Visual micromethod for assay of fungal growth

A visual micromethod for measuring antifungal effects on germination and growth is described. The antifungal agent griseofulvin and the fungus Trichophyton mentagrophytes were used as materials to compare the micromethod with a standard assay based on dry mycelial weight. The micromethod was more sensitive than the weight method in detecting the minimum inhibitory concentration of griseofulvin (0.18 and 35 microgram/ml, respectively). At higher concentrations of griseofulvin (22.5 microgram/ml), the micromethod measured minimal fungal growth that was undetectable on a weight basis. The micromethod showed that griseofulvin does not change the number of spores forming germ tubes. Progressively severe alterations in fungal morphology occured as the concentration of griseofulvin was increased from 0.09 to 22.5 microgram/ml.     

Cellular chemiluminescence of activated phagocytes of whole blood

Both, the phagocytic process and the activation of phagocytes with soluble stimuli are accompanied by increased production of reactive oxygen species (ROS). Chemiluminescence (CL) measurement is a simple and sensitive method for the detection of ROS generation. Phagocytes (mainly polymorphonuclear leukocytes, PMNL) were stimulated with soluble stimulus or via phagocytosis in diluted whole blood, and the generation of Luminol-enhanced CL was registered. The time dependence of CL, determined in whole blood, corresponds to the CL from isolated leukocytes. A relationship between peak CL and the number of leukocytes as well as of PMNL was observed. The specific CL, i.e. the CL response related to a defined PMNL number, increases with the age of investigated healthy individuals. No correlations were found between CL and the capacity of PMNL to ingest zymosan particles. Relations between CL and spontaneous platelet aggregation suggest, that reactivity of blood platelets may be a contributing factor to the kinetics of the CL signal in our test system. The inhibition of CL by the sulphydryl reagents diamide and fever few extract indicate the role of cellular sulphydryl groups for phagocyte function. Measurement of CL in whole blood is proved to be a simple assay for assessment of PMNL function and allows measurements in very small blood samples (greater than or equal to 10 ul).     

Chemiluminescence induced by phagocytosis of Escherichia coli by polymorphonuclear leucocytes

Chemiluminescence emitted by phagocytosing human polymorphonuclear leucocytes stimulated by Escherichia coli was measured using a liquid scintillation counter equipped with a multichannel analyser. In the presence of the amplifying agent luminol, light emission can be divided into two channels, one of which ('high energy') appears to correlate directly with phagocytic activity of the PMNL, and the other ('low energy') with the background luminol dioxygenation by the cells. Measuring in the 'high energy' window also eliminates the normal 'out of coincidence' background. The method is applicable to measuring opsonizing capacity of different sera, and responds to PMNL number, age, composition of assay medium and the integrity of the stimulating bacteria. Other bacterial strains produce a similar response, as does the artificial stimulator zymosan. Low temperature and anaerobiosis, which inhibit phagocytic killing, also suppress light emission.     

Estimation of viable biomass in aerobic biodegradation processes of organic fraction of municipal solid waste (MSW)

2-(p-Iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) dehydrogenase test and RNA assay were introduced to evaluate biomass in the processes of aerobic biodegradation of the organic fraction of municipal solid waste (MSW) in bioreactors. It was found that RNA quantification by KOH/UV method delivered reliable and repeatable results. Relative standard deviation (RSD) for INT test was significantly higher than for RNA assay and achieved values of 3-15%. Moreover, it occurred that the optimum temperature for the growth of autochthonic biomass, which takes part in the biodegradation process, was in the range from 25 to 37 degrees C.     

Detection of antigen of rickettsia of the tick group in bird droppings by antibody neutralization test]

The author presents methodical materials pointing to a possibility of using a micromethod of the antibody neutralization test for detection in bird pellets of the antigen of rickettsia referred to the Rocky Mountains spotted fever tick group. Specificity of the test, rickettsia antigen and sera was studied. The method is recommended for detection at the territory under study of circulation of the causative agent of tick-borne rickettsiosis by revealing the specific antigen in bird pellets.     

Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase

A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis.     

