Synthesis, monitoring and structure-function studies on some neurokinin A analogues.
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Amino Acid Sequence↗
Biomedical subjects
J M Allen
Publications and source records attributed to J M Allen.
Molecular structure of neuropeptide Y.
The original description of NPY following its isolation commented on its homology to the pancreatic polypeptide family of peptides. This homology is extended to the mRNA sequences and the genomic structure, suggesting that this family has arisen as a result of gene duplication. However, each member demonstrates remarkable specificity in its expression within tissue types. The expression of the NPY gene is thus controlled by, as yet undefined, factors regulating to neural cells. The level of expression within cells is dependent on nerve growth factor. However, other factors, such as the rate of processing of the precursor, may be involved in regulation of the stored concentrations of the peptide product.
Amino Acid Sequence↗
Nerve-mediated contractions of sheep mesenteric lymph node capsules.
1. Isometric tension was recorded in vitro from strips cut from the capsules of mesenteric lymph nodes of sheep. 2. One minute periods of field stimulation at frequencies of 1, 2, 4, 8 and 16 Hz (pulse duration, 0.3 ms) elicited tonic contractions of increasing force and duration. The stimulus frequency-response relationship began to flatten out at frequencies greater than 4 Hz, where the response was already 72% of that at 16 Hz. 3. The response to field stimulation was abolished by tetrodotoxin (1 microM). 4. Phentolamine, rauwolscine and prazosin (all 1 microM) reduced the response to field stimulation, while desipramine (1 microM), potentiated it. 5. Atropine (1 microM) was without effect on the response. 6. These results suggest that sheep mesenteric lymph node capsules have a noradrenergic innervation which modulates their tone via an action on alpha-adrenoceptors.
Adrenergic alpha-Antagonists↗
Nurses & child abuse/neglect reporting: duties, responsibilities, and issues.
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Child Abuse↗
Sex differences in vasoactive intestinal peptide (VIP) concentrations in the anterior pituitary and hypothalamus of rats.
Recent evidence suggests that vasoactive intestinal peptide (VIP), a putative prolactin (PRL)-releasing factor, is both synthesized and released by anterior pituitary cells, to act as a paracrine or autocrine factor. We have investigated the hypothesis that hypothalamic or pituitary VIP levels differ in male and female rats, since neuroendocrine control of PRL is sexually differentiated. Opposite sex differences were found in the hypothalamus and anterior pituitary. Random-cycle female rats had one-third higher VIP levels in the hypothalamus than males. In contrast, anterior pituitary VIP levels were 3 times as high in male rats as in females. Median eminence VIP levels were similarly low in both sexes. These results support a possible role of VIP in the sexually dimorphic regulatory mechanisms of PRL secretion. Moreover, demonstration that hypothalamic and pituitary VIP levels vary in opposite directions suggests that VIP is differentially regulated at the two sites.
Animals↗
Neuropeptide Y (NPY) and vasopressin (AVP) in the hypothalamo-neurohypophysial axis of salt-loaded or Brattleboro rats.
A close anatomical relationship between nerve terminals containing neuropeptide Y (NPY) and vasopressin (AVP) has been demonstrated in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). Furthermore, injections of NPY into the SON increased plasma concentrations of AVP in the rat. These data suggest a potential involvement of hypothalamic NPY in fluid homeostasis in the rat. Therefore, we have studied the effect of elevated plasma osmolality on the concentration of NPY and AVP in the hypothalamus and neurointermediate lobe (NIL) of the pituitary gland. Furthermore, we measured the concentration of NPY in the AVP-deficient Brattleboro rat, which suffers from diabetes insipidus and hyperosmolality. Salt-loading increased plasma osmolality and the concentration of AVP from 2.0 +/- 0.5 to 4.1 +/- 0.6 pg/ml after 7 days. The concentration of NPY in the NIL doubled after 7 days of salt-loading, from 7.9 +/- 0.6 ng/mg protein to 15.2 +/- 1.4 ng/mg protein, whereas AVP concentrations fell from 2285.7 +/- 210.9 ng/mg protein to 187.5 +/- 2.5 ng/mg protein. AVP concentrations in the ME increased transiently after 2 days of salt-loading and returned to control levels after 7 days. In contrast, NPY concentrations in the ME were unchanged at 2 days and were increased 61% after 7 days. NPY concentrations also were significantly elevated after 7 days of salt-loading in the preoptic area (POA) and mediobasal hypothalamus (MBH). The concentration of NPY in the NIL of the homozygous Brattleboro rat was 2-fold greater than in the heterozygous Brattleboro rat and 4-fold greater than in Sprague-Dawley rats used as controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Isolation and expression of functional high-affinity Fc receptor complementary DNAs.
