HPLC method for determination of fluorescence derivatives of cortisol,cortisone and their tetrahydro- and allo-tetrahydro-metabolites in biological fluids

11β-Hydroxysteroid dehydrogenase isoform 2 (11β-HSD2) is responsible for conversion of cortisol (F) to inactive cortisone (E). Disturbance of its activity can cause hypertension. To estimate 11β-HSD2 activity, besides F and E, their tetrahydro- (THF, THE) as well allo-tetrahydro- (allo-THF, allo-THE) metabolites should be determined. This study describes HPLC-FLD method for the quantitative determination of endogenous glucocorticoids (GCs) in plasma and urine (total and free) and their metabolites in urine. Following extraction at pH 7.4 using dichloromethane, GCs (F, E, THF, allo-THF, THE, allo-THE and internal standard – prednisolone) were derivatized with 9-anthroyl nitrile and purified by SPE using C₁₈ cartridges. The enzymatic hydrolysis of conjugated steroids was provided using β-glucuronidase. The influence of organic bases on 9-AN derivatization of steroids was investigated. The best yield of the derivatization was obtained in presence of the mixture of 10.0% triethylamine (TEA) and 0.1% quinuclidine (Q). Chromatographic separation was accomplished in the Chromolith RP-18e monolithic column. The elaborated method was validated. Calibration curves were linear in the ranges: for F, E and THF 5.0–1000.0 ng mL^?1, for allo-THF and THE + allo-THE 10.0–1000.0 ng mL^?1. LOD (S/N = 3:1) for all analytes amounted 3.0 ng mL^?1. Recoveries of GCs exceeded 90%. The method was precise and accurate, intra- and inter-day precision were 3.0–12.1% and 9.2–14.0%, respectively. Accuracy ranged from 0.2 to 15.1%. The method was applied for estimating endogenous GCs in plasma and urine. Plasma levels of F and E were in the ranges: 133.0–174.5 ng mL^?1 and 17.4–35.9 ng mL^?1, respectively. Free urinary steroids were in the ranges: 12.0–54.1 μg/24 h (UFF) and 37.8–76.2 μg/24 h (UFE). The ratio of (THF + allo-THF)/(THE + allo-THE) amounted from 1.01 to 1.23. The obtained results confirmed utility of the elaborated method in the assessment of 11β-HSD2 activity in man.     

The role of GABA(A) and GABA(B) receptors in the control of GnRH release in anestrous ewes

The paper reviews data concerning the involvement of GABA(A) and GABA(B) receptors in the control of GnRH secretion in anestrous ewes. Generally, GABA influences the GnRH release through GABA(A) and GABA(B) receptors located on perikaria of the GnRH neurons in the preoptic area (MPOA) or through the influence on beta-endorphinergic and catecholaminergic systems activity in MPOA and in ventromedial-infundibular region of the hypothalamus (VEN/NI). Stimulation of GABA(A) receptors in VEN/NI and MPOA attenuates GnRH release, while activation of GABA(B) receptors in MPOA decreases GnRH secretion, and in VEN/NI increases concentration of GnRH. The different neural mechanisms could be involved in this process: direct ligand action on the GABA(A) and GABA(B) receptors located on GnRH cells and axon terminals or indirect effect through the changes in the beta-endorphinergic and catecholaminergic systems activity in these structures of the brain.     

Cellular source in ewes of prostaglandin-endoperoxide synthase-2 in uterine arteries following stimulation with lipopolysaccharide.

Prostaglandin-endoperoxide synthase (PTGS) (also known as cyclooxygenase) converts arachidonic acid into several prostaglandins, many of which have roles in vasodilation and vasoconstriction under normal and pathological conditions. There are two isoforms of PTGS: PTGS-1 and PTGS-2; PTGS-1 is constitutively expressed in many tissues and is believed to be involved in the homeostatic maintenance of the body. In contrast, PTGS-2 is believed to have a "differentiative" role in the cells and is highly inducible during inflammation and in response to lipopolysaccharide (LPS). Endothelial cells as well as vascular smooth muscle cells can be a source of PTGS within the artery. The objective of this study was to determine the cell population(s) in uterine arteries that respond to LPS with an increase in PTGS-2 protein expression. Uterine arteries collected from ewes during the follicular (Day 0, Day 0 = estrus, n = 4) or luteal (Day 10, n = 4) phase were treated in vitro with LPS as intact artery segments, cut-open artery segments, or cut-open and denuded (endothelial cells absent) artery segments. After 24 h of LPS treatment, intact, cut-open, and denuded uterine artery segments were collected into homogenization buffer for determination of PTGS-2 protein levels by Western blot analysis. The culture medium was collected and used for detection of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), the stable metabolite of prostacyclin, using an enzyme immunoassay. In addition, the location of PTGS-2 after LPS treatment was analyzed by immunohistochemistry in intact artery segments. Denuded arteries (endothelium absent) did not show increases in PTGS-2 protein in the homogenates or 6-keto-PGF(1alpha) in the culture medium after LPS exposure. In contrast, cut uterine arteries responded to LPS stimulation with a significant increase in PTGS-2 protein in homogenates and 6-keto-PGF(1alpha) in culture medium. Immunohistochemical staining for PTGS-2 was associated with both endothelial cells and vascular smooth muscle cells. These results suggest that while both endothelial cells and vascular smooth muscle cells are associated with PTGS-2, after LPS exposure it is the endothelial cells that are essential in uterine artery increases in PTGS-2 and prostacyclin in response to LPS stimulation.     

