A robust procedure for removing background damage in assays of radiation-induced DNA fragment distributions

The non-random distribution of DNA breakage in PFGE (pulsed-field gel electrophoresis) experiments poses a problem of proper subtraction of the background DNA damage to obtain a fragment-size distribution due to radiation only. A naive bin-to-bin subtraction of the background signal will not result in the right DNA mass distribution histogram. This problem could become more pronounced for high-LET (linear energy transfer) radiation, because the fragment-size distribution manifests a higher frequency of smaller fragments. Previous systematic subtraction methods have been based on random breakage, appropriate for low-LET radiation. Moreover, an investigation is needed to determine whether the background breakage is itself random or non-random. We consider two limiting cases: (1) the background damage is present in all cells, and (2) it is present in only a small subset of cells, while other cells are not contributing to the background DNA fragmentation. We give a generalized formalism based on stochastic processes for the subtraction of the background damage in PFGE experiments for any LET and apply it to two sets of PFGE data for iron ions.     

Regulation of the Ste20-like kinase, SLK: involvement of activation segment phosphorylation

Expression and activation of the Ste20-like kinase, SLK, is increased during kidney development and recovery from ischemic acute kidney injury. SLK promotes apoptosis, and it may regulate cell survival during injury or repair. This study addresses the role of phosphorylation in the regulation of kinase activity. We mutated serine and threonine residues in the putative activation segment of the SLK catalytic domain and expressed wild type (WT) and mutant proteins in COS-1 or glomerular epithelial cells. Compared with SLK WT, the T183A, S189A, and T183A/S189A mutants showed reduced in vitro kinase activity. SLK WT, but not mutants, increased activation-specific phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. Similarly, SLK WT stimulated activator protein-1 reporter activity, but activation of activator protein-1 by the three SLK mutants was ineffective. To test if homodimerization of SLK affects phosphorylation, the cDNA encoding SLK amino acids 1-373 (which include the catalytic domain) was fused with a cDNA for a modified FK506-binding protein, Fv (Fv-SLK 1-373). After transfection, the addition of AP20187 (an FK506 analog) induced regulated dimerization of Fv-SLK 1-373. AP20187-stimulated dimerization enhanced the kinase activity of Fv-SLK 1-373 WT. In contrast, kinase activity of Fv-SLK 1-373 T183A/S189A was weak and was not enhanced after dimerization. Finally, apoptosis was increased after expression of Fv-SLK 1-373 WT but not T183A/S189A. Thus, phosphorylation of Thr-183 and Ser-189 plays a key role in the activation and signaling of SLK and could represent a target for novel therapeutic approaches to renal injury.     

Synthesis and antibacterial activity of desosamine-modified macrolide derivatives

Structural factors behind erm macrolide resistance were studied through synthesis of new macrolide derivates possessing truncated desosamine sugar moieties and subsequent determination of their antibacterial activity. Synthesized compounds with 2'-deoxy and 3'-desmethyl desosamine rings demonstrated decreased antibacterial activity on the native Staphylococcus aureus strain and were inactive against constitutively resistance S. aureus. The obtained results indicate that steric repulsion between the dimethylated A2058 and desosamine ring cannot be considered as a primary reason for erm-resistance.     

Fibrils connect microtubule tips with kinetochores: a mechanism to couple tubulin dynamics to chromosome motion

Kinetochores of mitotic chromosomes are coupled to spindle microtubules in ways that allow the energy from tubulin dynamics to drive chromosome motion. Most kinetochore-associated microtubule ends display curving "protofilaments," strands of tubulin dimers that bend away from the microtubule axis. Both a kinetochore "plate" and an encircling, ring-shaped protein complex have been proposed to link protofilament bending to poleward chromosome motion. Here we show by electron tomography that slender fibrils connect curved protofilaments directly to the inner kinetochore. Fibril-protofilament associations correlate with a local straightening of the flared protofilaments. Theoretical analysis reveals that protofilament-fibril connections would be efficient couplers for chromosome motion, and experimental work on two very different kinetochore components suggests that filamentous proteins can couple shortening microtubules to cargo movements. These analyses define a ring-independent mechanism for harnessing microtubule dynamics directly to chromosome movement.     