Human and murine mononuclear phagocytes express a high-affinity receptor for immunoglobulin G that plays a central role in macrophage antibody-dependent cellular cytotoxicity and clearance of immune complexes. The receptor (FcRI) may also be involved in CD4-independent infection of human macrophages by human immunodeficiency virus. This report describes the isolation of cDNA clones encoding the human FcRI by a ligand-mediated selection technique. Expression of the cDNAs in COS cells gave rise to immunoglobulin G binding of the expected affinity and subtype specificity. RNA blot analysis revealed expression of a 1.7-kilobase transcript in macrophages and in cells of the promonocytic cell line U937 induced with interferon-gamma. The extracellular region of FcRI consists of three immunoglobulin-like domains, two of which share homology with low-affinity receptor domains.
Amino Acid Sequence↗
The effects of cholinergic and serotoninergic drugs on motility in vitro of Haplometra cylindracea (Trematoda: Digenea).
The spontaneous activity of the somatic muscle of the amphibian lung fluke, Haplometra cylindracea has been recorded in vitro, using an isometric force transducer system. Normal movement consisted of a continuous series of regular contractions which were maintained for over 4 h. Acetylcholine (ACh) inhibited motility at a concentration of 1 X 10(-3)M induced a flaccid paralysis. A similar response occurred with the cholinomimetric drugs, carbachol and nicotine, although in these instances the inhibition was less easily reversed by washing with frog Ringer. The inhibitory effect of ACh was blocked by d-tubocurarine but not by atropine. Serotonin (5-HT) caused an increase in the frequency and amplitude of contractions and the effect was blocked by methysergide. No uniform response was observed with 3-hydroxytryptamine (3-HT), but both reserpine and fluoxetine inhibited worm movement. Results suggest that the somatic musculature of the worm is controlled, at least in part, by an inhibitory cholinergic and an excitatory serotoninergic system.
Acetylcholine↗
Beta-adrenoceptor-mediated facilitation of [3H]-noradrenaline release from the intramural nerves of bovine mesenteric lymphatic vessels.
1. Isolated segments of bovine mesenteric lymphatic vessels were loaded with [3H]-noradrenaline and its efflux in response to field stimulation at 2 Hz (0.3 ms pulses, 1 min train) examined. 2. Isoprenaline (10(-6) M) increased evoked fractional 3H efflux (3H released as a percentage of total tissue 3H at the onset of stimulation) from its control value of 0.67 +/- 0.09 to 1.2 +/- 0.18% (s.e.mean; n = 5). 3. Propranolol (10(-6) M) alone had no effect on evoked fractional 3H efflux but blocked the increase in transmitter overflow induced by isoprenaline (10(-6) M). 4. In the presence of the alpha-adrenoceptor antagonist phentolamine (10(-6) M) the facilitatory effect of isoprenaline (10(-6) M) on transmitter efflux was enhanced. In 5 experiments isoprenaline increased evoked fractional 3H efflux from its control value of 1.07 +/- 0.17 to 2.5 +/- 0.37% when phentolamine was present. 5. Adrenaline (10(-8) M) increased evoked fractional 3H efflux from its control value of 0.76 +/- 0.09 to 1.13 +/- 0.17% (n = 7). 6. These findings are consistent with the presence of presynaptic beta-adrenoceptors in this preparation. There is no evidence that these receptors play any role in feedback regulation of transmitter release.
