The Drosophila innexin 7 gap junction protein is required for development of the embryonic nervous system

The Drosophila genome encodes eight members of the innexin family of gap junction proteins. Most of the family members are expressed in complex and overlapping expression patterns during Drosophila development. Functional studies and mutant analysis have been performed for only few of the innexin genes. The authors generated an antibody against Innexin7 and studied its expression and functional role in embryonic development by using transgenic RNA interference (RNAi) lines. The authors found Innexin7 protein expression in all embryonic epithelia from early to late stages of development, including in the developing epidermis and the gastrointestinal tract. In early embryonic stages, the authors observed a nuclear localization of Innexin7, whereas Innexin7 was found in a punctuate pattern in the cytoplasm and at the membrane of most epithelial tissues at later stages of development. During central nervous system (CNS) development, Innexin7 was expressed in cells of the neuroectoderm and the mesectoderm and at later stages of embryogenesis, its expression was largely restricted to a segmental pattern of few glia and neuronal cells derived from the midline precursors. Coimmunostaining experiments showed that Innexin7 is expressed in midline glia, and in two different neuronal cells, the pCC and MP2 neurons, which are pioneer cells for axon guidance. RNAi-mediated knock down was used to gain insight into the embryonic function of innexin7. Down-regulation of innexin7 expression resulted in a severe disruption of embryonic nervous system development. Longitudinal, posterior, and anterior commissures were disrupted and the outgrowth of axon fibers of the ventral nerve cord was aberrant, causing peripheral nervous system defects. The results suggest an essential role for innexin7 for axon guidance and embryonic nervous system development in Drosophila.     

Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A^–/– PCD remains elusive. Here, we present the native three-dimensional structure of RSPH4A^–/– human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo–electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and RSPH4A^–/– cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1^–/– cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A^–/– cilia. However, RSPH4A^–/– cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A^–/– PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.     

Ecological Genomics of Marine Picocyanobacteria

Summary: Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primary production. Prochlorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecotypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45°N to 40°S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton clade, but are distinguishable by their possession of dissimilar light-harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized by phylogenetically discrete lineages with a wide range of pigmentation. In this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level.     

Learning to get along despite struggling to get by

How cooperation can evolve by natural selection is important for understanding the evolutionary transition from unicellular to multicellular life. Here we review the evolutionary theories for cooperation, with emphasis on the mechanisms that can favor cooperation and reduce conflict in multicellular organisms.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Histone H3 lysine 27 acetylation is altered in colon cancer

Background

Histone post-translational modifications (PTMs) play an important role in the regulation of the expression of genes, including those involved in cancer development and progression. However, our knowledge of PTM patterns in human tumours is limited.

Methods

MS-based analyses were used to quantify global alterations of histone PTMs in colorectal cancer (CRC) samples. Histones isolated from 12 CRCs and their corresponding normal mucosa by acidic extraction were separated by SDS-PAGE and analysed by liquid chromatography-mass spectrometry.

Results

Among 96 modified peptides, 41 distinct PTM sites were identified, of which 7, 13, 11, and 10 were located within the H2A, H2B, H3, and H4 sequences, respectively, and distributed among the amino-terminal tails and the globular domain of the four histones. Modification intensities were quantified for 33 sites, of which 4 showed significant (p-value ≤ 0.05) differences between CRC tissues and healthy mucosa samples. We identified histone H3 lysine 27 acetylation (H3K27Ac) as a modification upregulated in CRC, which had not been shown previously.

Conclusions

The present results indicate the usefulness of a bottom-up proteomic approach for the detection of histone modifications at a global scale. The differential abundance of H3K27Ac mark in CRC, a PTM associated with active enhancers, suggests its role in regulating genes whose expression changes in CRC.     

The role of the p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand-induced dendritic cell activation and expansion of virus-specific CD8+ T cell memory responses

Mature dendritic cells (DCs) are central to the development of optimal T cell immune responses. CD40 ligand (CD40L, CD154) is one of the most potent maturation stimuli for immature DCs. We studied the role of three signaling pathways, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and phosphoinositide-3-OH kinase (PI3K), in CD40L-induced monocyte-derived DC activation, survival, and expansion of virus-specific CD8(+) T cell responses. p38 MAPK pathway was critical for CD40L-mediated up-regulation of CD83, a marker of DC maturation. CD40L-induced monocyte-derived DC IL-12 production was mediated by both the p38 MAPK and PI3K pathways. CD40L-mediated DC survival was mostly mediated by the PI3K pathway, with smaller contributions by p38 MAPK and ERK pathways. Finally, the p38 MAPK pathway was most important in mediating CD40L-stimulated DCs to induce strong allogeneic responses as well as expanding virus-specific memory CD8(+) T cell responses. Thus, although the p38 MAPK, PI3K, and ERK pathways independently affect various parameters of DC maturation induced by CD40L, the p38 MAPK pathway within CD40L-conditioned DCs is the most important pathway to maximally elicit T cell immune responses. This pathway should be exploited in vivo to either completely suppress or enhance CD8(+) T cell immune responses.     

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg²⁺ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K _m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k _cat/K _m = 682,608.7 and 5080 s⁻¹ M⁻¹, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.     

Isocitrate dehydrogenase 1 R132C mutation occurs exclusively in microsatellite stable colorectal cancers with the CpG island methylator phenotype

The CpG Island Methylator Phenotype (CIMP) is fundamental to an important subset of colorectal cancer; however, its cause is unknown. CIMP is associated with microsatellite instability but is also found in BRAF mutant microsatellite stable cancers that are associated with poor prognosis. The isocitrate dehydrogenase 1 (IDH1) gene causes CIMP in glioma due to an activating mutation that produces the 2-hydroxyglutarate oncometabolite. We therefore examined IDH1 alteration as a potential cause of CIMP in colorectal cancer. The IDH1 mutational hotspot was screened in 86 CIMP-positive and 80 CIMP-negative cancers. The entire coding sequence was examined in 81 CIMP-positive colorectal cancers. Forty-seven cancers varying by CIMP-status and IDH1 mutation status were examined using Illumina 450K DNA methylation microarrays. The R132C IDH1 mutation was detected in 4/166 cancers. All IDH1 mutations were in CIMP cancers that were BRAF mutant and microsatellite stable (4/45, 8.9%). Unsupervised hierarchical cluster analysis identified an IDH1 mutation-like methylation signature in approximately half of the CIMP-positive cancers. IDH1 mutation appears to cause CIMP in a small proportion of BRAF mutant, microsatellite stable colorectal cancers. This study provides a precedent that a single gene mutation may cause CIMP in colorectal cancer, and that this will be associated with a specific epigenetic signature and clinicopathological features.