Antimicrobial and cytolytic peptides of venomous arthropods

As a response to invading microorganisms, the innate immune system of arthropods has evolved a complex arrangement of constitutive and inducible antimicrobial peptides that immediately destroy a large variety of pathogens. At the same time, venomous arthropods have developed an additional offensive system in their venom glands to subdue their prey items. In this complex venom system, several enzymes, low-molecular-mass compounds, neurotoxins, antimicrobial and cytolytic peptides interact together, resulting in extremely rapid immobilization and/or killing of prey or aggressors. This review provides an overview of antimicrobial peptides identified in the hemolymph of venomous arthropods, and especially of cytolytic peptides in their venom. For these peptides a dual role is proposed: acting as antimicrobials as well as increasing the potency of the venom by influencing excitable cells.Received 17 March 2003; received after revision 11 June 2003; accepted 17 June 2003     

The Sushi peptides: structural characterization and mode of action against Gram-negative bacteria

The compositional difference in microbial and human cell membranes allows antimicrobial peptides to preferentially bind microbes. Peptides which specifically target lipopolysaccharide (LPS) and palmitoyl-oleoyl-phosphatidylglycerol (POPG) are efficient antibiotics. From the core LPS-binding region of Factor C, two 34-mer Sushi peptides, S1 and S3, were derived. S1 functions as a monomer, while S3 is active as a dimer. Both S1 and S3 display detergent-like properties in disrupting LPS aggregates, with specificity for POPG resulting from electrostatic and hydrophobic forces between the peptides and the bacterial lipids. During interaction with POPG, the S1 transitioned from a random coil to an α-helix, while S3 resumed a mixture of α-helix and β-sheet structures. The unsaturated nature of POPG confers fluidity and enhances insertion of the peptides into the lipid bilayer, causing maximal disruption of the bacterial membrane. These parameters should be considered in designing and developing new generations of peptide antibiotics with LPS-neutralizing capability. Received 2 October 2007; received after revision 2 November 2007; accepted 4 December 2007 J. L. Ding, B. Ho: Co-senior authors.     

海洋活性肽研究的回顾与展望

海洋环境资源丰富,物种多样,是活性肽开发与研究的巨大宝库。本文分析了活性肽丰富的海洋来源及生物功用,并且对活性肽的自然来源途径和人工合成来源途径进行了论述,包括活性肽的分离、纯化、合成、筛选等方面,并强调了海洋活性肽在食品工业、医药学、畜牧业、水产养殖业、尤其是在环境保护中的应用现状与潜力,还从多角度、多层面对海洋活性肽的研究作出展望．     

Distribution patterns of mammalian-like peptide immunoreactive cells in the midgut ofAeshna cyanea (Insecta,Odonata)

Summary The distribution of cells reacting with antisera to cholecystokinin, substance P, gonadoliberin, methionine-enkephalin, and vasoactive intestinal peptide, demonstrated by the indirect immunoperoxidase method, was studied along the entire midgut of an insect,Aeshna cyanea. For each antiserum, the number of reacting cells increased from the middle part to the end of the midgut. Only a few cells reacted to somatoliberin, leucin-enkephalin and somatostatin antisera. In the connective sheath surrounding the midgut epithelium, nerve fibers were stained by antisera to serotonin, somatostatin, cholecystokinin, vasoactive intestinal peptide and methionine-enkephalin.     

Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin

This review discusses the state-of-the-art in molecular research on the most prominent and widely applied lantibiotic, i.e., nisin. The developments in understanding its complex biosynthesis and mode of action are highlighted. Moreover, novel applications arising from engineering either nisin itself, or from the construction of totally novel dehydrated and/or lanthionine-containing peptides with desired bioactivities are described. Several challenges still exist in understanding the immunity system and the unique multiple reactions occurring on a single substrate molecule, carried out by the dehydratase NisB and the cyclization enzyme NisC. The recent elucidation of the 3-D structure of NisC forms the exciting beginning of further 3-D-structure determinations of the other biosynthetic enzymes, transporters and immunity proteins. Advances in achieving in vitro activities of lanthionine-forming enzymes will greatly enhance our understanding of the molecular characteristics of the biosynthesis process, opening up new avenues for developing unique and novel biocatalytic processes. Received 9 April 2007; received after revision 31 August 2007; accepted 28 September 2007     

