Co-administration of chicken IL-7 or NK-lysin peptide 2 enhances the efficacy of Eimeria elongation factor-1α vaccination against Eimeria maxima infection in broiler chickens.

This study was conducted to develop a recombinant Eimeria elongation factor-1α (EF-1α)-vaccination strategy against Eimeria maxima (E. maxima) infection by co-administering with chicken IL-7 (chIL-7) or chicken NK-lysin peptide 2 (cNK-2) in commercial broiler chickens. Chickens were divided into the following 5 groups: control (CON, no Eimeria infection), nonimmunized control (NC, PBS plus Montanide ISA 78 VG), Vaccination 1 (VAC1, 100 µg of recombinant EF-1α plus Montanide ISA 78 VG), Vaccination 2 (VAC2, VAC1 plus 1 µg of chIL-7), and Vaccination 3 (VAC3, VAC2 plus 5 µg of cNK-2 peptide). The first immunization except the cNK-2 injection was performed intramuscularly on day 4, and the secondary immunization was given with the same concentration of components as the primary immunization 1 wk later. All chickens except the CON group were orally inoculated with freshly prepared E. maxima (1.0 × 104 oocysts per chicken) oocysts on Day 19. The results of the in vivo vaccination trial showed that chickens of all groups immunized with recombinant EF-1α antigen (VAC1, VAC2, and VAC3) showed higher serum antibody levels to EF-1α, and co-injection with chIL-7 further increased the serum IL-7 level in the VAC2 and VAC3 groups. Chickens in the VAC2 group showed significantly (P < 0.01) higher body weight gains at 6 and 9 d post-E. maxima challenge infection (dpi) with reduced gut lesions in the jejunum at 6 dpi. The VAC3 group showed reduced fecal oocyst shedding compared to the nonimmunized and infected chickens (NC). At 4 dpi, E. maxima infection significantly (P < 0.05) up-regulated the expression levels of proinflammatory cytokines (IL-ß and IL-17F) and type Ι cytokines (IFN-γ and IL-10) in the jejunum (NC), but the expression of these cytokines was significantly (P < 0.05) down-regulated in the VAC1, VAC2, and VAC3 groups. Furthermore, E. maxima challenge infection significantly (P < 0.05) down-regulated the expressions of jejunal tight junction (TJ) proteins (Jam2 and Occludin) at 4 dpi, but their expression was up-regulated in the VAC2 and VAC3 groups. Collectively, these results show the protective effects of the EF-1α recombinant vaccine, which can be further enhanced by co-injection with chIL-7 or cNK-2 peptide against E. maxima infection.

Bovine NK-lysin peptides exert potent antimicrobial activity against multidrug-resistant Salmonella outbreak isolates.

Multidrug-resistant (MDR) Salmonella is a threat to public health. Non-antibiotic therapies could serve as important countermeasures to control MDR Salmonella outbreaks. In this study, antimicrobial activity of cationic α-helical bovine NK-lysin-derived antimicrobial peptides was evaluated against MDR Salmonella outbreak isolates. NK2A and NK2B strongly inhibited MDR Salmonella growth while NK1 and NK2C showed minimum-to-no growth inhibition. Scrambled-NK2A, which is devoid of α-helicity but has the same net positive charge as NK2A, also failed to inhibit bacterial growth. Incubation of negatively charged MDR Salmonella with NK2A showed increased Zeta potential, indicating bacterial-peptide electrostatic attraction. Confocal and transmission electron microscopy studies revealed NK2A-mediated damage to MDR Salmonella membranes. LPS inhibited NK2A-mediated growth suppression in a dose-dependent response, suggesting irreversible NK2A-LPS binding. LPS-NK2A binding and bacterial membrane disruption was also confirmed via electron microscopy using gold nanoparticle-NK2A conjugates. Finally, NK2A-loaded polyanhydride nanoparticles showed sustained peptide delivery and anti-bacterial activity. Together, these findings indicate that NK2A α-helicity and positive charge are prerequisites for antimicrobial activity and that MDR Salmonella killing is mediated by direct interaction of NK2A with LPS and the inner membrane, leading to bacterial membrane permeabilization. With further optimization using nano-carriers, NK2A has the potential to become a potent anti-MDR Salmonella agent.

Cooperative inhibition of SNARE-mediated vesicle fusion by α-synuclein monomers and oligomers.

