Ramipril and risk of hyperkalemia in chronic hemodialysis patients

Angiotensin converting enzyme (ACE) inhibitors provide well known cardiorenal-protective benefits added to antihypertensive effects in chronic renal disease. These agents are underused in management of patients receiving hemodialysis (HD) because of common concern of hyperkalemia. However, few studies have investigated effect of renin angiotensin aldosterone system (RAAS) blockade on serum potassium in hemodialysis patients. We assessed the safety of ramipril in patients on maintenance HD. We enrolled 28 adult end stage renal disease (ESRD) patients treated by maintenance HD and prescribed them ramipril in doses of 1.25 to 5 mg per day. They underwent serum potassium concentration measurements before ramipril introduction and in 1 to 3 months afterwards. No significant increase in kalemia was found. Results of our study encourage the use of ACE inhibitors in chronically hemodialyzed patients, but close potassium monitoring is mandatory.     

Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H2O2 in Brown Rot Decay of Wood

A novel alcohol oxidase (AOX) has been purified from mycelial pellets of the wood-degrading basidiomycete Gloeophyllum trabeum and characterized as a homooctameric nonglycosylated protein with native and subunit molecular masses of 628 and 72.4 kDa, containing noncovalently bonded flavin adenine dinucleotide. The isolated AOX cDNA contained an open reading frame of 1,953 bp translating into a polypeptide of 651 amino acids displaying 51 to 53% identity with other published fungal AOX amino acid sequences. The enzyme catalyzed the oxidation of short-chain primary aliphatic alcohols with a preference for methanol (K_m = 2.3 mM, k_cat = 15.6 s⁻¹). Using polyclonal antibodies and immunofluorescence staining, AOX was localized on liquid culture hyphae and extracellular slime in sections from degraded wood and on cotton fibers. Transmission electron microscopy immunogold labeling localized the enzyme in the hyphal periplasmic space and wall and on extracellular tripartite membranes and slime, while there was no labeling of hyphal peroxisomes. AOX was further shown to be associated with membranous or slime structures secreted by hyphae in wood fiber lumina and within the secondary cell walls of degraded wood fibers. The differences in AOX targeting compared to the known yeast peroxisomal localization were traced to a unique C-terminal sequence of the G. trabeum oxidase, which is apparently responsible for the protein's different translocation. The extracellular distribution and the enzyme's abundance and preference for methanol, potentially available from the demethylation of lignin, all point to a possible role for AOX as a major source of H₂O₂, a component of Fenton's reagent implicated in the generally accepted mechanisms for brown rot through the production of highly destructive hydroxyl radicals.     

Incarnation of classical pro- and eukaryotic mechanisms of mutagenesis in hypermutagenesis and immunity of vertebrates

M.E. Lobashev has brilliantly postulated in 1947 that error-prone repair contribute to mutations in cells. This was shown to be true once the mechanisms of UV mutagenesis in Escherichia coli were deciphered. Induced mutations are generated during error-prone SOS DNA repair with the involvement of inaccurate DNA polymerases belonging to the Y family. Currently, several distinct mutator enzymes participating in spontaneous and induced mutagenesis have been identified. Upon induction of these proteins, mutation rates increase by several orders of magnitude. These proteins regulate the mutation rates in evolution and in ontogeny during immune response. In jawed vertebrates, somatic hypermutagenesis occurs in the variable regions of immunoglobulin genes, leading to affinity maturation of antibodies. The process is initiated by cytidine deamination in DNA to uracil by AID (Activation-Induced Deaminase). Further repair of uracil-containing DNA through proteins that include the Y family DNA polymerases causes mutations, induce gene conversion, and class switch recombination. In jawless vertebrates, the variable lymphocyte receptors (VLR) serve as the primary molecules for adaptive immunity. Generation of mature VLRs most likely depends on agnathan AID-like deaminases. AID and its orthologs in lamprey (PmCDA1 and PMCDA2) belong to the AID/APOBEC family of RNA/DNA editing cytidine deaminases. This family includes enzymes with different functions: APOBEC1 edits RNA, APOBEC3 restricts retroviruses. The functions of APOBEC2 and APOBEC4 have not been yet determined. Here, we report a new member of the AID/APOBEC family, APOBEC5, in the bacterium Xanthomonas oryzae. The widespread presence of RNA/DNA editing deaminases suggests that they are an ancient means of generating genetic diversity.     