Effect of antisense peptide binding on the dimerization of human cystatin C--gel electrophoresis and molecular modeling studies

Human cystatin C (HCC) shows a tendency to dimerize. This process is particularly easy in the case of the L68Q HCC mutant and might lead to formation of amyloid deposits in brain arteries of young adults. Our purpose was to find ligands of monomeric HCC that can prevent its dimerization. Eleven antisense peptide ligands of monomeric HCC were designed and synthesized. The influence of these ligands on HCC dimerization was studied using gel electrophoresis and molecular modeling methods. The results suggest that all the designed peptides interact with monomeric HCC facilitating its dimerization rather than preventing it.     

Phenotypic characteristics of coagulase-negative Staphylococci colonizing pleural drains in patients with lung cancer after thoracic surgery

Coagulase-negative staphylococci (CNS) represent an important group of etiologic agents of infections associated with plastic biomaterials, e.g. drains. In the present paper 33 strains of CNS were characterized. All of them were isolated from fluid of pleural drains in patients with lung cancer after pulmonary resection under conditions of antimicrobial prophylaxis. The most frequently isolated species were Staphylococcus epidermidis and S. warneri. The majority of CNS strains showed ability to produce slime and possessed hydrophobic properties of cell surface. Strains of CNS resistant to penicillin and oxacillin, but sensitive to amoxicillin/clavulanate were isolated most frequently. Only two methicillin-resistant strains, belonging to S. haemolyticus, were found. The obtained data indicate that CNS strains colonizing pleural drains had potential ability to adhere to smooth surfaces. Most of isolated strains were susceptible to antibiotics and chemotherapeutics routinely used in staphylococcal infections.     

Schalenmerkmale verkieselter Ostracoden: Sind die ordovizischen Duplikaturen nur Artefakte oder eher evolutionäre Vorversuche?

The post-Silurian, podocopine duplicature (calcified inner lamella) is reviewed, its phylogeny and ecological importance are shown. Taking the relevant literature into account, the phenomenon ofGramm’s Ordovician, “mesostene duplicature” is discussed. Features of the free margin shown in some Ordovician, podocopine species which were obtained from silicified (or silicified and calcium fluorite converted) shells, are either artefacts or results of optical illusions.Gramm’s conception of the “mesoplatic duplicature”, i.a. the marginal thickening in palaeocopine species, however, corresponds toBecker’s model of evolutionary dead-ends, after which the species level is determined to be the favoured experimental stage of the evolution (in concrete terms, the development of the free margin) in times of increased phylogenetic stress.     

Investigation of the salt tolerance of new Polish bread and durum wheat cultivars

In some regions of the world, low annual precipitation necessitates irrigation of crop plants which usually leads to soil salinity. Due to climatic changes this effect is also expected in the countries of Central Europe, and so in Poland. The aim of the study was (1) to compare tolerance to salt stress of Polish Triticum aestivum cvs. ‘Bogatka’ and ‘Banderola’ with T. durum cv. ‘Komnata’ and breeding line 121, and (2) to indicate the physiological parameter/parameters most suitable for such comparison. The investigation was performed in two experiments. In the first one, the germination ability of caryopses and coleoptiles’ growth were estimated at 0–250 mM of NaCl. The second experiment was conducted on plants grown in a glasshouse in saline soil at 0–150 mM of NaCl for 6 weeks. Salt tolerance was evaluated on the basis of following parameters: chlorophyll fluorescence, net photosynthesis rate (P _N), transpiration rate (E), stomatal conductance (g _s), cell membrane permeability (EL), proline content, fresh weight (FW), dry weight (DW), and relative water content (RWC). Highest germination of caryopses of durum cultivars was recorded at all the salinity levels; however, their coleoptiles were shorter than coleoptiles of bread wheat cultivars. Analysis of chlorophyll fluorescence showed that applied salt doses did not disturb the light phase of photosynthesis in all cultivars under study. Plants of durum wheat showed the higher dissipation of energy excess at the level of the antenna chlorophyll (DIo/CSm) under salinity as compared to plants of bread wheat. Both ‘Komnata’ and line 121 showed stronger P _N reduction as an effect of salinity. A decline of P _N was closely connected with a decrease in g _s. The P _N correlated with a decrease in DW in all studied cultivars except ‘Bogatka’. Control plants of ‘Komnata’ and line 121 were characterized by higher EL and proline level than bread wheat cultivars. An increasing cell membrane permeability correlated with a decrease of RWC in ‘Banderola’ and ‘Komnata’. The content of proline under the increasing salinity correlated with changes of RWC in ‘Banderola’, ‘Komnata’ and line 121, which indicate protectoral role of proline against dehydration of tissue. Dry weight and RWC seem to be the parameters most useful in the salt-tolerance estimation of wheat plants. Taking into account the studied parameters ‘Banderola’ could be recognized as more salt tolerant, the degree of salinity tolerance of ‘Bogatka’ is the same as line 121, while ‘Komnata’ seems to be the most salt sensitive. The salt tolerance of T. aestivum and T. durum depends on the cultivar rather than the wheat species.