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RNA viruses are critically dependent upon virally encoded proteases to cleave the viral polyproteins into functional proteins. Many of these proteases exhibit a similar fold and contain an essential catalytic cysteine, offering the opportunity to inhibit these enzymes with electrophilic small molecules. Here we describe the successful application of quantitative irreversible tethering (qIT) to identify acrylamide fragments that target the active site cysteine of the 3C protease (3C

Objective The purpose of this report is to describe a patient with scapholunate advanced collapse (SLAC) of the wrist. Clinical Features A 38-year-old man presented to a chiropractic teaching clinic with right wrist pain after falling off of the tailgate of a truck 7 years prior. The mechanism of injury was a fall on an outstretched hand. Ultrasonography and radiography were performed, which demonstrated abnormal lunate kinematics and scapholunate interval diastasis associated with a clenched-fist maneuver. These findings were consistent with SLAC. Intervention and Outcome Following the diagnosis of SLAC, the wrist was splinted. Conservative care consisting of physical therapy included paraffin dips, therapeutic ultrasound, and stretching. The patient received only minimal alleviation of pain, and a surgical consultation was obtained. The patient elected surgical intervention, utilizing the proximal row carpectomy procedure. Conclusion This case demonstrates a patient with chronic wrist pain, with progression to carpal instability, which ultimately manifested as SLAC. We demonstrate, utilizing multiple imaging modalities, both preoperative and postoperative findings. To our knowledge, this is the first case to describe the use of diagnostic ultrasonography in the evaluation of the proximal row carpectomy procedure.

The genus Theileria includes tick-transmitted apicomplexan parasites of ruminants with substantial economic impact in endemic countries. Some species, including Theileria parva and Theileria annulata, infect leukocytes where they induce phenotypes that are shared with some cancers, most notably immortalization, hyperproliferation, and dissemination. Despite considerable research into the affected host signaling pathways, the parasite proteins directly responsible for these host phenotypes remain unknown. In this review we outline current knowledge on the manipulation of host cells by transformation-inducing Theileria, and we propose that comparisons between cancer biology and host-Theileria interactions can reveal chemotherapeutic targets against Theileria-induced pathogenesis based on cancer treatment approaches.

Several mammalian forkhead transcription factors have been shown to impact on cell cycle regulation and are themselves linked to cell cycle control systems. Here we have investigated the little studied mammalian forkhead transcription factor FOXK2 and demonstrate that it is subject to control by cell cycle-regulated protein kinases. FOXK2 exhibits a periodic rise in its phosphorylation levels during the cell cycle, with hyperphosphorylation occurring in mitotic cells. Hyperphosphorylation occurs in a cyclin-dependent kinase (CDK)·cyclin-dependent manner with CDK1·cyclin B as the major kinase complex, although CDK2 and cyclin A also appear to be important. We have mapped two CDK phosphorylation sites, serines 368 and 423, which play a role in defining FOXK2 function through regulating its stability and its activity as a transcriptional repressor protein. These two CDK sites appear vital for FOXK2 function because expression of a mutant lacking these sites cannot be tolerated and causes apoptosis.

Cyclin-dependent kinase 2 (cdk2) is a central regulator of the mammalian cell cycle. Here we describe the properties of a mutant form of cdk2 identified during large-scale sequencing of protein kinases from cancerous tissue. The mutation substituted a leucine for a proline in the PSTAIRE helix, the central motif in the interaction of the cdk with its regulatory cyclin subunit. We demonstrate that whilst the mutant cdk2 is considerably impaired in stable cyclin association, it is still able to generate an active kinase that can functionally complement defective cdks in vivo. Molecular dynamic simulations and biophysical measurements indicate that the observed biochemical properties likely stem from increased flexibility within the cyclin-binding helix.

Four enantiopure cytisine-inspired scaffolds can be accessed via a versatile pyrrolidine template derived from a stereocontrolled [3+2] azomethine ylide-alkene cycloaddition. Differential ester protection allows for the selective formation of either a bridged bicyclic or tricyclic scaffold via pyridone cyclization. Solid-phase diversification of the pyridone scaffolds yielded a diverse library of 15,000 compounds enabling the discovery of a novel class of Bcl-2 inhibitors.