The primary hallmark of Parkinson's disease (PD) is the generation of Lewy bodies of which major component is α-synuclein (α-Syn). Because of increasing evidence of the fundamental roles of α-Syn oligomers in disease progression, α-Syn oligomers have become potential targets for therapeutic interventions for PD. One of the potential toxicities of α-Syn oligomers is their inhibition of SNARE-mediated vesicle fusion by specifically interacting with vesicle-SNARE protein synaptobrevin-2 (Syb2), which hampers dopamine release. Here, we show that α-Syn monomers and oligomers cooperatively inhibit neuronal SNARE-mediated vesicle fusion. α-Syn monomers at submicromolar concentrations increase the fusion inhibition by α-Syn oligomers. This cooperative pathological effect stems from the synergically enhanced vesicle clustering. Based on this cooperative inhibition mechanism, we reverse the fusion inhibitory effect of α-Syn oligomers using small peptide fragments. The small peptide fragments, derivatives of α-Syn, block the binding of α-Syn oligomers to Syb2 and dramatically reverse the toxicity of α-Syn oligomers in vesicle fusion. Our findings demonstrate a new strategy for therapeutic intervention in PD and related diseases based on this specific interaction of α-Syn.

Phospholamban and sarcolipin prevent thermal inactivation of sarco(endo)plasmic reticulum Ca2+-ATPases.

Na+-K+-ATPase from mice lacking the γ subunit exhibits decreased thermal stability. Phospholamban (PLN) and sarcolipin (SLN) are small homologous proteins that regulate sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs) with properties similar to the γ subunit, through physical interactions with SERCAs. Here, we tested the hypothesis that PLN and SLN may protect against thermal inactivation of SERCAs. HEK-293 cells were co-transfected with different combinations of cDNAs encoding SERCA2a, PLN, a PLN mutant (N34A) that cannot bind to SERCA2a, and SLN. One-half of the cells were heat stressed at 40°C for 1 h (HS), and one-half were maintained at 37°C (CTL) before harvesting the cells and isolating microsomes. Compared with CTL, maximal SERCA activity was reduced by 25-35% following HS in cells that expressed either SERCA2a alone or SERCA2a and mutant PLN (N34A) whereas no change in maximal SERCA2a activity was observed in cells that co-expressed SERCA2a and either PLN or SLN following HS. Increases in SERCA2a carbonyl group content and nitrotyrosine levels that were detected following HS in cells that expressed SERCA2a alone were prevented in cells co-expressing SERCA2a with PLN or SLN, whereas co-expression of SERCA2a with mutant PLN (N34A) only prevented carbonyl group formation. In other experiments using knock-out mice, we found that thermal inactivation of SERCA was increased in cardiac left ventricle samples from Pln-null mice and in diaphragm samples from Sln-null mice, compared with WT littermates. Our results show that both PLN and SLN form a protective interaction with SERCA pumps during HS, preventing nitrosylation and oxidation of SERCA and thus preserving its maximal activity.

Fortetropin inhibits disuse muscle atrophy in dogs after tibial plateau leveling osteotomy.

OBJECTIVE: To determine if a commercial myostatin reducer (Fortetropin®) would inhibit disuse muscle atrophy in dogs after a tibial plateau leveling osteotomy. DESIGN: A prospective randomized, double-blinded, placebo-controlled clinical trial. ANIMALS: One hundred client-owned dogs presenting for surgical correction of cranial cruciate ligament rupture by tibial plateau leveling osteotomy. PROCEDURES: Patients were randomly assigned into the Fortetropin® or placebo group and clients were instructed to add the assigned supplement to the dog's normal diet once daily for twelve weeks. Enrolled patients had ultrasound measurements of muscle thickness, tape measure measurements of thigh circumference, serum myostatin level assays, and static stance analysis evaluated at weeks 0, 8, and 12. RESULTS: From week 0 to week 8, there was no change for thigh circumference in the Fortetropin® group for the affected limb (-0.54cm, P = 0.31), but a significant decrease in thigh circumference for the placebo group (-1.21cm, P = 0.03). There was no significant change in serum myostatin levels of dogs in the Fortetropin® group at any time point (P>0.05), while there was a significant rise of serum myostatin levels of dogs in placebo group during the period of forced exercise restriction (week 0 to week 8; +2,892 pg/ml, P = 0.02). The percent of body weight supported by the affected limb increased in dogs treated with Fortetropin® (+7.0%, P<0.01) and the placebo group (+4.9%, P<0.01) at the end of the period of forced exercise restriction. The difference in weight bearing between the Fortetropin® and placebo groups was not statistically significant (P = 0.10). CONCLUSION: Dogs receiving Fortetropin® had a similar increase in stance force on the affected limb, no significant increase in serum myostatin levels, and no significant reduction in thigh circumference at the end of the period of forced exercise restriction compared to the placebo. These findings support the feeding of Fortetropin® to prevent disuse muscle atrophy in canine patients undergoing a tibial plateau leveling osteotomy.

