Changes in growth hormone secretion and leptin receptor mRNA expression under the influence of leptin and adrenocorticotropin in pituitary cells of early weaned ewe lambs.

Early weaning of ewe lambs strongly stimulates the hypothalamic-pituitary-adrenal axis and is associated with suppressed growth rate despite the increased food intake. At the same time, plasma leptin concentration increases only slightly or undetectably. To better understand this atypical interdependence among somatic stress, leptin, and lamb growth rate, we analyzed impact of leptin and/or adrenocorticotropic hormone (ACTH) on growth hormone (GH) secretion as well as the effect of ACTH on mRNA expression of two splice variants of leptin receptor (LEPRa, LEPRb) in pituitary cells isolated from early weaned ewe lambs. The GH secretion under the influence of leptin and/or ACTH depended on the timing of exposure and hormone concentration. After 6 - 30 h, GH secretion increased under 10-11 - 10-8 M leptin (P ≤ 0.05). However, after 24 - 30 h, GH secretion significantly increased only in cells exposed to both leptin and ACTH compared to culture with leptin only. Simultaneously, there was a significant (P ≤ 0.05) decrease in leptin receptor mRNA expression under the influence of ACTH at 10-8 - 10-6 M after 12 - 30 and 24 - 30 h for LEPRa and LEPRb, respectively. ACTH-related downregulation of LEPR mRNA was associated with a significant (P ≤ 0.05) reduction in leptin-stimulated GH secretion, also after 24 - 30 hours. Thus, the timing of ACTH exposure, followed by decreased leptin receptor mRNA, converged with the timing of decreased GH secretion under the influence of leptin with ACTH. The ACTH-induced downregulation of LEPR mRNA therefore may underlie the decrease in GH. These results show a direct role for leptin, ACTH, and leptin receptor expression in modulation of pituitary GH secretion in early weaned ewe lambs. During the early weaning-induced stress response, the ACTH-mediated decrease in sensitivity of pituitary cells to leptin may abolish a stimulatory effect of leptin on GH secretion and explain in part, the reduction in lamb growth rate.

Association of CAST gene polymorphism with carcass value and meat quality in two synthetic lines of sheep.

The aim of study was to estimate genotype frequencies of the polymorphism in the intron 12 of CAST gene of growing lambs, and to evaluate associations between the CAST genotype and carcass and meat traits. A total number of 317 rams representing the synthetic BCP (163) and SCP (154) meaty lines were genotyped by the RFLP/MspI/NcoI/Hin6I. Three genotypes including "aa", "ae" and "ac" with most frequencies of 0.48, 0.27 and 0.19, were observed in whole population. Lambs with the "aa" CAST genotype showed the highest muscle percentage and the lowest fat percentage in the hind legs. On the other hand, the loins of rams with "ac" genotype had the highest intramuscular fat content, indicative of beneficial health-related properties and technological usefulness. However, differences between CAST genotype stated for the sensory properties, texture and thawing and cooking losses of the meat were insignificant. Breeding efforts aimed at the improvement of lamb meat quality should prioritize the selection of the "ac" genotype.

Comparative characterization of surface sludge deposits from fourteen French Vertical Flow Constructed Wetlands sewage treatment plants using biological, chemical and thermal indices.

Due to their design and mode of operation, French Vertical Flow Constructed Wetlands (VFCWs) accumulate suspended solids from the inflow wastewater in the form of a sludge layer at the surface of the first filter. In order to maintain the treatment performance over the long term, the characteristics of the sludge deposits and their evolution have to be well described. In this objective, a panel of sludge deposit samples taken from 14 French VFCW sewage treatment plants was investigated. Elemental composition and organic matter content, nature and reactivity were analyzed. Results clearly revealed two categories of sludge deposits, namely the "young-age plants" type (1 year of operation and less) and the "mature plants" type (3 years of operation and more). Sludge deposits from the "mature plants" exhibited same biological, physical and chemical properties. Their organic matter was globally less abundant, more humified and less biodegradable than in the young-age plants type. Their overall contents in trace metals were also higher, although in a limited manner. The effect of additional treatments, particularly FeCl3 injection for phosphorus precipitation, was observable in the "young-age plants" group. Finally, the sludge deposits sampled from one particular plant with specific operating conditions were found to exhibit very different characteristics from those of either groups identified. This observation underlined the influence of local conditions on the typology of the sludge deposits.

