Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci

When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.     

Effect of antioxidants on chemiluminescence produced by reactive oxygen species

Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.     

One-step homogeneous non-stripping chemiluminescence metal immunoassay based on catalytic activity of gold nanoparticles

The catalytic activity of gold nanoparticles (AuNPs) on a luminol–H₂O₂ chemiluminescence (CL) system is found to be greatly enhanced after its crosslinking aggregation induced by immunoreaction. Based on this observation, a one-step homogeneous non-stripping CL metalloimmunoassay was designed. In the presence of corresponding antigen (Ag), the immunoreaction caused the aggregation of antibody (Ab)-modified AuNPs, and these crosslinking aggregated AuNPs could catalyze luminol–H₂O₂ CL reaction to produce a much stronger CL signal than dispersed Ab-modified AuNPs. The assay, including immunoreaction and detection, can be accomplished in homogeneous solution. In the assay, no tedious and strict stripping of metal nanoparticles, difficult synthesis of labels, multiple steps of immunoreactions and washings, and complicated magnetic separation process were required. The detection limit of human immunoglobulin G (IgG, 3σ) was estimated to be as low as 3.2 × 10⁻¹¹ g ml⁻¹. The sensitivity was increased by two orders of magnitude over that of other AuNP-based CL immunoassay. The current CL metalloimmunoassay offers the advantages of being simple, cheap, rapid, and sensitive.     

Bacteriostatic activity of serum against staphylococci

Cybulska, Janina (State Institute of Hygiene, Warsaw, Poland), and J. Jeljaszewicz. Bacteriostatic activity of serum against staphylococci. J. Bacteriol. 91:953-962. 1966.-Antistaphylococcal activity of normal serum against strains exhibiting various patterns of coagulase, clumping-factor, and staphylokinase production is not connected with the presence of these factors. Purified coagulase does not influence this property of serum. Coagulase-negative strains with clumping-factor activity grow in normal serum as typical pathogenic staphylococci. Serum bacteriostatic activity against staphylococci may be reversed by several nonspecific factors, such as sterile broth, supernatant fluids of coagulase-negative strains, and ammonium sulfate precipitates of culture supernatant fluids of various staphylococci. Immune sera with a high agglutinating titer for staphylococcal cells do not prevent growth of serum-resistant strains; serum-susceptible strains are inhibited as in normal serum control. Activation or blocking of the serum fibrinolytic system does not influence serum bacteriostatic activity. The growth rate of serum-resistant strains is identical in serum and in Todd-Hewitt broth; serum-susceptible strains are inhibited to the inoculum level, but decreases and increases in viable count are noted during a 24-hr observation period. Observations made with sera of 10 animal species clearly demonstrated differences in serum bacteriostatic activity, mouse serum being completely noninhibitory and cat serum only weakly inhibitory. The technique of quantitative determination of serum susceptibility of staphylococci is described, and the importance of serum antistaphylococcal activity in vitro is discussed. Experimental staphylococcal infection produced in rabbits by intravenous injection of different Staphylococcus aureus strains did not result in significant changes in serum antistaphylococcal activity. The technique of experimental infection used caused chronic infection, with a peak on the 14th day; this was proved by means of a newly developed 5'-nucleotidase test. At the same time, sera of infected animals exhibited slight inhibitory properties, which returned to initial values 1 week later. Infection was produced by strains recognized as nonpathogenic and was inhibited in vitro by sera from both normal and infected rabbits. It is concluded that antistaphylococcal activity of serum should be considered as an "in vitro" phenomenon, which seems to have no importance in defense mechanisms of rabbits infected intravenously with staphylococci.     

Comparative study of antioxidants as quenchers or scavengers of reactive oxygen species based on quenching of MCLA-dependent chemiluminescence.

The quenching or scavenging effect of non-enzymatic antioxidants against reactive oxygen species (ROS) was studied by comparing the degree of suppression of chemiluminescence (CL) caused by the oxidation of MCLA (methoxylated Cypridina luciferin analogue) by ROS. MCLA-dependent CL caused by O2- was effectively quenched by ascorbic acid, beta-carotene, lycopene and astaxanthin, while it was enhanced by alpha-tocopherol. The CL by 1O2 was quenched effectively by beta-carotene, lycopene and astaxanthin, moderately by ascorbic acid, and slightly by alpha-tocopherol. beta-Carotene and alpha-tocopherol remarkably suppressed the CL when ROS was HO*. The present study revealed that MCLA-dependent CL assay provides a simple and rapid method for the evaluation of antioxidants as a quencher or scavenger against any kind of ROS.     

Acetyl-CoA-synthetase activity of pigmented staphylococci]

In 19 strains of staphylococci a study was made of the activity of acetyl-CoA-synthetase reaction. All the strains possessed an active enzymatic system transforming the acetate into an active form. The activity of acetyl-CoA-synthetase proved to be much greater in the pigmented staphyloccus strains than in the nonpigmented ones. It is supposed that there existed an association between the acetyl-CoA-synthetase and the biogenesis of carotinoid pigments in Staph. aureus.     