Cultivation at high osmotic pressure confers ubiquinone 8-independent protection of respiration on Escherichia coli.

Ubiquinone 8 (coenzyme Q8 or Q8) mediates electron transfer within the aerobic respiratory chain, mitigates oxidative stress, and contributes to gene expression in Escherichia coli In addition, Q8 was proposed to confer bacterial osmotolerance by accumulating during growth at high osmotic pressure and altering membrane stability. The osmolyte trehalose and membrane lipid cardiolipin accumulate in E. coli cells cultivated at high osmotic pressure. Here, Q8 deficiency impaired E. coli growth at low osmotic pressure and rendered growth osmotically sensitive. The Q8 deficiency impeded cellular O2 uptake and also inhibited the activities of two proton symporters, the osmosensing transporter ProP and the lactose transporter LacY. Q8 supplementation decreased membrane fluidity in liposomes, but did not affect ProP activity in proteoliposomes, which is respiration-independent. Liposomes and proteoliposomes prepared with E. coli lipids were used for these experiments. Similar oxygen uptake rates were observed for bacteria cultivated at low and high osmotic pressures. In contrast, respiration was dramatically inhibited when bacteria grown at the same low osmotic pressure were shifted to high osmotic pressure. Thus, respiration was restored during prolonged growth of E. coli at high osmotic pressure. Of note, bacteria cultivated at low and high osmotic pressures had similar Q8 concentrations. The protection of respiration was neither diminished by cardiolipin deficiency nor conferred by trehalose overproduction during growth at low osmotic pressure, but rather might be achieved by Q8-independent respiratory chain remodeling. We conclude that osmotolerance is conferred through Q8-independent protection of respiration, not by altering physical properties of the membrane.

Proteoliposome-based model for screening inhibitors targeting histidine kinase AgrC.

AgrC, as an integral membrane receptor protein with histidine kinase activity, is an important component of the agr quorum-sensing system of Staphylococcus aureus. AgrC acts as a sensor for the recognition of environmental signals and transduction of the signals into the cytoplasm. Therefore, AgrC is considered to be a compelling target for the development of novel quorum-sensing inhibitors. Here, we constructed a proteoliposome-based model for screening inhibitors targeting AgrC by incorporating AgrC into liposomes. We demonstrated that the dissolution state of the liposome was a critical factor in the reconstruction of the AgrC proteoliposome, in which AgrC maintained similar orientation and function as those in natural biological membranes. Two monomers, namely, rhein and aloeemodin, were successfully screened out as inhibitors targeting AgrC by the proteoliposome-based model from 14 traditional Chinese medicine monomers. The inhibitory effects of these compounds on the growth of suspended bacteria was dose dependent, and subinhibitory concentrations of these compounds significantly reduced the expression of three virulence factors (hla, clfA, and clpP), that are regulated by the agr system. The results preliminarily indicated that rhein and aloeemodin can inhibit the agr signaling pathway and also indirectly confirmed the feasibility and effectiveness of the AgrC proteoliposome as a drug screening model.

Phototheranostics: Active Targeting of Orthotopic Glioma Using Biomimetic Proteolipid Nanoparticles.

Advances in phototheranostics revolutionized glioma intraoperative fluorescence imaging and phototherapy. However, the lack of desired active targeting agents for crossing the blood-brain barrier (BBB) significantly compromises the theranostic efficacy. In this study, biomimetic proteolipid nanoparticles (NPs) with U.S. Food and Drug Administration (FDA)-approved indocyanine green (ICG) were constructed to allow fluorescence imaging, tumor margin detection, and phototherapy of orthotopic glioma in mice. By embedding glioma cell membrane proteins into NPs, the obtained biomimetic ICG-loaded liposome (BLIPO-ICG) NPs could cross BBB and actively reach glioma at the early stage thanks to their specific binding to glioma cells due to their excellent homotypic targeting and immune escaping characteristics. High accumulation in the brain tumor with a signal to background ratio of 8.4 was obtained at 12 h post-injection. At this time point, the glioma and its margin were clearly visualized by near-infrared fluorescence imaging. Under the imaging guidance, the glioma tissue could be completely removed as a proof of concept. In addition, after NIR laser irradiation (1 W/cm2, 5 min), the photothermal effect exerted by BLIPO-ICG NPs efficiently suppressed glioma cell proliferation with a 94.2% tumor growth inhibition. No photothermal damages of normal brain tissue and treatment-induced side effects were observed. These results suggest that the biomimetic proteolipid NP is a promising phototheranostic nanoplatform for brain-tumor-specific imaging and therapy.

