Nutrient Inadequacy in Korean Young Adults with Depression: A Case Control Study.

The role of diet is gaining attention among the modifiable factors associated with depression; thus, this case-control study examined the association between nutrition and depression in young Korean adults. Dietary surveys in individuals with depression (n = 39) and age- and gender-matched controls (n = 76) were conducted using food records and food frequency questionnaires. Men with depression consumed less mushrooms and meat, while women consumed significantly less grains (p < 0.05). Overall, the depression group consumed less energy and nutrients, and the difference was more pronounced in men. The male depression group had lower nutrient adequacy ratio (NAR) for energy, protein, vitamin A, thiamine, niacin, folate, and phosphorus, whereas the female depression group had lower NARs for energy, protein, niacin, and vitamin B12. The depression group had a significantly lower mean adequacy ratio in both genders. Furthermore, the proportion of inappropriate nutrient intake was higher in both genders of the depression group, exhibiting significant differences in energy, protein, niacin, folate, and zinc in men and energy, riboflavin, folate, and vitamin C in women. Hence, both men and women in the depression group had poor nutrient intake and high rates of nutrient inadequacy and improper consumption. This suggests that the quantity and quality of meals should be improved for individuals with depressive symptoms.

Ferulic Acid as a Protective Antioxidant of Human Intestinal Epithelial Cells.

The intestinal epithelial barrier is the primary and most significant defense barrier against ingested toxins and pathogenic bacteria. When the intestinal epithelium barrier is breached, inflammatory response is triggered. GWAS data showed that endoplasmic reticulum (ER) stress markers are elevated in Inflammatory Bowel Disease (IBD) patients, which suggests ER stress regulation might alleviate IBD symptoms. Ferulic acid (FA) is a polyphenol that is abundant in plants and has antioxidant and anti-inflammatory properties, although it is unclear whether FA has these effects on the intestine. Therefore, we investigated the effect of FA in vitro and in vivo. It was found that FA suppressed ER stress, nitric oxide (NO) generation, and inflammation in polarized Caco-2 and T84 cells, indicating that the ER stress pathway was implicated in its anti-inflammatory activities. The permeability of polarized Caco-2 cells in the presence and absence of proinflammatory cytokines were decreased by FA, and MUC2 mRNA was overexpressed in the intestines of mice fed a high-fat diet (HFD) supplemented with FA. These results suggest that FA has a protective effect on intestinal tight junctions. In addition, mouse intestine organoids proliferated significantly more in the presence of FA. Our findings shed light on the molecular mechanism responsible for the antioxidant effects of FA and its protective benefits on the health of the digestive system.

Atherogenic Index of Plasma and Its Association with Risk Factors of Coronary Artery Disease and Nutrient Intake in Korean Adult Men: The 2013-2014 KNHANES.

Coronary artery disease (CAD) has been linked to one of the highest death rates globally. The atherogenic index of plasma (AIP) may be an important predictor of atherosclerosis and cardiovascular disease, superior to the standard atherosclerotic lipid profile. This study investigated the relationship between AIP and obesity indices, blood glucose, lipid profile, and nutrient intake status in Korean adult men. The study included 1292 males aged ≥19 years old who participated in the Korea National Health and Nutrition Examination Survey, 2013-2014. Participants were divided into four groups according to AIP quartiles, calculated as log (triglyceride (TG)/high-density lipoprotein cholesterol (HDL-C)). Body mass index, waist circumference, fasting blood glucose, hemoglobin A1c, total cholesterol, TG, and low-density lipoprotein cholesterol levels increased as AIP levels increased, whereas HDL-C level declined. As the level of AIP increased, intake of saturated fatty acid, calcium, phosphorus, riboflavin, milk, and dairy product decreased significantly, and the contribution rate of milk and dairy products to fat intake decreased. AIP was linked to obesity indices, blood glucose, and blood lipid profile in Korean men, suggesting that it could predict CAD.

Antioxidant and Anti-Inflammatory Effects of Korean Black Ginseng Extract through ER Stress Pathway.