Animals↗
Beta 2-adrenergic receptor regulation after transfection into a cell line deficient in the cAMP-dependent protein kinase.
A mouse beta 2-adrenergic receptor (beta 2AR) DNA clone was transfected and expressed in a mouse adrenocortical tumor cell line (Kin8) that lacks both beta 2AR and cAMP-dependent protein kinase (PKA). The receptor displayed a characteristic beta 2AR agonist binding profile that was similar to that observed in beta 2AR-transfected PKA+ mouse adrenocortical tumor cells (Y1). Isoproterenol treatment of beta 2AR-transfected Kin8 and Y1 cells resulted in a rapid loss of surface beta 2AR, as determined by the binding of the hydrophilic beta 2AR radioligand [3H]CGP 12177 [( 3H]CGP), followed by a decrease in adenylate cyclase activity. Sequestration of beta 2AR in Kin8 cells was beta 2AR agonist specific, temperature dependent, and rapidly reversible. Repeated treatment and recovery from isoproterenol incubation resulted in a cycling of surface [3H]CGP binding. The reappearance of [3H]CGP binding following short isoproterenol treatment was not affected by cycloheximide treatment of the cells. Prolonged incubation of beta 2AR-transfected Kin8 cells with isoproterenol resulted in the down-regulation of beta 2AR protein without a change in beta 2AR mRNA levels. Polysome profiles of control and down-regulated cells revealed that translation of beta 2AR mRNA is inefficient and does not change upon prolonged agonist treatment. Protein synthesis was required to reverse the down-regulation of beta 2AR. These results indicate that neither sequestration nor down-regulation of beta 2AR depends on PKA.
Adenylyl Cyclases↗
Nucleotide sequence of three cDNAs for the human high affinity Fc receptor (FcRI).
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Amino Acid Sequence↗
3-Hydroxy-3-methylglutaryl-coenzyme A reductase of the sea urchin embryo. Deduced structure and regulatory properties.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity is developmentally regulated in the sea urchin Strongylocentrotus purpuratus (Woodward, H. D., Allen, J. M. C., and Lennarz, W. J. (1988) J. Biol. Chem. 263, 2513-2517). To study the structural and regulatory properties of this enzyme, we isolated and sequenced a 3-kb cDNA encoding the sea urchin embryo reductase. The deduced amino acid sequence of this cDNA predicted a protein structure consisting of a hydrophobic N-terminal region containing seven potential membrane-spanning domains and a somewhat less hydrophobic C-terminal domain joined by a hydrophilic linker region. Comparison with reductase from mammalian sources revealed that the N-terminal membrane domain and the C-terminal cytoplasmic domain exhibited high sequence similarity, whereas the domain that linked these two showed little or no sequence similarity. We investigated the possibility that sterols or sterol derivatives might be involved in the marked change that occurs in the level of reductase activity over development. Enzyme activity and reductase mRNA levels measured in extracts from embryos cultured in the presence of cholesterol, 25-hydroxycholesterol, dolichol, or mevalonic acid were found to be virtually unchanged as compared to control embryos. Similar experiments with mevinolin, a competitive inhibitor of reductase, failed to show a drug-induced change in enzyme or mRNA level. Thus, despite structural similarities the sea urchin embryo enzyme differs markedly from the mammalian enzyme with respect to regulation, since its level is neither repressed by sterols nor induced by mevinolin. Moreover, it appears unlikely that sterols or sterol derivatives play a role in the striking change in the level of this enzyme that occurs during development.
Amino Acid Sequence↗
3-Hydroxy-3-methylglutaryl coenzyme A reductase in the sea urchin embryo is developmentally regulated.
The activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, an enzyme which plays a regulatory role in the synthesis of cholesterol, dolichol, and coenzyme Q, has been measured in the developing embryo of the sea urchin. Enzyme activity increased at least 200-fold during development from the unfertilized egg to the pluteus stage embryo. Mixing experiments suggested that the low level of enzyme activity found at early stages was not due to the presence of inhibitor(s) in the egg or zygote. The enzyme in the sea urchin embryo exhibited properties different from that found in mammals: only a fraction of the activity could be solubilized from microsomes, and mild trypsinization inactivated the enzyme without releasing any of it from the microsomes in soluble form. To further study the sea urchin HMG-CoA reductase, a genomic clone was identified by hybridization to a cDNA encoding hamster HMG-CoA reductase. Sequence analysis of this clone revealed a coding region that shares a high degree of homology with the carboxyl-terminal domain of hamster HMG-CoA reductase. Analysis of sea urchin embryo HMG-CoA reductase mRNA levels using a restriction fragment derived from the genomic clone revealed a 5.5-kilobase poly(A)+ mRNA that increased 15-fold during development from the egg to the gastrula stage and then decreased 1.5-fold at the pluteus stage. Since the relative increase in HMG-CoA reductase mRNA was less than the increase in enzyme activity (15-fold versus 200-fold) factors in addition to the level of mRNA may control the activity of this enzyme during embryogenesis.
Animals↗
Isoproterenol response following transfection of the mouse beta 2-adrenergic receptor gene into Y1 cells.
The beta 2-adrenergic receptor (beta 2AR) gene was isolated from a mouse genomic library, sequenced and shown to share 93% identity with the hamster beta 2AR cDNA at the amino acid level. This mouse beta 2AR genomic clone was transfected into the Y1 mouse adrenal cortex tumor cell line. Northern blot and S1 nuclease analysis showed that the beta 2AR-transfected cells expressed an mRNA of the appropriate size to encode the receptor. Membrane receptor number and affinities for various beta-adrenergic agonists demonstrated that the transfected clone encoded a beta 2AR protein product. Incubation of the transfected Y1 cells, which do not normally possess beta 2AR, with the beta 2AR agonist, isoproterenol, resulted in an increase in the rate of steroid secretion by these cells as well as a rapid change in cell morphology. This response was fully blocked by the beta 2AR antagonist, propranolol. Prolonged incubation of the cells with isoproterenol resulted in agonist insensitivity and an 80% reduction in membrane receptor number.
Amino Acid Sequence↗
The effect of known K+-channel blockers on the electrical activity of bovine lymphatic smooth muscle.
The effects of known K+-channel blockers on the electrical properties of bovine lymphatic smooth muscle were investigated using the double sucrose-gap technique. Constant current anodal pulses elicited hyperpolarizing electrotonic potentials (EP's) which were characterised by a "sag" in the potential record. Current/voltage relationship (I/V), which were examined by measuring EP amplitude at the end of 5 s anodal pulses (less than 30 microA), showed an apparent increase in conductance with increasing hyperpolarization. In the presence of caesium (10 mM), 4-aminopyridine (10 mM) or in the absence of external K+ the sag in the EP was lost and the inward rectification characteristic of the control I/V relationship was abolished. Barium (2.5 mM) also abolished in sag in the EP although TEA (10 mM) had no effect on either EP shape or I/V relationship. Thus it would appear that lymphatic smooth muscle shows inward rectification which is slowly activating and is blocked by some of the known K+-channel blockers or by the removal of external K+.
4-Aminopyridine↗
Introns increase transcriptional efficiency in transgenic mice.
Experiments were designed to test the effect of introns on gene expression in transgenic mice. Four different pairs of gene constructs, which were identical except that one member of each pair lacked all introns, were compared for expression of mRNA after introduction into the murine germ line by microinjection of fertilized eggs. The expression of two chimeric genes, made by fusing either the mouse metallothionein I or the rat elastase 1 promoter/enhancer to the rat growth hormone gene, was assayed in fetal liver or pancreas, respectively, while two natural genes, an oligonucleotide-marked mouse metallothionein I gene and the human beta-globin gene, were assayed in fetal liver. In each case there was, on average, 10- to 100-fold more mRNA produced from the intron-containing construct. Moreover, mRNA levels were proportional to the relative rates of transcription that were measured in isolated nuclei. However, when the expression of the two mouse metallothionein I gene-based constructs was tested after transfection into cultured cells, little difference was observed. These observations suggest that introns play a role in facilitating transcription of microinjected genes and that this effect may be manifest only on genes exposed to developmental influences.
