Synergistic hematopoietic growth factors

A number of growth factors acting on hematopoietic stem cells have now been purified and characterized. These include erythropoietin, granulocyte-macrophage colony-stimulating activity (GM-CSA), granulocyte colony-stimulating activity and colony-stimulating factor-1 (CSF-1). Factors which act in concert with these defined factors and appear to act relatively early in the hematopoietic stem cell lineage are currently under study. Interleukin 3 appears to have both the characteristics of a differentiating hormone and the ability to generate proliferation of relatively early stem cells. Interleukin 3 acts in concert with at least CSF-1 and erythropoietin to enhance their effect on stem cell proliferation and differentiation. A new class of hematopoietic growth factor activities termed synergizing activities also exist. These activities appear to have no intrinsic capacity to stimulate hematopoietic colony formation by themselves but enhance the effects of other differentiating hormones such as GM-CSA and CSF-1. Activities which appear to represent synergizing activities have now been found to evolve from a human bladder carcinoma line, a cell line derived from murine marrow adherent cells and normal murine marrow and thymic cells. These activities may act on very primitive hematopoietic progenitors to allow them to express receptors to various differentiating hormones or alternatively they may act as commitment factors in a commitment-progression model of stem cell regulation.     

Tetanus toxin action: inhibition of neurotransmitter release linked to synaptobrevin proteolysis.

Tetanus toxin is a potent neurotoxin that inhibits the release of neurotransmitters from presynaptic nerve endings. The mature toxin is composed of a heavy and a light chain that are linked via a disulfide bridge. After entry of tetanus toxin into the cytoplasm, the released light chain causes block of neurotransmitter release. Recent evidence suggests that the L-chain may act as a metalloendoprotease. Here we demonstrate that blockade of neurotransmission by tetanus toxin in isolated nerve terminals is associated with a selective proteolysis of synaptobrevin, an integral membrane protein of synaptic vesicles. No other proteins appear to be affected by tetanus toxin. In addition, recombinant light chain selectively cleaves synaptobrevin when incubated with purified synaptic vesicles. Our data suggest that cleavage of synaptobrevin is the molecular mechanism of tetanus toxin action.     

The honey-bee “dance language” hypothesis and the foundations of biology and behavior

We have carried out a detailed analysis of the region of the hormone, which can be called an “active site”, that is essential for receptor binding and/or agonist activity, on the basis of structure-activity data of peptide hormones published in the literature. We find that one or more aromatic residues, often in a cluster, is present at the active sites of insulin, glucagon, adrenocorticotropin, gastrin, endorphins and angiotensin. Recognition of the functional importance of aromatic residues, combined with sequence comparisons and secondary structural predictions, enable us to identif active sites of nerve growth factor, somatostatin, calcitonin, parathyroid hormone and luteinizing hormone releasing hormone. Aromatic residues also appear to be essential for the function of nonpeptide hormones that act at the plasma membrane, and opiates. The apparently ubiquitous presence of aromatic groups at the active site of peptide hormones may facilitate understanding of the mechanism of action of hormones and provide insights into the design of hormone analogues.     

A role for the divergent actin gene, ACT2, in nuclear pore structure and function.

We have identified a temperature-sensitive allele of the yeast divergent actin gene ACT2, act2-1, which displays defects in nuclear pore complex (NPC) structure and nuclear import at the restrictive temperature. Although defective in nuclear import, act2-1 cells still selectively retain reporter proteins in the nucleus, and by indirect immunofluorescence the actin cytoskeleton appears normal. Previous studies in Acanthamoeba and Saccharomyces cerevisiae reported that the cellular location of Act2p partially overlaps that of conventional actin, indicating that it has a cytoskeletal function. In this study, both immunofluorescence localization and cellular fractionation of different epitope-tagged versions of Act2p also reveal an association with the nucleus, suggesting an independent nuclear function for Act2p. Analysis of act2-1 by electron microscopy, 30 min after a shift to the restrictive temperature (37 degrees C), reveals a striking aberration in NPC morphology; NPCs appear as abnormal densities on either side of, rather than spanning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically lethal in combination with a deletion in the XFXFG nucleoporin gene, NUP1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting that the proteins exist in a complex. Together our data argue that Act2p plays an important role in NPC structure and function.     

Advances in antihypertensive therapy: Non-peptide angiotensin II receptor antagonists as potent therapeutic agents

Summary The development of drugs that selectively block angiotensin receptors has resulted largely from a process of trialand-error medicinal chemistry on an early lead. The new generation of angiotensin antagonists or angiotensin mimetics are essentially devoid of side effects and are set to replace the angiotensin converting enzyme inhibitors for the treatment of cardiovascular diseases.     