Insights into pathophysiology of dystropy through the analysis of gene networks: an example of bronchial asthma and tuberculosis

Co-existence of bronchial asthma (BA) and tuberculosis (TB) is extremely uncommon (dystropic). We assume that this is caused by the interplay between genes involved into specific pathophysiological pathways that arrest simultaneous manifestation of BA and TB. Identification of common and specific genes may be important to determine the molecular genetic mechanisms leading to rare co-occurrence of these diseases and may contribute to the identification of susceptibility genes for each of these dystropic diseases. To address the issue, we propose a new methodological strategy that is based on reconstruction of associative networks that represent molecular relationships between proteins/genes associated with BA and TB, thus facilitating a better understanding of the biological context of antagonistic relationships between the diseases. The results of our study revealed a number of proteins/genes important for the development of both BA and TB.     

Biophysical and Hormonal Changes Linked to Postharvest Needle Abscission in Balsam Fir

Efforts are being made to determine significant biophysical and physiological events related to postharvest needle abscission. It is known that initial postharvest average water consumption is 0.2 mL g^?1 day^?1 (based on dry shoot tissue), but gradually decreases by up to 75 %. It is hypothesized that some degree of water deficit is manifested through changes in several biophysical and hormonal factors. Parameters including needle loss, water use, relative water content, electrical capacitance, membrane injury, and xylem pressure potential were measured once every 5 days on balsam fir branches collected from a clonal orchard. In addition, needles were sampled at the beginning of the experiment and during peak needle abscission which were then subjected to endogenous hormonal analysis. Peak needle abscission occurred within 24 days. During this time water use decreased by 70 %, relative water content decreased by 23 %, capacitance decreased by 64 %, membrane injury increased by 100 %, needle break strength decreased by 50 %, and xylem pressure potential decreased fourfold. Abscisic acid increased by 32-fold and trans-zeatin riboside increased by fourfold during peak abscission compared to fresh branches. Other cytokinins, such as cis-zeatin riboside, isopentenyl adenosine, trans-zeatin-O-glucoside, and dihydrozeatin riboside all doubled during abscission. Finally, there was a 95 % decrease in indole-3-acetic acid. Observed changes in all biophysical parameters, as well as abscisic acid, could be indicative of a possible postharvest water stress or dehydration. It is possible that dehydration-induced changes in biophysical and hormonal factors trigger and/or modulate postharvest needle abscission.     

Celastrol analogues as inducers of the heat shock response. Design and synthesis of affinity probes for the identification of protein targets

The natural product celastrol (1) possesses numerous beneficial therapeutic properties and affects numerous cellular pathways. The mechanism of action and cellular target(s) of celastrol, however, remain unresolved. While a number of studies have proposed that the activity of celastrol is mediated through reaction with cysteine residues, these observations have been based on studies with specific proteins or by in vitro analysis of a small fraction of the proteome. In this study, we have investigated the spatial and structural requirements of celastrol for the design of suitable affinity probes to identify cellular binding partners of celastrol. Although celastrol has several potential sites for modification, some of these were not synthetically amenable or yielded unstable analogues. Conversion of the carboxylic acid functionality to amides and long-chain analogues, however, yielded bioactive compounds that induced the heat shock response (HSR) and antioxidant response and inhibited Hsp90 activity. This led to the synthesis of biotinylated celastrols (23 and 24) that were used as affinity reagents in extracts of human Panc-1 cells to identify Annexin II, eEF1A, and β-tubulin as potential targets of celastrol.     

