Peptide Aa Research Articles

Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin.

MsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP-promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP-promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.

Laquinimod exerts strong clinical and immunomodulatory effects in Lewis rat experimental autoimmune neuritis

Laquinimod is an immunomodulatory drug with neuroprotective potential. We used the animal model of experimental autoimmune neuritis (EAN) in the Lewis rat to study the effects of laquinimod treatment. After immunization with the neuritogenic peptide aa 53–78 of P2 myelin protein, preventive therapy with 12.5mg/kg laquinimod once daily inhibited neuritis in clinical and electrophysiological terms. Histology corroborated a lower degree of inflammatory lesions and demyelination in the sciatic nerve. The proportion of FoxP3-positive regulatory T cells in the peripheral lymph nodes of treated rats remained unchanged. We conclude that laquinimod may represent a therapeutic option in human autoimmune neuropathies.

Evaluation of CNS and peripheral antitumor activity of ANG1005 in patients with brain metastases from breast tumors and other advanced solid tumors.

2523 Background: ANG1005 consists of 3 paclitaxel molecules covalently linked to a proprietary 19 AA peptide that targets the LRP1 receptor. This receptor is highly expressed on endothelial cells o...

Different expression profiles of Interleukin 11 (IL-11), Intelectin (ITLN) and Purine nucleoside phosphorylase 5a (PNP 5a) in crucian carp (Carassius auratus gibelio) in response to Cyprinid herpesvirus 2 and Aeromonas hydrophila

Interleukin 11 (IL-11), Intelectin (ITLN) and Purine nucleoside phosphorylase 5a (PNP5a) play important roles in innate immunity. In a previous study to identify differentially expressed immune-related genes, suppression subtractive hybridization (SSH) assay was used to characterize differentially expressed genes in crucian carp (Carassius auratus gibelio) infected with Cyprinid herpesvirus 2 (CyHV-2) in which IL-11, ITLN and PNP5a were identified to be the three most significantly up-regulated genes (Xu et al., Archives of Virology, 2014, http://dx.doi.org/10.1007/s00705-014-2011-9). In this study, the complete open reading frames (ORF) of IL-11, ITLN and PNP5a genes were cloned and sequenced. The full-length cDNAs of the three genes contained an ORF of 597, 945 and 882 bp, encoding a polypeptide of 198, 314 and 293 amino acids, respectively. Phylogenetic analysis indicated that the three genes shared high homology to other bony fish species including Zebrafish. Interestingly, the ITLN gene of crucian carp lacked a 10 aa peptide that was found in the C-terminal of other fish species. A real-time RT-PCR assay was developed to quantitatively examine their tissue distribution. We found that IL-11, ITLN and PNP5a were expressed at low levels in all of the tissues examined. To monitor the response of these genes to CyHV- 2 or Aeromonas hydrophila (A. hydrophila) infection, we determined the expression level of IL-11, ITLN and PNP5a at different time points after infection in kidney. Significant up-regulation of IL-11, ITLN and PNP5a was only observed 72 h post-CyHV-2 injection (P < 0.01), whereas significant up-regulation was observed as early as 6 h after infection with A. hydrophila (P < 0.01). Our results demonstrated that host innate immune response to CyHV-2, at least in which IL-11, ITLN and PNP5a were involved, was slow in comparison to that induced by A. hydrophila. It suggested that CyHV-2 might suppress host innate response during early infection. The lack of a C-terminal peptide of crucian carp ITLN gene implied a possible functional difference of this gene during evolution, which merit further investigation.

Immunoglobulin E epitope mapping by microarray immunoassay reveals differences in immune response to genetic variants of caseins from different ruminant species

