Kentsin: tetrapeptide from hamster embryos produces naloxone-sensitive effects without binding to opioid receptors

The opioid nature of kentsin (Thr-Pro-Arg-Lys) and its ability to alter pain perception and intestinal transit were examined. Kentsin (30,000 nM) did not inhibit electrically stimulated contractions of the guinea pig ileum (GPI) or mouse vas deferens (MVD), nor did it cause a rightward displacement of the inhibitory concentration-response curves of the mu-selective opioid agonist PL017 in the GPI or the delta-selective agonist DPDPE in the MVD. Kentsin (10,000 nM) did not displace [3H] naloxone from rat brain homogenates. These results indicate that kentsin lacks opioid agonist and mu and delta opioid antagonist properties and does not bind to opioid receptors. In vivo, kentsin produced dose-dependent analgesia in both the hotplate and abdominal stretch tests when administered intracerebroventricularly (ICV) and intrathecally but not intravenously. The central analgesic effect of kentsin was partially antagonized by the opioid antagonist naloxone. Kentsin inhibited intestinal transit in a dose-dependent manner after ICV administration only. The intestinal antitransit effect of kentsin was not blocked by pretreatment with naloxone. These results suggest that kentsin acts centrally to produce both opioid and non-opioid effects. Further, the opioid-mediated analgesic effects of kentsin involve mechanisms other than direct interaction with opioid receptors.     

Comparing applesnails with oranges: the need to standardize measuring techniques when studying <Emphasis Type="Italic">Pomacea</Emphasis>

Although invaders come in all shapes and sizes, several mollusks have recently achieved notoriety as both economically and ecologically costly invaders. Applesnails of the genus Pomacea get their name from reaching the size of an apple. Native to South America, the species P. insularum has recently established reproducing, and potentially invasive, populations in Texas, Alabama, Georgia, and Florida. In contrast to the widely invasive golden applesnail (P. canaliculata), few studies of the channeled species P. insularum exist. In studying similar invasive applesnail species, scientists use several methods of measurement. We have explored the relationships among shell height, operculum width, and weight among juvenile and adult P. insularum and tested their inter-measurer reliability. We also investigated the use of shell height, shell length, and operculum width measurements in P. canaliculata studies and observed whether or not those studies defined their measurements. We found that operculum width served as a significantly more reliable measure among researchers. Furthermore, operculum width better predicted weight than shell height. The majority of articles that measured P. canaliculata did not define their measurements, which may cause problems when comparing studies between native and exotic populations or when comparing the two species. We recommend that future studies of P. insularum use operculum width to measure snails and explore a possible sex dimorphism in the operculum width of adult P. insularum.     

The phylogenetic potential of entire 26S rDNA sequences in plants

18S ribosomal RNA genes are the most widely used nuclear sequences for phylogeny reconstruction at higher taxonomic levels in plants. However, due to a conservative rate of evolution, 18S rDNA alone sometimes provides too few phylogenetically informative characters to resolve relationships adequately. Previous studies using partial sequences have suggested the potential of 26S or large-subunit (LSU) rDNA for phylogeny retrieval at taxonomic levels comparable to those investigated with 18S rDNA. Here we explore the patterns of molecular evolution of entire 26S rDNA sequences and their impact on phylogeny retrieval. We present a protocol for PCR amplification and sequencing of entire (approximately 3.4 kb) 26S rDNA sequences as single amplicons, as well as primers that can be used for amplification and sequencing. These primers proved useful in angiosperms and Gnetales and likely have broader applicability. With these protocols and primers, entire 26S rDNA sequences were generated for a diverse array of 15 seed plants, including basal eudicots, monocots, and higher eudicots, plus two representatives of Gnetales. Comparisons of sequence dissimilarity indicate that expansion segments (or divergence domains) evolve 6.4 to 10.2 times as fast as conserved core regions of 26S rDNA sequences in plants. Additional comparisons indicate that 26S rDNA evolves 1.6 to 2.2 times as fast as and provides 3.3 times as many phylogenetically informative characters as 18S rDNA; compared to the chloroplast gene rbcL, 26S rDNA evolves at 0.44 to 1.0 times its rate and provides 2.0 times as many phylogenetically informative characters. Expansion segment sequences analyzed here evolve 1.2 to 3.0 times faster than rbcL, providing 1.5 times the number of informative characters. Plant expansion segments have a pattern of evolution distinct from that found in animals, exhibiting less cryptic sequence simplicity, a lower frequency of insertion and deletion, and greater phylogenetic potential.      

