Tamas Koszegi

Oral magnesium load test in patients with migraine

Objective.—To determine whether migraineurs may have a systemic deficiency of magnesium.

Substance P released from sensory nerve endings influences tear secretion and goblet cell function in the rat

The aim of this study was to present morphological and functional evidence to evaluate whether tear secretion is influenced by neuropeptides released from sensory nerve endings of the conjunctiva. Following unilateral electrical stimulation of the trigeminal ganglion, tears were collected at both sides and assessed for volume and protein concentration; as well as gel electrophoresis and luminol chemiluminescence with immunostaining to immunoglobulin A and lysozyme measurements. Goblet cell density (goblet cells/100 basal cells) was recorded during histopathological examination of removed lids. Rats were pretreated with atropine to block parasympathetic; guanethidine to block sympathetic neuronal pathways; or hexamethonium to block synaptic transmission in ganglia. Capsaicin was used to deplete neurotransmitters from sensory nerve endings or SR140333 to block substance P tachykinin NK1 receptor mediated responses. Effects of inadequate electrode position or incidental lesion of trigeminal ganglion were examined by placing the electrode in false position, or no stimulation at a correct position. Electrical stimulation resulted in 380% increase of tear secretion (p < 0.001) and 30% decrease of goblet cell density (p < 0.001) on the the stimulated side compared to the unstimulated side. Atropine, guanethidine and hexamethonium pretreatments had no effect (p > 0.05), but capsaicin and SR140333 inhibited the effect of stimulation (by 96% and 72%, respectively, p < 0.001). Inadequate stimulation did not increase the tear secretion (p < 0.05). Protein concentration decreased, whilst tear volume and total secreted protein increased (p < 0.005) after stimulation. Electrophoresis showed no difference in protein pattern between stimulated and control side and analysis of equivalent amount of tear protein with luminol chemiluminescence indicated no difference in immunoglobulin A and lysozyme ratio following stimulation (p>0.05). We conclude that antidromic electrical activation of conjunctival sensory nerve endings significantly increases water, mucus and protein phases of tear. It is suggested that the sensory neuropeptide substance P plays a pivotal role in this neurogenic regulatory mechanism.

Immunoluminometric detection of human procalcitonin

Procalcitonin is a normal precursor of the active hormone calcitonin; however, its level in blood is normally undetectable. Dramatically increased levels of procalcitonin have been found in severe systemic bacterial infections and, today, this molecule is considered to be one of the earliest inflammatory markers of sepsis. In spite of the large body of data, it is still uncertain how and from which tissues procalcitonin is released into the circulation. In our experiments, we tested the analytical performance of a flash-type chemiluminescent immunoassay. The precision and accuracy of the assay was acceptable for early detection of sepsis and procalcitonin levels showed predictive information on the outcome of the infections. Using isolated leukocyte subpopulations and acridinium-labelled anti-calcitonin monoclonal antibody, we made attempts to detect procalcitonin inside the cells or in surrounding medium by measuring the chemiluminescent signal triggered after the specific binding of the antibodies had occurred. Our preliminary data showed that lymphocytes did not contain detectable amounts of procalcitonin nor neutrophils secreted it after stimulation. However, neutrophils expressed chemiluminescence of intracellular origin. This finding suggests that neutrophil leukocytes might be a potential source of serum procalcitonin under in vivo conditions.

Optimization of conditions for culture of the test bacteria used for direct bioautographic detection. 1. The gram-positive test bacterium Bacillus subtilis

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.

MAINTENANCE OF IONS, PROTEINS AND WATER IN LENS FIBER CELLS BEFORE AND AFTER TREATMENT WITH NON-IONIC DETERGENTS

If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks ’ balanced salt solution in either absence or presence of non‐ionic detergents (0.2 % Triton X‐100 or 0.2 % Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K+, Mg2 +, Na+, Ca2 +, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X‐100 caused a faster flux rate of all solutes than did Brij 58. The Triton X‐100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 °C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co‐operative ATP‐dependent association between K+, Mg2 +, water and cytomatrix proteins.

Effect of glutamine in patients with esophagus resection.

