Ultra-deformable free fatty acid based nano-carriers for topical delivery of Luteolin: A potential paradigm for management of Methicillin-Resistant Staphylococcus aureus skin infections.

The incidences of antimicrobial resistance in particular, Methicillin-Resistant Staphylococcus aureus (MRSA) have increased during the last two decades. However, conventional dosage forms are unable to evade the barrier effect of the stratum corneum to permit deep penetration of the skin to resolve deep skin infections. There is, therefore, an urgent need for an advanced drug delivery system. Thus the study reported herein was aimed to fabricate a novasome-loaded luteolin (LUT) to improve its topical delivery and to enhance its antibacterial activity. The system was investigated for the impact of the type of surfactant, stearic acid concentration (g %), cholesterol amount (mg) and Brij 52 amount (mg) on the percent entrapment efficiency, particle size, poly-dispersity index and zeta potential. Statistical optimization of these factors was conducted using the Design-Expert® software. The optimum formulation was further in-vitro characterized by release study, differential scanning calorimetry, transmission electron microscope, x-ray diffraction and antibacterial activity. Formulation F2 composed of Span 60, 0.4 g % of stearic acid, 100 mg cholesterol and 30 mg Brij 52 was selected as the optimum formula based on the highest desirability value (0.634). F2 demonstrated enhanced antimicrobial activity with lower minimum inhibitory concentrations against a panel of MRSA clinical isolates when compared to LUT dispersion. Furthermore, the F2 formula exhibited higher anti-virulence activity by effectively inhibiting biofilm formation and suppressing α-hemolysin activity in MRSA isolates. It also demonstrated improved biosafety based on cytotoxicity assessment on human skin fibroblasts (HSF). Finally, when assessed in an in vivo skin infection mouse model, the F2 formula and commercially available fusidic acid preparation significantly reduced the microbial load of infected skin lesions compared to both the negative control and LUT dispersion-treated groups. Based on the aforementioned results, the validity of novasomes as a nano-carrier to boost in vitro and in vivo anti-MRSA activity of LUT could be affirmed.

ATP serves as an endogenous inhibitor of UDP-glucuronosyltransferase (UGT): a new insight into the latency of UGT.

We have suggested that adenine-related compounds are allosteric inhibitors of UGT in rat liver microsomes (RLM) treated with detergent. To clarify whether the same occurs with a pore-forming peptide, alamethicin, the effects of adenine-related compounds on 4-metylumbelliferone (4-MU) glucuronidation were examined using RLM and human liver microsomes (HLM). ATP inhibited 4-MU glucuronidation when polyoxyethylene cetyl alcohol ether (Brij-58)-treated RLM were used (IC(50) = approximately 70 µM). However, alamethicin-treated RLM exhibited a lower susceptibility (IC(50) = approximately 460 µM) than Brij-58-treated RLM. A similar phenomenon was observed when pooled HLM were used. Then, the endogenous ATP content of RLM was determined in the presence and absence of alamethicin or detergent, and although no ATP remained in the microsomal pellets after Brij-58 treatment, more than half of the microsomal ATP remained even after treatment with alamethicin. Furthermore, the V(max) in the absence of an adenine-related compound was approximately three times higher in Brij-58-treated than in alamethicin-treated RLM. The difference in the inhibitory potency observed was due to the difference in remaining endogenous ATP and the accessibility of exogenous ATP to the luminal side of the endoplasmic reticulum (ER), where the active site of UDP-glucuronosyltransferase (UGT) is located. Gefitinib (Iressa), a protein tyrosine kinase inhibitor, markedly inhibited human UGT1A9 activity. It is interesting to note that AMP antagonized Gefitinib-provoked inhibition of UGT1A9, and ATP exhibited an additive inhibitory effect at a lower concentration. Therefore, Gefitinib inhibits UGT1A9 at the common ATP-binding site shared with ATP and AMP. Releasing adenine nucleotide from the ER is suggested to be one of the mechanisms that explain the "latency" of UGT.

