Lucifensin, the long-sought antimicrobial factor of medicinal maggots of the blowfly Lucilia sericata

A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.     

The mode of antifungal action of plant, insect and human defensins

Defensins are small (~5 kDa), basic, cysteine-rich antimicrobial peptides that fulfill an important role in the innate immunity of their host by combating pathogenic invading micro-organisms. Defensins can inhibit the growth or virulence of microorganisms directly or can do so indirectly by enhancing the host's immune system. Because of their wide distribution in nature, defensins are believed to be ancient molecules with a common ancestor that arose more than a billion years ago. This review summarizes current knowledge concerning the mode of antifungal action of plant, insect and human defensins.     

Expression of defensins in non-infected araneomorph spiders

Defensins are a major family of antimicrobial peptides found throughout the phylogenetic tree. From the spider species: Cupiennius salei, Phoneutria reidyi, Polybetes pythagoricus, Tegenaria atrica, and Meta menardi, defensins belonging to the ‘ancestral’ class of invertebrate defensins were cloned and sequenced. The deduced amino acid sequences contain the characteristic six cysteines of this class of defensins and reveal precursors of 60 or 61 amino acid residues. The mature peptides consist of 37 amino acid residues, showing up to 70% identities with tick and scorpion defensins. In C. salei, defensin mRNA was found to be constitutively expressed in hemocytes, ovaries, subesophageal nerve mass, hepatopancreas, and muscle tissue. This is the first report presenting and comparing antimicrobial peptides belonging to the family of defensins from spiders.     

The role of mammalian antimicrobial peptides and proteins in awakening of innate host defenses and adaptive immunity

Since we live in a dirty environment, we have developed many host defenses to contend with microorganisms. The epithelial lining of our skin, gastrointestinal tract and bronchial tree produces a number of antibacterial peptides, and our phagocytic neutrophils rapidly ingest and enzymatically degrade invading organisms, as well as produce peptides and enzymes with antimicrobial activities. Some of these antimicrobial moieties also appear to alert host cells involved in both innate host defense and adaptive immune responses. The epithelial cells are a source of constitutively produced beta defensin (HBD1) and proinflammatory cytokine-inducible beta defensins (HBD2 and -3) and cathelicidin (LL37). The neutrophils-derived antimicrobial peptides are released on demand from their cytoplasmic granules. They include the enzymes cathepsin G and chymase, azurocidin, a defensins and cathelicidin. In contrast, C5a and C3b are produced by activation of the serum complement cascade. The antimicrobial moieties direct the migration and activate target cells by interacting with selected G-protein-coupled seven-transmembrane receptors (GPCRs) on cell surfaces. The beta defensins interact with the CCR6 chemokine GPCRs, whereas cathelicidins interact with the low-affinity FPRL-1 receptors. The neutrophil-derived cathepsin G acts on the high-affinity FMLP receptor (GPCR) known as FPR, while the receptors for chymase and azurocidin have not been identified as yet. The serum-derived C5a uses a GPCR known as C5aR to mediate its chemotactic and cell-activating effects. Consequently, all these ligand-receptor interactions in addition to mediating chemotaxis also activate receptor-expressing cells to produce other mediators of inflammation.     

Hedgehog signaling in vertebrate and invertebrate limb patterning

Invertebrate and vertebrate limbs have very different anatomies and modes of development. Despite these differences, recent studies demonstrate that a significant overlap exists in the signals used to pattern invertebrate and vertebrate limbs. One of these signaling molecules is Hedgehog, a secreted protein that functions to coordinate growth and proliferation along the anterior-posterior axis of developing limbs. Recent studies indicate that the mechanism of action, regulation and function of Hedgehog signaling in Drosophila and vertebrate limb development are often quite similar, yet at other times are distinct. Here we highlight the similarities and differences between the use of Hedgehog signaling in these two systems.     

On the existence of cytokines in invertebrates

Based on the assumption that invertebrates, like vertebrates, possess factors regulating responses to infection or wounding, studies dealing with the evolution of immunity have focussed on the isolation and characterisation of putative cytokine-related molecules from invertebrates. Until recently, most of our knowledge of cytokine- and cytokine receptor-like molecules in invertebrates relies on functional assays and similarities at the physicochemical level. As such, a phylogenetic relationship between invertebrate cytokine-like molecules and vertebrate counterparts could not be convincingly demonstrated. Recent genomic sequence analyses of interleukin-1-receptor-related molecules, that is Toll-like receptors, and members of the transforming growth factor-β superfamily suggest that the innate immune system of invertebrates and vertebrates evolved independently. In addition, data from protochordates and annelids suggest that invertebrate cytokine-like molecules and vertebrate factors do not have the same evolutionary origin. We propose instead that the convergence of function of invertebrate cytokine-like molecules and vertebrate counterparts involved in innate immune defences may be based on similar lectin-like activities. Received 27 November 2000; received after revision 11 December 2000; accepted 13 December 2000     

