Growth inhibition of the cereal root pathogens Rhizoctonia solani AG8, R. oryzae and Gaeumannomyces graminis var. tritici by a recombinant 42-kDa endochitinase from Trichoderma harzianum

The objective of this study was to determine the effectiveness of a 42-kDa endochitinase coded by the ThEn42 gene from Trichoderma harzianum as a potential source of transgenic resistance to Rhizoctonia root rot of barley caused by Rhizoctonia solani AG8 and/or R. oryzae. The gene cThEn42 was codon optimized (GC content increased from 53.3 to 65.1%) and then synthesized to produce the modified cThEn42GC in Pichia pastoris for in vitro tests. Two expression vectors were constructed: one with the fungal signal peptide and the fungal activation peptide [FSP-FAP-cThEn(GC)] and the other with barley chitinase 26 signal peptide followed by the fungal signal and activation peptides [SP(HVChi26)-FSP-FAP-cThEn(GC)]. N-terminal sequencing showed that, of two proteins secreted into liquid medium, FSP was cleaved off faithfully in one protein and both FSP and FAP were cleaved from the other protein. Purified endochitinase provided strong in vitro inhibition of both R. solani AG8 and R. oryzae. The enzyme had an intermediate inhibitory activity against Gaeumannomyces graminis var. tritici, and no inhibitory activity against Fusarium graminearum, F. pseudograminearum, and F. culmorum.     

DSIP affects adrenergic stimulation of rat pineal N-acetyltransferase in vivo and in vitro

The natural occurrence, sleep, and extra-sleep effects of delta sleep-inducing peptide (DSIP) have been shown by different laboratories. However, neither an in vitro assay system nor a probable mechanism of action of the peptide have been conclusively demonstrated so far. The recent finding that DSIP influences the nocturnal rise of N-acetyltransferase (NAT) activity in rat pineal led us to investigate a possible effect on pharmacologically induced NAT activity in vivo and in vitro. Stimulation of the enzyme with adrenergic drugs such as isoproterenol and phenylephrine was reduced by DSIP at doses of 150 and 300 μg/kg injected subcutaneously. In vitro, 6, 150 and 300 nM DSIP attenuated isoproterenol stimulation of the enzyme in cultured pineals, whereas 150 nM DSIP effectively reduced stimulation induced by a combination of the two drugs. The peptide alone did not influence NAT activity in vitro, but produced a slight stimulation in vivo. To our knowledge, these results represent the first report of a direct interaction of DSIP with adrenergic transmission. The in vitro system could prove useful for establishing possible mechanism(s) of action of the ‘sleep peptide.’     

The central nervous system of Bulla gouldiana: peptide localization

In the present study, we describe the structure of the central nervous system (CNS) of the marine gastropod Bulla gouldiana, and compare it with the structure of the CNS of the related mollusc, Aplysia californica. In addition, we performed an immunohistochemical analysis of a series of peptides, and the synaptic vesicle protein, synapsin I, in the central nervous system of B. gouldiana. The most common peptide in the B. gouldiana nervous system is the molluscan cardioexcitatory peptide (FMRFamide), which is present in a significant proportion of B. gouldiana neurons. A smaller number of neurons exhibit immunoreactivity to antisera raised against the calcitonin gene related peptide, vasopressin, vasoactive intestinal peptide, cholecystokinin, galanin and enkephalin. In some instances there is colocalization of two or more peptides. Very few neurons or axons exhibit synapsin I-like immunoreactivity. The patterns of immunoreactivity to these antisera is quite similar to the patterns that have been described in other gastropods, including Lymnaea stagnalis and Aplysia californica. These observations emphasize the importance of FMRFamide-like compounds in phylogenetically old nervous systems and indicate that compounds similar to mammalian peptides are present in the gastropod. Thus, the production of a wide variety of peptide molecules and their use in neuronal function appears to be a highly conserved phylogenetic process.     

