Effect of organic N-halamines on selected membrane functions in intact Staphylococcus aureus cells

Two N-halamine compounds, 3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone, were compared with free chlorine as to their effects on selected membrane functions of intact Staphylococcus aureus cells. Free chlorine was found to cause a loss of permeability control, as measured by the efflux of potassium from the cells and a dramatic increase in hydrogen ion permeability, and to affect cell respiration in a nonreversible fashion, as measured by oxygen uptake. The two N-halamines were found to have very little effect on permeability to either potassium or hydrogen ions but were both found to dramatically inhibit respiration in a reversible manner. It is proposed that the first step in the disinfection process by these N-halamines is an inhibition of respiratory enzymes that, if not reversed, ultimately leads to a loss of cell viability.     

Effect of organic N-halamines on selected membrane functions in intact Staphylococcus aureus cells.

Two N-halamine compounds, 3-chloro-4,4-dimethyl-2-oxazolidinone and 1,3-dichloro-4,4,5,5-tetramethyl-2-imidazolidinone, were compared with free chlorine as to their effects on selected membrane functions of intact Staphylococcus aureus cells. Free chlorine was found to cause a loss of permeability control, as measured by the efflux of potassium from the cells and a dramatic increase in hydrogen ion permeability, and to affect cell respiration in a nonreversible fashion, as measured by oxygen uptake. The two N-halamines were found to have very little effect on permeability to either potassium or hydrogen ions but were both found to dramatically inhibit respiration in a reversible manner. It is proposed that the first step in the disinfection process by these N-halamines is an inhibition of respiratory enzymes that, if not reversed, ultimately leads to a loss of cell viability.     

Bactericidal properties of an organic N-chloramine formed in situ

Agent I (3-chloro-4,4-dimethyl-2-oxazolidinone) formed in situ was compared with pre-formed agent I as a disinfectant against Staphylococcus aureus. In situ formation involved combining the non-chlorinated oxazolidinone precursor with calcium hypochlorite to form 5 and 10 mg/l total chlorine concentrations of agent I. The variables included in the study were temperature, pH and concentration. Overall the bacteria were killed more rapidly at 22 degrees than at 4 degrees C. The in situ formation appeared to occur most rapidly at pH 7.0, slightly slower at pH 9.5, and very slowly at pH 4.5 as evidenced by the presence of residual free chlorine. In the in situ experimental runs the 5 and 10 mg/l concentrations were equally effective in obtaining a six log decline in cfu/ml. This study indicates the potential for using the organic N-chloramine as a general purpose disinfectant while omitting the laboratory synthesis of the final product.     

Bactericidal properties of an organic N-chloramine formed in situ

Agent I (3-chloro-4,4-dimethyl-2-oxazolidinone) formed in situ was compared with pre-formed agent I as a disinfectant against Staphylococcus aureus. In situ formation involved combining the non-chlorinated oxazolidinone precursor with calcium hypochlorite to form 5 and 10 mg/l total chlorine concentrations of agent I. The variables included in the study were temperature, pH and concentration. Overall the bacteria were killed more rapidly at 22° than at 4°C. The in situ formation appeared to occur most rapidly at pH 7˙0, slightly slower at pH 9˙5, and very slowly at pH 4˙5 as evidenced by the presence of residual free chlorine. In the in situ experimental runs the 5 and 10 mg/l concentrations were equally effective in obtaining a six log decline in cfu/ml. This study indicates the potential for using the organic N-chloramine as a general purpose disinfectant while omitting the laboratory synthesis of the final product.     

Bactericidal and bacteriostatic antigonococcal activities produced by urogenital staphylococci

We have overcome some of the difficulties in obtaining soluble antigonococcal activity produced by staphylococci by using a very sensitive detection method. This method is based on the light absorbance determinations of liquid cultures of the gonococcus incubated for 6 h in the presence of serial dilutions of the inhibitor as compared to the absorbance of uninhibited control cultures. Antigonococcal activity was detected in the liquid phase prepared from semisolid agar cultures of all twenty two staphylococcal isolates tested. Sixteen supernatants from liquid cultures were also found to be active. The antigonococcal activity detected was differentiated by colony forming units counts into two types, bacteriostatic and bactericidal. After 6 h of incubation of the gonococcus in the presence of five arbitrary units (AU)/ml of the bactericidal activity produced by one of the strains of staphylococci, isolate 37, the loss of viability was over 99.9%, while 10 AU/ml of the bacteriostatic activity produced by isolate 66 did not cause any loss of viability of the gonococcus.     

Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin

Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.     

Prediction of biodegradability for selected organic chemicals

Summary The 5-day BODs of 45 organic chemicals were determined using acclimated mixed microbial cultures. These chemicals included alcohols, acids, esters, ketones, aromatics and miscellaneous compounds. The BOD data were correlated with (1) water solubilities, (2) log of 1-octanol/water partition coefficients, (3) molar refractivities and volumes, (4) melting (m.p.) and boiling points, (5) number of carbon (C No.), hydrogen and oxygen atoms, (6) molecular weights, and (7) theoretical (Th) BODs of chemicals. Linear and secondorder polynomial regression analyses were used; the latter was also attempted with two or more independent variables. All prediction equations were compared for statistical merits. The equations, one from each regression type, with the highest prediction power were: log 5-day mmol BOD/mmol chemical=(1)–0.183+0.813 (log ThBOD), (2)–0.391+1.560 (log ThBOD) –0.532 (log ThBOD)², and (3) –0.4060+0.2470 (C No.) –0.0133 (C No.)²–0.0005 (m.p.). The measured BOD data for 43 additional chemicals were compared with the predicted values calculated through the above equations. The three equations predicted the BODs for 84–88% of the test chemicals within 80% of the experimental values. The mean percent relative standard deviations between predicted and experimental BOD values were statistically compared for these equations, and no significant difference (P0.01) in their predictive utility was found. The acclimation potential of an autochthonous microbial community cannot yet be predicted, but this study demonstrates that the process of active biodegradation for structurally dissimilar chemicals appears to correlate quantitatively with certain physicochemical parameters.     

Comparison of ligninolytic activities of selected white-rot fungi

Summary Six fast growing ligninolytic white-rot fungi were compared with Phanerochaete chrysosporium. The results showed that the fungi have similar ligninolytic systems, although minor differences exist. Like in P. chrysosporium the ligninolytic system could be induced by veratryl alcohol in Coriolus versicolor and Chrysosporium pruinosum. These three lignin peroxidase producing fungi were the fastest lignin degraders in stationary cultures, whereas in agitated cultures Bjerkandera adusta showed highest lignin degradation rates. Metabolites accumulating during the degradation of veratryl alcohol were analyzed and compared. Peroxidase production seems to be a common feature of all the tested fungi. Polyclonal antibodies against the lignin peroxidase with pl of 4.65 from P. chrysosporium reacted with the extracellular peroxidases of C. pruinosum, C. versicolor and B. adusta, but not with those of Pleurotus ostreatus.Dedicated to Professor Dr. Hans-Jürgen Rehm on the occasion of his 60th birthday     

Effect of selected dimethylaminochalcones on some mitochondrial activities

Chalcones and their synthetic cyclic analogues have been shown to possess a full scale of biological activities in a variety of experimental systems. They were assessed to be mostly effective in defense against free radicals in the organism, but several compounds exhibited cytotoxic pro-oxidant activities. The respiratory response and antioxidant status in mitochondria were investigated upon addition of 4′-dimethylaminochalcone (1a) and its cyclic analogues, (E)-2-(4′-((CH₃)₂?N)-benzylidene)-1-indanone (1b), -1-tetralone (1c), and -1-benzosuberone (1d). Selected structures were able to change the respiratory response of mitochondria and showed an ability to modify mitochondrial metabolic and redox efficiency, though they did not indicate redox reactivity towards glutathione in adduct-free incubations. The results of the study indicate that -chalcone and -tetralone derivatives cause suppression of reactive oxygen species affecting mitochondrial respiration by mild uncoupling. In addition, (E)-2-(4′-((CH₃)₂?N)-indanone (1b), and to a greater extent, -benzosuberone (1d), showed pro-oxidant effects, which partially explain their cytotoxicity.     

