Solid-phase synthesis of neuropeptides by Fmoc strategies.
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Automation↗
Biomedical subjects
M O'Shea
Publications and source records attributed to M O'Shea.
Behavioral role for nitric oxide in chemosensory activation of feeding in a mollusc.
A role for the NO-cGMP pathway in mediating chemosensory activation of feeding is suggested by intense NADPH diaphorase staining observed in nerve fibers that project from sensory cells in the lips to the CNS and by the presence in the CNS of a NO-activated guanylyl cyclase. In preparations reduced to isolated lips and CNS, intracellular recordings were made from motoneurons driven by the interneurons of the central pattern generator (CPG) for feeding. Fictive feeding in such preparations can be recorded from these motoneurons following the application of sucrose to the lips. Sucrose activation of fictive feeding is inhibited by the NO scavenger hemoglobin, the NO synthase inhibitor N omega-Nitro-L-Arginine Methyl Ester (L-NAME) and by methylene blue, an inhibitor of guanylyl cyclase. Fictive feeding in isolated lip-CNS preparations can be activated without sucrose by superfusion of NO donor molecules such as SNAP and hydroxylamine and by the nonhydrolyzable analog of cGMP, 8-bromo-cGMP. The feeding CPG can also be activated centrally by depolarizing a modulatory interneuron, the slow oscillator (SO). When the CPG is activated in this way, fictive feeding is not susceptible to inhibition by hemoglobin, the most potent of the inhibitors of sucrose-activated fictive feeding. Behavioral experiments on intact snails confirm the findings from in vitro experiments and show that hemoglobin prevents feeding and methylene blue significantly delays the onset of feeding. These results indicate (1) that NO is a putative chemosensory transmitter in the snail L. stagnalis, (2) that the NO-cGMP pathway can mediate chemosensory activation of specific patterns of centrally generated behavior, (3) that NO is not involved in transmission within the central network of neurons responsible for the behavior, and more generally (4) that a freely diffusing and highly reactive gaseous signalling molecule can have restricted and specific behavioral functions.
Animals↗
Regulation of matrix metalloproteinase activity.
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Amino Acid Sequence↗
Different domain interactions are involved in the binding of tissue inhibitors of metalloproteinases to stromelysin-1 and gelatinase A.
The matrix metalloproteinases gelatinase A and stromelysin-1 have definable N-terminal (catalytic) and C-terminal domains. In order to analyze their interactions with the N- and C-terminal domains of the tissue inhibitors of metalloproteinases TIMP-1 and -2, mutants of both the enzymes and the inhibitors were prepared in which the C-terminal domains had been deleted. Since the Ki values for TIMP inhibition of the matrix metalloproteinases are in the picomolar range, it was not possible to measure these accurately within the sensitivity of available activity assays. Rate constants for the association of the wild-type proteins were therefore determined and systematically compared with those for the deletion mutants. It was found that TIMP-1 binds more rapidly than TIMP-2 to stromelysin-1 and that the C-terminal domain of the enzyme does not affect the rate of association of enzyme and inhibitor. This is in contrast to gelatinase A, where the C-terminal domain has been shown to play an important role in increasing the rate of complex formation with the TIMPs (Willenbrock et al., 1993). The TIMPs are also comprised of an N- and C-terminal domain. By deletion mutagenesis, we found that the C-terminal domain of both TIMPs contributed less to the rate of complex formation with stromelysin-1 than to that with gelatinase A. Hybrids of the N- and C-terminal domains of gelatinase A and stromelysin-1 were prepared and used to analyze further the differences in domain interactions with the TIMPs. They demonstrated that the interactions between the C-terminal domains of enzyme and inhibitor can occur irrespective of the nature of the N-terminal domain. We can conclude that the TIMPs have two major binding regions which associate in different ways with the domains of the enzymes gelatinase A and stromelysin-1. The N-terminal domains of the TIMPs bind to the enzyme catalytic domains to inhibit activity. The TIMP C-terminal domain acts to increase the association rate constant by binding to the N-terminal domain of stromelysin or the C-terminal domain of gelatinase A.
Amino Acid Sequence↗
Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing.