Animals↗
Release of [3H]-noradrenaline from the sympathetic nerves to bovine mesenteric lymphatic vessels and its modification by alpha-agonists and antagonists.
1. Isolated segments of bovine mesenteric lymphatic vessels were loaded with [3H]-noradrenaline and its efflux in response to field stimulation examined. Vessels were attached to an isometric force transducer for the simultaneous recording of mechanical activity. 2. Field stimulation at 1, 4 and 8 Hz (0.3 ms pulses, 1 min train) increased spontaneous contraction rate and evoked 3H release up to a maximum of 4.5% of total tissue 3H at 8 Hz. Output per pulse was maximal at 4 Hz. 3. Tetrodotoxin (3 x 10(-6) M) blocked the release of 3H in response to field stimulation although the drug did not attenuate release evoked by high K+ (65 mM) solution. Field-evoked release of 3H was also absent in Ca2+ -free solution containing EGTA (1 mM). 4. When vessels were preincubated with labelled transmitter plus cocaine (5 x 10(-5) M) evoked release of 3H was absent. After preloading with [3H]-noradrenaline, cocaine (10(-6) M) potentiated both the mechanical response to field stimulation and evoked 3H release. 5. The relatively non selective alpha-adrenoceptor antagonist phentolamine (3 x 10(-6) M) and the alpha 2-antagonists yohimbine (10(-8) M) and rauwolscine (10(-6) M) significantly increased evoked 3H release at both of the frequencies examined (1 and 4 Hz). In contrast, the selective alpha 1-antagonist prazosin (10(-6) M) failed to alter 3H release to 4 Hz stimulation although release at 1 Hz was potentiated in the presence of the drug. 6. The postsynaptic excitatory response to field stimulation remained in the presence of prazosin (10(-6) M), but was converted to an inhibitory effect in the presence of phentolamine (3 x 10(-6) M), yohimbine (10(-6) M) or rauwolscine (10(-6) M). 7. Evoked 3H efflux was significantly reduced by clonidine (10(-6) M), xylazine (10(-6) M) and exogenous noradrenaline (5 x 10(-7) M), although phenylephrine (10(-6) M) reduced release only at the lower of the two frequencies tested (1 Hz). 8. These findings suggest that release of 3H by field stimulation reflects endogenous transmitter release and that this is subject to autoinhibition via feedback onto inhibitory prejunctional alpha 2-adrenoceptors. The postjunctional excitatory response is mediated via postjunctional alpha 2-adrenoceptors.
Adrenergic alpha-Agonists↗
Transient excitatory responses to sustained stimulation of intramural nerves in isolated bovine lymphatic vessels.
Spontaneous isometric contractions were measured in 2 cm segments of bovine mesenteric lymphatics. Field stimulation at 4 Hz was applied for a 20 min period. This caused a threefold increase in contraction frequency initially but the response faded rapidly so that, within 6 min of the beginning of stimulation, contraction frequency had returned to a value which was not significantly different from control. In contrast to the effect on frequency 3H efflux from vessels pre-loaded with [3H]noradrenaline was maintained at a value significantly higher than control for the entire stimulation period. The fade in response to exogenous noradrenaline was similar to that seen with field stimulation but with a slightly slower time course. Vessels desensitized with noradrenaline showed a greatly diminished response to field stimulation but responded normally to other agonists. Increasing the extracellular KCl concentration to 18 mM also caused a threefold increase in contraction frequency but, unlike the response to noradrenaline and field stimulation, this was maintained for the 20 min period during which [K+]o was raised. The results suggest that the adrenoceptors on lymphatic smooth muscle exhibit very rapid homologous desensitization.
Animals↗