A bilateral comparison study of pimecrolimus cream 1% and a topical medical device cream in the treatment of patients with atopic dermatitis.

Corticosteroids are the mainstay of therapy for atopic dermatitis, but long-term use is associated with adverse effects. We sought to evaluate the clinical efficacy of two steroid-sparing creams for atopic dermatitis. Twenty patients were enrolled in an investigator-blinded, bilateral comparison study. Patients applied pimecrolimus cream twice daily to a target lesion on one side of the body and also applied a topical medical device cream three times daily on a symmetrical target lesion on the opposite side of the body for four weeks. Clinical assessments including Physician Global Assessment (PGA), Target Lesion Symptom Score (TLSS), subject self-assessment and digital photography were performed at the baseline, 2 week, and 4 week visits. Seventy-five percent of patients (pimecrolimus, 15 of 20; topical medical device, 15 of 20) were rated "clear" (0) or "almost clear" (1) by PGA for both medications after four weeks. Percent improvement of the PGA from randomization for pimecrolimus cream and the topical medical device cream were 72.50 and 71.67 respectively (P=0.9283). PGA scores decreased significantly from baseline for both treatments (P=0.004). Overall, there was no statistically significant difference between treatment groups for PGA scores throughout the study (P=0.8236). No cutaneous side effects were noted. Our study was limited by a small sample size and lack of double-blinding; however, both treatments were found to be safe and effective in treating atopic dermatitis over four weeks. Significant improvements were noted for all efficacy variables. In conclusion, a lipid-rich, non-steroidal, topical medical device cream was as effective in improving atopic dermatitis as pimecrolimus cream.

The effect of detergents on trimeric G-protein activity in isolated plasma membranes from rat brain cortex: correlation with studies of DPH and Laurdan fluorescence.

The effect of non-ionic detergents on baclofen (GABAB-R agonist)-stimulated G-protein activity was measured as a [(35)S]GTPgammaS binding assay in the plasma membranes (PM) isolated from the brain tissue. The effect was clearly biphasic--a decrease in the activity was followed by an activation maximum and finally, at high concentrations, drastic inhibition of the G-protein activity was noticed. Contrarily, specific radioligand binding to GABAB-receptor was inhibited in the whole range of detergent concentrations step by step, i.e. it was strictly monophasic. The magnitude of both detergent effects was decreased in the same order of potency: Brij58>Triton X-100>Digitonin. The identical order was found when comparing detergents ability to alter fluorescence anisotropy of the membrane probe 1,6-diphenyl-1,3,5-hexatriene (rDPH) incorporated into the hydrophobic PM interior. Decrease of rDPH, in the order of Brij58>Triton X-100>Digitonin, was reflected as decrease of the S-order parameter and rotation correlation time phi paralleled by an increase of diffusion wobbling constant Dw (analysis by time-resolved fluorescence according to "wobble-in-cone" model). The influence of the detergents on the membrane organization at the polar headgroup region was characterized by Laurdan generalized polarization (GP). As before, the effect of detergents on GP parameters proceeded in the order: Brij58>Triton X-100>Digitonin.

Eradication of meticillin-resistant Staphylococcus aureus from human skin by the novel LL-37-derived peptide P10 in four pharmaceutical ointments.

Skin bacterial colonization/infection is a frequent cause of morbidity in patients with chronic wounds and allergic/inflammatory skin diseases. This study aimed to develop a novel approach to eradicate meticillin-resistant Staphylococcus aureus (MRSA) from human skin. To achieve this, the stability and antibacterial activity of the novel LL-37-derived peptide P10 in four ointments was compared. Results indicate that P10 is chemically stable and antibacterial in hypromellose gel and Softisan-containing cream, but not in Cetomacrogol cream (with or without Vaseline), at 4 °C for 16 months. Reduction in MRSA counts on Leiden human epidermal models (LEMs) by P10 in hypromellose gel was greater than that of the peptide in Cetomacrogol cream or phosphate buffered saline. P10 did not show adverse effects on LEMs irrespective of the ointment used, while Cetomacrogol with Vaseline and Softisan cream, but not hypromellose gel or Cetomacrogol cream, destroyed MRSA-colonized LEMs. Taking all this into account, P10 in hypromellose gel dose-dependently reduced MRSA colonizing the stratum corneum of the epidermis as well as biofilms of this bacterial strain on LEMs. Moreover, P10 dose-dependently reduced MRSA counts on ex-vivo human skin, with P10 in hypromellose gel being more effective than P10 in Cetomacrogol and Softisan creams. P10 in hypromellose gel is a strong candidate for eradication of MRSA from human skin.