We have used a complete, synthetic precursor to adipokinetic hormone I (AKH I) and oligopeptides derived from this precursor as substrates for prohormone-processing enzymes extracted from AKH-synthesizing neurosecretory cells to reconstitute the post-translational steps in AKH biosynthesis in vitro. The results demonstrate the existence of endoproteolytic activity which cleaves the precursor only at the appropriate processing site (at the C-terminal side of Arg13). Further proteolytic processing of C-terminally extended AKH I (AKH-Gly-Lys-Arg) by a carboxypeptidase H-like activity removes the basic residues producing AKH-Gly-Lys, followed by AKH-Gly. Finally, a peptidylglycine-alpha-amidating-monooxygenase activity produces the amidated bioactive product from the glycine-extended peptide in a two-step process, the first of which requires ascorbate and Cu2+. Our results show that all steps in AKH precursor processing can be reconstituted and studied in vitro, providing a system to characterize the processing enzymes and to investigate the development of enzyme inhibitors for use as potential insecticides.
Amino Acid Sequence↗
A model for the structure of a homodimeric prohormone: the precursor to the locust neuropeptide AKH I.
We have determined the structure in solution of a homodimeric protein that is a precursor to the locust neuropeptide adipokinetic hormone I using nuclear magnetic resonance spectroscopy. This precursor, called P1, is comprised of two 41 residue strands joined by a single inter-chain disulphide at Cys39. We have also determined the structure of an end product of P1 processing, called APRP1; this is a homodimer comprised of residues 14-41 of P1. Nuclear Overhauser Effect (nOe) data indicate that in both P1 and APRP1, residues 22-37 (numbered with respect to P1) form pairs of alpha-helices, with no evidence for any other secondary structure.
Amino Acid Sequence↗
Outbreak of measles in a teenage school population: the need to immunize susceptible adolescents.
An outbreak of measles occurred in a community school and the surrounding area in Crowborough, East Sussex, UK, from December 1992 to February 1993. There were 96 suspected cases reported: 66 cases among 1673 students at one school and 30 community cases. The majority of suspected cases were in those aged 11-17 (78%), 2 cases occurred in infants < 1 year old and 8 cases in adults aged 18 years or over. Data collected on 60 (91%) of the 66 suspect school cases showed 56 (93%) had an illness which met a case definition of measles. Eighteen had confirmatory IgM measles antibody. Two cases were hospitalized. The local percentage uptake for measles immunization for the school age years affected varied between 64% and 84%. A survey of parents showed that approximately 74% of the students attending the school had a history of measles immunization. The immunization rates reported by parents for children who developed measles was 21%, (29% based on GP records) compared with 77% for those who remained well. Vaccine efficacy was estimated to be 92%. This outbreak, along with others recently reported in older unimmunized children in the UK, reinforces the need for catch-up immunization programmes to reach this susceptible group of adolescents.
Adolescent↗
Effects of IGF-I on renal function in end-stage chronic renal failure.
To determine whether insulin-like growth factor I (IGF-I) affects kidney function in patients with end-stage chronic renal failure, we administered recombinant human IGF-I (rhIGF-I) (100 micrograms/kg body wt subcutaneously twice daily) to nine individuals with baseline inulin clearances below 21 ml/min/1.73 m2. Four patients were treated for four days (short-term treatment) and five for periods between 13 and 27 days (long-term treatment). Administration of rhIGF-I increased inulin clearance, p-aminohippurate (PAH) clearance and the percent tubular reabsorption of filtered phosphate, and decreased plasma creatinine, blood urea nitrogen (BUN) and plasma phosphate during short-term administration. Kidney volume was unchanged in patients receiving the growth factor. rhIGF-I did not cause weight gain, proteinuria or hypoglycemia. Inulin clearance was not increased significantly above baseline after 13 or 20 days of IGF-I administration. PAH clearance remained elevated after 13 days, but not after 20 days of IGF-I. Levels of total circulating IGF-I were elevated above basal levels during the entire course of long-term IGF-I administration. In contrast, levels of circulating IGF binding protein 3 (IGFBP3) declined over time. Side effects related to IGF-I forced discontinuation of its use in two of five patients undergoing long-term treatment, and side-effects possibly related to IGF-I prompted discontinuation of its use in two others. We conclude that rhIGF-I can enhance glomerular filtration rate and renal plasma flow when administered short-term to humans with end-stage chronic renal renal failure. Further studies will be required to define its efficacy and usefulness long-term.