Study of aggregation in surface sludge deposits from 14 full-scale French constructed wetlands using particle size distribution and dynamic vapor sorption analyses.

French vertical flow constructed wetlands (French VFCWs) are widely used for the treatment of wastewaters from small communities. In the system, unsettled wastewater is percolated through two successive stages of filter-cells planted with reeds. This causes the formation of a surface sludge layer. This layer plays positive roles in the treatment performance, but also leads to clogging. The objective of this study was to contribute to the description of the sludge deposits characteristics and their dynamics of evolution, which may control the development of clogging. Representative samples of sludge deposits were taken from 14 French VFCWs full-scale plants and analyzed for particle size, dynamic vapor sorption and other parameters of composition to compare their structure and evaluate the factors of influence. Results showed that ageing of the surface deposits layer over the years of operation in each plant induced the formation and integration of microaggregates within the initial macrostructure of fresh organic matter (OM). The humification process of the OM was found to play a key role in the aggregation process. The injection of FeCl3 operated to precipitate phosphates before filtration was found to accelerate the aggregation process in the early phase (<1 year) of operation of the sludge.

Analytical indicators to characterize Particulate Organic Matter (POM) and its evolution in French Vertical Flow Constructed Wetlands (VFCWs).

The design of French VFCWs leads to the formation of a sludge layer at the surface of the first filters due to the retention of suspended solids from the percolation of unsettled wastewater. This layer plays a major role in the system but still little is known on its characteristics and evolutions. In this study, suspended solids and sludge deposits sampled from two French VFCW plants were analyzed by different methods in the objective to assess the evolution of particulate organic matter (POM) along the treatment chain and within the sludge layer, and identify relevant analytical indicators of these phenomena. The treatment chain included an aerobic trickling filter followed by FeCl3 injection and two successive stages of filters. Thermal analyses showed that OM contents of suspended solids decreased along the treatment chain. POM in inflow suspended solids was predominantly composed of reactive, biodegradable compounds which were partly hydrolyzed and mineralized notably at the trickling filter stage. 3D fluorescence spectra collected from aqueous POM extracts confirmed the evolution of organic matter from low-molecular reactive compounds to more complex and stable structures such as humic-like substances. FTIR confirmed the mineralization of POM's reactive constituents along the treatment chain by the decrease in the intensities of the characteristics bands of aliphatic compounds or proteins, and its humification in the sludge deposits through the relative increase of the bands at 1634cm-1 (vC=O) and 1238cm-1 (δC=O and/or δOH). Isotopic ratios δ2H/1H and δ15N/14N were found to be good indicators of POM evolutions. The higher values of δ2H/1H and δ15N/14N ratios measured in sludge deposits as compared to inflow suspended solids were related to POM humification and to microbial processes of POM hydrolysis and mineralization, respectively.

Improved robust T-wave alternans detectors.

New statistical and spectral detectors, the modified matched pairs t test, the extended spectral method and the modified spectral method, were proposed for T-wave alternans (TWA) detection gaining robustness according to trend and single-frequency interferences. They were compared to classic detectors such as matched pairs t test, unpaired t test, spectral method, generalized likelihood ratio test and estimated TWA amplitude within a simulation framework and applied to real data. The optimal detection threshold was selected by using a full Monte-Carlo simulation where signals, with and without alternans episodes, were corrupted by Gaussian noise with different power and single-frequency interferences with different tones. All the combinations of noise and frequency were selected and repeated 500 times in order to compute probability of detection ([Formula: see text]) and the false alarm probability ([Formula: see text]), providing ROC curves. The study group consisted of 50 patients with implantable cardioverter-defibrillator (age: [Formula: see text]; LVEF: [Formula: see text]), who were paced (ventricular pacing) at 100 bpm. Two-minute recordings were analyzed. The XYZ orthogonal lead system was used. The best performance was reached by using the modified matched pairs t test (in comparison with the spectral method and other reference methods).

A simulation of T-wave alternans vectocardiographic representation performed by changing the ventricular heart cells action potential duration.