The excessive release of reactive oxygen species (ROS) can result in the development of chronic inflammation. The mechanisms involved in inflammation are various, with endoplasmic reticulum (ER) stress known to be among them. We have previously shown that black ginseng (BG) reduced lipid accumulation in and enhanced the antioxidant function of the liver in vitro and in vivo mostly due to ginsenoside Rb1, Rg3 and Rk1 components. Therefore, this study investigated the antioxidant effect of BG on the intestines and its possible mechanistic pathway through ER stress. The results showed that BG extract decreased ROS and nitric oxide (NO) production and reduced inducible nitric oxide synthase (iNOS) expression levels in vitro, and these results were confirmed by zebrafish embryos in vivo. However, this phenotype was abolished in the absence of inositol-requiring enzyme 1 (IRE1α) but not in the absence of protein kinase RNA (PKR)-like ER-resistant kinase (PERK) or X-box-binding protein 1 (XBP1) in the mouse embryo fibroblast (MEF) knockout (KO) cells, suggesting that BG elicits an antioxidant effect in an IRE1α-dependent manner. Antioxidant and anti-inflammatory effects were assessed in the liver and intestines of the mouse model affected by nonalcoholic fatty liver disease (NAFLD), which was induced by a high-fat/high-fructose diet. In the liver, BG treatment rescued NAFLD-induced glutathione (GSH), catalase (CAT), tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 expression. In the intestines, BG also rescued NAFLD-induced shortened villi, inflammatory immune cell infiltration, upregulated IL-6, cytosine-cytosine-adenosine-adenosine-thymidine (CCAAT)/enhancer-binding homologous protein (CHOP) and binding immunoglobulin protein (BiP) expression. In conclusion, our results show that BG reduces ROS and NO production followed by inflammation in an IRE1α-dependent and XBP1-independent manner. The results suggest that BG provides antioxidant and anti-inflammatory effects through an ER stress mechanism.

Antioxidant Effect of Lycium barbarum Leaf through Inflammatory and Endoplasmic Reticulum Stress Mechanism.

Although the prevalence and incidence of inflammatory bowel disease (IBD), a defective immune response of the gastrointestinal tract, has been increasing in North America and Western Europe, recent studies have shown that this disease is also increasing rapidly in Asia. Several studies have been searching for functional foods that can prevent or reduce IBD symptoms because the drug treatments for IBD are expensive with complications. Genome-Wide Association Study (GWAS), an observational study of a genome-wide set of genetic variants in different individuals, showed that endoplasmic reticulum (ER) stress is one of the causes of IBD. Previously, we reported the effects of Lyciumbarbarum fruit and this study investigated the effects of Lycium barbarum leaf (LL) on inflammation and ER stress of the intestine. The paracellular permeability, antioxidant, and anti-inflammatory response were measured on polarized Caco-2 cells. The ER stress pathway and pro-inflammatory cytokines were evaluated on MEF-knockout cell lines, and on the intestines of the mice fed a high-fat diet with lipopolysaccharide-induced inflammation. Our data showed that the LL pretreatment strengthened the tight junction integrity and reduced NO production both in the presence and in the absence of inflammation. Furthermore, LL inhibited ER stress and inflammation via IRE1α and XBP1 in vitro as well as in the inflamed intestines of mice, highlighting the antioxidant and anti-inflammatory function of LL in an IRE1α-XBP1-dependent manner.

Cell cycle profile data on splenocytes of high fat diet induced obese mice treated with ferulic acid.

The reported data are related to the article entitled "Ferulic acid maintains the self-renewal capacity of embryo stem cells and adipose-derived mesenchymal stem cells in high fat diet-induced obese mice" [1]. Ferulic acid is a natural bioactive compound and demonstrated potential to serve as a self-renewing biomarker in an alkaline phosphate assay and caused increased Nanog mRNA levels in embryonic stem cells. In these data, we examined another functional aspect of ferulic acid, namely the effect of ferulic acid on the cell cycle of splenocytes. These data were collected from the splenocytes of C57BL/6 J male mice that were fed either a high fat diet (HFD) alone or an HFD diet supplemented with ferulic acid (5 g/kg diet) for 8 weeks. As expected, the HFD resulted in a significant increase in mouse body weight, liver weight, and epididymal fat tissue weight compared to the control diet (Cho and Park, 2020). The cell cycle profile of mouse splenocytes in HFD-induced obese mice was evaluated by FACS. Since the G1 checkpoint is the point at which cells enter the cell cycle, an internal or external stimulation can cause the cell to delay passing G1 and instead enter a quiescent state known as G0 without proceeding past the restriction checkpoint. DNA damage is the main trigger that can cause a cell to "restrict" itself and not enter the cell cycle [2]. These results show that ferulic acid helps attenuate G1/S arrest in splenocytes in HFD-induced obese mice.

