Probing the disulfide folding pathway of insulin-like growth factor-I

The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin-like growth factor (IGF)-I which when refolded in vitro produces the native three-disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF-I which contains a 13-amino acid N-terminal extension and a charge mutation at position 3 (Long-[Arg3]IGF-I). Unlike IGF-I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long-[Arg3]IGF-I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long-[Arg3]IGF-I and IGF-I, we acid-trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non-native intermediates, three native-like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long-[Arg3]IGF-I; a single-disulfide Cys18-Cys61 intermediate, an intermediate with Cys18-Cys61 and Cys6-Cys48 disulfide bonds and another with Cys18-Cys61 and Cys47-Cys52 disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys18-Cys61, Cys6-Cys48 intermediate forms the native structure, not by the direct formation of the last (Cys47-Cys52) disulfide bond, but by rearrangement via the Cys18-Cys61 intermediate and a productive Cys18-Cys61, Cys47-Cys52 intermediate. In this pathway, the last disulfide bond to form involves Cys6 and Cys48. Finally, we apply this pathway to IGF-I and conclude that the divergence in the in vitro folding pathway of IGF-I is caused by non-native interactions involving Glu3 that stabilize the alternative structure.     

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) shows significant differences to that found in solution

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) has been solved and refined at 2.5 A resolution. The refinement procedure converged at R = 0.181 for all reflections in the range 20.0-2.5 A. In the crystal, the RNA/DNA hybrid duplex has an A' conformation with all but one of the nucleotide sugar moieties adopting a C3'- endo (N) conformation. Both strands in the double helix adopt a global conformation close to the A-form and the width of the minor groove is typical of that found in the crystal structures of other A-form duplexes. However, differences are observed between the RNA and DNA strands that make up the hybrid at the local level. In the central portion of the duplex, the RNA strand has backbone alpha, beta and gamma torsion angles that alternate between the normal gauche -/ trans / gauche + conformation and an unusual trans / trans / trans conformation. Coupled with this so-called 'alpha/gamma flipping' of the backbone torsion angles, the distance between adjacent phosphorous atoms on the RNA strand systematically varies. Neither of these phenomena are observed on the DNA strand. The structure of the RNA/DNA hybrid presented here differs significantly from that found in solution for this and other sequences. Possible reasons for these differences and their implications for the current model of RNase H activity are discussed.     

Clathrin-coated pit-associated proteins are required for alveolar macrophage phagocytosis

During phagocytosis, phagocytic receptors and membrane material must be inserted in the pseudopod membrane as it extends over the phagocytic target. This may require a clathrin-mediated recycling mechanism similar to that postulated for leading edge formation during cell migration. To investigate this possibility, liposomes were used to deliver to intact rat alveolar macrophages (AMs): 1) Abs to clathrin, clathrin adaptor AP-2, and hsc70, and 2) amantadine. Phagocytosis was assayed by fluorometric and colorimetric techniques. Liposome-delivered Abs to clathrin and AP-2 inhibited AM phagocytosis of zymosan-coated, fluorescent liposomes from 16.3+/-0.3 to 5.8+/-0.3, and 10.1+/-0.9 to 4.8+/-0.2 liposomes/cell (p<0.01). Similarly, liposome-delivered Ab to clathrin also inhibited AM phagocytosis of IgG-opsonized RBCs from 11.7+/-1.7 to 3.8+/-0.7 RBCs/cell (p<0.01). Amantadine, which blocks the budding of clathrin-coated vesicles, inhibited phagocytosis from 13.8+/-0.8 to 5.7+/-0.6 (p<0.01). Ab blockade of hsc70, which catalyzes clathrin turnover, also inhibited phagocytosis from 9.1+/-0.5 to 4.3+/-0.2 (p<0.01). These findings suggest that clathrin-mediated receptor/membrane recycling is required for phagocytosis.     

Superionic and metallic states of water and ammonia at giant planet conditions

The phase diagrams of water and ammonia were determined by constant pressure ab initio molecular dynamic simulations at pressures (30 to 300 gigapascal) and temperatures (300 to 7000 kelvin) of relevance for the middle ice layers of the giant planets Neptune and Uranus. Along the planetary isentrope water and ammonia behave as fully dissociated ionic, electronically insulating fluid phases, which turn metallic at temperatures exceeding 7000 kelvin for water and 5500 kelvin for ammonia. At lower temperatures, the phase diagrams of water and ammonia exhibit a superionic solid phase between the solid and the ionic liquid. These simulations improve our understanding of the properties of the middle ice layers of Neptune and Uranus.     

VEGI, a novel cytokine of the tumor necrosis factor family, is an angiogenesis inhibitor that suppresses the growth of colon carcinomas in vivo

A novel member of the tumor necrosis factor (TNF) family has been identified from the human umbilical vein endothelial cell cDNA library, named vascular endothelial growth inhibitor (VEGI). The VEGI gene was mapped to human chromosome 9q32. The cDNA for VEGI encodes a protein of 174 amino acid residues with the characteristics of a type II transmembrane protein. Its amino acid sequence is 20-30% identical to other members of the TNF family. Unlike other members of the TNF family, VEGI is expressed predominantly in endothelial cells. Local production of a secreted form of VEGI via gene transfer caused complete suppression of the growth of MC-38 murine colon cancers in syngeneic C57BL/6 mice. Histological examination showed marked reduction of vascularization in MC-38 tumors that expressed soluble but not membrane-bound VEGI or were transfected with control vector. The conditioned media from soluble VEGI-expressing cells showed marked inhibitory effect on in vitro proliferation of adult bovine aortic endothelial cells. Our data suggest that VEGI is a novel angiogenesis inhibitor of the TNF family and functions in part by directly inhibiting endothelial cell proliferation. The results further suggest that VEGI maybe highly valuable toward angiogenesis-based cancer therapy.     

