A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence

BackgroundThe polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.ResultsHere, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.ConclusionsWe identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.

Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance

Abstract Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated Streptococcus pneumoniae (Non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if Non-Ec-Sp evolve sporadically, if they have high antibiotic non-susceptiblity rates and a unique, specific gene content. Here, whole genome sequencing of 131 Non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multi-locus sequences types ST344 (n=39) and ST448 (n=40). All ST344 and nine ST448 isolates had high non-susceptiblity rates to β-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic Non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic Non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic Non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P=0.005). In contrast, sporadic Non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, Non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of pneumococcal conjugate vaccines, Non-Ec-Sp may become more prevalent.

Novel oxazolo-oxazole derivatives of FTY720 reduce endothelial cell permeability, immune cell chemotaxis and symptoms of experimental autoimmune encephalomyelitis in mice

The immunomodulatory FTY720 (fingolimod) is presently approved for the treatment of relapsing-remitting multiple sclerosis. It is a prodrug that acts by modulating sphingosine 1-phosphate (S1P) receptor signaling. In this study, we have developed and characterized two novel oxazolo-oxazole derivatives of FTY720, ST-968 and the oxy analog ST-1071, which require no preceding activating phosphorylation, and proved to be active in intact cells and triggered S1P1 and S1P3, but not S1P2, receptor internalization as a result of receptor activation. Functionally, ST-968 and ST-1071 acted similar to FTY720 to abrogate S1P-triggered chemotaxis of mouse splenocytes, mouse T cells and human U937 cells, and reduced TNFa- and LPS-stimulated endothelial cell permeability. The compounds also reduced TNFα-induced ICAM-1 and VCAM-1 mRNA expression, but restored TNFα-mediated downregulation of PECAM-1 mRNA expression. In an in vivo setting, the application of ST-968 or ST-1071 to mice resulted in a reduction of blood lymphocytes and significantly reduced the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice comparable to FTY720 either by prophylactic or therapeutic treatment. In parallel to the reduced clinical symptoms, infiltration of immune cells in the brain was strongly reduced, and in isolated tissues of brain and spinal cord, the mRNA and protein expressions of ICAM-1 and VCAM-1, as well as of matrix metalloproteinase-9 were reduced by all compounds, whereas PECAM-1 and tissue inhibitor of metalloproteinase TIMP-1 were upregulated. In summary, the data suggest that these novel butterfly derivatives of FTY720 could have considerable implication for future therapies of multiple sclerosis and other autoimmune diseases.

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage.

TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.

Use of cardiac glycosides for treatment of cancer: Determinants of cancer cell sensitivity

Cardiac glycoside compounds have traditionally been used to treat congestive heart failure. Recently, reports have suggested that cardiac glycosides may also be useful for treatment of malignant disease. Our research with oleandrin, a cardiac glycoside component of Nerium oleander, has shown it to be a potent inducer of human but not murine tumor cell apoptosis. Determinants of tumor sensitivity to cardiac glycosides were therefore studied in order to understand the species selective cytotoxic effects as well as explore differential sensitivity amongst a variety of human tumor cell lines. ^ An initial model system involved a comparison of human (BRO) to murine (B16) melanoma cells. Human BRO cells were found to express both the sensitive α3 as well as the less sensitive α1 isoform subunits of Na+,K +-ATPase while mouse B16 cells expressed only the α1 isoform. Drug uptake and inhibition of Na+,K+-ATPase activity were also different between BRO and B16 cells. Partially purified human Na+,K+-ATPase enzyme was inhibited by cardiac glycosides at a concentration that was 1000-fold less than that required to inhibit mouse B16 enzyme to the same extent. In addition, uptake of oleandrin and ouabain was 3–4 fold greater in human than murine cells. These data indicate that differential expression of Na+,K+-ATPase isoform composition in BRO and B16 cells as well as drug uptake and total enzyme activity may all be important determinants of tumor cell sensitivity to cardiac glycosides. ^ In a second model system, two in vitro cell culture model systems were investigated. The first consisted of HFU251 (low expression of Na+,K+-ATPase) and U251 (high Na+ ,K+-ATPase expression) cell lines. Also investigated were human BRO cells that had undergone stable transfection with the α1 subunit resulting in an increase in total Na+,K+-ATPase expression. Data derived from these model systems have indicated that increased expression of Na+,K+-ATPase is associated with an increased resistance to cardiac glycosides. Over-expression of Na +,K+-ATPase in tumor cells resulted in an increase of total Na+,K+-ATPase activity and, in turn, a decreased inhibition of Na+,K+-ATPase activity by cardiac glycosides. However, of interest was the observation that increased enzyme expression was also associated with an elevated basal level of glutathione (GSH) within cells. Both increased Na+,K+-ATPase activity and elevated GSH content appear to contribute to a delayed as well as diminished release of cytochrome c and caspase activation. In addition, we have noted an increased colony forming ability in cells with a high level of Na+,K+-ATPase expression. This suggests that Na+,K+-ATPase is actively involved in tumor cell growth and survival. ^

