The dimerization of glucagon-like peptide-2 MIMETIBODY™ is linked to leucine-17 in the glucagon-like peptide-2 region

Glucagon-like peptide-2 (GLP-2) is a member of the glucagon multigene family that is produced by intestinal enteroendocrine cells in response to food intake. GLP-2 stimulates growth of the intestinal epithelium, enhances its barrier functions, and increases nutrient uptake. Therefore, a GLP-2 agonist may be efficacious in human diseases characterized by malabsorption or injury to the gastrointestinal epithelium. MIMETIBODY? refers to a proprietary scaffold developed to extend the half-life of rapidly cleared peptides. It consists of a peptide linked to a scaffold that contains sequence elements from a human immunoglobulin G including those that allow recycling through the FcRn. The GLP-2 sequence was engineered into the MIMETIBODY? scaffold. The primary state of both GLP-2 and the GLP-2 MIMETIBODY? in DPBS was a noncovalently associated dimer indicative of self-interaction. The increased heterogeneity and the decreased lot-to-lot reproducibility caused by the self-interaction of therapeutic proteins are a challenge to drug development. A similar protein, GLP-1 MIMETIBODY?, contains the related GLP-1 peptide and does not form a dimer under similar conditions. Therefore, to minimize or abrogate dimerization, several variants were made by substituting GLP-2 amino acids with the corresponding amino acids from GLP-1. Molecular weight and secondary structure analyses reveal that substituting leucine for glutamine at position 17 (L17Q) reduces dimerization and α-helix content yet retains bioactivity.     

Metal effects on the membrane interactions of amyloid-β peptides

Aβ(1–42) peptide, found as aggregated species in Alzheimer’s disease brain, is linked to the onset of dementia. We detail results of ³¹P and ²H solid-state NMR studies of model membranes with Aβ peptides and the effect of metal ions (Cu²⁺ and Zn²⁺), which are found concentrated in amyloid plaques. The effects on the lipid bilayer and the peptide structure are different for membrane incorporated or associated peptides. Copper ions alone destabilise the lipid bilayer and induce formation of smaller vesicles, but not when Aβ(1–42) is associated with the bilayer membrane. Aβ(25–35), a fragment from the C-terminal end of Aβ(1–42), which lacks the metal coordinating sites found in the full length peptide, is neurotoxic to cortical cortex cell cultures. Addition of metal ions has little effect on membrane bilayers with Aβ(25–35) peptides. ³¹P magic angle spinning NMR data show that Aβ(1–42) and Aβ(1–42)-Cu²⁺ complexes interact at the surface of anionic phospholipid membranes. Incorporated peptides, however, appear to disrupt the membrane more severely than associated peptides. Solid-state ¹³C NMR was used to compare structural changes of Aβ(1–42) to those of Aβ(25–35) in model membrane systems of anionic phospholipids and cholesterol. The Aβ peptides appeared to have an increase in β-strand structure at the C-terminus when added to phospholipid liposomes. The inclusion of Cu²⁺ also influenced the observed chemical shift of residues from the C-terminal half, providing structural clues for the lipid-associated Aβ/metal complex. The results point to the complex pathway(s) for toxicity of the full-length peptide. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

The ordered secretion of bioactive peptides: oldest or newest first?

The order of secretion of newly synthesized and older bioactive peptides was investigated using primary rat intermediate pituitary melanotropes, which synthesize, store, and secrete peptides derived from pro-ACTH/endorphin (PAE; also POMC). PAE-derived peptides produced by the cells were biosynthetically labeled by incubating the cells with radioactive amino acids at various times preceding the period during which secretion was examined; secreted and cellular peptides were characterized and quantitated by immunoprecipitation, using affinity-purified antibodies to selected regions of PAE, followed by polyacrylamide gel electrophoretic analysis. Release in the absence of secretagogues (basal or constitutive release) was compared to release in the presence of maximally effective levels of 8-bromo-cAMP and BaCl2 (stimulated or regulated release). Both cell types showed short-lived preferential basal release of newly synthesized and not fully mature peptides (less than 2-3 h old). Conversely, the cells showed preferential stimulated secretion of older peptides. A process of maturation occurred, taking 2-4 h, after which the secretion of newly synthesized and older peptides in response to secretagogues was nearly indistinguishable for the smallest product peptides. The data support a model of gradual processing of peptides from precursors into smaller products and maturation from molecules only available for basal release into peptides available for stimulated secretion as well as for basal release. Basal secretion was found to include mature peptides as well as intermediates and precursor molecules. The data do not support the existence of any preferential regulated secretion of newly synthesized peptides.     

