IL-4, but not IL-5, can act synergistically with B cell activating factor (BCAF) to induce proliferation of resting B cells

B cell activating factor (BCAF) was initially identified in the supernatant of the murine T helper cell clone 52-3 (52-3 SN) because of its ability to promote activation and proliferation of resting B cells in the absence of any other costimulus. In this paper, we show that 52-3 T helper cells also secrete IL-4 and IL-5 and we have analyzed the influence of these two lymphokines on B cell proliferation induced by BCAF-containing 52-3 SN. Using the neutralizing anti-IL-4 monoclonal antibody 11B11, we observed partial inhibition of B cell proliferation. 52-3 SN free of IL-4 prepared using an immunoabsorbent column was still able to induce significant B cell proliferation. Although recombinant IL-4 alone does not induce B cell proliferation, it increased the proliferation induced by IL-4-free 52-3 SN. Kinetic studies showed that IL-4 is required at the start of B cell cultures in order to exert optimal synergistic effects. In contrast, anti-IL-5 monoclonal antibody NC17 did not affect the B cell proliferative activity of 52-3 SN whether or not IL-4 was present. When 52-3 SN was tested on dextran-sulfate-activated B cells, IL-5 and BCAF activities were detected but only the IL-5 activity was neutralized by monoclonal antibody NC17. These results demonstrate that (i) BCAF-containing SN can induce proliferation of resting B cells independently of IL-4 and IL-5, and (ii) IL-4, but not IL-5, can act synergistically with BCAF to induce B cell proliferation.     

DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.     

TORC2 Is Required to Maintain Genome Stability during S Phase in Fission Yeast

DNA damage can occur due to environmental insults or intrinsic metabolic processes and is a major threat to genome stability. The DNA damage response is composed of a series of well coordinated cellular processes that include activation of the DNA damage checkpoint, transient cell cycle arrest, DNA damage repair, and reentry into the cell cycle. Here we demonstrate that mutant cells defective for TOR complex 2 (TORC2) or the downstream AGC-like kinase, Gad8, are highly sensitive to chronic replication stress but are insensitive to ionizing radiation. We show that in response to replication stress, TORC2 is dispensable for Chk1-mediated cell cycle arrest but is required for the return to cell cycle progression. Rad52 is a DNA repair and recombination protein that forms foci at DNA damage sites and stalled replication forks. TORC2 mutant cells show increased spontaneous nuclear Rad52 foci, particularly during S phase, suggesting that TORC2 protects cells from DNA damage that occurs during normal DNA replication. Consistently, the viability of TORC2-Gad8 mutant cells is dependent on the presence of the homologous recombination pathway and other proteins that are required for replication restart following fork replication stalling. Our findings indicate that TORC2 is required for genome integrity. This may be relevant for the growing amount of evidence implicating TORC2 in cancer development.     

Pyrido[2,3-d]pyrimidines: Discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.     

Markov Model Predicts Changes in STH Prevalence during Control Activities Even with a Reduced Amount of Baseline Information

Background

Estimating the reduction in levels of infection during implementation of soil-transmitted helminth (STH) control programmes is important to measure their performance and to plan interventions. Markov modelling techniques have been used with some success to predict changes in STH prevalence following treatment in Viet Nam. The model is stationary and to date, the prediction has been obtained by calculating the transition probabilities between the different classes of intensity following the first year of drug distribution and assuming that these remain constant in subsequent years. However, to run this model longitudinal parasitological data (including intensity of infection) are required for two consecutive years from at least 200 individuals. Since this amount of data is not often available from STH control programmes, the possible application of the model in control programme is limited. The present study aimed to address this issue by adapting the existing Markov model to allow its application when a more limited amount of data is available and to test the predictive capacities of these simplified models.

Method

We analysed data from field studies conducted with different combination of three parameters: (i) the frequency of drug administration; (ii) the drug distributed; and (iii) the target treatment population (entire population or school-aged children only). This analysis allowed us to define 10 sets of standard transition probabilities to be used to predict prevalence changes when only baseline data are available (simplified model 1). We also formulated three equations (one for each STH parasite) to calculate the predicted prevalence of the different classes of intensity from the total prevalence. These equations allowed us to design a simplified model (SM2) to obtain predictions when the classes of intensity at baseline were not known. To evaluate the performance of the simplified models, we collected data from the scientific literature on changes in STH prevalence during the implementation of 26 control programmes in 16 countries. Using the baseline data observed, we applied the simplified models and predicted the onward prevalence of STH infection at each time-point for which programme data were available. We then compared the output from the model with the observed data from the programme.

Results

The comparison between the model-predicted prevalence and the observed values demonstrated a good accuracy of the predictions. In 77% of cases the original model predicted a prevalence within five absolute percentage points from the observed figure, for the simplified model one in 69% of cases and for the simplified model two in 60% of cases. We consider that the STH Markov model described here could be an important tool for programme managers to monitor the progress of their control programmes and to select the appropriate intervention. We also developed, and made freely available online, a software tool to enable the use of the STH Markov model by personnel with limited knowledge of mathematical models.     

