Effects of Na+, H+, and Cl− on alanine transport by lobster hepatopancreatic brush border membrane vesicles

Summary Epithelial brush border membrane vesicles (BBMV) of lobster hepatopancreas were formed by a magnesium precipitation technique previously described (Ahearn et al. 1985).³H-l-alanine transport by these vesicles was sodium and potassium insensitive, in contrast to a strong Na-dependency exhibited by³H-d-glucose transport. Initial alanine entry rates (15 s uptake) were stimulated and transient alanine uptake overshoots were observed when external pH was acidic (e. g. pH 4.0, 5.0 or 6.0) and a Cl gradient was imposed across the vesicular wall; at pH_o=7.4 alanine uptake was reduced in rate and hyperbolic in character. Alanine uptake from an acidic extravesicular environment in the absence of Cl responded to a transmembrane electrical potential difference created by an outwardly-directed, valinomycin-induced, potassium diffusion potential, suggesting that the alanine molecule alone carried sufficient charge under these conditions to respond to the electrical gradient. External 5.0 mMl-lysine andl-serine similarly inhibited the influx and overshoot properties of 0.05 mM³H-l-alanine uptake, whereas 5.0 mMl-leucine had virtually no effect. Trans-stimulation of alanine initial uptake rates and an enhancement of alanine accumulation against a concentration gradient were observed by vesicles preloaded with 1 mMl-lysine, but not by vesicles lacking amino acids or those containing 1 mMl-leucine orl-serine.³H-l-alanine influx from acidic external environments in the presence of a Cl gradient occurred by a combination of carrier-mediated transfer and apparent diffusion. Decreasing pH_o from 6.0 to 4.0 elevated alanineK _t from 0.55 to 2.64 mM, while alanineJ _M increased from 55 to 550 pmol/mg protein· 15 s. Apparent diffusional permeability of the membranes to alanine under these conditions increased slightly. These results suggest, but do not conclusively prove, that alanine transport across BBMV of lobster hepatopancreas may occur by way of a classical y+ transprot protein at acidic pH. The extent of this transport is determined by the magnitude of the transmembrane chloride gradient which serves as a powerful driving force for cationic amino acids in this tissue.     

Functional roles of Na+ and H+ in SO 4 2− transport by rabbit ileal brush border membrane vesicles

Summary Sulphate uptake by rabbit ileal brush border membrane vesicles was stimulated by a transmembrane sodium gradient ([Na⁺] _o >[Na⁺] _i ), but not by a similar potassium gradient.³⁵SO ₄ ^2– influx (J _oi ^SO4 ) from outside (o) to inside (i) these vesicles was a hyperbolic function of [SO ₄ ^2– ] _o and the affinity constant for anion transport was strongly influenced by [Na⁺] _o (100mm Na⁺,K _t ^SO4 =0.52mm SO ₄ ^2– ; 10mm Na⁺,K _t ^SO4 =4.32mm SO ₄ ^2– ).J _t ^SO4 was a sigmoidal function of [Na⁺] _o at pH 7.4 for both low (0.2m) and high (4.0mm) [SO ₄ ^2– ] _o . The Na⁺-dependency ofJ _t ^SO4 was examined at pH 6.0, 7.4, and 8.0 (same pH inside and outside). At pH 6.0 and 7.4 sigmoidal Na⁺-dependentJ _t ^SO4 exhibited nonlinear Eadie-Hofstee plots indicative of a transport mechanism capable of binding a variable number of sodium ions over the [Na⁺] _o range used. Hill plots of anion transport under these conditions displayed slopes near unity at low [Na⁺] _o and slopes approximating 2.0 at higher cation concentrations. At pH 8.0, Na⁺-dependentJ _t ^SO4 was hyperbolic and showed linear Eadie-Hofstee and Hill plots, the latter with a single slope near 1.0. When a H⁺ gradient was imposed across the vesicle wall (pH _i =8.0, pH _o =6.0), Na⁺-dependentJ _t ^SO4 was hyperbolic and significantly increased at each [Na⁺] _o over values observed using bilateral pH 8.0. In contrast, a H⁺ gradient oriented in the opposite direction (pH _i =6.0, pH _o =8.0) led to Na⁺-dependentJ _t ^SO4 that was sigmoidal and significantly lower at each [Na⁺] _o than values found using bilateral pH 6.0. Electrogenicity ofJ _t ^SO4 at pH 8.0 for both high and low [Na⁺] _o was demonstrated by using a valinomycin-induced transmembrane electrical potential difference. At pH 6.0, electrogenicJ _t ^SO4 occurred only at low [Na⁺] _o (5mm); anion transfer was electroneutral at 50mm Na⁺. A model is proposed for proton regulation of sodium sulphate cotransport where flux stoichiometry is controlled by [H⁺] _i and sodium binding affinity is modified by [H⁺] _o . Preliminary experiments with rabbit proximal tubular brush border membrane vesicles disclosed similarJ _t ^SO4 kinetic properties and a common transport mechanism may occur in both tissues.     

