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Brain signalling pathways involved in subclinical anxiety and depressed mood can be modulated via the gut brain axis (GBA), providing the potential for diet and dietary components to affect mood. We investigated behavioural, physiological and gut microbiome responses to the Lacticaseibacillus rhamnosus strain HN001 (LactoB HN001™), which has been shown to reduce postpartum anxiety and depression, and a milk fat globule membrane-enriched product, Lipid 70 (SurestartTM MFGM Lipid 70), which has been implicated in memory in stress-susceptible Wistar Kyoto rats. We examined behaviour in the open field, elevated plus maze and novel object recognition tests in conjunction with the expression of host genes in neuro-signalling pathways, and we also assessed brain lipidomics. Treatment-induced alterations in the caecal microbiome and short-chain fatty acid (SCFA) profiles were also assessed. Neither ingredient induced behavioural changes or altered the brain lipidome (separately or when combined). However, with regard to brain gene expression, the L. rhamnosus HN001 + Lipid 70 combination produced a synergistic effect, reducing GABAA subunit expression in the amygdala (Gabre, Gat3, Gabrg1) and hippocampus (Gabrd). Treatment with L. rhamnosus HN001 alone altered expression of the metabotropic glutamate receptor (Grm4) in the amygdala but produced only minor changes in gut microbiota composition. In contrast, Lipid 70 alone did not alter brain gene expression but produced a significant shift in the gut microbiota profile. Under the conditions used, there was no observed effect on rat behaviour for the ingredient combination. However, the enhancement of brain gene expression by L. rhamnosus HN001 + Lipid 70 implicates synergistic actions on region-specific neural pathways associated with fear, anxiety, depression and memory. A significant shift in the gut microbiota profile also occurred that was mainly attributable to Lipid 70. [ABSTRACT FROM AUTHOR]

Colostrum plays an important role in initiating the development of the intestinal barrier in newborn mammals. Given its bioactivity, there is much interest in the potential use of bovine colostrum to improve human gastrointestinal health throughout the life span. There is evidence that bovine colostrum is effective at improving small intestinal barrier integrity and some indication that it may alter colonic motility. However, for colostrum to be used as a product to improve intestinal health, it needs to be bioactive after processing. The aim of this study was to determine whether industrial processing of bovine colostrum affects its ability to improve small intestinal barrier integrity or alter distal colon motility. Three colostrum sample types were compared; raw whole colostrum powder (WCP), raw skim colostrum powder (SCP), and industrially produced colostrum milk protein concentrate (CMPC). To determine whether these colostrum powders had different effects on small intestinal barrier integrity, their effects on the transepithelial electrical resistance across an in vitro intestinal epithelial layer (Caco-2 cells) were measured, both with and without a challenge from the proinflammatory cytokine tumor necrosis factor-α. These results showed that CMPC enhanced transepithelial electrical resistance across unchallenged epithelial cell layers, whereas the raw colostrum samples, WCP and SCP, did not have an effect. The colostrum samples were also compared to determine how they affect contractility in the distal colon isolated from the rat. Skim colostrum powder was the only sample to act directly on colonic tissue to modulate motility, increasing the amplitude of contractions. The results show that bovine colostrum is able to improve small intestinal barrier integrity and alter colon motility, and they implicate different components. The barrier integrity enhancement was apparent only in the industrial CMPC, which may have been due to the increase in protein concentration or the release of small peptides as a result of processing. The ability to alter colon motility was present in SCP but absent in WCP, again implying that an increase in protein concentration is responsible for the effect. However, this effect was not apparent for the industrially processed CMPC, suggesting denaturation or degradation of the active component. The beneficial effect of colostrum on small intestinal barrier integrity was present after processing, confirming that it is feasible to industrially produce an active product for gut health. [ABSTRACT FROM AUTHOR]