A bioassay for the simultaneous measurement of metabolic activity, membrane integrity, and lysosomal activity in cell cultures

The aim of this study was the development of an in vitro bioassay that combines several endpoints of general cytotoxicity for the screening of compounds or mixtures of compounds with potential bioactivity. The Alamar Blue assay was employed to assess metabolic activity, the Neutral Red assay was used for the assessment of membrane function and lysosomal activity, and the lactate dehydrogenase leakage assay was employed for the assessment of membrane integrity. Each assay was performed separately and in combination using a human fibroblast cell line (MRC-5). Three fungal secondary metabolites of different chemistry that affect different cellular targets were tested as model compounds: deoxynivalenol, enniatin B1, and 2-amino-14,16-dimethyloctadecan-3-ol. The obtained inhibitive compound concentrations for the assays performed separately and in combination were not significantly different (P < 0.05, n = 9). The combination of several cytotoxicity endpoints in a single assay increases the chance that potential bioactive/cytotoxic compounds are discovered during the screening of mixtures of natural compounds (e.g., extracts from fungal cultures or plants) when one endpoint fails and, at the same time, might give some basic information on the cellular target.     

Affinity maturation generates greatly improved xyloglucan-specific carbohydrate binding modules

Background  

Molecular evolution of carbohydrate binding modules (CBM) is a new approach for the generation of glycan-specific molecular probes. To date, the possibility of performing affinity maturation on CBM has not been investigated. In this study we show that binding characteristics such as affinity can be improved for CBM generated from the CBM4-2 scaffold by using random mutagenesis in combination with phage display technology.     

Increased permeability of the blood-brain barrier following administration of glyceryl trinitrate in common carp (Cyprinus carpio L.)

Nitric oxide (NO) has been implicated in the pathogenesis of migraine and treatment with its exogenous donor glyceryl trinitrate (GTN) represents widely accepted experimental "migraine model". In this study, glyceryl trinitrate was administered intraperitoneally to carps, serum nitrite and nitrate levels were determined, permeability of blood-brain barrier was investigated, and histological changes of brain tissue were analyzed. Serum nitrite and nitrate levels displayed characteristic biphasic pattern with moderate initial increase and maximal terminal increase, suggesting the GTN-induced endogenous NO synthesis. Increased permeability of the blood-brain barrier in GTN-treated animals was determined based on Evans blue capillary leakage into the brain tissue. Histological analysis revealed changes consistent with vasodilatation and oedema. Our study strongly supports the importance of the NO role in the pathogenesis of migraine attacks and increase in blood-brain barrier permeability during the attack. The study has also provided evidence that this mechanism of action is conserved to the lower vertebrate.     

Glucagon receptor recycling: role of carboxyl terminus, beta-arrestins, and cytoskeleton

Glucagon receptor (GR) activity and expression are altered in several diseases, including Type 2 diabetes. Previously, we investigated the mechanism of GR desensitization and internalization. The present study focused on the fate of internalized GR. Using both hamster hepatocytes and human embryonic kidney (HEK)-293 cells, we showed that internalized GR recycled to the plasma membrane within 30-60 min following stimulation of the cells with 100 nM glucagon. In HEK-293 cells and during recycling, GR colocalized with Rab4, Rab11, beta-arrestin1, beta-arrestin2, and actin filaments, in the cytosolic and/or perinuclear domains. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol. GR coimmunoprecipitated with beta-actin in both hepatocytes and HEK-293 cells. Downregulation of beta-arrestin1 and beta-arrestin2 or disruption of the cytoskeleton inhibited recycling, but not internalization of GR. Deletion of the GR carboxyl-terminal 70 amino acids abolished internalization of GR in response to glucagon while deletion of the last 40 amino acids only did not affect GR internalization and recycling. After exposure of the cells to either high concentrations or prolonged duration of glucagon, GR colocalized with lysosomes. GR degradation was inhibited by lysosomal, but not proteosomal, inhibitors. In conclusion, GR recycles through Rab4- and Rab11- positive vesicles. The actin cytoskeleton, beta-arrestin1, beta-arrestin2, and the receptor's carboxyl terminus are involved in recycling. Prolonged stimulation with glucagon targets GR for degradation in lysosomes. Therefore, the present study provides a better understanding of the GR recycling mechanism, which could become useful in the treatment of certain diseases, including diabetes.