Sex chromosomes and sex-determining genes: insights from marsupials and monotremes

Comparative studies of the genes involved in sex determination in the three extant classes of mammals, and other vertebrates, has allowed us to identify genes that are highly conserved in vertebrate sex determination and those that have recently evolved roles in one lineage. Analysis of the conservation and function of candidate genes in different vertebrate groups has been crucial to our understanding of their function and positioning in a conserved vertebrate sex-determining pathway. Here we review comparisons between genes in the sex-determining pathway in different vertebrates, and ask how these comparisons affect our views on the role of each gene in vertebrate sex determination.     

Convergent evolution of defensin sequence,structure and function

Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly diverse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other diverse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive divergent evolution in their sequence, structure and function. Referred to as the cis- and trans-defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing examples from across the eukaryotes.     

Vertebrate circadian rhythms: Retinal and extraretinal photoreception

Summary ERRs Both the pineal and the SCN are elements of the vertebrate multioscillator system although the relative importance of these 2 areas probably varies between, and possibly within, the different vertebrate classes. Extraretinal photoreception is a universal feature of submammalian vertebrates, and possibly of neonatal mammals, but is absent in adult mammals. Although the pineal systems of sumammalian vertebrates are photosensitive, the pineal system has been directly implicated as an extraocular site for the perception of entraining light cycles only in amphibians. In all other submammalian vertebrates extraretinal entrainment can occur in the absence of the pineal system although it is certainly conceivable that the pineal system may act as an alternate route of photoreception. These extraretinal-extrapineal receptors are located within the brain but the exact location(s) of these receptors within the brain is unknown. The hypothalamus would be likely area for this extraretinal photoreception, however, for several reasons: 1. Neurophysiological studies have identified light sensitive neurons in the frog's hypothalamus⁴³. 2. The avian hypothalamus is a site of photoperiodic photoreception^100–103. 3. The only other light sensitive structures known in vertebrates—the pineal system and the lateral eyes—are all derived embryologically from the hypothalamus. 4. The hypothalamus appears to be the site of a circadian clock and there may be advantages in having the photoreceptors and the clock anatomically close to one another. These considerations, of course, do not exclude the possibility that other brain areas may be involved as well. The reason behind the loss of extraretinal photoreception in mammals is uncertain. The shift to exclusive retinal photoreception in mammals may have been dictated by the extensive reorganization that occurred during the evolution of the mammalian brain. Or, perhaps, the increased size of the mammalian skull and overlying tissue made direct photoreception difficult and necessitated a shift to retinal photoreception. The persistence of extraretinal photoreceptors in submammalian vertebrates, however, underscores their importance in the sensory repertoire of vertebrates.     

Evolution of nonclassical MHC-dependent invariant T cells

TCR-mediated specific recognition of antigenic peptides in the context of classical MHC molecules is a cornerstone of adaptive immunity of jawed vertebrate. Ancillary to these interactions, the T cell repertoire also includes unconventional T cells that recognize endogenous and/or exogenous antigens in a classical MHC-unrestricted manner. Among these, the mammalian nonclassical MHC class I-restricted invariant T cell (iT) subsets, such as iNKT and MAIT cells, are now believed to be integral to immune response initiation as well as in orchestrating subsequent adaptive immunity. Until recently the evolutionary origins of these cells were unknown. Here we review our current understanding of a nonclassical MHC class I-restricted iT cell population in the amphibian Xenopus laevis. Parallels with the mammalian iNKT and MAIT cells underline the crucial biological roles of these evolutionarily ancient immune subsets.     

The genetic and evolutionary basis of colour variation in vertebrates

Variation in pigmentation is one of the most conspicuous phenotypic traits in vertebrates. Although mammals show less variation in body pigmentation than other vertebrate groups, the genetics of colour determination and variation is best understood for them. More than 150 genes have been identified that influence pigmentation, and in many cases, the cause for variation in pigmentation has been identified down to the underlying nucleotide changes. These studies show that while some genes are often responsible for deviating pigmentation, similar or almost identical phenotypes even in the same species may be due to mutations in different genes. In this review we will first discuss the current knowledge about the genes and their functions underlying the biochemical pathways that determine pigmentation and then give examples where the mutations responsible for colour variation have been determined. Finally, we will discuss potential evolutionary causes for and consequences of differences in pigmentation between individuals.     

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     

Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification

G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs. However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available. This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells. After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results.     