α-Glutamyl-β-naphthylamide hydrolase of rabbit small intestine: Localization in the brush border and separation from other brush border peptidases

About 70% of the total mucosal enzymatic activity hydrolyzing α-l-glutamyl-β-naphthylamide in the rabbit small intestine is present in the brush border; the specific activity in this subcellular fraction is 7 times higher than that of the homogenate. Similar results are obtained for l-leucyl-β-naphthylamide hydrolase.The enzyme activity is efficiently solubilized by papain digestion and is clearly separated from l-leucyl-β-naphthylamide hydrolase by chromatography on concanavalin A-Sepharose. It probably represents a digestive peptidase, different from the other known peptide hydrolases of the digestive surface of the small intestine.     

Mechanisms of Cl− uptake through brush border membranes isolated from the posterior intestine of the freshwater trout,Oncorhynchus mykiss,R.

Summary This paper presents a study of the mechanisms of Cl^– transport through the brush border membranes of the posterior part of the intestine in the freshwater trout, Oncorhynchus mykiss. The mechanisms for Cl^– transport in the posterior intestine are distinct from those in the middle intestine; an inwardly directed pH gradient stimulates Cl^– uptake by bursh border membrane vesicles, indicating a Cl^–/OH^– exchange. A pH-regulated Cl^– conductance is present, which is not activated at normal intracellular pH. Cl^– uptake is stimulated by an outwardly directed HCO ₃ ^– gradient revealing the presence of a Cl^–/HCO ₃ ^– exchange but, conversely, Cl^– is not exchanged against SO ₄ ^2- . In addition, carbonic anhydrase activities have been detected in both the intracellular and extracellular leaflets of the bursh border membranes which favour the establishment of a bicarbonate gradient. A model of Cl^– transport mechanisms through the brush-border membranes of the posterior intestine of the freshwater trout is proposed.Abbreviations BBM brush border membrane - CA carbonic anhydrase - EGTA ethylene-bis(oxyethylenenitrilo)tetra-acetic acid - FW fresh water - Hepes N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulphonic acid - Mes 2-(N-morpholino)ethane sulphonic acid - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid - TEA triethanolamine - TMA tetramethylammonium - TRIS tris(hydroxymethyl)aminomethane     

Binding of Cry δ-endotoxins to brush border membrane vesicles of Helicoverpa armigera and Helicoverpa punctigera （Lepidoptera： Noctuidae）