UNLABELLED Glutamine is the most abundant amino-acid in the extra- and intracellular compartments of the human body, which accounts for over 50% of its free amino-acid content. Utilization of glutamine peptides is explicitly useful, resulting in a decrease in the number of postoperative infectious complications, period of hospitalization, and therapeutic costs. This article aims to study the effects of glutamine on systemic inflammatory response, morbidity, and mortality after esophagectomy. A prospective, randomized, double-blind, and controlled trial was used. Following sealed-envelope block randomization, the patients were divided into two groups. Members of the glutamine group (group G) received glutamine (Dipeptiven, Fresenius) as continuous infusion for 6 hours at 0.5 g/kg for 3 days prior to, and 7 days following surgery; while patients of the control group were given placebo. We examined 30 patients in group G, and 25 patients as controls. In both patient groups, the levels of total protein, albumin, pre-albumin, retinol binding protein, transferrin, transferring-saturation, C-reactive protein, procalcitonin, lymphocte, Interleukin-6, Interleukin-8, tumor necrosis factor alpha, and serum lactate were determined prior to surgery (t(0)), directly after surgery (t(u)), following surgery on day 1 (t(1)), day 2 (t(2)), and day 7 (t(7)). For statistical analysis Mann-Whitney U test and chi-square test were used. There was no significant difference between the two groups regarding age, male/female ratio, and SAPS II scores. Intensive care unit morbidity and mortality was similar in both groups (group G: 24 survivors/6 nonsurvivors; CONTROL 17 survivors/8 nonsurvivors; P= 0.607). Daily Multiple Organ Dysfunction Score did not differ significantly between the two groups. The observed inflammatory markers followed the pattern we described without significant difference. Based on our study, the glutamine supplementation that we used had no influence on morbidity, mortality, or postoperative inflammatory response after esophagectomy.

HPTLC and HPLC determination of alkaloids in poppies subjected to stress

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’, Budakalasz, 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.

Investigating the clinical potential for 14-3-3 zeta protein to serve as a biomarker for epithelial ovarian cancer

ObjectiveRecently, 14-3-3 zeta protein was identified as a potential serum biomarker of epithelial ovarian cancer (EOC). The goal of this study was to investigate the clinical potential of 14-3-3 zeta protein for monitoring EOC progression compared with CA-125 and HE4.DesignProspective follow-up study.SettingUniversity of Pecs Medical Center Department of Obstetrics and Gynecology/Oncology (Pecs, Hungary).PopulationThirteen EOC patients with advanced stage (FIGO IIb-IIIc) epithelial ovarian cancer that underwent radical surgery and received six consecutive cycles of first line chemotherapy (paclitaxel, carboplatin) in 21-day intervals.MethodsPre- and post-chemotherapy computed tomography (CT) scans were performed. Serum levels of CA-125, HE4, and 14-3-3 zeta protein were detected by enzyme-linked immunosorbent assay (ELISA) and quantitative electrochemiluminescence assay (ECLIA).Main outcome measuresSerum levels of CA-125, HE4, and 14-3-3 zeta protein, as well as lesion size according to pre- and post-chemotherapy CT scans.ResultsSerum levels of CA-125 and HE4 were found to significantly decrease following chemotherapy, and this was consistent with the decrease in lesion size detected post-chemotherapy. In contrast, 14-3-3 zeta protein levels did not significantly differ in healthy postmenopausal patients versus EOC patients.ConclusionsDetermination of CA-125 and HE4 serum levels for the determination of the risk of ovarian malignancy algorithm (ROMA) represents a useful tool for the prediction of chemotherapy efficacy for EOC patients. However, levels of 14-3-3 zeta protein were not found to vary significantly as a consequence of treatment. Therefore we question if 14-3-3 zeta protein is a reliable biomarker, which correlates with the clinical behavior of EOC.

Optimization of conditions for culture of test bacteria used for direct bioautographic TLC detection. 2. Gram-negative test bacterium: Escherichia coli

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, ATPassay 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, OD600nm = 0.1–0.4) in the bacterial suspension used for dipping. TLC plates immersed in the logphase 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 OD600nm = 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. – 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 OD600nm = 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.

Optimization of Growth Conditions for Test Fungus Cultures used in Direct Bioautographic TLC Detection. 3. Test Fungus: Candida albicans

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 (OD600nm = 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 microbio-cidal (bactericidal or fungicidal) effects on TLC plates. Our micrographs clearly show the borders of inhibition zones in bioauto-grams. 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.