The allergenicity of the caseins (CN), one of the major allergens in cow milk, is well characterized and their immunoglobulin E (IgE)-binding epitopes have been identified. However, investigations about the allergenic potential of the genetic variants occurring in the caseins are lacking. Therefore, this study determined the influence of the genetic polymorphism on IgE binding to epitopes of bovine casein variants. Furthermore, differences in IgE binding between epitopes of goats and water buffaloes were analyzed. A set of 187 peptides, covering the previously identified sequential IgE-binding epitopes of αS1-, αS2-, β-, and κ-CN variants from cows and the corresponding homologous peptides of water buffaloes and goats, were synthesized and tested by means of peptide microarray for IgE binding, using sera from 16 cow milk-sensitized individuals. Seven of the 16 sera samples showed positive signals on microarrays and were included in this study. In 5 αS1-CN variants (A, B, C, E, and I), the AA substitution or deletion affected the immunoreactivity of epitopes AA 4 to 23, AA 17 to 36, AA 83 to 102, AA 173 to 192, and AA 175 to 194, as well as of the variant-specific peptides AA 184 to 196, AA 187 to 199, AA 174 to 193, and AA 179 to 198, which were found to resist gastrointestinal digestion. Variation in IgE binding was further detected for peptides AA 103 to 123 and AA 108 to 129 of 3 β-CN variants (A1, A2, and B). The majority of sera showed IgE binding to αS1-CN peptides of cows and the homologous counterpart of goats and water buffaloes. However, αS1- and β-CN epitopes from goats and water buffaloes had lower immunoreactivity than those of cows, but, in some cases, higher or exclusive IgE binding was observed. The results of this study indicate that genetic variants of the caseins differ in their allergenicity. This might be useful in the search for a suitable protein source for cow milk-allergic patients. In addition, milk from water buffaloes and goats harbor an allergenic potential due to cross-reactivity of IgE antibodies with cow milk caseins and are, therefore, not an acceptable alternative in the nutrition of cow milk-allergic patients.

Characterization and antigenicity of recombinant Campylobacter jejuni flagellar capping protein FliD

Campylobacter jejuni, a flagellated, spiral-rod, Gram-negative bacterium, is the leading pathogen of human acute bacterial gastroenteritis worldwide, and chickens are regarded as a major reservoir of this micro-organism. Bacterial flagella, composed of more than 35 proteins, play important roles in colonization and adhesion to the mucosal surface of chicken caeca. In this study, the flagellar capping protein, FliD, encoded by the fliD gene, from the Campylobacter jenuni D1-39 isolate was expressed and characterized, and its antigenicity determined. The fliD gene comprised 1929 nt, potentially encoding a 642 aa peptide with a calculated molecular mass of 69.6 kDa. This gene was PCR amplified and overexpressed in Escherichia coli. The recombinant FliD protein was purified by cobalt-chelating affinity chromatography and confirmed by nucleotide sequencing of the expression plasmid, SDS-PAGE analysis, His tag detection and matrix-assisted laser desorption/ionization time of flight mass spectrometry. The immunoblot data showed that the purified recombinant FliD protein reacted strongly to sera from broiler chickens older than 4 weeks, indicating that anti-FliD antibody may be prevalent in the poultry population. These results provide a rationale for further evaluation of the FliD protein as a vaccine candidate for broiler chickens to improve food safety for poultry.

Human CD8+ T Cells from TB Pleurisy Respond to Four Immunodominant Epitopes in Mtb CFP10 Restricted by HLA-B Alleles

CD8+ T cells are essential for host defense to Mycobacterium tuberculosis (Mtb) infection and identification of CD8+ T cell epitopes from Mtb is of importance for the development of effective peptide-based diagnostics and vaccines. We previously demonstrated that the secreted 10-KDa culture filtrate protein (CFP10) from Mtb is a potent CD8+ T cell antigen but the repertoire and dominance pattern of human CD8 epitopes for CFP10 remained poorly characterized. In the present study, we undertook to define immunodominant CD8 epitopes involved in CFP10 using a panel of CFP10-derived 13–15 amino acid (aa) peptides overlapping by 11 aa. Four peptides in CFP10 were observed to induce significant CD8+ T cell responses and we further determined the size of the epitopes involved in each individual peptide tested. Four 9 aa CD8 epitopes were finally identified and deleting a single amino acid from the N or C terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimum of CD8 epitopes. In the individuals tested, each epitope represented a single immunodominant response in CD8+ T cells. The epitope-specific CD8+ T cells displayed effector or effector memory phenotypes and could upregulate the expression of CD107a/b upon antigen stimulation. In addition, we found that epitope-specific CD8+ T cells shared biased usage of T cell receptor (TCR) variable region of β chain (Vβ) 12, 9, 7.2 or Vβ4 chains. As judged from HLA-typing results and using bioinformatics technology for prediction of MHC binding affinity, we found that the epitope-specific CD8+ T cells are all restricted by HLA-B alleles. Our findings suggest that the four epitopes in CFP10 recognized by CD8+ T cells might be of importance for the development of Mtb peptide-based vaccines and for improved diagnosis of TB in humans.