The relatively low susceptibility ofHelicoverpa armigera to CrylAc, its history of resistance to chemical insecticides and the seasonal decline in expression of CrylAc in transgenic cotton necessitated the development of cotton expressing two insecticidal proteins to provide sustainable control of this multinational pest. To manage the resistance issue, it was essential that the second insecticidal protein have a significantly different mode of action to CrylAc. A common feature of resistance to CrylA proteins in several species as well as H. armigera has been a change in the binding site. A study of binding sites for some Cry proteins in the brush border membrane vesicles （BBMV） ofH. armigera and Helicoverpa punctigera was undertaken. The binding affinity for CrylAc was higher than for CrylAb, matching their relative toxicities, and CrylAc and CrylAb were found to share at least one binding site in both I-1. armigera and I-1. punctigera. However Cry2Aa did not compete with CrylAc for binding and so could be used in transgenic cotton in combination with CrylAc to control H. armigera and manage resistance. Variation in the susceptibilities of three different H. armigera strains to CrylAc correlated with the parameter Bmax/Kcom.     

β-Lactamase export across the outer membrane of Acinetobacter calcoaceticus:perturbation of the outer membrane lipopolysaccharide leaflet by enzyme overproduction

Acinetobacter calcoaceticus is able to produce a β-lactamase which was found in the periplasm and to be released into the extracellular culture medium. β-Lactamase export was dependent on enzyme over-production in a cooperative manner. Furthermore, it was accompanied by a steadily increasing release of lipopolysaccharide, an outer membrane constituent, and by an increase in the susceptibility to hydrophobic antibiotics. The data point towards a self-promoted perturbation of the outer membrane by overproduction of the enzyme, leading to a semi-selective increase in membrane permeability.     

Thyroid hormones increase Na+–Pi co-transport activity in intestinal brush border membrane: Role of membrane lipid composition and fluidity

In the present study, we documented the promising role of thyroid hormones status in animals in modulation of Na⁺–P_i transport activity in intestinal brush border membrane vesicles (BBMV) which was accompanied with alterations in BBM lipid composition and fluidity. Augmentation of net P_i balance in hyperthyroid (Hyper-T) rats was fraternized with accretion of P_i transport across BBMV isolated from intestine of Hyper-T rats as compared to hypothyroid (Hypo-T) and euthyroid (Eu-T) rats while Na⁺–P_i transport across BBMV was decreased in Hypo-T rats relative to Eu-T rats. Increment in Na⁺–P_i transport in intestinal BBMV isolated from Hyper-T rats was manifested as an increase in the maximal velocity (V_max) of Na⁺–P_i transport system. Furthermore, BBMV lipid composition profile in intestinal BBM from Hyper-T was altered to that of Hypo-T rats and Eu-T rats. The molar ratio of cholesterol/phospholipids was higher in intestinal BBM from Hypo-T rats. Fluorescence anistropy of diphenyl hexatriene (rDPH) and microviscosity were significantly decreased in the intestinal BBM of Hyper-T rats and decreased in Hypo-T rats as compared to Eu-T rats which corroborated with the alteration in membrane fluidity in response to thyroid hormone status of animals. Therefore, thyroid hormone mediated change in membrane fluidity might play an important role in modulating Na⁺–P_i transport activity of intestinal BBM. (Mol Cell Biochem 278: 195–202, 2005)     

Does a fibroblast membrane proteinase affect the binding, uptake and metabolism of epidermal growth factor?

Pre-treatment of human fibroblasts to inhibit a cell-surface growth-related proteinase inhibits the mitogenic action of epidermal growth factor. It also reduces the binding of epidermal growth factor to these cells, and lowers the rate of internalisation and degradation of the growth factor, but quantitative considerations render it unlikely that these parameters contribute directly to the inhibition of mitogenesis.     

Measurements of K+-driven Cl− uptake in thylakoids: Inhibition of uptake by antibodies raised to the major polypeptides of the Cl−-efflux active particle(s)

The mechanism of a K⁺-driven Cl^– accumulation against a concentration gradient was investigated by flow dialysis after addition of K⁺-Hepes. Non-specific chloride binding, measured in the presence of choline-Hepes, accounted for approximately 50% of the observed uptake in this system. The K⁺-Hepesdriven Cl^– uptake was inhibited by poly-l-lysine and by two antibodies raised to the major polypeptides of the Cl^–-efflux active particle. Poly-l-lysine had no effect on Cl^– binding estimated with choline-Hepes.     