Biocatalysis of lipoxygenase in selected organic solvent media

The biocatalysis of purified soybean lipoxygenase (LOX) (EC 1.13.11.12), using linoleic acid as a substrate model, was investigated in selected organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene. The results indicated that there was a 2.6-fold increase in LOX activity in the monophasic iso-octane medium compared to that obtained in the aqueous medium. The results also showed that there was an increase of 2.2- and 1.8-fold in LOX activity in the monophasic reaction media of octane and hexane, respectively. However, an inhibitory effect on enzyme activity was observed when the monophasic reaction media of toluene, chloroform and dichloromethane were used. In addition, the results showed that the optimum concentration of octane and iso-octane in the biphasic medium containing the organic solvent and Tris–HCl buffer solution, was determined to be 3.5% and 4%, respectively, for LOX activity. Moreover, the biocatalysis of LOX in a ternary micellar system, containing either 3.5% octane or 4% iso-octane, Tris–HCl buffer solution and an emulsifier, resulted in an overall increase in enzyme activity. The K_m and V_max values, substrate specificity, optimum protein concentration, optimum reaction temperature as well as the enzymatically catalyzed end-products were investigated for LOX biocatalysis in both ternary micellar systems.     

Investigation of inorganic deposits in selected organic matrices

Inorganic deposits in the wall of human and animal arteries and in experimental tumor (Morris hepatoma 7777) were examined using proton induced X-ray emission (PIXE) and PIXE in combination with proton microprobe (micro-PIXE) techniques. The sections adjacent to the irradiated ones part were submitted to histological investigations and one part of the material was additionally investigated by infrared (IR) spectroscopy. For identification of mineral deposits, the micro-PIXE method appeared the most sensitive. The mineral deposits were detected in the artery samples, even in those without visible morphological changes, as well as in tumor samples. The deposites showed different localization and composition, depending on age and type of vessel. There were also differences between human and animal arteries. IR spectroscopy revealed the presence of carbonate apatite within the artery samples from old individuals. Matching of histological observations with data obtained by micro-PIXE method allows a better correlation of morphological and analytical results.     

Growth yields of bacteria on selected organic compounds

Cell yields were determined for two bacterial soil isolants grown aerobically in minimal media on a variety of synthetic organic compounds. 1-Dodecanol, benzoic acid, phenylacetic acid, phenylglyoxylic acid, and diethylene, triethylene, and tetraethylene glycols were tested. Two “biochemicals,” succinate and acetate, were also tested for comparison. Yields were calculated on the basis of grams of cells obtained per mole of substrate utilized, gram atom of carbon utilized, mole of oxygen consumed, and equivalent of “available electrons” in the substrates. This latter value appears to be nearly constant at 3 g of cells per equivalent of “available electrons.” Yields predicted on this basis for other bacteria and for yeasts on other substrates are in fair agreement with reported values.     

Bactericidal activity of alkyl peroxyl radicals generated by heme-iron-catalyzed decomposition of organic peroxides.

To clarify the nature of cytocidal molecular species among the radicals generated in the iron-catalyzed reactions of peroxides (ROOH), we examined the cytocidal effects of these radicals against gram-positive and gram-negative bacteria in the presence or absence of various radical scavengers. Three organic peroxides, t-butyl hydroperoxide (t-BuOOH), methyl ethyl ketone peroxide (MEKOOH), and cumene hydroperoxide, were used. Each radical generated from these peroxides was identified and quantitated by electron spin resonance (ESR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The major cytotoxic radical species generated in the mixtures of various peroxides and heme iron, especially methemoglobin, metmyoglobin, or hemin, was the alkyl peroxyl radical (ROO.). Strong bactericidal action against gram-positive bacteria was observed in the peroxide-heme iron system, especially in the case of t-BuOOH and MEKOOH. Killing curves for gram-positive bacteria showed an initial lag period, which may indicate the multihit/multitarget kinetics of cell killing. When the diethylenetriamine pentaacetic acid (DTPA)-Fe2+ complex was used as a catalyst for decomposition of various peroxides, alkyl, alkoxyl, and alkyl peroxyl radicals were identified by spin-trapping analysis. However, study of the time course of alkyl peroxyl radical production in the DTPA-Fe2+ complex system revealed that radical species generated in this system were very short lived: a maximal level was achieved within 1 min and then declined sharply, and no bactericidal activity was observed after 10 min. In contrast, the alkyl peroxyl radical level generated by the organic peroxide-heme iron system remained high for 30 min or longer. The generation of alkyl peroxyl radicals quantified by ESR correlated quite well with the bactericidal effect of the system of peroxide plus iron. In addition, bactericidal activity was completely inhibited by the addition of the spin trap DMPO, as well as of other various radical scavengers (alpha-tocopherol and L-ascorbic acid), into the peroxide-heme iron system, but this effect was not observed with superoxide dismutase, beta-carotene, dimethyl sulfoxide, diphenylamine, or butylated hydroxyltoluene. In view of these results, it is assumed that alkyl peroxyl radicals are the potent molecular species that are cytotoxic against bacteria, whereas alkoxyl radicals (RO.) generated in this system do not affect bacterial viability.     

