Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.     

Structure, synthesis, and activity of dermaseptin b, a novel vertebrate defensive peptide from frog skin: relationship with adenoregulin

A novel antimicrobial peptide, designated dermaseptin b, was isolated from the skin of the arboreal frog Phyllomedusa bicolor. This 27-residue peptide amide is basic, containing 3 lysine residues that punctuate an alternating hydrophobic and hydrophilic sequence. In helix-inducing solvent, dermaseptin b adopts an amphipathic alpha-helical conformation that most closely resembles class L amphipathic helixes, with all lysine residues on the polar face of the helix. The peptide exhibits growth inhibition activity in vitro against a broad spectrum of pathogenic microorganisms including yeast and bacteria as well as various filamentous fungi that are responsible for severe opportunistic infections accompanying acquired immunodeficiency syndrome and the use of immunosuppressive agents. Maximized pairwise sequence alignment of dermaseptin b and dermaseptin s, a 34-residue antimicrobial peptide previously isolated from Phyllomedusa sauvagii, reveals 81% amino acid identity. No other significant similarity was found between dermaseptin b and any prokaryotic or eukaryotic protein, but similarity was found with adenoregulin (38% amino acid postional identity), a 33-residue peptide that enhances binding of agonists to the A1 adenosine receptor. The synthetic replicates of dermaseptin b and adenoregulin displayed similar but nonidentical spectra of antimicrobial activity, and both peptides were devoid of lytic effect on mammalian cells. Accordingly, the observation that adenoregulin enhances binding of agonists to the adenosine receptor may in fact be a consequence of its ability to alter the structure of biological membranes and to produce signal transduction via interactions with the lipid bilayer, bypassing cell surface receptor interactions.     

Culture and early infancy among central African foragers and farmers

A 27-residue stretch of amino acids encompassing two putative 13-residue amphiphilic helical segments is an important determinant of activity in the 47-residue antibacterial peptide bovine seminalplasmin. Synthetic peptides corresponding to the 27-residue stretch (P27) SLSRYAKLANRLANPKLLETFLSKWIG as well as the 13-residue segments PKLLETFLSKWIG (SPF),exhibit antimicrobial activity. An analog of SPF where E has been replaced by K(SPFK) showed improved antimicrobial properties as compared to SPF. The peptides have the ability to bind and permeabilize membranes. We have modeled helical bundles of P27 and the two 13-residue peptides SPF and SPFK using simulated annealing via molecular dynamics. Octameric but not hexameric aggregates of P27 can form channels which would allow the passage of ions. In the case of 13-residue peptides, aggregates formed by 6 monomers can conceivably form ion conducting channels. Since the ability to form channels which would allow the passage of ions across the membranes is an important determinant of the biological activities of these peptides, knowledge of the pore forming structures should help in the design of analogs with improved activities.     

DNA ploidy analysis in breast carcinoma. Comparison of unfixed and fixed tissue analyzed by image and flow cytometry

We present here the isolation and characterization of four antimicrobial peptides produced by a European bumblebee Bombus pascuorum. A 51-residue insect defensin was characterized which, like the Apis mellifera defensins, had a highly conserved 12-residue extension to its C-terminal compared to defensins from other insects. Monoisotopic mass analysis of the C-terminal of B. pascuorum defensin confirmed that this molecule was C-terminally amidated. This defensin showed strong anti-Gram-positive activity and some anti-fungal activity; also, in contrast to other insect defensins, it showed anti-Gram-negative activity. A 17-residue apidaecin was characterized, showing anti-Gram-negative activity, and differing by a single amino acid substitution from the A. mellifera apidaecin. A 39-residue abaecin was isolated, the largest proline-rich antimicrobial peptide characterized to date, which showed activity against both Gram-negative and Gram-positive bacteria. Finally, we isolated an N-terminally blocked molecule, with a molecular mass of 10,122 Da, which showed activity against Gram-negative bacteria only. These characteristics are reminiscent of hymenoptaecin from the honeybee A. mellifera, but a definitive characterization of this molecule awaits further work. No evidence of lysozyme activity was found in the haemolymph of challenged or naive B. pascuorum.     