The novel peptide NbPPI1 identified from Nicotiana benthamiana triggers immune responses and enhances resistance against Phytophthora pathogens

In plants, recognition of small secreted peptides, such as damage/danger‐associated molecular patterns (DAMPs), regulates diverse processes, including stress and immune responses. Here, we identified an SGPS (Ser‐Gly‐Pro‐Ser) motif‐containing peptide, Nicotiana tabacum NtPROPPI, and its two homologs in Nicotiana benthamiana, NbPROPPI1 and NbPROPPI2. Phytophthora parasitica infection and salicylic acid (SA) treatment induced NbPROPPI1/2 expression. Moreover, SignalP predicted that the 89‐amino acid NtPROPPI includes a 24‐amino acid N‐terminal signal peptide and NbPROPPI1/2‐GFP fusion proteins were mainly localized to the periplasm. Transient expression of NbPROPPI1/2 inhibited P. parasitica colonization, and NbPROPPI1/2 knockdown rendered plants more susceptible to P. parasitica. An eight‐amino‐acid segment in the NbPROPPI1 C‐terminus was essential for its immune function and a synthetic 20‐residue peptide, NbPPI1, derived from the C‐terminus of NbPROPPI1 provoked significant immune responses in N. benthamiana. These responses led to enhanced accumulation of reactive oxygen species, activation of mitogen‐activated protein kinases, and up‐regulation of the defense genes Flg22‐induced receptor‐like kinase (FRK) and WRKY DNA‐binding protein 33 (WRKY33). The NbPPI1‐induced defense responses require Brassinosteroid insensitive 1‐associated receptor kinase 1 (BAK1). These results suggest that NbPPI1 functions as a DAMP in N. benthamiana; this novel DAMP provides a potentially useful target for improving plant resistance to Pytophthora pathogens.     

Binding of Ala-scanning analogs of omega-conotoxin MVIIC to N- and P/Q-type calcium channels

We report the isolation and characterization of a novel human peptide with antimicrobial activity, termed LEAP-1 (liver-expressed antimicrobial peptide). Using a mass spectrometric assay detecting cysteine-rich peptides, a 25-residue peptide containing four disulfide bonds was identified in human blood ultrafiltrate. LEAP-1 expression was predominantly detected in the liver, and, to a much lower extent, in the heart. In radial diffusion assays, Gram-positive Bacillus megaterium, Bacillus subtilis, Micrococcus luteus, Staphylococcus carnosus, and Gram-negative Neisseria cinerea as well as the yeast Saccharomyces cerevisiae dose-dependently exhibited sensitivity upon treatment with synthetic LEAP-1. The discovery of LEAP-1 extends the known families of mammalian peptides with antimicrobial activity by its novel disulfide motif and distinct expression pattern.     

Molecular biology of rat steroid 11 beta-hydroxylase [P450(11 beta)] and aldosterone synthase [P450(11 beta, aldo)].

The molecular features of rat steroid 11ß-hydroxylase [P450(11ß)] and aldosterone synthase [P450(11ß, aldo)] are discussed. P450(11ß) is biosynthesized as a precursor form composed of 499 amino acids, having a 24-amino acid extension peptide. Two species of P450(11ß, aldo) were identified; a precursor form of P450(11ß, aldo)-1 is 510 amino acids long and has a 34-amino acid extension peptide, while that of P450(11ß, aldo)-2 is 500 amino acids long and has a 24-amino acid extension peptide. The 286th amino acid of P450(11ß, aldo)-1 is Glu, while that of P450(11ß, aldo)-2 is Lys. The cDNA-expression studies showed that P450(11ß, aldo)-1 had the aldosterone producing activity whereas P450(11ß, aldo)-2 had no activity, suggesting that Glu286 of P450(11ß, aldo) plays an important role in the catalysis. The amino acid sequence of a region in P450(11ß) from Leu337 through Pro352 is highly conserved among the steroidogenic P450s. Functional expression studies on the cDNAs for two P450(11ß)s showed that P450(11ß) catalyzes the 11ß-, 18- and 19-hydroxylations of 11-deoxycorticosterone, but not the aldosterone synthesis. P450(11ß, aldo), on the other hand, catalyzes the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. The two P450(11ß)s were also shown to catalyze the conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol and cortisone.     

