Analysis of scale energy budgets in wall turbulence using dual plane PIV

The scale energy budget in the near wall region is a subject of great interest in turbulent flows since it combines concepts from independent analysis in physical space and scale space. Earlier, this energy budget was studied numerically using Direct Numerical Simulation (DNS) data and experimentally using low resolution dual plane Particle Image Velocimetry (PIV) data. It was observed that the low resolution PIV data were not sufficient to accurately capture the dynamics of the energy balance and hence high resolution experiments were conducted in similar experimental conditions. The results from these high resolution data conducted in two locations of the logarithmic layer of the boundary layer indicate that the resolution of these experiments is sufficient to capture the scale energy budget in the near wall region. Predictions of the cross-over scale, which is related to the relative importance of production and transfer of turbulent kinetic energy, are found to match expected trends, and illustrate that the experimental technique provides a powerful tool for the scale energy budget analysis

Purification, crystallization and preliminary X-ray studies of Mycobacterium tuberculosis RRF

The ribosome recycling factor from Mycobacterium tuberculosis has been crystallized. The monoclinic crystals, with 52.5% solvent content, contain one protein molecule in the asymmetric unit

Structure of Mycobacterium smegmatis single-stranded DNA-binding protein and a comparative study involving homologus SSBs: biological implications of structural plasticity and variability in quaternary association

The structure of Mycobacterium smegmatis single-stranded DNA-binding protein (SSB) has been determined using three data sets collected from related crystals. The structure is similar to that of its homologue from Mycobacterium tuberculosis, indicating that the clamp arrangement that stabilizes the dimer and the ellipsoidal shape of the tetramer are characteristic features of mycobacterial SSBs. The central OB fold is conserved in mycobacterial SSBs as well as those from Escherichia coli, Deinococcus radiodurans and human mitochondria. However, the quaternary structure exhibits considerable variability. The observed plasticity of the subunit is related to this variability. The crystal structures and modelling provide a rationale for the variability. The strand involved in the clamp mechanism, which leads to higher stability of the tetramer, appears to occur in all high-G+C Gram-positive bacteria. The higher stability is perhaps required by these organisms. The mode of DNA binding of mycobacterial SSBs is different from that of E. coli SSB partly on account of the difference in the shape of the tetramers. Another difference between the two modes is that the former contains additional ionic interactions and is more susceptible to salt concentration

Domain closure and action of uracil DNA glycosylase (UDG): structures of new crystal forms containing the Escherichia coli enzyme and a comparative study of the known structures involving UDG

The structures of a new crystal form of free Escherichia coli uracil DNA glycosylase (UDG), containing four molecules in the asymmetric unit, and two forms of its complex with the proteinaceous inhibitor Ugi, containing two and four crystallographically independent complexes, have been determined. A comparison of these structures and the already known crystal structures containing UDG shows that the enzyme can be considered to be made up of two independently moving structural entities or domains. A detailed study of free and DNA-bound human enzyme strengthens this conclusion. The domains close upon binding to uracil-containing DNA, whereas they do not appear to do so upon binding to Ugi. The comparative study also shows that the mobility of the molecule involves the rigid-body movement of the domains superposed on flexibility within domains

Translocation-coupled DNA cleavage by the Type ISP restriction-modification enzymes

Endonucleolytic double-strand DNA break production requires separate strand cleavage events. Although catalytic mechanisms for simple dimeric endonucleases are available, there are many complex nuclease machines which are poorly understood in comparison. Here we studied the single polypeptide Type ISP restriction-modification (RM) enzymes, which cleave random DNA between distant target sites when two enzymes collide following convergent ATP-driven translocation. We report the 2.7 Angstroms resolution X-ray crystal structure of a Type ISP enzyme-DNA complex, revealing that both the helicase-like ATPase and nuclease are unexpectedly located upstream of the direction of translocation, inconsistent with simple nuclease domain-dimerization. Using single-molecule and biochemical techniques, we demonstrate that each ATPase remodels its DNA-protein complex and translocates along DNA without looping it, leading to a collision complex where the nuclease domains are distal. Sequencing of single cleavage events suggests a previously undescribed endonuclease model, where multiple, stochastic strand nicking events combine to produce DNA scission

BAY61-3606 Affects the Viability of Colon Cancer Cells in a Genotype-Directed Manner

Background: K-RAS mutation poses a particularly difficult problem for cancer therapy. Activating mutations in K-RAS are common in cancers of the lung, pancreas, and colon and are associated with poor response to therapy. As such, targeted therapies that abrogate K-RAS-induced oncogenicity would be of tremendous value. Methods: We searched for small molecule kinase inhibitors that preferentially affect the growth of colorectal cancer cells expressing mutant K-RAS. The mechanism of action of one inhibitor was explored using chemical and genetic approaches. Results: We identified BAY61-3606 as an inhibitor of proliferation in colorectal cancer cells expressing mutant forms of K-RAS, but not in isogenic cells expressing wild-type K-RAS. In addition to its anti-proliferative effects in mutant cells, BAY61-3606 exhibited a distinct biological property in wild-type cells in that it conferred sensitivity to inhibition of RAF. In this context, BAY61-3606 acted by inhibiting MAP4K2 (GCK), which normally activates NFκβ signaling in wild-type cells in response to inhibition of RAF. As a result of MAP4K2 inhibition, wild-type cells became sensitive to AZ-628, a RAF inhibitor, when also treated with BAY61-3606. Conclusions: These studies indicate that BAY61-3606 exerts distinct biological activities in different genetic contexts