Sendai virus C proteins must interact directly with cellular components to interfere with interferon action

Sendai virus (SeV) infection of interferon (IFN)-competent cells is one of the most efficient ways of inducing IFN production. Virus replication is nevertheless largely unaffected, since SeV infection also interfers with IFN action, a prerequisite for the establishment of an antiviral state. This property has been mapped by reverse genetics to the viral C gene, which is also known to act as a promoter-specific inhibitor of viral RNA synthesis. Using luciferase reporter plasmids containing IFN-responsive promoters, we have found that all four C proteins effectively interdict IFN signaling when expressed independently of SeV infection. The C proteins must therefore interact directly with cellular components to carry this out. The C gene in the context of an SeV infection was also found to induce STAT1 instability in some cells, whereas in other cells it apparently acts to prevent the synthesis of STAT1 in response to the virus infection or IFN treatment. The SeV C proteins appear to act in at least two ways to counteract the IFN induced by SeV infection.     

THE INFLUENCE OF SECRETIN, GLUCAGON AND OTHER PEPTIDES, OF AMINO ACIDS, PROSTAGLANDIN ENDOPEROXIDE ANALOGUES AND DIAZEPAM ON THE LEVEL OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN NEUROBLASTOMA GLIOMA HYBRID CELLS

Abstract— Neuroblastoma glioma hybrid cells display many properties of neurons. A series of compounds, among them a number of amino acids, peptides and peptide hormones were tested for their ability to influence the level of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in the hybrid cells. Two prostaglandin endoperoxide analogues exhibit a weak stimulatory action, if applied in at least micromolar concentrations. At nanomolar concentrations, only the gastrointestinal hormones secretin and glucagon stimulate the formation of cyclic AMP, as detected in the presence of a phosphodiesterase inhibitor. The effect of secretin but not that of glucagon is antagonized by secretin-(5–27), suggesting that secretin and glucagon act on the cells via different receptors. These results appear to be noteworthy since (a) an effect of secretin or glucagon on a cell with neuronal characteristics has not yet been described, and (b) many peptide hormones have been detected both in the gastrointestinal tract and the nervous system.     

Epigenetics and the power of art

This review presents an epigenetic view on complex factors leading to development and perception of "genius." There is increasing evidence which indicates that artistic creativity is influenced by epigenetic processes that act both as targets and mediators of neurotransmitters as well as steroid hormones. Thus, perception and production of art appear to be closely associated with epigenetic contributions to physical and mental health.     

Hormonal regulation of the number and activity of ribosomes in mammary gland explants of mice

Possible effects of insulin, hydrocortisone and prolactin on the number and activity of ribosomes enganged in protein biosynthesis in mammary gland explants were explored. The rate and extent of [3H]-puromycin attachment to nascent peptides was used to assess, respectively, the activity and number of ribosomes engaged in protein biosynthesis. None of the hormones altered the number of ribosomes engaged in protein biosynthesis. In addition, of the hormones tested, only insulin appeared to accelerate the rate at which ribosomes carried out the translocation process. The early (1 hr) effect of insulin on protein biosynthesis in the mammary gland would therefore appear to occur via an activation of ribosomal activity. In contrast, the early (6 hr.) effect of prolactin on protein biosynthesis would appear to be exclusively via an RNA-DNA dependent mechanism.     

Iodinated neurohypophyseal hormones as potential ligands for receptor binding and intermediates in synthesis of tritiated hormones.

[3-Iodo-Tyr2]oxytocin (MIOT), [3,5-diiodo-Tyr2]oxytocin (DIOT), [3-iodo-Tyr2,Lys8]vasopressin (MILVP), [3,5-diiodo-Tyr2,Lys8]vasopressin (DILVP), [3-iodo-Tyr2,Arg8]vasopressin (MIAVP), and [3,5-diiodo-Tyr2,Arg8]vasopressin (DIAVP) were synthesized by iodination of the respective hormones, pruified, and characterized. All the monoiodo hormones had to be freshly prepared prior to bioassays, since on storage they gave rise to hormonal-like biological activity. The biological activities of these iodo analogues were measured in an adenylate cyclase assay employing neurohypophyseal hormone (NHH) sensitive bovine renal medullary membranes, and/or the rat oxytocic assay. In the cyclase assay, DIOT, DILVP, and DIAVP were inactive as agonists or antagonists. MIOT shows no agonistic activity in the renal cyclase system and uterus, but is a weak reversible inhibitor of oxytocin (OT) in both systems. When MIOT (10(-4) M) was preincubated with renal membranes for 10 min at 37 degrees C before addition of OT, it behaved as a noncompetitive inhibitor of NHH-stimulated adenylate cyclase. MILVP and MIAVP appear to be partial agonists with Km (half maximal response) 3 X 10(-6) and 3 X 10(-7) M, respectively, as determined in the cyclase assay. Upon preincubation with renal medullary membranes, MILVP (10(-6) M) behaves as a more potent noncompetitive inhibitor of OT than MIOT. Accordingly, iodo derivatives of NHH do not exhibit sufficient affinity to serve an specific ligands to measure OT, LVP, or AVP receptors in the uterus and kidney. Study of the specificity of inhibition produced by MIOT revealed that this analogue does not act selectively upon NHH receptors. Thus, MIOT modified adenylate cyclase systems which do not have NHH receptors, e.g., the PTH-sensitive adenylate cyclase in bovine renal cortex and the glucagon-sensitive adenylate cyclase in rat liver. DIOT, DILVP, and DIAVP were subjected to catalytic tritiation (employing carrier free tritium) and were converted to [3H]OT (25, 31, and 25 Ci/mmol), [3H]LVP (26 and 23 Ci/mmol), and [3H]AVP (17 Ci/mmol), respectively. These tritiated ligands have been successfully used to measure NHH receptor sites both in kidney and uterine membranes as described in other studies.     

