Gap-Directed Translesion DNA Synthesis of an Abasic Site on Circular DNA Templates by a Human Replication Complex

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged “minicircle” DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.     

Coexpression and Heteromerization of Two Neuronal K-Cl Cotransporter Isoforms in Neonatal Brain

The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters. The KCC2 gene codes for two isoforms, KCC2a and KCC2b, which differ in their N termini. The relative expression and cellular distribution of the two KCC2 protein isoforms are unknown. Here, we characterize an antibody against the KCC2a isoform and show that a previously described antibody against KCC2 is specific for the KCC2b isoform (Hubner, C. A., Stein, V., Hermans-Borgmeyer, I., Meyer, T., Ballanyi, K., and Jentsch, T. J. (2001) Neuron 30, 515–524). Immunostaining of dissociated hippocampal cultures confirms that both KCC2 isoforms are neuron-specific. Immunoblot analysis indicates that KCC2b is the major KCC2 isoform in the adult brain, whereas in the neonatal mouse central nervous system, half of total KCC2 protein is KCC2a. At this stage, the two KCC2 isoforms are largely colocalized and show similar patterns of distribution in the brain. When coexpressed in HEK293 cells, KCC2a and KCC2b proteins form heteromeric complexes. Moreover, the two isoforms can be coimmunoprecipitated from the neonatal brain, suggesting the presence of endogenous KCC2a-KCC2b heteromers. Consistent with this, native gel analysis shows that a substantial part of endogenous KCC2 isoforms in the neonatal brain constitute dimers.The neuron-specific K⁺-Cl^- cotransporter KCC2 extrudes potassium and chloride ions from neurons, thus maintaining the low intracellular chloride concentration [Cl]_i necessary for the fast hyperpolarizing actions of inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine (1). KCC2 is an ∼140-kDa plasma membrane protein that belongs to the cation chloride cotransporter (CCC)⁴ family (2, 3). CCCs, including KCC2, are thought to exist in the plasma membrane as functional oligomers, although the mechanisms whereby oligomerization affects their transport activity are unclear (4–8).We have recently shown that the KCC2 gene (alias Slc12a5) generates two mRNAs, KCC2a and KCC2b, by an alternative promoter and first exon usage (9). The difference between the KCC2a and KCC2b proteins lies in the most N-terminal part; the 40 unique amino acids in KCC2a include a putative binding sequence for the Ste20-related proline-alanine-rich kinase (SPAK). KCC2 null mutant mice deficient for both KCC2 isoforms show a disrupted breathing rhythm and die immediately after birth (10, 11), whereas selective KCC2b isoform knock-out mice exhibit spontaneous seizures but can survive up to 3 weeks after birth (9, 12). Thus, KCC2a obviously supports some vital functions of lower brain structures.In general, KCC2 expression in the CNS follows neuronal maturation; it is first detected in the postmitotic neurons of the spinal cord and brainstem and is then gradually increased in higher brain structures (13, 14). Our previous work has shown that during postnatal development, KCC2a mRNA expression remains relatively constant, whereas KCC2b mRNA is strongly up-regulated in the cortex during postnatal development (9). This indicates that KCC2b is responsible for the developmental shift from depolarizing to hyperpolarizing GABAergic responses.Here, we study the relative expression and cellular distribution of KCC2a and KCC2b proteins in the mouse brain. We characterize a new antibody specific for the KCC2a isoform and demonstrate that a previously described antibody against KCC2 (11) is specific for the KCC2b isoform. The relative expression of the KCC2 protein isoforms determined by immunoblot analysis correlates well with their mRNA levels (9). KCC2a and KCC2b proteins have a similar level and pattern of expression in the neonatal mouse brain and are colocalized in most neurons in non-cortical lower brain areas. Coimmunoprecipitation (coIP) experiments and coexpression followed by native gel analysis indicate that KCC2a-KCC2b heteromers can form in vitro and may exist in vivo.     

