Afrotherian phylogeny as inferred from complete mitochondrial genomes

Afrotheria is a huge assemblage of various mammals encompassing six orders that were once classified as distantly related groups. This superordinal relationship may have resulted from the break-up of Gondowanaland followed by the isolation of the African continent between 105 and 40 million years ago. Although the monophyly of Afrotheria is well supported by recent molecular studies, the interrelationships within afrotherian mammals remain unclarified. In this study, we determined the sequence of the complete mitochondrial genomes of hyrax, golden mole, and elephant shrew. These sequences were compared with those of other eutherians to analyze the phylogenetic relationships among afrotherians and, in particular, those among paenungulates. Our mitochondrial genome analysis supports the monophyly of Tethytheria.     

Conversion of embryonic stem cells into neuroectodermal precursors in adherent monoculture

Mouse embryonic stem (ES) cells are competent for production of all fetal and adult cell types. However, the utility of ES cells as a developmental model or as a source of defined cell populations for pharmaceutical screening or transplantation is compromised because their differentiation in vitro is poorly controlled. Specification of primary lineages is not understood and consequently differentiation protocols are empirical, yielding variable and heterogeneous outcomes. Here we report that neither multicellular aggregation nor coculture is necessary for ES cells to commit efficiently to a neural fate. In adherent monoculture, elimination of inductive signals for alternative fates is sufficient for ES cells to develop into neural precursors. This process is not a simple default pathway, however, but requires autocrine fibroblast growth factor (FGF). Using flow cytometry quantitation and recording of individual colonies, we establish that the bulk of ES cells undergo neural conversion. The neural precursors can be purified to homogeneity by fluorescence activated cell sorting (FACS) or drug selection. This system provides a platform for defining the molecular machinery of neural commitment and optimizing the efficiency of neuronal and glial cell production from pluripotent mammalian stem cells.     

TIP,a T-cell factor identified using high-throughput screening increases survival in a graft-versus-host disease model

A coordinated effort combining bioinformatic tools with high-throughput cell-based screening assays was implemented to identify novel factors involved in T-cell biology. We generated a unique library of cDNAs encoding predicted secreted and transmembrane domain-containing proteins generated by analyzing the Human Genome Sciences cDNA database with a combination of two algorithms that predict signal peptides. Supernatants from mammalian cells transiently transfected with this library were incubated with primary T cells and T-cell lines in several high-throughput assays. Here we describe the discovery of a T cell factor, TIP (T cell immunomodulatory protein), which does not show any homology to proteins with known function. Treatment of primary human and murine T cells with TIP in vitro resulted in the secretion of IFN-gamma, TNF-alpha, and IL-10, whereas in vivo TIP had a protective effect in a mouse acute graft-versus-host disease (GVHD) model. Therefore, combining functional genomics with high-throughput cell-based screening is a valuable and efficient approach to identifying immunomodulatory activities for novel proteins.     

PCAS – a precomputed proteome annotation database resource

Background  

Many model proteomes or "complete" sets of proteins of given organisms are now publicly available. Much effort has been invested in computational annotation of those "draft" proteomes. Motif or domain based algorithms play a pivotal role in functional classification of proteins. Employing most available computational algorithms, mainly motif or domain recognition algorithms, we set up to develop an online proteome annotation system with integrated proteome annotation data to complement existing resources.     

Imaging the distribution and secondary structure of immobilized enzymes using infrared microspectroscopy

Synchrotron infrared microspectroscopy (SIRMS) was used for the first time to image the distribution and secondary structure of an enzyme (lipase B from Candida antarctica, CALB) immobilized within a macroporous polymer matrix (poly(methyl methacrylate)) at 10 microm resolution. The beads of this catalyst (Novozyme435) were cut into thin sections (12 microm). SIRMS imaging of these thin sections revealed that the enzyme is localized in an external shell of the bead with a thickness of 80-100 microm. Also, the enzyme was unevenly distributed throughout this shell. Furthermore, by SIRMS-generated spectra, it was found that CALB secondary structure was not altered by immobilization. Unlike CALB, polystyrene molecules of similar molecular weight diffuse easily throughout Novozyme435 beads. Scanning electron micrograph (SEM) images of the Novozyme435 beads showed that the average pore size is 10 times larger than CALB or polystyrene molecules, implying that there is no physical barrier to enzyme or substrate diffusion throughout the bead. Thus, the difference between polystyrene and enzyme diffusivity suggests that protein-matrix and protein-protein interactions govern the distribution of the enzyme within the macroporous resin.     

