Ortho-hydroxybenzoates may act at the protein fraction to enhance membrane permeability

Experimental observation suggests that the ability of ortho-hydroxybenzoates to alter the permeability of rat rectal epithelial membrane may involve an interaction between the ortho-isomer and the membrane protein. Such an interaction may cause a temporary structural modification of the protein rendering the membrane permeable to compounds normally poorly absorbed from the rectal compartment.     

A Note on the Anti-Pseudomonas Activity of Cephalosporins with Chlorhexidine

Thirty clinical isolates of Pseudomonas aeruginosa were tested for susceptibility to cephaloridine, cefuroxime, cefotaxime and cefoxitin with and without chlorhexidine in agar dilution 'chequerboard' titrations and antibiotic disc tests. A synergistic response (σFIC = < 0.7) in combined titration with chlorhexidine was seen in 50% of the strains with cefuroxime and cefoxitin and 13% with cefotaxime. Cephaloridine MICs mostly exceeded the tested range but a definite reduction in combination was shown with 33% of strains. Susceptibility to cephaloridine, cefuroxime and cefoxitin was not enhanced to clinically useful levels and the effect was seen only in chlorhexidine concentrations close to the MIC. Disc tests failed to show the antibacterial activity of the combinations. It is suggested that the level of the principal lesion induced by chlorhexidine in the inner membrane might be too deep to affect the penetration of β-lactam antibiotics to their site of action.     

Microviscosity of mouse splenocyte cytoplasmic membrane modified by some antibiotics

This study has assessed the changes in the polarization fluorescence of 1,6-diphenyl-1,3,5-hexatriene in the splenic membrane of mice treated with clindamycin, penicillin, cefoxitin, cefamandole, cephaloridine, tetracycline, and erythromycin. The data show that most of the antibiotics tested modified membrane activity, as was shown by the changes in membrane microviscosity parameters.     

Activation of the Na-K-Cl cotransporter NKCC1 detected with a phospho-specific antibody

The Na-K-Cl cotransporter NKCC1 is activated by phosphorylation of a regulatory domain in its N terminus. In the accompanying paper (Darman, R. B., and Forbush, B. (2002) J. Biol. Chem. 277, 37542-37550), we identify three phosphothreonines important in this process. Using a phospho-specific antibody (anti-phospho-NKCC1 antibody R5) raised against a diphosphopeptide containing Thr(212) and Thr(217) of human NKCC, we were readily able to monitor the cotransporter activation state. In (32)P phosphorylation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of (32)P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation. Immunofluorescence analysis of shark rectal gland shows activation-dependent R5 antibody staining along the basolateral membrane. In perfused rat parotid glands, isoproterenol induced staining of both acinar and ductal cells along the basolateral membrane. Isoproterenol also induced basolateral staining of the epithelial cells in rat trachea, whereas basal cells in the subepithelial tissue displayed heavy, non-polarized staining of the cell membrane. In rat colon, agonist stimulation induced staining along the basolateral membrane of crypt cells. These data provide direct evidence of NKCC1 regulation in these tissues, and they further link phosphorylation of NKCC1 with its activation in transfected cells and native tissue. The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo.     

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.     

Light and electron microscopic study of the hindgut of the ant (Formica nigricans, hymenoptera): II. Structure of the rectum

The rectum of the ant Formica nigricans is composed of six ovoid rectal papillae inserted into a rectal pouch. The wall of the rectal pouch is made up of a flat epithelium of simple rectal cells lined by cuticle, and surrounded by a circular muscle layer. Each rectal papilla is comprised by a simple columnar epithelium of principal cells facing the lumen, and a simple cuboid epithelium of secondary cells towards the hemolymph; a group of 20-25 slender junctional cells lies laterally between both epithelia enclosing an intrapapillar sinus. The muscle layer of the rectal wall also surrounds the base of the papillae. Principal cells do not exhibit extensive infoldings at the apical and basal plasma membranes. Lateral membranes, in contrast, develop highly folded mitochondria-scalariform junction complexes enclosing very narrow intercellular canaliculi between adjacent cells. These canaliculi open to wider intercellular sinuses that ultimately drain into the intrapapillar sinus at the sites of entry of tracheal cells. The lateral plasma membranes do not link to the apical or basal plasma membrane, thus originating a syncytium throughout the principal cells. The apical plasma membrane of secondary cells shows invaginations in relation with an apical tubulovacuolar system, bearing portasomes to the cytoplasmic side of the membrane. Secondary cells unite by convoluted septate junctions, and basolateral infoldings are also developed. These ultrastructural traits, some of them different from those found in other insects, are discussed and examined in relation to their role in water and solute absorption. A route for rectal transport in F. nigricans is proposed.     