Effects of paraquat on selected microbial activities in soil

Paraquat, applied as Gramoxone, to a nonamended sandy loam soil at five times the suggested field application rate (10 lb/A 115g/cm²) increased the numbers of bacteria, actinomycetes, and fungi during a 14-day incubation at 25°C. This increase was attributed to the use of compounds in the Gramoxone formulation rather than the use of paraquat. Treatment at one and five times the normal rate reduced CO₂ evolution by 44% and 67%, respectively, in soil amended with 2% glucose during a 12-day incubation. Similar treatments reduced CO₂ evolution in 1% straw-amended soil by 39% and 58%, respectively, during a 28-day incubation. Cellulose decomposition of cotton duck containing 13 and 176g of paraquat per milligram of material was inhibited for 15 and 28 days, respectively, in soil containing a large population of cellulolytic microorganisms. A concentration of 5000g/gm of paraquat was necessary to inhibit nitrification in soil by 44% druing a 28-day incubation at 20°C. Paraquat inhibited C₂H₂ reduction in artificial aggregates of soil amended with 2% glucose and incubated anaerobically at 25°C. Nitrogenase activity in aggregates was inhibited by 43% and 52% at concentrations of 580 and 720g/gm of paraquat respectively. The inhibitory effects of the herbicide were reduced when soil was amended with organic matter in the form of peat or straw. The availability of paraquat controlled the toxicity of the herbicide to soil microorganisms.     

Antioxidative and free radical scavenging activities of selected medicinal herbs

The antioxidative and superoxide- and hydroxyl radical-scavenging activities and pro-oxidant effect of twelve selected medicinal herbs were studied. The aqueous extracts of Coptis chinensis, Paeonia suffruticosa, Prunella vulgaris and Senecio scandens exhibited the highest potency in inhibiting rat erythrocyte hemolysis and lipid peroxidation in rat kidney and brain homogenates. The aforementioned four herbs also demonstrated strong superoxide- and hydroxyl radical-scavenging activity, but exerted only a slight pro-oxidant effect.     

Immobilization and biocatalysis of chlorophyllase in selected organic solvent systems

Chlorophyllase extract from Phaeodactylum tricornutum was immobilized by physical adsorption on DEAE-cellulose and silica gel as well as by covalent binding on Eupergit C, Eupergit C250L, Eupergit C/ethylenediamine (EDA) and Eupergit C250L/EDA. Although the highest immobilization yield (83-93%) and efficiency (51-53%) were obtained when chlorophyllase extract was immobilized on DEAE-cellulose and silica gel, there was no improvement in the thermal stability of chlorophyllase as compared to that of the free one. The immobilization of chlorophyllase extract on Eupergit C250L/EDA resulted by a high recovery of enzymatic activity, with an immobilization efficiency of 44%, and promoted a higher stabilization of chlorophyllase (four times) in the aqueous/miscible organic solvent medium. On the other hand, the inhibitory effect of refined bleached deodorized (RBD) canola oil was reduced by immobilization of chlorophyllase extract onto silica gel as compared to those obtained with other enzyme preparations. However, the re-cycled chlorophyllase extract immobilized on Eupergit C250L/EDA retained more than 75% of its initial enzyme activity after 6 cycles, whereas that immobilized on silica gel was completely inactivated. The highest catalytic efficiency, for both free and immobilized chlorophyllase on Eupergit C250L/EDA, was obtained in the ternary micellar system as compared to the aqueous/miscible organic solvent and biphasic media.