The presence of T wave alternans (TWA) in the surface ECG signals has been recognized as a marker of electrical instability, and is hypothesized to be related to patients at increased risk for ventricular arrhythmias. In this paper we present a TWA simulation study. The TWA phenomenon was simulated by changing the duration of the ventricular heart cells action potential. The magnitude was calculated in the surface ECG with the use of the time domain method. The spatially concordant TWA, where during one heart beat all ventricular cells display a short-duration action potential and during the next beat they exhibit a long-duration action potential, as well as the discordant TWA, where at least one region is out of phase, was simulated. The vectocardiographic representation was employed. The obtained results showed a high level of T-loop pattern and location disturbances connected to the discordant TWA simulation in contrast to the concordant one. This result may be explained by the spatial heterogeneity of the ventricular repolarization process, which could be higher for the discordant TWA than for the concordant TWA.

Nanoscale domains with nematic order in supercooled vitamin-A acetate: molecular dynamics studies.

Vitamin-A acetate is one of the most versatile vitamins. It is applied in medicine because of its antioxidative properties, in tumor therapy because of its cytostatic activity, and in cosmetics because of its nutritional additives. Herein, using broadband dielectric spectroscopy, the molecular dynamics of supercooled and glassy vitamin-A acetate was investigated. It was shown that dielectric measurements carried out at ambient and elevated pressures reveal a number of relaxation processes associated with different types of molecular motions: α, δ, and ν processes-observed above the glass transition temperature and the next two modes: ß and γ identified in the glassy state. The occurrence of the δ mode in the dielectric spectrum may imply the existence of nanoscale domains with nematic order. This hypothesis is further checked by atomic force microscopy measurements. Finally, we have determined the value of the glass transition temperature (T(g)) as well as the steepness index (m(P)) at various T-P conditions.

T waves segmentation and analysis using inverse normalized integrals.

Stress tests are typical protocols that exhibit T waves changes for healthy and ischemic subjects. ST-T elevation is one example among others that characterizes ischemic patient. A more complex description of T wave changes needs extended models with a cost of higher identification pitfalls. We propose here to work in the inverse normalized integrals domain. This domain permits us an estimation of all pertinent parameters for the characterization of T waves shape. These parameters stand for the delay, the width (defined in the paper as the scaling factor) and the offset. In contrast, direct application of PCA on the data (defined in the paper as time-domain approach) assumes perfectly aligned waves with identical widths. Time-varying parameters values corresponding to exercise tests are estimated by using both approaches. It is shown that results are consistent with clinical knowledge.

Kinetic processes in supercooled monosaccharides upon melting: Application of dielectric spectroscopy in the mutarotation studies of D-ribose.

Dielectric spectroscopy has been recently used to monitor mutarotation in undercooled D-fructose. This method can be viewed as a universal method to study mutarotation phenomenon in the whole family of monosaccharides. In this paper, we studied kinetics of mutaration of anhydrous D-ribose at ambient pressure as well as pressure effect on the rate constant of this process. Ribose mutarotation behavior is compared to the one obtained for D-fructose. In addition, we attempted to determine the "direction" of mutarotation in undercooled monosaccharides after quenching the melted sample. To this end, analysis of dipole moments of different tautomers of D-fructose and D-ribose have been performed. Conformational analysis of studied carbohydrates was done with use of density functional theory. Geometry optimizations as well as calculations of dipole moments were done on the 6-311++G(d,p)/B3LYP level. Finally, it turned out that data obtained from the mutarotation experiment might be helpful in understanding the origin of gamma-process occurring in the whole family of carbohydrates.

Multifocal type of pilomatrixoma.

Pilomatrixoma is a benign skin neoplasm that arises from hair follicle matrix cells. The skin lesion occurs usually as a solitary tumor and the multifocal types are very rare. Skin changes can be described as a firm to hard, non-painful, oval-shaped tumor that is covered by normal skin. It commonly occurs on a scalp, face, neck and rarely back and extremities. Complete surgical excision with the proper margin is the treatment of choice, what guaranteed the radical therapy of pilomatrixoma. In this paper case of 16-years-old male patient with many solid tumors in subcutaneous tissue on both arms will be reported. The first skin lesion appeared on the left arm 6 years ago. Clinically the disturbance was diagnosed as an atheroma, and it was excised. One year after surgical procedure the patient observed the appearance of new nodules on both arms. In the therapy surgical excision was performed with histopathological examination of the tissues. Histopathological test has proved the clinical diagnosis of pilomatrixoma. The case of multifocal pilomatrixoma, which is rarely diagnosed and described in professional literature, will be presented.