Scrolls: novel microparticulate systems for enhanced delivery to/across the skin.

We describe the scroll system as a new microparticulate structured delivery system for enhanced delivery to/across the skin. The basic components of the scroll system are non-ionic surface active of the type of alkyl polyglycol ethers and a glycol. The unique structures are preserved with addition of various ingredients such as polymers, vegetable oils, pharmaceuticals, and permeation enhancers but are dismissed when amphiphile is withdrawn. The microparticles have a unique scroll structure with multiple "wrapping." Besides enabling superior permeation of drugs into/across the skin, the drugs delivered by scroll systems were more effective in vitro and in vivo compared to controls. Model drugs presented high entrapment capacity in scroll systems. The systems are stable over time and are safe for skin application. In order to form, they require a small number of ingredients, simple preparation method, and are environment friendly. The scroll systems may be new potential tools in the dermal/transdermal pharmaceutical and cosmetic industry.

Hemolysis caused by polyoxyethylene-derived surfactants. Evidence for peroxide participation.

The hemolytic properties of nonionic surfactants of the series CH3(CH2)15-17-O-(CH2CH2O)nCH2CH2OH were investigated and compared to those of saponins, sapogenins and H2O2. Antioxidants and anaerobic conditions were shown to inhibit the hemolysis, while glycyrrhizin was found to enhance it. Similar effects were obtained for H2O2 hemolysis, but not for saponin and sapogenin hemolysis. It is proposed that peroxides and free radicals are mainly responsible for the polyoxyethylene derived surfactants induced hemolysis.

Activation of plant plasma membrane H(+)-ATPase by the non-ionic detergent Brij 58.

Plasma membrane vesicles were purified from tobacco callii and the modulation of H(+)-ATPase by detergents was investigated. The nonionic detergent Brij 58 not only activated ATP hydrolysis (2-fold) but also proton pumping (more than 4-fold). Triton X-100, within a more limited concentration range, produced a similar effect. The simultaneous activation of ATP hydrolysis and proton pumping is not compatible with current interpretations of effects of nonionic detergents on the H(+)-ATPase based on latency of the enzyme and opening of vesicles.

Modulation of plant plasma membrane H+-ATPase by phytotoxic lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae.

Pseudomonas fuscovaginae produces the lipodepsipeptides syringotoxin, fuscopeptin A and fuscopeptin B concurrently. These phytotoxins inhibit acidification of the external medium by fusicoccin-treated rice leaf sheath discs. When tested in vitro on H+-ATPase of rice shoot plasma membranes, syringotoxin and its structural analogue syringomycin, produced by P. syringae pv. syringae, displayed a double effect. At low concentrations they stimulated the ATPase activity of native right-side-out membrane vesicles in a detergent-like manner. At higher concentrations, however, this stimulation was reversed. With membranes treated with the detergent Brij 58, inhibition of ATPase activity was observed at low concentrations of the nonapeptides. The latter effect required the presence of an intact lactone ring formed by the nonapeptide head of these molecules. In contrast, fuscopeptins A and B inhibited enzyme activity regardless of the orientation of the vesicles. These observations were confirmed using plasma membranes from a yeast strain whose own H+-ATPase had been replaced by a single plant H+-ATPase isoform, PMA2, from Nicotiana plumbaginifolia. The kinetics of inhibition induced by the most active compound fuscopeptin B, showed a non-competitive pattern, with a Ki of about 1 microM. The combination of syringotoxin (or syringomycin) with the more hydrophobic fuscopeptins, in amounts with little or no effect, resulted in strong inhibition of the enzyme activity of rice membranes, suggesting a synergistic effect for the two types of toxins.