Evaluation of methods for detecting oxacillin resistance in coagulase-negative staphylococci including cefoxitin disc diffusion

The aim of this study was to evaluate the accuracy of cefoxitin disc diffusion as a prediction of oxacillin resistance in coagulase-negative staphylococci (CoNS), and also to compare genotypic and phenotypic methods for detecting this resistance property. A total of 151 clinical CoNS isolates were tested by PCR for the presence of the mecA gene (gold standard method). The isolate susceptibilities were determined by the disc diffusion method with oxacillin (1 microg) and cefoxitin (30 microg) and by the agar dilution method for cefoxitin and oxacillin. Although none of the techniques showed 100% sensitivity and 100% specificity, the cefoxitin disc diffusion and oxacillin agar dilution were the best methods for detecting resistance to oxacillin among CoNS as these methods produced the best negative and positive predictive values. A combination of methods can be used routinely to identify resistance to oxacillin in CoNS.     

Salicylate-promoted permeation of cefoxitin, insulin and phenylalanine across red cell membrane. Possible mechanism

Uptake of sodium cefoxitin, D-phenylalanine and insulin into human red blood cells was significantly enhanced by the presence of salicylate and 5-methoxysalicylate in the medium. The mechanism of adjuvant action appeared to depend on an affinity between the adjuvant and the protein fraction in the erythrocyte membrane. The inhibitory effect of DIDS and phlorizin on the salicylate-enhanced uptake of these compounds strongly suggests that the ability of salicylate to permeate the membrane may be essential for it to act as an adjuvant.     

Ultrastructure of the type “B” cells in the rectal pad epithelium of Locusta migratoria

The electron microscopical structure of the type “B” cells in the rectal pad epithelium of Locusta is described. The type “B” cells occur singly in the distal region of the rectal pad epithelium. They are characteristically goblet shaped and join with contiguous type “A” or rectal pad cells, near the apical surface by means of a restricted region of septate desmosomes. Type “B” cells possess a microvillate apical membrane, with the villi arranged as a rosette overlying the apical inaginations of adjacent type “A” cells. Large numbers of microtubules and vacuoles of various sizes containing an assortment of inclusions are present in the apical region of the type “B” cells. Many of the microtubules insert distally on hemidesmosomes located in the apical plasma membrane. Rough endoplasmic reticulum and mitochondria are also present but neither are abundant. The possible significance of these findings is discussed.     

Functional Characterization of a ClC-2-Like Cl− Conductance in Surface Epithelial Cells of Rat Rectal Colon

ClC-2, a member of the voltage-gated Cl⁻ channel family, is expressed in the distal colonic surface epithelial cells of various species, but its functional significance remains unclear. Here, by means of electrophysiological and molecular biological techniques, we have identified and characterized a ClC-2-like conductance naturally expressed by surface epithelial cells acutely dissociated from rectal colon of rats fed a standard diet. Whole-cell patch-clamp experiments showed that the surface cells, whether an amiloride-sensitive Na⁺ conductance was present or not, displayed a strong hyperpolarization-activated, inwardly rectifying Cl⁻ current. Analysis both by in situ hybridization and immunohistochemistry confirmed the expression of ClC-2 in the rectal surface epithelium. The native Cl⁻ current shared common electrophysiological properties including voltage-dependent activation, anion selectivity sequence, and Zn²⁺ sensitivity with that recorded from HEK293 cells transfected with ClC-2 cloned from rat rectal colon (rClC-2). Cell-attached patch recordings on the surface cells revealed that native ClC-2-like currents activated only at potentials at least 40 mV more negative than resting membrane potentials. In Ussing chamber experiments with rat rectal mucosa, either basolateral or apical application of Zn²⁺ (0.1 mM), which inhibited both native ClC-2-like currents and recombinant rClC-2 currents, had little, if any, effects on basal amiloride-sensitive short-circuit current. Collectively, these results not only demonstrate that a functional ClC-2-type Cl⁻ channel is expressed in rat rectal surface epithelium, but also suggest that the channel activity may be negligible and thus nonessential for controlling electrogenic Na⁺ transport in this surface epithelium under basal physiological conditions.     

热应激时大鼠肺组织中β—肾上腺素受体的变化与膜磷...