Exercise and Curcumin in Combination Improves Cognitive Function and Attenuates ER Stress in Diabetic Rats.

Type 2 diabetes mellitus (T2DM) is a metabolic disease associated with chronic low-grade inflammation that is mainly associated with lifestyles. Exercise and healthy diet are known to be beneficial for adults with T2DM in terms of maintaining blood glucose control and overall health. We investigated whether a combination of exercise and curcumin supplementation ameliorates diabetes-related cognitive distress by regulating inflammatory response and endoplasmic reticulum (ER) stress. This study was performed using male Otsuka Long-Evans Tokushima Fatty (OLETF) rats (a spontaneous diabetes Type 2 model) and Long-Evans Tokushima Otsuka (LETO) rats (LETO controls) by providing them with exercise alone or exercise and curcumin in combination. OLETF rats were fed either a diet of chow (as OLETF controls) or a diet of chow containing curcumin (5 g/kg diet) for five weeks. OLETF rats exercised with curcumin supplementation exhibited weight loss and improved glucose homeostasis and lipid profiles as compared with OLETF controls or exercised OLETF rats. Next, we examined cognitive functions using a Morris water maze test. Exercise plus curcumin improved escape latency and memory retention compared to OLETF controls. Furthermore, OLETF rats exercised and fed curcumin had lower IL6, TNFα, and IL10 levels (indicators of inflammatory response) and lower levels of ER stress markers (BiP and CHOP) in the intestine than OLETF controls. These observations suggest exercise plus curcumin may offer a means of treating diabetes-related cognitive dysfunction.

Spirulina Enhances Bone Modeling in Growing Male Rats by Regulating Growth-Related Hormones.

In recent years, growth hormone deficiency in children has been treated with hormone therapy despite the possible significant side effects. Therefore, it was deemed beneficial to develop functional foods or dietary supplements for safely improving children's growth. Spirulina platensis is known for its high antioxidant, anti-aging, anti-cancer, and immunity-enhancing properties, as well as its high digestibility and high protein content, but little has been reported about its influence on bone development in children with a normal supply of protein. In this study, we evaluated the effects of spirulina on the bone metabolism and antioxidant profiles of three-week-old growing male rats. The animals were divided into four groups (n = 17 per group) and were fed AIN93G diets with 0% (control), 30% (SP30), 50% (SP50), and 70% (SP70) of casein protein replaced by spirulina, respectively, for seven weeks. We observed that spirulina enhanced bone growth and bone strength by stimulating parathyroid hormone and growth hormone activities, as well its increased antioxidant activity. These results indicate that spirulina provides a suitable dietary supplement and alternative protein source with antioxidant benefits for growth improvement in early developmental stages.

Au nanozyme-driven antioxidation for preventing frailty.

From senescence and frailty that may result from various biological, mechanical, nutritional, and metabolic processes, the human body has its own antioxidant defense enzymes to remove by-products of oxygen metabolism, and if unregulated, can cause several types of cell damage. Herein, an antioxidant, artificial nanoscale enzyme, called nanozyme (NZs), is introduced that is composed of Au nanoparticles (NPs) synthesized with a mixture of two representative phytochemicals, namely, gallic acid (GA) and isoflavone (IF), referred to as GI-Au NZs. Their unique antioxidant and anti-aging effects are monitored using Cell Counting Kit-8 and senescence-associated ß-galactosidase assays on neonatal human dermal fibroblasts (nHDFs). Furthermore, alterations in epidermal thickness and SOD activity are measured under ultraviolet light to investigate the effects of the topical application of NZs on the histological structure and antioxidant activity in hairless mice skin. Then, hepatotoxicity and nephrotoxicity in the hairless mice are monitored. It is concluded that the NZs can effectively prevent serial passage-induced senescence in nHDFs, as well as oxidative stress in mice skin, suggesting a range of strategies to further develop novel therapeutics for acute frailty.