Klenow fragment and DNA polymerase alpha-primase fromserva calf thymus in water-in-oil microemulsions.

The activity of DNA polymerase alpha-primase complex from calf thymus and Klenow fragment of E. coli DNA polymerase 1 has been studied in reverse microemulsions formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecylsulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), polyoxyethylene 20 cetyl ether (Brij 58), and Triton X-114 in decane. DNA polymerases were not active in AOT, CTAB, and SDS reverse microemulsions, but these enzymes catalyzed DNA synthesis in Brij 58 and its mixture with other surfactants. We have also found the system composed from the Triton X-114, SDS, CTAB, and Brij 58 (concentration of 128, 25, 15, and 10 mM, respectively) in hexanol-decane (1:12 v/v), in which DNA polymerases revealed maximum activity. The above system was optically transparent, fluid, and stable during a few hours with a water-surfactants molar ratio up to 160. The pH dependence of DNA polymerase activity was not significantly different in comparison with water; however, DNA polymerase was sensitive to ionic strength in microemulsions. The dependence of DNA polymerase activity on w0 was the curve with a few optima. DNA polymerases synthesized more products in water than in reverse microemulsions, and the processivity of Klenow fragment decreased. An increase of the water content resulted in an increase of DNA polymerase processivity.

Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent.

Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.

High Efficacy but Low Potency of δ-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments.

PRINCIPAL FINDINGS: HEK293 cells stably expressing PTX-insensitive δ-opioid receptor-Gi1α (C351I) fusion protein were homogenized, treated with low concentrations of non-ionic detergent Brij-58 at 0°C and fractionated by flotation in sucrose density gradient. In optimum range of detergent concentrations (0.025-0.05% w/v), Brij-58-treated, low-density membranes exhibited 2-3-fold higher efficacy of DADLE-stimulated, high-affinity [32P]GTPase and [35S]GTPγS binding than membranes of the same density prepared in the absence of detergent. The potency of agonist DADLE response was significantly decreased. At high detergent concentrations (>0.1%), the functional coupling between δ-opioid receptors and G proteins was completely diminished. The same detergent effects were measured in plasma membranes isolated from PTX-treated cells. Therefore, the effect of Brij-58 on δ-opioid receptor-G protein coupling was not restricted to the covalently bound Gi1α within δ-opioid receptor-Gi1α fusion protein, but it was also valid for PTX-sensitive G proteins of Gi/Go family endogenously expressed in HEK293 cells. Characterization of the direct effect of Brij-58 on the hydrophobic interior of isolated plasma membranes by steady-state anisotropy of diphenylhexatriene (DPH) fluorescence indicated a marked increase of membrane fluidity. The time-resolved analysis of decay of DPH fluorescence by the "wobble in cone" model of DPH motion in the membrane indicated that the exposure to the increasing concentrations of Brij-58 led to a decreased order and higher motional freedom of the dye. SUMMARY: Limited perturbation of plasma membrane integrity by low concentrations of non-ionic detergent Brij-58 results in alteration of δ-OR-G protein coupling. Maximum G protein-response to agonist stimulation (efficacy) is increased; affinity of response (potency) is decreased. The total degradation plasma membrane structure at high detergent concentrations results in diminution of functional coupling between δ-opioid receptors and G proteins.

Glucuronidation of thyroid hormone in rat liver: effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions.

