(1A) adrenergic receptor, having good selectivity over the alpha(1B), alpha(1D) and alpha(2) sub-types. A key structural motif to impart selectivity is a methylene spacer between the indane and a pendant substituent, which includes heterocycles, sulphones and ethers. Introduction of an ortho-halogen to this group led to a lowering of intrinsic efficacy (E(max)).     

Evolution of the insulin molecule: insights into structure-activity and phylogenetic relationships.

The conformation of insulin in the crystalline state has been known for more than 30 years but there remains uncertainty regarding the biologically active conformation and the structural features that constitute the receptor-binding domain. The primary structure of insulin has been determined for at least 100 vertebrate species. In addition to the invariant cysteines, only ten amino acids (GlyA1, IleA2, ValA3, TyrA19, LeuB6, GlyB8, LeuB11, ValB12, GlyB23 and PheB24) have been fully conserved during vertebrate evolution. This observation supports the hypothesis derived from alanine-scanning mutagenesis studies that five of these invariant residues (IleA2, ValA3, TyrA19, GlyB23, and Phe24) interact directly with the receptor and five additional conserved residues (LeuB6, GlyB8, LeuB11, GluB13 and PheB25) are important in maintaining the receptor-binding conformation. With the exception of the hagfish, only conservative substitutions are found at B13 (Glu --> Asp) and B25(Phe --> Tyr). In contrast, amino acid residues that were also considered to be important in receptor binding based upon the crystal structure of insulin (GluA4, GlnA5, AsnA21, TyrB16, TyrB26) have been much less well conserved and are probably not components of the receptor-binding domain. The hypothesis that LeuA13 and LeuB17 form part of a second receptor-binding site in the insulin molecule finds some support in terms of their conservation during vertebrate evolution, although the site is probably absent in some hystricomorph insulins. In general, the amino acid sequences of insulins are not useful in cladistic analyses especially when evolutionary distant taxa are compared but, among related species in a particular order or family, the presence of unusual structural features in the insulin molecule may permit a meaningful phylogenetic inference. For example, analysis of insulin sequences supports monophyletic status for Dipnoi, Elasmobranchii, Holocephali and Petromyzontiformes.     

Molecular evolution of peptide tyrosine--tyrosine: primary structure of PYY from the lampreys Geotria australis and Lampetra fluviatilis, bichir, python and desert tortoise

Peptide tyrosine-tyrosine (PYY) has been isolated from the intestines of two species of reptile, the desert tortoise Gopherus agassizii (Testudines) and the Burmese python Python molurus (Squamata), from the primitive Actinopterygian fish, the bichir Polypterus senegalis (Polypteriformes) and from two agnathans, the Southern-hemisphere lamprey Geotria australis (Geotriidae) and the holarctic lamprey Lampetra fluviatilis (Petromyzontidae). The primary structure of bichir PYY is identical to the proposed ancestral sequence of gnathostome PYY (YPPKPENPGE10/DAPPEELAKY20/YSALR HYINL30/ITRQRY). Tortoise and python PYY differ by six and seven residues, respectively, from the ancestral sequence consistent with the traditional view that the Testudines represent an earlier divergence from the primitive reptilian stock than the Squamates. The current views of agnathan phylogeny favor the hypothesis that the Southern-hemisphere lampreys and the holarctic lampreys arose from a common ancestral stock but their divergence is of a relatively ancient (pre-Tertiary) origin. The Geotria PYY-related peptide shows only two amino acid substitutions (Pro10-->Gln and Leu22-->Ser) compared with PYY from the holarctic lamprey Petromyzon marinus. This result was unexpected as Petromyzon PYY differs from Lampetra PYY deduced from the nucleotide sequence of a cDNA (S?derberg et al. J. Neurosci. Res. 1994;37:633-640) by 10 residues. However, a re-examination of an extract of Lampetra intestine revealed the presence of a PYY that differed in primary structure from Petromyzon PYY by only one amino acid residue (Pro10-->Ser). This result suggests that the structure of PYY has been strongly conserved during the evolution of Agnatha and that at least two genes encoding PYY-related peptides are expressed in Lampetra tissues.