A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays

A simplified method for inorganic phosphate determination has been developed. The method is sensitive, easy, economic, and applicable for estimation of phosphate released in both enzymatic and nonenzymatic reactions. A mixture of hydrazine sulfate and ascorbic acid was used as the reducing agent and the conditions for the development of the molybdenum blue color were optimized. Thus in the 4.0 ml assay system, 0.4 ml of the reducing agent solution containing 20 mg each of hydrazine sulfate and ascorbic acid per milliliter of 1.0 N H2SO4 gave a rapid optimum color development with absorption maximum at 820 nm. Color development showed a linear relationship up to 10 microg Pi concentration. Thus the method has a 2.5x higher range of Pi estimation than that of the Bartlett method. The molar extinction coefficient at 820 nm was higher than that obtained in the Bartlett procedure. Also the molybdenum blue color formed was stable up to 24 h. Under the standard assay conditions, interference from acid-labile phosphate as in the case of Na+,K+ ATPase was at the minimum. The applicability of the method for assay of microsomal Na+,K+ ATPase and glucose-6-phosphatase was checked in microassays (final volume 0.1 ml) in comparison to the conventional procedures which use 3-4 times higher volumes. Likewise the applicability of the method for phospholipid analysis was compared with that of the conventional Bartlett method. Under both test systems the results obtained by the micromethod were identical to those obtained by the conventional methods. In general the method, which rapidly produces quantitatively molybdenum blue color, not only is rapid economical, and convenient but also has wide applicability.     

Effect of stobadine on opsonized zymosan stimulated generation of reactive oxygen species in human blood cells

To predict more precisely the effect of stobadine, a pyridoindole antioxidant agent, in the whole organism, we studied its effect on opsonized zymosan-stimulated free radical generation in whole blood, on superoxide generation in the mixture of PMNL : platelets (1:50), as well as on superoxide generation and myeloperoxidase release in isolated PMNL. Without stimulation, stobadine had no effect on reactive oxygen species (ROS) generation and myeloperoxidase release. Stobadine in a concentration of 10 or 100 micromol/l significantly decreased luminol-enhanced chemiluminescence in opsonized zymosan-stimulated whole blood. In concentrations of 10 and 100 micromol/l, it reduced myeloperoxidase release from isolated neutrophils. Stobadine significantly decreased superoxide generation in isolated neutrophils in 100 micromol/l concentration. Its effect was much less pronounced in the mixture of neutrophils and platelets in the ratio close to physiological conditions (1:50). Our results suggest that stobadine might exert a beneficial effect in diseases or states where superfluous ROS generation could be deleterious.     

Rapid enumeration of viable Legionella pneumophila serogroup 1

A rapid assay for the detection of viable Legionella pneumophila serogroup 1 was evaluated. A total of 431 environmental water samples were examined using an immunofluorescent assay (IFA) combined with the cell respiration stain iodonitrotetrazolium violet (INT) and the results compared with conventional culture. The IFA/INT assay was at least as sensitive and much quicker than culture for the detection of viable Legionella pneumophila serogroup 1 in most types of sample.     

A micromethod for the quantitation of cellular proteins in Percoll with the Coomassie brilliant blue dye-binding assay

A simple procedure for the determination of cellular proteins in Percoll-containing samples is described. Percoll precipitated when particulate proteins were solubilized by dilution of the samples in a NaOH-Triton X-100 mixture. After centrifugation at high speed (12,000g), the supernatant was assayed for proteins with the Coomassie brilliant blue dye-binding assay. With an automatic spectrophotometer, 50-microliter aliquots gave a linear response between 0 and 3 micrograms of bovine serum albumin. After a fivefold dilution in the alkali-detergent mixture, proteins in samples containing up to at least 60% Percoll can be accurately quantitated on a standard curve prepared in the absence of Percoll. Because the sensitivity of the assay was better than 100 ng, the procedure outlined in this paper can also be used as a general protein micromethod.     

An NADH-tetrazolium-coupled sensitive assay for malate dehydrogenase in mitochondria and crude tissue homogenates

A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.     