Conformationally constrained cyclic enkephalin analogs with pronounced delta opioid receptor agonist selectivity

The enkephalin analogs, [D-Pen2,L-Cys5]- and [D-Pen2,D-Cys5]-enkephalin are cyclic compounds, conformationally constrained by virtue of their 14-membered, disulfide containing rings and by the rigidizing effect of the beta, beta dimethyl substituents of the penicillamine side chain. The analogs exhibit profound delta receptor specificity as assessed by their relative potencies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, exhibiting, respectively, 666 and 215 times higher potency in the latter assay system. By contrast, the receptor selectivities measured in rat brain binding assays in the absence of sodium were much more modest, the cyclic analogs being, respectively, 15.2 and 6.0 times more effective at displacing [3H] [D-Ala2,D-Leu5]enkephalin than [3H]naloxone. However, for binding assays performed in the presence of a sodium concentration equivalent to that used in the GPI and MVD assays, these binding selectivities increased to 167 and 49, respectively.     

Neuropeptides in sensory neurons

Substance P, somatostatin, VIP, CCK, angiotensin II, and bombesin have all been localized by immunohistochemical or radioimmunological means in neurons of sensory ganglia or in the dorsal horn of the spinal cord. Most of these neuropeptides have electrophysiological effects on spinal neurons and for substance P and somatostatin, these effects have been associated with particular sensory modalities. Newer investigations using the compound capsaicin are consistent with the hypothesis that substance P is an important neurochemical mediator of certain kinds of noxious peripheral stimuli. The newly described substance P antagonists promise to be important pharmacological tools for investigation of the long-neglected neurochemical bases of sensory neuron function. Elaboration of the roles of these sensory neuropeptides will no doubt shed light on many disease states in which there seems to be sensory neuron involvement.     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

Coronavirus isolates SK and SD from multiple sclerosis patients are serologically related to murine coronaviruses A59 and JHM and human coronavirus OC43, but not to human coronavirus 229E.

Two coronaviruses (SK and SD), isolated from fresh autopsy brain tissue from two multiple sclerosis patients, were compared with known human and murine coronaviruses. In plaque neutralization assays, antisera prepared against multiple sclerosis isolates SK and SD demonstrated significant cross-reactivity to each other and to murine coronavirus A59, weak cross-reactivity to murine coronavirus JHM, but no cross-reactivity to the human coronavirus 229E. Antiserum to SK or SD failed to inhibit hemagglutination of chicken erythrocytes by the human coronavirus OC43. However, OC43 antiserum neutralized both SD and SK. Specific coronavirus polypeptides were identified and compared by immunoprecipitation and polyacrylamide gel electrophoresis. Infected and mock-infected 17Cl-1 cells were pretreated with actinomycin D and labeled with [35S]methionine. Polypeptides in Nonidet P-40 cytoplasmic extracts were immunoprecipitated with homologous and heterologous antisera. Identical polypeptides were precipitated from A59-, SD-, or SK-infected cell extracts by SD, SK, OC43, or A59 antisera. The polypeptides of human virus 229E were antigenically distinct, with the exception of weak recognition of a polypeptide of 50,000 molecular weight. We conclude that the two multiple sclerosis virus isolates SK and SD are closely related serologically to the murine coronavirus A59 and the human coronavirus OC43.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

NMR solution structure of a potent cyclic nonapeptide inhibitor of ICAM-1-mediated leukocyte adhesion produced by homologous amino acid substitution.

We have previously described a disulfide-linked cyclic nonapeptide (inhibitory peptide-01, IP01), with the sequence CLLRMRSIC, which binds to intercellular adhesion molecule-1 (ICAM-1), and blocks binding to its counter-structure, the integrin alphaLbeta2 (leukocyte functional antigen-1, LFA-1) (Sillerud et al., J. Peptide Res. 62, 2003: 97). We now report the optimization of this peptide by means of single homologous amino acid substitutions to yield a new peptide (IP02-K6; CLLRMKSAC) which shows an approximately sixfold improvement in inhibitory activity of multivalent leukocyte binding (inhibition constant for 50% inhibition, IC50 = 90 microm) compared with IP01 (IC50 = 580 microm). This improvement in activity gives IP02-K6 potent in vivo activity in a murine model of ischemia reperfusion injury (Merchant et al., Am. J. Physiol. Heart Circ. 284, 2003: H1260). In order to determine the structural features relevant to ICAM-1-binding, we have determined the structure of IP02-K6 using proton nuclear magnetic resonance (NMR) spectroscopy and restrained molecular modeling. In our previously reported study of solution models of IP01, we observed three interconverting conformations during low-temperature molecular dynamics simulation. In the present study, we find a single conformation of IP02-K6 similar to one of the previously found conformations of IP01 (family C). In particular, an R4-S7 beta-turn is present in similar proportions in both conformation C of IP01 and in IP02-K6; this motif is important in binding to ICAM-1 because this turn enables the IP02-K6 backbone to drape over proline-36 on ICAM-1. The NMR-derived solution model of IP02-K6 was found to dock at the alphaLbeta2-binding site on ICAM-1 with no changes in peptide backbone conformation. This docking model displaced five of the 15 alphaLbeta2 residues at the ICAM-1-binding site and provided a rationale for understanding the quantitative relationship between IP02-K6 structure and biologic activity.