Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses

Antimicrobial polypeptides (AMPPs), consisting of peptides and small proteins with antimicrobial activity, are an integral component of innate immunity. Their often potent properties and widespread prevalence in fish suggests that designing means of manipulating their levels has considerable potential for maintaining or improving fish health. There is evidence that a number of chronic stresses lead to significant downregulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak. Conversely, upregulation of AMPP expression could be used to enhance disease resistance in stressful environments, as well as improve the efficacy of traditional antimicrobial drugs. However, further work is required in linking levels of a number of AMPPs to physiological function since, while a number of studies have documented the down- or upregulation of AMPPs via gene expression, relatively few studies have quantitatively examined changes in protein expression. In addition, not all AMPPs appear to be expressed at microbicidal levels in vivo, suggesting that at least some may have functions other than being directly protective. Nonetheless, in fish, there is evidence that some constitutively expressed AMPPs, such as piscidins and histone-like proteins, are expressed at microbicidal levels and that they decline with stress. Furthermore, certain AMPPs derived from hemoglobin-β are upregulated to microbicidal levels after experimental challenge. The likely widespread distribution of these three AMPP groups in fish provides the opportunity to design strategies to greatly improve the health of cultured fish populations.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Research progress on the immune mechanism of the silkworm Bombyx mori

The silkworm Bombyx mori L., representing an important economic insect and one of the best models for studying insect immunity, possesses an efficient and sophisticated innate immune system against invasive microorganisms. The innate immune system basically includes humoural immunity and cellular immunity. The humoural immunity, which functions via molecules including humoural factors, lysozymes, phenoloxidase, hemolin, lectins and, in particular, antimicrobial peptides, plays a central role in eliminating the invading pathogens. The cellular immunity is primarily carried out and mediated by plasmatocytes and granular cells of haemocytes in the haemolymph, usually followed by melanization. Additionally, apoptosis, a primary viral defence for insects lacking adaptive immunity, comprises an important part of the silkworm immune system. Currently, there is still the lack of a comprehensive and systematic understanding of the molecular mechanisms of silkworm immunity. We review the latest research progress on silkworm immune mechanisms, including phenoloxidase‐dependent melanization and apoptosis, which is conducive to improving our understanding of the silkworm immune mechanism, clarifying the relationship of various immune mechanisms, and also providing a theoretical basis and reference for the future research of insect immunity.     

A “Love” Dart Allohormone Identified in the Mucous Glands of Hermaphroditic Land Snails

Animals have evolved many ways to enhance their own reproductive success. One bizarre sexual ritual is the “love” dart shooting of helicid snails, which has courted many theories regarding its precise function. Acting as a hypodermic needle, the dart transfers an allohormone that increases paternity success. Its precise physiological mechanism of action within the recipient snail is to close off the entrance to the sperm digestion organ via a contraction of the copulatory canal, thereby delaying the digestion of most donated sperm. In this study, we used the common garden snail Cornu aspersum to identify the allohormone that is responsible for this physiological change in the female system of this simultaneous hermaphrodite. The love dart allohormone (LDA) was isolated from extracts derived from mucous glands that coat the dart before it is stabbed through the partner''s body wall. We isolated LDA from extracts using bioassay-guided contractility measurement of the copulatory canal. LDA is encoded within a 235-amino acid precursor protein containing multiple cleavage sites that, when cleaved, releases multiple bioactive peptides. Synthetic LDA also stimulated copulatory canal contractility. Combined with our finding that the protein amino acid sequence resembles previously described molluscan buccalin precursors, this indicates that LDA is partially conserved in helicid snails and less in other molluscan species. In summary, our study provides the full identification of an allohormone that is hypodermically injected via a love dart. More importantly, our findings have important consequences for understanding reproductive biology and the evolution of alternative reproductive strategies.     

