A Structural View on the Functional Importance of the Sugar Moiety and Steroid Hydroxyls of Cardiotonic Steroids in Binding to Na,K-ATPase

The Na,K-ATPase is specifically inhibited by cardiotonic steroids (CTSs) like digoxin and is of significant therapeutic value in the treatment of congestive heart failure and arrhythmia. Recently, new interest has arisen in developing Na,K-ATPase inhibitors as anticancer agents. In the present study, we compare the potency and rate of inhibition as well as the reactivation of enzyme activity following inhibition by various cardiac glycosides and their aglycones at different pH values using shark Na,K-ATPase stabilized in the E2MgP_i or in the E2BeF_x conformations. The effects of the number and nature of various sugar residues as well as changes in the positions of hydroxyl groups on the β-side of the steroid core of cardiotonic steroids were investigated by comparing various cardiac glycoside compounds like ouabain, digoxin, digitoxin, and gitoxin with their aglycones. The results confirm our previous hypothesis that CTS binds primarily to the E2-P ground state through an extracellular access channel and that binding of extracellular Na⁺ ions to K⁺ binding sites relieved the CTS inhibition. This reactivation depended on the presence or absence of the sugar moiety on the CTS, and a single sugar is enough to impede reactivation. Finally, increasing the number of hydroxyl groups of the steroid was sterically unfavorable and was found to decrease the inhibitory potency and to confer high pH sensitivity, depending on their position on the steroid β-face. The results are discussed with reference to the recent crystal structures of Na,K-ATPase in the unbound and ouabain-bound states.     

Analysis of Cultured Methanogenic Archaea from the Tarkhankut Peninsula Coastal Methane Seeps

Microbiology - The major substrates for methanogenesis were used for investigation of cultured methanogenic archaea from coastal methane seeps near the Tarkhankut Peninsula, Black Sea. Analysis of...     

Method for spiking soil samples with organic compounds

We examined the harmful side effects on indigenous soil microorganisms of two organic solvents, acetone and dichloromethane, that are normally used for spiking of soil with polycyclic aromatic hydrocarbons for experimental purposes. The solvents were applied in two contamination protocols to either the whole soil sample or 25% of the soil volume, which was subsequently mixed with 75% untreated soil. For dichloromethane, we included a third protocol, which involved application to 80% of the soil volume with or without phenanthrene and introduction of Pseudomonas fluorescens VKI171 SJ132 genetically tagged with luxAB::Tn5. For both solvents, application to the whole sample resulted in severe side effects on both indigenous protozoa and bacteria. Application of dichloromethane to the whole soil volume immediately reduced the number of protozoa to below the detection limit. In one of the soils, the protozoan population was able to recover to the initial level within 2 weeks, in terms of numbers of protozoa; protozoan diversity, however, remained low. In soil spiked with dichloromethane with or without phenanthrene, the introduced P. fluorescens VKI171 SJ132 was able to grow to a density 1,000-fold higher than in control soil, probably due mainly to release of predation from indigenous protozoa. In order to minimize solvent effects on indigenous soil microorganisms when spiking native soil samples with compounds having a low water solubility, we propose a common protocol in which the contaminant dissolved in acetone is added to 25% of the soil sample, followed by evaporation of the solvent and mixing with the remaining 75% of the soil sample.     

Proteolytic activity and general characteristics of a marine bacterium, Aeromonas proteolytica sp. N

Merkel, Joseph R. (Fort Johnson Marine Biological Laboratory, College of Charleston, Charleston, S.C.), Eugene D. Traganza, Barid B. Mukherjee, Travis B. Griffin, and J. M. Prescott. Proteolytic activity and general characteristics of a marine bacterium, Aeromonas proteolytica sp. n. J. Bacteriol. 87:1227-1233. 1964.-A highly proteolytic bacterial species was isolated from the alimentary canal of the marine borer, Limnoria. The morphological and biochemical characteristics of the organism indicated that it was a new Aeromonas species, for which the name A. proteolytica is proposed. When freshly isolated, the organism required seawater for growth; but, upon prolonged culture in the laboratory, it was able to grow in media of greatly reduced salt concentration, provided that relatively large amounts of peptone were supplied. Peptone or hydrolysates of casein were capable of supplying all organic nutrients required for growth and proteinase production. Certain individual amino acids were also able to furnish all energy, carbon, and nitrogen requirements. Inorganic nitrogen was utilized in the presence of citrate, but could not serve as the only source of nitrogen in the presence of glucose. The organism was facultatively anaerobic, but best growth and proteinase production occurred only with vigorous aeration. The amount of growth obtained in 24 hr increased rapidly as the incubation temperature was increased up to a maximum of 40 C, but no growth occurred at 42 C.     

Temperature-dependent structural changes in intrinsically disordered proteins: Formation of α-helices or loss of polyproline II?

Structural characterization of intrinsically disordered proteins (IDPs) is mandatory for deciphering their potential unique physical and biological properties. A large number of circular dichroism (CD) studies have demonstrated that a structural change takes place in IDPs with increasing temperature, which most likely reflects formation of transient α-helices or loss of polyproline II (PPII) content. Using three IDPs, ACTR, NHE1, and Spd1, we show that the temperature-induced structural change is common among IDPs and is accompanied by a contraction of the conformational ensemble. This phenomenon was explored at residue resolution by multidimensional NMR spectroscopy. Intrinsic chemical shift referencing allowed us to identify regions of transiently formed helices and their temperature-dependent changes in helicity. All helical regions were found to lose rather than gain helical structures with increasing temperature, and accordingly these were not responsible for the change in the CD spectra. In contrast, the nonhelical regions exhibited a general temperature-dependent structural change that was independent of long-range interactions. The temperature-dependent CD spectroscopic signature of IDPs that has been amply documented can be rationalized to represent redistribution of the statistical coil involving a general loss of PPII conformations.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

Population fragmentation in the southern rock agama, Agama atra: more evidence for vicariance in Southern Africa

Mitochondrial DNA sequence data derived from two genes were used to infer phylogeographical relationships between 13 Agama atra populations. Three distinct geographical assemblages were found among the lizard populations. The first occurs in southern Namibia, the second is restricted to the western dry arid regions of South Africa, whereas the third is distributed throughout the more mesic southern and eastern parts of the subcontinent. Geographically structured differences among populations within Agama clades are probably the result of dispersal and historic isolations among populations. At the broader scale, there were marked congruences between the Agama genetic discontinuities and those described previously in other rock-dwelling vertebrates such as Pronolagus rupestris and Pachydactylus rugosus. This suggests vicariance, probably as a response to natural climatic changes during the past three million years.