Evolution, expression and effectiveness in a cluster of novel bovine β-defensins

The anti-microbial peptides β-defensins constitute a large family of innate immune effector molecules, conserved across a wide species range. In this paper, we describe a systematic search of the sequenced bovine genome to characterise this extensive gene family in Bos taurus, providing an insight into the pattern of conservation of β-defensin genes between species. We have sequenced a sub-set of these newly discovered bovine β-defensin genes and also report expression data for these genes across a range of tissues. We have synthesised the peptide product of one of these genes, bovine β-defensin 123, and found it to be a potent inhibitor of several pathogenic microbes, particularly Escherichia coli and Listeria monocytogenes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Tat system proofreads FeS protein substrates and directly initiates the disposal of rejected molecules

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane, including FeS proteins that receive their cofactors in the cytoplasm. We have studied two Escherichia coli Tat substrates, NrfC and NapG, to examine how, or whether, the system exports only correctly folded and assembled FeS proteins. With NrfC, substitutions in even one of four predicted FeS centres completely block export, indicating an effective proofreading activity. The FeS mutants are rapidly degraded but only if they interact with the Tat translocon; they are stable in a tat deletion strain and equally stable in wild-type cells if the signal peptide twin-arginine motif is removed to block targeting. Basically similar results are obtained with NapG. The Tat apparatus thus proofreads these substrates and directly initiates the turnover of rejected molecules. Turnover of mutated FeS substrates is completely dependent on the TatA/E subunits that are believed to be involved in the late stages of translocation, and we propose that partial translocation triggers substrate turnover within an integrated quality control system for FeS proteins.     

Prevalence and misdiagnosis of chronic heart failure in nursing home residents: the role of B-type natriuretic peptides

Background/objectives. Without knowing the exact CHF prevalence, chronic heart failure (CHF) occurs frequently in elderly people both inside and outside nursing homes. For a diagnosis we have to rely on physical examination and additional tests. We therefore run the risk of missing CHF diagnoses or of diagnosing CHF when we should not. Natriuretic peptide assays have emerged as a diagnostic test but their use in nursing home residents is limited. We examined the number of misdiagnoses, the CHF prevalence and the role of natriuretic peptide. Method. Residents in one centre without aphasia, cognitive impairments or metastatic cancer were screened for CHF; the natriuretic peptide levels were measured separately. Results. Of the 150 residents, 103 (64%) were included (79±11 years). The diagnosis of CHF was established in 24 of these 103 residents with NTproBNP 1871 (IQR 539 to 4262) and BNP 194 (IQR 92 to 460) pg/ml. A striking result was that of the 24 residents found to have CHF after the screening, 15 (66%) had previously been undetected: NT-proBNP 1146 (interquartile range (IQR) 228 to 3341) and BNP 200 (IQR 107 to 433) pg/ml. Moreover, in 13 out of 22 residents (62%) who had previously been thought to have CHF, the diagnosis was rejected: NT-proBNP 388 (IQR 174 to 719) and BPN 90 (IQR 35 to 128) pg/ml). Regarding the diagnostic accuracy of NT-proBNP and BNP, the optimal cut-off level of NT-proBNP was 450 pg/ml with a sensitivity of 0.71 and specificity of 0.67, and for BNP it was 100 pg/ml with a sensitivity of 0.71 and specificity of 0.70. Conclusion. Both undetected and incorrect diagnoses of CHF were common. NT-proBNP and BNP were moderately accurate at diagnosing CHF. CHF prevalence was 23%. (Neth Heart J 2008;16: 123-8.)     

Microwave-assisted Solid Phase Peptide Synthesis on High Loaded Resins

Kinetic of diffusion of reactants from the solution into the resin beads is a key factor of the efficiency of reaction in solid phase synthesis. We demonstrated in this paper that stirring is required to obtain homogeneous solution during the short coupling steps in microwave-assisted SPPS. Different types of resin, loading rate and coupling reactants were investigated to highlight this observation.     

甾短杆菌胆固醇氧化酶基因在大肠杆菌中的表达

为了实现胆固醇氧化酶在大肠杆菌中的表达,将甾短杆菌Brevibacterium sp．DGCDC-82胆固醇氧化酶基因用PCR的方法去掉信号肽序列,连接到质粒pTrc99a,遗过筛选得到了表达胆固醇氧化酶的重组菌JMl09／pTrc99a—COD。经IPTG诱导后表达出相对分子质量约为5．5×10^4的蛋白质。分别考察了诱导温度、时间、IPTG浓度等因素对重组菌表达的胆固醇氧化酶的影响。在优化条件下,该胆固醇氧化酶的酶活可以达到700U／L。酶学特性分析表明其反应的最适pH为7．5,最适温度为40℃。     

High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage

Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.     

Interaction of DDSDEEN peptide with N-CAM protein. Possible mechanism enhancing neuronal differentiation

DDSDEEN chromatin peptide, after dansylation, was studied for its ability to bind N-CAM protein. The binding causes a quenching of the Dns-peptide fluorescence emission. Dose- and time-dependent binding of Dns-peptide with N-CAM has been shown. Fluorescence quenching is completely lost if the Dns-peptide is subjected to carboxypeptidase digestion. Moreover the undansylated peptide pEDDSDEEN competes with the DnsDDSDEEN peptide for the binding with the N-CAM protein. The Dns-peptide-N-CAM bond has been related to the peptide biological activity probably involved in the promotion of neuronal differentiation. An attempt to recognize a possible N-CAM binding site for Dns-peptide was performed by alignment of N-CAM from various sources with some sequences that have been previously reported as binding sites for the pEDDSDEEN and DDSDEEN peptides. Interestingly, the alignment of N-CAM from various sources with the peptides WHPREGWAL and WFPRWAGQA recognizes on rat and human N-CAM a unique sequence that could be the specific binding site for chromatin peptide: WHSKWYDAK. This sequence is present in fibronectin type-III domain of N-CAM. In addition molecular modeling studies indicate the N-CAM sequence WHSKWYDAK as, probably, the main active site for DnsDDSDEEN (or pEDDSDEEN) peptide ligand. Accordingly the binding experiments show a high affinity between WHSKWYDAK and DnsDDSDEEN peptides.     

Glucagon-like peptide 1 (7-36) amide (GLP-1) and exendin-4 stimulate serotonin release in rat hypothalamus

Glucagon-like peptide 1 (7-36) amide (GLP-1) and exendin-4 are gastrointestinal hormones as well as neuropeptides involved in glucose homeostasis and feeding regulation, both peripherally and at the central nervous system (CNS), acting through the same GLP-1 receptor. Aminergic neurotransmitters play a role in the modulation of feeding in the hypothalamus and we have previously found that peripheral hormones and neuropeptides, which are known to modulate feeding in the central nervous system, are able to modify catecholamine and serotonin release in the hypothalamus. In the present paper we have evaluated the effects of GLP-1 and exendin-4 on dopamine, norepinephrine, and serotonin release from rat hypothalamic synaptosomes, in vitro. We found that glucagon-like peptide 1 (7-36) amide and exendin-4 did not modify either basal or depolarization-induced dopamine and norepinephrine release; on the other hand glucagon-like peptide 1 (7-36) amide and exendin-4 stimulated serotonin release, in a dose dependent manner. We can conclude that the central anorectic effects of GLP-1 agonists could be partially mediated by increased serotonin release in the hypothalamus, leaving the catecholamine release unaffected.     

The angiotensin converting enzyme inhibitory tripeptides Ile-Pro-Pro and Val-Pro-Pro show increasing permeabilities with increasing physiological relevance of absorption models

Transepithelial transport of the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro was studied in different models of absorption. Apparent permeability (P(app)) values for absorptive transport across Caco-2 monolayers were 1.0+/-0.9 x 10(-8) (Ile-Pro-Pro) and 0.5+/-0.1 x 10(-8)cms(-1) (Val-Pro-Pro). Ex vivo transport across jejunal segments in the Ussing chamber was 5-times (Ile-Pro-Pro) to 10-times (Val-Pro-Pro) higher with no significant differences (p>0.05) observed between both peptides. The peptidase inhibitor bestatin increased permeability for the absorptive direction for Ile-Pro-Pro by twofold. Neither a transepithelial pH gradient nor increased apical tripeptide concentration nor longitudinal localization of the intestinal segment influenced P(app) in the ex vivo experiments. Val-Pro-Pro transport across Peyer's patches, however, was 4-times higher (P(app)=21.0+/-9.3 x10(-8)cms(-1)) as compared to duodenum (P(app)=4.8+/-1.4 x 10(-8)cms(-1)). In the in situ perfusion experiments P(app) values varied greatly among different animals ranging from 0.5 to 24.0 x10(-8)cms(-1) (Ile-Pro-Pro) and from 1.0 to 15.6 x 10(-8)cms(-1) (Val-Pro-Pro). In summary, Caco-2 and ex vivo absorption models differ considerably regarding their peptide permeability. The in situ model seems to be less appropriate because of the observed large variability in peptide permeability. The results of this study demonstrate that the ACE inhibitory peptides Ile-Pro-Pro and Val-Pro-Pro are absorbed partially undegraded.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.