A fullness in sensorial response is sought in low fat dairy foods to improve palatability, which may be achieved through activation of the calcium sensing receptor (CaSR). Free l -amino acids (FAA) are known kokumi substances that bind to the CaSR and impart attributes of creaminess and mouthfullness. In this study, skim milk (SM) was fermented using five different lactic acid bacteria (LAB) strains and their ability to activate the CaSR was compared. Our results demonstrated that fermented milks (FM) activated the CaSR with varying potency and efficacy in rank order: Leuconostoc pseudomesenteroides (CL3ST), Lactococcus lactis (BL1) > Streptococcus thermophilus (ST), Leuconostoc mesenteroides (CL3) > Lactobacillus helveticus (LH). [ABSTRACT FROM AUTHOR]

Abstract: This report describes a procedure for purification of large conductance calcium-activated potassium (BK, maxi-K) channels using immobilised metal affinity chromatography (IMAC) under non-denaturing conditions. An amino-terminal histidine fusion tag was added to hSlo, the human BK channel, and expressed in Sf9 insect cells. Following IMAC purification and production of proteoliposomes, protein function was assessed electrophysiologically in planar bilayer lipid membranes. Single channel openings had conductances of 250–300pS and were inhibited by paxilline, demonstrating that the BK channels remained functional following IMAC purification. This method to obtain functional human ion channels will be useful in assays to screen potential pharmaceuticals. [Copyright &y& Elsevier]

Abstract: The effects of the mycotoxin lolitrem B on the function of hSlo large conductance calcium-activated potassium channels expressed in HEK293 cells have been investigated using inside-out membrane patches. Lolitrem B potently inhibited hSlo potassium currents activated by depolarising voltage pulses in the presence of 10μM free calcium. At a concentration of 100nM, lolitrem B rapidly and completely inhibited outward potassium currents. The concentration that produced half-maximal inhibition was 3.7nM, indicating a high apparent affinity for hSlo channels. This is the first time that a molecular site of action has been identified for a compound of the lolitrem structural class of indole diterpene and identifies a novel BK channel blocker. [Copyright &y& Elsevier]

We used the open-channel blocker, penicillin (10 mM), as a tool to investigate if the human α1β1 or α1β1γ2S γ-aminobutyric acid type A (GABAA) receptor channels opened in the absence of GABA. Application of penicillin to cells expressing the receptors resulted in a transient inward whole-cell current, the off-current, upon penicillin removal. The amplitude of the off-current was dependent on the duration of the penicillin application, it reversed in polarity at depolarized potentials and exhibited “run-down” similar to the GABA-activated currents. Bicuculline (100 μM) blocked the off-current response. Pentobarbital (50 μM) enhanced the peak off-current amplitude by 2.8 and 3.4 in α1β1 and α1β1γ2S receptors, respectively. Diazepam (1 μM) only enhanced the off-current peak response in α1β1γ2S receptors (1.6) and induced the development of an inward current when applied alone. The results are consistent with that the α1β1 or α1β1γ2S GABAA receptors can open in the absence of GABA and raise the question of what role spontaneous channel openings have in the function of GABAA receptors. [Copyright &y& Elsevier]

• Neurons of the ENS control propagating colonic contractions. • Resident and probiotic bacteria and their components modulate ENS function and motility. • Enterocytes are the gateway through which luminal content may signal or access afferent neurons. Sensory nerve endings within the wall of the gastrointestinal (GI) tract may respond to bacterial signalling, providing the basis for key biological processes that underlie intestinal motility and microbial homeostasis. Enteric neurons and smooth muscle cells are well known to express an array of receptors, including G-protein coupled receptors and ligand-gated ion channels, that can sense chemical ligands and other bacterially-derived substances. These include short chain fatty acids, secondary bile acids and lipopolysaccharide. For neural detection of microbial activators to occur, luminal substances must first interact with enterocytes for direct signalling or cross paracellularly. Recent studies indicate that bacterial-derived microvesicles can cross the gut epithelial barrier and affect motility. This suggests a possible intercellular communication pathway between the GI tract and the ENS. We explore the idea that bacterial microvesicles can behave as a delivery package for communication between microbe and host. [ABSTRACT FROM AUTHOR]