Anti-inflammatory effect of Lycium barbarum on polarized human intestinal epithelial cells.

BACKGROUND/OBJECTIVES: Inflammatory Bowel Disease (IBD) has rapidly escalated in Asia (including Korea) due to increasing westernized diet patterns subsequent to industrialization. Factors associated with endoplasmic reticulum (ER) stress are demonstrated to be one of the major causes of IBD. This study was conducted to investigate the effect of Lycium barbarum (L. barbarum) on ER stress. MATERIALS/METHODS: Mouse embryonic fibroblast (MEF) cell line and polarized Caco-2 human intestinal epithelial cells were treated with crude extract of the L. chinense fruit (LF). Paracellular permeability was measured to examine the effect of tight junction (TJ) integrity. The regulatory pathways of ER stress were evaluated in MEF knockout (KO) cell lines by qPCR for interleukin (IL) 6, IL8 and XBP1 spliced form (XBP1s). Immunoglobulin binding protein (BiP), XBP1s and CCAAT/enhancer-binding homologous protein (CHOP) expressions were measured by RT-PCR. Scanning Ion Conductance Microscopy (SICM) at high resolution was applied to observe morphological changes after treatments. RESULTS: Exposure to LF extract strengthened the TJ, both in the presence and absence of inflammation. In polarized Caco-2 pretreated with LF, induction in the expression of proinflammatory marker IL8 was not significant, whereas ER stress marker XBP1s expression was significantly increased. In wild type (wt) MEF cells, IL6, CHOP and XBP1 spliced form were dose-dependently induced when exposed to 12.5-50 µg/mL extract. However, absence of XBP1 or IRE1α in MEF cells abolished this effect. CONCLUSION: Results of this study show that LF treatment enhances the barrier function and reduces inflammation and ER stress in an IRE1α-XBP1-dependent manner. These results suggest the preventive effect of LF on healthy intestine, and the possibility of reducing the degree of inflammatory symptoms in IBD patients.

Targeting friend and foe: Emerging therapeutics in the age of gut microbiome and disease.

Mucosal surfaces that line our gastrointestinal tract are continuously exposed to trillions of bacteria that form a symbiotic relationship and impact host health and disease. It is only beginning to be understood that the cross-talk between the host and microbiome involve dynamic changes in commensal bacterial population, secretion, and absorption of metabolites between the host and microbiome. As emerging evidence implicates dysbiosis of gut microbiota in the pathology and progression of various diseases such as inflammatory bowel disease, obesity, and allergy, conventional treatments that either overlook the microbiome in the mechanism of action, or eliminate vast populations of microbes via wide-spectrum antibiotics need to be reconsidered. It is also becoming clear the microbiome can influence the body's response to therapeutic treatments for cancers. As such, targeting the microbiome as treatment has garnered much recent attention and excitement from numerous research labs and biotechnology companies. Treatments range from fecal microbial transplantation to precision-guided molecular approaches. Here, we survey recent progress in the development of innovative therapeutics that target the microbiome to treat disease, and highlight key findings in the interplay between host microbes and therapy.

Application of the biotin-labeled toxin mutant for affinity isolation of associated proteins in the mammalian cells.