Serum Amyloid A3 Binds MD-2 To Activate p38 and NF-κB Pathways in a MyD88-Dependent Manner

Serum amyloid A (SAA) 3 is a major component of the acute phase of inflammation. We previously reported that SAA3 served as an endogenous peptide ligand for TLR4 to facilitate lung metastasis. Because these experiments were performed with SAA3 recombinant proteins purified from Escherichia coli or mammalian cells, we could not rule out the possibility of LPS contamination. In this study, we used SAA3 synthetic peptides to eliminate the presence of LPS in SAA3. We found that the SAA3 synthetic peptide (aa 20-86) (20-86) stimulated cell migration and activated p38 in a manner dependent on TLR4, MD-2, and MyD88. SAA3 (20-86) also activated NF-κB and Rho small GTPase. Using surface plasmon resonance analysis, the binding constant KD values between SAA3 (20-86) or SAA3 (43-57) and TLR4/MD-2 protein highly purified by the baculovirus system were 2.2 and 30 μM, respectively. FLAG-tagged SAA3 tightly bound to protein A-tagged MD-2, but not to TLR4 in baculovirus coinfection experiments. Although SAA3 (20-86) caused a low, but appreciable level of endocytosis in TLR4, it induced the upregulation of both IL-6 and TNF-α, but not IFN-β1. An i.v. injection of SAA3 (43-57) induced the lung recruitment of CD11b(+)Gr-1(+) cells at an estimated serum concentration around its KD value toward TLR4/MD-2. Taken together, these results suggest that SAA3 directly binds MD-2 and activates the MyD88-dependent TLR4/MD-2 pathway.

Incorrectly annotated keratin derived peptide sequences lead to misleading MS/MS data interpretation

In the course of the nanoLC-nanoESI-MS/MS analysis of Chelidonium majus proteins we detected an extremely abundant 12 aa peptide (sequence: TNAENEFVTIKK). The Mascot search against NCBInr database with Viridiplantae taxonomic restriction revealed its complete identity to Unknown protein 18 from Pseudotsuga menziesii (P85925). The same sequence has also been submitted as Unknown protein 1 (P86104) from Vitis rotundifolia and as a part of the oxygen-evolving enhancer protein 1 (OEE1) from Pinus strobus (P84718). After careful inspection the record P84718 turned out to comprise a set of peptides of unconfirmed origin rather than complete protein sequence. In this paper we present extensive data indicating that the peptide in question may originate from type II cytoskeletal keratin - a common contaminant in protein samples. We found empirical evidence that it can be detected in several types of keratin-contaminated samples and its sequence is identical to one of the proteotypic peptides commonly observed for keratins. Nevertheless, the peptide has been annotated as plant protein and thus leads to data misinterpretation. We advise extreme caution when dealing with sequences of Unknown protein 18 (P. menziesii), Unknown protein 1 (V. rotundifolia) and OEE1 (P. strobus) since they are at best poorly annotated, if not artifactual. Biological significance To our knowledge, this is the first report indicating that the peptide TNAENEFVTIKK, which is identical to Unknown protein 18 from P. menziesii (P85925), Unknown protein 1 (P86104) from V. rotundifolia and a part of the oxygen-evolving enhancer protein 1 (OEE1) from P. strobus (P84718), may originate from type II cytoskeletal keratin - a common contaminant in protein samples. We found empirical evidence that it can be detected in several types of keratin-contaminated samples and its sequence is identical to one of the proteotypic peptides commonly observed for keratins. Nevertheless, the peptide has been annotated as plant protein and thus, without proper quality assessment, leads to biological misinterpretation of proteomic data.

