Interactions of trimeric purine nucleoside phosphorylases with ground state analogues--calorimetric and fluorimetric studies

Binding enthalpies, dissociation constants and stoichiometry of binding for interaction of trimeric calf spleen and Cellulomonas sp. purine nucleoside phosphorylases with their ground state analogues (substrates and inhibitors) were studied by calorimetric and spectrofluorimetric methods. Data for all ligands, with possible exception of hypoxanthine, are consistent with three identical non-interacting binding sites.     

Biochemical and immunohistochemical study on physiological activity and distribution of hepatic cathepsin D

Cathepsin D (EC 3.4.23.5) is a lysosomal endopeptidase physiologically present at very low concentration in different tissues. The aim of the study was to estimate the physiological activity and distribution of cathepsin D in the liver. Four groups of ten-week-old male Wistar rats were raised without xenobiotics and sacrificed on day 4, 42, 47 and 84 of the experiment, and their livers were taken for immunohistochemical and biochemical investigation. Immunostaining for cathepsin D was evaluated by light microscope. Activity of the free and bound fractions of hepatic cathepsin D was measured spectrophotometrically. Immunohistochemical staining for cathepsin D was positive in Browicz-Kupffer cells in some but not in all rat liver specimens of each experimental group. The staining pattern was cytoplasmic and granular. Occasionally the positive stained endothelial cells were also found. No activity of cathepsin D in hepatocytes was detected. The positive immunostaining was found in livers with high enzyme activity in the biochemical investigation. No significant differences in activity of the free and bound fractions of cathepsin D among the different age groups were noted. However, the higher, age-dependent activity (p>0.05) of the free fraction was observed in the youngest and the two-middle groups of rats that were sacrificed on day 42 and 47 than in the oldest one. The bound fraction did not reveal such changes. It could be concluded that there were no differences in the activity of hepatic free and bound fractions of cathepsin D in male Wistar rats of various reproductive age. The rat Browicz-Kupffer cells revealed the highest activity of cathepsin D.     

Activity of cathepsin B,D and L in rat cerebrum after cimetidine and famotidine administration

Cathepsins are lysosomal enzymes that are used a sensitive markers in various toxicological investigations. The purpose of this study was to evaluate and compare the influence of cimetidine and famotidine on the cerebral cortex, particularly on the activity of cortical cathepsin B, D and L in the frontal lobe of rat brain. The drugs were administered intraperitoneally, twice a day, for six weeks to male Wistar rats in two doses. The initial dose was 2.85 mg/kg for cimetidine and 0.285 mg/kg for famotidine. The second dose was 10 times higher. Control animals were injected with 0.9% NaCl. Half of the animals from each of the drug-treated and control groups were sacrificed on the 42nd day of the experiment. The remaining animals were raised for another 6 weeks without any xenobiotics, and sacrificed on the 84th day. The frontal lobe of the right cerebral hemisphere was taken for biochemical investigation. The activities of free and bound fractions of cathepsin B, D and L were evaluated spectrophotometrically in cortical homogenates. The activity of bound fraction of cathepsin D and L decreased significantly in animals exposed to the higher dose of cimetidine and sacrificed on the 42nd day. Also significant elevation of the free fraction of cathepsin L was noted in the same group of rats. Cathepsin activities were normalized during the next six weeks. No behavioural changes were noted among the observed animals. Unlike cimetidine, famotidine did not change profiles of the cerebral cathepsins.     

Expression profiling in reference bacteria: dreams and reality

Profiling of gene expression in bacteria is now being used to uncover unknown genes expressed in particular genetic backgrounds or environmental conditions. Obtaining the best possible information from the expected avalanche of such experiments will require standardization of both experimental approach and statistical analysis. The first such experiments reveal challenges, pitfalls and reasonable solutions.     

Effects of Aluminum and Calcium on Acetyl-CoA Metabolism in Rat Brain Mitochondria

Abstract: Al complexes are known to accumulate in extra- and intracellular compartments of the brain in the course of different encephalopathies. In this study possible effects of Al accumulation in the cytoplasmic compartment on mitochondrial metabolism were investigated. Al, like Ca, inhibited pyruvate utilization as well as citrate and oxoglutarate accumulation by whole brain mitochondria. Potencies of Ca²⁺_total effects were 10–20 times stronger than those of Al. Al decreased mitochondrial acetyl-CoA content in a concentration-dependent manner, along with an equivalent rise of free CoA level, whereas Ca caused loss of both intermediates from mitochondria. In the absence of P_i in the medium, Ca had no effect on mitochondrial metabolism, whereas Al lost its ability to suppress pyruvate utilization and acetyl-CoA content in Ca-free conditions. Verapamil potentiated, whereas ruthenium red reversed, Ca-evoked suppression of mitochondrial metabolism. On the other hand, in Ca-supplemented medium, Al partially overcame the inhibitory influence of verapamil. Accordingly, verapamil increased mitochondrial Ca levels much more strongly than Al. However, Al partially reversed the verapamil-evoked rise of Ca²⁺_total level. These data indicate that Al accumulated in cytoplasm in the form of the Al(PO₄)OH⁻ complex may inhibit mitochondrial functions by an increase of intramitochondrial [Ca²⁺]_total resulting from the Al-evoked rise of cytoplasmic [Ca²⁺]_free, as well as from inhibitory interference with the verapamil binding site on the Na⁺/Ca²⁺ antiporter.     

New Chimeric Analogues of Galanin: Synthesis and Effects on the Glucose-Induced Insulin Secretion

The solid-phase synthesis and in vitro assays on the glucose-induced insulin secretion from rat pancreatic islets of Langerhans with six new chimeric peptides were performed. All the peptides were built up of the N-terminal galanin (GAL) fragment or its analogues, linked to the C-terminal portion of substance P (SP) analogues or scyliorhinin I (SCY-I) analogues. Two strong antagonists of the inhibitory effect of galanin on the glucose-induced insulin release were found: [cycloleucine⁴]GAL(1-13)-SP(5-11)-amide and GAL(1-13)-[L-norleucine¹⁰]SCY-I(3-10)-amide.     

A comparative study of hourly and daily relationships between selected meteorological parameters and airborne fungal spore composition

Air sampling was conducted in Szczecin (Poland) throughout April–September 2013. The final data set included 177 daily and 4248 hourly samples. The total of 21 types of spores, which occurred in a number >10 in the season, were taken into account. The following meteorological parameters were analyzed: air temperature, relative humidity, precipitation and wind speed. Effects of individual weather parameters on hourly and daily concentrations of different fungal spore types were examined using Spearman’s rank association test, whereas effects of complex of meteorological factors on hourly and daily compositions of spore were assessed using detrended correspondence analysis (DCA) and redundancy analysis (RDA). Airborne fungal spore distribution patterns in relation to meteorological variables were determined by RDA, after DCA results detected a linear structure of the spore data. The RDA results obtained indicated that all the applied variables accounted for 20 and 22% of the total variance in the hourly and daily spore data, respectively. The results of stepwise forward selection of variables revealed all included hourly and daily meteorological variables were statistically significant. The largest amount of the total variance in the spore composition was explained by the air temperature in both cases (16%). Multivariate ordination did not show large differences between the hourly and daily relationships (with exception of wind speed impact), while the differences between simple hourly and daily correlations were more clear. Correlations between daily values of variables were in most cases higher than between hourly values of variables.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.