High‐resolution crystal structure of human Mapkap kinase 3 in complex with a high affinity ligand

The Mapkap kinases 2 and 3 (MK2 and MK3) have been implicated in intracellular signaling pathways leading to the production of the pro‐inflammatory cytokine tumor necrosis factor alpha. MK2 has been pursued by the biopharmaceutical industry for many years for the development of a small molecule anti‐inflammatory treatment and drug‐like inhibitors have been described. The development of some of these compounds, however, has been slowed by the absence of a high‐resolution crystal structure of MK2. Herein we present a high‐resolution (1.9 Å) crystal structure of the highly homologous MK3 in complex with a pharmaceutical lead compound. While all of the canonical features of Ser/Thr kinases in general and MK2 in particular are recapitulated in MK3, the detailed analysis of the binding interaction of the drug‐like ligand within the adenine binding pocket allows relevant conclusions to be drawn for the further design of potent and selective drug candidates.     

Enhanced cell viability and cell adhesion using low conductivity medium for negative dielectrophoretic cell patterning

Negative dielectrophoretic (n-DEP) cell manipulation is an efficient way to pattern human liver cells on micro-electrode arrays. Maintaining cell viability is an important objective for this approach. This study investigates the effect of low conductivity medium and the optimally designed microchip on cell viability and cell adhesion. To explore the influence of conductivity on cell viability and cell adhesion, we have used earlier reported dielectrophoresis (DEP) buffer with a conductivity of 10.2 mS/m and three formulated media with conductivity of 9.02 (M1), 8.14 (M2), 9.55 (M3) mS/m. The earlier reported isotonic sucrose/dextrose buffer (DEP buffer) used for DEP manipulation has the drawback of poor cell adhesion and cell viability. A microchip prototype with well-defined positioning of titanium electrode arrays was designed and fabricated on a glass substrate. The gap between the radial electrodes was accurately determined to achieve good cell patterning performance. Parameters such as dimension of positioning electrode, amplitude, and frequency of voltage signal were investigated to optimize the performance of the microchip.     

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing

Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53−/−:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53−/− mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.     

Generation of CD4CreERT2 transgenic mice to study development of peripheral CD4‐T‐cells

After thymic emigration CD4‐T‐cells continue to differentiate into multiple effector and suppressor sublineages in peripheral lymphoid organs. In vivo analysis of peripheral CD4‐T‐cell differentiation has relied on animal models with targeted gene mutations. These are expressed either constitutively or conditionally after Cre mediated recombination. Available Cre transgenic strains to specifically target T‐cells act at stages of thymocyte development that precede thymic selection. Tracing gene functions in CD4‐T‐cell development after thymic exit becomes complicated when the targeted gene is essential during thymic development. Other approaches to conditionally modify gene functions in peripheral T‐cells involve infection of in vitro activated cells with Cre expressing lenti‐, retro‐, or adenoviruses, which precludes in vivo analyses. To study molecular mechanisms of peripheral CD4‐T‐cell differentiation in vivo and in vitro we generated transgenic mice expressing a tamoxifen inducible Cre recombinase (CreER^T2) under the control of the CD4 gene promoter. We show here that in CD4CreER^T2 mice Cre is inducibly and selectively activated in CD4‐T‐cells. Tamoxifen treatment both in vivo and in vitro results in efficient recombination of loci marked by LoxP sites. Moreover, this strain shows no abnormalities related to transgene insertion. Therefore it provides a valuable tool for studying gene function during differentiation of naïve peripheral CD4‐T‐cells into effector or suppressor sub‐lineages. genesis 50:908–913, 2012. © 2012 Wiley Periodicals, Inc.     

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.     