Transgenic expression of plant-specific insert of potato aspartic proteases (StAP-PSI) confers enhanced resistance to Botrytis cinerea in Arabidopsis thaliana.

The plant-specific insert of Solanum tuberosum aspartic proteases (StAP-PSI) has high structural similarity with NK-lysin and granulysin, two saposin-like proteins (SAPLIPs) with antimicrobial activity. Recombinant StAP-PSI and some SAPLIPs show antimicrobial activity against pathogens that affect human and plants. In this work, we transformed Arabidopsis thaliana plants with StAP-PSI encoding sequence with its corresponding signal peptide under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Results obtained show that StAP-PSI significantly enhances Arabidopsis resistance against Botrytis cinerea infection. StAP-PSI is secreted into the leaf apoplast and acts directly against pathogens; thereby complementing plant innate immune responses. Data obtained from real-time PCR assays show that the constitutive expression of StAP-PSI induces the expression of genes that regulate jasmonic acid signalling pathway, such as PDF1.2, in response to infection due to necrotrophic pathogens. On the other hand, according to the data described for other antimicrobial peptides, the presence of the StAP-PSI protein in the apoplast of A. thaliana leaves is responsible for the expression of salicylic acid-associated genes, such as PR-1, irrespective of infection with B. cinerea. These results indicate that the increased resistance demonstrated by A. thaliana plants that constitutively express StAP-PSI owing to B. cinerea infection compared to the wild-type plants is a consequence of two factors, i.e., the antifungal activity of StAP-PSI and the overexpression of A. thaliana defense genes induced by the constitutive expression of StAP-PSI. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the use of pesticides. We suggest that the use of this protein would help in minimizing the ecological and health risks that arise from the spreading of resistance of agriculturally important pathogens.

Antibacterial poly(ethylene oxide) electrospun nanofibers containing cinnamon essential oil/beta-cyclodextrin proteoliposomes.

A novel antibacterial packaging material was engineered by incorporating cinnamon essential oil/ß-cyclodextrin (CEO/ß-CD) proteoliposomes into poly(ethylene oxide) (PEO) nanofibers by electrospinning technique. Herein, PEO was a stabilizing polymer and used as electrospinning polymeric matrix for the fabrication of CEO/ß-CD proteoliposomes nanofibers. The nanoliposomes were inlaid with protein are defined as proteoliposomes. Taking advantage of bacterial protease secreted from Bacillus cereus (B. cereus), the controlled release of CEO from proteoliposomes was achieved via proteolysis of protein in proteoliposomes. The CEO/ß-CD inclusion complex was prepared by the aqueous solution method and characterized by Raman and FTIR spectroscopy. After the treatment of CEO/ß-CD proteoliposomes nanofibers packaging, the satisfactory antibacterial efficiency against B. cereus on beef was realized without any impact on sensory quality of beef. This study demonstrated that the CEO/ß-CD proteoliposomes nanofibers can significantly extend the shelf life of beef and have potential application in active food packaging.

Functional reconstitution of cell-free synthesized purified Kv channels.

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active Kv1.1 and Kv1.3 channels and their efficient insertion into liposomes. Two complementary methods for the determination of the electrical activity of the proteoliposome-embedded channels were compared using Kv1.1 as a model system: (1) single channel recordings in droplet interface bilayers (DIB) and (2) measurement of the membrane voltage potential generated by a potassium ion diffusion potential using the voltage-sensitive fluorescent dye oxonol VI. Single channel recordings in DIBs proved unreliable because of the non-reproducible fusion of proteoliposomes with an artificial membrane. Therefore, the use of the optical indicator oxonol VI was adapted for 96 well microtiter plates using the ionophore valinomycin as a positive control. The activity of Kv1.1 and Kv1.3 channels was then monitored in the absence and presence of different venom toxins, demonstrating that fluorescent dyes can be used very efficiently when screening small molecules for their channel blocking activity.

The Arabidopsis Golgi-localized GDP-L-fucose transporter is required for plant development.