Adult↗
Increased frequency of obstructive airway abnormalities with long-term tracheostomy.
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Airway Obstruction↗
Structural requirements for processing of pro-adipokinetic hormone I.
We found that a seven-residue sequence in pro-adipokinetic hormone I (proAKH I) which precedes the endopeptidase cleavage site is predicted to form an omega loop. Molecular modelling experiments indicated that a stable omega loop may form at this site, and suggested that loop stability may depend on the C-terminal loop residue, Lys12. The importance of this residue in proAKH I processing was confirmed by the observation that replacement of Lys12 by thialysine, a Lys analog with an altered side chain, prevented processing in vivo. In addition we showed by molecular modelling that this side-chain alteration may prevent formation of an omega loop. Together, these approaches lead us to propose that an omega loop may serve as a recognition motif in proAKH I processing.
Amino Acid Sequence↗
Nitric oxide synthesis and action in an invertebrate brain.
Nitric oxide (NO) is synthesized in mammalian neurons by Ca2+/calmodulin activated NO synthase and functions as a signalling molecule by activating soluble guanylyl cyclases in target cells. We demonstrate here that both NO synthase and NO-activated guanylyl cyclase are present in the brain of the locust Schistocerca gregaria. Our observations indicate, for the first time, that the NO-cyclic GMP signalling pathway exists in invertebrate nervous systems.
Amino Acid Oxidoreductases↗
The 28-kDa N-terminal domain of mouse stromelysin-3 has the general properties of a weak metalloproteinase.
The putative matrix metalloproteinase mouse stromelysin-3 was expressed from Escherichia coli and from a mouse myeloma cell line. In the former case a single major protein of 58-kDa was detectable by immunoblotting, but no proteolytic activity could be elicited by zymography or trypsin or organomercurial treatment as would be expected for a typical matrix metalloproteinase. In the latter case immunodetectable proteins of 55-58 and 27-28-kDa were produced. The effect of trypsin or organomercurial treatment of the 55-58-kDa forms was to generate a 51-kDa form and lower molecular mass fragments. Upon zymographic analysis only the 27-28-kDa forms showed caseinolytic activity. N-terminal sequencing and immunoblotting analysis with antibodies specific to distinct domains of stromelysin-3 indicated that the 27-28-Da stromelysin-3 forms had lost the predicted propeptide and the majority of the C-terminal domain. The purified 28-kDa form of stromelysin-3 could weakly degrade a number of extracellular matrix proteins and was inhibited by TIMP. However, the evidence that mature full-length stromelysin-3 is a metalloproteinase could not be substantiated and the precise role of this protein in vivo remains to be elucidated. By partial analogy with interstitial collagenase, one hypothesis is that stromelysin-3 with an intact C-terminal domain has specific properties for an as yet undefined substrate.
Amino Acid Sequence↗
The activity of the tissue inhibitors of metalloproteinases is regulated by C-terminal domain interactions: a kinetic analysis of the inhibition of gelatinase A.
The cloning and expression of the full-length tissue inhibitor of metalloproteinase 2 (TIMP-2), delta 187-194TIMP-2, and delta 128-194TIMP-2 and the purification of these inhibitors and a cleaved version of TIMP-2 lacking nine C-terminal amino acids (delta 186-194TIMP-2) are described. The mechanism of inhibition of gelatinase A by the TIMPs was investigated by comparing the kinetics of association of TIMP-1, TIMP-2, the C-terminal deletions, and the mutants of both TIMPs which consisted of the N-terminal domain only. The full-length TIMPs inhibited gelatinase A rapidly with association constants of 3.2 x 10(6) M-1 s-1 for TIMP-1 and 2.1 x 10(7) M-1 s-1 for TIMP-2 at I = 0.2. The C-terminal peptide of TIMP-2 is proposed to exist as an exposed "tail" responsible for binding to progelatinase A and for increasing the rate of inhibition of active gelatinase A through electrostatic interactions with the C-terminal domain of the enzyme. The C-terminal domains of both TIMP-1 and TIMP-2 participate in low-affinity interactions with the C-terminal domain of gelatinase A which increase the rate of association by a factor of about 100 in both cases.
Amino Acid Sequence↗
Crystallization and preliminary X-ray analysis of a truncated tissue metalloproteinase inhibitor delta 128-194 TIMP-2.
