Dynamic compression inhibits the synthesis of nitric oxide and PGE(2) by IL-1beta-stimulated chondrocytes cultured in agarose constructs

Both mechanical loading and interleukin-1beta (IL-1beta) are known to regulate metabolic processes in articular cartilage through pathways mediated by nitric oxide ((*)NO) and PGE(2). This study uses a well-characterized model system involving isolated chondrocytes cultured in agarose constructs to test the hypothesis that dynamic compression alters the synthesis of (*)NO and PGE(2) by IL-1beta-stimulated articular chondrocytes. The data presented demonstrate for the first time that dynamic compression counteracts the effects of IL-1beta on articular chondrocytes by suppressing both (*)NO and PGE(2) synthesis. Inhibitor experiments indicated that the dynamic compression-induced inhibition of PGE(2) synthesis and stimulation of proteoglycan synthesis were (*)NO mediated, while compression-induced stimulation of cell proliferation was (*)NO independent. The inhibition of (*)NO and PGE(2) by dynamic compression is a finding of major significance that could contribute to the development of novel strategies for the treatment of cartilage-degenerative disorders.     

The coenzyme b12 analog 5'-deoxyadenosylcobinamide-gdp supports catalysis by methylmalonyl-coa mutase in the absence of trans-ligand coordination

Methylmalonyl-CoA mutase is an 5'-adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to cobalt in solution, is replaced by His(610) donated by the active site. The role of the lower axial ligand in the trillion-fold labilization of the upper axial cobalt-carbon bond has been the subject of enduring debate in the model inorganic literature. In this study, we have used a cofactor analog, 5'deoxyadenosylcobinamide GDP (AdoCbi-GDP), which reconstitutes the enzyme in a "histidine-off" form and which allows us to evaluate the contribution of the lower axial ligand to catalysis. The k(cat) for the enzyme in the presence of AdoCbi-GDP is reduced by a factor of 4 compared with the native cofactor AdoCbl. The overall deuterium isotope effect in the presence of AdoCbi-GDP ((D)V = 7.2 +/- 0.8) is comparable with that observed in the presence of AdoCbl (5.0 +/- 0.6) and indicates that the hydrogen transfer steps in this reaction are not significantly affected by the change in coordination state of the bound cofactor. These surprising results are in marked contrast to the effects ascribed to the corresponding lower axial histidine ligands in the cobalamin-dependent enzymes glutamate mutase and methionine synthase.     

Hoechst 33258 binds to G-quadruplex in the promoter region of human c-myc

In vitro binding of Hoechst 33258 to the promoter region of human c-myc, d(GG GGAGGG TGG GGA GGG TGG GGA AGG TGG GG) which forms G-quadruplex, both in vitro and in vivo in the presence of metal ions, was investigated by equilibrium absorption, fluorescence, and kinetic surface plasmon resonance methods. Hypochromic effect in UV absorption spectra and blue shift in fluorescence emission maxima of Hoechst in the presence of quadruplex revealed that Hoechst binds to the quadruplex. Analysis of UV and fluorescence titration data revealed that Hoechst binds to quadruplex with binding affinity of the order of 10(6). Anisotropy measurements and higher lifetime obtained from time-resolved decay experiments revealed that quadruplex-bound Hoechst is rotationally restricted in a less polar environment than the bulk buffer medium. From surface plasmon resonance studies, we obtained kinetic association (k(a)) and dissociation (k(d)) of 1.23+/-0.04 x 10(5)M(-1)s(-1) and 0.686+/-0.009 s(-1), respectively. As Hoechst is known to bind A-T-rich region of duplex DNA, here we propose the likelihood of Hoechst interacting with the AAGGT loop of the quadruplex.     

A nanosecond molecular dynamics study of antiparallel d(G)7 quadruplex structures: effect of the coordinated cations

Nanosecond scale molecular dynamics simulations have been performed on antiparallel Greek key type d(G7) quadruplex structures with different coordinated ions, namely Na+ and K+ ion, water and Na+ counter ions, using the AMBER force field and Particle Mesh Ewald technique for electrostatic interactions. Antiparallel structures are stable during the simulation, with root mean square deviation values of approximately 1.5 A from the initial structures. Hydrogen bonding patterns within the G-tetrads depend on the nature of the coordinated ion, with the G-tetrad undergoing local structural variation to accommodate different cations. However, alternating syn-anti arrangement of bases along a chain as well as in a quartet is maintained through out the MD simulation. Coordinated Na+ ions, within the quadruplex cavity are quite mobile within the central channel and can even enter or exit from the quadruplex core, whereas coordinated K+ ions are quite immobile. MD studies at 400K indicate that K+ ion cannot come out from the quadruplex core without breaking the terminal G-tetrads. Smaller grooves in antiparallel structures are better binding sites for hydrated counter ions, while a string of hydrogen bonded water molecules are observed within both the small and large grooves. The hydration free energy for the K+ ion coordinated structure is more favourable than that for the Na+ ion coordinated antiparallel quadruplex structure.     

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.