Neuroprotection by pramipexole against dopamine- and levodopa-induced cytotoxicity

Pramipexole, a novel non-ergoline dopamine (DA) agonist, has been applied successfully for treatment of Parkinson's disease (PD). We report here that pramipexole can protect dopaminergic cell line Mes23.5 against dopamine- and levodopa-induced cytotoxicity possibly through a mechanism related to antioxidant activity. In the MES 23.5 cultures, DA and L-DOPA induce a dose- and time-dependent cytotoxicity, as determined by tetrazolium salt and trypan blue assays. Furthermore, an in situ terminal deoxynucleotidyl transferase assay demonstrates that DA-induced cell death is apoptotic. Pretreatment with pramipexole in a concentration range (4-100 microM) significantly attenuates DA- or L-DOPA-induced cytotoxicity and apoptosis, an action which is not blocked by D3 antagonist U-99194 A or D2 antagonist raclopride. Pramipexole also protects MES 23.5 cells from hydrogen peroxide-induced cytotoxicity in a dose-dependent manner. In cell-free system, pramipexole can effectively inhibit the formation of melanin, an end product resulting from DA or L-DOPA oxidation. These results indicate that pramipexole exerts its neuroprotective effect possibly through a mechanism, which is independent of DA receptors but related to antioxidation or scavenging of free radicals (e.g. hydrogen peroxide). As a direct DA agonist and potentially neuroprotective agent, pramipexole remains attractive in the treatment of PD.     

The epithelial-mesenchymal transition of the Drosophila mesoderm requires the Rho GTP exchange factor Pebble

Drosophila pebble (pbl) encodes a Rho-family GTP exchange factor (GEF) required for cytokinesis. The accumulation of high levels of PBL protein during interphase and the developmentally regulated expression of pbl in mesodermal tissues suggested that the primary cytokinetic mutant phenotype might be masking other roles. Using various muscle differentiation markers, we found that Even skipped (EVE) expression in the dorsal mesoderm is greatly reduced in pbl mutant embryos. EVE expression in the dorsalmost mesodermal cells is induced in response to DPP secreted by the dorsal epidermal cells. Further analysis revealed that this phenotype is likely to be a consequence of an earlier defect. pbl mutant mesodermal cells fail to undergo the normal epithelial-mesenchymal transition (EMT) and dorsal migration that follows ventral furrow formation. This phenotype is not a secondary consequence of failed cytokinesis, as it is rescued by a mutant form of pbl that does not rescue the cytokinetic defect. In wild-type embryos, newly invaginated cells at the lateral edges of the mesoderm extend numerous protrusions. In pbl mutant embryos, however, cells appear more tightly adhered to their neighbours and extend very few protrusions. Consistent with the dependence of the mesoderm EMT and cytokinesis on actin organisation, the GTP exchange function of the PBL RhoGEF is required for both processes. By contrast, the N-terminal BRCT domains of PBL are required only for the cytokinetic function of PBL. These studies reveal that a novel PBL-mediated intracellular signalling pathway operates in mesodermal cells during the transition from an epithelial to migratory mesenchymal morphology during gastrulation.     

Development of a size-dependent aerosol deposition model utilising human airway epithelial cells for evaluating aerosol drug delivery

Aerosol delivery to the airways of the human respiratory tract, followed by absorption, constitutes an alternative route of administration for compounds unsuitable for delivery by conventional oral and parenteral routes. The target for aerosol drug delivery is the airways epithelium, i.e. tracheal, bronchial, bronchiolar and alveolar cells, which become the site of drug deposition. These epithelial layers also serve as a barrier to the penetration of inhaled material. An in vitro model for aerosol deposition and transport across epithelia in the human airways may be a good predictor of in vivo disposition. The present preliminary studies begin an investigation that blends the dynamics of aerosol delivery and the basis of an in vitro simulated lung model to evaluate the transport properties of a series of molecular weight marker compounds across human-derived bronchiolar epithelial cell monolayers. An Andersen viable cascade impactor was used as a delivery apparatus for the deposition of size-segregated particles onto monolayers of small airway epithelial cells and Calu-3 cells. It was shown that these cell layers can withstand placement in the impactor, and that permeability can be tested subsequent to removal from the impactor.     

Age at onset in two common neurodegenerative diseases is genetically controlled

To identify genes influencing age at onset (AAO) in two common neurodegenerative diseases, a genomic screen was performed for AAO in families with Alzheimer disease (AD; n=449) and Parkinson disease (PD; n=174). Heritabilities between 40%–60% were found in both the AD and PD data sets. For PD, significant evidence for linkage to AAO was found on chromosome 1p (LOD = 3.41). For AD, the AAO effect of APOE (LOD = 3.28) was confirmed. In addition, evidence for AAO linkage on chromosomes 6 and 10 was identified independently in both the AD and PD data sets. Subsequent unified analyses of these regions identified a single peak on chromosome 10q between D10S1239 and D10S1237, with a maximum LOD score of 2.62. These data suggest that a common gene affects AAO in these two common complex neurodegenerative diseases.