The role of AT1 receptor autoantibodies in the pathophysiology of preeclampsia

Preeclampsia is a disease that affects 3–5% of all pregnancies. The cause is unknown and there is currently no treatment. The disease poses significant health risks to both the mother and the fetus. To date, research on the topic has not produced a convincing cause for the development of the hallmark symptoms of preeclampsia. The hypothesis of an agonistic autoimmune response to the AT1 receptor is presented. Immunoglobulin fractions from normotensive and preeclampsia patients were prepared for experimental tests. Model systems were tested in three categories to determine if AT 1 receptor specific activation and receptor-ligand interaction was caused by a suspected autoantibody. Activation was found in rat neonatal cardiornyocytes that caused an increased contraction rate. This activity was found in preeclampsia patients, absent in normotensive patients. The activation was antagonized by losartan, an AT1 receptor antagonist, and by epitope peptide competition of the receptor-ligand type interaction. This epitope was the 7 amino acid peptide fragment, AFHYESQ, a sequence present in the second extracellular loop of the AT1 receptor. The patterns of AT1 receptor activation were also found in a human trophoblast cell line, HTR8, with an effect on Pai-1 secretion, a factor that plays a role in preventing hypercoagulation. In human mesangial cells, the AT1 receptor autoantibody present in the immunoglobulin fraction from preeclampsia patients was found to stimulate the secretion of Pai-1, and IL-6, a factor that plays a role in the activation of an inflammatory response. This activity was found in samples from preeclampsia patients, but absent in normotensive patients. Tests including losartan, AFHYESQ, and a non-competitive peptide demonstrated that the secretion of Pai-1 and IL-6 met the criteria for AT1 receptor activation by the suspected agonistic autoantibody. These three model systems address relevant pathophysiology for preeclampsia patients, including increased cardiac output, abnormal placentation, and renal damage. The AT1 receptor agonistic autoantibody is potentially a key player in the development of the pathology and symptoms of preeclampsia. ^

The role of interleukin-6 in the angiogenesis of ovarian carcinoma

The purpose of this study was to characterize the effects of IL-6 on endothelial cells and to investigate the role of IL-6 in the angiogenesis of ovarian carcinomas. We evaluated human ovarian carcinoma clinical specimens and determined that high expression of IL-6 was associated with increased tumor vascularization. Additionally, endothelial cells derived from the ovary and mesentery expressed the IL-6 receptor (IL-6R), and their stimulation with the exogenous ligand activated downstream signaling molecules and enhanced cell migration. Dual immunohistochemical staining for CD-31 and IL-6R revealed IL-6R expression on human endothelial cells within normal ovary and ovarian carcinomas. To further investigate the possible proangiogenic function of IL-6, Gelfoam sponges containing IL-6 or bFGF were implanted into the subcutis of BALB/c mice. IL-6 containing sponges were vascularized to the same extent as bFGF containing sponges. ^ Chronic stress can adversely affect disease progression. Stimulation of ovarian carcinoma cell lines with concentrations of catecholamines achieved in individuals experiencing chronic stress resulted in a substantial increase in IL-6 production. It was determined that stress mediators regulate IL-6 expression through the β-adrenergic receptor and Src. These data illustrate one mechanism by which chronic stress may influence tumor progression. ^ To investigate whether IL-6 contributes to the angiogenesis of ovarian carcinomas, we isolated low IL-6 expressing clones from the SKOV3.ip1 cell line and transfected them with a plasmid encoding the IL-6 gene. We observed no difference in tumor weight between high and low IL-6 expressing cells. However, while low IL-6 expressing tumors were highly vascularized, high IL-6 expressing tumors appeared hypervascularized. Immunohistochemical analysis revealed that all tumors exhibited robust expression of additional proangiogenic molecules. ^ Collectively, these studies indicate that IL-6 secreted by ovarian cancer cells is a highly proangiogenic cytokine. However, IL-6 is but one of several proangiogenic molecules produced by ovarian cancer, and its inhibition may not be sufficient to inhibit angiogenesis of ovarian carcinoma. The findings presented in this dissertation provide insight into the function of IL-6 as a regulator of angiogenesis. Understanding of the role of proangiogenic molecules such as IL-6 in ovarian carcinoma may have important implications for therapy directed at the vascular component of this disease. ^

Identification and functional characterization of novel phosphotyrosine and phosphoserine residues in the interleukin-2 receptor complex