Genetic variants of bovine β- and κ-casein result in different immunoglobulin E-binding epitopes after in vitro gastrointestinal digestion

Immunoglobulin E-mediated allergy to cow milk is a common allergy in industrialized countries, mainly affecting young children and infants. β-Casein (CN) and κ-CN belong to the major allergens in cow milk. Within these milk proteins, genetic polymorphisms occur, which are characterized by substitutions or deletions of AA, resulting in different variants for each protein. Until now, these variants have not been considered when discussing the allergenic potential of bovine milk. In this study, the focus was placed on the arising peptide pattern after in vitro gastrointestinal digestion of several β- and κ-CN variants to determine resistant fragments containing IgE-binding epitopes and to identify potential differences between these variants. β-Casein A1, A2, and B, as well as κ-CN A, B, and E, were separated and isolated from milk of cows homozygous for these variants and digested with an in vitro gastrointestinal digestion model. The resulting peptides were identified using mass spectrometry and compared with previously determined epitopes. Seven β-CN and 4 κ-CN peptides, common in all β- or κ-CN variants, remained of sufficient size to harbor IgE-binding epitopes. In addition, some peptides and, consequently, epitopes differ from each other due to the AA substitution occurring in the individual variants. The distinct peptides AA 108 to 129 of β-CN A1 and A2, AA 103 to 123 of β-CN B, as well as AA 59 to 72, AA 59 to 80, and AA 58 to 80 of all 3 β-CN variants correspond to the IgE-binding epitopes AA 107 to 120 and AA 55 to 70, respectively. In κ-CN, the 2 variant-specific peptides AA 136 to 149 (κ-CN A, E) and AA 134 to 150 (κ-CN B) are congruent with the IgE-binding epitope AA 137 to 148. The present study shows that genetic polymorphisms affected the arising peptide pattern of the caseins and thus modifications in the IgE-binding epitopes occurred. As a consequence, the casein variants could show differences in their allergenicity. Studies investigating the allergenic potential of these different peptides are currently in progress.

A Huntingtin Peptide Inhibits PolyQ-Huntingtin Associated Defects

BackgroundHuntington’s disease (HD) is caused by the abnormal expansion of the polyglutamine tract in the human Huntingtin protein (polyQ-hHtt). Although this mutation behaves dominantly, huntingtin loss of function also contributes to HD pathogenesis. Indeed, wild-type Huntingtin plays a protective role with respect to polyQ-hHtt induced defects.Methodology/Principal FindingsThe question that we addressed here is what part of the wild-type Huntingtin is responsible for these protective properties. We first screened peptides from the Huntingtin protein in HeLa cells and identified a 23 aa peptide (P42) that inhibits polyQ-hHtt aggregation. P42 is part of the endogenous Huntingtin protein and lies within a region rich in proteolytic sites that plays a critical role in the pathogenesis process. Using a Drosophila model of HD, we tested the protective properties of this peptide on aggregation, as well as on different polyQ-hHtt induced neuronal phenotypes: eye degeneration (an indicator of cell death), impairment of vesicular axonal trafficking, and physiological behaviors such as larval locomotion and adult survival. Together, our results demonstrate high protective properties for P42 in vivo, in whole animals. These data also demonstrate a specific role of P42 on Huntington’s disease model, since it has no effect on other models of polyQ-induced diseases, such as spinocerebellar ataxias.Conclusions/SignificanceAltogether our data show that P42, a 23 aa-long hHtt peptide, plays a protective role with respect to polyQ-hHtt aggregation as well as cellular and behavioral dysfunctions induced by polyQ-hHtt in vivo. Our study also confirms the correlation between polyQ-hHtt aggregation and neuronal defects. Finally, these results strongly suggest a therapeutic potential for P42, specific of Huntington’s disease.

An Enhanced Heterologous Virus-Like Particle for Human Papillomavirus Type 16 Tumour Immunotherapy

Cervical cancer is caused by high-risk, cancer-causing human papillomaviruses (HPV) and is the second highest cause of cancer deaths in women globally. The majority of cervical cancers express well-characterized HPV oncogenes, which are potential targets for immunotherapeutic vaccination. Here we develop a rabbit haemorrhagic disease virus (RHDV) virus-like particle (VLP)-based vaccine designed for immunotherapy against HPV16 positive tumours. An RHDV-VLP, modified to contain the universal helper T cell epitope PADRE and decorated with an MHC I-restricted peptide (aa 48–57) from the HPV16 E6, was tested for its immunotherapeutic efficacy against the TC-1 HPV16 E6 and E7-expressing tumour in mice. The E6-RHDV-VLP-PADRE was administered therapeutically for the treatment of a pre-existing TC-1 tumour and was delivered with antibodies either to deplete regulatory T cells (anti-CD25) or to block T cell suppression mediated through CTLA-4. As a result, the tumour burden was reduced by around 50% and the median survival time of mice to the humane endpoint was almost doubled the compared to controls. The incorporation of PADRE into the RHDV-VLP was necessary for an E6-specific enhancement of the anti-tumour response and the co-administration of the immune modifying antibodies contributed to the overall efficacy of the immunotherapy. The E6-RHDV-VLP-PADRE shows immunotherapeutic efficacy, prolonging survival for HPV tumour-bearing mice. This was enhanced by the systemic administration of immune-modifying antibodies that are commercially available for use in humans. There is potential to further modify these particles for even greater efficacy in the path to development of an immunotherapeutic treatment for HPV precancerous and cancer stages.

