The biologically active conformation of ergot alkaloids

Summary Molecular mechanics and NMR studies of the D ring conformation of ergot alkaloids demonstrate that both D₁ and D₂ forms may exist in solution. The comparison of the geometric parameters defining the spatial relations between the aromatic moieties and the basic nitrogen of conformationally restricted dopamine analogs, and that of ergolene, shows the D₁ conformation to be the bioactive one.     

A possible new role for the anti-ageing peptide carnosine

The naturally occurring dipeptide carnosine (β-alanyl-L-histidine) is found in surprisingly large amounts in long-lived tissues and can delay ageing in cultured human fibroblasts. Carnosine has been regarded largely as an anti-oxidant and free radical scavenger. More recently, an anti-glycating potential has been discovered whereby carnosine can react with low-molecular-weight compounds that bear carbonyl groups (aldehydes and ketones). Carbonyl groups, arising mostly from the attack of reactive oxygen species and ow-molecular-weight aldehydes and ketones, accumulate on proteins during ageing. Here we propose, with supporting evidence, that carnosine can react with protein carbonyl groups to produce protein-carbonyl-carnosine adducts (‘carnosinylated’ proteins). The various possible cellular fates of the carnosinylated proteins are discussed. These proposals may help explain anti-ageing actions of carnosine and its presence in non-mitotic cells of long-lived mammals. Received 29 November 1999; accepted 27 December 1999     

NMR structures of the C-terminal end of human complement serine protease C1s

Synthetic peptides derived from the C-terminal end of the human complement serine protease C1s were analysed by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism indicates that peptides 656-673 and 653-673 are essentially unstructured in water and undergo a coil-to-helix transition in the presence of increasing concentrations of trifluoroethanol. Two-dimensional NMR analyses performed in water/trifluoroethanol solutions provide evidence for the occurrence of a regular α-helix extending from Trp659 to Ser668 (peptide 656-673), and from Tyr656 to Ser668 (peptide 653-673), the C-terminal segment of both peptides remaining unstructured under the conditions used. Based on these and other observations, we propose that the serine protease domain of C1s ends in a 13-residue α-helix (656Tyr-Ser668) followed by a five-residue C-terminal extension. The latter appears to be flexible and is probably locked within C1s through a salt bridge involving Glu672. Received 19 November 1997; accepted 24 November 1997     

Natriuretic peptide hormones promote radial water movements from the xylem of Tradescantia shoots

Immunological evidence suggests that plants, like vertebrates, contain natriuretic peptides (NPs) and that rat atrial NP (rANP) binds specifically to plant membranes and promotes concentration and conformation-dependent stomatal opening. Stomatal opening and specific increases in cGMP levels were also observed in response to immunoreactive plant NP (irPNP). Here we report that both 1 μM rANP and irPNP (100 ng total protein/100 μL) significantly increase radial water movements out of the xylem of shoots of Tradescantia multiflora. Enhanced radial water movements are also observed in response to the cell permeant cGMP analogue 8-Br-cGMP (100 nM). The water channel inhibitor mercuric chloride (HgCl₂) significantly inhibits radial water movements at concentrations of 50 μM, while the presence of 10 μM 2-hydroxyethylmercaptoethanol (ME) prevents the inhibitory effect of the mercurial. The guanylate cyclase inhibitor LY 83583 at a concentration of 20 μM and sodium azide (NaN₃) at concentrations of ≥ 1 μM both also reduce radial water movements. We therefore conclude that the regulation of radial water movement out of the xylem involves modulation of cGMP levels, water channels and respiration-dependent processes. In addition, we propose that NPs have a critical role to play in radial water movements out of the xylem and speculate that as in vertebrates, NP effects might, at least in part, be mediated via the regulation of guanylate cyclases and water channels. Received 15 June 1998; received after revision 7 August 1998; accepted 26 August 1998     

