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Peptidergic innervation of the crop and the effects of an ingested nonpeptidal agonist on longevity in female Musca domestica (Diptera: Muscidae).

Dromyosuppressin (DMS) immunoreactive neurons were discovered running along the crop duct and on the surface of the crop in the house fly, Musca domestica L. DMS is a myoinhibitory neuropeptide that has been shown to inhibit crop contractions, in vitro, in the blow fly, Phormia regina (Meigen), and in Drosophila melanogaster Meigen. Various concentrations of benzethonium chloride (Bztc), an agonist of DMS with shown inhibitory effects on blow fly crop contractions, were fed to adult female M. domestica. Flies exhibited a dose-dependent mortality; avoidance and subsequent dehydration are probably the cause of the low survivorship at higher Bztc concentrations.

Animal Feed↗

[Appraisal of fungistatic and fungicidal activities of quaternary compounds towards yeasts belonging to the genera Candida and Cryptococcus].

The fungistatic and fungicidal activities of five quaternary ammonium compounds towards Candida and Cryptococcus strains were determined using a bacteriological method adapted to mycological experiments. It could be deduced from this study that Cryptococcus in spite of the capsule around the cell were more sensitive than Candida and that the benzethonium chloride (BC) exhibited the most effective action against the whole yeasts. No resistance appeared, only Cryptococcus strain was less sensitive than the others.

Anti-Infective Agents, Local↗

[Protein determination in urine--a critical review].

Seven methods for protein determination in urine were systematically checked, viz. turbidimetric assay with benzethonium chloride, turbidimetry with trichloroacetic acid of various concentrations, micellar complex formation with Coomassie Brilliant Blue G-250, formation and gel filtration of protein complexes with copper, reaction of protein tannin compounds with iron, and biuret reaction after different precipitation procedures. The evaluation was mainly based on the following criteria: identical reaction with various proteins, stable and linear absorbance with concentration, complete recovery, and greatest possible freedom from interferences. On this basis, the biuret reaction must now be regarded as the method of choice. Albumin and gamma-globulins are reliably estimated after precipitation by perchloric acid or by an ethanol-diethylether mixture, whereas all proteins, including uromucoid, may be measured after precipitation by Tsuchiya's reagent. Interference from urinary pigments can be avoided by reading versus a sample blank.

Benzethonium↗

Improved Ellman procedure for erythrocyte cholinesterase.

The procedure of Dietz et al. (Clin. Chem. 19: 1309-1313, 1973) for plasma cholinesterase (EC 3.1.1.7) gives a background absorbance of 1.4 A when extended to erythrocyte cholinesterase (EC 3.1.1.8) measurement, because the peak absorbance of the reaction product, 5-thionitrobenzoate, coincides with the hemoglobin Soret band at 410 nm. Consequently, the precision of erythrocyte cholinesterase measurements is poor, and the test is restricted to laboratories with a spectrophotometer having a high signal-to-noise ratio. Use of the detergent benzethonium chloride (Hyamine 1622) instead of quinidine sulfate to stop enzyme action allows readings to be made at 440 nm because the hemoglobin band is shifted to 405 nm and its peak intensity is decreased. Moreover, detergent micelle interactions shift the peak absorbance of the 5-thionitrobenzoate from 410 to 435 nm. Overall, the blank absorbance is decreased to about 0.4 A. This results in an assay that is twice as precise as the previous version and is suited for use in a routine laboratory with a moderate-quality spectrophotometer. Thus erythrocyte cholinesterase measurements can readily be made, to complement plasma cholinesterase in the investigation of exposure to organophosphates.

Benzethonium↗

Effects of intrathecal saline and ketamine with and without preservative on the spinal nerve roots of monkeys.

Ketamine, a cataleptic analgesic substance which theoretically may block pain receptors in the spinal cord, providing pain relief without respiratory depression, was injected intrathecally with or without benzethonium chloride 0.1 mg/ml (a preservative) into 8 monkeys under anaesthesia. Two monkeys received saline (control group). No assessment as to relief from experimental pain was possible. All Monkeys recovered normally from anaesthesia and were found moving about in their enclosures without gross evidence of neurological impairment. No adverse reactions were noted. Ten days after the subarachnoid injection the monkeys are sacrificed. Autopsy was performed within 30 minutes. No macroscopic abnormality of the cord was noticed. Microscopic examination revealed oedema of a few nerve roots in all animals, irrespective of whether ketamine or saline was injected intrathecally. Focal degeneration with loss of myelin and axoplasm was observed within a solitary nerve root in 2 monkeys that had received ketamine, 1 with and 1 without preservative. However, in these animals lumbar puncture proved difficult and bloody taps ensued. Hence trauma could have been a contributing factor in these 2 cases. None of the other monkeys showed these changes, regardless of group.