Conformation-dependent effects of VIP on nociception in rats

The purpose of this study was to determine whether intrathecal injection of aqueous (random coil) vasoactive intestinal peptide (VIP) and VIP self-associated with sterically stabilized phospholipid micelles (alpha-helix VIP) at the lower lumbar vertebral level modulates foot withdrawal latency to low and high rate noxious radiant skin heating in anesthetized rats. We found that intrathecal random coil VIP evoked a significant bimodal, concentration-dependent response, early potent antinociception followed by hyperalgesia, during exposure to low and high rates of skin heating (P<0.05). Intrathecal alpha-helix VIP elicited a qualitatively similar response to that of random coil VIP except that the rate of decay of antinociception was faster and slower at low and high rates of skin heating, respectively. In addition, a low concentration of alpha-helix VIP evoked a potent late antinociception not observed with random coil VIP. Taken together, these data indicate that VIP modulates somatosensory processing in the lumbosacral spinal cord of rats in a complex fashion, and that this response is dependent, in part, on the conformation of VIP in the vicinity of target cells in the peripheral nervous system.     

PROSPECT II: protein structure prediction program for genome-scale applications

A new method for fold recognition is developed and added to the general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/). The new method (PROSPECT II) has four key features. (i) We have developed an efficient way to utilize the evolutionary information for evaluating the threading potentials including singleton and pairwise energies. (ii) We have developed a two-stage threading strategy: (a) threading using dynamic programming without considering the pairwise energy and (b) fold recognition considering all the energy terms, including the pairwise energy calculated from the dynamic programming threading alignments. (iii) We have developed a combined z-score scheme for fold recognition, which takes into consideration the z-scores of each energy term. (iv) Based on the z-scores, we have developed a confidence index, which measures the reliability of a prediction and a possible structure-function relationship based on a statistical analysis of a large data set consisting of threadings of 600 query proteins against the entire FSSP templates. Tests on several benchmark sets indicate that the evolutionary information and other new features of PROSPECT II greatly improve the alignment accuracy. We also demonstrate that the performance of PROSPECT II on fold recognition is significantly better than any other method available at all levels of similarity. Improvement in the sensitivity of the fold recognition, especially at the superfamily and fold levels, makes PROSPECT II a reliable and fully automated protein structure and function prediction program for genome-scale applications.     

Forskolin up-regulates metastasis-related phenotypes and molecules via protein kinase B,but not PI-3K,in H7721 human hepato-carcinoma cell line

Forskolin (FSK) is known as an up-regulator of intracellular cAMP and inhibitor of cancer growth and metastasis. The effects of FSK on the metastasis potential and its mechanisms were studied using a human hepatocarcinoma cell line, H7721. It was found that FSK stimulated cell growth, increased cAMP in the cells, and enhanced the metastasis-related phenotypes, including adhesion to laminin (Ln) and human umbilical vein epithelial cells (HUVEC), chemotactic migration and invasion. These effects were supposed to result from the increase of the SLe^x expression induced by FSK, since only the monoclonal antibody of SLe^x showed a significant attenuation of the enhanced metastasis-associated phenotypes. Using H7721 cells transfected with the sense or antisense cDNA of protein kinase B (PKB) and some inhibitors of signal transduction, it was discovered that FSK up-regulated the expression of SLe^x via PKB, but it was independent of phosphotidylinositide-3-kinase (PI-3K). A subtype of atypical protein kinase C (-PKC) might also participate in the up-regulation of SLe^x expression by FSK, and cAMP/PKA pathway is a negative regulator of SLe^x expression on H7721 cells. It can be concluded that FSK shows a metastasis-promoting effect ex vivo.     

Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta)

In order to investigate the possible impacts of increased atmospheric CO₂ levels on algal growth and photosynthesis, the influence of CO₂ concentration was tested on three planktonic algae (Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Scenedesmus obliquus). Increased CO₂ concentration enhanced significantly the growth rate of all three species. Specific growth rates reached maximal values at 30, 100, and 60 M CO₂ in C. reinhardtii, C. pyrenoidosa, and S. obliquus, respectively. Such significant enhancement of growth rate with enriched CO₂ was also confirmed at different levels of inorganic N and P, being more profound at limiting levels of N inC. pyrenoidosa and P in S. obliquus. The maximal rates of net photosynthesis, photosynthetic efficiency and light-saturating point increased significantly (p < 0.05) in high-CO₂-grown cells. Elevation of the CO₂ levels in cultures enhanced the photoinhibition of C. reinhardtii, but reduced that of C. pyrenoidosa and S. obliquus when exposed to high photon flux density. The photoinhibited cells recovered to some extent (from 71% to 99%) when placed under dim light or in darkness, with better recovery in high-CO₂-grownC. pyrenoidosa and S. obliquus. Although pH and pCO₂ effects cannot be distinguished from this study, it can be concluded that increased CO₂ concentrations with decreased pH could affect the growth rate and photosynthetic physiology of C. reinhardtii, C. pyrenoidosa, and S. obliquus.