Activation of Guanylate Cyclase-C Attenuates Stretch Responses and Sensitization of Mouse Colorectal Afferents

Irritable bowel syndrome (IBS) is characterized by altered bowel habits, persistent pain and discomfort, and typically colorectal hypersensitivity. Linaclotide, a peripherally restricted 14 aa peptide approved for the treatment of IBS with constipation, relieves constipation and reduces IBS-associated pain in these patients presumably by activation of guanylate cyclase-C (GC-C), which stimulates production and release of cyclic guanosine monophosphate (cGMP) from intestinal epithelial cells. We investigated whether activation of GC-C by the endogenous agonist uroguanylin or the primary downstream effector of that activation, cGMP, directly modulates responses and sensitization of mechanosensitive colorectal primary afferents. The distal 2 cm of mouse colorectum with attached pelvic nerve was harvested and pinned flat mucosal side up for in vitro single-fiber recordings, and the encoding properties of mechanosensitive afferents (serosal, mucosal, muscular, and muscular-mucosal; M/M) to probing and circumferential stretch studied. Both cGMP (10-300 μM) and uroguanylin (1-1000 nM) applied directly to colorectal receptive endings significantly reduced responses of muscular and M/M afferents to stretch; serosal and mucosal afferents were not affected. Sensitized responses (i.e., increased responses to stretch) of muscular and M/M afferents were reversed by cGMP, returning responses to stretch to control. Blocking the transport of cGMP from colorectal epithelia by probenecid, a mechanism validated by studies in cultured intestinal T84 cells, abolished the inhibitory effect of uroguanylin on M/M afferents. These results suggest that GC-C agonists like linaclotide alleviate colorectal pain and hypersensitivity by dampening stretch-sensitive afferent mechanosensitivity and normalizing afferent sensitization.

Effects of Dietary Supplementation with Hainanmycin on Protein Degradation and Populations of Ammonia-producing Bacteria In vitro.

An in vitro fermentation was conducted to determine the effects of hainanmycin on protein degradation and populations of ammonia-producing bacteria. The substrates (DM basis) for in vitro fermentation consisted of alfalfa hay (31.7%), Chinese wild rye grass hay (28.3%), ground corn grain (24.5%), soybean meal (15.5%) with a forage: concentrate of 60:40. Treatments were the control (no additive) and hainanmycin supplemented at 0.1 (H0.1), 1 (H1), 10 (H10), and 100 mg/kg (H100) of the substrates. After 24 h of fermentation, the highest addition level of hainanmycin decreased total VFA concentration and increased the final pH. The high addition level of hainanmycin (H1, H10, and H100) reduced (p<0.05) branched-chain VFA concentration, the molar proportion of acetate and butyrate, and ratio of acetate to propionate; and increased the molar proportion of propionate, except that for H1 the in molar proportion of acetate and isobutyrate was not changed (p>0.05). After 24 h of fermentation, H10 and H100 increased (p<0.05) concentrations of peptide nitrogen and AA nitrogen and proteinase activity, and decreased (p<0.05) NH3-N concentration and deaminase activity compared with control. Peptidase activitives were not affected by hainanmycin. Hainanmycin supplementation only inhibited the growth of Butyrivibrio fibrisolvens, which is one of the species of low deaminative activity. Hainanmycin supplementation also decreased (p<0.05) relative population sizes of hyper-ammonia-producing species, except for H0.1 on Clostridium aminophilum. It was concluded that dietary supplementation with hainanmycin could improve ruminal fermentation and modify protein degradation by changing population size of ammonia-producing bacteria in vitro; and the addition level of 10 mg/kg appeared to achieve the best results.