Indel PDB: A database of structural insertions and deletions derived from sequence alignments of closely related proteins

Background  

Insertions and deletions (indels) represent a common type of sequence variations, which are less studied and pose many important biological questions. Recent research has shown that the presence of sizable indels in protein sequences may be indicative of protein essentiality and their role in protein interaction networks. Examples of utilization of indels for structure-based drug design have also been recently demonstrated. Nonetheless many structural and functional characteristics of indels remain less researched or unknown.     

Matrix Rigidity-Modulated Cardiovascular Organoid Formation from Embryoid Bodies

Stem cell clusters, such as embryoid bodies (EBs) derived from embryonic stem cells, are extensively studied for creation of multicellular clusters and complex functional tissues. It is common to control phenotypes of ES cells with varying molecular compounds; however, there is still a need to improve the controllability of cell differentiation, and thus, the quality of created tissue. This study demonstrates a simple but effective strategy to promote formation of vascularized cardiac muscle - like tissue in EBs and form contracting cardiovascular organoids by modulating the stiffness of a cell adherent hydrogel. Using collagen-conjugated polyacrylamide hydrogels with controlled elastic moduli, we discovered that cellular organization in a form of vascularized cardiac muscle sheet was maximal on the gel with the stiffness similar to cardiac muscle. We envisage that the results of this study will greatly contribute to better understanding of emergent behavior of stem cells in developmental and regeneration process and will also expedite translation of EB studies to drug-screening device assembly and clinical treatments.     

Voltammetric studies of glutathione transfer across arrays of liquid–liquid microinterfaces for sensing applications

Amino Acids - The simple and facilitated transfer of tripeptide glutathione across the water/2-nitrophenyl octhyl ether interface was studied via cyclic voltammetry at interface between two...     

Structure-based design, synthesis, and SAR evaluation of a new series of 8-hydroxyquinolines as HIF-1alpha prolyl hydroxylase inhibitors

A new series of potent 8-hydroxyquinolines was designed based on the newly resolved X-ray crystal structure of EGLN-1. Both alkyl and aryl 8-hydroxyquinoline-7-carboxyamides were good HIF-1alpha prolyl hydroxylase (EGLN) inhibitors. In subsequent VEGF induction assays, these exhibited potent VEGF activity. In addition, this class of compounds did show the ability to stabilize HIF-1alpha.     

Molecular architecture of leishmania EF-1alpha reveals a novel site that may modulate protein translation: a possible target for drug development

Elongation factor-1alpha plays an essential role in eukaryotic protein biosynthesis. Recently, we have shown by protein structure modeling the presence of a hairpin-loop of 12 amino acids in mammalian EF-1alpha that is absent in the leishmania homologue [D. Nandan, A. Cherkasov, R. Sabouti, T. Yi, N.E. Reiner, Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue, Biochem. Biophys. Res. Commun. 302 (2003) 646-652]. As a consequence of this deletion, an exposed region is available on the main body of leishmania EF-1alpha. Here we report the generation of an anti-EF-1alpha antibody (DN-3) which bound selectively to the exposed region of leishmania EF-1alpha, with no reactivity with human EF-1alpha. In a leishmania cell-free protein translation system, DN-3 substantially inhibited protein translation. A similar inhibitory effect was observed when a specific peptide based on the exposed region was used in the cell-free protein translation assay. The application of structure-based in silico methods to identify potential ligands to target the exposed region identified a small molecule that selectively attenuated in vitro translation using leishmania extracts. Moreover, this small molecule showed selective suppressive effect on multiplication of leishmania in culture. Taken together, these findings identify a novel, exposed region in leishmania EF-1alpha that may be involved in protein synthesis and a potential site for drug targeting.