Characterization of lens fiber cell triton insoluble fraction reveals ERM (ezrin, radixin, moesin) proteins as major cytoskeletal-associated proteins

To understand lens fiber cell elongation- and differentiation-associated cytoskeletal remodeling, here we identified and characterized the major protein components of lens fiber cell Triton X-100 insoluble fraction by mass spectrometry and immunoblot analysis. This analysis identified spectrin, filensin, vimentin, tubulin, phakinin, and β-actin as major cytoskeletal proteins in the lens fibers. Importantly, ezrin, radixin, and moesin (ERM), heat-shock cognate protein 70, and β/γ-crystallins were identified as major cytoskeletal-associated proteins. ERM proteins were confirmed to exist as active phosphorylated forms that exhibited intense distribution in the organelle free-zone fibers. Furthermore, ERM protein phosphorylation was found to be dramatically reduced in Rho GTPase-targeted transgenic mouse lenses. These data identify the ERM proteins, which cross-link the plasma membrane and actin, as major and stable cytoskeletal-associated proteins in lens fibers, and indicate a potential role(s) for the ERMs in fiber cell actin cytoskeletal and membrane organization.     

Functionalization of carbon nanotubes enables non-covalent binding and intracellular delivery of small interfering RNA for efficient knock-down of genes

The lipophilic nature of biological membranes restricts the direct intracellular delivery of potential drugs and molecular probes and makes intracellular transport one of the key problems in gene therapy. Because of their ability to cross cell membranes, single walled carbon nanotubes (SWNTs) are of interest as carriers of biologically active molecules, such as small interfering RNAs (siRNAs). We developed a strategy for chemical functionalization of SWNTs with hexamethylenediamine (HMDA) and poly(diallyldimethylammonium)chloride (PDDA) to obtain a material that was able to bind negatively charged siRNA by electrostatic interactions. PDDA-HMDA-SWNTs exhibited negligible cytotoxic effects on isolated rat heart cells at concentrations up to 10 mg/l. PDDA-HMDA-SWNTs loaded with extracellular signal-regulated kinase (ERK) siRNA were able to cross the cell membrane and to suppress expression of the ERK target proteins in primary cardiomyocytes by about 75%. PDDA-functionalized SWNTs thus present an effective carrier system for applications in siRNA-mediated gene silencing.     

Differential expression and activation of Rab27A in human eosinophils: relationship to blood eosinophilia

Eosinophil degranulation is thought to play a pathophysiological role in asthma. Rab27A is a GTP-binding protein that is known to be essential for the degranulation of several leukocyte subsets and thus may be essential for eosinophil granule exocytosis. Here, we show that Rab27A mRNA and protein are expressed in human eosinophils. We have developed a novel assay to assess Rab27A activation and have found a similar activation pattern of this protein upon stimulation of eosinophils, neutrophils and NK cells suggesting a similar function in these cell types. Interestingly, Rab27A expression was elevated in eosinophils from asthmatic donors. Furthermore, eosinophils from eosinophilic donors displayed more rapid Rab27A activation kinetics than those from donors with lower eosinophil counts. Given that elevated blood eosinophil numbers correlate with increased priming of eosinophils, this pattern of Rab27A activation suggests differential protein expression in activated cells may allow eosinophils to degranulate more rapidly upon stimulation.     

Trafficking of cGMP-dependent protein kinase II via interaction with Rab11

cGMP-dependent protein kinase II (cGK-II) is implicated in several physiological functions including intestinal secretion, bone growth, and learning and memory, but the detailed mechanisms are still unclear. To identify proteins that are involved in cGMP/cGK-II signaling, we performed yeast two-hybrid screening and identified Rab11b as a cGK-II-interacting protein that regulates the slow-recycling pathway. Interestingly, cGK-II interacted with the GDP-bound form of Rab11b (Rab11b S25N), but not the GTP-bound form, in mammalian cells. Immunofluorescence staining revealed that Rab11b S25N promoted the translocation of cGK-II from the plasma membrane to the cytoplasm and that the localization of cGK-II extensively overlapped with Rab11b. Furthermore, treatment with a membrane-permeable cGMP analog caused the rapid retranslocation of cGK-II and Rab11b S25N to the membrane. These data indicate that Rab11b is necessary for the trafficking of cGK-II and that the cGMP/cGK-II signaling pathway is closely related to Rab11b recycling pathway.     

Babesia divergens: identification and characterization of BdHSP-20, a small heat shock protein

This study describes the identification and characterization of the Babesia divergens α-crystallin/small heat shock protein 20 (BdHSP-20). BdHSP-20 was recognized by the DG7 monoclonal antibody (DG7 mAb) originally produced by Precigout et al. [Precigout, E., Valentin, A., Carcy, B., Gorenflot, A., Nakamura, K., Aikawa, M., Schrevel, J. 1993. Babesia divergens: characterization of a 17-kDa merozoite membrane protein. Experimental Parasitology 77, 425-434] against B. divergens merozoites. We used DG7 mAb to immunoscreen a B. divergens cDNA library to clone the gene encoding the small heat shock protein. Bdhsp-20 is a single copy gene interrupted by one intron. The deduced gene product (BdHSP-20) clearly belongs to the α-crystallin family and shows significant homology to Babesia bovis, Plasmodiumfalciparum and Toxoplasma gondii sHSPs, with the highest degree of sequence identity around the catalytic domain. Nutritient stress (serum depletion) treatment of the parasites induced the upregulation of BdHSP-20 gene expression observed by semi-quantitative PCR and immunoprecipitation. This regulation pattern suggests that BdHSP-20 could probably be of importance for parasite survival in the case of environmental stress. BdHSP-20 has previously been shown to be highly conserved among different strains and antibodies against the protein drastically reduce parasitemia in vitro.     

Large nucleotide-dependent conformational change in Rab28

Rab GTPases are essential regulators of membrane trafficking. We report crystal structures of Rab28 in the active (GppNHp-bound) and inactive (GDP-3′P-bound) forms at 1.5 and 1.1 Å resolution. Rab28 is a distant member of the Rab family. While the overall fold of Rab28 resembles that of other Rab GTPases, it undergoes a larger nucleotide-dependent conformational change than other members of this family. Added flexibility resulting from a double-glycine motif at the beginning of switch 2 might partially account for this observation. The double-glycine motif, which is conserved in the Arf family, only occurs in Rab28 and Rab7B of the Rab family, and may have a profound effect on their catalytic activities.     

The ice cover on small and large lakes: scaling analysis and mathematical modelling

Lake ice cover is described by its thickness, temperature, stratigraphy and overlying snow layer. When the ratio of ice thickness to lake size is above ～10^?5, the ice cover is stable; otherwise, mechanical forcing breaks the ice cover, and ice drifting takes place with lead-opening and ridging. This transition enables a convenient distinction to be made between small and large lakes. The evolution of the ice cover on small lakes is solved by a wholly thermodynamic model, but a coupled mechanical–thermodynamic model is needed for large lakes. The latter indicates a wide distribution of ice thickness, and frazil ice may be formed in openings. Ecological conditions in large lakes differ markedly from those in small lakes because vertical mixing and oxygen renewal may take place during the ice season, and the euphotic zone penetrates well into the water column in thin ice regions. Mesoscale sea ice models are applicable to large lakes with only minor tuning of the key parameters. These model systems are presented and analysed using Lake Peipsi as an example. As the climate changes, the transition size between small and large lake ice cover will change.     

Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha-(t-butoxycarbonyl)-l-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.