NMR investigations of structural and dynamics features of natively unstructured drug peptide – salmon calcitonin: implication to rational design of potent sCT analogs

Backbone dynamics and conformational properties of drug peptide salmon calcitonin have been studied in aqueous solution using nuclear magnetic resonance (NMR). Although salmon calcitonin (sCT) is largely unfolded in solution (as has been reported in several circular dichroism studies), the secondary H^α chemical shifts and three bond H^N–H^α coupling constants indicated that most of the residues of the peptide are populating the α‐helical region of the Ramachandran (?, ψ) map. Further, the peptide in solution has been found to exhibit multiple conformational states exchanging slowly on the NMR timescale (10²–10³ s^?1), inferred by the multiple chemical shift assignments in the region Leu4–Leu12 and around Pro23 (for residues Gln20–Tyr22 and Arg24). Possibly, these slowly exchanging multiple conformational states might inhibit symmetric self‐association of the peptide and, in part, may account for its reduced aggregation propensity compared with human calcitonin (which lacks this property). The ¹⁵N NMR‐relaxation data revealed (i) the presence of slow (microsecond‐to‐millisecond) timescale dynamics in the N‐terminal region (Cys1–Ser5) and core residues His17 and Asn26 and (ii) the presence of high frequency (nanosecond‐to‐picosecond) motions in the C‐terminal arm. Put together, the various results suggested that (i) the flexible C‐terminal of sCT (from Thr25–Thr31) is involved in identification of specific target receptors, (ii) whereas the N‐terminal of sCT (from Cys1–Gln20) in solution – exhibiting significant amount of conformational plasticity and strong bias towards biologically active α‐helical structure – facilitates favorable conformational adaptations while interacting with the intermembrane domains of these target receptors. Thus, we believe that the structural and dynamics features of sCT presented here will be useful guiding attributes for the rational design of biologically active sCT analogs. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Improving oral bioavailability of cyclic peptides by N-methylation

The renaissance of peptides in pharmaceutical industry results from their importance in many biological functions. However, low metabolic stability and the lack of oral availability of most peptides is a certain limitation. Whereas metabolic instability may be often overcome by development of small cyclic peptides containing d-amino acids, the very low oral availability of most peptides is a serious limitation for some medicinal applications. The situation is complicated because a twofold optimization – biological activity and oral availability – is required to overcome this problem. Moreover, most simple “rules” for achieving oral availability are not general and are applicable only to limited cases. Many structural modifications for increasing biological activities and metabolic stabilities of cyclic peptides have been described, of which N-alkylation is probably the most common. This mini-review focuses on the effects of N-methylation of cyclic peptides in strategies to optimize bioavailabilities.     

Genetic selection for protein solubility enabled by the folding quality control feature of the twin-arginine translocation pathway

One of the most vexing problems facing structural genomics efforts and the biotechnology enterprise in general is the inability to efficiently produce functional proteins due to poor folding and insolubility. Additionally, protein misfolding and aggregation has been linked to a number of human diseases, such as Alzheimer's. Thus, a robust cellular assay that allows for direct monitoring, manipulation, and improvement of protein folding could have a profound impact. We report the development and characterization of a genetic selection for protein folding and solubility in living bacterial cells. The basis for this assay is the observation that protein transport through the bacterial twin-arginine translocation (Tat) pathway depends on correct folding of the protein prior to transport. In this system, a test protein is expressed as a tripartite fusion between an N-terminal Tat signal peptide and a C-terminal TEM1 beta-lactamase reporter protein. We demonstrate that survival of Escherichia coli cells on selective medium expressing a Tat-targeted test protein/beta-lactamase fusion correlates with the solubility of the test protein. Using this assay, we isolated solubility-enhanced variants of the Alzheimer's Abeta42 peptide from a large combinatorial library of Abeta42 sequences, thereby confirming that our assay is a highly effective selection tool for soluble proteins. By allowing the bacterial Tat pathway to exert folding quality control on expressed target protein sequences, we have generated a powerful tool for monitoring protein folding and solubility in living cells, for molecular engineering of solubility-enhanced proteins or for the isolation of factors and/or cellular conditions that stabilize aggregation-prone proteins.     