Development of human papillomavirus chimaeric L1/L2 candidate vaccines

Recombinant human papillomavirus (HPV) virus-like particle (VLP) vaccines based on the L1 capsid protein have been shown to be efficient prophylactic vaccines, albeit type-specific. As a first step to investigate the feasibility of extending protection against non-vaccine types, HPV-16 L1 chimaeras were generated. The region downstream of L1 amino acid (aa) 413 was replaced with selected cross-neutralising epitopes (aa 108-120; 56-81 and 17-36) derived from the HPV-16 L2 protein, generating proteins designated SAF, L2.56 and L2.17, respectively. The chimaera L1BPV containing BPV-1 L2 peptide aa 1-88 was similarly constructed. The chimaeras were evaluated for expression in insect cells; their ability to form particles was studied by electron microscopy, and their immunogenicity was evaluated in mice. SAF, L2.56 and L2.17 proteins were expressed to high concentrations in insect cells and elicited HPV-16 pseudovirus-neutralising anti-L1 antibodies. L2.56 and L2.17 also elicited anti-L2 antibodies. L1BPV was a poor vaccine candidate due to low levels of expression with concomitant lack of immunogenicity. All chimaeras assembled into tertiary structures. The results indicate that chimaeric L1 vaccines incorporating cross-neutralising L2 peptides could be promising second-generation prophylactic HPV vaccine candidates.

A herpes simplex virus scaffold peptide that binds the portal vertex inhibits early steps in viral replication.

Previous experiments identified a 12-amino-acid (aa) peptide that was sufficient to interact with the herpes simplex virus 1 (HSV-1) portal protein and was necessary to incorporate the portal into capsids. In the present study, cells were treated at various times postinfection with peptides consisting of a portion of the Drosophila antennapedia protein, previously shown to enter cells efficiently, fused to either wild-type HSV-1 scaffold peptide (YPYYPGEARGAP) or a control peptide that contained changes at positions 4 and 5. These 4-tyrosine and 5-proline residues are highly conserved in herpesvirus scaffold proteins and were previously shown to be critical for the portal interaction. Treatment early in infection with subtoxic levels of wild-type peptide reduced viral infectivity by over 1,000-fold, while the mutant peptide had little effect on viral yields. In cells infected for 3 h in the presence of wild-type peptide, capsids were observed to transit to the nuclear rim normally, as viewed by fluorescence microscopy. However, observation by electron microscopy in thin sections revealed an aberrant and significant increase of DNA-containing capsids compared to infected cells treated with the mutant peptide. Early treatment with peptide also prevented formation of viral DNA replication compartments. These data suggest that the antiviral peptide stabilizes capsids early in infection, causing retention of DNA within them, and that this activity correlates with peptide binding to the portal protein. The data are consistent with the hypothesis that the portal vertex is the conduit through which DNA is ejected to initiate infection.

Neuropeptide F regulates male reproductive processes in the desert locust, Schistocerca gregaria

Although Neuropeptide F (NPF) has been identified in different insect species, its function has mainly been studied in the fruit fly, Drosophila melanogaster, where it regulates diverse physiological processes, such as learning, stress responses and male courtship behavior. In locusts, only a truncated form of the “full-length” NPF (the biologically active “trNPF”) has been isolated. This 9 AA peptide stimulates oocyte maturation, food intake and weight increase in adult desert locusts (Schistocerca gregaria [Forskål]). In this study, we investigated whether this peptide is also involved in the regulation of male reproductive physiology in this orthopteran species. Daily injections of trNPF in adult males resulted in proportionally heavier testes and seminal vesicles, while RNAi-mediated knockdown of the Schgr-NPF precursor transcript gave rise to proportionally lighter testes and seminal vesicles. Furthermore, adult males precociously displayed courtship behavior when injected daily with trNPF, while this behavior was inhibited or delayed by RNAi knockdown of the Schgr-NPF precursor transcript. In order to further analyze these effects of trNPF on male reproductive physiology, fertility of males was tested by analyzing progeny numbers following copulation with untreated females. In this way, we showed that daily trNPF injection in adult males resulted in a larger egg pod size and a higher percentage of hatched eggs per egg pod after copulation, while RNAi knockdown caused the opposite effects. Taken together, we provide clear evidence for a role of trNPF in the regulation of reproductive physiology in adult males of the desert locust, S. gregaria. Possible modes of action of trNPF in influencing these reproductive processes in male locusts are discussed.