Interleukin-2 (IL-2) is a major T cell growth factor and plays an essential role in the development of normal immune responses. The Janus kinases (Jaks) and Signal transducers and activators of transcription (Stats) are critical for transducing signals from the IL-2 receptors (IL2Rs) to the nucleus to control cell growth and differentiation. In recent years there has been increasing evidence to indicate that the IL-2 activated Jak3/Stat5 pathway provides a new molecular target for immune suppression. Thus, understanding the regulation of this effector cascade has important therapeutic potential.^ One objective of this work was to identify and define the role and molecular mechanism of novel phosphorylation sites in Jak3. Using functional proteomics, three novel Jak3 phosphorylation sites, Y904, Y939 and S574 were identified. Phosphospecific antibodies confirmed that phosphorylation of Y904 and Y939 were mediated by IL-2 and other IL-2 family cytokines in distinct cell types. Biochemical analysis demonstrated that phosphorylation of both Y904 and Y939 positively regulated Jak3 enzymatic activity, while phosphorylation of S574 did not affect Jak3 in vitro kinase activity. However, a gain-of-function mutation of S574 in Jak3 abrogated IL-2 mediated Stat5 activation, suggesting that phosphorylation of this residue might serve a negative role to attenuate IL-2 signaling. Furthermore, mechanistic analysis suggested that phosphorylation of Y904 in Jak3 affects the KmATP of Jak3, while phosphorylation of Y939 in Jak3 was required to bind one of its substrates, Stat5.^ The second objective was to determine the role of serine/threonine phosphatases in the regulation of the IL2R complex. Activation of Jak3 and Stat5 by IL-2 is a transient event mediated by phosphorylation. Using a specific PP1/PP2A inhibitor, we observed that inhibition of PP1/PP2A negatively regulated the IL-2 activated Jak3/Stat5 signaling pathway in a human NK cell line (YT) and primary human T cells. More importantly, coimmunoprecipitation assays indicated that inhibition of PP1/PP2A blocked the formation of an active IL2R complex. Pretreatment of cells with the inhibitor also reduced the electrophoretic mobility of the IL2Rβ and IL2Rγ subunits in YT cells, suggesting that inhibition of PP1/PP2A directly or indirectly regulates undefined serine/threonine kinases which phosphorylate these proteins. Based on these observations, a model has emerged that serine/threonine phosphorylation of the IL2Rβ and IL2Rγ subunits causes a conformational change of these proteins, which disrupts IL2R dimerization and association of Jak3 and Stat5 to these receptors.^

Proteomic identification and functional characterization of prohibitin 1 and 2 in T cell activation, expansion, survival and disease

Several immune pathologies are the result of aberrant regulation of T lymphocytes. Pronounced T cell proliferation can result in autoimmunity or hematologic malignancy, whereas loss of T cell activity can manifest as immunodeficiency. Thus, there is a critical need to characterize the signal transduction pathways that mediate T cell activation so that novel and rational strategies to detect and effectively control T cell mediated disease can be achieved. ^ The first objective of this dissertation was to identify and characterize novel T cell regulatory proteins that are differentially expressed upon antigen induced activation. Using a functional proteomics approach, two members of the prohibitin (Phb) family of proteins, Phb1 and Phb2, were determined to be upregulated upon activation of primary human T cells. Furthermore, their regulated expression was dependent upon CD3 and CD28 signaling pathways which synergistically increased their expression. In contrast to previous reports of Phb nuclear localization, both proteins were determined to localize to the mitochondrial inner membrane of human T cells. Additionally, novel Phb phosphorylation sites were identified and characterized using mass spectrometry, phosphospecific antibodies and site directed mutagenesis. ^ Prohibitins have been proposed to play important roles in cancer development however the mechanism of action has not been elucidated. The second objective of this dissertation was to define the functional role of Phbs in T cell activity, survival and disease. Compared to levels in normal human T cells, Phb expression was higher in the human tumor T cell line Kit225 and subcellularly localized to the mitochondrion. Ablation of Phb expression by siRNA treatment of Kit225 cells resulted in disruption of mitochondrial membrane potential and significantly enhanced their sensitivity to cell death, suggesting they serve a protective function in T cells. Furthermore, Q-RT-PCR analysis of human oncology cDNA expression libraries indicated the Phbs may represent hematological cancer biomarkers. Indeed, Phb1 and Phb2 protein levels were 6-10 fold higher in peripheral blood mononuclear cells isolated from malignant lymphoma and multiple myeloma patients compared to healthy individuals. ^ Taken together, Phb1 and Phb2 are novel phosphoproteins upregulated during T cell activation and transformation to function in the maintenance of mitochondrial integrity and perhaps energy metabolism, thus representing previously unrecognized intracellular biomarkers and therapeutic targets for regulating T cell activation and hematologic malignancies. ^