Regional Dissociation of Cyclic AMP and Inositol Phosphate Formation in Response to Thyrotropin-Releasing Hormone in the Rat Brain

The present study was undertaken to define effects of thyrotropin-releasing hormone (TRH) on formation of cyclic AMP (cAMP) and inositol phosphates (IPs) in rat brain regions. The brain of male Wistar rats was dissected into seven discrete regions, and each region was sliced. The slices were incubated in Krebs-Henseleit glucose buffer containing varying doses of TRH. TRH caused a significant and consistent increase in cAMP level, but not in formation of IPs, in the hypothalamus, striatum, and midbrain. TRH stimulated formation of IPs in the cerebellum, where the tripeptide did not change the cAMP level. In contrast, formation of neither cAMP nor IPs was affected by TRH in the cortex, hippocampus, or pons-medulla. These data suggest that TRH possesses two distinct types of brain intracellular signaling systems, which vary with brain regions.     

Oxygen-binding Heme Complexes of Peptides Designed to Mimic the Heme Environment of Myoglobin and Hemoglobin

Development of effective resuscitation agents for blood-loss replacement in trauma or surgery is extremely important despite substantial improvements in screening methods of blood from human donors. This paper reports the design and synthesis of peptides that mimic the natural environment of the heme group in myoglobin (Mb) and in the - and -subunits of human adult hemoglobin (Hb). The designs were based on the fact that the heme group in the aforementioned proteins is sandwiched between helices E and F. Fifteen test peptides and six control peptides were synthesized, and their ability to form stable complexes with heme was investigated. It was found that none of the control peptides or proteins was able to bind heme. However, each of the peptides that were designed to mimic the E--F helices, and even shorter designs, which removed from this region residues that do not contribute to contacts with the heme group, were each able to bind one mole of heme per mole of peptide forming peptide–heme complexes that were stable to manipulation and behaved as single molecular species. Oxygen binding measurements on the reduced peptide–heme complexes showed that these compounds bind oxygen and give visible spectra that were typical of oxygenated heme-proteins. In oxygen binding measurements done under different partial pressures of oxygen, the heme–peptide complexes gave hyperbolic oxygen-saturation curves, but showed slight differences in their P₅₀ values. The P₅₀ values ranged from 3.8 mmHg for the heme–peptide B7 complex to 13.7 mmHg for the heme–peptide D13 complex (under the same conditions, P₅₀ values for Hb and Mb were 34.0 and 5.5 mmHg, respectively). It is concluded that peptide constructs designed to mimic the heme-binding regions of Mb or the Hb subunits were able to form coordinate 1:1 complexes with heme, and these complexes bind oxygen in a manner expected for single subunit heme proteins.     

Effects of cyclic irrigation on water and nitrogen mass balances in a paddy field

Cyclic irrigation is considered an effective water management practice for reducing pollutant loads from a paddy-field district. The objective of this study was to clarify the effects of cyclic irrigation on mass balance in paddy plots. At the study site, cyclic irrigation with a high cyclic irrigation ratio (% reused water in irrigation water) was conducted from late April to late June. We found a complementary relationship between the volume of irrigation water and rainfall, which together totaled about 1400-1600 mm during the irrigation period each year. We concluded that a cyclic irrigation system that enables the paddy-field district to use a high cyclic irrigation ratio may lead to more efficient use of rainfall for crop irrigation. Nitrogen concentrations in both irrigation water and ponded water tended to be higher during the cyclic irrigation period than during the lake water irrigation period. Nitrogen input from irrigated water accounted for about 8-16% of the total input of nitrogen. It is suggested that fertilizer application of nitrogen can be reduced by its return through cyclic irrigation.