Structure-activity relationships of an inhibitory nematode FMRFamide-related peptide,SDPNFLRFamide (PF1), on Ascaris suum muscle

FMRFamide-related peptides are widespread among the Nematoda. Among them is a family of extended PNFLRFamide peptides encoded on the flp-1 peptide precursor gene in Caenorhabditis elegans. The most studied peptide from this series is SDPNFLRFamide (PF1). Each residue in this peptide was sequentially substituted with either alanine or the corresponding d-isomer of the native amino acid in order to define structure–function relationships in this peptide using an Ascaris suum muscle tension assay. In general, substitutions in the N-terminal tetrapeptide had only minor consequences for efficacy, while substitutions in the C-terminal tetrapeptide caused more dramatic changes. Such substitutions typically markedly diminished efficacy, but d-isomer substitution at either position 5 (Phe) or 6 (Leu) converted the inhibitory activity of the prototype into excitation. In addition, it has been evident that KPNFLRFamide and SDPNFLRFamide, though encoded on flp-1 and sharing a PNFLRFamide hexapeptide, act through different receptors. KPNFLRFamide directly gates a chloride channel in A. suum muscle cells, while SDPNFLRFamide acts through nitric oxide synthase to open K⁺ channels in the same tissue. The use of K⁺ channel blockers and nitric oxide synthase inhibitors in electrophysiological experiments employing A. suum muscle membranes allowed the unambiguous conclusion that the N-terminal lysine is absolutely required for activation of the chloride channel and excludes interaction with the SDPNFLRFamide receptor.     

On the interaction of the synthetic 53–58 fragment of lac repressor with DNA

The hexapeptide fragment 53–58 of the lac repressor protein (Ala-Gln-Gln-Leu-Ala-Gly) was synthesized. Circular dichroic spectrum of the peptide has negative bands at 195 and 225 nm (weak). The hexapeptide, which has no basic residues, stabilized the AT rich Clostridium perfringens DNA, but had no effect, significantly, on the melting profile of either poly dA · poly dT or poly d(A-T)·poly d(A-T). The melting temperature of Micrococcus lysodeikticus DNA with a high GC content, was unaffected by the peptide.     

New potent antimicrobial peptides from the venom of Polistinae wasps and their analogs

Four new peptides of the mastoparan family, characterized recently in the venom of three neotropical social wasps collected in the Dominican Republic, Polistes major major, Polistes dorsalis dorsalis and Mischocyttarus phthisicus were synthesized and tested for antimicrobial potency against Bacillus subtilis, Staphylococcus aureus, Escherichia coli (E.c.) and Pseudomonas aeruginosa, and for hemolytic and mast cells degranulation activities. As these peptides posses strong antimicrobial activity (minimal inhibitory concentration (MIC) values against Bacillus subtillis and E.c. in the range of 5–40 μM), we prepared 40 of their analogs to correlate biological activities, especially antimicrobial, with the net positive charge, hydrophobicity, amphipathicity, peptide length, amino acid substitutions at different positions of the peptide chain, N-terminal acylation and C-terminal deamidation. Circular dichroism spectra of the peptides measured in the presence of trifluoroethanol or SDS showed that the peptides might adopt -helical conformation in such anisotropic environments.     

Production of peptide leukotrienes in endotoxin shock

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 mg/kg) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase-HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC₄ and LTD₄ in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC₄ and LTD₄ which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin-induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.     