In addition to the well characterized phosphoinositide second messengers derived from the plasma membrane, increasing evidence supports the existence of a nuclear phosphoinositide signaling network. The aim of this investigation was to dissect the role played by nuclear phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in cell cycle progression and to determine the cell cycle regulatory component(s) that are involved. A number of cytosolic/nuclear PtdIns(4,5)P2-deficient Swiss 3T3 cell lines were established, and their G 0/G 1/S cell cycle phase transitions induced by defined mitogens were examined. Our results demonstrate that nuclear PtdIns(4,5)P2 down-regulation caused a delay in phorbol ester-induced S phase entry and that this was at least in part channeled through cyclin A2 at the transcriptional level. In summary, these data identify cyclin A2 as a downstream effector of the nuclear PtdIns(4,5)P2 signaling network and highlight the importance of nuclear PtdIns(4,5)P2 in the regulation of mammalian mitogenesis.

Members of the γ2-herpesvirus family encode cyclin-like proteins that have the ability to deregulate mammalian cell cycle control. Here we report the key features of the viral cyclin encoded by Murine Herpesvirus 68, M cyclin. M cyclin preferentially associated with and activated cdk2; the M cyclin/cdk2 holoenzyme displayed a strong reliance on phosphorylation of the cdk T loop for activity. cdk2 associated with M cyclin exhibited substantial resistance to the cdk inhibitor proteins p21 Cip and p27 Kip . Furthermore, M cyclin directed cdk2 to phosphorylate p27 Kip1 on threonine 187 (T187) and cellular expression of M cyclin led to down-regulation of p27 Kip1 and the partial subversion of the associated G1 arrest. Mutation of T187 to a non-phosphorylatable alanine rendered the p27 Kip1 -imposed G1 arrest resistant to M cyclin expression. Unlike the related K cyclin, M cyclin was unable to circumvent the G1 arrest associated with p21 Cip1 and was unable to direct its associated catalytic subunit to phosphorylate this cdk inhibitor. These results imply that M cyclin has properties that are distinct from other viral cyclins and that M cyclin expression alone is insufficient for S phase entry.

EtCRK2, a cyclin-dependent kinase from the coccidian parasite, Eimeria tenella is closely related to eukaryotic cyclin-dependent kinases that regulate progression of the cell cycle and to several cyclin-dependent kinases identified in the Apicomplexa. Northern blot analyses revealed that EtCRK2 is transcribed during both asexual (first-generation schizogony) and sexual (oocyst sporulation) replicative phases of the parasite life cycle. In addition, it appears to be transcriptionally regulated during meiosis. Recombinant EtCRK2 produced in Escherichia coli has kinase activity which is significantly stimulated by the addition of vertebrate cyclin A. This cyclin-dependent kinase may play a significant role in regulating critical cell cycle events during both asexual proliferation and sexual development of the parasite.

The cyclin-dependent kinase (cdk) inhibitor p27(Kip1) is a central mediator in the imposition and maintenance of quiescence through the sequestration of G(1)-specific cyclin-cdk complexes. Previous studies have implicated the c-Jun co-activator protein Jab1 as a regulator of intracellular p27(Kip1) levels. Jab1 has been reported to interact with p27(Kip1) and cause its translocation to the cytoplasm as a prelude to the degradation of the cdk inhibitor. Here we describe experiments that showing phosphorylation of p27(Kip1) at serine 10 leads to the suppression of Jab1 levels with the concomitant inhibition of c-Jun-dependent transcription. This repression is minimized upon quiescence exit through the rapid and preferential loss of the serine 10-phosphorylated form of p27(Kip1) following serum stimulation. Our results, therefore, demonstrate an additional role for p27(Kip1) in the modulation of c-Jun-dependent transcription via Jab1.

