Occupancy of lymphocyte LFA-1 by surface-immobilized ICAM-1 is critical for TCR- but not for chemokine-triggered LFA-1 conversion to an open headpiece high-affinity state

Lymphocyte arrest and spreading on ICAM-1-expressing APCs require activation of lymphocyte LFA-1 by TCR signals, but the conformational switches of this integrin during these critical processes are still elusive. Using Ab probes that distinguish between different LFA-1 conformations, we found that, unlike strong chemokine signals, potent TCR stimuli were insufficient to trigger LFA-1 extension or headpiece opening in primary human lymphocytes. Nevertheless, LFA-1 in these TCR-stimulated T cells became highly adhesive to both anchored and mobile surface-bound ICAM-1, although it failed to bind soluble ICAM-1 with measurable affinity. Rapid rearrangement of LFA-1 by immobilized ICAM-1 switched the integrin to an open headpiece conformation within numerous scattered submicron focal dots that did not readily collapse into a peripheral LFA-1 ring. Headpiece-activated LFA-1 microclusters were enriched with talin but were devoid of TCR and CD45. Notably, LFA-1 activation by TCR signals as well as subsequent T cell spreading on ICAM-1 took place independently of cytosolic Ca(2+). In contrast to LFA-1-activating chemokine signals, TCR activation of LFA-1 readily took place in the absence of external shear forces. LFA-1 activation by TCR signals also did not require internal myosin II forces but depended on intact actin cytoskeleton. Our results suggest that potent TCR signals fail to trigger LFA-1 headpiece activation unless the integrin first gets stabilized by surface-bound ICAM-1 within evenly scattered actin-dependent LFA-1 focal dots, the quantal units of TCR-stimulated T cell arrest and spreading on ICAM-1.     

IL-23 is increased in dendritic cells in multiple sclerosis and down-regulation of IL-23 by antisense oligos increases dendritic cell IL-10 production

IL-23 is a heterodimeric cytokine comprising a p19 subunit associated with the IL-12/23p40 subunit. Like IL-12, IL-23 is expressed predominantly by activated dendritic cells (DCs) and phagocytic cells, and both cytokines induce IFN-gamma secretion by T cells. The induction of experimental autoimmune encephalitis, the animal model of multiple sclerosis (MS), occurs in mice lacking IL-12, but not in mice with targeted disruption of IL-23 or both IL-12 and IL-23. Thus, IL-23 expression in DCs may play an important role in the pathogenesis of human autoimmune diseases such as MS. We quantified the expression of IL-23 in monocyte-derived DCs in MS patients and healthy donors and found that DCs from MS patients secrete elevated amounts of IL-23 and express increased levels of IL-23p19 mRNA. Consistent with this abnormality, we found increased IL-17 production by T cells from MS patients. We then transfected monocyte-derived DCs from healthy donors with antisense oligonucleotides specific for the IL-23p19 and IL-12p35 genes and found potent suppression of gene expression and blockade of bioactive IL-23 and IL-12 production without affecting cellular viability or DCs maturation. Inhibition of IL-23 and IL-12 was associated with increased IL-10 and decreased TNF-alpha production. Furthermore, transfected DCs were poor allostimulators in the MLR. Our results demonstrate that an abnormal Th1 bias in DCs from MS patients related to IL-23 exists, and that antisense oligonucleotides specific to IL-23 can be used for immune modulation by targeting DC gene expression.     

Pollen-specific pectin methylesterase involved in pollen tube growth

Pollen tube elongation in the pistil is a crucial step in the sexual reproduction of plants. Because the wall of the pollen tube tip is composed of a single layer of pectin and, unlike most other plant cell walls, does not contain cellulose or callose, pectin methylesterases (PMEs) likely play a central role in the pollen tube growth and determination of pollen tube morphology. Thus, the functional studies of pollen-specific PMEs, which are still in their infancy, are important for understanding the pollen development. We identified a new Arabidopsis pollen-specific PME, AtPPME1, characterized its native expression pattern, and used reverse genetics to demonstrate its involvement in determination of the shape of the pollen tube and the rate of its elongation.     

