Urea-dependent unfolding of HIV-1 protease studied by circular dichroism and small-angle X-ray scattering

HIV-1 protease is responsible for the maturation of infective virions, and is one of the targets of drugs against AIDS. It is an aspartic protease with a 99-resiude polypeptide dimerized. Previous study with fluorescence and sedimentation measurements revealed that the protein was unfolded with concomitant dissociation of the subunits. In the present study, we investigated urea-dependent unfolding of HIV-1 protease with CD and SAXS in order to monitor the secondary structure and the global size and shape of the molecule, respectively. The unfolding parameters estimated by both methods were almost the same, indicating that the dissociation of the subunits accompanied the disruption of their internal structures. This is in line with the previous results, and moreover some residual structures were suggested to be present in the unfolded state. The distinct difference, as compared with the unfolding of pepsin, was interpreted from the point of their molecular architectures.     

Desert hedgehog‐patched 2 expression in peripheral nerves during Wallerian degeneration and regeneration

Hedgehog proteins are important in the development of the nervous system. As Desert hedgehog (Dhh) is involved in the development of peripheral nerves and is expressed in adult nerves, it may play a role in the maintenance of adult nerves and degeneration and regeneration after injury. We firstly investigated the Dhh‐receptors, which are expressed in mouse adult nerves. The Dhh receptor patched(ptc)2 was detected in adult sciatic nerves using RT‐PCR, however, ptc1 was undetectable under the same experimental condition. Using RT‐PCR in purified cultures of mouse Schwann cells and fibroblasts, we found ptc2 mRNA in Schwann cells, and at much lower levels, in fibroblasts. By immunohistochemistry, Ptc2 protein was seen on unmyelinated nerve fibers. Then we induced crush injury to the sciatic nerves of wild‐type (WT) and dhh‐null mice and the distal stumps of injured nerves were analyzed morphologically at different time points and expression of dhh and related receptors was also measured by RT‐PCR in WT mice. In dhh‐null mice, degeneration of myelinated fibers was more severe than in WT mice. Furthermore, in regenerated nerves of dhh‐null mice, minifascicular formation was even more extensive than in dhh‐null intact nerves. Both dhh and ptc2 mRNA levels were down‐regulated during the degenerative phase postinjury in WT mice, while levels rose again during the phase of nerve regeneration. These results suggest that the Dhh‐Ptc2 signaling pathway may be involved in the maintenance of adult nerves and may be one of the factors that directly or indirectly determines the response of peripheral nerves to injury. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Nuclear compartmentalization is abolished during fission yeast meiosis

In eukaryotic cells, the nuclear envelope partitions the nucleus from the cytoplasm. The fission yeast Schizosaccharomyces pombe undergoes closed mitosis in which the nuclear envelope persists rather than being broken down, as in higher eukaryotic cells. It is therefore assumed that nucleocytoplasmic transport continues during the cell cycle. Here we show that nuclear transport is, in fact, abolished specifically during anaphase of the second meiotic nuclear division. During that time, both nucleoplasmic and cytoplasmic proteins disperse throughout the cell, reminiscent of the open mitosis of higher eukaryotes, but the architecture of the S. pombe nuclear envelope itself persists. This functional alteration of the nucleocytoplasmic barrier is likely induced by spore wall formation, because ectopic induction of sporulation signaling leads to premature dispersion of nucleoplasmic proteins. A photobleaching assay demonstrated that nuclear envelope permeability increases abruptly at the onset of anaphase of the second meiotic division. The permeability was not altered when sporulation was inhibited by blocking the trafficking of forespore-membrane vesicles from the endoplasmic reticulum to the Golgi. The evidence indicates that yeast gametogenesis produces vesicle transport-mediated forespore membranes by inducing nuclear envelope permeabilization.     

Determination of iodide in urine using electrospray ionization tandem mass spectrometry

A rapid and sensitive electrospray ionization (ESI) tandem mass spectrometry (MS–MS) procedure was developed for the determination of iodide (I⁻). A gold (Au) and I⁻ complex was formed immediately after the addition of the chelating agent NaAuCl₄ to I⁻ solution, and was extracted with methyl isobutyl ketone. One to five microliters of the extract were injected directly into an ESI–MS–MS instrument. I⁻ quantification was performed by selecting reaction monitoring of the product ion I⁻ at m/z 127 derived from the precursor ion ¹⁹⁷AuI₂⁻ at m/z 451. I⁻ concentration was measured in the quantification range from 10⁻⁷ to 10⁻⁵ M using 50 μL of solution within 10 min. Iodate was reduced to I⁻ with ascorbic acid and determined. I⁻ concentration in reference urine 2670a was measured after treatments.     

Thymic Alterations in GM2 Gangliosidoses Model Mice

Background

Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb ^−/− mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies.

Methodology/Principal Findings

In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb ^−/− mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4⁺/CD8⁺ T cells and a significantly increased number of CD4⁺/CD8⁻ T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb ^−/− mice.

Conclusions/Significance

These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.     

Determination of platinum derived from cisplatin in human tissues using electrospray ionization mass spectrometry

Determination of platinum (Pt) derived from cisplatin in tissues was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as the internal standard. Pt and Ag reacted with diethyldithiocarbamate (DDC), and were extracted using isoamylalcohol and acidified with oxalic acid. The compounds were termed Pt(DDC)(3)(+) and Ag(DDC)(2)(+), based on their m/z values exhibiting the highest peaks at m/z 639 and m/z 405, respectively. The limit of detection was 30 pg and the quantitation range was from 100 to 10,000 pg using 5 mg tissue. The present method allowed the determination of Pt in wet-ashed tissue in 10 min.     

Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex

Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8-5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 degrees C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.     

Characterization of a novel C. elegans RGS protein with a C2 domain: evidence for direct association between C2 domain and Galphaq subunit

RGS (regulator of G protein signaling) proteins are GTPase-activating proteins (GAPs) for heterotrimeric G protein alpha subunits and negatively regulate G protein-mediated signal transduction. In this study, we determined the cDNA sequence of a novel Caenorhabditis elegans (C. elegans) RGS protein. The predicted protein, termed C2-RGS, consists of 782 amino acids, and contains a C2 domain and an RGS domain. C2 domains are typically known to be Ca(2+) and phospholipid binding sites, found in many proteins involved in membrane traffic or signal transduction, and most of their biological roles are not identified. To study the function of C2-RGS protein, a series of six truncated versions of C2-RGS were constructed. When the full-length protein of C2-RGS was expressed transiently in AT1a-293T cells, ET-1-induced Ca(2+) responses were strongly suppressed. When each of the mutants with either RGS domain or C2 domain was expressed, the Ca(2+) responses were suppressed moderately. Furthermore, we found that C2 domain of PLC-beta1 also had a similar moderate inhibitory effect. RGS domain of C2-RGS bound to mammalian and C. elegans Galphai/o and Galphaq subunits only in the presence of GDP/AlF(4)(-), and had GAP activity to Galphai3. On the other hand, C2 domains of C2-RGS and PLC-beta1 also bound strongly to Galphaq subunit, in the presence of GDP, GDP/AlF(4)(-), and GTPgammaS, suggesting the stable persistent association between these C2 domains and Galphaq subunit at any stage during GTPase cycle. These results indicate that both the RGS domain and the C2 domain are responsible for the inhibitory effect of the full-length C2-RGS protein on Galphaq-mediated signaling, and suggest that C2 domains of C2-RGS and PLC-beta1 may act as a scaffold module to organize Galphaq and the respective whole protein molecule in a stable signaling complex, both in the absence and presence of stimulus.