Abstract: Human voltage-gated sodium ion channels are major sites of action for drugs and toxins that modulate cellular excitability, and are therefore key molecular targets for ion channel research, high throughput screening for new drugs, and toxin detection. Protein suitable for these applications must be produced in a functionally active form. We report the successful use of ion metal affinity chromatography (IMAC) to purify C-terminal polyhistidine tagged human skeletal muscle voltage-gated sodium (hSkM1-HT) channels from Sf9 insect cells; hSkM1 channels were pharmacologically functional when reconstituted into liposomes and incorporated into planar bilayer lipid membranes. hSkM1-HT single channel currents activated by veratridine had a conductance of 21pS and those activated by brevetoxin, 16pS. Channel activity was inhibited by tetrodotoxin and saxitoxin. This protein is suitable for the development of biosensor and high throughput screening technologies. [Copyright &y& Elsevier]

Largeconductance Ca2+- and voltage-activated K+ (BK) channels play prominent roles in shaping muscle and neuronal excitability. In the cardiovascular system, BK channels promote vascular relaxation and protect against ischemic injury. Recently, inhibition of BK channels has been shown to lower heart rate in intact rodents and isolated hearts, suggesting a novel role in heart function. However, the underlying mechanism is unclear. In the present study, we recorded ECGs from mice injected with paxilline (PAX), a membrane-permeable BK channel antagonist, and examined changes in cardiac conduction. ECGs revealed a 19 ± 4% PAX-induced reduction in heart rate in wild-type but not BK channel knockout (Kcnma1-/-) mice. The heart rate decrease was associated with slowed cardiac pacing due to elongation of the sinus interval. Action potential firing recorded from isolated sinoatrial node cells (SANCs) was reduced by 55 ± 15% and 28 ± 9% by application of PAX (3 μM) and iberiotoxin (230 nM), respectively. Furthermore, baseline firing rates from Kcnma1-/-SANCs were 33% lower than wild-type SANCs. The slowed firing upon BK current inhibition or genetic deletion was due to lengthening of the diastolic depolarization phase of the SANC action potential. Finally, BK channel immunoreactivity and PAX-sensitive currents were identified in SANCs with HCN4 expression and pacemaker current, respectively, and BK channels cloned from SANCs recapitulated similar activation as the PAX-sensitive current. Together, these data localize BK channels to SANCs and demonstrate that loss of BK current decreases SANC automaticity, consistent with slowed sinus pacing after PAX injection in vivo. Furthermore, these findings suggest BK channels are potential therapeutic targets for disorders of heart rate. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to compare the mode of action of the commonly used BK inhibitor paxilline with that of the more recently discovered lolitrem B. Similarities and differences in characteristics of inhibition between the two compounds were investigated. We have previously shown that lolitrem B does not affect the BK channel G–V, in contrast to the rightward shift produced by paxilline. These different effects on the voltage-dependence of activation suggest different modes of action for these two compounds. In this study we show that inhibition by both paxilline and lolitrem B is characterized by an open state preference for BK (hSlo) channels. Both compounds had a 3-fold higher apparent affinity under conditions likely to favour the open state, suggesting they have a similar BK conformational preference for binding. Furthermore, both compounds had a calcium concentration-dependence to their inhibitory effects. The G–V shift induced by paxilline was calcium concentration-dependent. [Copyright &y& Elsevier]

To identify materials suitable as membrane supports for ion channel biosensors, six filter materials of varying hydrophobicity, tortuosity, and thickness were examined for their ability to support bilayer lipid membranes as determined by electrical impedance spectroscopy. Bilayers supported by hydrophobic materials (PTFE, polycarbonate, nylon, and silanised silver) had optimal resistance (14-19GΩ) and capacitance (0.8-1.6 µF) values whereas those with low hydrophobicity did not form BLMs (PVDF) or were short-lived (unsilanised silver). The ability of ion channels to function in BLMs was assessed using a method recently reported to improve the efficiency of proteoliposome incorporation into PTFE-supported bilayers. Voltage-gated sodium channel activation by veratridine and inhibition by saxitoxin showed activity for PTFE, nylon, and silanised silver, but not polycarbonate. Bilayers on thicker, more tortuous, and hydrophobic materials produced higher current levels. Bilayers that self-assembled on PTFE filters were the longest lived and produced the most channel activity using this method. [ABSTRACT FROM AUTHOR]