To explore the relationship between the change of beta-adrenoceptor and the metabolism of phospholipids in lung tissue from acute heat stressed rats, the Bmax of beta-adrenoceptors, the activity of phospholipase A2 (PLA2), the content of phosphatidylcholine (PC) and phosphatidylserine (PS), and membrane fluidity in lung tissue of normal and heat stressed rats were investigated. The relevant parameter values mentioned above were 479 +/- 94 fmol/mg protein, 78.5 +/- 8.2 U, 53.5 +/- 1.7 mg/g.wet. w. and 425.1 +/- 68.1 micrograms/g.wet. w. respectively. Whereas in the heat stressed rats with rectal temperature raised to 42 degrees C for 15 min, the Bmax of beta-adrenoceptor was decreased by 43% (P less than 0.01), the activity of PLA2 increased by 83% (P less than 0.01), the contents of PC and PS decreased by 50% and 47% (P less than 0.01) respectively. A lower membrane fluidity in lung tissue for heat stressed rats was also demonstrated. The results suggest that the decreased binding sites of beta-adrenoceptor in lung tissue of rat during hyperthermia may be contributed to the activation of PLA2, which then accelerated the catabolism of phospholipids such as PC and PS in the cell plasma membrane, with a consequent alteration of membrane fluidity.     

Effects of opioid antagonists and their quaternary analogs on temperature changes in morphine-dependent rats

Subcutaneous administration of three opioid antagonists; naloxone, naltrexone and nalmefene, produced a significant rise in tail skin temperature and a subsequent fall in rectal temperature in morphine dependent rats. However, subcutaneous administration of equimolar concentrations of the quaternary derivatives of these opioid antagonists (naloxone methobromide, naltrexone methobromide and n-methylnalmefenium iodide) failed to produce any significant alterations in either tail skin or rectal temperatures in the morphine dependent rat. At doses of naloxone methobromide 6 to 9 times greater than naloxone, there was a slight reduction of rectal temperatures with no significant elevation of skin temperature. However, the fall in rectal temperature was still significantly less than that achieved with administration of naloxone. When each of these six agents were administered centrally (20 micrograms/5 microliter, icv) in the morphine dependent rat, similar increases in tail skin temperature and decreases in rectal temperature were observed. These temperature changes were similar to those observed following systemic administration of the opioid antagonist. Previously, we have suggested that acute withdrawal in the morphine-dependent rat may serve as an animal model for the mechanism of the menopausal hot flush. Collectively, these results suggest that the temperature changes associated with morphine-withdrawal in our rat model for studying the mechanisms of the menopausal hot flush are centrally mediated.     

Cellular differentiation in relation to water vapour absorption in the rectal complex of the mealworm, Tenebrio molitor

Larvae of the mealworm beetle Tenebrio molitor, are able to absorb water vapour from subsaturated air, but adults cannot. This functional difference is paralleled by structural differences in the cryptonephridial rectal complex, the site of vapour absorption in the larva. Very distinctive differences occur in the rectal pad epithelium and these are reported in detail for the first time. The cells of the larval rectal pad are very closely apposed, forming a structural, and presumably functional, unit, whereas the cells of the adult rectal pad are more clearly separate. Intracellular organization also shows clear differences. These differences indicate that the rectal epithelium may play a more important role in vapour absorption than recently ascribed to it. Other, less striking, differences in the cryptonephric Malpighian tubules and perinephric membrane as previously recorded have largely been confirmed. Morphometric analysis suggests that diffusion alone could account for the observed absorption of water vapour across the larval system from rectal lumen to the lumen of the cryptonephric tubules, but this does not rule out the possibility that other transporting mechanisms are also involved. Radial diffusion and antero-posterior gradients may be facilitated by the predominently radial and circumferential arrangement of the rectal pad cells and the surrounding cryptonephric tubules. Reinvestigation of the isolating perinephric membrane and its insertion onto the rectal cuticle supports the conclusions that insertion occurs only posteriorly. The model incorporating anterior as well as posterior insertion does not apply. The membrane posteriorly encloses a single perirectal space between rectum and tubules and in this region no perinephric or peritubular space is found between inner and outer regions of the membrane. This is the region where maximal gradients occur across the system.     

Resistance of Providencia stuartii to chlorhexidine: a consideration of the role of the inner membrane

Spheroplasts of three strains of Providencia stuartii (one sensitive, one moderately sensitive and one resistant to chlorhexidine) were induced by cefoxitin, glycine or a lysozyme-tris-EDTA combination, and their susceptibility to chlorhexidine-induced lysis investigated. Maximum lysis of spheroplasts occurred at a low (2.5-5 micrograms/ml) concentration of chlorhexidine and was greatest with the most sensitive strain, Pv2. The possible role of the inner membrane in chlorhexidine resistance is considered in the light of the findings obtained.     