A phase II trial of pemetrexed plus gemcitabine in locally advanced and/or metastatic transitional cell carcinoma of the urothelium.

BACKGROUND: Both pemetrexed and gemcitabine have single-agent activity in bladder cancer, but the combination of these two drugs has not been previously evaluated for safety and efficacy in this disease. Thus, the objectives in the current study were to determine overall response rate (ORR), progression-free survival, overall survival and safety and toxicity in chemonaive patients with locally advanced and/or metastatic transitional cell carcinoma of the urothelium. PATIENTS AND METHODS: Gemcitabine 1250 mg/m2 was administered over 30 min i.v. on days 1 and 8, and pemetrexed 500 mg/m2 over 10 min i.v. on day 8 after gemcitabine, every 21 days. RESULTS: Sixty-four patients were enrolled, 11 female and 53 male, median age 65 years (range 38-81), median WHO performance status of 1. Visceral metastases were present in 55% of patients. ORR among 47 patients evaluable for response was 28% (95% CI 16% to 43%) and ORR for the intention-to-treat population was 20% (95% CI 11% to 32%) with three CR and 10 PR. Median response duration was 11.2 months and median overall survival 10.3 months (95% CI 8.1-14.6 months). CTC grade 3/4 hematologic toxicities included anemia (19%), thrombocytopenia (9%), neutropenia (38%), febrile neutropenia (17%) and neutropenic sepsis (3%). Grade 3/4 non-hematologic toxicities included elevated transaminases (12%), dyspnea (8%), fatigue (8%) and stomatitis (5%). There was one toxic death due to neutropenic sepsis. CONCLUSIONS: The combination of pemetrexed and gemcitabine had a manageable safety profile. However, efficacy was apparently not superior to that of single-agent gemcitabine.

Pyoderma gangrenosum coexisting with acute myelogenous leukaemia.

The frequency of occurrence of malignant neoplasms in the cases of pyoderma gangrenosum is not exactly determined, but it can be assessed to be at 7%. The aim of the study was to report a 26-year-old male patient with pyoderma gangrenosum coexisting with acute myelogenous leukaemia. The first skin lesions on both tibia occurred in June 2001. Prior to the proper diagnosis of pyoderma gangrenosum, the patient was treated surgically. Because of the dramatic dermatological and general condition in November 2001, the patient was admitted to the Dermatological Department of the Silesian Medical Academy in Katowice where the diagnosis of pyoderma gangrenosum was established. On the clinical and biochemical picture, the diagnosis of pyoderma gangrenosum within acute myelogenous leukaemia was made. Initially, cyclosporin A 200 mg orally per day in the therapy of pyoderma gangrenosum was administered to achieve a slight clinical improvement. Although chemotherapy leukaemia was performed, the patient died after 4 months of the confirmation of the acute myelogenous leukaemia diagnosis.

Surface plasmon resonance sensor for domoic acid based on grafted imprinted polymer.

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BIAcore 3000. The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4'-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethylamino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BIAcore 3000. Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BIAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months.

[Levels of carnitine and homocysteine in plasma of long-term hemodialysis patients with chronic renal failure]. / Stezenie karnityny i homocysteiny w osoczu u poddawanych hemodializie chorych na przewlekla niewydolnosc nerek.

Homocysteine and carnitine are the metabolic products of exogenous amino acids. Increased plasma concentration of homocysteine but decreased or normal carnitine concentration are usual findings in haemodialysis patients with chronic renal failure. This study aimed to evaluate the interrelationship (if any) between the above mentioned metabolites in haemodialysis uraemic patients. 44 haemodialyzed patients with chronic renal failure--HD (25 female, 19 male, mean age 47 +/- 12 years) were enrolled into this study. Blood samples for estimation of plasma glucose, cholesterol, triglycerides, albumin, folic acid, vitamin B12, homocysteine (HC), total (TC) and free (FC) carnitine were withdrawn after overnight fasting before subsequent haemodialysis session. In all subjects whole body total fat mass (TFM) and lean mass (TLM) were assessed by dual X-ray absorptiometry (DEXA). Hyperhomocysteinaemia was found in 90.9% patients, while carnitine deficiency in 22.7% of all analysed subjects. Both hyperhomocysteinaemia and carnitine deficiency was found in 18.2% of haemodialysis patients. Folic acid deficiency regardless of prescribed supplementation was observed in 9.5% patients. A significant positive correlation was found between plasma concentration of TC or FC and TLM (tau = 0.332, p < 0.001; tau = 0.298, p < 0.01 respectively). A negative correlation was observed between plasma concentration of folic acid and homocysteine (tau = -0.201, p < 0.05). No significant relationship was noted between homocysteinaemia, total and free plasma carnitine levels and anthropometrical parameters. In conclusion, plasma concentration of homocysteine and carnitine are independent indicators of abnormal amino acid metabolism in uraemic patients.

Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii beta-lactamase: a time-resolved FTIR spectroscopic study.

Time-resolved infrared difference spectroscopy has been used to show that the carbonyl group of the acylenzyme reaction intermediate in the Citrobacter freundii beta-lactamase-catalyzed hydrolysis of methicillin can assume at least four conformations. A single-turnover experiment shows that all four conformations decline during deacylation with essentially the same rate constant. The conformers are thus in exchange on the reaction time scale, assuming that deacylation takes place only from the conformation which is most strongly hydrogen bonded or from a more minor species not visible in these experiments. All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethyl ester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to free solution. The polarity of the carbonyl group environment in the conformers varies from 'ether-like' to strongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme. From the absorption intensities, it is estimated that the conformers are populated approximately proportional to the hydrogen bonding strength at the carbonyl oxygen. A change in the difference spectrum at 1628 cm-1 consistent with a perturbation (relaxation) of protein beta-sheet occurs slightly faster than deacylation. Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation in the acyl group is a plausible explanation of the change seen at 1628 cm-1. A turnover reaction supports the above conclusions and shows that the conformational relaxation occurs as the substrate is exhausted and the acylenzymes decline. The observation of multiple conformers is discussed in relation to the poor specificity of methicillin as a substrate of this beta-lactamase and in terms of X-ray crystallographic structures of acylenzymes where multiple forms are not apparently observed (or modeled). Infrared spectroscopy has shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactions in physiological turnover conditions as well as for determination of ordering, hydrogen bonding, and protein perturbation.

Structure-based design of beta-lactamase inhibitors. 1. Synthesis and evaluation of bridged monobactams.

Bridged monobactams are novel, potent, mechanism-based inhibitors of class C beta-lactamases, designed using X-ray crystal structures of the enzymes. They stabilize the acyl-enzyme intermediate by blocking access of water to the enzyme-inhibitor ester bond. Bridged monobactams are selective class C beta-lactamase inhibitors, with half-inhibition constants as low as 10 nM, and are less effective against class A and class B enzymes (half-inhibition constants > 100 microM) because of the different hydrolysis mechanisms in these classes of beta-lactamases. The stability of the acyl-enzyme complexes formed with class C beta-lactamases (half-lives up to 2 days were observed) enabled determination of their crystal structures. The conformation of the inhibitor moiety was close to that predicted by molecular modeling, confirming a simple reaction mechanism, unlike those of known beta-lactamase inhibitors such as clavulanic acid and penam sulfones, which involve secondary rearrangements. Synergy between the bridged monobactams and beta-lactamase-labile antibiotics could be observed when such combinations were tested against strains of Enterobacteriaceae that produce large amounts of class C beta-lactamases. The minimal inhibitory concentration of the antibiotic of more than 64 mg/L could be decreased to 0.25 mg/L in a 1:4 combination with the inhibitor.

26S proteasome structure revealed by three-dimensional electron microscopy.

In 26S proteasomes, "19S cap complexes" associate with either one or both ends of the barrel-shaped 20S core complex. These regulatory complexes which comprise about 20 different subunits, including 6 ATPases of the AAA family, are thought to recognize ubiquitinated substrate proteins, to dissociate and unfold them before threading them into the 20S core where they are degraded. Here, we examine the structure of 26S proteasomes from Drosophila embryos and Xenopus oocytes by electron microscopy. Image analysis reveals a rather flexible linkage between the 19S caps and the 20S core, with a peculiar wagging-type movement of the caps relative to the core. At this stage of the analysis, it is not clear whether this movement is relevant in terms of function. Three-dimensional reconstructions, taking this into account, provide first insights into the remarkably complex structure of the 19S caps and allows us to put forward a composite model of the entire 26S complex.