Acid-base versus structural properties of an aminoglycoside antibiotic--sisomicin: NMR and potentiometric approach

Aminoglycoside antibiotics constitute a class of the drugs of high interest, whose therapeutic action is based upon the electrostatic interaction with the variety of RNA molecules. The positive charge of these drugs molecules, located at their amino functions, has a prevailing influence on this process. The potentiometry and (1)H NMR spectroscopy are applied hereby to achieve the characteristics of the acid-base properties of particular protonating groups. We found that the pK values of deprotonation processes cover a wide values range 6-9.8. The correlation spectra of sisomicin, both COSY and TOCSY, allowed attributing unambiguously individual signals to the corresponding protons. These spectra involve a lot of the cross-peaks originating from the B and C rings protons, while the analogous signals originating from A rings protons are less numerous. Molecular modeling provided that the methylated amino group of A ring is located too far from the protonated functions of the remaining rings to affect their pK values. The phenomena observed herein are discussed in line of strength of the analogous processes observed for other aminoglycosides. As the result, four types of amino groups consisted within these antibiotics are distinguished.     

Species assortment or habitat filtering: a case study of spider communities on lake islands

Competition theory predicts that species of similar ecological niches are less likely to coexist than species with different niches, a process called species assortment. In contrast, the concept of habitat filtering implies that species with similar ecological requirements should co-occur more often than expected by chance. Here we use environmental and ecological data to assess patterns of co-occurrence of regional communities of spiders distributed across two assemblies of lake islands in northern Poland. We found aggregated and random co-occurrences of species of the same genus and a significant tendency of species segregation across genera. We also found that species of the same genus react similarly to important environmental variables. A comparison of ecological traits of species of the local communities with those expected from a random sample from the regional Polish species pool corroborated partly the habitat filtering hypothesis. On the other hand, we did not find evidence for species assortment. Our results also imply that at least some observed species co-occurrences result from niche differentiation.     

Sphingomyelin-rich domains are sites of lysenin oligomerization: Implications for raft studies

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.     

Facies development and sequence stratigraphy of the Ludfordian (Upper Silurian) deposits in the Zbruch River Valley, Podolia, western Ukraine: local facies overprint on the δ13Ccarb record of a global stable carbon isotope excursion

The Ludfordian (Upper Silurian) succession in Podolia, western Ukraine, represents a Silurian carbonate platform developed in an epicontinental sea on the shelf of the paleocontinent of Baltica. Coeval deposits throughout this basin record a positive stable carbon isotope excursion known as the Lau excursion. The record of this excursion in Podolia exhibits an unusual amplitude from highly positive (+6.9 ‰) to highly negative (?5.0 ‰) δ¹³C_carb values. In order to link δ¹³C_carb development with facies, five sections in the Zbruch River Valley were examined, providing microfacies characterization and revised definitions of the Isakivtsy, Prygorodok, and Varnytsya Formations. The Isakivtsy Fm. is developed as dolosparite replacing originally bioclastic limestone. The Prygorodok Fm., recording strongly depleted (down to ?10.53 ‰) to near zero (0.12 ‰) δ¹³C_carb values is developed as laminated, organic-rich dolomicrite with metabentonite and quartz siltstone beds. The Varnytsya Fm. is characterized by peritidal deposition with consistent, slightly negative δ¹³C_carb values (?0.57 to ?3.20 ‰). It is proposed that dolomitization of the Isakivtsy Fm. is associated with a sequence boundary and erosional surface. The overlying Prygorodok Fm. represents the proximal part of a TST deposited in restricted and laterally extremely variable environments dominated by microbial carbonate production. The transition to the overlying Varnytsya Fm. facies is marked by a maximum flooding surface. The SB and MFS are potentially correlative within the basin and support a global rapid sea-level fall previously proposed for this interval. The interpretation of the Prygorodok Fm. as coastal lake deposits may explain the unusual δ¹³C_carb values and constitute one of the few records of this type of environment identified in the early Paleozoic.     

Expression and activity of cGMP-dependent phosphodiesterases is up-regulated by lipopolysaccharide (LPS) in rat peritoneal macrophages