We investigated the effects of in vivo treatment with different microsomal enzyme inducers, including clofibrate (CLOF), hexachlorobenzene (HCB), 3-methylcholanthrene (MC), 3,3',4,4'-tetrachlorobiphenyl (TCB), and 2,3,7,8-tetrachloro-p-dioxin, as well as of in vitro addition of the detergent Brij 56 on the glucuronidation of T4, T3, and rT3 by UDP-glucuronyltransferase (UGT) activities of rat liver microsomes. The results were compared with measurements of UGT activities for bilirubin, p-nitrophenol (PNP), and androsterone. In general, glucuronidation rates were 5-fold or more higher with rT3 than with T4 or T3 as substrate. In liver microsomes from untreated rats, T4 UGT activity was stimulated by Brij 56 to a maximum of about 2-fold at 0.025% detergent. Treatment of Wistar rats for 4 days with CLOF (200 mg/kg BW.day) resulted in significant increases in UGT activities for T4 (to 154%), rT3 (to 155%), and bilirubin (to 194%), in particular if assayed in the presence of 0.025% Brij 56, but had little effect on the UGT activities for T3, PNP, and androsterone. The CLOF-induced increases in T4 and rT3 UGT activities were not observed in Gunn rats, which have a complete lack of bilirubin UGT activity and greatly impaired PNP UGT activity. Treatment of Wistar rats with a single injection of MC (50 mg/kg BW), TCB (50 mg/kg BW), or 2,3,7,8-tetrachloro-p-dioxin (6.25 micrograms/kg BW) resulted, after 4 days, in 6.3- to 7.3-fold increases in T4 UGT activity and 15.1- to 16.7-fold increases in rT3 UGT activity if determined in the absence of Brij 56, whereas T4 UGT activity was only increased by 33-68% when assayed in the presence of Brij 56. T3 glucuronidation was not affected (with Brij 56) or was increased by only 33-68% (without Brij 56) after treatment with these MC-type inducers. PNP UGT activity was induced 3.6- to 4.3-fold, whereas bilirubin and androsterone UGT activities were changed little by these treatments. Similar findings regarding T4, rT3, PNP, and bilirubin UGT activities were obtained after chronic treatment of WAG rats with HCB, another MC-type inducer. However, WAG rats lack androsterone UGT and show low T3 UGT activity, which was increased about 2.3-fold by HCB treatment. On the basis of these and previous findings it is concluded that at least three UGT isoenzymes are involved in the glucuronidation of thyroid hormone.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct and indirect antibody-induced TX-100 resistance of cell surface antigens.

Translocation into detergent-insoluble microdomains (rafts) represents one of the earliest events in the process of cell activation, which follows the binding of surface receptor with natural ligand or mimicking antibody. In this study, the antibody-induced TX-100 resistance of surface antigens has been studied utilizing flow cytometry on TX-100 extracted cells. TX-100 resistance was evaluated by the ratio of antigen retained on the cells after detergent extraction compared with mAb-pretreated and untreated cells. All the antigens under study except CD98 demonstrated antibody-induced TX-100 resistance if the cells were treated with monoclonal antibodies and further cross-linked with secondary antibodies prior to lysis. CD20, CD5, and sIgM molecules were capable of transferring into TX-100-insoluble state in the absence of additional cross-linking. The experiments on modification of raft and cytoskeletal components of the cell, as well as the data on co-localization of TX-100-resistant antigens with raft and cytoskeletal markers strongly indicate that antibody-induced TX-100 resistance of antigens is mainly related to the translocation of antigens into lipid microdomains.

Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes.

A specific high performance liquid chromatographic assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol glucuronidation kinetics in human livers microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction. Mean apparent Km and Vmax values were 7.37 +/- 0.99 mM and 4.76 +/- 1.35 nmol/min/mg, respectively. Addition of the non-ionic detergent Brij 58 to microsomal incubations resulted in approximately 50% activation of microsomal paracetamol UDPGT-activity. This contrasts to the approximately three-fold activation of 4-methylumbelliferone, morphine and 4-nitrophenol glucuronidation observed following Brij 58 treatment of human liver microsomes. The glucuronidated xenobiotics chloramphenicol, digitoxigenin monodigitoxoside, 4-hydroxybiphenyl, 4-methylumbelliferone, morphine, 1-naphthol and 4-nitrophenol were screened for inhibitory effects on paracetamol glucuronidation. Of these compounds, only digitoxigenin monodigitoxoside and 1-naphthol were found to cause significant inhibition of paracetamol UDPGT activity. Along with the results of previous studies of the kinetics and inhibitor profile of human liver glucuronidation reactions (Miners et al., Biochem Pharmacol 37: 665-671, 1988 and 37: 2839-2845, 1988), these data indicate that the model glucuronidated substrates paracetamol, morphine and 4-methyllumbelliferone may be used to differentiate at least four human liver UDPGT isozyme activities.