Cholera toxin (CT), one of the AB5 bacterial toxin families, is produced by Vibrio cholerae, breeches the intestinal epithelial barrier and enters host epithelial cells to cause the massive secretory diarrhea. This study focused on understanding the retro-translocation machinery of the bacterial toxin using biotin-avidin technology to explain toxin trafficking from the endoplasmic reticulum (ER) to the cytosol. Because the association between the A1 chain of CT and other components of the retro-translocation machinery is likely transient or very weak, the successful bioengineering of such a mutant to be trapped as an intermediate in ER is essential for affinity isolation and further analysis. Here, we prepared a mutant toxin that 15 amino acid Biotin Acceptor Peptide (BAP) was fused to the C-terminal of A1 chain of CT. Biotinylation efficiency of the BAP-inserted cholera toxin (BT) was nearly 100%. Moreover, BT was functionally toxic and successfully pulled down by NeutrAvidin in vitro and in vivo. However, NeutrAvidin-bound biotinylated BT was not toxic. These results suggest the possibility of a plug effect of the biotin-NeutrAvidin-BT complex stuck in the ER without retro-translocation to the cytosol. Therefore, this model might identify the interacting proteins with A1 chain of CT in the host cells by holding the moment of retro-translocation of the bacterial toxin. In conclusion, this study established the model using biotin-avidin technology to elucidate the molecular basis for retro-translocation of bacterial toxin from within the lumen of ER to the cytosol.

Innate immunity at mucosal surfaces: the IRE1-RIDD-RIG-I pathway.

Recent studies have linked the ER stress sensor IRE1α with the RIG-I pathway, which triggers an inflammatory response upon detection of viral RNAs. In response to ER dysfunction, IRE1α cleaves mRNA into single-strand fragments that lack markers of self, which activate RIG-I. Certain microbial products from mucosal pathogens activate this pathway by binding IRE1α directly, and the discovery that IRE1 is amplified at mucosal surfaces by gene duplication suggests an important role for IRE1 in mucosal immunity. Here, we review evidence in support of this hypothesis, and propose a model wherein IRE1 surveys the integrity of the ER, acting as a guard receptor and a pattern recognition receptor, capable both of sensing cellular stress caused by microbial infection and of responding to pathogens directly.

Curcumin utilizes the anti-inflammatory response pathway to protect the intestine against bacterial invasion.

BACKGROUND/OBJECTIVES: Curcumin, a major component of the Curcuma species, contains antioxidant and anti-inflammatory properties. Although it was found to induce apoptosis in cancer cells, the functional role of curcumin as well as its molecular mechanism in anti-inflammatory response, particularly in intestinal cells, has been less investigated. The intestine epithelial barrier is the first barrier and the most important location for the substrate coming from the lumen of the gut. SUBJECTS/METHODS: We administered curcumin treatment in the human intestinal epithelial cell lines, T84 and Caco-2. We examined endoplasmic reticulum (ER) stress response by thapsigargin, qPCR of XBP1 and BiP, electrophysiology by wild-type cholera toxin in the cells. RESULTS: In this study, we showed that curcumin treatment reduces ER stress and thereby decreases inflammatory response in human intestinal epithelial cells. In addition, curcumin confers protection without damaging the membrane tight junction or actin skeleton change in intestine epithelial cells. Therefore, curcumin treatment protects the gut from bacterial invasion via reduction of ER stress and anti-inflammatory response in intestinal epithelial cells. CONCLUSIONS: Taken together, our data demonstrate the important role of curcumin in protecting the intestine by modulating ER stress and inflammatory response post intoxication.

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2.

Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro- translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endo-somal sorting of the toxin-GM1 complex.

Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum.

Cholera toxin (CT), an AB(5)-subunit toxin, enters host cells by binding the ganglioside GM1 at the plasma membrane (PM) and travels retrograde through the trans-Golgi Network into the endoplasmic reticulum (ER). In the ER, a portion of CT, the enzymatic A1-chain, is unfolded by protein disulfide isomerase and retro-translocated to the cytosol by hijacking components of the ER associated degradation pathway for misfolded proteins. After crossing the ER membrane, the A1-chain refolds in the cytosol and escapes rapid degradation by the proteasome to induce disease by ADP-ribosylating the large G-protein Gs and activating adenylyl cyclase. Here, we review the mechanisms of toxin trafficking by GM1 and retro-translocation of the A1-chain to the cytosol.

The inactive 44-kDa processed form of membrane type 1 matrix metalloproteinase (MT1-MMP) enhances proteolytic activity via regulation of endocytosis of active MT1-MMP.

