Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced strand separation of double helical DNA

Escherichia coli RNA polymerase is able to site-specifically melt 12 bp of promoter DNA at temperatures far below those normally associated with DNA melting. Here we consider several models to explain how RNA polymerase destabilizes duplex DNA. One popular model proposes that upon binding to the promoter, RNA polymerase untwists the spacer DNA between the –10 and –35 regions, which results in a destabilization of the –10 region at a TA base step where melting initiates. Promoter untwisting may result, in part, from extensive wrapping of the DNA around RNA polymerase. Formation of the strand-separated open complex appears to be facilitated by specific protein-DNA interactions which occur predominantly on the non-template strand. Recent evidence suggests that these include important contacts with Sigma factor region 2.3, which we propose binds the displaced single strand of DNA.     

Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger

Summary The subcellular localization of glucose oxidase (E.C. 1.1.3.4) in mycelia of Aspergillus niger has been investigated using cytochemical staining techniques. Mycelia from fermenter cultures, which produced gluconic acid from glucose, contained elevated levels of glucose oxidase and catalase. Both enzymes were located in microbodies. In addition, when the organism was grown on glucose with methylamine as a nitrogen source, amine oxidase activity was detected in the microbodies. These organelles can therefore be designated as peroxisomes.     

Close linkage of the Wilsons's disease locus to D13S12 in the chromosomal region 13q21 and not to ESD in 13q14

Summary Wilson's disease (WD) is an autosomal recessive disorder resulting in copper accumulation notably in liver and brain tissue. Linkage of the WD locus (WND) to ESD at 13q14 was first shown by studies in families of Middle Eastern origin using the isozymic polymorphism of esterase D. Using RFLPs detected by the ESD cDNA we could not confirm this reported close linkage in an analysis of 17 WD families of northwest European origin. A tight linkage was detected, however, to the marker D13S12, located more distally at 13q21. No obligate cross-overs were detected in 63 gametes informative for this marker. Our data confirm an assignment of WND to 13q14-21. Its localization, however, seems to be more distal to ESD than previously reported. Although genetic heterogeneity cannot be excluded, the observed differences between the two populations are probably due to random variation.     

Unique Features of the Loblolly Pine (Pinus taeda L.) Megagenome Revealed Through Sequence Annotation

The largest genus in the conifer family Pinaceae is Pinus, with over 100 species. The size and complexity of their genomes (∼20–40 Gb, 2n = 24) have delayed the arrival of a well-annotated reference sequence. In this study, we present the annotation of the first whole-genome shotgun assembly of loblolly pine (Pinus taeda L.), which comprises 20.1 Gb of sequence. The MAKER-P annotation pipeline combined evidence-based alignments and ab initio predictions to generate 50,172 gene models, of which 15,653 are classified as high confidence. Clustering these gene models with 13 other plant species resulted in 20,646 gene families, of which 1554 are predicted to be unique to conifers. Among the conifer gene families, 159 are composed exclusively of loblolly pine members. The gene models for loblolly pine have the highest median and mean intron lengths of 24 fully sequenced plant genomes. Conifer genomes are full of repetitive DNA, with the most significant contributions from long-terminal-repeat retrotransposons. In depth analysis of the tandem and interspersed repetitive content yielded a combined estimate of 82%.     

Development and validation of a 5-day-ahead hay fever forecast for patients with grass-pollen-induced allergic rhinitis