Dissection of events in the resistance to β-lactam antibiotics mediated by the protein BlaR1 from Staphylococcus aureus

A heterologous expression system was used to evaluate activation of BlaR1, a sensor/signal transducer protein of Staphylococcus aureus with a central role in resistance to β-lactam antibiotics. In the absence of other S. aureus proteins that might respond to antibiotics and participate in signal transduction events, we documented that BlaR1 fragmentation is autolytic, that it occurs in the absence of antibiotics, and that BlaR1 directly degrades BlaI, the gene repressor of the system. Furthermore, we disclosed that this proteolytic activity is metal ion-dependent and that it is not modulated directly by acylation of the sensor domain by β-lactam antibiotics.     

Synergistic evaluation of Moringa oleifera extract and ß-lactam antibiotic to restore the susceptibility of methicillin-resistant Staphylococcus aureus

Molecular Biology Reports - The antibiotic resistance has become a major threat to global health. The combinatorial use of two or more compounds to develop a new formulation may overcome the...     

Selenium Attenuates Staphylococcus aureus Mastitis in Mice by Inhibiting the Activation of the NALP3 Inflammasome and NF-κB/MAPK Pathway

Mastitis is one of the most important diseases affecting the dairy industry in the world, and it also poses a great threat to human food safety. In this study, we explored whether selenium can inhibit the activation of the NALP3 inflammasome and NF-κB/MAPK pathway to achieve anti-inflammatory effects. Sixty BALB/c female mice were randomly divided into three groups according to diets of different selenium concentrations (high, normal, and low). After 90 days, mice fed the same selenium concentration were randomly divided into two smaller groups, one of which was inoculated with Staphylococcus aureus and the other injected with saline as a control. Through histopathologic examination staining, western blot, qPCR, and ELISA, the results showed that with increasing selenium concentrations, the expression levels of IL-1β, TNF-α, NALP3, caspase-1, and ASC were decreased in mouse mammary tissue. Therefore, this study revealed that selenium can attenuate S. aureus mastitis by inhibiting the activation of the NALP3 inflammasome and NF-κB/MAPK pathway.

     

K+ and Cl− conductances in the apical membrane from secreting oxyntic cells are concurrently inhibited by divalent cations

Summary This study concerns the properties of rapid K⁺ and Cl transport pathways that are present in the (H⁺+K⁺)-ATPase membrane from stimulated, and secreting, gastric oxyntic cells. Ion permeabilities in the isolated stimulation-associated vesicles were monitored via the rates of H⁺ efflux under conditions of exclusive H⁺/K⁺ counterflux or H⁺–Cl co-efflux, as well as by comparison of equilibration rates for⁸⁶Rb and³⁶Cl under conditions of equilibrium exchange and unidirectional salt flux. These latter studies suggest that Rb⁺ and Cl pathways are conductive and independent. In spite of the functional independence of the ion pathways, several divalent cations inhibit Rb⁺ and Cl isotopic exchange as well as the H⁺ efflux that is dependent on either K⁺ or anion (Cl, SCN, NO₂) fluxes. Zn²⁺ is the more potent inhibitor, reducing by 50% the sensitive component of K⁺, Cl, and NO₂ fluxes at about 20 m; Mn²⁺ has a similar effect at 200 m. Ni²⁺ and Co²⁺ were roughly equipotent to Mn²⁺ while Mg²⁺ and Ca²⁺ had not inhibitory effect. These results suggest that the stimulation-induced permeabilities, while functioning independently, may be physically linked, i.e., residing within a single entity. In similar studies carried out in (H⁺+K⁺)-ATPase vesicles obtained from nonstimulated cells, no vestiges of sensitivity to the inhibitory divalent cations could be detected. The implications of these findings for the physiology of the oxyntic cell in the context of a model for membrane fusion are discussed.