A micromethod for agglutination and antiglobulin tests for antibody to Brucella abortus

A micromethod has been developed for the standard agglutination and antihuman globulin tests for serum antibodies to Brucella abortus. The test uses a haematoxylin-stained antigen. The test is simple and quick to perform and, in a comparison at two centres involving 367 sera, the micromethod was found to be more sensitive than the classical test. It is highly suitable both for routine serological screening and for large-scale surveys of occupationally exposed industrial groups.     

Quantitative analysis of cholesterol in 5 to 20 microliter of plasma

A gas-liquid chromatographic micromethod for quantitation of cholesterol in 20 micro l of plasma was developed using 5alpha-cholestane as an internal standard, saponification with tetramethylammonium hydroxide-isopropanol, and extraction with tetrachloroethylene-methyl butyrate. Cholesterol levels in plasma samples were calculated by comparing cholesterol-cholestane peak height ratios with those of preassayed reference plasma. Over a plasma cholesterol range of 44 to 468 mg/100 ml, the gas-liquid chromatographic micromethod and the automated ferric chloride colorimetric method gave nearly identical results (r = 0.99) in duplicate aliquots of 131 plasma samples.     

Depression of polymorphonuclear leukocyte functions by purified influenza virus hemagglutinin and sialic acid-binding lectins

Infection of polymorphonuclear leukocytes (PMNL) with influenza virus causes depression of PMNL metabolic and bactericidal activities. The studies reported here were undertaken to determine whether the hemagglutinin (HA) glycoprotein of influenza virus mediates this depression. PMNL were incubated with purified HA and the oxidative responses to exogenous stimuli were measured. The results indicate that HA, in either liposomes or protein aggregates referred to as rosettes, depressed PMNL oxidative responses. Depression was observed within 2 min of initial interaction of HA with PMNL and lasted more than 2 h. The membrane fusion activity of HA requires proteolytic cleavage of the HA, whereas the receptor binding activity does not. There was no difference in the ability of virions with cleaved or uncleaved HA to depress PMNL responses suggesting that the fusion event is not required for PMNL dysfunction. Inasmuch as the HA glycoprotein binds to sialic acid-containing receptors on the surface of the PMNL, we tested whether other sialic acid-specific binding proteins can mediate the reduction of PMNL responses. Sialic acid-specific lectins from Limulus polyphemus or Limax flavus were incubated with PMNL before measuring their responses to secondary stimulus. Depression was observed upon incubation with the lectins similar to that seen upon incubation with the HA or influenza virus. These results suggest that attachment of influenza virus to sialic acid-containing receptors is responsible at least in part, for suppressing PMNL oxidative responses.     

Melphalan-induced DNA damage in p53(+/-) and wild type mice analysed by the comet assay

Melphalan is an alkylating substance used as a therapeutic agent; its mutagenicity is related to its ability to produce monoadducts and to form DNA cross-links. The alkaline comet assay is a useful test for the detection of DNA lesions. However, cross-links are not easily detected under standard conditions. Recently, modifications to the test have been introduced to measure cross-links by evaluating the reduction in induced DNA migration.

In this work, the standard comet assay and an assay modified by prolonging the electrophoresis time have been applied to evaluate DNA lesions induced by single, 4 or 26 weekly oral administrations of melphalan to p53^+/− knockout and to isotype parental mice. Cells were analysed from the liver, bone marrow, peripheral blood and the distal intestine. Moreover, a further protocol in which the presence of cross-links was inferred by the reduction in X-ray-induced DNA migration was applied to bone marrow cells and the sensitivity of the different methods was compared.

The majority of groups examined by the standard protocol showed no difference compared to controls, while the modified protocol (prolonged electrophoresis time) could detect a retarded DNA migration in cells from all the organs analysed with the exception of bone marrow cells.

Only the protocol based on X-ray in vitro irradiation showed the presence of melphalan-induced cross-links in bone marrow cells exposed to 2 mg/kg for 4 weeks, demonstrating that this was the most sensitive approach for detecting this type of lesion.