Animals↗

Intrathecal ketamine with preservative - histological effects on spinal nerve roots of baboons.

Ketamine, an analgesic-cataleptic drug, provides pain relief without respiratory depression. Ketamine with the preservative benzethonium chloride 0.1 mg/ml was injected intrathecally into 4 baboons under Ketamine anaesthesia. A control group of 2 baboons received intrathecal saline. No assessment as to relief from experimental pain was possible, but all the baboons recovered normally from anaesthesia and were found moving about in their enclosures without gross evidence of neurological impairment. No adverse reactions were noted. One month after the intrathecal injection the baboons were sacrificed, and an autopsy was performed within 30 minutes. No macroscopic abnormally of the cord was noticed. Microscopic examination revealed oedema of a few nerve roots in all animals irrespective of whether ketamine or saline had been injected intrathecally. No other changes attributable to ketamine were seen, and its is therefore concluded that, in the doses given, intrathecally injected ketamine would seem to be safe.

Animals↗

New micro-turbidimetric method for determination of protein in cerebrospinal fluid and urine.

We report a new micro-scale (0.1-mL sample) turbidimetric method for determination of protein by use of benzethonium chloride in alkali. The method is highly specific for protein, has a higher sensitivity than the classic method of Lowry et al., and shows satisfactory reproducibility and recovery. The turbidity produced in our method is the same for albumin and gamma-globulin and is more stable than in Meulemans' method (in which sulfosalicylic acid is used) or in the method of Bossak et al. (in which trichloracetic acid is used). In contrast to Pesce and Strande's method, there is no manipulative loss of protein.

Benzethonium↗

Application of an improved biuret method to the determination of total protein in urine and cerebrospinal fluid without concentration step by use of Hitachi 7170 auto-analyzer.

A biuret automated colorimetric assay for total protein in urine and cerebrospinal fluids was established. The procedures were as follows. Acidify all urine sample before analysis. Add precipitant Na(2)WO(4) to urine samples. After 10 min, centrifuge, decant the supernatant fluid, drain the inverted tubes on absorbent tissue, dissolve the precipitation with 0.1 mol/L NaOH, and finally adapt the reconstituted urine to the Hitachi 7170 analyzer. A cell-free cerebrospinal fluid sample produced by centrifugation can be inserted in an auto-analyzer for protein measurement directly. The program: mix 35 microl sample (CSF or reconstituted urine) and standard with 0.2 mol/L NaOH; incurable at 37 degrees C for 5 min, and real A1. Add concentrated biuret reagent, and 10 min later measure absorbance A2 at 546 nm vs. reagent blank. Secondary wavelength was 700 nm. The test results were calculated against a one-point standard. This biuret colorimetric method was relatively simple, fast, and accurate for the determination of protein in urine and cerebrospinal fluid, with a wide linearity extending from 0.125 g/L up to 6 g/L, had a good correlation with Benzethonium chloride turbidimetry technique, and was a practical routine method.

Albumins↗

Enhancement of ampicillin partition behavior.

Several ion-pair or adduct forming additives tha" enhanced ampicillin partition behavior were identified and evaluated. At pH 3, picric acid and trichloroacetic acid increased the ampicillin aqueous-octanol partition coefficient 250 and 30 times, respectively. At pH 7, quaternary compounds gave the most significant increases in the partition coefficient. Values for an aqueous pH 7 chloroform system increased from zero in the absence of additives to 2.28, 1.86, 1.82, and 1.70 for equimolar amounts of benzalkonium, tetraheptylammonium, benzethonium, and cetalkonium chlorides, respectively. Extraction of ampicillin from aqueous pH 7 solution was possible by adding a quaternary agent in an equimolar amount. However, extraction of ampicillin from plasma required large molar excesses. Tetraheptylammonium chloride was added at a molar concentration 10(3) times greater than that of the ampicillin. Plasma samples spiked at the 3-microgram/ml level gave 93% recovery (CV 6.7%, n = 16) when extracted three times. The extracts were quantitated by TLC.

Ampicillin↗

Purification and properties of dipeptidase from Escherichia coli AJ005.

A Co2+-dependent dipeptidase from E. coli strain AJ005, a peptidase-deficient mutant, was purified with streptomycin sulfate, ammonium sulfate and DEAE-cellulose. The purified dipeptidase increased by about 106-fold in specific activity, with dilysine as a substrate. The dipeptidase cleaved dilysine to two lysines among the lysine homopolymers, the possibility remaining that it is active toward peptides other than dilysine, since it was investigated in the present study only for activity toward lysine homopolymers. Activity was inhibited 54% by 10(-3) M KCN and completely by 10(-3) M PCMB, EDTA and benzethonium chloride, but not at all by soybean trypsin inhibitors. 78% and 95% of its activity was lost with 30 minutes' treatment at 45 degrees C and 50 degrees C, respectively. The apparent Km value was 6.7 X 10(-4) M for dilysine. It is probable that the dipeptidase differs from dipeptidase DP.