Expression kinetics and plasmid stability of recombinant E. coli encoding urease-derived peptide with bioinsecticide activity

The nucleotide sequence encoding for an insecticidal peptide derived from the Canavalia ensiformis urease gene jbureII (AF 468788), was cloned and expressed in the pET101/Escherichia coli expression system. Bacterial cultivation in shaker with lactose as inducer produced 1.26 μg of recombinant peptide/mg protein, after 8 h of growth. The plasmid stability and the expression of the recombinant peptide were studied in bioreactor. Expression of the recombinant peptide was strongly affected by pH of cultures, with a decrease of more than 50% when acidification was freely allowed. Likewise, peptide production and plasmid stability were shown to be affected by aeration and agitation speed, both decreasing for higher values of oxygen mass transfer rates. Despite these difficulties, in bioreactor cultures carried out with controlled pH, low oxygen mass transfer rates and using lactose as inducer, we were able to achieve a total peptide production of 7.14 μg/mg protein, which represents approximately 2% of total cell protein. Bioassays were carried out using the purified peptide on insect models. The peptide fed to Dysdercus peruvianus nymphs produced 100% mortality after 11 days, deaths starting with a lag phase of 3–4 days, confirming that the bioreactor-produced peptide retained its biological activity.     

Rainbow trout (Oncorhynchus mykiss) neuropeptide Y.

Neuropeptide Y (NPY) has been isolated from brain extracts of the rainbow trout (Oncorhynchus mykiss) and subjected to structural analyses. Plasma desorption mass spectroscopy estimated the molecular mass of the purified peptide as 4303.9 Da. Automated Edman degradation unequivocally established the sequence of a 36 amino acid residue peptide as: Tyr-Pro-Pro-Lys-Pro-Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Pro-Glu-Glu-Leu-Ala-Lys-Tyr-Tyr-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr. The molecular mass calculated from this sequence (4304 Da) is consistent with that obtained by mass spectroscopy. The presence of a C-terminal amide was established by radioimmunoassay. Rainbow trout NPY is identical in primary structure to coho salmon (Oncorhynchus kisutch) pancreatic polypeptide (PP). These data may indicate that, in this group of salmonid fishes, a single member of the NPY/PP peptide family is expressed in both neurons and peripheral endocrine cells.     

A library of linear undecapeptides with bactericidal activity against phytopathogenic bacteria

A 125-member library of synthetic linear undecapeptides was prepared based on a previously described peptide H-K¹KLFKKILKF¹⁰L-NH₂ (BP76) that inhibited in vitro growth of the plant pathogenic bacteria Erwinia amylovora, Xanthomonas axonopodis pv. vesicatoria, and Pseudomonas syringae pv. syringae at low micromolar concentrations. Peptides were designed using a combinatorial chemistry approach by incorporating amino acids possessing various degrees of hydrophobicity and hydrophilicity at positions 1 and 10 and by varying the N-terminus. Library screening for in vitro growth inhibition identified 27, 40 and 113 sequences with MIC values below 7.5 μM against E. amylovora, P. syringae and X. axonopodis, respectively. Cytotoxicity, bactericidal activity and stability towards protease degradation of the most active peptides were also determined. Seven peptides with a good balance between antibacterial and hemolytic activities were identified. Several analogues displayed a bactericidal effect and low susceptibility to protease degradation. The most promising peptides were tested in vivo by evaluating their preventive effect of inhibition of E. amylovora infection in detached apple and pear flowers. The peptide H-KKLFKKILKYL-NH₂ (BP100) showed efficacies in flowers of 63–76% at 100 μM, being more potent than BP76 and only less effective than streptomycin, currently used for fire blight control.     

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.     

cDNA cloning of halocidin and a new antimicrobial peptide derived from the N-terminus of Ci-META4