Influence of morphine concentration on detergent activation of rat liver morphine-UDP-glucuronosyltransferase.

The effect of two detergents, Triton X-100 and Brij 58, on the production rate of morphine-3-glucuronide by rat hepatic microsomes has been investigated over a range of detergent and substrate concentrations, using a specific HPLC assay. Activation of morphine-UDP-glucuronosyltransferase (morphine-UDPGT) by Triton X-100 was more complex than that shown by Brij 58. At the optimal concentration of Triton X-100 (0.1-0.125 mg Triton X-100/mg microsomal protein), relative metabolic activity (activity of morphine-UDPGT in the activated state/activity of morphine-UDPGT in the native state; RMA) was 0.9, 1.3 and 2.5 at morphine concentrations of 0.05, 0.5 and 2.5 mM, respectively. Analysis of results from six individual rats in the native and maximally activated state (0.125 mg Triton X-100/mg microsomal protein) showed that RMA was highly dependent upon substrate concentration (P less than 0.0001). Activation produced by the optimal concentration of Brij 58 (0.15 mg Brij 58/mg microsomal protein) was also dependent upon substrate concentration with values for RMA of 3.3, 6.4 and 9.3 at morphine concentrations of 0.05, 0.5 and 2.5 mM, respectively. Analysis of kinetic data is complicated by substrate concentration-dependent detergent activation. It is proposed that factors contributing to substrate concentration-dependent variable activation may include micellar solubilization of substrate by detergent and/or the presence of at least two enzyme forms capable of glucuronidating morphine with differential effects of detergents on these forms.

Effect of Brij 58 on the hydrolysis of methyl butyrate by lipase from Pseudomonas fluorescens.

Purified Pseudomonas fluorescens lipase [EC 3.1.1.3] exhibited slight activity on water-soluble esters such as methyl butyrate, and this activity was increased on addition of Brij 58 (20 oxyethylene hexadecyl ether) to the solution. This stimulating effect of Brij 58 on hydrolysis of various esters (dimethyl succinate, butyl n-acetate, and tributyrin) in aqueous solution was unspecific. Hydrolysis of methyl butyrate depended on the molecular ratio of Brij 58 to lipase, being maximal (about 8 times the basal level at 37 degrees C with 80 mM substrate in 0.1 M NaCl solution) with 30 mol of Brij 58 per mol of lipase. Comparative studies showed that all polyoxyethylene (POE) alkyl ethers tested, stimulated the methyl butyrate hydrolyzing activity and that the Adekatol SO series (dihydric normal alcohol ethoxylate) also stimulated the appreciably active, whereas Triton X-100, sodium cholate, sodium deoxycholate, sodium dodecylsulfate, POE, and fatty acids had no effect. Comparison of the effects of Brij 58 on the methyl butyrate hydrolyzing activities of various lipolytic enzymes indicated that its effect was specific for this lipase. Brij 58 had no detectable effect with emulsified esters, such as supersaturated methyl butyrate and triolein.

Modulation of the Ca(2+) release channel of sarcoplasmic reticulum by amiloride analogs.