Nucleotide sugar transport across Golgi membranes is essential for the luminal biosynthesis of glycan structures. Here we identify GDP-fucose transporter 1 (GFT1), an Arabidopsis nucleotide sugar transporter that translocates GDP-L-fucose into the Golgi lumen. Using proteo-liposome-based transport assays, we show that GFT preferentially transports GDP-L-fucose over other nucleotide sugars in vitro, while GFT1-silenced plants are almost devoid of L-fucose in cell wall-derived xyloglucan and rhamnogalacturonan II. Furthermore, these lines display reduced L-fucose content in N-glycan structures accompanied by severe developmental growth defects. We conclude that GFT1 is the major nucleotide sugar transporter for import of GDP-L-fucose into the Golgi and is required for proper plant growth and development.

The Effects of Fortetropin Supplementation on Body Composition, Strength, and Power in Humans and Mechanism of Action in a Rodent Model.

OBJECTIVE: The purpose of this study was to investigate the effects of Fortetropin on skeletal muscle growth and strength in resistance-trained individuals and to investigate the anabolic and catabolic signaling effects using human and rodent models. METHODS: In the rodent model, male Wistar rats (250 g) were gavage fed with either 1.2 ml of tap water control (CTL) or 0.26 g Fortetropin for 8 days. Then rats participated in a unilateral plantarflexion exercise bout. Nonexercised and exercised limbs were harvested at 180 minutes following and analyzed for gene and protein expression relative to mammalian target of rapamycin (mTOR) and ubiquitin signaling. For the human model, 45 (of whom 37 completed the study), resistance-trained college-aged males were divided equally into 3 groups receiving a placebo macronutrient matched control, 6.6 or 19.8 g of Fortetropin supplementation during 12 weeks of resistance training. Lean mass, muscle thickness, and lower and upper body strength were measured before and after 12 weeks of training. RESULTS: The human study results indicated a Group × Time effect (p ≤ 0.05) for lean mass in which the 6.6 g (+1.7 kg) and 19.8 g (+1.68 kg) but not placebo (+0.6 kg) groups increased lean mass. Similarly, there was a Group × Time effect for muscle thickness (p ≤ 0.05), which increased in the experimental groups only. All groups increased equally in bench press and leg press strength. In the rodent model, a main effect for exercise (p ≤ 0.05) in which the control plus exercise but not Fortetropin plus exercise increased both ubiquitin monomer protein expression and polyubiquitination. mTOR signaling was elevated to a greater extent in the Fortetropin exercising conditions as indicated by greater phosphorylation status of 4EBP1, rp6, and p70S6K for both exercising conditions. CONCLUSIONS: Fortetropin supplementation increases lean body mass (LBM) and decreases markers of protein breakdown while simultaneously increasing mTOR signaling.

Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins.

This study compared detergent-solubilised (soluble) and lipid-reconstituted (proteoliposome) protein to establish a high-throughput method for identifying membrane protein inhibitors. We identified inhibitors of the membrane-bound type II NADH dehydrogenase with lower lipophilicity and better potency, suggesting proteoliposome systems may be advantageous over detergent-solubilised systems for respiratory membrane proteins.

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis.

Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP-l-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP-l-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP-l-Rha/UDP-d-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP-l-Rha and UDP-d-Gal for matrix polysaccharide biosynthesis.

Measurement of lecithin-cholesterol acyltransferase activity with the use of a Peptide-proteoliposome substrate.

Lecithin-cholesterol acyltransferase (LCAT) is the major enzyme responsible for the esterification of free cholesterol on plasma lipoproteins, which is a key step in the reverse cholesterol transport pathway. The measurement of plasma LCAT activity not only is important in the diagnosis of patients with genetic or acquired LCAT deficiency but is also valuable in calculating cardiovascular risk, as well as in research studies of lipoprotein metabolism. In this chapter, we describe a convenient LCAT assay based on the use of an apoA-I mimetic peptide. The proteoliposome substrate used in this assay for LCAT is easily made with the peptide and can be stored by deep freezing without significant loss of activity.

Pilot scale production of the vaccine adjuvant Proteoliposome derived Cochleates (AFCo1) from Neisseria meningitidis serogroup B.

