Individual mRNA expression profiles reveal the effects of specific microRNAs

BACKGROUND: MicroRNAs (miRNAs) are oligoribonucleotides with an important role in regulation of gene expression at the level of translation. Despite imperfect target complementarity, they can also significantly reduce mRNA levels. The validity of miRNA target gene predictions is difficult to assess at the protein level. We sought, therefore, to determine whether a general lowering of predicted target gene mRNA expression by endogenous miRNAs was detectable within microarray gene expression profiles. RESULTS: The target gene sets predicted for each miRNA were mapped onto known gene expression data from a range of tissues. Whether considering mean absolute target gene expression, rank sum tests or 'ranked ratios', many miRNAs with significantly reduced target gene expression corresponded to those known to be expressed in the cognate tissue. Expression levels of miRNAs with reduced target mRNA levels were higher than those of miRNAs with no detectable effect on mRNA expression. Analysis of microarray data gathered after artificial perturbation of expression of a specific miRNA confirmed the predicted increase or decrease in influence of the altered miRNA upon mRNA levels. Strongest associations were observed with targets predicted by TargetScan. CONCLUSION: We have demonstrated that the effect of a miRNA on its target mRNAs' levels can be measured within a single gene expression profile. This emphasizes the extent of this mode of regulation in vivo and confirms that many of the predicted miRNA-mRNA interactions are correct. The success of this approach has revealed the vast potential for extracting information about miRNA function from gene expression profiles.

Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column

The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

Impact of cough across different chronic respiratory diseases: comparison of two cough-specific health-related quality of life questionnaires

Background: Cough is a prominent symptom across a range of common chronic respiratory diseases and impacts considerably on patient health status.

Methods: We undertook a cross-sectional comparison of scores from two cough-specific health-related quality of life (HRQoL) questionnaires, the Leicester Cough Questionnaire (LCQ), and the Cough Quality of Life Questionnaire (CQLQ), together with a generic HRQoL measure, the EuroQol. Questionnaires were administered to and spirometry performed on 147 outpatients with chronic cough (n = 83), COPD (n = 18), asthma (n = 20), and bronchiectasis (n = 26).

Results: There was no significant difference in the LCQ and CQLQ total scores between groups (p = 0.24 and p = 0.26, respectively). Exploratory analyses of questionnaire subdomains revealed differences in psychosocial issues and functional impairment between the four groups (p = 0.01 and p = 0.05, respectively). CQLQ scores indicated that chronic coughers have more psychosocial issues than patients with bronchiectasis (p = 0.03) but less functional impairment than COPD patients (p = 0.04). There was a significant difference in generic health status across the four disease groups (p = 0.04), with poorest health status in COPD patients. A significant inverse correlation was observed between CQLQ and LCQ in each disease group (chronic cough r = - 0.56, p < 0.001; COPD r = - 0.49, p = 0.04; asthma r = - 0.94, p < 0.001; and bronchiectasis r = - 0.88, p < 0.001). There was no correlation between cough questionnaire scores and FEV1 in any group, although a significant correlation between EuroQol visual analog scale component and FEV1 (r = 0.639, p = 0.004) was observed in COPD patients.

Conclusion: Cough adversely affects health status across a range of common respiratory diseases. The LCQ and CQLQ can each provide important additional information concerning the impact of cough.

Isoform-specific inhibition of TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.

Full-length transient receptor potential (TRP) cation channel TRPC4alpha and shorter TRPC4beta lacking 84 amino acids in the cytosolic C terminus are expressed in smooth muscle and endothelial cells where they regulate membrane potential and Ca(2+) influx. In common with other "classical" TRPCs, TRPC4 is activated by G(q)/phospholipase C-coupled receptors, but the underlying mechanism remains elusive. Little is also known about any isoform-specific channel regulation. Here we show that TRPC4alpha but not TRPC4beta was strongly inhibited by intracellularly applied phosphatidylinositol 4,5-bisphosphate (PIP(2)). In contrast, several other phosphoinositides (PI), including PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), had no effect or even potentiated TRPC4alpha indicating that PIP(2) inhibits TRPC4alpha in a highly selective manner. We show that PIP(2) binds to the C terminus of TRPC4alpha but not that of TRPC4beta in vitro. Its inhibitory action was dependent on the association of TRPC4alpha with actin cytoskeleton as it was prevented by cytochalasin D treatment or by the deletion of the C-terminal PDZ-binding motif (Thr-Thr-Arg-Leu) that links TRPC4 to F-actin through the sodium-hydrogen exchanger regulatory factor and ezrin. PIP(2) breakdown appears to be a required step in TRPC4alpha channel activation as PIP(2) depletion alone was insufficient for channel opening, which additionally required Ca(2+) and pertussis toxin-sensitive G(i/o) proteins. Thus, TRPC4 channels integrate a variety of G-protein-dependent stimuli, including a PIP(2)/cytoskeleton dependence reminiscent of the TRPC4-like muscarinic agonist-activated cation channels in ileal myocytes.

