Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

Effect of helix-promoting strategies on the biological activity of novel analogues of the B-chain of INSL3

Insulin-like 3 (INSL3) is a novel circulating peptide hormone that is produced by testicular Leydig cells and ovarian thecal and luteal cells. In males, INSL3 is responsible for testicular descent during foetal life and suppresses germ cell apoptosis in adult males, whereas in females, it causes oocyte maturation. Antagonists of INSL3 thus have significant potential clinical application as contraceptives in both males and females. Previous work has shown that the INSL3 receptor binding region is largely confined to the B-chain central α-helix of the hormone and a conformationally constrained analogue of this has modest receptor binding and INSL3 antagonist activity. In the present study, we have employed and evaluated several approaches for increasing the α-helicity of this peptide in order to better present the key receptor binding residues and increase its affinity for the receptor. Analogues of INSL3 with higher α-helicity generally had higher receptor binding affinity although other structural considerations limit their effectiveness.     

Human gene 2 relaxin chain combination and folding

Relaxin is a small 6 kD two-chain peptide member of the insulin superfamily that is principally produced in the corpus luteum of the ovary and which plays a key role in connective tissue remodeling during parturition. Like insulin, it is produced on the ribosome as preprohormone that undergoes oxidative folding and subsequent proteolytic processing to yield the mature insulin-like peptide. In contrast to the now considerable insight into insulin chain folding and oxidation, comparatively little is known about the folding pathway of relaxin. A series of synthetic pairwise serine substituted relaxin A-chain cysteine analogues was prepared, and their oxidation behavior was studied both on their own and in the presence of native relaxin B-chain. It was observed that native S-reduced A-chain oxidized rapidly to a bicyclic product, whereas individual formation of each of the intramolecular disulfide bonds between Cys11 and Cys24 and the native Cys10 and Cys15 was considerably slower. Curiously, the non-native, isomeric Cys11-Cys15 disulfide bond formed most rapidly, although circular dichroism spectroscopy analysis showed this product to be devoid of secondary structure. This suggested that it may in fact be an intermediate in the subsequent formation of the native Cys10-Cys15 intramolecular disulfide. Combination of the native A-chain with the B-chain proceeded rapidly as compared with the A-chain analogue that lacked the intramolecular disulfide bond suggesting that this latter element is required as a first step in the folding process. It is therefore probable that relaxin is generated from its constituent A- and B-chains in a stepwise organization manner similar to that of insulin chain combination and folding. Further studies showed that the efficiency of combination of A-chain to B-chain was not markedly influenced by reaction temperature and that a reasonable yield of relaxin could be obtained on combination of the preoxidized A-chain with the S-reduced B-chain.     

Mitochondrial growth and division during the cell cycle in HeLa cells

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Rapid determination of transgene copy number in stably-transfected mammalian cells by competitive PCR

We describe here an application of the competitive PCR technique to the analysis of copy number of recombinant rat parathyroid hormone-related protein (rPTHrP) gene in stably-transfected murine erythroleukemia (MEL) cell lines. A single-copy reference gene (endogenous mouse PTHrP gene or mPTHrP) is used as an internal control. This control gene, present in the genome of MEL cells, shares the same primer binding sites as the rPTHrP cDNA but contains an internal PvuII site, which allows resolution of the amplified products after restriction enzyme digestion by polyacrylamide gel electrophoresis (PAGE). The transgene copy number is determined by the ratio of band intensity of the rPTHrP product to that of the mPTHrP product. Using this method, we have determined the copy number of the rPTHrP transgene from isolated genomic DNA, and compared the results with those obtained from Southern blot analysis. In addition, we have demonstrated that the procedure can be applied very simply to whole MEL cells without DNA extractions and that as few as 10⁴ cells are required for the analysis.     

Analysis of expressed sequence tags from oil palm (Elaeis guineensis)

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm.     

Global expression profiling of theophylline response genes in macrophages: evidence of airway anti-inflammatory regulation

Background

Theophylline has been used widely as a bronchodilator for the treatment of bronchial asthma and has been suggested to modulate immune response. While the importance of macrophages in asthma has been reappraised and emphasized, their significance has not been well investigated. We conducted a genome-wide profiling of the gene expressions of macrophages in response to theophylline.

Methods

Microarray technology was used to profile the gene expression patterns of macrophages modulated by theophylline. Northern blot and real-time quantitative RT-PCR were also used to validate the microarray data, while Western blot and ELISA were used to measure the levels of IL-13 and LTC4.

Results

We identified dozens of genes in macrophages that were dose-dependently down- or up-regulated by theophylline. These included genes related to inflammation, cytokines, signaling transduction, cell adhesion and motility, cell cycle regulators, and metabolism. We observed that IL-13, a central mediator of airway inflammation, was dramatically suppressed by theophylline. Real-time quantitative RT-PCR and ELISA analyses also confirmed these results, without respect to PMA-treated THP-1 cells or isolated human alveolar macrophages. Theophylline, rolipram, etazolate, db-cAMP and forskolin suppressed both IL-13 mRNA expression (~25%, 2.73%, 8.12%, 5.28%, and 18.41%, respectively) and protein secretion (<10% production) in macrophages. These agents also effectively suppressed LTC4 expression.

Conclusion

Our results suggest that the suppression of IL-13 by theophylline may be through cAMP mediation and may decrease LTC4 production. This study supports the role of theophylline as a signal regulator of inflammation, and that down regulation of IL-13 by theophylline may have beneficial effects in inflammatory airway diseases.