Eleven novel polymorphic microsatellite DNA markers from the green-lipped mussel, <Emphasis Type="Italic">Perna viridis</Emphasis>

We report on the characterization of 11 polymorphic microsatellite loci in P. viridis, the first set of such markers developed and characterized for this species. The number of alleles per locus ranged from 2 to 7, whereas the observed heterozygosity ranged from 0.0447 to 0.4837. These markers should prove useful as powerful genetic markers for this species. This article was submitted by the authors in English.     

Evolutionary trace analysis at the ligand binding site of laccase

Laccase belongs to the family of blue multi-copper oxidases and are capable of oxidizing a wide range of aromatic compounds. Laccases have industrial applications in paper pulping or bleaching and hydrocarbon bioremediation as a biocatalyst. We describe the design of a laccase with broader substrate spectrum in bioremediation. The application of evolutionary trace (ET) analysis of laccase at the ligand binding site for optimal design of the enzyme is described. In this attempt, class specific sites from ET analysis were mapped onto known crystal structure of laccase. The analysis revealed 162PHE as a critical residue in structure function relationship studies.     

Activation of TRPP2 through mDia1-dependent voltage gating

The TRPP2 cation channel is directly responsible for approximately 15% of all cases of autosomal dominant polycystic kidney disease. However, the mechanisms underlying fundamental properties of TRPP2 regulation, such as channel gating and activation, are unknown. We have shown that TRPP2 was activated by EGF and physically interacted with the mammalian diaphanous-related formin 1 (mDia1), a downstream effector of RhoA. Now, we show that mDia1 regulates TRPP2 by specifically blocking its activity at negative but not positive potentials. The voltage-dependent unblock of TRPP2 by mDia1 at positive potentials is mediated through RhoA-induced molecular switching of mDia1 from its autoinhibited state at negative potentials to its activated state at positive potentials. Under physiological resting potentials, EGF activates TRPP2 by releasing the mDia1-dependent block through the activation of RhoA. Our data reveal a new role of mDia1 in the regulation of ion channels and suggest a molecular basis for the voltage-dependent gating of TRP channels.     

Inhibition of polysome assembly enhances imatinib activity against chronic myelogenous leukemia and overcomes imatinib resistance

Dysregulated mRNA translation is implicated in the pathogenesis of many human cancers including chronic myelogenous leukemia (CML). Because our prior work has specifically implicated translation initiation in CML, we tested compounds that could modulate translation initiation and polysomal mRNA assembly. Here, we evaluated the activity of one such compound, CGP57380, against CML cells and explored its mechanisms of action. First, using polysomal mRNA profiles, we found that imatinib and CGP57380 could independently, and cooperatively, impair polysomal mRNA loading. Imatinib and CGP57380 also synergistically inhibited the growth of Ba/F3-Bcr-Abl and K562 cells via impaired cell cycle entry and increased apoptosis. Mechanistically, CGP57380 inhibited efficient polysomal assembly via two processes. First, it enhanced imatinib-mediated inhibition of eukaryotic initiation factor 4F induction, and second, it independently impaired phosphorylation of ribosomal protein S6 on the preinitiation complex. We also identified multiple substrates of the mTOR, Rsk, and Mnk kinases as targets of CGP57380. Finally, we found a novel negative-feedback loop to the mitogen-activated protein kinase/Mnk pathway that is triggered by CGP57380 and demonstrated that an interruption of the loop further increased the activity of the combination against imatinib-sensitive and -resistant CML cells. Together, this work supports the inhibition of translation initiation as a therapeutic strategy for treating cancers fueled by dysregulated translation.     

The SH3 domain of Lck modulates T-cell receptor-dependent activation of extracellular signal-regulated kinase through activation of Raf-1

Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-γ, and Ras. We determined that the signaling lesion in W97ALck-expressing cells lies at the level of Raf-1 activation and is dependent on the presence of tyrosines 340/341 in the Raf-1 sequence. These data demonstrate a second function for Lck in TCR-mediated signaling to ERK. Additionally, we found that a significant fraction of Lck is localized to the Golgi apparatus and that, compared with wild-type Lck, W97ALck displays aberrant Golgi membrane localization. Our results support a model where under conditions of weak stimulation through the TCR, in addition to activated Ras, Golgi apparatus-localized Lck is needed for the full activation of Raf-1.     

Eleven novel polymorphic microsatellite DNA markers from the green-lipped mussel Perna viridis

We report on the characterization of 11 polymorphic microsatellite loci in P. viridis, the first set of such markers developed and characterized for this species. The number of alleles per locus ranged from 2 to 7, whereas the observed heterozygosity ranged from 0.0447 to 0.4837. These markers should prove useful as powerful genetic markers for this species.     

