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Summary
Arthritis of major joints, especially osteoarthritis of the knee is a very frequent disease of human beings mainly in the developed countries. The pathology of osteoarthritis has been subject of many publications before, using a wide spectrum of different methods to evaluate degenerative changes of hyaline cartilage. The authors examined osteoarthritic human knee joint hyaline cartilage with differential scanning calorimetry. The different stages of cartilage degeneration have been verified by histological examinations. The research group demonstrated thermal differences between various stages of osteoarthritis. Besides explaining possible causes for experienced thermodynamic effects, the authors reflect upon future research ways and the possibilities of applying the method in practice.
Papaver somniferum produces secondary metabolites which have important roles in self-defense processes, in plant biochemistry, and in allelochemistry. By performing experiments to determine how irregular stress effects changed the alkaloid content of poppies we have shown that different types of stress affect the quantities of alkaloids. Papaver somniferum (cv. ‘Kék Duna’, Budakalász, Hungary) plants were grown for 2 months, from seeds, in quartz-sand (natural light, temperature 24–28°C, in Knopf ’s nutritive solution). In this work we studied alkaloids in poppies subjected to two kinds of stress — mycotoxin and drought. Amounts of alkaloids were measured by different separation and detection procedures — thin-layer chromatography (TLC and HPTLC) with fluorescence detection, and high-performance liquid chromatography (HPLC). HPLC proved superior for identification and approximate estimation of the morphine alkaloids, but the effects of stress on poppy plants can be detected by use of either method. Drought stress resulted in higher levels of the alkaloids whereas mycotoxin stress did not result in significant differences.
The main purpose of this study was to determine optimum conditions for culture of a test microbe Bacillus subtilis (ATCC 6633) which enabled us to establish its use for direct bioautography. The viability of the bacteria on TLC plates was measured on the basis of their adenosine-5′-triphosphate (ATP) content as determined by bioluminescent luciferin/luciferase assay, the data being referred to values for total bacterial protein. In the first experiments, we used a ‘20-h’ culture of B. subtilis prepared by dilution of an optical density ( OD ) ≫ 0.4 culture to furnish a culture of OD = 0.4 (Method A). Later, on the basis of our optimization experiments we found that a ‘5–9-h’ broth culture of B. subtilis was suitable. Under these conditions the bacteria remained in the log phase ( OD = 0.2–0.4) for 5–9 h (Method B) in immersion bacterial suspension. Because the test bacteria were in the log phase a much shorter incubation time (4–8 h) was sufficient for TLC plates instead of the original 18 h in a previous study. One advantage of this method, in addition to the shorter incubation time, is that we can use TLC plates coated with adsorbents other than silica.
Direct bioautography is a potent means of obtaining information about the antimicrobial activity of a compound separated from a complex mixture. In this process the developed TLC plate is dipped into a broth culture of a test bacterium and the bacterium will grow directly on the plate. Optimum experimental conditions must, however, be used for each test bacterium.The main purpose of this study was to find optimum culture conditions for a Gram-negative test bacterium, Escherichia coli (ATCC 25922) enabling us to establish a direct bioautographic method with the shortest possible performance time. Because the intracellular adenosine-5′-triphosphate (ATP) level is a direct and sensitive measure of bacterial well-being, ATP assay was used for this purpose. As far as we know this is the first report of the use of an ATP method for optimization of direct bioautography with E. coli . Our optimizing experiments on E. coli culture showed that the bacteria had to be in the log phase (optical density, OD 600nm = 0.1–0.4) in the bacterial suspension used for dipping. TLC plates immersed in the log-phase culture needed a shorter incubation time for bacterial growth on the TLC plate (3 h) than for the original ‘overnight’ culturing suggested in studies by others.In this paper we will show that:
- ATP assay is a valid method for optimizing E. coli direct bioautography. Bacterial ATP level oscillates during the growth phase in culture media. TLC plates should be immersed in E. coli dipping suspension with OD 600nm = 0.1–0.4. Dipping a developed TLC plate for 10 s gave acceptable results. Incubation of the seeded TLC plate at 37°C for 3 h was found to be optimum. An ATP/protein ratio of 10–15 nmol mg −1 in dipping culture and ∼5 nmol mg −1 on seeded TLC plates were the minimum threshold values for visualization of living bacteria by means of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reaction. With our optimized coditions the total performance time of E. coli direct bioautography is 9.6 h instead of the originally reported 11.5 h. Our procedure results in much sharper contrast of the inhibition zone than that without optimization.