Dermaseptin, a peptide antibiotic, stimulates microbicidal activities of polymorphonuclear leukocytes

Dermaseptin (DRs S1), a 34-amino acid residue cationic antimicrobial peptide was studied for its effects on the production of reactive oxygen species (respiratory burst) and exocytosis of polymorphonuclear leukocytes (PMN). Treatment of PMN with DRs S1 (10-100 nM) stimulated significant production of reactive oxygen species (approximately a 2-fold increase relative to control) and release of myeloperoxidase. In addition, low DRs S1 concentrations (1-10 nM) primed the stimulation of respiratory burst induced by zymosan particles. In contrast to the native peptide, a dermaseptin fragment without either the COOH-terminal (DRs 1-10) or NH2 terminal (DRs 16-34) portion was inactive. The DRs S1-induced respiratory burst was inhibited by a selective protein kinase C inhibitor, GF 109203X, and was associated with early signalling events such as a rapid and transient elevation of cytosolic-free calcium concentration and phospholipase D activity. These data provide the first evidence of stimulating and priming properties of a peptide antibiotic on microbicidal activities of neutrophils, suggesting a potential role of dermaseptin in modulating host-defense mechanisms.     

Physicians'' patterns of using a computerized collection of guidelines for primary care

Conserved amino acid motifs are found in numerous expressed genes. Proteins and peptides with functional relationships may be identified using probes designed to hybridize with these motifs. An oligonucleotide probe was prepared to match the sequence of the expected active region of a frog corticotropin-releasing factor-like peptide sauvagine and used to screen a sheep brain cDNA library. A novel 1331-bp cDNA encoding a putative 328-residue protein with a theoretical mass of 36 kDa was identified. The presence of a strong signal sequence indicates that it is a secreted protein. The amino- and carboxy-terminal regions are characterized by several potential phosphorylation sites and binding motifs, suggesting a role in intracellular signal transduction. Although the protein possesses a 7-residue sequence identical to that found in sauvagine, its overall primary structure most closely resembles those of the alpha-carbonic anhydrases (alpha-CAs). Moreover, the detection of the human and mouse orthologues in the EST databases, together with an evolutionary analysis, indicates that the protein represents a new member of the alpha-CA gene family, which we designate carbonic anhydrase-related protein XI (CA-RP XI), encoded by CA11 (human) and Car11 (mouse, rat). The human CA11 gene appears to be located between the secretor type alpha(1,2)-fucosyltransferase gene cluster (FUT1-FUT2-FUT2P) and the D-site binding protein gene (DBP) on chromosome 19q13.3. Despite potentially inactivating changes in the active-site residues, CA-RP XI is evolving very slowly in mammals, a property indicative of an important function, which has also been observed in the two other "acatalytic" CA isoforms, CA-RP VIII and CA-RP X, whose functions are unknown.     

Membrane thinning caused by magainin 2

Magainin 2 is a 23-residue antibiotic peptide found in the skin of Xeonpus laevis (African clawed frog). It belongs to a broad class of alpha-helical peptides which interact directly with the lipid bilayer. Very little is presently known about the nature of this peptide/lipid interaction on the molecular level. We have performed a sequence of lamellar X-ray diffraction experiments to provide some insight into the nature of this interaction. We have found that, at concentrations below the critical concentration for lysis, the peptide causes the membrane thickness to decrease roughly in proportion to the peptide concentration. We further show that this thinning is consistent with a model where the peptide adsorbs within the headgroup region of the lipid bilayer at these concentrations. The energy cost of this thinning may also explain why the peptide inserts at high concentrations. We have already shown that a similar interaction exists for alamethicin interacting with diphytanoylphosphatidylcholine, and it should hold for a wide variety of peptide/lipid systems.     

