Tumour necrosis factor activates the mitogen-activated protein kinases p38α and ERK in the synovial membrane in vivo

Tumour necrosis factor (TNF) is considered to be a major factor in chronic synovial inflammation and is an inducer of mitogen-activated protein kinase (MAPK) signalling. In the present study we investigated the ability of TNF to activate MAPKs in the synovial membrane in vivo. We studied human TNF transgenic mice – an in vivo model of TNF-induced arthritis – to examine phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) and p38MAPKα in the inflamed joints by means of immunoblot and immunohistochemistry. In addition, the effects of systemic blockade of TNF, IL-1 and receptor activator of nuclear factor-κB (RANK) ligand on the activation of MAPKs were assessed. In vivo, overexpression of TNF induced activation of p38MAPKα and ERK in the synovial membrane, whereas activation of JNK was less pronounced and rarely observed on immunohistochemical analysis. Activated p38MAPKα was predominantly found in synovial macrophages, whereas ERK activation was present in both synovial macrophages and fibroblasts. T and B lymphocytes did not exhibit major activation of any of the three MAPKs. Systemic blockade of TNF reduced activation of p38MAPKα and ERK, whereas inhibition of IL-1 only affected p38MAPKα and blockade of RANK ligand did not result in any decrease in MAPK activation in the synovial membrane. These data indicate that TNF preferentially activates p38MAPKα and ERK in synovial membrane exposed to TNF. This not only suggests that targeted inhibition of p38MAPKα and ERK is a feasible strategy for blocking TNF-mediated effects on joints, but it also shows that even currently available methods to block TNF effectively reduce activation of these two MAPKs.     

A Src-Tks5 pathway is required for neural crest cell migration during embryonic development

In the adult organism, cell migration is required for physiological processes such as angiogenesis and immune surveillance, as well as pathological events such as tumor metastasis. The adaptor protein and Src substrate Tks5 is necessary for cancer cell migration through extracellular matrix in vitro and tumorigenicity in vivo. However, a role for Tks5 during embryonic development, where cell migration is essential, has not been examined. We used morpholinos to reduce Tks5 expression in zebrafish embryos, and observed developmental defects, most prominently in neural crest-derived tissues such as craniofacial structures and pigmentation. The Tks5 morphant phenotype was rescued by expression of mammalian Tks5, but not by a variant of Tks5 in which the Src phosphorylation sites have been mutated. We further evaluated the role of Tks5 in neural crest cells and neural crest-derived tissues and found that loss of Tks5 impaired their ventral migration. Inhibition of Src family kinases also led to abnormal ventral patterning of neural crest cells and their derivatives. We confirmed that these effects were likely to be cell autonomous by shRNA-mediated knockdown of Tks5 in a murine neural crest stem cell line. Tks5 was required for neural crest cell migration in vitro, and both Src and Tks5 were required for the formation of actin-rich structures with similarity to podosomes. Additionally, we observed that neural crest cells formed Src-Tks5-dependent cell protrusions in 3-D culture conditions and in vivo. These results reveal an important and novel role for the Src-Tks5 pathway in neural crest cell migration during embryonic development. Furthermore, our data suggests that this pathway regulates neural crest cell migration through the generation of actin-rich pro-migratory structures, implying that similar mechanisms are used to control cell migration during embryogenesis and cancer metastasis.     

Metabolomics: quantification of intracellular metabolite dynamics

The rational improvement of microbial strains for the production of primary and secondary metabolites ('metabolic engineering') requires a quantitative understanding of microbial metabolism. A process by which this information can be derived from dynamic fermentation experiments is presented. By applying a substrate pulse to a substrate-limited, steady state culture, cellular metabolism is shifted away from its metabolic steady state. With the aid of a rapid sampling and quenching routine it is possible to take 4-5 samples per second during this process, thus capturing the metabolic response to this stimulus. Over 30 metabolites, nucleotides and cofactors from Escherichia coli metabolism can be extracted and analysed using a range of different techniques, for example enzymatic assays, HPLC and LC-MS methods. Using different substrates as limiting and pulse-substrates (glucose, glycerol), different metabolic pathways and substrate uptake systems are investigated. The resulting plots of intracellular metabolite concentrations against time serve as a data basis for modelling microbial metabolic networks.     

Negative Regulation of Bone Formation by the Transmembrane Wnt Antagonist Kremen-2

Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2) is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2) results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.     