Profile of cognitive functioning in women with the fragile X mutation.

Two studies tested the specificity of the neurocognitive profile of women with fragile X syndrome (FXS). First, women with an FXS full mutation were compared with women with a premutation and women without FXS who grew up in FXS families. Women with FXS had a significantly lower IQ than the other groups, and analyses of subtest profiles showed they had a relative weakness on Arithmetic and strength on Picture Completion. Women with FXS performed worse than the other groups on executive function, spatial ability, and visual memory. Next, women with FXS were compared with women without FXS matched on age and IQ. A similar IQ profile was found, but women with FXS were worse than controls only on executive function. The authors also examined which neurocognitive indices were related to the underlying biology of the disorder. Overall, the results indicated that executive rather than visuospatial deficits were primary in the neurocognitive profile of FXS. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sirt3, Mitochondrial ROS, Ageing, and Carcinogenesis

One fundamental observation in cancer etiology is that the rate of malignancies in any mammalian population increases exponentially as a function of age, suggesting a mechanistic link between the cellular processes governing longevity and carcinogenesis. In addition, it is well established that aberrations in mitochondrial metabolism, as measured by increased reactive oxygen species (ROS), are observed in both aging and cancer. In this regard, genes that impact upon longevity have recently been characterized in S. cerevisiae and C. elegans, and the human homologs include the Sirtuin family of protein deacetylases. Interestingly, three of the seven sirtuin proteins are localized into the mitochondria suggesting a connection between the mitochondrial sirtuins, the free radical theory of aging, and carcinogenesis. Based on these results it has been hypothesized that Sirt3 functions as a mitochondrial fidelity protein whose function governs both aging and carcinogenesis by modulating ROS metabolism. Sirt3 has also now been identified as a genomically expressed, mitochondrial localized tumor suppressor and this review will outline potential relationships between mitochondrial ROS/superoxide levels, aging, and cell phenotypes permissive for estrogen and progesterone receptor positive breast carcinogenesis.     

Modulation of the cleavage of glycosylphosphatidylinositol-anchored proteins by specific bacterial phospholipases

Many enzymes are tethered to the extracellular face of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. These proteins can be released in soluble form by the action of GPI-specific phospholipase. Little is currently known about the factors modulating this release. We investigated the effects of several experimental variables on the cleavage of the GPI-anchored proteins 5'nucleotidase, acetylcholinesterase, and alkaline phosphatase by phospholipases from Bacillus thuringiensis and Staphylococcus aureus. Phospholipase activity was not inhibited by isotonic salt and was relatively unaffected by buffer type and concentration. In both cases, the optimum pH for cleavage was approximately 6.5. Over 80% of 5'-nucleotidase activity present in the lymphocyte plasma membrane was cleaved by the B. thuringiensis enzyme, and the initial rate of release was linear with phospholipase concentration. All three GPI-anchored proteins were released from lymphocyte plasma membrane at comparable phospholipase concentrations, suggesting that they have similar anchor structures. The catalytic activity of 5'-nucleotidase appeared to increase following conversion to the soluble form. The relative surface charge of the host plasma membrane modulated catalytic activity towards GPI-anchored proteins, depending on the net charge of the phospholipase. Studies on purified lymphocyte 5'-nucleotidase reconstituted into bilayers of dimyristoylphosphatidylcholine indicated that the efficiency of phospholipase cleavage was 12- to 50-fold lower when compared with the native plasma membrane. The ability of the phospholipase to cleave the GPI anchor was further reduced when the bilayer was in the gel phase.     

Effects of argon laser curing on dentin shear bond strengths

Previous studies have demonstrated the ability of the argon laser to polymerize light-activated materials and improve enamel shear bond strengths. This study was conducted to evaluate the effects of the argon laser on dentin shear bond strengths of current dentin bonding systems. Argon laser (HGM Model 8) at 231 and 280 mW, 5 sec bonding agent, 10 sec composite, and a conventional curing light (Translux EC/Kulzer) at 10 sec bonding agent, 20 sec composite were used to polymerize samples of dentin bonding systems [Scotchbond Multi-Purpose Plus (3M) and Prime Bond (Dentsply/Caulk), both with TPH (Dentsply/Caulk) composite]. A flat dentin bonding site (600 grit) was prepared on the buccal surface of extracted human teeth. Twelve samples were made for each set of parameters for both laser and conventional light totaling 48 samples. Samples were stored in distilled water in light-proof containers for 24 h at 37 degrees C. Shear bond strengths (MPa) were determined for each sample on the Instron testing machine. Mean values were calculated for each set of data and ANOVA with Fisher PLSD were used for statistical analysis. The argon laser provided bond strengths that were 21-24% greater than those of the conventional curing light system.