Cloning and expression of sialidase L, a NeuAcalpha2-->3Gal-specific sialidase from the leech, Macrobdella decora

Sialidase L is a NeuAcalpha2-->3Gal linkage-specific sialidase that releases 2,7-anhydro-NeuAc instead of NeuAc from sialoglycoconjugates (Chou, M.-Y., Li, S.-C., Kiso, M., Hasegawa, A., and Li, Y.-T.(1994) J. Biol. Chem. 269, 18821-18826). A 2. 5-kilobase cDNA of sialidase L was cloned by a combination of methods based on polymerase chain reactions. The composite cDNA sequence reveals an open reading frame coding for 762 amino acids, including a putative 28-residue signal peptide at the N terminus that is similar to the signal sequence of the Clostridium septicum sialidase. The result suggests that sialidase L is a secretory enzyme. The coding sequence excluding the putative signal peptide of sialidase L was overexpressed in Escherichia coli. The purified recombinant enzyme was characterized to be as active as the enzyme isolated from the leech. It also possessed the strict NeuAcalpha2-->3Gal linkage specificity and released the unique cleavage product, 2,7-anhydro-NeuAc from sialoglycoconjugates. The deduced amino acid sequence of sialidase L exhibits little similarity with other reported sialidases. However, sialidase L contains a conserved "FRIP region" and four repeating "Asp box" motifs that align well with the corresponding positions of bacterial sialidases. The predicted beta-strand structures near the conserved motifs of sialidase L are similar to those of Salmonella typhimurium sialidase. Several conserved single amino acid residues of bacterial sialidases, including those known to be involved in the active site of Salmonella enzyme, are conserved in the deduced amino acid sequence of sialidase L. This observation suggests that part of the catalytic mechanism of sialidase L may be similar to the ordinary sialidase.     

Production of biologically active recombinant tilapia insulin-like growth factor-II polypeptides in Escherichia coli cells and characterization of the genomic structure of the coding region

Insulin-like growth factor-II (IGF-II) is a fetal growth factor in humans, but has not been clearly identified in fish up to now. For a detailed understanding of the physiological response of fish IGF-II, the first step was to clone tilapia IGF-II cDNA from the brain cDNA library, coding the region of genomic DNA, and also expressing tilapia IGF-II polypeptides from Escherichia coli. Tilapia cDNA sequences total 1,977 bp, and predicted nucleotide sequences and amino acid sequences of tilapia share 77.9% and 90.7% homology identity with rainbow trout IGF-II, respectively. The genomic structure of the tilapia prepro-IGF-II coding region is very difficult to sequence in mammals and birds. The cloned tilapia IGF-II gene coding region appears much more complex than in other vertebrates. In tilapia IGF-II, the first coding exon I encoding part of the signal peptide sequence is 25 amino acids shorter than the first coding exon of mammals and birds. The other 23 amino acids of the signal peptide, and the first amino acids of the B domain and C domain are encoded by tilapia coding exon 2. The C, A, and D domains, and the first 20 amino acids of the E peptide are encoded by tilapia coding exon 3. The other E peptides and the 3' untranslated region (UTR) region are encoded by tilapia coding exon 4. These data show that the IGF-II genes have significantly differing structures in vertebrate evolution, and there are differences of interrupting introns in the IGF-I genomic structure compared with mammals. To obtain recombinant biologically active polypeptides, tilapia IGF-II B-C-A-D domains were amplified using the polymerase chain reaction (PCR), then ligated with glutathione S-transferase (GST, pGEX-2T vector). Tilapia recombinant IGF-II protein was purified and characterized in E. coli. The fusion protein was also digested with thrombin and appeared as a recombinant IGF-II polypeptide single band with a molecular mass of 7 kD. The recombinant tilapia IGF-II protein biological function was measured by stimulation of [3H]thymidine incorporation. The assay concentration was set up from 0 to 120 nM to stimulate tilapia ovary cell line (TO-2) significantly to uptake thymidine. The results suggest that the recombinant IGF-II protein was dose dependent.     