The effects of β motor stimulation on the interpretation of signals from muscle spindles

A previously proposed method for the interpretation of the signals in sensory nerve fibres is extended to incorporate activation of muscle spindles. Simulations, based on previous experimental observations, of muscle spondles subjected to ramp and hold stretches are used as input to an interpreter, where the simulated trains of action potentials are reconverted to a length change interpretation. The interpreted signals are compared with the original length change inputs to observe the effects of -stimulation and stochastic variability.     

The effects of Leu or Val residues on cell selectivity of α-helical peptides

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.     

Effects of metal-binding loop mutations on ligand binding to calcium- and integrin-binding protein 1. Evolution of the EF-hand?

Calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous, multifunctional regulatory protein consisting of four helix-loop-helix EF-hand motifs. Neither EF-I nor EF-II binds divalent metal ions; however, EF-III is a mixed Mg2+/Ca2+-binding site, and EF-IV is a higher-affinity Ca2+-specific site. Through the generation of several CIB1 mutant proteins, we have investigated the importance of the last (-Z) metal-coordinating position of EF-III (D127) and EF-IV (E172) with respect to the binding of CIB1 to Mg2+, Ca2+, and its biological target, the cytoplasmic domain of the platelet alphaIIb integrin. A D127N mutant had reduced Mg2+ and Ca2+ affinity at EF-III but retained affinity for the alphaIIb domain. A D127E mutant had increased Mg2+ and Ca2+ affinity at EF-III, but unexpectedly, the affinity for the alphaIIb domain was too low for binding to be observed. E172Q and E172D mutants showed no and weak Mg2+ binding at EF-IV, respectively, and each mutant had reduced Ca2+ affinity at EF-IV and showed moderate metal-dependent differences in affinity for the alphaIIb domain. Finally, a D127Q mutant bound Mg2+ and Ca2+ in a manner similar to that of D127N, but like that of D127E, the affinity for the alphaIIb domain was reduced below the detection limit. These data, combined with a NMR-based structural comparison of the Mg2+- and Ca2+-loaded CIB1-alphaIIb peptide complexes, suggest that the D127E and D127Q mutations have a disruptive effect on alphaIIb binding since they expand the metal-binding loop and change the alpha-helix positions in EF-III. Conversely, upon replacement of the ancestral Glu with Asp at the -Z position of EF-III, CIB1 gained affinity for alphaIIb, and the Ca2+ affinity of CIB1 shifted into a range where the protein is able to act as an intracellular Ca2+ sensor.     

Effect of hydrocarbon stapling on the properties of α-helical antimicrobial peptides isolated from the venom of hymenoptera

The impact of inserting hydrocarbon staples into short α-helical antimicrobial peptides lasioglossin III and melectin (antimicrobial peptides of wild bee venom) on their biological and biophysical properties has been examined. The stapling was achieved by ring-closing olefin metathesis, either between two S-2-(4′-pentenyl) alanine residues (S ₅) incorporated at i and i + 4 positions or between R-2-(7′-octenyl) alanine (R ₈) and S ₅ incorporated at the i and i + 7 positions, respectively. We prepared several lasioglossin III and melectin analogs with a single staple inserted into different positions within the peptide chains as well as analogs with double staples. The stapled peptides exhibited a remarkable increase in hemolytic activity, while their antimicrobial activities decreased. Some single stapled peptides showed a higher resistance against proteolytic degradation than native ones, while the double stapled analogs were substantially more resistant. The CD spectra of the singly stapled peptides measured in water showed only a slightly better propensity to form α-helical structure when compared to native peptides, whereas the doubly stapled analogs exhibited dramatically enhanced α-helicity.     

The effects of dispersal patterns on marine reserves: does the tail wag the dog?

The concept of marine reserves as a method of improving management of fisheries is gaining momentum. While the list of benefits from reserves is frequently promoted, precise formulations of theory to support reserve design are not fully developed. To determine the size of reserves and the distances between reserves an understanding of the requirements for persistence of local populations is required. Unfortunately, conditions for persistence are poorly characterized, as are the larval dispersal patterns on which persistence depends. With the current paucity of information regarding meroplanktonic larval transport processes, understanding the robustness of theoretical results to larval dispersal is of key importance. From this formulation a broad range of dispersal patterns are analyzed. Larval dispersal is represented by a probability distribution that defines the fraction of successful settlers from an arbitrary location, the origin of the distribution, to any other location along the coast. While the effects of specific dispersal patterns have been investigated for invasion processes, critical habitat size and persistence issues have generally been addressed with only one or two dispersal types. To that end, we formulate models based on integrodifference equations that are spatially continuous and temporally discrete. We consider a range of dispersal distributions from leptokurtic to platykurtic. The effect of different dispersal patterns is considered for a single isolated reserve of varying size receiving no external larvae, as well as multiple reserves with varying degrees of connectivity. While different patterns result in quantitative differences in persistence, qualitatively similar effects across all patterns are seen in both single- and multiple reserve models. Persistence in an isolated reserve requires a size that is approximately twice the mean dispersal distance and regardless of the dispersal pattern the population in a patch is not persistent if the reserve size is reduced to just the mean dispersal distance. With an idealized coastline structure consisting of an infinite line of equally spaced reserves separated by regions of coastline in which reproduction is nil, the relative settlement as a function of the fraction of coastline and size of reserve is qualitatively very similar over a broad range of dispersal patterns. The upper limit for the minimum fraction of coastline held in reserve is about 40%. As the fraction of coastline is reduced, the minimum size of reserve becomes no more than 1.25 times the mean dispersal distance.     