Evaluation of six process‐based forest growth models using eddy‐covariance measurements of CO2 and H2O fluxes at six forest sites in Europe

Reliable models are required to assess the impacts of climate change on forest ecosystems. Precise and independent data are essential to assess this accuracy. The flux measurements collected by the EUROFLUX project over a wide range of forest types and climatic regions in Europe allow a critical testing of the process‐based models which were developed in the LTEEF project. The ECOCRAFT project complements this with a wealth of independent plant physiological measurements. Thus, it was aimed in this study to test six process‐based forest growth models against the flux measurements of six European forest types, taking advantage of a large database with plant physiological parameters. The reliability of both the flux data and parameter values itself was not under discussion in this study. The data provided by the researchers of the EUROFLUX sites, possibly with local corrections, were used with a minor gap‐filling procedure to avoid the loss of many days with observations. The model performance is discussed based on their accuracy, generality and realism. Accuracy was evaluated based on the goodness‐of‐fit with observed values of daily net ecosystem exchange, gross primary production and ecosystem respiration (gC m^?2 d^?1), and transpiration (kg H₂O m^?2 d^?1). Moreover, accuracy was also evaluated based on systematic and unsystematic errors. Generality was characterized by the applicability of the models to different European forest ecosystems. Reality was evaluated by comparing the modelled and observed responses of gross primary production, ecosystem respiration to radiation and temperature. The results indicated that: Accuracy. All models showed similar high correlation with the measured carbon flux data, and also low systematic and unsystematic prediction errors at one or more sites of flux measurements. The results were similar in the case of several models when the water fluxes were considered. Most models fulfilled the criteria of sufficient accuracy for the ability to predict the carbon and water exchange between forests and the atmosphere. Generality. Three models of six could be applied for both deciduous and coniferous forests. Furthermore, four models were applied both for boreal and temperate conditions. However, no severe water‐limited conditions were encountered, and no year‐to‐year variability could be tested. Realism. Most models fulfil the criterion of realism that the relationships between the modelled phenomena (carbon and water exchange) and environment are described causally. Again several of the models were able to reproduce the responses of measurable variables such as gross primary production (GPP), ecosystem respiration and transpiration to environmental driving factors such as radiation and temperature. Stomatal conductance appears to be the most critical process causing differences in predicted fluxes of carbon and water between those models that accurately describe the annual totals of GPP, ecosystem respiration and transpiration. As a conclusion, several process‐based models are available that produce accurate estimates of carbon and water fluxes at several forest sites of Europe. This considerable accuracy fulfils one requirement of models to be able to predict the impacts of climate change on the carbon balance of European forests. However, the generality of the models should be further evaluated by expanding the range of testing over both time and space. In addition, differences in behaviour between models at the process level indicate requirement of further model testing, with special emphasis on modelling stomatal conductance realistically.     

Collagen-induced arthritis in common marmosets: a new nonhuman primate model for chronic arthritis

Introduction  

There is an ever-increasing need for animal models to evaluate efficacy and safety of new therapeutics in the field of rheumatoid arthritis (RA). Particularly for the early preclinical evaluation of human-specific biologicals targeting the progressive phase of the disease, there is a need for relevant animal models. In response to this requirement we set out to develop a model of collagen-induced arthritis (CIA) in a small-sized nonhuman primate species (300 to 400 g at adult age); that is, the common marmoset (Callithrix jacchus).     

Meta-analysis identified the TNFA -308G > A promoter polymorphism as a risk factor for disease severity in patients with rheumatoid arthritis

Introduction

The goal of this study is to investigate whether the -308G > A promoter polymorphism in the tumor necrosis factor alpha (TNFA) gene is associated with disease severity and radiologic joint damage in a large cohort of patients with rheumatoid arthritis (RA).

Methods

A long-term observational early RA inception cohort (n = 208) with detailed information about disease activity and radiologic damage after 3, 6 and 9 years of disease was genotyped for the TNFA -308G > A promoter polymorphism (rs1800629). A longitudinal regression analysis was performed to assess the effect of genotype on RA disease severity and joint damage. Subsequently, a meta-analysis, including all publically available data, was performed to further test the association between joint erosions and the TNFA polymorphism. To learn more about the mechanism behind the effect of the polymorphism, RNA isolated from peripheral blood from RA patients (n = 66) was used for TNFA gene expression analysis by quantitative PCR.

Results

Longitudinal regression analysis with correction for gender and disease activity showed a significant difference in total joint damage between GG and GA+AA genotype groups (P = 0.002), which was stable over time. The meta-analysis, which included 2,053 patients, confirmed an association of the genetic variant with the development of erosions (odds ratio 0.78, 95% CI 0.62, 0.98). No significant differences in TNFA gene expression were observed for the different genotypes, confirming earlier findings in healthy individuals.

Conclusions

Our data confirm that the TNFA -308G > A promoter polymorphism is associated with joint damage in patients with RA. This is not mediated by differences in TNFA gene expression between genotypes.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.     