The role of glycosylation in protein antigenic properties

Glycosylation of proteins is a common event and contributes to protein antigenic properties. Most data have been obtained from model studies on glycoprotens with well-defined structure or synthetic glycopeptides and their respective monoclonal antibodies. Antibodies raised against glycoprotein antigens may be specific for their carbohydrate units which are recognized irrespective of the protein carrier (carbohydrate epitopes), or in the context of the adjacent amino acid residues (glycopeptidic epitopes). Conformation or proper exposure of peptidic epitopes of glycoproteins is also frequently modulated by glycosylation due to intramolecular carbohydrate-protein interactions. The effects of glycosylation are broad: glycosylation may 'inactivate' the peptidic epitope or may be required for its reactivity with the antibody, depending on the structure of the antigenic site and antibody fine specificity. Evidence is increasing that similar effects of glycosylation pertain to T cell-dependent cellular immune responses. Glycosylated peptides can be bound and presented by MHC class I or II molecules and elicit glycopeptide-specific T cell clones. Received 5 July 2001; received after revision 9 October 2001; accepted 11 October 2001     

The role of myosin phosphorylation in the contraction-relaxation cycle of smooth muscle

Summary Considerable evidence from a variety of experimental procedures indicates that the phosphorylation of myosin is involved in the regulation of contractile activity in smooth muscle. Phosphorylation of the 20,000-dalton myosin light chains is required to initiate crossbridge cycling and this is consistent with the observation that the actin-activated Mg²⁺-ATPase activity of myosin is phosphorylation-dependent. In the simplest interpretation of this process it may be proposed that phosphorylation acts as an on-off switch. Clearly this cannot explain the observed complexity of smooth muscle contractile behavior and such may imply either that additional mechanisms are involved or that the role of myosin phosphorylation is not fully appreciated. Recently it has been shown that monomeric smooth muscle myosin can exist in a folded and an extended conformation and that each form is characterized by distinct enzymatic properties. Under appropriate solvent conditions phosphorylation of myosin favors the extended conformation. It is tentatively suggest that this, or an analogous, transition might be involved in the regulation of the smooth muscle contractile apparatus, and this possibility is discussed.The authors are supported by grants HL 23615 and HL 20984 from the National Institutes of Health.     

Kinetics of chemo-attraction of polymorphonuclear leukocytes towards N-formyl peptide studied with a novel polycarbonate (Nuclepore) membrane in the Boyden chamber

Summary The motile responses of human polymorphonuclear leukocytes (PMN) to N-formyl-methionyl-leucyl-phenylalanine (FMLP) in the Boyden chamber using a new sparse-pore polycarbonate membrane (pores 3 m in diameter and occupying 0.1% of surface area) were compared with those demonstrated by using a standard polycarbonate (Nuclepore) filtration membrane (pores 3 m in diameter and occupying 5% of surface area). Motility of PMN in gradients of FMLP using the new membrane was not influenced by chemokinetic effects of the factor, and the background migration of the cells was minimal. However, motility of PMN in gradients of FMLP using the standard membrane was found to be influenced by chemokinetic effects of the chemotactic factor, and the background or control migration (in the absence of chemotactic factor) of the cells was substantial. Greater directional migration of PMN according to steepness of the gradient of chemotactic factor was demonstrated with the use of the new membrane. The new membrane may be of considerable value in the further study of the chemotactic responses of PMN.     

A new semisynthetic ergot peptide alkaloid: DCN 203-922

Summary We report the synthesis, stereochemistry and preliminary pharmacological evaluation of DCN 203-922, a novel ergot alkaloid of the cyclol type, which contains in its peptide moiety the uncommon amino acid L-allo-isoleucine.Part of this paper was reported by this author at the Herbstversammlung der Schweizerischen Chemischen Gesellschaft, Bern, in October 1986.     

Identification of the bioactive peptide PEC-60 in brain

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60may play a role in the central nervous system disorders involving dopamine dysregulation. Received 6 December 2002; received after revision 10 December 2002; accepted 11 December 2002 RID="*" ID="*"Corresponding author.     

Release of calcitonin gene-related peptide from a human medullary carcinoma of the thyroid in vitro

Summary The secretion of human calcitonin gene-related peptide was examined in perifusates of medullary carcinoma of the thyroid with a sensitive radioreceptor assay. Calcitonin gene-related peptide was released after the addition of calcium (25–100 mM), in a dose-dependent manner. The results indicate that human medullary carcinomas of the thyroid secrete the calcitonin gene-related peptide as well as calcitonin.     