Halocidin is an antimicrobial peptide, which is isolated from hemocytes from the tunicate, Halocynthia aurantium. In this study, we cloned the full-length cDNA of halocidin from pharyngeal tissue, using a combination of RT-PCR and 5′-RACE-PCR. The observed cDNA structure indicated that halocidin is synthesized as a 10.37 kDa prepropeptide. Based on the cDNA structure and the known amino acid sequence of the mature peptide, it was concluded that the precursor of halocidin contains a 21-residue signal peptide, followed by the 18 residues of the mature peptide, and a 56-residue anionic C-terminal extension, which is removed later on in the process. The signal sequence of halocidin exhibited a high degree of similarity with the corresponding portion of the Ci-META4 protein, which had been previously discovered in the coelomic cells of another tunicate, Ciona intestinalis, and is considered to play a role in metamorphosis. However, in several respects, the cDNA structure of Ci-META4 suggested that it might constitute a precursor for an antimicrobial peptide. Thus, we prepared a synthetic peptide, which was comprised of 19 N-terminal amino acid residues in the predicted mature region of Ci-META4, and tested it with regard to its antimicrobial activity. As a result, we confirmed that the synthetic peptide exhibited potent antimicrobial activity against Gram (+) and (−) bacteria, while evidencing no hemolytic activity toward human erythrocytes.     

Expression of mRNA for PACAP and its receptors in intra- and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis

Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagons/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) mRNAs in pheochromocytomas. Methods: The levels of the mRNA for PACAP and vasoactive intestinal peptide (VIP), and their receptors, and for TH and PNMT were measured by RT-PCR or real-time PCR analysis, and the concentrations of catecholamines were measured by HPLC in 24 intra-adrenal and six extra-adrenal pheochromocytomas. Results: mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of the PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH (p=0.0018) and PNMT (p=0.05) mRNA expression, as well as epinephrine (p=0.0342) levels in 16 intra-adrenal pheochromocytomas. Conclusion: Our findings support a possible role for PACAP in the regulation of expression of genes encoding catecholamine-synthesizing enzymes in intra-adrenal pheochromocytomas.     

美国白蛾丝氨酸蛋白酶基因HcSP1的克隆、时空表达及对取食不同寄主植物的表达响应

【目的】为探究美国白蛾Hyphantria cunea在寄主转换过程中的消化生理机制奠定基础。【方法】通过筛选美国白蛾cDNA文库,克隆美国白蛾丝氨酸蛋白酶基因。荧光定量PCR检测该基因在美国白蛾不同发育阶段的表达特性;半定量RT-PCR和荧光定量PCR分别检测该基因在美国白蛾5龄幼虫体内不同组织中的分布及表达特性;荧光定量PCR检测取食不同寄主植物(美洲黑杨Populus deltoides,日本晚樱Cerasus serrulata var.lannesiana,山樱花Cerasus serrulata,喜树Camptotheca acuminata和法国梧桐Platanus orientalis)叶片后美国白蛾4龄幼虫中该基因的表达量。【结果】克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1(GenBank登录号:MH663425),开放阅读框长882 bp,编码293个氨基酸,预测分子量为30.5 kD,理论等电点预测为9.86。编码蛋白N末端疏水区包含15个氨基酸组成的信号肽;具有丝氨酸蛋白酶的典型特征,即氨基酸序列中具有组氨酸(His)、天门冬氨酸(Asp)以及丝氨酸(Ser)残基组成的酶活性催化中心三元件;具有明显的胰蛋白酶前体的特征,即具有信号肽、激活肽以及胰蛋白酶N末端保守的起始氨基酸序列(IVGG)。NCBI BLAST比对结果表明美国白蛾HcSP1与其他鳞翅目昆虫丝氨酸蛋白酶的氨基酸序列一致性在50%～70%之间。荧光定量PCR结果显示,HcSP1在美国白蛾幼虫不同发育阶段的相对表达量呈现动态的变化,并随着幼虫虫龄的增长呈现上升趋势。半定量RT-PCR及荧光定量PCR结果显示,HcSP1在美国白蛾5龄幼虫头部、唾液腺、中肠、脂肪体、表皮、马氏管和血淋巴等组织中均有表达且在幼虫中肠中表达量极高。与取食其他寄主植物叶片相比,美国白蛾取食喜树叶片后HcSP1的相对表达量明显升高,并显著高于取食其他寄主植物。【结论】本研究克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1,检测了其在美国白蛾不同发育阶段、不同组织以及取食不同寄主植物叶片后的表达量,为探究美国白蛾在寄主转换过程中消化生理的机制奠定基础,也为美国白蛾的防治提供新的思路。