The heart generates and propagates action potentials through synchronized activation of ion channels allowing inward Na+ and Ca2+ and outward K+ currents. There are a number of K+ channel types expressed in the heart that play key roles in regulating the cardiac cycle. Large conductance calcium-activated potassium (BK) ion channels are not thought to be directly involved in heart function. Here we present evidence that heart rate can be significantly reduced by inhibiting the activity of BK channels. Agents that specifically inhibit BK channel activity, including paxilline and lolitrem B, slowed heart rate in conscious wild-type mice by 30% and 42%, respectively. Heart rate of BK channel knock-out mice (Kcnma1-/-) was not affected by these BK channel inhibitors, suggesting that the changes to heart rate were specifically mediated through BK channels. The possibility that these effects were mediated through BK channels peripheral to the heart was ruled out with experiments using isolated, perfused rat hearts, which showed a significant reduction in heart rate when treated with the BK channel inhibitors paxilline (1 μM), lolitrem B (1 μM), and iberiotoxin (0.23 μM), of 34%, 60%, and 42%, respectively. Furthermore, paxilline was shown to decrease heart rate in a dose-dependent manner. These results implicate BK channels located in the heart to be directly involved in the regulation of heart rate. [ABSTRACT FROM AUTHOR]

Abstract: Lolitrem B is an indole-diterpenoid neurotoxin which is the main causative agent of ryegrass staggers, an animal disease associated with tremors and incoordination. It is also a potent inhibitor of large conductance calcium-activated potassium (BK) channel activity (IC50 =4 nM). Furthermore, we have recently shown that the motor function deficits induced by lolitrem B are specifically mediated by BK channels, making the toxin a valuable tool for investigating the molecular function and physiological roles of these channels. To determine what structural features of BK channel agents are required for high potency, the effect of lolitrem B and seven structurally-related lolitrems on BK channel activity has been measured. Concentration–responses and conductance–voltage (G–V) relationships were determined for each compound and related to the different structure types. This study has identified seven new BK channel inhibitors and has allowed the identification of two key structural features required for high potency BK channel activity by lolitrems. [Copyright &y& Elsevier]

• Peptides from in vitro digestion of cow, goat and sheep products were different. • β-CN and β-Lg were digested faster in yogurt samples. • Yoghurts produced a greater amount of bioactive peptides than milks. • The majority of bioactive peptides had antihypertensive activity. Fermentation of milk is commonly used throughout the world to produce a variety of foods with different health benefits. We hypothesised that due to differences in physicochemical properties and protein sequences among milk from different species and their fermented yogurt samples, their protein digestion and resulting peptide profiles would differ. Cow, goat and sheep milk and yogurt were compared at designated timepoints throughout in vitro gastric and intestinal digestion for differences in peptide profiles and peptide bioactivities. The results showed that most proteins in all milk and yogurt samples were digested within the early phase of gastric digestion. β-Lg and β-CN were digested faster in yogurt than milk, which was most evident for sheep products. Regardless of species, in vitro gastric and intestinal digestion released a higher concentration of specific peptides, particularly anti-hypertensives, from yogurt compared with their milk counterparts. [ABSTRACT FROM AUTHOR]

Stress negatively impacts gut and brain health. Individual differences in response to stress have been linked to genetic and environmental factors and more recently, a role for the gut microbiota in the regulation of stress-related changes has been demonstrated. However, the mechanisms by which these factors influence each other are poorly understood, and there are currently no established robust biomarkers of stress susceptibility. To determine the metabolic and microbial signatures underpinning physiological stress responses, we compared stress-sensitive Wistar Kyoto (WKY) rats to the normo-anxious Sprague Dawley (SD) strain. Here we report that acute stress-induced strain-specific changes in brain lipid metabolites were a prominent feature in WKY rats. The relative abundance of Lactococcus correlated with the relative proportions of many brain lipids. In contrast, plasma lipids were significantly elevated in response to stress in SD rats, but not in WKY rats. Supporting these findings, we found that the greatest difference between the SD and WKY microbiomes were the predicted relative abundance of microbial genes involved in lipid and energy metabolism. Our results provide potential insights for developing novel biomarkers of stress vulnerability, some of which appear genotype specific. [ABSTRACT FROM AUTHOR]