Abstract
Meniscus degeneration is a very frequent disease of human beings mainly in the developed countries. The ability of the meniscus to participate in load bearing, shock absorption, joint lubrication, and joint stability depends on the maintenance of its structural integrity. Therefore the pathology of the degeneration has been subject of many publications before. These studies all agreed that the grade of the degeneration correlated with the patient’s age, weight, profession, and athletic activity [1]. These reviews also described the biochemical changes in the structure, too [2, 3]. In the current study authors examined degenerated human meniscus with differential scanning calorimetry and demonstrated thermal differences between healthy and intraoperatively removed pathological samples.
Summary
Resection and subsequent end-to-end anastomosis of the windpipe is a tried-and-tested acceptable method for the surgical treatment of segmental defects. There are a variety of different techniques for tracheal end-to-end anastomosis, but controversial reports highlight the fact that the suturing technique of the anastomosis is still subject of debate. We aimed to show the postoperative effects of the continuous and simple interrupted suturing technique respectively on the tracheal cartilage using differential scanning calorimetry. Transsection and subsequent reanastomosis of the cervical trachea was performed in 14 adult beagle dogs. The trachea was anastomized with continuous or simple interrupted sutures respectively depict no change in microcirculation after the resection of the trachea, but significant decrease following the completion the anastomosis with continuous sutures. Conventional histological analysis did not show any marked postoperative change in the tracheal cartilage but our DSC scans clearly demonstrated the differences between the intact cartilages and the ones involved in the anastomosis.
Abstract
Melanoma malignum (MM) is a common type of skin cancer, and its incidence is increasing in the general population. We aimed to detect blood plasma components with differential scanning calorimetry (DSC) in 15 white adult MM patients, who had histopathologically diagnosed, operable cutaneous MM without any distant metastases. We observed that thermal changes (second T m , calorimetric enthalpy) in blood plasma showed correlation with tumor thickness and the extent of regional invasion. Further studies are needed to elucidate these relationships, but our preliminary work has provided DSC should be a new tool for the early diagnosis and monitoring of MM patients.
The chemical composition of the oils of four thyme (Lamiaceae) chemotypes ( Thymus vulgaris L., Thymus serpyllum L., Thymus x citriodorus (Pers.) Schreb., and Thymus x citriodorus “Archer’s Gold”) has been determined by gas chromatography (GC). Thymol was the main component of the oils of Thymus vulgaris and Thymus serpyllum , geraniol was the main component of the oil of Thymus x citriodorus , and carvacrol was the main component of the oil of Thymus x citriodorus “Archer’s Gold”. The bioactivity of the volatile oil of Thymus vulgaris and of the three main components of the oils against Gram negative plant pathogenic bacteria was examined by direct bioautography. They had an inhibitory effect on all of the test microorganisms. Two bacterial strains ( Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. phaseolicola ) were most sensitive in the bioautographic system and use of these bacteria would considerably shorten the process of bioautographic detection. The two antibiotics gentamycin and streptomycin were used as controls.
Optimum conditions have been established for culture of the fungus Candida albicans (ATCC 90028) for microbial detection of zones in direct bioautographic TLC. Bioluminescent ATP assay is a highly sensitive method for optimizing the viability of Candida albicans test fungus in bioautographic TLC detection. A suspension of microbes (OD 600nm = 0.5–0.7, in Mueller-Hinton broth with 5% glucose) in the log phase of growth can be used for dipping TLC plates. On the basis of our results with Candida albicans , we can differentiate between microbiostatic (bacteriostatic or fungistatic) and microbiocidal (bactericidal or fungicidal) effects on TLC plates. Our micrographs clearly show the borders of inhibition zones in bioautograms. This technique leads to new possibilities in studies of the interactions between microbes and antimicrobial compounds on bioautographic silica gel TLC plates by using scanning electron microscopy. In this paper we describe an optimization procedure for bioautographic TLC detection.