X-ray structural studies of Mycobacterium tuberculosis RRF and a comparative study of RRFs of known structure. Molecular plasticity and biological implications

The crystal structure of Mycobacterium tuberculosis ribosome recycling factor has been determined and refined against three X-ray diffraction data sets, two collected at room temperature and the other at 100 K. The two room-temperature data sets differ in the radiation damage suffered by the crystals before the data used for processing were collected. A comparison between the structures refined against the two data sets indicates the possibility of radiation-induced conformational change. The L-shaped molecule is composed of a long three-helix bundle domain (domain I) and a globular domain (domain II) connected by a linker region. The main difference between the room-temperature structure and the low temperature structure is in the rotation of domain II about an axis close to its libration axis. This observation and a detailed comparative study of ribosome recycling factors (RRFs) of known structures led to an elaboration of the present understanding of the structural variability of RRF. The variability involves a change in the angle between the two arms of the molecule, a rotation of domain II in a plane nearly perpendicular to the axis of the helix bundle and an internal rotation of domain II. Furthermore, the domains and the linker could be delineated into fixed and variable regions in a physically meaningful manner. The relative mobility of the domains of the molecule in the crystal structure appears to be similar to that in the ribosome-RRF complex. That permits a meaningful discussion of the structural features of RRF in terms of ribosome-RRF interactions. The structure also provides insights into the results of inter-species complementation studies

Substitutions at tyrosine 66 of Escherichia coli uracil DNA glycosylase lead to characterization of an efficient enzyme that is recalcitrant to product inhibition

Uracil DNA glycosylase (UDG), a ubiquitous and highly specific enzyme, commences the uracil excision repair pathway. Structural studies have shown that the tyrosine in a highly conserved GQDPY water-activating loop of UDGs blocks the entry of thymine or purines into the active site pocket. To further understand the role of this tyrosine (Y66 in Escherichia coli UDG), we have overproduced and characterized Y66F, Y66H, Y66L and Y66W mutants. The complexes of the wild-type, Y66F, Y66H and Y66L UDGs with uracil DNA glycosylase inhibitor (Ugi) (a proteinaceous substrate mimic) were stable to 8 M urea. However, some dissociation of the complex involving the Y66W UDG occurred at this concentration of urea. The catalytic efficiencies (V(max) / K(m)) of the Y66L and Y66F mutants were similar to those of the wild-type UDG. However, the Y66W and Y66H mutants were ∼7- and ∼173-fold compromised, respectively, in their activities. Interestingly, the Y66W mutation has resulted in an enzyme which is resistant to product inhibition. Preferential utilization of a substrate enabling a long range contact between the –5 phosphate (upstream to the scissile uracil) and the enzyme, and the results of modeling studies showing that the uracil-binding cavity of Y66W is wider than those of the wild type and other mutant UDGs, suggest a weaker interaction between uracil and the Y66W mutant. Furthermore, the fluorescence spectroscopy of UDGs and their complexes with Ugi, in the presence of uracil or its analog, 5-bromouracil, suggests compromised binding of uracil in the active site pocket of the Y66W mutant. Lack of inhibition of the Y66W UDG by apyrimidinic DNA (AP-DNA) is discussed to highlight a potential additional role of Y66 in shielding the toxic effects of AP-DNA, by lowering the rate of its release for subsequent recognition by an AP endonuclease

X-ray structural studies of Mycobacterium tuberculosis RRF and a comparative study of RRFs of known structure. Molecular plasticity and biological implications

The crystal structure of Mycobacterium tuberculosis ribosome recycling factor has been determined and refined against three X-ray diffraction data sets, two collected at room temperature and the other at 100K. The two room-temperature data sets differ in the radiation damage suffered by the crystals before the data used for processing were collected. A comparison between the structures refined against the two data sets indicates the possibility of radiation-induced conformational change. The L-shaped molecule is composed of a long three-helix bundle domain (domain I) and a globular domain (domain II) connected by a linker region. The main difference between the room-temperature structure and the low temperature structure is in the rotation of domain II about an axis close to its libration axis. This observation and a detailed comparative study of ribosome recycling factors (RRFs) of known structures led to an elaboration of the present understanding of the structural variability of RRF. The variability involves a change in the angle between the two arms of the molecule, a rotation of domain II in a plane nearly perpendicular to the axis of the helix bundle and an internal rotation of domain II. Furthermore, the domains and the linker could be delineated into fixed and variable regions in a physically meaningful manner. The relative mobility of the domains of the molecule in the crystal structure appears to be similar to that in the ribosome--RRF complex. That permits a meaningful discussion of the structural features of RRF in terms of ribosome--RRF interactions. The structure also provides insights into the results of inter-species complementation studies