Dichlorobenzamil, phenamil and other amiloride analogs (1-100 microM) elicit transient tension in rabbit skinned muscle fibers. Tension requires preloading of Ca(2+) into the sarcoplasmic reticulum, is facilitated by low-[Mg(2+)] solutions, abolished by ruthenium red or by functional disruption of the sarcoplasmic reticulum, and is followed by inhibition of the caffeine-evoked tension. Bilayer recording of Cs(+) currents through the sarcoplasmic reticulum Ca(2+) release channel reveals that phenamil (10-100 microM) increases the open channel probability, whereas dichlorobenzamil affects the channel activity in a complex concentration- and time-dependent manner: stimulation occurs throughout exposure to 10 microM, but is followed by channel blockade when 100 microM dichlorobenzamil is used. It is concluded that stimulation of the sarcoplasmic reticulum Ca(2+) release channel accounts for the dichlorobenzamil- or phenamil-induced tension in skinned fibers, whereas depletion of sarcoplasmic reticulum Ca(2+) stores and channel block (with dichlorobenzamil) explains the inhibition of the caffeine-evoked tension by amiloride analogs.

Biochemical basis for deficient paracetamol glucuronidation in cats: an interspecies comparison of enzyme constraint in liver microsomes.

Unlike most other mammalian species, domestic cats glucuronidate phenolic compounds poorly and are therefore highly susceptible to the toxic side effects of many drugs, including paracetamol. In this study, we evaluated the role of enzyme constraint, a characteristic that limits the activity of all uridine 5'-diphosphoglucuronosyltransferase (UGT) enzymes, in the aetiology of this species-dependent defect of drug metabolism. Detergent activation experiments were performed using hepatic microsomes from cats (4), dogs (4), man (4), and 6 other mammalian species (1 liver each). In addition, we used microsomes from Gunn rats which are sensitive to paracetamol toxicity because of a genetic defect affecting all family 1 UGTs. Increase in paracetamol-UGT activity at optimum concentrations of detergent was used as an index of enzyme constraint. Native activity (measured in the absence of detergent) was less than one-sixth in cats compared with other species. Optimum detergent treatment tended to enhance rather than abolish this difference, however, indicating relatively lower levels of constraint of paracetamol-UGT in cats compared with other species. Similarly, detergent treatment failed to reduce the native activity difference between homozygous mutant and normal Gunn rats. Initially CHAPS (3-(3-cholamidopropyl)-dimethylammonio-1-propanesulphonic acid) was used as the detergent activator; in 3 of 4 microsomal preparations from man, however, inhibition rather than activation was observed at all detergent concentrations used. Studies were repeated using the non-ionic detergent, Brij 58 (polyoxyethylene 20-cetyl ether), which resulted in similar although more profound activation and no inhibition. We conclude that deficient paracetamol glucuronidation in cats does not result from increased paracetamol-UGT constraint in this species compared with other mammalian species. Other causes, such as differences in enzyme protein concentration or substrate affinity might be responsible.

The effects of detergent on the contractility and ultrastructure of frog skeletal muscle.

The contractility of detergent-treated frog skeletal muscle and its ultrastructure were studied. Triton X-100 and Brij-58 were used as nonionic surface active substances. The concentrations of the detergent solutions were adjusted so that the trains of the twitch tensions would continue for about 10 min. The duration of the twitch tensions was about 10 min when the muscles were soaked in 0.1 mM (0.006%) Triton or in 2.5 mM (0.280%) Brij solution. The twitch tensions did not recover upon returning the muscles to the normal Ringer solution after treatment of the detergents. Rapid cooling of muscle that had been soaked in a 1.0 mM caffeine Ringer solution for 10 min provoked a marked contracture (RCC) in 0.1-0.2 mM Triton-treated preparation. No such contracture was obtained in 1.0-2.5 mM Brij-treated preparation. In ultrastructural findings of the Triton-treated muscle, some structural changes were observed in the transverse tubule and the junctional gap, but not in the terminal cisternae. Brij-treated muscle showed damage of each of these membranes, including the terminal cisternae. These findings suggest that the two detergents have different surface activities on the internal membrane system of the skeletal muscle. The biological activities of the membrane remained even after the treatment of Triton with a suitable concentration.