Does additional brachytherapy improve the effect of external irradiation? A prospective, randomized study in central lung tumors

A 28-residue peptide (peptide G), derived from the C-terminal (W643-S670) of the beta-adrenergic receptor kinase (betaARK), was previously identified as the critical domain for binding to the betagamma subunits of the heterotrimeric guanine-nucleotide-binding regulatory protein (G betagamma). We observed that the 18-amino-acid core of this domain is poorly conserved between betaARK1 and betaARK2 and so may provide the basis for differences in G betagamma-binding properties. Specific antibodies raised against 18-residue peptides derived from the divergent sequences (peptides P1 and P2 for betaARK1 and betaARK2, respectively) competitively inhibited G betagamma-activation of the related betaARK subtype, confirming the involvement of this region in binding to G betagamma. Peptides P1 and P2 inhibited G betagamma-stimulated activity of both betaARK1 and betaARK2, with P2 being significantly more potent than P1 (IC50 of 179+/-5 microM for P2 and >500 microM for P1). The 28-residue peptides G showed the same relative inhibitory activities (IC50 = 48+/-5 microM for G2 and 146+/-8 microM for G1). This relative order of potency G2 > G1 approximately P2 > P1 was confirmed in a direct G betagamma-binding assay. No binding selectivity for the beta1, beta2, beta3 and beta4 G beta subtypes was observed. The EC50 value for G betagamma-activation of betaARK1 was about double of that for betaARK2, indicating a higher affinity between G betagamma and betaARK2, which is the expected result based on the findings with the peptides. These findings show that the 18-residue peptides P represent the shortest sequence of betaARK that can bind to G betagamma and provide a demonstration of a functional difference between the G betagamma binding domains of betaARK1 and betaARK2.     

Selective cytotoxicity of dermaseptin S3 toward intraerythrocytic Plasmodium falciparum and the underlying molecular basis

The antimicrobial activity of various naturally occurring microbicidal peptides was reported to result from their interaction with microbial membrane. In this study, we investigated the cytotoxicity of the hemolytic peptide dermaseptin S4 (DS4) and the nonhemolytic peptide dermaseptin S3 (DS3) toward human erythrocytes infected by the malaria parasite Plasmodium falciparum. Both DS4 and DS3 inhibited the parasite's ability to incorporate [3H]hypoxanthine. However, while DS4 was toxic toward both the parasite and the host erythrocyte, DS3 was toxic only toward the intraerythrocytic parasite. To gain insight into the mechanism of this selective cytotoxicity, we labeled the peptides with fluorescent probes and investigated their organization in solution and in membranes. In Plasmodium-infected cells, rhodamine-labeled peptides interacted directly with the intracellular parasite, in contrast to noninfected cells, where the peptides remained bound to the erythrocyte plasma membrane. Binding experiments to phospholipid membranes revealed that DS3 and DS4 had similar binding characteristics. Membrane permeation studies indicated that the peptides were equally potent in permeating phosphatidylserine/phosphatidylcholine vesicles, whereas DS4 was more permeative with phosphatidylcholine vesicles. In aqueous solutions, DS4 was found to be in a higher aggregation state. Nevertheless, both DS3 and DS4 spontaneously dissociated to monomers upon interaction with vesicles, albeit with different kinetics. In light of these results, we propose a mechanism by which dermaseptins permeate cells and affect intraerythrocytic parasites.     

Alternatives to percussion and postural drainage. A review of mucus clearance therapies: percussion and postural drainage, autogenic drainage, positive expiratory pressure, flutter valve, intrapulmonary percussive ventilation, and high-frequency chest compression with the ThAIRapy Vest

Four avian beta-defension prepropeptide cDNA sequences [gallinacins: Gal 1 (synonym CHP 1, chicken heterophil peptide 1), and Gal 2; turkey heterophil peptides: THP 1 and THP 2] were amplified from chicken or turkey bone marrow mRNA samples, respectively. Partial chicken beta-defensin cDNA sequences were obtained using degenerate primers based on chicken peptide sequences (Gal 1/CHP 1 and Gal 2). The complete cDNA sequences of the chicken beta-defensins were then determined by designing specific intrapeptidal primers, from the newly acquired sequence, and pairing one primer with a specific poly A primer tail sequence (3' end) and the other primer with an adapter primer in a 5' rapid amplification of cDNA ends (RACE) reaction. The two, turkey beta-defensins were amplified from turkey marrow using primers designed from chicken beta-defensin preproregions. The complete amino acid sequences for the prepropeptides were deduced for all four avian beta-defensins. Previously, only partial mature peptide sequences for the turkey beta-defensins and complete mature peptide sequences for the chicken beta-defensins were known. All sequences obtained translated accurately to complete and partial amino acid sequences reported for beta-defensins purified from chicken and turkey heterophil granules except for one additional amino acid for Gal 1/CHP 1. The four deduced beta-defensin proregions lack the long, negatively charged propiece reported in classical defensin proregions. These regions are thought to stabilize and inactivate the positively charged mature peptide and target the propeptide to the storage granule. Instead, these beta-defensin proregions are shorter and similar to storage granule-free beta-defensins proregions reported for bovine tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP). These are the first prepropeptide beta-defensins from leukocyte granules to be completely characterized.     

Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis

We have isolated from the blood of immune-challenged and untreated mussels (Mytilus edulis) antibacterial and antifungal peptides. We have characterized two isoforms of a novel 34-residue, cysteine-rich, peptide with potent bactericidal activity and partially characterized a novel 6.2-kDa antifungal peptide containing 12 cysteines. We report the presence of two members of the insect defensin family of antibacterial peptides and provide a phylogenetic analysis that indicates that mollusc and arthropod defensins have a common ancestry. Our data argue that circulating antimicrobial peptides represent an ancient host defense mechanism that predated the separation between molluscs and arthropods at the root of the Cambrian, about 545 million years ago.     

Regulation of sorting and post-Golgi trafficking of rhodopsin by its C-terminal sequence QVS(A)PA

Several mutations that cause severe forms of the human disease autosomal dominant retinitis pigmentosa cluster in the C-terminal region of rhodopsin. Recent studies have implicated the C-terminal domain of rhodopsin in its trafficking on specialized post-Golgi membranes to the rod outer segment of the photoreceptor cell. Here we used synthetic peptides as competitive inhibitors of rhodopsin trafficking in the frog retinal cell-free system to delineate the potential regulatory sequence within the C terminus of rhodopsin and model the effects of severe retinitis pigmentosa alleles on rhodopsin sorting. The rhodopsin C-terminal sequence QVS(A)PA is highly conserved among different species. Peptides that correspond to the C terminus of bovine (amino acids 324-348) and frog (amino acids 330-354) rhodopsin inhibited post-Golgi trafficking by 50% and 60%, respectively, and arrested newly synthesized rhodopsin in the trans-Golgi network. Peptides corresponding to the cytoplasmic loops of rhodopsin and other control peptides had no effect. When three naturally occurring mutations: Q344ter (lacking the last five amino acids QVAPA), V345M, and P347S were introduced into the frog C-terminal peptide, the inhibitory activity of the peptides was no longer detectable. These observations suggest that the amino acids QVS(A)PA comprise a signal that is recognized by specific factors in the trans-Golgi network. A lack of recognition of this sequence, because of mutations in the last five amino acids causing autosomal dominant retinitis pigmentosa, most likely results in abnormal post-Golgi membrane formation and in an aberrant subcellular localization of rhodopsin.     

Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin

Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is approximately 10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin (Ca2+.CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 micrM Ca2+.CaM. Our results suggest that Ca2+.CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4, 5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).     

Small, anionic, and charge-neutralizing propeptide fragments of zymogens are antimicrobial

Some inactive precursor proteins, or zymogens, contain small, amino terminus, homopolymeric regions of Asp that neutralize the cationic charge of the active protein during synthesis. After posttranslational cleavage, the anionic propeptide fragment may exhibit antimicrobial activity. To demonstrate this, ovine trypsinogen activation peptide, and frog (Xenopus laevis) PYL activation peptide, both containing homopolymeric regions of Asp, were synthesized and tested against previously described surfactant-associated anionic peptide. Peptides inhibited the growth of both gram-negative (MIC, 0.08 to 3.00 mM) and gram-positive (MIC, 0.94 to 2.67 mM) bacteria. Small, anionic, and charge-neutralizing propeptide fragments of zymogens form a new class of host-derived antimicrobial peptides important in innate defense.     

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore formation in bacterial cytoplasmic membranes

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic alpha-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.