Depth-dependent strain of patellofemoral articular cartilage in unconfined compression

This biomechanical study reports strain gradients in patellofemoral joint cross-sections of seven porcine specimens in response to 1% unconfined axial compression subsequent to specific amounts of off-set strain. Strain distributions were quantified with a customized laser-based electronic speckle pattern interferometry (ESPI) system in a non-contact manner, delivering high-resolution, high-sensitivity strain maps over entire patellofemoral cartilage cross-sections. Strain reports were evaluated to determine differences in strain magnitudes between the superficial, middle, and deep cartilage layers in femoral and patellar cartilage. In addition, the effect of 5%, 10%, 15%, and 20% off-set strain on depth-dependent strain gradients was quantified. Regardless of the amount of off-set strain, the superficial layer of femoral cartilage absorbed the most strain, and the deep layer absorbed the least strain. These depth-dependent strain gradients were most pronounced for 5% off-set strain, at which the superficial layer absorbed on average 5.7 and 23.7 times more strain as compared to the middle and deep layers, respectively. For increased off-set strain levels, strain gradients became less pronounced. At 20% off-set strain, differences in layer-specific strain were not statistically significant, with the superficial layer showing a 1.4 fold higher strain as the deep layer. Patellar cartilage exhibited similar strain gradients and effects of off-set strain, although the patellar strain was on average 19% larger as compared to corresponding femoral strain reports. This study quantified for the first time continuous strain gradients over patellofemoral cartilage cross-sections. Next to provision of a detailed functional characterization of normal diarthrodial joints, this novel experimental approach holds considerable attraction to investigate joint degenerative processes.     

Structural and Thermodynamic Characterization of the TYK2 and JAK3 Kinase Domains in Complex with CP-690550 and CMP-6

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.     

Recruitment and activation of PLCgamma1 in T cells: a new insight into old domains

Engagement of the T-cell antigen receptor leads to recruitment of phospholipase Cgamma1 (PLCgamma1) to the LAT-nucleated signaling complex and to PLCgamma1 activation in a tyrosine phosphorylation-dependent manner. The mechanism of PLCgamma1 recruitment and the role of PLCgamma1 Src homology (SH) domains in this process remain incompletely understood. Using a combination of biochemical methods and real-time fluorescent imaging, we show here that the N-terminal SH2 domain of PLCgamma1 is necessary but not sufficient for its recruitment. Either the SH3 or C-terminal SH2 domain of PLCgamma1, with the participation of Vav1, c-Cbl and Slp76, are required to stabilize PLCgamma1 recruitment. All three PLCgamma1 SH domains are required for phosphorylation of PLCgamma1 Y783, which is critical for enzyme activation. These novel findings entailed revision of the currently accepted model of PLCgamma1 recruitment and activation in T lymphocytes.     

Effect of benzene and its closed ring metabolites on intrachromosomal recombination in Saccharomyces cerevisiae

Genome rearrangements, such as DNA deletions, translocations and duplications, are associated with cancer in rodents and humans, and clastogens are capable of inducing such genomic rearrangements. The clastogen benzene and several of its toxic metabolites have been shown to cause cancer in animals. Benzene is associated with leukemia and other blood related disorders in humans. Benzene and metabolites tested negative in short-term bacterial mutation assays such as the Salmonella Mutagenicity Test and the Escherichia coli Tryptophan Reversion Assay. These assays, while reliable for the detection of point-mutagenic carcinogens, are incapable of detecting DNA strand break inducing xenobiotics. The yeast DEL assay is based on intrachromosomal recombination events resulting in deletions and is very sensitive in detecting DNA strand breaks. In previous results the DEL assay detected 17 Salmonella positive as well as 25 Salmonella negative carcinogens [Bishop, Schiestl, Hum. Mol. Genet. 9 (2000) 2427-2434]. The carcinogen benzene and its metabolites including phenol, catechol, p-benzoquinone and hydroquinone induced DEL recombination. The benzene metabolite 1,2,4-benzenetriol was negative. Interestingly, p-benzoquinone induced DEL recombination at a dose 300-fold lower than any of the other metabolites, suggesting that it might be responsible for much of benzene's genotoxicity. In addition, an excision repair deficient strain was used, but no difference was detected compared to the wildtype, indicating that DNA adducts subject to excision repair were not formed by benzene or its metabolites.     

DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion

DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions.     

Processing of DNA replication and repair intermediates by the concerted action of RecQ helicases and Rad2 structure-specific nucleases

Processing of DNA replication and repair intermediates is a critical aspect of genome stability maintenance. The coordinated action of RecQ-like helicases with structure-specific nucleases such as Flap Endonuclease 1 plays an important role in the processing of certain DNA structures associated with the replication fork, DNA repair, or telomeres. We will summarize our current understanding of how and in what context these interactions take place, with a particular emphasis on the mechanisms of RecQ helicases in processing of key DNA replication and repair intermediates by their protein interactions with FEN-1 and related structure-specific nucleases.