Cell-specific processing of chromogranin A in endocrine cells of the rat stomach

The rat stomach is rich in endocrine cells. The acid-producing (oxyntic) mucosa contains ECL cells, A-like cells, and somatostatin (D) cells, and the antrum harbours gastrin (G) cells, enterochromaffin (EC) cells and D cells. Although chromogranin A (CgA) occurs in all these cells, its processing appears to differ from one cell type to another. Eleven antisera generated to different regions of rat CgA, two antisera generated to a human (h) CgA sequences, and one to a bovine Ib) CgA sequence, respectively, were employed together with antisera directed towards cell-specific markers such as gastrin (G cells), serotonin (EC cells), histidine decarboxylsae (ECL cells) and somatostatin (D cells) to characterize the expression of CgA and CgA-derived peptides in the various endocrine cell populations of the rat stomach. In the oxyntic mucosa, antisera raised against CgA(291-319) and CGA(316-321) immunostained D cells exclusively, whereas antisera raised against bCgA(82-91) and CgA(121-128) immunostained A-like cells and D cells. Antisera raised against CgA(318-349) and CgA(437-448) immunostained ECL cells and A-like cells, but not D cells. In the antrum, antisera against CgA(291-319) immunostained D cells, and antisera against CgA(351-356) immunostained G cells. Our observations suggest that each individual endocrine cell type in the rat stomach generates a unique mixture of CgA-derived peptides, probably reflecting cell-specific differences in the post-translational processing of CgA and its peptide products. A panel of antisera that recognize specific domains of CgA may help to identify individual endocrine cell populations.

Sensory-specific satiety and flavor amplification of foods

The effect of flavor amplification on sensory-specfic satiety was investigated. Nineteen young adults (mean age = 25 years) and 19 elderly adults (mean age = 72 years) rated the sensory properties of six foods, and were then asked to consume normal-flavored or flavor-amplified strawberry yogurt until comfortably full. The participants then re-rated the sensory properties of the six foods. There were no cl differences in the amount of yogurt consumed in either age group. Moreover flavor-fortifying the yogurt had no effect on the amount consumed in either age group. The consumption of both yogurts caused a reduction in rated pleasantness of the yogurt among young adults, but no change in the rated pleasantness of the uneaten foods. However, the elderly did not show a decrease in the rated pleasantness of any of the foods contained in the taste trays This study indicates that sensations of sensory-specific satiety were significantly reduced in the elderly, and these sensations were not induced by the addition of strawberry flavor to the yogurt.

Specific and sensitive detection of Nosema bombi (Microsporidia : Nosematidae) in bumble bees (Bombus spp.; Hymenoptera : Apidae) by PCR of partial rRNA gene sequences

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees. Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N. bombi (323 bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 107 down to 10 spores diluted in 100 mu l of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 10(6) spores down to 1 spore per PCR). Though the N. bombi-specific primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi-non-specific primer pair ITS-f2/r2, which amplifies a short fragment of similar to 120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to b more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy. (c) 2005 Elsevier Inc. All rights reserved.

Supported and liquid phase task specific ionic liquids for base catalysed Knoevenagel reactions

A series of Hunig's base tethered ammonium ionic liquids have been used to catalyse the Knoevenagel condensation of aldehydes/ketones with malononitrile and ethyl cyanoacetate. The reactions were performed under homogeneous and under biphasic, liquid-liquid and liquid-silica supported ionic liquid, conditions with the biphasic systems employing cyclohexene as the second phase. By increasing the distance between the ammonium head group and Hunig's base the activity of the catalyst was found to increase. Higher activity, in general, was found under homogeneous reaction conditions; however, the recyclability of the catalyst was improved by supporting the BIL under biphasic conditions. (c) 2007 Elsevier B.V. All rights reserved.