One-third of the Dutch population suffers from allergic rhinitis, including hay fever. In this study, a 5-day-ahead hay fever forecast was developed and validated for grass pollen allergic patients in the Netherlands. Using multiple regression analysis, a two-step pollen and hay fever symptom prediction model was developed using actual and forecasted weather parameters, grass pollen data and patient symptom diaries. Therefore, 80 patients with a grass pollen allergy rated the severity of their hay fever symptoms during the grass pollen season in 2007 and 2008. First, a grass pollen forecast model was developed using the following predictors: (1) daily means of grass pollen counts of the previous 10 years; (2) grass pollen counts of the previous 2-week period of the current year; and (3) maximum, minimum and mean temperature (R ²?=?0.76). The second modeling step concerned the forecasting of hay fever symptom severity and included the following predictors: (1) forecasted grass pollen counts; (2) day number of the year; (3) moving average of the grass pollen counts of the previous 2 week-periods; and (4) maximum and mean temperatures (R ²?=?0.81). Since the daily hay fever forecast is reported in three categories (low-, medium- and high symptom risk), we assessed the agreement between the observed and the 1- to 5-day-ahead predicted risk categories by kappa, which ranged from 65 % to 77 %. These results indicate that a model based on forecasted temperature and grass pollen counts performs well in predicting symptoms of hay fever up to 5 days ahead.     

The Role of Dyadic Organization in Regulation of Sarcoplasmic Reticulum Ca2+ Handling during Rest in Rabbit Ventricular Myocytes

The dyadic organization of ventricular myocytes ensures synchronized activation of sarcoplasmic reticulum (SR) Ca²⁺ release during systole. However, it remains obscure how the dyadic organization affects SR Ca²⁺ handling during diastole. By measuring intraluminal SR Ca²⁺ ([Ca²⁺]_SR) decline during rest in rabbit ventricular myocytes, we found that ∼76% of leaked SR Ca²⁺ is extruded from the cytosol and only ∼24% is pumped back into the SR. Thus, the majority of Ca²⁺ that leaks from the SR is removed from the cytosol before it can be sequestered back into the SR by the SR Ca²⁺-ATPase (SERCA). Detubulation decreased [Ca²⁺]_SR decline during rest, thus making the leaked SR Ca²⁺ more accessible for SERCA. These results suggest that Ca²⁺ extrusion systems are localized in T-tubules. Inhibition of Na⁺-Ca²⁺ exchanger (NCX) slowed [Ca²⁺]_SR decline during rest by threefold, however did not prevent it. Depolarization of mitochondrial membrane potential during NCX inhibition completely prevented the rest-dependent [Ca²⁺]_SR decline. Despite a significant SR Ca²⁺ leak, Ca²⁺ sparks were very rare events in control conditions. NCX inhibition or detubulation increased Ca²⁺ spark activity independent of SR Ca²⁺ load. Overall, these results indicate that during rest NCX effectively competes with SERCA for cytosolic Ca²⁺ that leaks from the SR. This can be explained if the majority of SR Ca²⁺ leak occurs through ryanodine receptors in the junctional SR that are located closely to NCX in the dyadic cleft. Such control of the dyadic [Ca²⁺] by NCX play a critical role in suppressing Ca²⁺ sparks during rest.     

Development of an oral mucosa model to study host-microbiome interactions during wound healing

Crosstalk between the human host and its microbiota is reported to influence various diseases such as mucositis. Fundamental research in this area is however complicated by the time frame restrictions during which host-microbe interactions can be studied in vitro. The model proposed in this paper, consisting of an oral epithelium and biofilm, can be used to study microbe-host crosstalk in vitro in non-infectious conditions up to 72 h. Microbiota derived from oral swabs were cultured on an agar/mucin layer and challenged with monolayers of keratinocytes grown on plastic or collagen type I layers embedded with fibroblasts. The overall microbial biofilm composition in terms of diversity remained representative for the oral microbiome, whilst the epithelial cell morphology and viability were unaffected. Applying the model to investigate wound healing revealed a reduced healing of 30 % in the presence of microbiota, which was not caused by a reduction of the proliferation index (52.1–61.5) or a significantly increased number of apoptotic (1–1.13) or necrotic (32–30.5 %) cells. Since the model allows the separate study of the microbial and cellular exometabolome, the biofilm and epithelial characteristics after co-culturing, it is applicable for investigations within fundamental research and for the discovery and development of agents that promote wound healing.