Alpha-complementation as a probe for dual localization of mitochondrial proteins

There are a growing number of proteins which are reported to reside in multiple compartments within the eukaryotic cell. However, lack of appropriate methods limits our knowledge on the true extent of this phenomenon. In this study, we demonstrate a novel application of beta-galactosidase alpha-complementation to study dual distribution of proteins in yeast cells. Using a simple colony color phenotype, we show that alpha-complementation depends on co-compartmentalization of alpha and omega fragments and exploit this to probe dual localization of proteins between the cytosol and mitochondria in yeast. The quality of our assay was assessed by analysis of the known dual targeted enzyme fumarase and several mutant derivatives, which are exclusively localized to one or the other of these subcellular compartments. Addition of the alpha fragment did not abolish the enzymatic activity of the tagged proteins nor did it affect their localization. By examining 10 yeast gene products for distribution between the cytosol and the mitochondria, we demonstrate the potential of alpha-complementation to screen the mitochondrial proteome for dual distribution. Our data indicate the distribution of two uncharacterized proteins--Bna3 and Nif3--between the cytosol and the mitochondria.     

Relaxation of selection with equalization of parental contributions in conservation programs: an experimental test with Drosophila melanogaster

Equalization of parental contributions is one of the most simple and widely recognized methods to maintain genetic diversity in conservation programs, as it halves the rate of increase in inbreeding and genetic drift. It has, however, the negative side effect of implying a reduced intensity of natural selection so that deleterious genes are less efficiently removed from the population with possible negative consequences on the reproductive capacity of the individuals. Theoretical results suggest that the lower fitness resulting from equalization of family sizes relative to that for free contribution schemes is expected to be substantial only for relatively large population sizes and after many generations. We present a long-term experiment with Drosophila melanogaster, comparing the fitness performance of lines maintained with equalization of contributions (EC) and others maintained with no management (NM), allowing for free matings and contributions from parents. Two (five) replicates of size N = 100 (20) individuals of each type of line were maintained for 38 generations. As expected, EC lines retained higher gene diversity and allelic richness for four microsatellite markers and a higher heritability for sternopleural bristle number. Measures of life-history traits, such as egg-to-adult viability, mating success, and global fitness declined with generations, but no significant differences were observed between EC and NM lines. Our results, therefore, provide no evidence to suggest that equalization of family sizes entails a disadvantage on the reproductive capacity of conserved populations in comparison with no management procedures, even after long periods of captivity.     

Exploring the role of the active site cysteine in human muscle creatine kinase

All known guanidino kinases contain a conserved cysteine residue that interacts with the non-nucleophilic eta1-nitrogen of the guanidino substrate. Site-directed mutagenesis studies have shown that this cysteine is important, but not essential for activity. In human muscle creatine kinase (HMCK) this residue, Cys283, forms part of a conserved cysteine-proline-serine (CPS) motif and has a pKa about 3 pH units below that of a regular cysteine residue. Here we employ a computational approach to predict the contribution of residues in this motif to the unusually low cysteine pKa. We calculate that hydrogen bonds to the hydroxyl and to the backbone amide of Ser285 would both contribute approximately 1 pH unit, while the presence of Pro284 in the motif lowers the pKa of Cys283 by a further 1.2 pH units. Using UV difference spectroscopy the pKa of the active site cysteine in WT HMCK and in the P284A, S285A, and C283S/S285C mutants was determined experimentally. The pKa values, although consistently about 0.5 pH unit lower, were in broad agreement with those predicted. The effect of each of these mutations on the pH-rate profile was also examined. The results show conclusively that, contrary to a previous report (Wang et al. (2001) Biochemistry 40, 11698-11705), Cys283 is not responsible for the pKa of 5.4 observed in the WT V/K(creatine) pH profile. Finally we use molecular dynamics simulations to demonstrate that, in order to maintain the linear alignment necessary for associative inline transfer of a phosphoryl group, Cys283 needs to be ionized.     