DNA lesions were evident in all the organs analysed. However, results suggest that the kinetics of cross-link repair could be different in bone marrow cells compared to other organs tested. After comparison between genotype-matched treated and control groups, a significant effect was shown more frequently in p53^+/− than in wild type groups.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Effects of statins on oxidative stress and primed polymorphonuclear leukocytes in hyperlipidemic patients

Inflammation and oxidative stress are among the factors that have been implicated in the pathogenesis of hyperlipidemia. In metabolic syndrome and hyperlipidemic patients, peripheral polymorphonuclear leukocytes (PMNL) are primed and they release uncontrolled superoxide that contributes to oxidative stress and inflammation. Recent studies have demonstrated that the anti-hyperlipidemic drug, Atrovastatin effects improvement in endothelial function, exhibits anti-oxidative characteristics and reduces lipid markers of oxidation. To evaluate possible nontraditional effects of treatment with Atrovastatin on PMNL priming, oxidative stress and inflammation in hyperlipidemia, 50 non-smoking hyperlipidemic patients were treated for 6 months with Atrovastatin and compared to age and gender-matched healthy controls. PMNL priming was assessed by the rate of superoxide release from separated, phorbol ester-stimulated PMNL and by PMNL-CD11b levels. Inflammation was reflected by blood inflammatory markers including albumin, transferrin, C-reactive protein (CRP) and fibrinogen levels, white blood cells (WBC), PMNL counts and PMNL apoptosis. Atrovastatin treatment showed a reduction in PMNL priming, PMNL apoptosis, fibrinogen and CRP levels concomitant with decreased lipid levels. Atrovastatin may be preferred for hyperlipidemic patients owing to its combined anti-PMNL priming and anti-inflammatory effects in addition to its anti-atherogenic effects.     

Measurement of whole blood phagocyte chemiluminescence in the Wistar rat

The factors influencing the rat whole blood chemiluminescence (CL): concentrations of blood, luminol, zymosan or opsonized zymosan, volume of the reaction mixture, storage time of blood samples and the presence of anticoagulants were evaluated. The CL micromethod described provides a fast and sensitive tool for the determination of metabolic activity of phagocytes in the microlitre range of rat whole blood. © 1997 John Wiley & Sons, Ltd.     

Subpopulations of neutrophils with increased oxidative product formation in blood of patients with infection

Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2-dependent oxidative product formation, the intracellular oxidation of 2',7'-dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely between patients (range 0 to 80%). This variable subpopulation may explain the variability of mean responsiveness of the PMNL of patients reported previously. Moreover, the marked increase in oxidative metabolic capability of the primed PMNL may be a significant component of the host response to acute infection. It could also contribute to the damage to host tissues such as pulmonary vascular endothelium during bacteremia.     

Two short, daily activity bouts vs. one long bout: are health and fitness improvements similar over twelve and twenty-four weeks?

This study sought to determine whether a 12-week intermittent (INT; 2 x 15 min.d(-1)) exercise program yielded similar improvements in cardiovascular health and fitness, compared with a traditional 12-week, 30-minute continuous (CON; 1 x 30 min.d(-1)) exercise program. A second purpose was to determine the effects of switching exercise programs and continuing training for an additional 12 weeks. Twenty women and 17 men, (age 48.8 +/- 9.0 years) were divided randomly into 2 groups: INT (n = 20) and CON (n = 17). Aerobic exercise was performed 4 d.wk(-1) for 12 weeks. Subjects then crossed over to the opposite training program for an additional 12 weeks of training. Subjects exercised incrementally for weeks 1-4 and training was conducted at 70-80% heart rate reserve for weeks 5-24. Both groups showed comparable exercise adherence, completing 96.6 +/- 12.2% (CON) and 96.3% +/- 17.7% (INT) of the prescribed exercise time. The INT walked at a lower percentage of Vo(2)max, maximum heart rate, systolic blood pressure, and diastolic blood pressure (p < 0.05). Maximal oxygen consumption increased by 4.5% in CON and by 8.7% in INT. Following the second 12 weeks, Vo(2)max increased by 3.6 and 7.7% in CON and INT, respectively. Treadmill test time increased by 41 seconds in CON (p < 0.05) and 71 seconds in INT (p < 0.05) after 12 weeks of training. High-density lipoproteins significantly increased in the INT group following the first 12 weeks of training. This study suggests that an INT exercise program, which is incremental in nature, provides comparable, and in some cases greater, health and fitness benefits than those expected following traditional CON exercise training.