Membrane type 1 (MT1) matrix metalloproteinase (MMP-14) is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recombinant 44-MT1 (Gly(285)-Val(582)) in HT1080 fibrosarcoma cells results in enhanced pro-MMP-2 activation, proliferation within a three-dimensional collagen I matrix, and tumor growth and lung metastasis in mice. Stimulation of pro-MMP-2 activation and growth in collagen I was also observed in other cell systems. Expression of 44-MT1 in HT1080 cells is associated with a delay in the rate of active MT1-MMP endocytosis resulting in higher levels of active enzyme at the cell surface. Consistently, deletion of the cytosolic domain obliterates the stimulatory effects of 44-MT1 on MT1-MMP activity. In contrast, deletion of the hinge turns the 44-MT1 form into a negative regulator of enzyme function in vitro and in vivo, suggesting a key role for the hinge region in the functional relationship between active and processed MT1-MMP. Together, these results suggest a novel role for the 44-kDa form of MT1-MMP generated during autocatalytic processing in maintaining the pool of active enzyme at the cell surface.

MT4-(MMP17) and MT6-MMP (MMP25), A unique set of membrane-anchored matrix metalloproteinases: properties and expression in cancer.

The process of cancer progression involves the action of multiple proteolytic systems, among which the family of matrix metalloproteinases (MMPs) play a pivotal role. The MMPs evolved to accomplish their proteolytic tasks in multiple cellular and tissue microenvironments including lipid rafts by incorporation and deletions of specific structural domains. The membrane type-MMPs (MT-MMPs) incorporated membrane anchoring domains that display these proteases at the cell surface, and thus they are optimal pericellular proteolytic machines. Two members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP), are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor, which confers these enzymes a unique set of regulatory and functional mechanisms that separates them from the rest of the MMP family. Discovered almost a decade ago, the body of work on GPI-MT-MMPs today is still surprisingly limited when compared to other MT-MMPs. However, new evidence shows that the GPI-MT-MMPs are highly expressed in human cancer, where they are associated with progression. Accumulating biochemical and functional evidence also highlights their distinct properties. In this review, we summarize the structural, biochemical, and biological properties of GPI-MT-MMPs and present an overview of their expression and role in cancer. We further discuss the potential implications of GPI-anchoring for enzyme function. Finally, we comment on the new scientific challenges that lie ahead to better understand the function and role in cancer of these intriguing but yet unique MMPs.

Calcium drives fusion of SNARE-apposed bilayers.

N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) has been proposed to play a critical role in the membrane fusion process. The SNARE complex was suggested to be the minimal fusion machinery. However, there is mounting evidence for a major role of calcium in membrane fusion. Hence, the role of calcium in SNARE-induced membrane fusion was the focus of this study. It revealed that recombinant v-SNARE and t-SNARE, reconstituted into separate liposomes, interact to bring lipid vesicles into close proximity, enabling calcium to drive fusion of opposing bilayers. Exposure to calcium triggered vesicle fusion at both, high potency and efficacy. The half-time for calcium-induced fusion of SNARE-reconstituted vesicles was determined to be approximately 10 s, which is two orders of magnitude faster than in its absence. Calcium acts downstream of SNAREs, since the presence of SNAREs in bilayers increases the potency of calcium-induced vesicle fusion, without significantly influencing its efficacy. Hence, this study suggests that in the physiological state in cells, both SNAREs and calcium operate as the minimal fusion machinery.

SNAREs in opposing bilayers interact in a circular array to form conducting pores.

The process of fusion at the nerve terminal is mediated via a specialized set of proteins in the synaptic vesicles and the presynaptic membrane. Three soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion. The structure and arrangement of these SNAREs associated with lipid bilayers were examined using atomic force microscopy. A bilayer electrophysiological setup allowed for measurements of membrane conductance and capacitance. Here we demonstrate that the interaction of these proteins to form a fusion pore is dependent on the presence of t-SNAREs and v-SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane. However when t-SNARE vesicles were added to a v-SNARE membrane, SNAREs assembles in a ring pattern and a stepwise increase in capacitance, and increase in conductance were observed. Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion.