Proteomic analysis of a eukaryotic cilium

Cilia and flagella are widespread cell organelles that have been highly conserved throughout evolution and play important roles in motility, sensory perception, and the life cycles of eukaryotes ranging from protists to humans. Despite the ubiquity and importance of these organelles, their composition is not well known. Here we use mass spectrometry to identify proteins in purified flagella from the green alga Chlamydomonas reinhardtii. 360 proteins were identified with high confidence, and 292 more with moderate confidence. 97 out of 101 previously known flagellar proteins were found, indicating that this is a very complete dataset. The flagellar proteome is rich in motor and signal transduction components, and contains numerous proteins with homologues associated with diseases such as cystic kidney disease, male sterility, and hydrocephalus in humans and model vertebrates. The flagellum also contains many proteins that are conserved in humans but have not been previously characterized in any organism. The results indicate that flagella are far more complex than previously estimated.     

Cross-talk between integrin α6β4 and insulin-like growth factor-1 receptor (IGF1R) through direct α6β4 binding to IGF1 and subsequent α6β4-IGF1-IGF1R ternary complex formation in anchorage-independent conditions

Integrin αvβ3 plays a role in insulin-like growth factor-1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk). The specifics of the cross-talk are, however, unclear. In a current model, "ligand occupancy" of αvβ3 (i.e. the binding of extracellular matrix proteins) enhances signaling induced by IGF1 binding to IGF1R. We recently reported that IGF1 directly binds to αvβ3 and induces αvβ3-IGF1-IGF1R ternary complex formation. Consistently, the integrin binding-defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, but it still binds to IGF1R. Like αvβ3, integrin α6β4 is overexpressed in many cancers and is implicated in cancer progression. Here, we discovered that α6β4 directly bound to IGF1, but not to R36E/R37E. Grafting the β4 sequence WPNSDP (residues 167-172), which corresponds to the specificity loop of β3, to integrin β1 markedly enhanced IGF1 binding to β1, suggesting that the WPNSDP sequence is involved in IGF1 recognition. WT IGF1 induced α6β4-IGF1-IGF1R ternary complex formation, whereas R36E/R37E did not. When cells were attached to matrix, exogenous IGF1 or α6β4 expression had little or no effect on intracellular signaling. When cell-matrix adhesion was reduced (in poly(2-hydroxyethyl methacrylate-coated plates), IGF1 induced intracellular signaling and enhanced cell survival in an α6β4-dependent manner. Also IGF1 enhanced colony formation in soft agar in an α6β4-dependent manner. These results suggest that IGF binding to α6β4 plays a major role in IGF signaling in anchorage-independent conditions, which mimic the in vivo environment, and is a novel therapeutic target.     

SBA immobilized phosphomolybdic acid: Efficient hybrid mesostructured heterogeneous catalysts

SBA-15 and SBA-3 mesoporous silicas are synthesised by P123 and CTAB surfactants via hydrothermal and liquid phase deposition procedures, respectively. An inorganic-organic hybrid mesoporous material is then synthesised by functionalization of SBA-15 with aminopropyl functional groups via grafting method. After characterization, effect of immobilizing support and functional groups on intercalation of phosphomolybdic acid (H₃PMo₁₂O₄₀) is taken into consideration. The immobilization pattern is discussed and supported H₃PMo₁₂O₄₀ catalysts are characterized using X-ray diffraction (XRD), scanning electron microscopy (SEM), EDX, inductively coupled plasma (ICP), FT-IR, and UV-vis analysis. The newly synthesised hybrid catalysts are investigated for epoxidation of cyclooctene in presence of hydrogen peroxide as oxidant. The reaction mechanism is discussed. Furthermore, effects of different immobilizing supports and functionalization on catalyst activity, stability, and reusability are taken into consideration. Similar catalytic reactions are carried out with pristine supports and neat H₃PMo₁₂O₄₀ (homogeneous). Results reveal that the mesostructured phosphomolybdic acid based catalysts are shown to be efficient and selective heterogeneous catalysts for oxidation of alkenes.     

