Tachykinin receptors mediating airway macromolecular secretion.

Three tachykinin receptor types, termed NK1, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with 3H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). All the tachykinins tested stimulated HMWG release to an approximately equal degree. Stimulation was concentration-related, with log concentrations giving half-maximal effects (EC50) as follows: SP -9.47, NKA -7.37, NKB -5.98, PHY -8.08, and ELE -7.68. This rank order of potency (SP greater than PHY greater than or equal to ELE greater than or equal to NKA greater than NKB) is most consistent with NK1 receptors. To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers. The potency of SP was not diminished by pretreatment with atropine, propranolol, or chlorpheniramine, and atropine actually increased the magnitude of the secretory response. The SP receptor antagonist [D-Arg1,D-Phe5, D-Trp7,9, Leu11]-SP blocked SP-induced secretion. These findings indicate that SP is a potent stimulus of airway macromolecular secretion. This effect occurs through the action of NK1 receptors, and is not dependent upon cholinergic, beta-adrenergic, or H-1 histamine receptors. The facilitation by atropine of SP stimulation suggests the existence of a mechanism of cholinergic inhibition of SP-induced stimulation.     

Paleobiology of the first Americans

The majority of scholars studying the human colonization of the New World agree that the first Americans, traditionally identifed as Paleoindians, entered near the end of the Pleistocene via the Bering land bridge from northeast Asia. But this is where agreement ends. Questions about the number and timing of migrations, the physical appearance of the colonists, and the manner in which they lived have been examined since the turn of the century^1–7 and still engender lively debate.^8–16 Curiously lacking from the growing body of data on the peopling of the Americas is evidence from the physical remains of the first Americans. We summarize research on the earliest human remains from North America and discuss how these remains shed new light on these unanswered questions.     

Transforming growth factor beta 1: importance of glycosylation and acidic proteases for processing and secretion

The role of glycosylation of the transforming growth factor-beta 1 (TGF-beta 1) precursor was investigated by treating a transfected Chinese hamster ovary (CHO) cell line expressing high levels of recombinant TGF-beta 1 (TGF-beta 3-2000 cells) with a series of glycosylational inhibitors. Tunicamycin, a nucleoside antibiotic which prevents the formation of the dolichol intermediate necessary for oligosaccharide addition of the nascent polypeptide chain, appeared to block secretory exit and led to an increase in the cellular associated, nonglycosylated pro-TGF-beta 1 form. 1-Deoxymannojirimycin and swainsonine, inhibitors of the mannosidases I and II, respectively, blocked complete glycoprotein processing of the TGF-beta 1 precursor as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by sensitivity to glycosidases. However, the abnormal TGF-beta 1 polypeptides containing the altered carbohydrate side chains were secreted readily by the CHO cells. In contrast, inhibitors of the glucosidases at the first step in glycoprotein remodeling, 1-deoxynojirimycin and castanospermine, markedly inhibited secretion of the TGF-beta 1 polypeptides from transfected CHO cells. In all cases, these inhibitors did not appear to affect proteolytic processing of the TGF-beta 1 polypeptides. Furthermore, inhibitor treatment did not affect mannose-6-phosphorylation of the TGF-beta 1 polypeptides. These results suggest that glycosylation and early stage remodeling of oligosaccharide side chains are necessary for secretion of TGF-beta 1. Treatment of the transfected CHO cells with weak bases (NH4Cl and chloroquine), or a monovalent ionophore (monensin), prevented proteolytic processing of the TGF-beta 1 precursor indicating that cleavage occurs by proteases in an acidic cellular compartment.     

Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood

Background aimsOligodendrocyte precursor cells (OPC) hold promise as a cellular therapy for demyelinating diseases. The feasibility of using OPC-based therapies in humans depends upon a reliable, readily available source. We have previously described the isolation, expansion and characterization of oligodendrocyte-like cells from fresh human umbilical cord blood (UCB). We now describe the isolation and expansion of OPC from thawed, cryopreserved UCBMethodsWe thawed cryopreserved UCB units employing a standard clinical protocol, then isolated and plated mononuclear cells under previously established culture conditions. All OPC cultures were trypsinized at 21 days, counted, then characterized by flow cytometry after fixation, permeablization and labeling with the following antibodies: anti-oligodendrocyte marker 4 (O4), anti-oligodendrocyte marker 1 (O1) and anti-myelin basic protein (MBP). OPC were also placed in co-culture with shiverer mouse neuronal cells then stained in situ for beta tubulin III (BT3) and MBP as a functional assay of myelination.ResultsThe average OPC yield per cryopreserved UCB unit was 64% of that seen with fresh UCB. On flow cytometric analysis, 74% of thawed UCB units yielded cells with an O4-expression level of at least 20% of total events, compared with 95% of fresh UCB units. We observed myelination of shiverer neurons in our functional assay, which could be used as a potency assay for release of OPC cells in phase I human clinical trials.ConclusionsOur results demonstrate that OPC can be derived reliably from thawed, cryopreserved UCB units, and support the feasibility of using these cells in human clinical trials.     

The Miocene differentiation of old world Pecora (Mammalia)

Dental and horn core/antler morphology is discussed for some representative lower and middle Miocene pecorans in a review of the phylogeny and early zoogeography of the modern Old World families. Giraffes and bovids are more closely related than either is to deer. Andegameryx, Walangania and Teruelia are related to giraffoids plus bovoids together; Amphimoschus and Hispanomeryx are a probable sister group of bovoids; Propalaeoryx is the sister group of the giraffids plus climacoceratids. Procervulus and Lagomeryx may be closer to modern deer than are Stephanocemas and Dicrocerus but all are included in the Cervidae. The horn structure of Palaeomeryx has resemblances to giraffids, but its teeth and postcranial bones are more like cervids and it is placed in the Cervoidea. Pecorans seem to have evolved in Eurasia and there gave rise to deer. By the early Miocene some pecorans had entered Arabo‐Africa where they gave rise to giraffids and probably bovids. The relationships and zoogeography of the early bovids Eotragus, Homoiodorcas and Caprotragoides are likely to be important for later bovid history.     

Methylglyoxal Evokes Pain by Stimulating TRPA1

Diabetic neuropathy is a severe complication of long-standing diabetes and one of the major etiologies of neuropathic pain. Diabetes is associated with an increased formation of reactive oxygen species and the electrophilic dicarbonyl compound methylglyoxal (MG). Here we show that MG stimulates heterologously expressed TRPA1 in CHO cells and natively expressed TRPA1 in MDCK cells and DRG neurons. MG evokes [Ca²⁺]_i-responses in TRPA1 expressing DRG neurons but is without effect in neurons cultured from Trpa1^−/− mice. Consistent with a direct, intracellular action, we show that methylglyoxal is significantly more potent as a TRPA1 agonist when applied to the intracellular face of excised membrane patches than to intact cells. Local intraplantar administration of MG evokes a pain response in Trpa1^+/+ but not in Trpa1^−/− mice. Furthermore, persistently increased MG levels achieved by two weeks pharmacological inhibition of glyoxalase-1 (GLO-1), the rate-limiting enzyme responsible for detoxification of MG, evokes a progressive and marked thermal (cold and heat) and mechanical hypersensitivity in wildtype but not in Trpa1^−/− mice. Our results thus demonstrate that TRPA1 is required both for the acute pain response evoked by topical MG and for the long-lasting pronociceptive effects associated with elevated MG in vivo. In contrast to our observations in DRG neurons, MG evokes indistinguishable [Ca²⁺]_i-responses in pancreatic β-cells cultured from Trpa1^+/+ and Trpa1^−/− mice. In vivo, the TRPA1 antagonist {"type":"entrez-nucleotide","attrs":{"text":"HC030031","term_id":"262060681"}}HC030031 impairs glucose clearance in the glucose tolerance test both in Trpa1^+/+ and Trpa1^−/− mice, indicating a non-TRPA1 mediated effect and suggesting that results obtained with this compound should be interpreted with caution. Our results show that TRPA1 is the principal target for MG in sensory neurons but not in pancreatic β-cells and that activation of TRPA1 by MG produces a painful neuropathy with the behavioral hallmarks of diabetic neuropathy.     

Spirocyclic replacements for the isatin in the highly selective,muscarinic M1 PAM ML137: The continued optimization of an MLPCN probe molecule

This Letter describes the further optimization of an MLPCN probe molecule (ML137) through the introduction of 5- and 6-membered spirocycles in place of the isatin ketone. Interestingly divergent structure–activity relationships, when compared to earlier M₁ PAMs, are presented. These novel spirocycles possess improved efficacy relative to ML137, while also maintaining high selectivity for the human and rat muscarinic M₁ receptor subtype.     

Laforin, a dual-specificity phosphatase involved in Lafora disease, is phosphorylated at Ser25 by AMP-activated protein kinase

Lafora progressive myoclonus epilepsy [LD (Lafora disease)] is a fatal autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in either the EPM2A gene, encoding the dual-specificity phosphatase laforin, or the EPM2B gene, encoding the E3-ubiquitin ligase malin. Previously, we and others showed that laforin and malin form a functional complex that regulates multiple aspects of glycogen metabolism, and that the interaction between laforin and malin is enhanced by conditions activating AMPK (AMP-activated protein kinase). In the present study, we demonstrate that laforin is a phosphoprotein, as indicated by two-dimensional electrophoresis, and we identify Ser(25) as the residue involved in this modification. We also show that Ser(25) is phosphorylated both in vitro and in vivo by AMPK. Lastly, we demonstrate that this residue plays a critical role for both the phosphatase activity and the ability of laforin to interact with itself and with previously established binding partners. The results of the present study suggest that phosphorylation of laforin-Ser(25) by AMPK provides a mechanism to modulate the interaction between laforin and malin. Regulation of this complex is necessary to maintain normal glycogen metabolism. Importantly, Ser(25) is mutated in some LD patients (S25P), and our results begin to elucidate the mechanism of disease in these patients.     

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as ‘topological poisons of topoisomerase I’.