The use of new adjuvants in vaccine formulations is a subject of current research. Only few parenteral adjuvants have been licensed. We have developed a mucosal and parenteral adjuvant known as AFCo1 (Adjuvant Finlay Cochleate 1, derived from proteoliposomes of N. meningitidis B) using a dialysis procedure to produce them on lab scale. The immunogenicity of the AFCo1 produced by dialysis has been already evaluated, but it was necessary to demonstrate the feasibility of a larger-scale manufacturing process. Therefore, we used a crossflow diafiltration system (CFS) that allows easy scale up to obtain large batches in an aseptic environment. The aim of this work was to produce AFCo1 on pilot scale, while conserving the adjuvant properties. The proteoliposomes (raw material) were resuspended in a buffer containing sodium deoxycholate and were transformed into AFCo1 under the action of a calcium forming buffer. The detergent was removed from the protein solution by diafiltration to a constant volume. In this CFS, we used a hollow fiber cartridge from Amicon (polysulfona cartridge of 10 kDa porosity, 1mm channel diameter of fiber and 0.45 m² area of filtration), allowing production of a batch of up to 20 L. AFCo1 were successfully produced by tangential filtration to pilot scale. The batch passed preliminary stability tests. Nasal immunization of BALB/c mice, induced specific saliva IgA and serum IgG. The induction of Th1 responses were demonstrated by the induction of IgG2a, IFNγ and not IL-5. The adjuvant action over Neisseria (self) antigens and with co-administered (heterologous) antigens such as ovalbumin and a synthetic peptide from haemolytic Streptococcus B was also demonstrated.

Testosterone regulates cardiac contractile activation by modulating SERCA but not NCX activity.

Alterations in intracellular Ca(2+) transients of cardiomyocytes in orchidectomized (ORX) rats could be a cause of cardiac dysfunction in the hypogonadal condition. To investigate the role of male sex hormones in intracellular Ca(2+) homeostasis during relaxation, Ca(2+)-handling activities by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and the Na(+)/Ca(2+) exchanger (NCX) were evaluated in the ventricular muscle of 10-wk-old ORX rats with and without testosterone supplementation (2.5 mg/kg testosterone propionate, 2 times/wk). ORX induced a 50% decrease in contraction force accompanied by a prolonged time to achieve 50% relaxation (T(50)) in isolated intact ventricular trabeculae, which was partially corrected by testosterone administration. Maximum active tension was also suppressed in ORX rats without changes in myofilament Ca(2+) sensitivity and passive stiffness of the heart. Using a sarcoplasmic reticulum-enriched membrane preparation, the maximum thapsigargin-sensitive SERCA activity of the ORX rat was 27% lower with an increased Ca(2+) sensitivity, which was prevented by testosterone treatment. However, neither changes in SERCA content nor its modulating components, sarcolipin and heat shock protein 20, were detected in the ORX rat, but there was a significant decrease in the phosphorylated Thr(17) form of phospholamban. Despite a lower level of NCX protein in the heart of ORX rats, prolonged T(50) disappeared after an incubation with thapsigargin (10 µM), implying a lack of effect of male sex hormone deficiency on NCX function. These findings indicate that male sex hormones can regulate cardiac relaxation by acting mainly through SERCA. However, a detailed mechanism of SERCA modulation under male sex hormone deficiency status remains to be explored.

Cochleates derived from Vibrio cholerae O1 proteoliposomes: the impact of structure transformation on mucosal immunisation.

Cochleates are phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. Proteoliposomes from bacteria may also be used as a source of negatively charged components, to induce calcium-cochleate formation. In this study, proteoliposomes from V. cholerae O1 (PLc) (sized 160.7±1.6 nm) were transformed into larger (16.3±4.6 µm) cochleate-like structures (named Adjuvant Finlay Cochleate 2, AFCo2) and evaluated by electron microscopy (EM). Measurements from transmission EM (TEM) showed the structures had a similar size to that previously reported using light microscopy, while observations from scanning electron microscopy (SEM) indicated that the structures were multilayered and of cochleate-like formation. The edges of the AFCo2 structures appeared to have spaces that allowed penetration of negative stain or Ovalbumin labeled with Texas Red (OVA-TR) observed by epi-fluorescence microscopy. In addition, freeze fracture electron microscopy confirmed that the AFCo2 structures consisted of multiple overlapping layers, which corresponds to previous descriptions of cochleates. TEM also showed that small vesicles co-existed with the larger cochleate structures, and in vitro treatment with a calcium chelator caused the AFCo2 to unfold and reassemble into small proteoliposome-like structures. Using OVA as a model antigen, we demonstrated the potential loading capacity of a heterologous antigen and in vivo studies showed that with simple admixing and administration via intragastric and intranasal routes AFCo2 provided enhanced adjuvant properties compared with PLc.

Flip-flop of phospholipids in proteoliposomes reconstituted from detergent extract of chloroplast membranes: kinetics and phospholipid specificity.

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6 ± 1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents.