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.     

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.     

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.     

Using evolutionary information and ancestral sequences to understand the sequence-function relationship in GLP-1 agonists

Glucagon-like peptide-1 (GLP-1) is an incretin hormone with therapeutic potential for type 2 diabetes. A variety of GLP-1 sequences are known from amphibian species, and some of these have been tested here and found to be able to bind and activate the human GLP-1 receptor. While little difference was observed for the in vitro potency for the human GLP-1 receptor, larger differences were found in the enzymatic stability of these peptides. Two peptides showed increased enzymatic stability, and they group together phylogenetically, though they originate from Amphibia and Reptilia. We have used ancestral sequence reconstruction to analyze the evolution of these GLP-1 molecules, including the synthesis of new peptides. We find that the increased stability could not be observed in the resurrected peptides from the common ancestor of frogs, even though they maintain the ability to activate the human GLP-1 receptor. Another method, using residue mapping on evolutionary branches yielded peptides that had maintained potency towards the receptor and also showed increased stability. This represents a new approach using evolutionary data in protein engineering.     

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1–42 (Aβ) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aβ boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aβ-induced effects.Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aβ and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites.As a whole, the collected data revealed that, the damaging path induced by Aβ in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.     

An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice

The aim of this study is to investigate the capability of an apoA-I mimetic with multiple amphipathic helices to form HDL-like particles in vitro and in vivo. To generate multivalent helices and to track the peptide mimetic, we have constructed a peptibody by fusing two tandem repeats of 4F peptide to the C terminus of a murine IgG Fc fragment. The resultant peptidbody, mFc-2X4F, dose-dependently promoted cholesterol efflux in vitro, and the efflux potency was superior to monomeric 4F peptide. Like apoA-I, mFc-2X4F stabilized ABCA1 in J774A.1 and THP1 cells. The peptibody formed larger HDL particles when incubated with cultured cells compared with those by apoA-I. Interestingly, when administered to mice, mFc-2X4F increased both pre-β and α-1 HDL subfractions. The lipid-bound mFc-2X4F was mostly in the α-1 migrating subfraction. Most importantly, mFc-2X4F and apoA-I were found to coexist in the same HDL particles formed in vivo. These data suggest that the apoA-I mimetic peptibody is capable of mimicking apoA-I to generate HDL particles. The peptibody and apoA-I may work cooperatively to generate larger HDL particles in vivo, either at the cholesterol efflux stage and/or via fusion of HDL particles that were generated by the peptibody and apoA-I individually.     

Targeted expression of redesigned and codon optimised synthetic gene leads to recrystallisation inhibition and reduced electrolyte leakage in spring wheat at sub-zero temperatures

Antifreeze proteins (AFPs) adsorb to ice crystals and inhibit their growth, leading to non-colligative freezing point depression. Crops like spring wheat, that are highly susceptible to frost damage, can potentially be made frost tolerant by expressing AFPs in the cytoplasm and apoplast where ice recrystallisation leads to cellular damage. The protein sequence for HPLC-6 α-helical antifreeze protein from winter flounder was rationally redesigned after removing the prosequences in the native protein. Wheat nuclear gene preferred amino acid codons were used to synthesize a recombinant antifreeze gene, rAFPI. Antifreeze protein was targeted to the apoplast using a Murine leader peptide sequence from the mAb24 light chain or retained in the endoplasmic reticulum using C-terminus KDEL sequence. The coding sequences were placed downstream of the rice Actin promoter and Actin-1 intron and upstream of the nopaline synthase terminator in the plant expression vectors. Transgenic wheat lines were generated through micro projectile bombardment of immature embryos of spring wheat cultivar Seri 82. Levels of antifreeze protein in the transgenic lines without any targeting peptide were low (0.06–0.07%). The apoplast-targeted protein reached a level of 1.61% of total soluble protein, 90% of which was present in the apoplast. ER-retained protein accumulated in the cells at levels up to 0.65% of total soluble proteins. Transgenic wheat line T-8 with apoplast-targeted antifreeze protein exhibited the highest levels of antifreeze activity and provided significant freezing protection even at temperatures as low as −7°C.     

The effect of fructose 2,6-bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase from rat liver

Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.