Isolation and detection of Leishmania species among naturally infected Rhombomis opimus, a reservoir host of zoonotic cutaneous leishmaniasis in Turkemen Sahara, North East of Iran

In Iran, three species of Leishmania have been incriminated as the causative agents of human leishmaniasis, Leishmania (L.) major, Leishmania tropica, and Leishmania infantum.Rhombomis opimus have been incriminated as a principal reservoirs of the parasitic protozoan Leishmania major, the causative agent of rural zoonotic cutaneous leishmaniasis (ZCL) in Iran. Rodents captured and examined to find Leishmania species using conventional methods including direct impression smear and microscopic observation inoculation samples to Balb/c and culture in NNN medium. Also molecular method was employed to detect Leishmania in rodents by amplifying a region of the ribosomal RNA amplicon of Leishmania (ITS1-5.8S rRNA–ITS2) using Nested PCR. Leshmania species were specified by DNA sequences. 36 (38.3%) of R. opimus were Leishmania positive using at least one conventional methods. Many more ITS-rDNA fragments were amplified from R. opimus but only 65 out of 74 PCR products contained enough DNA for direct sequencing or readable sequences. The PCR assays detected in Iranian R. opimus not only Leishmania major in 59 (79.7%) rodents but also Leishmania turanica in 6 (8.1%) rodents, another parasite of the great gerbil. These parasites were found in Turkemen Sahara, North East of Iran, in a focus of rural (ZCL). L. major and L. turanica in R. opimus firmly identified from Turkemen Sahara. Nine rodents with Leishmania infections unidentified which some were unreadable sequences, these could be mixed infections of L. major, L. turanica, Leishmania gerbillisensu lato and Leishmania close to L. gerbilli or a related species reported in sandflies previously from this location. The haplotypes of L. major and L. turanica were found to be identical to that of isolates of L. major and L. turanica from Iran and in GenBank elsewhere. R. opimus is probably the key reservoir in this ZCL focus because of its abundance and its infection rates with both L. major and L. turanica.     

Checkpoint kinase 1 negatively regulates somatic hypermutation

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.     

Direct Binding of the EGF-like Domain of Neuregulin-1 to Integrins (αvβ3 and α6β4) Is Involved in Neuregulin-1/ErbB Signaling

Integrin-growth factor receptor cross-talk plays a role in growth factor signaling, but the specifics are unclear. In a current model, integrins and growth factor receptors independently bind to their ligands (extracellular matrix and growth factors, respectively). We discovered that neuregulin-1 (NRG1), either as an isolated EGF-like domain or as a native multi-domain form, binds to integrins αvβ3 (with a K_D of 1.36 × 10⁻⁷ m) and α6β4. Docking simulation predicted that three Lys residues at positions 180, 184, and 186 of the EGF-like domain are involved in integrin binding. Mutating these residues to Glu individually or in combination markedly suppressed integrin binding and ErbB3 phosphorylation. Mutating all three Lys residues to Glu (the 3KE mutation) did not affect the ability of NRG1 to bind to ErbB3 but markedly reduced the ability of NRG1 to induce ErbB3 phosphorylation and AKT and Erk1/2 activation in MCF-7 and T47D human breast cancer cells. This suggests that direct integrin binding to NRG1 is critical for NRG1/ErbB signaling. Notably, stimulation of cells with WT NRG1 induced co-precipitation of ErbB3 with α6β4 and with αvβ3 to a much lower extent. This suggests that WT NRG1 induces integrin-NRG1-ErbB3 ternary complex formation. In contrast, the 3KE mutant was much less effective in inducing ternary complex formation than WT NRG1, suggesting that this process depends on the ability of NRG1 to bind to integrins. These results suggest that direct NRG1-integrin interaction mediates integrin-ErbB cross-talk and that α6β4 plays a major role in NRG-ErbB signaling in these cancer cells.     

Novel alkenal/one reductase activity of yeast NADPH:quinone reductase Zta1p. Prospect of the functional role for the ζ-crystallin family

ζ-Crystallins are a Zn(2+)-lacking enzyme group with quinone reductase activity, which belongs to the medium-chain dehydrogenase/reductase superfamily. It has been recently observed that human ζ-crystallin is capable of reducing the α,β-double bond of alkenals and alkenones. Here we report that this activity is also shared by the homologous Zta1p enzyme from Saccharomyces cerevisiae. While the two enzymes show similar substrate specificity, human ζ-crystallin exhibits higher activity with lipid peroxidation products and Zta1p is more active with cinnamaldehyde. The presence of Zta1p has an in vivo protective effect on yeast strains exposed to the toxic substrate 3-penten-2-one. Analysis of ZTA1 gene expression indicates an induction under different types of cellular stress, including ethanol and dimethylsulfoxide exposure and by reaching the stationary growth phase. The role of Zta1p in the yeast adaptation to some stress types and the general functional significance of ζ-crystallins are discussed.