Molecular aspects of pathogenesis in osteoarthritis: the role of inflammation

Arthritic diseases cause enormous burdens in terms of pain, crippling, and disability. Osteoarthritis (OA), the most common form of arthritis, is characterized by a slow progressive degeneration of articular cartilage. The exact etiology of OA is not known, but the degradation of cartilage matrix components is generally agreed to be due to an increased synthesis and activation of extracellular proteinases, mainly matrix metalloproteinases. Insufficient synthesis of new matrix macromolecules is also thought to be involved, possibly as a consequence of deficient stimulation by growth factors. Although OA is defined as a noninflammatory arthropathy, proinflammatory cytokines such as interleukin-1 have been implicated as important mediators in the disease. In response to interleukin-1, chondrocytes upregulate the production of nitric oxide and prostaglandin E₂, two factors that have been shown to induce a number of the cellular changes associated with OA. The generation of these key signal molecules depends on inducible enzymes and can be suppressed by pharmacological inhibitors.     

Role of bombesin-related peptides in the mediation or integration of the stress response

In addition to the relatively well established role of corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP) in the mediation of the stress response, there is reason to believe that bombesin-like peptides (BN-LPs) may also contribute to the mediation or integration of these responses and thus might be considered as putative 'stress peptides'. This review provides evidence supporting this contention by showing that (i) BN-LPs are present at brain sites known to be activated by stressors, (ii) stressor exposure alters utilization of BN-related peptides, (iii) exogenous BN administration mimics the endocrine, autonomic and/or behavioral effects elicited by stressors, and (iv) antagonism of BN action attenuates the behavioral and/or neurochemical effects of stressors or of exogenously administered peptide. The evidence presented also suggests that BN-LPs mediate their stress-relevant effects through activation of CRH and/or AVP neurons. Several hypothetical mechanisms for such peptidergic interactions are discussed as to the implications of considering BN-LPs as 'stress peptides'. Received 16 July 2001; received after revision 27 August 2001; accepted 28 August 2001     

The islet-acinar axis of the pancreas: Is there a role for glucagon or a glucagon-like peptide?

Intravenous glucagon inhibits exocrine pancreatic secretion in vivo, but exogenous glucagon does not affect exocrine secretion in vitro. Recent work, however, suggested that endogenous glucagon may be involved in the regulation of exocrine secretion even in vitro. We therefore investigated the effects of exogenous and endogenous glucagon on exocrine secretion by the isolated perfused rat pancreas in the presence of 1.8 mM glucose. Exogenous glucagon did not affect CCK-stimulated amylase output. 20 mM arginine stimulated glucagon release, but did not affect basal enzyme secretion. CCK-stimulated amylase output, however, was significantly inhibited in the presence of arginine. This inhibitory effect of arginine on exocrine pancreatic secretion could be blocked by glucagon antibodies, but not by nonspecific gammaglobulins. Thus exogenous glucagon failed to affect exocrine pancreatic secretion in vitro, but endogenously released glucagon or a glucagon-like peptide inhibited amylase release in the isolated perfused pancreas. We conclude that glucagon or a glucagon-like peptide may be a mediator in the islet-acinar axis.     

Molecular characterization and expression of the antimicrobial peptide defensin from the housefly (Musca domestica)

A 430-bp cDNA encoding the insect antimicrobial peptide defensin was cloned from the housefly, and designated Musca domestica defensin (Mdde). The open reading frame of the cDNA encoded a 92-amino acid peptide with an N-terminal signal sequence followed by a propeptide that is processed by cleavage to a 40-amino acid mature peptide. Northern analysis and in situ hybridization identified the corresponding mRNA in the fat body of bacterially challenged houseflies and in the epidermis of the body wall of naive and challenged houseflies. The Gram-negative bacterium (Escherichia coli) is a strong inducer of the gene. By RT-PCR, Mdde mRNA was also detected in naive and challenged insects. These findings suggest that the defensin gene is constitutively expressed in the epidermis of the housefly body wall. The predicted mature form of Mdde was expressed as a recombinant peptide in E. coli and Pichia pastoris. The recombinant Mdde expressed in Pichia was active against Gram-positive and some Gram-negative bacteria. Received 20 June 2006; received after revision 3 October 2006; accepted 30 October 2006     

Assembly of MHC class I peptide complexes from the perspective of disulfide bond formation

Assembly of functional major histocompatibility complex (MHC) class I peptide complexes within the endoplasmic reticulum is critically important for the development of an adaptive immune response. The highly regulated loading of peptides onto MHC class I molecules is controlled by a multi-component chaperone system called the MHC class I peptide loading complex. The recent identification of the thioredoxin family member ERp57 as a component of the loading complex led to an interesting question: Why is there a thiol-disulfide oxidoreductase inside a complex dedicated to inserting peptides into a receptor binding site? Most recently, specific ERp57-mediated disulfide bond rearrangements have been identified inside the loading complex. What these biochemical events mean for the peptide loading process remains a matter of conjecture. While several important questions wait to be answered, this review intends to summarize our current view of the oxidative folding of MHC class I molecules and addresses the question of how the receptor ligand interaction might be regulated by thiol-based redox reactions.     

