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     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Occurrence of ricinoleic acid in submerged cultures of variousClaviceps sp.

Summary Ricinoleic acid was found in differentClaviceps sp., indicating that it is neither specific chemotaxonomic marker ofClaviceps purpurea, nor a specific indicator of peptide alkaloid synthesis.     

Comparative aspects of structure and action of molluscan neuropeptides

A number of neuropeptides were isolated from the ganglia and muscles of molluscs, and their actions were examined. Diverse neuropeptides, in addition to several classical neurotransmitters, were suggested to be involved in the regulation of the anterior byssus retractor muscle ofMytilus. A wide structural variety of members of theMytilus inhibitory peptide family was observed in each of the generaMytilus, Achatina andHelix. Gly-Trp-NH₂, the C-terminal dipeptide fragment of the neuropeptide AGPWamide, showed a more potent action than the parent peptide in all of the muscles examined. Peptides related to some molluscan neuropeptides were found to be distributed interphyletically. Some neuropeptides containing ad-amino acid residue were found inAchatina andMytilus. These aspects of molluscan neuropeptides are thought not to be exceptional.     

Effect of inhibitors of protein synthesis from plants on tobacco mosaic virus infection

Summary Ricin, abrin, modeccin, gelonin andMomordica charantia inhibitor, as well as crude extracts ofBryonia dioica seeds and ofDianthus caryophyllus leaves, each inhibit protein synthesis in a rabbit reticulocyte lysate and reduce local lesion production by tobacco mosaic virus, thus resembling the effects of the pokeweed antiviral peptide.     

The never-ending story of peptide O-xylosyltransferase

In a journey lasting 40 years from the first reports on its activity in the 1960s to its purification and the cloning of relevant complementary DNAs, peptide O-xylosyltransferase has finally arrived at the same point as many other enzymes. This enzyme, whose systematic name is UDP--D-xylose:proteoglycan core protein -D-xylosyltransferase (EC 2.4.2.26), catalyses the first step in the biosynthesis of chondroitin, dermatan and heparan sulphates in the endoplasmic reticulum and/or the cis-Golgi cisternae. Analyses of their primary structure show that peptide O-xylosyltransferases are members of glycosyltransferase family 14 and so are homologous to 1,6-N-acetylglucosaminyltransferases involved in O-glycan and poly-N-acetyllactosamine branching. Furthermore, vertebrates appear to have two rather similar genes encoding xylosyltransferase I and xylosyltransferase II, but enzymatic activity was only detected for a recombinant form of the first isoform. On the other hand, Caenorhabditis and Drosophila have each only one peptide O-xylosyltransferase gene. In the worm sqv-6 mutant, a mutation of the xylosyltransferase gene is associated with defective vulval morphogenesis, indicative of the importance of the glycosaminoglycan chains of proteoglycans in animal development. There remain, however, open questions, for instance, on the enzymes intracellular localisation and structure-function relationships.Received 11 July 2003; received after revision 4 September 2003; accepted 24 September 2003     

Hydrolysis of tabtoxins by plant and bacterial enzymes

Summary Tabtoxins, dipeptides that induce chlorosis in plants, are produced by several closely related phytopathogenicPseudomonas spp. Culture filtrates from these bacteria and extracts prepared from leaves of their hosts contain multiple enzymes that hydrolyze the peptide bond of the toxins.     

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.     

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     

Lipopeptaibols, a novel family of membrane active, antimicrobial peptides

Lipopeptaibols are members of a novel group of naturally occurring, short peptides with antimicrobial activity, characterized by a lipophilic acyl chain at the N-terminus, a high content of the turn/helix forming α-aminoisobutyric acid and a 1,2-amino alcohol at the C-terminus. The amino acid sequences range from 6 to 10 residues and the fatty acyl moieties from 8 to 15 carbon atoms. The peptide portion of lipopeptaibols can be shorter than those of the nonlipidated peptaibols that range from 10 to 19 amino acid residues. The longest peptides fold into a mixed 3₁₀/α helix, whereas the shortest peptides tend to adopt a β-turn/sheet structure. Using solution methodologies, a series of analogues of trichogin GA IV was synthesized which allowed determination of the minimal lipid chain and peptide main-chain lengths for the onset of membrane activity and exploitation of a number of spectroscopic techniques aimed at determining its preferred conformation under a variety of conditions and investigating in detail its mode of interaction with, and its effect on, the phospholipid membranes. Received 26 January 2001; received after revision 7 March 2001; accepted 15 March 2001     

A possible method for improving the efficacy of dapsone

Summary The antileprosy drug dapsone is unable to penetrate intactMycobacterium leprae in vitro, as determined by its effect on o-diphenoloxidase in the bacilli. When combined with the peptide polylysine, the sulfone drug passes through the bacterial cell membranes, and penetrates the enzyme protein, resulting in a 100% inhibition of its activity.     

Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence

Phage display was used to identify new components of the mammalian mitochondrial receptor complex using Tom20 as a binding partner. Two peptides were identified. One had partial identity (SMLTVMA) with a bacterial signal peptide from Toho-1, a periplasmic protein. The other had partial identity with a mitochondrial inner membrane glutamate carrier. The bacterial signal peptide could carry a protein into mitochondria both in vivo and in vitro. The first six residues of the sequence, SMLTVM, were necessary for import but the two adjacent arginine residues in the 30-amino-acid leader were not critical for import. The signal peptides of Escherichia coli β-lactamase and Bacillsus subtilis lipase could not carry proteins into mitochondria. Presumably, the Toho-1 leader can adopt a structure compatible for recognition by the import apparatus.Received 29 April 2005; received after revision 8 June 2005; accepted 17 June 2005     

Myosin heavy chains in fast skeletal muscle of chick embryo

Summary The peptide map obtained by electrophoresis after digestion of purified myosin heavy chains from pectoralis muscle of embryonic chicken with theStaphylococcus aureus V8 protease, produces a peptide pattern very similar but not identical to that of adult fast myosin. In fact, some components that are present in a small amount in the map of slow adult myosin are visible in the embryonic pattern.Acknowledgments. I wish to acknowledge the enthusiastic encouragement of Dr Ugo Carraro and the help of Prof. Stefano Schiaffino's group and of Dr Marcello Cantini for embryonic tissue isolation. The skillful technical assistance of Mr Silvio Belluco is also gratefully acknowledged. This work was supported in part by a grant of Muscular Dystrophy Association of America to Prof. Massimiliano Aloisi.     

A new solid-phase synthesis of thymopoietin II by a mild procedure

Summary The solid-phase synthesis of a nonatetracontapeptide corresponding to the entire amino acid sequence of Thymopoietin II is described. Use of the recently developed, base-labile, fluorenylmethyloxycarbonyl-amino acids in combination withtert-butyl based side chain protecting groups and p-alkoxybenzyl ester peptide to resin linkage enabled the synthesis to be carried out under much milder reaction conditions than previously.     

Fibronectin peptide DRVPHSRNSIT and fibronectin receptor peptide DLYYLMDL arrest gastrulation ofRana pipiens

Gastrulation is characterized by dramatic cell migration which is thought to require the interaction of cell adhesion molecules with extracellular molecules. We have tested two novel peptides, a fibronectin peptide and a fibronectin receptor peptide, for their effects on gastrulation of the leopard frogRana pipiens. The fibronectin peptide DRVPHSRNSIT corresponds to residues 1373–1383 of the cell-binding domain of fibronectin; the receptor peptide DLYYLMDL corresponds to residues 124–131 of 1 subunit of a variety of integrins including 51. Either of these peptides significantly inhibited gastrulation after being microinjected into mid-blastulae. These results indicate that these sequences may correspond to the ligand/receptor interaction sites of fibronectin and its receptor(s).     

The accessory gland proteins in maleDrosophila: structural,reproductive, and evolutionary aspects

Recent results from biochemical and molecular genetic studies of the accessory gland proteins in maleDrosophila are reviewed. The most prominent feature is the species-specific variability. However, the analysis of the sex peptide inD. melanogaster shows that there is a strong homology in the molecular structure to the closely related sibling species, and that divergence increases with increasing phylogenetic distance. For this reason the sex peptide, after being transferred to the female genital tract during copulation, reduces receptivity and increases oviposition only in virgin females belonging to the same species group and subgroup. Even though studies were hitherto limited to a small number of the secretory components, it is evident that the accessory gland proteins play a key role in reproductive success of the fruit fly by changing female sexual behavior, supporting sperm transfer, storage and displacement. Thus, genes encoding the accessory gland proteins are apparently under strong evolutionary selection.     

The effect of corticotropin-releasing factor and pro-opiomelanocortin-derived peptides on the phagocytosis of molluscan hemocytes

The effect of corticotropin-releasing factor (CRF) and pro-opiomelanocortin (POMC)-derived peptides on hemocyte phagocytosis in two molluscs,Planorbarius corneus andViviparus ater was studied. The peptides and related fragments examined are those which have been shown to influence hemocyte motility in the two species. The results obtained revealed that the effects on phagocytosis are not directly correlated with previous findings on cell motility. Furthermore, the mode of action of an individual peptide could be species-specific and dose-dependent. The relationships between peptides, locomotion and phagocytosis in these molluscs are discussed.     

Relaxin family peptide systems and the central nervous system

Since its discovery in the 1920s, relaxin has enjoyed a reputation as a peptide hormone of pregnancy. However, relaxin and other relaxin family peptides are now associated with numerous non-reproductive physiologies and disease states. The new millennium bought with it the sequence of the human genome and subsequently new directions for relaxin research. In 2002, the ancestral relaxin gene RLN3 was identified from genome databases. The relaxin-3 peptide is highly expressed in a small region of the brain and in species from teleost to primates and has both conserved sequence and sites of expression. Combined with the discovery of the relaxin family peptide receptors, interest in the role of the relaxin family peptides in the central nervous system has been reignited. This review explores the relaxin family peptides that are expressed in or act upon the brain, the receptors that mediate their actions, and what is currently known of their functions.     

Amino acid composition and sequence of crinia-angiotensin,an angiotensin II-like endecapeptide from the skin of the Australian frogCrinia georgiana

Summary Methanol extracts of the skin of the Australian amphibianCrinia georgiana contain large amounts of crinia-angiotensin II, a new angiotensin II-like peptide. This differs sharply from the conventional octapeptide angiotensins II in having attached the tripeptide Ala-Pro-Gly- to the N-terminus, and having an Ile residue substituted for the Val residue at position 6 from the C-terminus. Small amounts of angiotensin-like peptides have been traced, by radioimmunoassay, in skin extracts of some otherCrinia species.Supported by grants from the Consiglio Nazionale delle Ricerche, Italy, and from the Ministry of Education, Japan.