When is bigger better? The effects of group size on the evolution of helping behaviours

Understanding the evolution of sociality in humans and other species requires understanding how selection on social behaviour varies with group size. However, the effects of group size are frequently obscured in the theoretical literature, which often makes assumptions that are at odds with empirical findings. In particular, mechanisms are suggested as supporting large‐scale cooperation when they would in fact rapidly become ineffective with increasing group size. Here we review the literature on the evolution of helping behaviours (cooperation and altruism), and frame it using a simple synthetic model that allows us to delineate how the three main components of the selection pressure on helping must vary with increasing group size. The first component is the marginal benefit of helping to group members, which determines both direct fitness benefits to the actor and indirect fitness benefits to recipients. While this is often assumed to be independent of group size, marginal benefits are in practice likely to be maximal at intermediate group sizes for many types of collective action problems, and will eventually become very small in large groups due to the law of decreasing marginal returns. The second component is the response of social partners on the past play of an actor, which underlies conditional behaviour under repeated social interactions. We argue that under realistic conditions on the transmission of information in a population, this response on past play decreases rapidly with increasing group size so that reciprocity alone (whether direct, indirect, or generalised) cannot sustain cooperation in very large groups. The final component is the relatedness between actor and recipient, which, according to the rules of inheritance, again decreases rapidly with increasing group size. These results explain why helping behaviours in very large social groups are limited to cases where the number of reproducing individuals is small, as in social insects, or where there are social institutions that can promote (possibly through sanctioning) large‐scale cooperation, as in human societies. Finally, we discuss how individually devised institutions can foster the transition from small‐scale to large‐scale cooperative groups in human evolution.     

The role of the β5-α11 loop in the active-site dynamics of acylated penicillin-binding protein A from Mycobacterium tuberculosis

Penicillin-binding protein A (PBPA) is a class B penicillin-binding protein that is important for cell division in Mycobacterium tuberculosis. We have determined a second crystal structure of PBPA in apo form and compared it with an earlier structure of apoenzyme. Significant structural differences in the active site region are apparent, including increased ordering of a β-hairpin loop and a shift of the SxN active site motif such that it now occupies a position that appears catalytically competent. Using two assays, including one that uses the intrinsic fluorescence of a tryptophan residue, we have also measured the second-order acylation rate constants for the antibiotics imipenem, penicillin G, and ceftriaxone. Of these, imipenem, which has demonstrable anti-tubercular activity, shows the highest acylation efficiency. Crystal structures of PBPA in complex with the same antibiotics were also determined, and all show conformational differences in the β5-α11 loop near the active site, but these differ for each β-lactam and also for each of the two molecules in the crystallographic asymmetric unit. Overall, these data reveal the β5-α11 loop of PBPA as a flexible region that appears important for acylation and provide further evidence that penicillin-binding proteins in apo form can occupy different conformational states.     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

What are the benefits of parental care? The importance of parental effects on developmental rate

The evolution of parental care is beneficial if it facilitates offspring performance traits that are ultimately tied to offspring fitness. While this may seem self‐evident, the benefits of parental care have received relatively little theoretical exploration. Here, we develop a theoretical model that elucidates how parental care can affect offspring performance and which aspects of offspring performance (e.g., survival, development) are likely to be influenced by care. We begin by summarizing four general types of parental care benefits. Care can be beneficial if parents (1) increase offspring survival during the stage in which parents and offspring are associated, (2) improve offspring quality in a way that leads to increased offspring survival and/or reproduction in the future when parents are no longer associated with offspring, and/or (3) directly increase offspring reproductive success when parents and offspring remain associated into adulthood. We additionally suggest that parental control over offspring developmental rate might represent a substantial, yet underappreciated, benefit of care. We hypothesize that parents adjust the amount of time offspring spend in life‐history stages in response to expected offspring mortality, which in turn might increase overall offspring survival, and ultimately, fitness of parents and offspring. Using a theoretical evolutionary framework, we show that parental control over offspring developmental rate can represent a significant, or even the sole, benefit of care. Considering this benefit influences our general understanding of the evolution of care, as parental control over offspring developmental rate can increase the range of life‐history conditions (e.g., egg and juvenile mortalities) under which care can evolve.     