Mercury toxicity in the shark (Squalus acanthias) rectal gland: apical CFTR chloride channels are inhibited by mercuric chloride

In the shark rectal gland, basolateral membrane proteins have been suggested as targets for mercury. To examine the membrane polarity of mercury toxicity, we performed experiments in three preparations: isolated perfused rectal glands, primary monolayer cultures of rectal gland epithelial cells, and Xenopus oocytes expressing the shark cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In perfused rectal glands we observed: (1) a dose-dependent inhibition by mercury of forskolin/3-isobutyl-1-methylxanthine (IBMX)-stimulated chloride secretion; (2) inhibition was maximal when mercury was added before stimulation with forskolin/IBMX; (3) dithiothrietol (DTT) and glutathione (GSH) completely prevented inhibition of chloride secretion. Short-circuit current (Isc) measurements in monolayers of rectal gland epithelial cells were performed to examine the membrane polarity of this effect. Mercuric chloride inhibited Isc more potently when applied to the solution bathing the apical vs. the basolateral membrane (23 +/- 5% and 68 +/- 5% inhibition at 1 and 10 microM HgCl2 in the apical solution vs. 2 +/- 0.9% and 14 +/- 5% in the basolateral solution). This inhibition was prevented by pre-treatment with apical DTT or GSH; however, only the permeant reducing agent DTT reversed mercury inhibition when added after exposure. When the shark rectal gland CFTR channel was expressed in Xenopus oocytes and chloride conductance was measured by two-electrode voltage clamping, we found that 1 microM HgCl2 inhibited forskolin/IBMX conductance by 69.2 +/- 2.0%. We conclude that in the shark rectal gland, mercury inhibits chloride secretion by interacting with the apical membrane and that CFTR is the likely site of this action.     

Identification and localization of a dogfish homolog of human cystic fibrosis transmembrane conductance regulator.

Chloride channels in the apical plasma membrane of cells in the dogfish rectal gland have served as a model system for the study of regulation of chloride flux by changes in intracellular cyclic AMP levels. Similar regulation by cyclic AMP has been described for channels in cells of human secretory epithelia where defective regulation by cyclic AMP-dependent protein phosphorylation is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). We have isolated a cDNA clone from the rectal gland encoding a protein that is 72% identical to the human CFTR. One of the major phosphorylation sites in CFTR is absent in the dogfish protein. The dogfish protein has, however, four additional putative substrate sites for the cyclic AMP-dependent protein kinase. A peptide antibody, which was raised against an amino acid sequence common to both the human and dogfish CFTR sequences, recognizes proteins with similar molecular masses (160 kDa) in the dogfish gland and in mammalian lung. Immunolocalization studies with this antibody show that the putative dogfish CFTR is localized to the apical membrane of cells lining the lumen of the rectal gland.     

Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis

TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an inhibition pattern opposite to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast upon trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardioprotectants or neuroprotectants.     

Hypothermia elicited by the intracerebral microinjection of neurotensin

Changes in rectal temperature were measured after the intracerebral microinjection of neurotensin (2.5 μg/0.5 μl) at 135 sites in the rat. At 63 of the 135 microinjection sites, the tridecapeptide produced a rapid onset of hypothermia ranging in magnitude from 0.8 to 2.3°C below the baseline rectal temperature. The drop in rectal temperature persisted for 2–4 hours following the microinjection. The greatest concentrations of neurotensin-sensitive sites were found in the medial preoptic region of the hypothalamus and in the periaqueductal gray area, both of which contain relatively large amounts of endogenous neurotensin. Other active sites were found in the ventral thalamus, the dorsomedial hypothalamus, and in the diagonal band of Broca. At no injection site did neurotensin evoke an increase in rectal temperature. These data support the proposition that neurotensin may act endogenously to mediate heat-loss mechanisms in the rat. The data provide further evidence indicating a potent neuromodulatory role for neurotensin.     

Resistance of Providencia stuartii to chlorhexidine: A consideration of the role of the inner membrane

Spheroplasts of three strains of Providencia stuartii (one sensitive, one moderately sensitive and one resistant to chlorhexidine) were induced by cefoxitin, glycine or a lysozyme-tris-EDTA combination, and their susceptibility to chlorhexidine-induced lysis investigated. Maximum lysis of spheroplasts occurred at a low (2·5–5 μg/ml) concentration of chlorhexidine and was greatest with the most sensitive strain, Pv2. The possible role of the inner membrane in chlorhexidine resistance is considered in the light of the findings obtained.