An Ancestral Recombination Graph for Diploid Populations with Skewed Offspring Distribution

A large offspring-number diploid biparental multilocus population model of Moran type is our object of study. At each time step, a pair of diploid individuals drawn uniformly at random contributes offspring to the population. The number of offspring can be large relative to the total population size. Similar “heavily skewed” reproduction mechanisms have been recently considered by various authors (cf. e.g., Eldon and Wakeley 2006, 2008) and reviewed by Hedgecock and Pudovkin (2011). Each diploid parental individual contributes exactly one chromosome to each diploid offspring, and hence ancestral lineages can coalesce only when in distinct individuals. A separation-of-timescales phenomenon is thus observed. A result of Möhle (1998) is extended to obtain convergence of the ancestral process to an ancestral recombination graph necessarily admitting simultaneous multiple mergers of ancestral lineages. The usual ancestral recombination graph is obtained as a special case of our model when the parents contribute only one offspring to the population each time. Due to diploidy and large offspring numbers, novel effects appear. For example, the marginal genealogy at each locus admits simultaneous multiple mergers in up to four groups, and different loci remain substantially correlated even as the recombination rate grows large. Thus, genealogies for loci far apart on the same chromosome remain correlated. Correlation in coalescence times for two loci is derived and shown to be a function of the coalescence parameters of our model. Extending the observations by Eldon and Wakeley (2008), predictions of linkage disequilibrium are shown to be functions of the reproduction parameters of our model, in addition to the recombination rate. Correlations in ratios of coalescence times between loci can be high, even when the recombination rate is high and sample size is large, in large offspring-number populations, as suggested by simulations, hinting at how to distinguish between different population models.     

Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader

Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca(2+) fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested--G(q)-coupled P2Y(2), G(s)-coupled dopamine D1 and D5, G(i)-coupled dopamine D2L, and G(q/11)-coupled muscarinic acetylcholine M1--yielded a significant rise in intracellular free [Ca(2+)] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response. Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca(2+) fluxes.     

Visualisation of Respiratory Tumour Motion and Co-Moving Isodose Lines in the Context of Respiratory Gating,IMRT and Flattening-Filter-Free Beams

Respiratory motion during percutaneous radiotherapy can be considered based on respiration-correlated computed tomography (4DCT). However, most treatment planning systems perform the dose calculation based on a single primary CT data set, even though cine mode displays may allow for a visualisation of the complete breathing cycle. This might create the mistaken impression that the dose distribution were independent of tumour motion. We present a movie visualisation technique with the aim to direct attention to the fact that the dose distribution migrates to some degree with the tumour and discuss consequences for gated treatment, IMRT plans and flattening-filter-free beams. This is a feasibility test for a visualisation of tumour and isodose motion. Ten respiratory phases are distinguished on the CT, and the dose distribution from a stationary IMRT plan is calculated on each phase, to be integrated into a movie of tumour and dose motion during breathing. For one example patient out of the sample of five lesions, the plan is compared with a gated treatment plan with respect to tumour coverage and lung sparing. The interplay-effect for small segments in the IMRT plan is estimated. While the high dose rate, together with the cone-shaped beam profile, makes the use of flattening-filter-free beams more problematic for conformal and IMRT treatment, it can be the option of choice if gated treatment is preferred. The different effects of respiratory motion, dose build-up and beam properties (segments and flatness) for gated vs. un-gated treatment can best be considered if planning is performed on the full 4DCT data set, which may be an incentive for future developments of treatment planning systems.     

SlyD proteins from different species exhibit high prolyl isomerase and chaperone activities

SlyD is a putative folding helper protein from the Escherichia coli cytosol, which consists of an N-terminal prolyl isomerase domain of the FKBP type and a presumably unstructured C-terminal tail. We produced truncated versions without this tail (SlyD) for SlyD from E. coli, as well as for the SlyD orthologues from Yersinia pestis, Treponema pallidum, Pasteurella multocida, and Vibrio cholerae. They are monomeric in solution and unfold reversibly. All SlyD variants catalyze the proline-limited refolding of ribonuclease T1 with very high efficiencies, and the specificity constants (kcat/KM) are equal to approximately 10(6) M(-1) s(-1). These large values originate from the high affinities of the SlyD orthologues for unfolded RCM-T1, which are reflected in low KM values of approximately 1 microM. SlyD also exhibits pronounced chaperone properties. Permanently unfolded proteins bind with high affinity to SlyD and thus inhibit its prolyl isomerase activity. The unfolded protein chains do not need to contain proline residues to be recognized and bound by SlyD. The conservation of prolyl isomerase activity and chaperone properties within the SlyD family suggests that these proteins might act as true folding helpers in the bacterial cytosol. The SlyD proteins are also well suited for biotechnological applications. As fusion partners they facilitate the refolding and increase the solubility of aggregation-prone proteins such as the gp41 ectodomain fragment of HIV-1.