Cryo-bioorganic chemistry: freezing effect on stereoselection of l- and dl-leucine cooligomerization in aqueous solution

The chirality of l-/dl-leucine (50–50%) cooligomerization was investigated in liquid and frozen aqueous solutions. Cooligomerization was carried out by carbonyldiimidazole activation without initiator at an ambient (+22°C) and frozen (−18°C) temperature, respectively. The separated samples obtained after different time intervals of treatment were completely hydrolyzed (HCl) and the diastereomeric l- and d-leucine derivates of Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-l-alanine amide) were then traced and evaluated by RP-HPLC analysis. After 9 days of oligomerization, the l-Leu content was slightly enhanced in the liquid (57%) and somewhat more enhanced in the frozen (64%) samples. After 17 days, however, the l-Leu content had decreased in the liquid (53%) and frozen (56%) conditions. These l-enantiomer amplifications indicate that an l-antipode is preferentially incorporated into the α-helical turn of the oligomer in the earlier stage of cooligomerization, while, later, the d-antipode is also incorporated. The role of ice in the improved stereoselection is discussed. This is the first recorded example of the effect of freezing on stereoselection. Received 27 October 2000; revised 11 December 2000; accepted 4 January 2000     

T-cell signal transduction and the role of protein kinase C

The T lymphocyte has a vital part to play in maintaining the host response to bacterial and viral infection and also appears to play a key pathological role in autoimmune diseases such as rheumatoid arthritis. In this review, we summarize the signalling pathways which trigger antigen-driven T-cell proliferation and examine the evidence which suggests that protein kinase C (PKC) is fundamental to this process. Finally, we discuss the therapeutic potential that PKC inhibitors may have in the treatment of autoimmune disease. Received 31 March 1998; received after revision 19 May 1998; accepted 19 May 1998     

New approaches to therapy of cancers of the stomach,colon and pancreas based on peptide analogs

Cancers of the stomach, colon and exocrine pancreas are major international health problems and result in more than a million deaths worldwide each year. The therapies for these malignancies must be improved. The effects of gastrointestinal (GI) hormonal peptides and endogenous growth factors on these cancers were reviewed. Some GI peptides, including gastrin and gastrin-releasing peptide (GRP) (mammalian bombesin), appear to be involved in the growth of neoplasms of the GI tract. Certain growth factors such as insulin-like growth factor (IGF)-I, IGF-II and epidermal growth factor and their receptors that regulate cell proliferation are also implicated in the development and progression of GI cancers. Experimental investigations on gastric, colorectal and pancreatic cancers with analogs of somatostatin, antagonists of bombesin/GRP, antagonists of growth hormone-releasing hormone as well as cytotoxic peptides that can be targeted to peptide receptors on tumors were summarized. Clinical trials on peptide analogs in patients with gastric, colorectal and pancreatic cancers were reviewed and analyzed. It may be possible to develop new approaches to hormonal therapy of GI malignancies based on various peptide analogs.Received 20 November 2003; accepted 6 January 2004     

Membrane fusion: the process and its energy suppliers

Membrane fusion constitutes a pivotal process in eukaryotic cell physiology. Both specialized proteins and membrane lipids play key roles in fusion. Here, our current understanding of the mechanism of membrane fusion is reviewed. The focus is on the relatively simple and well-understood proteinaceous fusion machinery of enveloped viruses and the physical properties of lipids that appear to be of great relevance for fusion progression. Recent observations suggest that viral fusion proteins use packed conformational energy and bilayer-destabilizing domains to (i) bring participating membranes into intimate contact, (ii) merge proximal lipid monolayers through highly curved stalk/hemifusion intermediates, and (iii) generate a lipid-containing fusion pore, thereby terminating the fusion process. Received 4 January 2002; received after revision 3 April 2002; accepted 5 April 2002