线粒体电子传递链电子漏的化学发光测定

本实验用差速离心法分离正常大鼠肝脏和心肌线粒体 ,以lucigenin (探测超氧阴离子 )与luminol (探测过氧化氢 )为探剂 ,用化学发光法测定METC电子漏的生成。在反应体系中加入外源底物 ,其发光强度明显高于空白对照 (体系中无线粒体 )。在肝线粒体体系中 ,无论是lucigenin还是luminol诱发的发光 ,琥珀酸底物引起的发光强均要高于丙酮酸 /苹果酸引起的发光强度。在心肌线粒体 luminol体系中也有与肝线粒体相似的结果 ,在心肌线粒体 lucigenin体系中 ,加入外源底物丙酮酸 /苹果酸诱发的发光强度高于琥珀酸诱发的发光强度     

Classification of staphylococci based on chemical and biochemical properties

Summary In the present work the chemical cell wall composition and some other biochemical characteristics were studied in staphylococci with the intention of utilizing the data obtained in their classification.According to the cell wall peptidoglycans and teichoic acids, the 130 strains of staphylococci studied are divided into 10 major groups. This division of staphylococci into groups is in good agreement with their present classification only in some cases. All of the 47Staphylococcus aureus strains contain a cell wall peptidoglycan of thel-Lys-Gly_5–6 type and ribitol teichoic acid. Coagulase-negative staphylococci are more heterogeneous and are divided according to their cell wall composition into 9 major groups. 21 strains of them are classified asS. epidermidis sensu stricto. They form a natural group and are distinguished by the occurrence of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 peptidoglycan type, glycerol teichoic acid and anl-lactate dehydrogenase which is activated by fructose-1,6-diphosphate. 8 strains with peptidoglycan of thel-Lys-Gly_4–5,l-Ser_0.5–1.8 type and ribitol teichoic acid are labeled asS. saprophyticus. The remaining groups have not been given species names and require further extensive comparative study.     

Detection of pregnancy in a hibernator based on activity data

Pregnancy and parturition are often difficult to detect in wild-living animals, especially in species that give birth to altricial young in burrows or caves. Current methods to detect pregnancy and birth in hibernating animals often entail disturbances that can affect the animals' reproductive success. We developed a new method to confirm pregnancy and parturition in hibernating brown bears (Ursus arctos) using activity data recorded in dual-axis motion sensors mounted on GPS–GSM neck collars on 30 brown bears during 52 hibernation seasons in Sweden. We adjusted recorded activity to the individual basal activity level for each bear. Pregnant females showed characteristic activity patterns during the period of pregnancy, with significantly higher daily activity levels and frequency of active periods during pregnancy than nonpregnant bears. Pregnant females were active on average 2.20 times more often during the pregnancy period than during the postpartum period, compared with 0.97 times for nonpregnant females. A pregnancy index was defined as the average of the proportion of mean daily activity levels and the proportion of activity events during the pregnancy period compared with the postpartum period. It averaged 2.61?±?1.73 (SD) for pregnant females and 0.94?±?0.24 for nonpregnant females. Using this method, we evaluated a group of adult females that had an unverified reproductive status but were classified as not pregnant because no cubs had been observed after den emergence. The results suggested that 35 % of those females had, in fact, been pregnant.     

An assessment of the inhibitory activity of rat serum on staphylococci

A study has been made on the effect of rat serum on staphylococci. By following the respiration and growth of 5 strains ofStaphylococcus aureus and an equal number ofS. epidermidis in fresh, normal rat serum, we found thatS. aureus grew in, and oxidized rat serum better thanS. epidermidis. Difference in growth was not correlated with nutritional requirements. The antibacterial agent of fresh, normal, undiluted rat serum was stable to heating at 56 C for 1 hr, but its activity was completely destroyed after heating at 60 C for 2 hr. A 50 per cent dilution of rat serum with nutrient broth significantly reduced the antibacterial activity and treatment of rat serum with 0.4m solutions of sodium citrate reduced it drastically. Once the serum had been treated with sodium citrate, or oxalate, addition of equimolar solutions of calcium chloride or magnesium chloride failed to restore the antibacterial activity. Addition of ferric ions in high concentrations allowed the coagulase-negative strains of staphylococci to grow well in this serum. The antibacterial agent of rat serum was absorbed by heat-killed cells ofStaphylococcus aureus andS. epidermidis but not byStreptococcus pyogenes andEscherichia coli. Treatment of rat serum with bentonite at a concentration of 100 mg per ml decreased its antibacterial activity.     

Quantitative indices of lysozyme activity in staphylococci]

The lysozyme activity of 354 lysozyme-positive and 100 lysozyme-negative (by the results of qualitative test) staphylococcus strains were studied quantitatively. The method was based on titration of the lysozyme in the culture fluid of 48-hour broth cultures of the strains under study. The quantitative method proved to be more sensitive than the qualitative one, and permitted to reveal the lysozyme production in 71% of the strains which were formerly considered to be lysozyme-negative. There were distinct species differences between the lysozyme-positive staphylococci: the mean lysozyme level in the S. aureus was significantly greater then in the S. epidermis. There was no regular association between the lysozyme activity, staphylococcus origin, bacteriophage reference and the antibiotic resistance.