Air temperature triggers the recovery of evergreen boreal forest photosynthesis in spring

The timing of the commencement of photosynthesis (P^*) in spring is an important determinant of growing‐season length and thus of the productivity of boreal forests. Although controlled experiments have shed light on environmental mechanisms triggering release from photoinhibition after winter, quantitative research for trees growing naturally in the field is scarce. In this study, we investigated the environmental cues initiating the spring recovery of boreal coniferous forest ecosystems under field conditions. We used meteorological data and above‐canopy eddy covariance measurements of the net ecosystem CO₂ exchange (NEE) from five field stations located in northern and southern Finland, northern and southern Sweden, and central Siberia. The within‐ and intersite variability for P^* was large, 30–60 days. Of the different climate variables examined, air temperature emerged as the best predictor for P^* in spring. We also found that ‘soil thaw’, defined as the time when near‐surface soil temperature rapidly increases above 0°C, is not a useful criterion for P^*. In one case, photosynthesis commenced 1.5 months before soil temperatures increased significantly above 0°C. At most sites, we were able to determine a threshold for air‐temperature‐related variables, the exceeding of which was required for P^*. A 5‐day running‐average temperature (T₅) produced the best predictions, but a developmental‐stage model (S) utilizing a modified temperature sum concept also worked well. But for both T₅ and S, the threshold values varied from site to site, perhaps reflecting genetic differences among the stands or climate‐induced differences in the physiological state of trees in late winter/early spring. Only at the warmest site, in southern Sweden, could we obtain no threshold values for T₅ or S that could predict P^* reliably. This suggests that although air temperature appears to be a good predictor for P^* at high latitudes, there may be no unifying ecophysiological relationship applicable across the entire boreal zone.     

Barriers and facilitators to implement shared decision making in multidisciplinary sciatica care: a qualitative study

Background

Venous thromboembolism (VTE) is a common preventable cause of mortality in hospitalized medical patients. Despite rigorous randomized trials generating strong recommendations for anticoagulant use to prevent VTE, nearly 40% of medical patients receive inappropriate thromboprophylaxis. Knowledge-translation strategies are needed to bridge this gap.

Methods

We conducted a 16-week pilot cluster randomized controlled trial (RCT) to determine the proportion of medical patients that were appropriately managed for thromboprophylaxis (according to the American College of Chest Physician guidelines) within 24 hours of admission, through the use of a multicomponent knowledge-translation intervention. Our primary goal was to determine the feasibility of conducting this study on a larger scale. The intervention comprised clinician education, a paper-based VTE risk assessment algorithm, printed physicians’ orders, and audit and feedback sessions. Medical wards at six hospitals (representing clusters) in Ontario, Canada were included; three were randomized to the multicomponent intervention and three to usual care (i.e., no active strategies for thromboprophylaxis in place). Blinding was not used.

Results

A total of 2,611 patients (1,154 in the intervention and 1,457 in the control group) were eligible and included in the analysis. This multicomponent intervention did not lead to a significant difference in appropriate VTE prophylaxis rates between intervention and control hospitals (appropriate management rate odds ratio = 0.80; 95% confidence interval: 0.50, 1.28; p = 0.36; intra-class correlation coefficient: 0.022), and thus was not considered feasible. Major barriers to effective knowledge translation were poor attendance by clinical staff at education and feedback sessions, difficulty locating preprinted orders, and lack of involvement by clinical and administrative leaders. We identified several factors that may increase uptake of a VTE prophylaxis strategy, including local champions, support from clinical and administrative leaders, mandatory use, and a simple, clinically relevant risk assessment tool.

Conclusions

Hospitals allocated to our multicomponent intervention did not have a higher rate of medical inpatients appropriately managed for thromboprophylaxis than did hospitals that were not allocated to this strategy.     

Nutritional regulation of proteinase production in the fungus, Tritirachium album

The fungus, Tritirachium album, produces a number of proteinases under proper conditions. We have studied the nutritional regulation mechanisms for proteinase production in the mold, i.e. the effects of carbon and nitrogen sources, and the influence of starvation. Proteinase production was induced when the nitrogen source was an exogenous protein or peptide, such as peptones or yeast extract. The production rate was affected by the amount of available inducing substrate. Inorganic nitrogen compounds, i.e., ammonium or nitrate salts, had a repressing effect on the production. Production was not induced if a detectable concentration of glucose or sucrose was present in the medium. Starvation did not trigger proteinase production. Journal of Industrial Microbiology & Biotechnology (2000) 24, 369–373. Received 20 January 1999/ Accepted in revised form 06 March 2000