Determination of ghrelin structure in the barfin flounder (Verasper moseri) and involvement of ingested fatty acids in ghrelin acylation

Ghrelin is a peptide hormone that is acylated with a fatty acid, usually n-octanoic acid, at the third amino acid (aa) residue (usually a serine or threonine), and this acylation is known to be essential for ghrelin activity not only in mammals but also in non-mammals, such as fish. However, the modification mechanisms of ghrelin modification in fish are not known. In this study, we elucidated the structure of ghrelin in a teleost, the barfin flounder (Verasper moseri), and determined whether ingested free fatty acids of various chain lengths participated in ghrelin acylation. Complementary DNA cloning revealed the barfin flounder prepro-ghrelin to be a 106-aa peptide and the mature ghrelin to be a 20-aa peptide (GSSFLSPSHKPPNKGKPPRA). However, purification of ghrelin peptides from stomach extracts demonstrated that the major form of the hormone was a 19-aa decanoylated peptide [GSS(C10:0)FLSPSHKPPNKGKPPR] missing the last alanine of the 20-aa peptide. Ingestion of feed enriched with n-heptanoic acid (C7), n-octanoic acid (C8), or n-non-anoic acid (C9) changed the modification status of the peptide: ingestion of C8 or C9 increased the amount of C8:0 or C9:0 19-aa ghrelin, respectively, but no C7:0 ghrelin was isolated after ingestion of C7. These results indicate that ingested free fatty acids are substrates for ghrelin acylation in the barfin flounder, but the types of free fatty acids utilized as substrates may be limited.

Prdm3 and Prdm16 are H3K9me1 Methyltransferases Required for Mammalian Heterochromatin Integrity

Heterochromatin serves important functions, protecting genome integrity and stabilizing gene expression programs. Although the Suv39h methyltransferases (KMTs) are known to ensure pericentric H3K9me3 methylation, the mechanisms that initiate and maintain mammalian heterochromatin organization remain elusive. We developed a biochemical assay and used in vivo analyses in mouse embryonic fibroblasts to identify Prdm3 and Prdm16 as redundant H3K9me1-specific KMTs that direct cytoplasmic H3K9me1 methylation. The H3K9me1 is converted in the nucleus to H3K9me3 by the Suv39h enzymes to reinforce heterochromatin. Simultaneous depletion of Prdm3 and Prdm16 abrogates H3K9me1 methylation, prevents Suv39h-dependent H3K9me3 trimethylation, and derepresses major satellite transcription. Most strikingly, DNA-FISH and electron microscopy reveal that combined impairment of Prdm3 and Prdm16 results in disintegration of heterochromatic foci and disruption of the nuclear lamina. Our data identify Prdm3 and Prdm16 as H3K9me1 methyltransferases and expose a functional framework in which anchoring to the nuclear periphery helps maintain the integrity of mammalian heterochromatin.

Antimicrobial Peptides from Musca Domestica Expressed in &lt;i&gt;Escherichia coli&lt;/i&gt;

Cecropins are cationic molecules with a wide range of antimicrobial activities. The native peptide cecropins from Musca domestica (Md-Cec) have antimicrobial activity against both Gram-positive and Gram-negative bacteria. In the present study, cDNA fragments encoding both the Md-Cec-L (62aa) and Md-Cec-S (40 aa) peptides of Md-Cec were respectively expressed using the pMAL-c4x expression vector. High level expression of Md-Cec-L was achieved in Escherichia coli, while expression of Md-Cec-S failed to reach a decent level due to its high level of toxicity to the host cells. Md-Cec-L was expressed as a soluble form using a maltose binding protein (MBP) system, whose product is a MBP-tagged fusion protein, and separated with the carrier amyrose resin. Heterologous expression in E. coli and antimicrobial activity assays showed that both the recombinant fusion protein Md-Cec-L and Md-Cec-S have exhibited antimicrobial activity in vivo; and Md-Cec-L also exhibited antimicrobial activity in vitro. Md-Cec has the potential to be developed as a novel type of antimicrobial drug or food preservative.