Do endogenous opioid peptides mediate the effects of photoperiod on release of luteinizing hormone and prolactin in ovariectomized ewes?

Three experiments were done to determine if endogenous opioid peptides (EOPs) mediate the effects of photoperiod on release of luteinizing hormone (LH) and prolactin (Prl) in ovariectomized (OVX) ewes. Intravenous infusions of 0.5 naloxone X h-1 X kg body weight-1 for 3.5 h increased (P less than 0.01) mean plasma concentrations of LH and decreased (P less than 0.025) mean interpulse interval (period) of LH pulses in OVX ewes exposed to long day lengths (16L:8D). Infusions of either 1.0 or 2.5 mg morphine-SO4 X h-1 X kg-1 for 3 h increased (P less than 0.005) the period of LH pulses and increased (P less than 0.005) concentrations of Prl in OVX ewes during the breeding season. In OVX ewes exposed to long (16L:8D) or short (8L:16D) day lengths infusions of naloxone increased (P less than 0.05) mean concentrations of LH, whereas morphine decreased (P less than 0.01) mean concentrations of LH. These effects were attributed to changes in period of LH pulses (P less than 0.001). The drug X photoperiod interactions were not significant for LH parameters. Naloxone did not affect Prl release in either long- or short-day groups, but morphine increased (P less than 0.001) Prl release during long and short day lengths. The effect of morphine on Prl release was more pronounced in ewes exposed to long day lengths than in those exposed to short day lengths. In conclusion, EOPs inhibit the LH pulse generator in OVX ewes. However, it is doubtful that the EOPs mediate the steroid-independent effects of photoperiod on LH release. The results also suggest that photoperiod may influence Prl release via opiate neurons.     

The effects of temperature on host-pathogen interactions in D. melanogaster: who benefits?

Drosophila melanogaster is widely used to study immune system function in insects. However, little work has been done in D. melanogaster on the effect of temperature on the immune system. Here we describe experiments that demonstrate that cooler temperatures enhance survival after infection and alter expression of immune-related genes in flies. This effect appears to be due not only to the fact that colder temperatures slow down bacterial growth, but also to the beneficial effects of cooler temperature on immune function. We explore the possibility that heat shock proteins, and in particular, Hsp83, may improve immune function at cool temperatures. We have long known that temperature can alter immune responses against microbial pathogens in insects. The approach described here allows us to determine whether this effect is due primarily to temperature-specific effects on the host or on its pathogen. These results suggest that both may be important.     

The effects of a load of phen?lalanine on glucose metabolism

The clinical and experimental hyperphenylalaninemic conditions seem associated with disorders of carbohydrates metabolism. Examples are the increased liver glucose turnover and the glycogen depletion under Phenylalanine load and the hyperexcretion of glucose in the urine of PKU children. We studied the effects of an acute load of Phenylalanine on Glucose-3-H3 kinetics and plasma glucose levels in adult male Wistar rats. The effects of the load were very slow and after 40 minutes we observed an increase in the rate of plasma glucose production (Ra), that in normal animals in an almost exclusive liver function. As result of this glucose production there was a modest increase in plasma glucose levels.     

Are mutations usually deleterious? A perspective on the fitness effects of mutation accumulation

Evolutionary Ecology - All adaptive alleles in existence today began as mutations, but a common view in ecology, evolution, and genetics is that non-neutral mutations are much more likely to be...     

Effect of guanabenz on the peristaltic reflex and on the spontaneous rhythmic movements of the ileum isolated from the rabbit: do the alpha-2-adrenoreceptors constitute a homogenous population?

The inhibitory effect of the predominantly alpha-2 adrenoceptor agonist, guanabenz, on the peristaltic reflex and on the pendular movements of the rabbit isolated ileum was investigated. Guanabenz depressed or abolished the peristaltic reflex as well as the pendular movements. These effects were concentration-dependent. Guanabenz is much more potent inhibiting the peristaltic reflex (IC50 1 X 10(-7) M) than the pendular movements (IC50 1 X 10(-5) M). The choline ester, acetylcholine restored the peristaltic reflex and the anticholinesterase, eserine, restored the pendular movements previously abolished by guanabenz. During the blockade of the peristaltic reflex produced by guanabenz, the pendular movements were virtually not changed. It is therefore reasonable to suppose that the inhibitory effect of guanabenz reflects the different properties of alpha-2 adrenoceptors associated with cholinergic nerve terminals within the myenteric plexus and the longitudinal smooth muscle subserving the peristaltic reflex and the pendular movements.