Nervous control of smooth muscle by transmitters,cotransmitters and modulators

Conclusion The part played by peripheral neuroeffector control mechanisms has been underestimated. These are additional to central and ganglionic control mechanisms and are much more elaborate than originally thought. While the classical view is that the autonomic nervous system consists largely of antagonistic cholinergic and adrenergic nerves, about sixteen putative neurotransmitters have been proposed in autonomic nerves in the past few years, including various monoamines, polypeptides, purines and amino acids. Modulatory transmitter mechanisms have also been recognized, including prejunctional inhibition or enhancement of transmitter release, postjunctional modulation of transmitter action, and the secondary involvement of locally synthesized hormones and prostaglandins. The existence of more than one transmitter substance in some nerves is now widely recognized, and suggestions have been made about the ways that this can lead to differential peripheral control mechanisms at nerve terminals themselves. The cotransmitters always have synergistic actions on postjunctional effector cells, but two different operating mechanisms are postulated. 1) If both substances are stored in the same vesicles (for example, ACh or NA with ATP), release is closely parallel at all impulse frequencies. Upon release, the cotransmitter, in addition to having a direct action on postjunctional cells, may facilitate the action of the other transmitter and/or act as an inhibitor of its release. Differential actions at different impulse frequencies are achieved post-junctionally by ATP and NA acting via EJP-spike and spike-independent mechanisms, respectively. 2) If the two substances are stored in separate vesicle types (for example ACh or NA with some peptides), then differential release is possible at different impulse frequencies; the peptides released at higher frequencies modulate the role of the classical transmitter, by both prejunctional enhancement of its release and post-junctional facilitation of its action.     

Precursors to regulatory peptides: their proteolytic processing

Summary Precursors to regulatory peptides undergo maturation processes which include protelytic processing. The enzymes involved in this process remove the hydrophobic peptide located at the amino-terminus of the precursor. Endoprotease cleavage also occurs at single and two adjacent basic residues, this is followed by a removal of basic residues located at the C-terminus of the peptides by a carboxypeptidase-like enzyme.     

Effects of enterally- and parenterally-administered bombesin on intestinal luminal tryptic activity and protein in the suckling rat

Summary Because of the presence of bombesin-like immunoreactivity in milk we investigated if enteral administration of bombesin affects the intestinal luminal content of trypsin and protein in 12-14-day-old rats. Bombesin (40 g/kg), given either orogastrically or subcutaneously, produced a significant elevation in the intestinal content of trypsin activity. Thus, enterally-administered bombesin can produce acute biologic effects in suckling rats.     

Co-localization of glucagon and pancreatic polypeptide in testudine pancreas

Summary Immunocytochemistry was carried out on sections of pancreas from the gopher tortoise,Gopherus polyphemus. Combined immunofluorescent and peroxidase-anti-peroxidase techniques showed unequivocally that some of the cells were immunoreactive for both glucagon and pancreatic polypeptide (PP). Antibodies directed against avian PP, bovine PP, and human PP all have a positive reaction. Co-localization of glucagon and PP in the pancreas of the gopher tortoise indicates that the occurrence of these hormones in the same cells is widespread in higher vertebrates.Acknowledgments. This work was supported in part by grants from the University of Cape Town, the M. Crossley bequest, the N. Atkinson bequest, and Herman and Caporn bequests. The generous gifts of antisera by Dr Chance and the late Dr Kimmel, and the gift of hormones by the Lilly Research Laboratories are gratefully acknowledged. The excellent technical assistance of J. Thompson and J. B. De Haan is also gratefully acknowledged. Please send all correspondence to the USA address.     

Participation of ACTH1–10 and ACTH4–10 on the melatonin modulation of benzodiazepine receptors in rat cerebral cortex