Hemolysins: Pore-forming proteins in invertebrates

Summary Invertebrates possess lytic molecules which lyse vertebrate erythrocytes. In all the species studied so far, hemolytic activity depends on proteins which possess a wide range of reactivity. It is generally calcium-dependent and heat-labile, although calcium-independent and heat-stable hemolysins have also been detected. The molecules interact with sugars or lipids which could represent the membrane receptors by which circular lesions on target membranes are produced.On the basis of some analogies with vertebrate lytic molecules it is conceivable that the hemolysins evolved from a common ancestral gene which also led to vertebrate pore-forming proteins.     

A synergistic factor of an insect granulosis virus agglutinates insect cells

Summary The synergistic factor (SF), a lipoprotein which enhances in vivo and in vitro baculovirus infections, occurs in the matrix of the occlusion body (capsule) of a granulosis virus of the armyworm,Pseudaletia unipuncta. The SF attaches to certain areas on the surfaces of cell plasma membranes of cultured insect cells, resulting in marked cellular agglutination. The minimal amount of SF detectable by agglutination is approximately 3 g/ml. Cultured cells of 6 out of 10 insect species in 3 orders are agglutinated by the SF, but not those of 2 mammalian species and the erythrocytes of 8 vertebrate species.We thank Dr M. Nagata and Dr T. Yamamoto for preparing the synergistic factor and R.T. Hess and E.M. Omi for technical assistance. We also thank the donors who provided the cell lines. This work was supported by the National Science Foundation under Grant No. PCM-8201247.     

Molecular basis of morphogenesis during vertebrate gastrulation

Gastrulation is a crucial step in early embryogenesis. During gastrulation, a set of morphogenetic processes takes place leading to the establishment of the basic body plan and formation of primary germ layers. A rich body of knowledge about these morphogenetic processes has been accumulated over decades. The understanding of the molecular mechanism that controls the complex cell movement and inductive processes during gastrulation remains a challenge. Substantial progress has been made recently to identify and characterize pathways and molecules implicated in the modulation of morphogenesis during vertebrate gastrulation. Here, we summarize recent findings in the analysis of signaling pathways implicated in gastrulation movements, with the aim to generalize the basic molecular principles of vertebrate morphogenesis.     

Intestinal epithelial barrier and mucosal immunity

The innate immune system plays a crucial role in maintaining the integrity of the intestine and protecting the host against a vast number of potential microbial pathogens from resident and transient gut microflora. Mucosal epithelial cells and Paneth cells produce a variety of antimicrobial peptides (defensins, cathelicidins, crytdinrelated sequence peptides, bactericidal/permeabilityincreasing protein, chemokine CCL20) and bacteriolytic enzymes (lysozyme, group IIA phospholipase A2) that protect mucosal surfaces and crypts containing intestinal stem cells against invading microbes. Many of the intestinal antimicrobial molecules have additional roles of attracting leukocytes, alarming the adaptive immune system or neutralizing proinflammatory bacterial molecules. Dysfunction of components of the innate immune system has recently been implicated in chronic inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, illustrating the pivotal role of innate immunity in maintaining the delicate balance between immune tolerance and immune response in the gut.     

Chitin in the epidermal cuticle of a vertebrate (Paralipophrys trigloides,Blenniidae, Teleostei)

Lectin binding, endo-chitinase binding and enzymatic degradation studies show that the epidermal cuticle of the bony fishParalipophrys trigloides (Blenniidae) is chitinous. This is the first evidence that a vertebrate species possesses a chitinous tissue. Recently aXenopus gene has been identified which has significant sequence similarity to the catalytic domain of yeast chitin synthase III, a chitin producing enzyme^1,2. Taken together these two findings imply that chitin synthesis capability may be a basic vertebrate feature.     

Hemolysins: pore-forming proteins in invertebrates

Invertebrates possess lytic molecules which lyse vertebrate erythrocytes. In all the species studied so far, hemolytic activity depends on proteins which possess a wide range of reactivity. It is generally calcium-dependent and heat-labile, although calcium-independent and heat-stable hemolysins have also been detected. The molecules interact with sugars or lipids which could represent the membrane receptors by which circular lesions on target membranes are produced. On the basis of some analogies with vertebrate lytic molecules it is conceivable that the hemolysins evolved from a common ancestral gene which also led to vertebrate pore-forming proteins.     

The tension/length relationship of an insect (Calliphora erythrocephala) supercontracting muscle

Summary The tension/length curves of an insect supercontracting striated muscle are described. Both vertebrate and invertebrate smooth (non-striated) muscles show a close similarity to these curves. Thus, although insects possess only striated muscle, some of these muscles can perform the function of smooth muscle of other animals.I would like to thank Dr.M. P. Osborne for supervizing this work which was supported by an S. R. C. studentship.