D-type cyclin homologs have been found in the genomes of herpesviruses associated with neoplasias. They appear to exploit features of G 1 cyclins but extend their properties to allow for deregulation of the cell cycle. Advances in the study of the molecular basis for these novel features as well as the potential role of viral cyclins in tumorigenesis are addressed.

A gene encoding a novel member of the PPP family of protein serine/threonine phosphatases, termed PPN 58A, was cloned from Drosophila melanogaster . The deduced amino acid sequence of PPN 58A exhibits 59–62% identity to D. melanogaster PP1 isoforms, 51% identity to D. melanogaster PPY 55A and ≤40% identity to other members of the PPP family. The single copy gene PPN 58A maps to chromosome 2 locus 58A . Analysis of PPN 58A mRNA reveals that, like PPY 55A, PPN 58A is a testis specific enzyme.

Phosphorylation and subsequent activation of p70 S6 kinase (S6K) are events that are highly conserved in the cellular response to mitogens. The neuropeptide bombesin, which is a potent mitogen for Swiss 3T3 cells, stimulated a time- and dose-dependent activation of p70S6K as determined by gel mobility shift and immune complex kinase assays. This effect was inhibited by the immunosuppressant rapamycin at 10 nM, which also completely abolished bombesin-stimulated DNA synthesis at identical concentrations. In striking contrast, the combination of bombesin and insulin in synergy stimulated maximum DNA synthesis in these cells despite persistent inhibition of p70S6K by rapamycin throughout G1. These results indicate that activation of p70S6K is not required for the transition of quiescent cells to the S phase of the cell cycle. The inhibitory effects of rapamycin on bombesin-stimulated cell cycle progression did not involve accumulation of the cyclin-dependent kinase inhibitor p27kip1 but a striking inhibition of the expression of cyclin D1. This effect was circumvented by bombesin with insulin, suggesting that a rapamycin-insensitive pathway stimulated by this combination leads to cyclin D1 expression. Thus, these findings, dissociating the mitogenic effects of bombesin in synergy with insulin from activation of p70S6K, support the hypothesis that this kinase is a component of one of the parallel pathways that can lead to DNA synthesis rather than an obligatory point of convergence in mitogenic signaling.

Background: The transition from G1 to S phase is the key regulatory step in the mammalian cell cycle. This transition is regulated positively by G1-specific cyclin-dependent kinases (cdks) and negatively by the product of the retinoblastoma tumour suppressor gene, pRb. Hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which controls the expression of genes required for S-phase progression. Hyperphosphorylation of pRb in late G1 phase results in the accumulation of active E2F, a critical event in the progression to S phase. The E2F factor is not a single entity, but rather represents a family of highly related molecules, all of which bind to pRb or the pRb-related proteins p107 and p130. Results In this study, we have used specific inhibitors of cdks to explore the requirements for cell-cycle progression from G1 to S phase. Expression of p16 Ink4 , which specifically inhibits cyclin D-directed cdks, blocks cells in G1 phase; this block can be overcome by expression of the viral proteins that inactivate pRb or by E2F-1. Importantly however, the G1 arrest is not overcome by overexpression of E2F-4. By using chimeric E2F proteins, containing amino-acid sequences from E2F-1 and E2F-4, we have shown that their differential abilities to overcome a p16-imposed arrest is determined by their respective amino-terminal regions. We also demonstrate that E2F-1 can promote entry into S phase without concomitant phosphorylation of pRb. In contrast to the p16-mediated G1 block, G1 arrest mediated by the cdk inhibitors p21 Cip1 or p27 Kip1 cannot be bypassed either by inactivation of pRb or overexpression of E2F family members. Conclusion These data demonstrate that the role of the cyclin D-directed cdks in promoting the progression of cells from G1 into S phase is wholly to activate an E2F-1-like activity through phosphorylation, thus preventing the formation of the E2F–pRb complex. The cyclin E–cdk2 complex is also required for the G1/S transition but has a different and as yet undefined role. We also provide evidence for a functional difference between E2F-1 and E2F-4, dependant upon the region that contains the DNA-binding and dimerization domains. These results indicate that these two E2F family members are likely to regulate the expression of different subsets of E2F-responsive promoters.