Crystals of an active truncated form of a human tissue metallproteinase inhibitor (delta 128-194 TIMP-2), a protein implicated in a number of diseases of the connective tissues, have been grown in a form suitable for study by X-ray diffraction analysis. Crystals grow using polyethylene glycol 600 as a precipitant and are in space group P2(1)2(1)2, with cell dimensions a = 75.28 A, b = 100.77 A, c = 31.45 A. The asymmetric unit appears to contain two molecules of delta TIMP-2 with a Vm value of 2.3 A3/dalton. The crystals diffract to Bragg-spacings beyond 2.5 A. Native data to 3.2 A have been collected and a search for heavy-metal derivatives is in progress.
Crystallization↗
Crystallization and preliminary X-ray analysis of nonglycosylated tissue inhibitor of metalloproteinases-1, N30QN78Q TIMP-1.
A nonglycosylated (N30QN78Q) form of the human tissue inhibitor of metalloproteinases, TIMP-1, has been prepared and crystallized in a form suitable for X-ray diffraction analysis. Small single crystals have been grown using sodium tartrate as a precipitant. The crystals are in space group P2(1), with cell dimensions a = 35.28, b = 53.95, c = 48.56, and beta = 96.0 degrees. There is a single molecule of TIMP-1 in the asymmetric unit. The crystals diffract to at least 2.3 A resolution. Complete data have been collected to 2.9 A and a search for heavy-metal derivatives is in progress.
Base Sequence↗
Roles of growth hormone and growth factors in the pathogenesis and treatment of kidney disease.
Growth hormone and a number of polypeptide growth factors exert actions on renal development, growth, and metabolism and on repair processes following renal injury. There is increasing evidence that under selected circumstances, these agents play roles in the pathogenesis of kidney disease and that under others, they may be useful in its treatment. Growth hormone, platelet-derived growth factor, or transforming growth factor-beta may be causative of glomerulosclerosis. The reduction in epidermal growth factor expression within the kidney in the setting of acute ischemic injury could delay regeneration, and replacement may be therapeutic. Insulin-like growth factor I may play a role in the regenerative response to acute renal injury. Pharmacologic properties of growth hormone or insulin-like growth factor I to enhance glomerular filtration rate and renal plasma flow and to increase skeletal growth may be harnessable for treating chronic renal failure and its complications. It is likely that strategies designed to employ growth hormone or growth factors as pharmacologic agents or to block their activities will assume increasingly important roles in therapy for renal disease.
Animals↗
Role of growth factors in regulation of renal growth.
It is clear that a number of growth factors are synthesized within the adult and the developing kidney. Compelling evidence exists that several of these agents orchestrate the nephrogenic process. A growing body of data supports roles for one or more of these agents in the adult kidney as regulators of renal function, growth, and repair processes. Growth factor expression may be the cause of glomerulosclerosis in one or more pathophysiological states. Our knowledge of the sites of growth factor synthesis and actions in the kidney and of the mechanisms by which growth factor synthesis is regulated and actions are exerted is rudimentary. Expansion of this knowledge base is likely to result in a greater understanding of renal disease mechanisms and thereby generate strategies by which kidney damage can be halted, prevented, or reversed.
Animals↗
Postembryonic proliferation of neuroendocrine cells expressing adipokinetic hormone peptides in the corpora cardiaca of the locust.
Neuroendocrine glands that synthesize and secrete peptide hormones regulate the levels of these peptide messengers during development. In this article we describe a mechanism for regulating neuropeptide levels in the corpora cardiaca of the locust Schistocerca gregaria, a neuroendocrine gland structurally analogous to the vertebrate adenohypophysis. A set of five colocalized peptide hormones of the adipokinetic hormone family is synthesized in intrinsic neurosecretory cells in the corpora cardiaca. During postembryonic development there are progressive changes in the absolute and relative levels of these five peptide hormones. We show that the ability of the gland to increase peptide synthesis is due to a 100-fold increase in the number of cells which make up the gland. The gland grows by the addition of new cells derived from symmetrical division of undifferentiated precursor cells within the corpora cardiaca. We show, using double-label immunocytochemistry, that cells born in the glandular lobe mature into cells that express adipokinetic hormone peptides. The pattern of cell birth and peptide expression can account for the dramatic increase in postembryonic peptide levels.
Animals↗