It has been shown that cyclic GMP (cGMP) modulates the inflammatory responses of macrophages, but the underlying molecular mechanisms are still poorly understood. Looking for proteins potentially regulated by cGMP in rat peritoneal macrophages (PMs), in this study we analyzed expression and activity of cGMP-hydrolyzing and cGMP-regulated phosphodiesterases (PDEs). It was found that freshly isolated peritoneal exudate macrophages (PEMs) express enzymes belonging to families PDE1-3, PDE5, PDE10, and PDE11. Analysis of substrate specificity, sensitivity to inhibitors, and subcellular localization showed that PDE2 and PDE3 are the main cGMP-regulated PDE isoforms in PEMs. The profile of PDE expression was altered by maintaining PEMs in culture and treatment with bacterial endotoxin (LPS). After 24 h culture, PDE5 was not present and the levels of PDE2, PDE3, and PDE11 were markedly decreased. However, their expression and activity was recovered after treatment of cultured cells with LPS. A similar pattern of changes was observed for the expression of TNFalpha, but not for guanylyl cyclase A (GC-A). LPS up-regulated PDE expression also in resident peritoneal macrophages (RPMs), although not all PDEs present in PEMs were detected in RPMs. Taken together, our results show that in rat PMs expression of cGMP-dependent PDEs positively correlates with the activation state of cells. Moreover, the fact that most of these PDEs hydrolyze also cAMP indicates that cGMP can play a role of potent regulator of cAMP signaling in macrophages.     

Up-regulation of NPY gene expression in hypothalamus of rats with experimental chronic renal failure

Anorexia is possibly one of the most important causes of malnutrition in uremic patients. The cause of this abnormality is still unknown. Considering that: (a) NPY is one of the most important stimulants of food intake; (b) eating is a central nervous system regulated process and (c) NPY is expressed in hypothalamus, we hypothesized that the decrease of NPY gene expression in the hypothalamus could be an important factor contributing to anorexia associated with uremic state. In contrast to the prediction, the results presented in this paper indicate that the NPY gene expression in the hypothalamus of chronic renal failure (CRF) rats was significantly higher than in the hypothalamus of control (pair-fed) rats. Moreover, we found that serum NPY concentration in CRF rats was higher than in control (pair-fed) animals. The increase of plasma NPY concentration in CRF rats may be due to the greater synthesis of the neuropeptide in liver, since higher level of NPY mRNA was found in liver of CRF rats. The results obtained revealed that experimental chronic renal failure is associated with the increase of NPY gene expression in hypothalamus and liver of rats.     

Interaction between yeast eukaryotic initiation factor eIF4E and mRNA 5' cap analogues differs from that for murine eIF4E

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (Kas) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5' cap than those of the murine and human proteins. The observed differences in the Kas values and the enthalpy changes of the association (deltaH(o)) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.     

The HI0073/HI0074 protein pair from Haemophilus influenzae is a member of a new nucleotidyltransferase family: structure,sequence analyses,and solution studies

The crystal structure of HI0074 from Haemophilus influenzae, a protein of unknown function, has been determined at a resolution of 2.4 A. The molecules form an up-down, four-helix bundle, and associate into homodimers. The fold is most closely related to the substrate-binding domain of KNTase, yet the amino acid sequences of the two proteins exhibit no significant homology. Sequence analyses of completely and incompletely sequenced genomes reveal that the two adjacent genes, HI0074 and HI0073, and their close relatives comprise a new family of nucleotidyltransferases, with 15 members at the time of writing. The analyses also indicate that this is one of eight families of a large nucleotidyltransferase superfamily, whose members were identified based on the proximity of the nucleotide- and substrate-binding domains on the respective genomes. Both HI0073 and HI0074 were annotated "hypothetical" in the original genome sequencing publication. HI0073 was cloned, expressed, and purified, and was shown to form a complex with HI0074 by polyacrylamide gel electrophoresis under nondenaturing conditions, analytic size exclusion chromatography, and dynamic light scattering. Double- and single-stranded DNA binding assays showed no evidence of DNA binding to HI0074 or to HI0073/HI0074 complex despite the suggestive shape of the putative binding cleft formed by the HI0074 dimer.     

Modulating the catalytic activity of AMPK has neuroprotective effects against α-synuclein toxicity

Background

Metabolic perturbations and slower renewal of cellular components associated with aging increase the risk of Parkinson’s disease (PD). Declining activity of AMPK, a critical cellular energy sensor, may therefore contribute to neurodegeneration.

Methods

Here, we overexpress various genetic variants of the catalytic AMPKα subunit to determine how AMPK activity affects the survival and function of neurons overexpressing human α-synuclein in vivo.

Results

Both AMPKα1 and α2 subunits have neuroprotective effects against human α-synuclein toxicity in nigral dopaminergic neurons. Remarkably, a modified variant of AMPKα1 (T172Dα1) with constitutive low activity most effectively prevents the loss of dopamine neurons, as well as the motor impairments caused by α-synuclein accumulation. In the striatum, T172Dα1 decreases the formation of dystrophic axons, which contain aggregated α-synuclein. In primary cortical neurons, overexpression of human α-synuclein perturbs mitochondrial and lysosomal activities. Co-expressing AMPKα with α-synuclein induces compensatory changes, which limit the accumulation of lysosomal material and increase the mitochondrial mass.

Conclusions

Together, these results indicate that modulating AMPK activity can mitigate α-synuclein toxicity in nigral dopamine neurons, which may have implications for the development of neuroprotective treatments against PD.