Comparative analysis of cis-regulation following stroke and seizures in subspaces of conserved eigensystems

Background  

It is often desirable to separate effects of different regulators on gene expression, or to identify effects of the same regulator across several systems. Here, we focus on the rat brain following stroke or seizures, and demonstrate how the two tasks can be approached simultaneously.     

Effect of mitochondrial genome rearrangement on respiratory activity, photosynthesis, photorespiration and energy status of MSC16 cucumber (Cucumis sativus) mutant

The effects of changes in mitochondrial DNA in cucumber ( Cucumis sativus L.) mosaic mutant (MSC16) on respiration, photosynthesis and photorespiration were analyzed under non-stressed conditions. Decreased respiratory capacity of complex I in MSC16 mitochondria was indicated by lower respiration rates of intact mitochondria with malate and by rotenone-inhibited NADH or malate oxidation in the presence of alamethicin. Moreover, blue native PAGE indicated decreased intensity of protein bands of respiratory chain complex I in MSC16 leaves. Concerning the redox state, complex I impairment could be compensated to some extent by increased external NADH dehydrogenases (ND_exNADH) and alternative oxidase (AOX) capacity, the latter presenting differential expression in the light and in the dark. Although MSC16 mitochondria have a higher AOX protein level and an increased capacity, the AOX activity measured in the dark conditions by oxygen discrimination technique is similar to that in wild-type (WT) plants. Photosynthesis induction by light followed different patterns in WT and MSC16, suggesting changes in feedback chloroplast ΔpH caused by different adenylate levels. At steady-state, net photosynthesis was only slightly impaired in MSC16 mutants, while photorespiration rate (PR) was significantly increased. This was the result of large decreases in both stomatal and mesophyll conductance to CO₂, which resulted in a lower CO₂ concentration in the chloroplasts. The observed changes on CO₂ diffusion caused by mitochondrial mutations open a whole new view of interaction between organelle metabolism and whole tissue physiology. The sum of all the described changes in photosynthetic and respiratory metabolism resulted in a lower ATP availability and a slower plant growth.     

An α-galactosidase with an essential function during leaf development

The putative α-galactosidase gene HvSF11 of barley, previously shown to be expressed during dark induced senescence, is expressed in the growing/elongating zone of primary foliage leaves of barley. The amino acid sequence deduced from the full length HvSF11 cDNA contains a hydrophobic signal sequence at the N-terminus. Phylogenetic relationship of the HvSF11 encoded barley α-galactosidase to other α-galactosidases revealed high homology with the α-galactosidase encoded by the gene At5g08370 from Arabidopsis thaliana. We have isolated two independent heterozygous At5g08370 T-DNA insertion mutants from Arabidopsis thaliana, both of which have a higher number of rosette leaves with a curly surface leaf morphology and delayed flowering time in comparison to wildtype plants. Localization of the Arabidopsis α-galactosidase protein via GUS-tag revealed that the protein is associated with the cell wall. This result was confirmed by immunological detection of the orthologous barley protein in a protein fraction derived from cell walls of barley leaves. It is concluded that the α-galactosidase proteins from barley and Arabidopsis might fulfill an important role in leaf development by functioning in cell wall loosening and cell wall expansion.

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Structure-activity relationships, and drug metabolism and pharmacokinetic properties for indazole piperazine and indazole piperidine inhibitors of ROCK-II

ROCK has been implicated in many diseases ranging from glaucoma to spinal cord injury and is therefore an important target for therapeutic intervention. In this study, we have designed a series of 1-(4-(1H-indazol-5-yl)piperazin-1-yl)-2-hydroxy(or 2-amino) analogs and a series of 1-(4-(1H-indazol-5-yl amino)piperidin-1-yl)-2-hydroxy(or 2-amino) inhibitors of ROCK-II. SR-1459 has IC(50)=13nM versus ROCK-II while the IC(50)s for SR-715 and SR-899 are 80nM and 100nM, respectively. Many of these inhibitors, especially the 2-amino substituted analogs for both series, are modest/potent CYP3A4 inhibitors as well. However, a few of these inhibitors (SR-715 and SR-899) show strong selectivity for ROCK-II over CYP3A4, but the overall potency of the 2-amino analogs (SR-1459) on CYP3A4 and the high clearance and volume of distribution of these compounds makes the in vivo utility of these analogs undesirable.     

Cholesterol oxidase is required for virulence of Mycobacterium tuberculosis

Recent reports have indicated that cholesterol plays a crucial role during the uptake of mycobacteria by macrophages. However, the significance of cholesterol modification enzymes encoded by Mycobacterium tuberculosis for bacterial pathogenicity remains unknown. Here, the authors explored whether the well-known cholesterol modification enzyme, cholesterol oxidase (ChoD), is important for virulence of the tubercle bacillus. Homologous recombination was used to replace the choD gene from the M. tuberculosis genome with a nonfunctional copy. The resultant mutant (delta choD) was attenuated in peritoneal macrophages. No attenuation in macrophages was observed when the same strain was complemented with an intact choD gene controlled by a heat shock promoter (delta choDP(hsp)choD). The mice infection experiments confirm the significance of ChoD in the pathogenesis of M. tuberculosis.     

Cardiovascular events in early RA are a result of inflammatory burden and traditional risk factors: a five year prospective study

Introduction  

Co-morbidity and mortality due to cardiovascular disease (CVD) are increased in patients with rheumatoid arthritis (RA). Most published studies in this field are retrospective or cross sectional. We investigated the presence of traditional and disease related risk factors for CVD at the onset of RA and during the first five years following diagnosis. We also evaluated their potential for predicting a new cardiovascular event (CVE) during the five-year follow-up period and the modulatory effect of pharmacological treatment.     

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses^1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)³. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity⁴, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method⁵. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses^6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide⁶, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)⁷. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera^7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only. Download video file.^{(80M, mov)}     

Infections caused by RSV among children and adults during two epidemic seasons

Respiratory Syncytial Virus (RSV) is one of the most common causes of lower respiratory tract infections in young children, immunocompromised patients (children and adults), patients with chronic respiratory diseases and elderly people. Reinfections occur throughout the life, but the severity of disease decreased with subsequent infection. The aim of this study was to analyze the frequency of RSV infections in two selected subpopulations: young children (below 5 y.) and adults with chronic respiratory diseases (25-87 y.). Nasopharyngeal swabs (334) collected from October 2008 to March 2010 were examined. The presence of RSV genome was determined by RT-PCR and the presence of RSV antigen by quick immunochromatographic test. Positive results of RT-PCR were found in 45.2% of all swabs: 48.6% samples in 2008; 41.5% in 2009; 50.8% in 2010. The highest frequency of RSV-positive samples was in fall-winter months, but differences in RSV epidemic seasons were found. In the first season (2008-2009) an increased number of RSV infections was observed from November 2008, but in the second season--from January 2010. Generally, the frequency of RSV-positive RT-PCR among children was 53%, among adults 25%. The highest difference was observed in the first three-month period of 2010. RT-PCR positive samples were found in 68.5% of children and 5.9% of adults. However, the RSV antigen was found in 44.4% of samples collected from adults in this period. Our results indicate that the contribution of RSV infections during epidemic season of respiratory tract infections in Poland was really high among children and adults.     

Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission‐FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional “digestion & extraction” method such as: non‐destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)