Contribution of lateral gene transfer to the gene repertoire of a gut-adapted methanogen

Methanobrevibacter smithii is the most abundant archaeon in the human colon. As most of its neighbors are bacterial species, it is expected that lateral gene acquisition from bacteria might have contributed to the evolution and adaptation of this archaeon. We performed a tree-based genome-wide survey of putative lateral gene transfer products in M. smithii, using a phylogenetic pipeline. Over 15% of the coding genes of M. smithii are inferred to be bacterial in origin, based on this analysis. Laterally acquired genes have had the largest contribution to surface functions, and encode glycosyl-transferases and adhesin-like proteins. In addition, several important ABC transporters, especially metal transporters are of bacterial origin. Thus, bacterial genes contributed to the host-adaptation by allowing a larger variety of surface structures and increasing the efficiency of metal ion uptake in the competitive gut niche.     

Research resource: Diagnostic and therapeutic potential of nuclear receptor expression in lung cancer

Lung cancer is the leading cause of cancer-related death. Despite a number of studies that have provided prognostic biomarkers for lung cancer, a paucity of reliable markers and therapeutic targets exist to diagnose and treat this aggressive disease. In this study we investigated the potential of nuclear receptors (NRs), many of which are well-established drug targets, as therapeutic markers in lung cancer. Using quantitative real-time PCR, we analyzed the expression of the 48 members of the NR superfamily in a human panel of 55 normal and lung cancer cell lines. Unsupervised cluster analysis of the NR expression profile segregated normal from tumor cell lines and grouped lung cancers according to type (i.e. small vs. non-small cell lung cancers). Moreover, we found that the NR signature was 79% accurate in diagnosing lung cancer incidence in smokers (n = 129). Finally, the evaluation of a subset of NRs (androgen receptor, estrogen receptor, vitamin D receptor, and peroxisome proliferator-activated receptor-γ) demonstrated the therapeutic potential of using NR expression to predict ligand-dependent growth responses in individual lung cancer cells. Preclinical evaluation of one of these receptors (peroxisome proliferator activated receptor-γ) in mouse xenografts confirmed that ligand-dependent inhibitory growth responses in lung cancer can be predicted based on a tumor's receptor expression status. Taken together, this study establishes NRs as theragnostic markers for predicting lung cancer incidence and further strengthens their potential as therapeutic targets for individualized treatment.     

Mitochondrial DNA mutations in respiratory complex-I in never-smoker lung cancer patients contribute to lung cancer progression and associated with EGFR gene mutation

Mitochondrial DNA (mtDNA) mutations were reported in different cancers. However, the nature and role of mtDNA mutation in never‐smoker lung cancer patients including patients with epidermal growth factor receptor (EGFR) and KRAS gene mutation are unknown. In the present study, we sequenced entire mitochondrial genome (16.5 kb) in matched normal and tumors obtained from 30 never‐smoker and 30 current‐smoker lung cancer patients, and determined the mtDNA content. All the patients' samples were sequenced for KRAS (exon 2) and EGFR (exon 19 and 21) gene mutation. The impact of forced overexpression of a respiratory complex‐I gene mutation was evaluated in a lung cancer cell line. We observed significantly higher (P = 0.006) mtDNA mutation in the never‐smokers compared to the current‐smoker lung cancer patients. MtDNA mutation was significantly higher (P = 0.026) in the never‐smoker Asian compared to the current‐smoker Caucasian patients' population. MtDNA mutation was significantly (P = 0.007) associated with EGFR gene mutation in the never‐smoker patients. We also observed a significant increase (P = 0.037) in mtDNA content among the never‐smoker lung cancer patients. The majority of the coding mtDNA mutations targeted respiratory complex‐I and forced overexpression of one of these mutations resulted in increased in vitro proliferation, invasion, and superoxide production in lung cancer cells. We observed a higher prevalence and new relationship between mtDNA alterations among never‐smoker lung cancer patients and EGFR gene mutation. Moreover, a representative mutation produced strong growth effects after forced overexpression in lung cancer cells. Signature mtDNA mutations provide a basis to develop novel biomarkers and therapeutic strategies for never‐smoker lung cancer patients. J. Cell. Physiol. 227: 2451–2460, 2012. © 2011 Wiley Periodicals, Inc.