Dipeptidases↗

Dipeptidyl peptidases in the soleus muscle of the rat before and after treatment with 5-hydroxytryptamine.

A moderate peptidase activity against L-lysyl-L-proline-4-methoxy-beta-napththylamide was detected histochemically in unfixed sections of soleus muscle fibres of inbred male Wistar rats using two variants of the semipermeable membrane technique. One variant involved simultaneous coupling with tetrazotised 3,3'-dimethoxybenzidine, the other post-coupling. The activity at pH 6 increased approximately three-fold in many fibres showing signs of insult in rats that had been given a single low dose of 5-hydroxytryptamine (10 mg/kg body weight) 48-72 h earlier. The hydroxytryptamine treatment was found to induce a selective myopathy. Some of the increased peptidase activity within insulted muscle fibres appeared to arise from invading mononuclear cells, but the majority seemed endogenous to muscle fibres. The peptidase activity persisted in some fibres 21-28 days after 5-hydroxytryptamine administration, by which time the whole muscle appeared histologically normal. The variation of the activity of the peptidase with pH in the presence of various inhibitors was investigated in both control and insulted muscle fibres. From its sensitivity and behaviour towards Zn2+, Hg2+, Cu2+, puromycin, benzethonium chloride and phenylmethylsulphonyl fluoride and its indifference towards Co2+, Cd2+, Mn2+ and o-phenanthroline, it is concluded that the activity can be attributed to a mixture of at least two peptidases, dipeptidyl peptidase II and an unidentified neutral dipeptidyl peptidase. The possible role of the peptidase(s) in muscle regeneration in discussed.

Animals↗

Acid proteinase of hypothalamus. Purification, some properties, and action on somatostatin and substance P.

In a continuing study of the physiological role of protein breakdown in the hypothalamus, acid proteinase from bovine hypothalamus was purified about 1000-fold. The molecular weight of the enzyme was approximately 50,000. Masimal activity against hemoglobin was obtained at pH 3.2-3.5; serum albumin was split much more slowly. Hypothalamus acid proteinase was partially inhibited by beta-phenyl pyruvate, or benzethonium Cl, and was completely inhibited by low concentrations of pepstatin. This proteinase splits somatostatin, substance P, and analogs of substance P. The probable sites of enzyme action on these peptides were determined by the end group dansyl technique. The enzyme, most likely cathepsin D, may play an important role in the formation and breakdown of peptide hormones in the hypothalamus.

Animals↗

Human and bovine brain cathepsin L and cathepsin H: purification, physico-chemical properties, and specificity.

Cathespin L (EC 3.4.22.15) and cathepsin H (EC 3.4.22.16) have been purified from brain cortex to apparent homogeneity by a simultaneous procedure involving acid extraction of homogenate at pH 4.2, ammonium sulfate fractionation (30-80%), chromatography on pepstatin-Sepharose, CM-Sephadex C-50, DEAE-Sephadex A-50, phenyl- and concanavalin A-Sepharose and isoelectric focusing. Cathepsin L and cathepsin H were assayed in the presence of dithiothreitol and Na2EDTA (2 mM each) with Z-Phe-Arg-NHMec (pH 5.5) and Lys-NNa (pH 6.5) respectively. Cathepsin L consists of 2 polypeptide chains with Mr 25,000 and 5,000, Mr of cathepsin H is 28,000. Cathepsin L exists in brain tissue in two multiple forms with pI values 5.7 and 5.9, pI of cathepsin H is 6.8. Substrate specificity of these thiol proteinases was tested with proteins (pyridoxyl-hemoglobin, azocasein) and low Mr naphthylamide and methylcoumarylamide substrates: Lys-NNa, Arg-NNa, Dz-Arg-NNa, Z-Arg-Arg-NNaOMe, Z-Phe-Arg-NHMec, Z-Phe, Val-Arg-NHMec, Z-Gly-Gly-Arg-NHMec. Z-Phe-Arg-NHMec is the best substrate for cathepsin L (KM = 5 microM, Kcat = 21 s-1), Arg-NNa--for cathepsin H (KM = 0.1 mM, Kcat = 1.93 s-1), being endoaminopeptidase cathepsin H also hydrolyses Bz-Arg-NNa (KM = 0.7 mM, Kcat = 1.3 s-1). Both proteinases are inhibited by traditional inhibitors of cysteine proteinases and E-64, but leupeptin turned to be more effective inhibitor of cathepsin L (Ki = 2.4 nM) than of cathepsin H (Ki = 9.2 microM), the latter enzyme being sensitive to puromycin and benzethonium chloride as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Influence of the oversulfation method and the degree of sulfation on the anticoagulant properties of dermatan sulfate derivatives.