In this study, we examined the effect of intracerebroventricular (i.c.v) injection of melatonin and/or ACTH_1–10 and ACTH_4–10 on [³H]flunitrazepam binding sites in the cerebral cortex of hypophysectomized rats. Hypophysectomy increased the B_max (maximum number of binding sites) of benzodiazepine (BNZ) receptors for at least 7 days after surgery, without changing K_D (dissociation constant). The i.c.v. injection of melatonin to hypophysectomized rats significantly increased B_max, whereas the same doses of melatonin were ineffective in sham-operated animals. In both cases, K_D values were unchanged. The i.c.v injection of ACTH_1–10 to hypophysectomized animals significantly increased B_max, an effect that was enhanced by simultaneous i.c.v. injection of ACTH_1–10+melatonin, reaching higher values of B_max than the i.c.v. injection of these hormones individually. No significant changes in K_D values were found after ACTH_1–10 and/or melatonin administration. However, the i.c.v. injection of ACTH_4–10 to hypophysectomized rats did not change B_max, although it significantly increased K_D values, indicating a decrease in the BNZ binding affinity. Melatonin injection counteracted this effect of ACTH_4–10, returning K_D to the control value. Moreover, although the lower dose of i.c.v. melatonin used, 10 ng, was unable to modify B_max of BNZ binding in the ACTH_4–10-injected group, the higher dose, 20 ng, significantly increased B_max. The results suggest that these ACTH-derived peptides can modulate the effect of melatonin on brain benzodiazepine receptors.     

The neurosecretory system of the octopus vena cava: A neurohemal organ

Summary Endings of about two million small neurones form a voluminous neuropil inside the vena cava of cephalopods, in direct contact with the blood. These nerve endings are filled with masses of typical neurosecretory granules. By immunocytochemistry we could distinguish three different populations of secretory endings in the vena cava neuropil ofOctopus vulgaris: 1) a population of endings which were immunoreactive with antibodies against the pentapeptide proctolin; 2) a population with oxytocin/vasopressin- and neurophysin-like immunoreactivity; 3) a population immunoreactive with antibodies which were raised against the molluscan cardioexcitatory peptide Phe-Met-Arg-Phe-amide, against -melanotropin, and against atriopeptin. Extracts of the octopus vena cava stimulated amplitude and frequency of the isolated octopus heart preparation. Similar effects were exerted by peptides with the C-terminal structure-Arg-Phe-amide. Recently, we could isolate and identify in vena cava extracts four peptides; Phe-Met-Arg-Phe-amide, Phe-Leu-Arg-Phe-amide, Ala-Phe-Leu-Arg-Phe-amide and Thr-Phe-Leu-Arg-Phe-amide. Other peptides have not yet been identified. The fact that the peptides against which the immunoreactive antibodies were raised affected, in different organisms, blood volume, blood pressure, renal function and heart contraction suggests that one of the main functions of the neurosecretory system of the vena cava is a hormonal control of circulation.     

Synthesis ofO 1.5-(β-D-galactopyranosyl) [DMet2, Hyp5] enkephalin amide,a new highly potent analgesic enkephalin-related glycosyl peptide

Summary A new series of O-glycosyl enkephalins has been prepared, following a convergent strategy, with high chemical yields. The galactosyl analogue,O ^1.5-(-D-galactopyranosyl) [DMet², Hyp⁵] enkephalin amide proved to be one of the most potent in vivo opioid agonists synthesized up to now.     

Positive selection targeting the cathelin-like domain of the antimicrobial cathelicidin family

The cathelin-like domain (CLD) of cathelicidins is grouped in the same superfamily with cystatins, natural cysteine protease inhibitors, due to their structural similarity. Intriguingly, human hCAP-18/LL37 and pig protegrin-3 (PG3) CLDs exhibit opposite effects against cathepsin L. Here, I evaluated the functional importance of the CLD through identifying whether positive selection has driven adaptive evolution of this domain. As a result, four positively selected sites were detected and three of them are located on a loop region previously recognized as a key determinant of the activating effect of the PG3 CLD. Analysis of amino acid variability of the CLD led to the discovery of a conserved region and three highly variable regions, in which two are subjected to positive selection. Positive selection targeting the variable regions provides a starting point for experimentally establishing a direct link between the observed amino acid changes and functional divergence of the CLD family. Received 8 February 2008; accepted 13 February 2008     

Novel cytotoxic peptides from the tropical marine cyanobacteriumHormothamnion enteromorphoides 1. Discovery,isolation and initial chemical and biological characterization of the hormothamnins from wild and cultured material

A Caribbean cyanobacterium,Hormothamnion enteromorphoides, was found to produce a complex mixture of ichthyotoxic peptides, perhaps explaining the apparent absence of predation upon these potentially palatable life forms. Bioassay-guided fractionation was used to isolate these toxic and antimicrobial natural products, and a variety of techniques including HR FAB mass spectrometry, 2D-NMR, traditional hydrolysis-amino acid analysis, and several chemical reactions were used to define the basic structural features of the major peptide, hormothamnin A. Hormothamnin A is a cyclic undecapeptide containing six common and five uncommon or new amino acid residues. HPLC analyses indicate that the relative proportions of these peptide natural products remain relatively constant between different collection locations and years, however, they do vary seasonally. Clonal isolates of this cyanobacterium in culture produce the full spectrum of toxic peptides.     