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

ANTI-TUMOR EFFECTS OF THE NOTCH PATHWAY IN GASTROINTESTINAL STROMAL TUMORS

Gastrointestinal Stromal Tumors (GIST) are sarcomas driven by gain-of-function mutations of KIT or PDGFRA. Although, the introduction of tyrosine kinase inhibitors has dramatically changed the history of this disease, evidences emerge that inhibition of KIT or PDGFRA are not sufficient to cure patients. The developmental pathway Notch has a critical role in the cell fate, regulating cell proliferation and differentiation. Dysregulation of Notch pathway has been implicated in a wide variety of cancers functioning as a tumor promoter or a tumor suppressor in a cell context dependent manner. Given that Notch activation deregulates the morphogenesis of mesenchymal cells in the GI track, that Notch acts as a tumor suppressor in neuroendocrine tumors, and finally that the cell of origin of GIST are the Interstitial Cell of Cajal that arise from a mesenchymal origin with some neuroendocrine features, we hypothesized that Notch pathway signaling may play a role in growth, survival and differentiation of GIST cells. To test this hypothesis, we genetically and pharmacologically manipulated the Notch pathway in human GIST cells. In this study, we demonstrated that constitutively active intracellular domain of Notch1 (ICN-1) expression potently induced growth arrest and downregulated KIT expression. We have performed a retrospective analysis of 15 primary GIST patients and found that high mRNA level of Hes1, a major target gene of Notch pathway, correlated with a significantly longer relapse-free survival. Therefore, we have established that treatment with the FDA approved histone deacetylase inhibitor SAHA (Vorinostat) caused dose-dependent upregulation of Notch1 expression and a parallel decrease in viability in these cells. Retroviral silencing of downstream targets of Notch with dominant negative Hes-1 as well as pharmacological inhibition of Notch pathway with a γ-secretase inhibitor partially rescued GIST cells from SAHA treatment. Taken together these results identify anti-tumor effect of Notch1 and a negative cross-talk between Notch1 and KIT pathways in GIST. Consequently, we propose that activation of this pathway with HDAC inhibitors may be a potential therapeutic strategy for GIST patients.

αvβ8 Integrin Interacts with RhoGDI1 to Regulate Rac1 and Cdc42 Activation and Drive Glioblastoma Cell Invasion

As an interface between the circulatory and central nervous systems, the neurovascular unit is vital to the development and survival of tumors. The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are major impediments to surgical resection and targeted therapies. Adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we have utilized human GBM cell lines, primary patient samples, and pre-clinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin associates with Rho GDP Dissociation Inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin-RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, suppresses activation of Rho proteins to promote GBM cell invasiveness. Hence, targeting the αvβ8 integrin-RhoGDI1 signaling axis may be an effective strategy for blocking GBM cell invasion.

Molecular and evolutionary genetics of the X -linked visual pigment genes in humans and New World monkeys

Normal humans have one red and at least one green visual pigment genes. These genes are tightly linked as tandem repeats on the X chromosome and each of them has six exons. There is only one X-linked visual pigment gene in New World monkeys (NWMs) but the locus has three polymorphic alleles encoding red, yellow and green visual pigments, respectively. The spectral properties of the squirrel monkey and the marmoset (both NWMs) have been studied and partial sequences of the three alleles are available. To study the evolutionary history of these X-linked opsin genes in humans and NWMs, coding and intron sequences of the three squirrel monkey alleles and the three marmoset alleles were amplified by PCR followed by subcloning and sequencing. Introns 2 and 4 of the human red and green pigment genes were also sequenced. The results obtained are as follows: (1) The sequences of introns 2 and 4 of the human red and green opsin genes are significantly more similar between the two genes than are coding sequences, contrary to the usual situation where coding regions are better conserved in evolution than are introns. The high similarities in the two introns are probably due to recent gene conversion events during evolution of the human lineage. (2) Phylogenetic analysis of both intron and exon sequences indicates that the phylogenetic tree of the available primate opsin genes is the same as the species tree. The two human genes were derived from a gene duplication event after the divergence of the human and NWM lineages. The three alleles in each of the two NWM species diverged after the split of the two NWMs but have persisted in the population for at least 5 million years. (3) Allelic gene conversion might have occurred between the three squirrel monkey alleles. (4) A model of additive effect of hydroxyl-bearing amino acids on spectral tuning is proposed by treating some unknown variables as groups. Under the assumption that some residues have no effect, it is found that at least five amino acid residues, at positions 178 (3 nm), 180 (5 nm), 230 ($-$4 nm), 277 (9 nm) and 285 (13 nm), have linear spectral tuning effects. (5) Adaptive evolution of the opsin genes to different spectral peaks was observed at four residues that are important for spectral tuning. ^