Maturation of Streptococcus pneumoniae lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence

Cell surface lipoproteins are important for the full virulence of several bacterial pathogens, including Streptococcus pneumoniae. Processing of prolipoproteins seems to be conserved among different bacterial species, and requires type II signal peptidase (Lsp) mediated cleavage of the N-terminal signal peptide to form the mature lipoprotein. Lsp has been suggested as a target for new antibiotic therapies, but at present there are only limited data on the function of Lsp for Gram-positive bacterial pathogens. We have investigated the function and role during disease pathogenesis of the S. pneumoniae Lsp, which, blast searches suggest, is encoded by the gene Sp0928. Expression of Sp0928 protected Escherichia coli against the Lsp antagonist globomycin, and proteomics and immunoblot analysis demonstrated that deletion of Sp0928 prevented processing of S. pneumoniae prolipoproteins to mature lipoproteins. These data strongly suggest that Sp0928 encodes the S. pneumoniae Lsp. However, immunoblots of membrane-associated proteins, immunoelectron microscopy and flow cytometry assays all confirmed that in the absence of Lsp, immature lipoproteins were still attached to the cell surface. Despite preservation of lipoprotein attachment to the cell membrane, loss of S. pneumoniae Lsp resulted in several phenotypes associated with impaired lipoprotein function and reduced S. pneumoniae replication in animal models of infection.     

Balamuthia mandrillaris, agent of amebic encephalitis: detection of serum antibodies and antigenic similarity of isolates by enzyme immunoassay

ABSTRACT. We report the development of an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to Balamuthia mandrillaris , a free-living ameba that is an etiologic agent of granulomatous amebic encephalitis (GAE). As part of the California Encephalitis Project (CEP), we have tested serum and cerebrospinal fluid (CSF) samples from a subgroup of 130 hospitalized encephalitis patients (out of ∼430 samples) over a 16-month period. Case criteria were based on clinical, laboratory, and occupational/recreational histories. All serum samples initially underwent screening by immunofluorescent antibody (IFA) staining with results ranging from no detectable ameba antibodies to titers of 1:256. In addition to the 130 samples tested prospectively, sera and/or CSF from 11 previously confirmed cases of balamuthiasis, six healthy individuals, and earlier CEP submissions with high IFA antibody titers were also tested retrospectively. Among the 130 samples, two cases of balamuthiasis were identified by ELISA and confirmed by the polymerase chain reaction (PCR). The availability of sera from human and animal cases and from varied geographic areas allowed comparisons of serologic similarities of the different Balamuthia strains and human sera. All sera, whether from human or other mammals, reacted with all strains of Balamuthia , as they did with Balamuthia amebae from different geographic areas. Enzyme-linked immunosorbent assay results were consistent with the IFA results. Differences between readings were likely due to cross-reactivity between Balamuthia antigens and unidentified antibodies in serum.     

Genomic targeting and mapping of tiller inhibition gene (<Emphasis Type="Italic">tin3</Emphasis>) of wheat using ESTs and synteny with rice

Changes in plant architecture have been central to the domestication of wild species. Tillering or the degree of branching determines shoot architecture and is a key component of grain yield and/or biomass. Previously, a tiller inhibition mutant with monoculm phenotype was isolated and the mutant gene (tin3) was mapped in the distal region of chromosome arm 3A^mL of Triticum monococcum. As a first step towards isolating a candidate gene for tin3, the gene was mapped in relation to physically mapped expressed sequence tags (ESTs) and sequence tag site (STS) markers developed based on synteny with rice. In addition, we investigated the relationship of the wheat region containing tin3 with the corresponding region in rice by comparative genomic analysis. Wheat ESTs that had been previously mapped to deletion bins provided a useful framework to identify closely related rice sequences and to establish the most likely syntenous region in rice for the wheat tin3 region. The tin3 gene was mapped to a 324-kb region spanned by two overlapping bacterial artificial chromosomes (BACs) of rice chromosome arm 1L. Wheat–rice synteny was exceptionally high at the tin3 region despite being located in the high-recombination, gene-rich region of wheat. Identification of tightly linked flanking EST and STS markers to the tin3 gene and its localization to highly syntenic rice BACs will assist in the future development of a high-resolution map and map-based cloning of the tin3 gene. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.