The diet and gut microflora influence the distribution of enteroendocrine cells in the rat intestine

Several functions of the gut are locally influenced by peptides and biogenic amines released from enteroendocrine cells. The aim of the present study was to assess whether the luminal stimulus of diet or microbial flora or diet-microbial interactions have an influence on the distribution of enteroendocrine cells along the crypt-surface axes of the small and large intestine. The effects of diet and indigenous flora were investigated by comparing the numbers of argyrophil and serotonin immunoreactive cells in the jejunum and colon of germ free and conventional rats fed either a purified diet containing fine ingredients or a commercial diet containing crude fibre of cereal origin. The effects of human flora were analysed in germ-free rats inoculated with human faecal organisms. 1. Feeding the commercial diet reduced the number of argyrophil endocrine cells in the jejunum and serotonin immunoreactive cells in the colon of gern-free animals but increased the serotonin immunoreactive cells in the colon of conventional animals. 2. The rat flora increased the serotonin immunoreactive cells in the colon of animals fed a commercial diet and decreased in those fed a purified diet. 3. Inculation of human flora increased the numbers of serotonin immunoreactive cells both in the jejunum and colon. The results provide evidence that the dietary changes and diet-microbial interactions can affect the regional number of enteroendocrine cells.     

Regulatory peptide immunocytochemistry at light- and electron microscopical levels

Summary Immunocytochemical techniques applied at both light- and electron microscopical levels are valuable in the study of regulatory peptide distribution in normal and diseased tissue, whether in the form of sections or whole cell preparations. Successful immunolocalisation depends on 1) adequate preservation of the peptide antigen and the tissue structure in which it resides; 2) a suitably specific and sensitive labelled antibody detecting system. In general, peptides are stable molecules, most of which retain their antigenicity after conventional cross-linking fixation and tissue processing, allowing standard immunocytochemical methods to be used for light- and electron microscopy. Regulatory peptides are derived from precursor molecules and several families of structurally similar peptides are now generally recognised. Region-specific antibodies may be needed to overcome problems of cross-reactivity or to identify a bioactive form in the presence of its precursor. Multiple co-localisation of different related and unrelated peptides in the same cell or even storage granule is now recognised and can be identified by immunocytochemistry.     

Ethanol ingestive behavior as a function of central neurotransmission

Summary Uncontrollable alcohol ingestive behavior has been linked to deficits of central neurotransmission. The pineal gland plays an important role in modulating ethanol intake in numerous animal species. The opioidergic (i.e. -endorphin, enkephalin, and dynorphin) system is involved in both the actions of alcohol and opiates, as well as craving and/or genetic predisposition towards abuse of these two agents. Furthermore, there is significant evidence to link ingestive behaviors with the ventral tegmental accumbens-hypothalamic axis, whereby the biogenic amines dopamine and serotonin are reciprocally involved. Evidence is presented which implicates the striatum and the hypothalamus as possible specific loci for regional differences between alcohol-preferring and alcohol-nonpreferring mice. We believe that photoperiod-induced alcohol ingestive behavior may involve alterations in both pineal and hypothalamic opioid peptides.     

Aspects of measurement and analysis of regulatory peptides

Summary Although almost all methods of mass measurement of regulatory peptides still depend on the high affinity antibody, the traditional Yalow and Berson radioimmunoassay technique is becoming outdated. Pure monoclonal antibodies allow excess antibody two site assay techniques with a variety of different labels (preferentially non-radioactive) of great sensitivity and speed. The large amounts of particular monoclonal antibodies available allow several different laboratories to use the same reagents and have increased comparability. Unfortunately many regulatory peptides exist in multiple molecular forms and attention must be paid to antibody region specificity. Improved methods of extraction of regulatory peptides from plasma tissue allow more accurate quantitation. New techniques for rapid high resolution chromatography make distinction of different molecular forms much easier than hitherto. Better education in techniques and/or attention to inter-assay standards are necessary to improve the comparability of regulatory peptide measurement in the future.