Effect of calcium ions on the density of lecithin and its effective molecular volume in lecithin-water dispersions

As the packing structure of lipid molecules in the liposomes will vary in the presence of ions, it is expected that the density of lipid and the effective volume of lipid molecules in the dispersions will also vary, albeit minutely. Density measurements of lipid-water dispersions with the addition of Ca(2+) ions were determined accurately. The effect of Ca(2+) ions on the molecular packing structure of the liposomes was elucidated from the results obtained. The results for the density of the lecithin in the dispersions with and without the addition of Ca(2+) ions are, respectively, 1.0782 and 1.0579 g cm(-3) at 25 degrees C; and 1.0048 and 0.9961 g cm(-3) at 50 degrees C. The average values of the effective molecular volume of lecithin in the dispersions with and without the addition of Ca(2+) ions are, respectively, 1.131E-21 and 1.152E-21 cm(3) at 25 degrees C; and 1.213E-21 and 1.224E-21 cm(3) at 50 degrees C.     

NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells

NOTCH signaling is a key regulator of cell fate decisions in prenatal skeletal development and is active during adult tissue renewal. In addition, its association with neoplasia suggests that it is a candidate therapeutic target. We find that attenuated NOTCH signaling enhances osteoclastogenesis and bone resorption in vitro and in vivo by a combination of molecular mechanisms. First, deletion of Notch1-3 in bone marrow macrophages directly promotes their commitment to the osteoclast phenotype. These osteoclast precursors proliferate more rapidly than the wild type in response to macrophage colony-stimulating factor and are sensitized to RANKL and macrophage colony-stimulating factor, undergoing enhanced differentiation in response to low doses of either cytokine. Conforming with a role for NOTCH in this process, presentation of the NOTCH ligand JAGGED1 blunts the capacity of wild-type bone marrow macrophages to become osteoclasts. Combined, these data establish that NOTCH suppresses osteoclastogenesis via ligand-mediated receptor activation. Although NOTCH1 and NOTCH3 collaborate in regulating osteoclast formation, NOTCH1 is the dominant paralog. In addition, NOTCH1 deficiency promotes osteoclastogenesis indirectly by enhancing the ability of osteoblast lineage cells to stimulate osteoclastogenesis. This is achieved by decreasing the osteoprotegerin/RANKL expression ratio. Thus, NOTCH1 acts as a net inhibitor of bone resorption, exerting its effect both directly in osteoclast precursors and indirectly via osteoblast lineage cells. These observations raise caution that therapeutic inhibition of NOTCH signaling may adversely accelerate bone loss in humans.     

Nuclear magnetic resonance structure and IgE epitopes of Blo t 5, a major dust mite allergen

A high incidence of sensitization to Blomia tropicalis, the predominant house dust mite species in tropical regions, is strongly associated with allergic diseases in Singapore, Malaysia, and Brazil. IgE binding to the group 5 allergen, Blo t 5, is found to be the most prevalent among all B. tropicalis allergens. The NMR structure of Blo t 5 determined represents a novel helical bundle structure consisting of three antiparallel alpha-helices. Based on the structure and sequence alignment with other known group 5 dust mite allergens, surface-exposed charged residues have been identified for site-directed mutagenesis and IgE binding assays. Four charged residues, Glu76, Asp81, Glu86, and Glu91 at around the turn region connecting helices alpha2 and alpha3 have been identified to be involved in the IgE binding. Using overlapping peptides, we have confirmed that these charged residues are located on a major putative linear IgE epitope of Blo t 5 from residues 76-91 comprising the sequence ELKRTDLNILERFNYE. Triple and quadruple mutants have been generated and found to exhibit significantly lower IgE binding and reduced responses in skin prick tests. The mutants induced similar PBMC proliferation as the wild-type protein but with reduced Th2:Th1 cytokines ratio. Mass screening on a quadruple mutant showed a 40% reduction in IgE binding in 35 of 42 sera of atopic individuals. Findings in this study further stressed the importance of surface-charged residues on IgE binding and have implications in the cross-reactivity and use of Blo t 5 mutants as a hypoallergen for immunotherapy.     

A mitochondrial protein compendium elucidates complex I disease biology

Mitochondria are complex organelles whose dysfunction underlies a broad spectrum of human diseases. Identifying all of the proteins resident in this organelle and understanding how they integrate into pathways represent major challenges in cell biology. Toward this goal, we performed mass spectrometry, GFP tagging, and machine learning to create a mitochondrial compendium of 1098 genes and their protein expression across 14 mouse tissues. We link poorly characterized proteins in this inventory to known mitochondrial pathways by virtue of shared evolutionary history. Using this approach, we predict 19 proteins to be important for the function of complex I (CI) of the electron transport chain. We validate a subset of these predictions using RNAi, including C8orf38, which we further show harbors an inherited mutation in a lethal, infantile CI deficiency. Our results have important implications for understanding CI function and pathogenesis and, more generally, illustrate how our compendium can serve as a foundation for systematic investigations of mitochondria.