'New' active steroids and an unforeseen mechanism of action

Steroid hormones mostly act via nuclear receptors. Dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulphate(s), classically known only as hormonal precursors, are also neurosteroidal efficacious signals, modulating neurotransmitter receptor function at the membrane level. An additional unforeseen mechanism of steroid action is reported here: PREG binds to neural microtubule-associated protein MAP2 and increases both the rate and extent of tubulin polymerization studied in vitro with purified tubulin and MAP2, forming microtubules of normal electron microscopic appearance. Progesterone and PREGS also bind to MAP2, but counteract PREG activity. In cultured neurons, PREG specifically increased immunostaining by an antiMAP2 monoclonal antibody and its extension into neurites. This novel mechanism may play a role in regulating microtubule formation and dynamics, which are altered in brain ageing and diseases.     

Activation of carboxylesterases in root cortical parenchymal cells of Pisum sativum during xylem induction in vitro

A quantitative cytochemical study of naphthol AS-D esterase activity in explants from roots of Pisum sativum grown on basal medium for 3 or 6 days showed similar levels of activity to those seen in sections of cortex and stele from intact roots of similar ages. Explants grown in the presence of auxins or cytokinin alone showed a threefold or twofold increase in cortical parenchymal activity, respectively. On adding both hormones to initiate xylem element formation, there was also a threefold increase in activity in the cortex. In all three cases, the stimulated activity was totally inhibited by either 10(-4) M diisopropylfluorophosphate (DFP) or 10(-4) M diethyl p-nitrophenylphosphate (E600), indicating carboxylesterase activity. The low level of activity normally present in cortical cells was inhibitor resistant, so indicating acetylesterase activity. Thus, auxins and cytokinins appear to activate mainly similar carboxylesterases during the initiation of xylem elements from cortical parenchyma cells.     

The mechanism of γ-Secretase dysfunction in familial Alzheimer disease

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid β (Aβ)42 relative to Aβ40 by an unknown, possibly gain‐of‐toxic‐function, mechanism. However, many PSEN mutations paradoxically impair γ‐secretase and ‘loss‐of‐function’ mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ε‐cleavage function is not generally observed among FAD mutants. On the other hand, γ‐secretase inhibitors used in the clinic appear to block the initial ε‐cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase‐like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aβ products, and suggest fundamental improvements for current drug development efforts.     

Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.     

Contractions of amphibian Wolffian duct in response to acetylcholine, norepinephrine, and arginine vasotocin

The regulation of sperm transport through the Wolffian duct of male amphibians is poorly understood. These experiments were conducted using rough-skinned newts (Taricha granulosa) to determine if Wolffian ducts are capable of contracting in vitro and, if so, to characterize the contractile responses to acetylcholine (ACh), norepinephrine (NE), and neurohypophysial hormones. Dose-response curves for NE and ACh, which were prepared by measuring isometric contractions, are similar to those reported for mammalian vas deferens. For NE, the minimum effective dose and ED50 were found to be 1 X 10(-5)M and 4.17 X 10(-5)M, respectively. For ACh, the minimum effective dose was 3.2 X 10(-8)M and the ED50 was 1.37 X 10(-5)M. Alpha-adrenoreceptors appear to mediate the contractile responses to NE because phentolamine (10(-5)M) blocked or attenuated the response to NE (10(-6)M, 10(-5)M or 10(-4) M). Beta-adrenoreceptors appear to mediate relaxation because dichloroisoproterenol (10(-5)M) enhanced the response to 10(-5)M NE. The contractile response to three neurohypophysial hormones were also investigated. Arginine vasotocin was more effective in eliciting contractions than oxytocin. The effect of lysine vasopressin was intermediate between arginine vasotocin and oxytocin. These experiments demonstrate that amphibian (Taricha) Wolffian ducts contract in vitro in response to neurotransmitters and neurohypophysial hormones. The contractile response to neurotransmitters occurs in a dose-dependent manner; the response to neurohypophysial hormones is hormone specific.