Six oversulfated dermatan sulfate (DS) derivatives, differing in their tissue origin (porcine skin, bovine and porcine intestinal mucosa), and the oversulfation method of preparation, have been tested for their anticoagulant properties. In the first method, the SO3-trimethylamine complex is added to a DS sodium salt dissolved in formamide while it is added to a DS-benzethonium salt dissolved in dimethyl formamide in the second method. The rate of sulfation of these compounds ranged from 7.8 to 11.5 percent of sulfur on a weight basis, whereas it is 6% and 12% for natural DS and for heparin respectively. The anticoagulant potency was assessed by determining the catalytic effect of each glycosaminoglycan on the inhibition of thrombin added to (i) plasma (ii) purified heparin cofactor II(HC II) or (iii) purified antithrombin III(AT III). The catalytic effect on Factor Xa inhibition in the presence of AT III has also been investigated. The increased sulfation is found to enhance the antithrombin activity of the native dermatan sulfate whatever the method used, while the Factor Xa inhibition by AT III could be catalysed only by the most sulfated derivative obtained by the second method. The two derivatives which were less oversulfated, by the first oversulfation method, exhibit equal or even higher catalytic effects on thrombin inhibition when compared to the four other derivatives. The use of the first oversulfation method provides slightly oversulfated derivatives which exhibit strong anticoagulant properties and may constitute effective antithrombotic drugs with no bleeding tendency, a side effect perhaps related to a high rate of sulfation.

Animals↗

Absence of role for plasmids in resistance to multiple disinfectants in three strains of bacteria.

Three chlorhexidine-resistant strains, two of Achromobacter xylosoxidans, one of Pseudomonas cepacia, were isolated from clinical sources. The three strains showed cross-resistance to benzethonium chloride, benzalkonium chloride and alkyldiaminoethylglycine. Resistance persisted after treatment of the strains with acridine orange (12.5 mg-25 mg l-1). Conjugation to appropriate recipients of Escherichia coli was unsuccessful and plasmid DNA could not be detected. These results suggest that plasmids do not play a role in resistance to multiple disinfectants in the strains of bacteria studied.

Alcaligenes↗

The analysis of quaternary ammonium compounds in human urine by direct inlet electron impact ionization mass spectrometry.

An accurate and rapid screening test for nine quaternary ammonium compounds (suxamethonium chloride, pancuronium bromide, ambenonium chloride, benzethonium chloride, distigmine bromide, methylbenactyzium bromide, neostigmine bromide, propantheline bromide and pyridostigmine bromide) by direct inlet electron impact ionization mass spectrometry (DI/EI-MS) was investigated. Each compound was extracted from urine as an ion pair with KI3 into dichloromethane. The reliability of the identification of these compounds was verified by the mass chromatographic analysis of their characteristic fragment ions. The analysis of these drugs by DI/EI-MS could be performed within 5 min. The detection limits were between 20-150 ng/ml for the nine compounds. This method appears to be efficient, rapid and suitable as a screening procedure for the quaternary ammonium compounds found in urine.

Gas Chromatography-Mass Spectrometry↗

Potentiometric titration of thiols, cationic surfactants and halides using a solid-state silver-silver sulphide electrode.

A rugged, low resistance silver-silver sulphide solid-state electrode for determining pharmaceuticals as authentic samples or in dosage forms by potentiometric titration is described. Sodium tetraphenylborate, mercury(II) acetate and silver nitrate (0.01) M were employed as titrants in the analysis of cationic surfactants (cetylpyridinium chloride, benzethonium chloride, benzalkonium chloride and chlorhexidine salts), antithyroid drugs (methimazole and propylthiouracil) or sodium halides respectively.

Journal Article↗

Determination of benzalkonium chloride in contact lens solutions by positive-ion fast atom bombardment mass spectrometry.

Under positive-ion fast atom bombardment (FAB) mass spectrometric conditions, benzalkonium chloride (BAK) afforded intense peaks at m/z 304 and 332, corresponding to the intact cations [M--Cl]+ of C12 and C14 homologues, respectively. The use of benzethonium chloride as an internal standard and thioglycerol as a FAB matrix allowed the direct and specific determination of the BAK content (0.004-0.020%) in commercial hard contact lens solutions through the individual assay of the two alkyl homologues. A linear relationship between the homologue concentration and the peak-area ratio was observed over the concentration range 3-180 micrograms ml-1.

Benzalkonium Compounds↗