Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assay

It is thought that sodium dodecyl sulfate (SDS), an anionic detergent, binds to hydrophobic moieties of peptide to destroy the conformational structure of protein. Because of this property, it is involved in many biochemical procedures such as separations of protein and proteolytic digestion. In the course of our study on a solid-phase protein assay, we found that SDS acts as an effective reagent for protein blotting onto a hydrophobic membrane of polyvinylidene difluoride with a manifold dot-blot apparatus. At least 0.1% SDS in an acid-ethanol blotting solution, while reducing the bias of pronounced interferers for protein assay to protein-membrane interaction, quantitatively retains protein on the membrane. Presumably, protein denatures by SDS to become an unfolded state and adsorbs into the membrane by hydrophobic interaction, even in the presence of excess SDS. Therefore, bolts stained with a pyrogallol red-molybdate complex (Pyromolex) reagent unreactive to the membrane allowed a precise protein determination without significant interference of materials, especially detergents in the sample solution. The filtration-blotting with SDS would be a crucial procedure for quantitative analyses such as immunoblotting in detergent-containing samples, together with the solid-phase protein assay with limited sample volumes, such as 20 microL or less.     

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.     

A role of a lymphocyte tryptase, granzyme A, in experimental ulcerative colitis

In the large-intestinal mucosae of rats orally administered dextran sulfate sodium, which induces an enteritis resembling ulcerative colitis (UC), the activity for granzyme A, a lymphocyte tryptase, increased at an earlier stage than that at which UC markers (growth-regulated gene product/cytokine-induced neutrophil chemoattractant-1 and caspase-3) increased. This suggests involvement of the enzyme in the exacerbation and perpetuation of enteritis.     

Engineering large viral DNA genomes using the CRISPR‐Cas9 system

Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus‐infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time‐consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat‐Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene‐ablated HSV but also gene knock‐in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein–Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.     

Significant Association of KIR2DL3-HLA-C1 Combination with Cerebral Malaria and Implications for Co-evolution of KIR and HLA

Cerebral malaria is a major, life-threatening complication of Plasmodium falciparum malaria, and has very high mortality rate. In murine malaria models, natural killer (NK) cell responses have been shown to play a crucial role in the pathogenesis of cerebral malaria. To investigate the role of NK cells in the developmental process of human cerebral malaria, we conducted a case-control study examining genotypes for killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen (HLA) class I ligands in 477 malaria patients. We found that the combination of KIR2DL3 and its cognate HLA-C1 ligand was significantly associated with the development of cerebral malaria when compared with non-cerebral malaria (odds ratio 3.14, 95% confidence interval 1.52–6.48, P = 0.00079, corrected P = 0.02). In contrast, no other KIR-HLA pairs showed a significant association with cerebral malaria, suggesting that the NK cell repertoire shaped by the KIR2DL3-HLA-C1 interaction shows certain functional responses that facilitate development of cerebral malaria. Furthermore, the frequency of the KIR2DL3-HLA-C1 combination was found to be significantly lower in malaria high-endemic populations. These results suggest that natural selection has reduced the frequency of the KIR2DL3-HLA-C1 combination in malaria high-endemic populations because of the propensity of interaction between KIR2DL3 and C1 to favor development of cerebral malaria. Our findings provide one possible explanation for KIR-HLA co-evolution driven by a microbial pathogen, and its effect on the global distribution of malaria, KIR and HLA.     

Granzyme A causes detachment of alveolar epithelial A549 cells accompanied by promotion of interleukin-8 release

Granzyme A (GrA) is a serine protease produced in cytotoxic lymphocytes, lung epithelial cells (alveolar type-II cells), and alveolar macrophages. In the present study, recombinant rat GrA (rGrA) was found to cause rounding and detachment of an alveolar type-II epithelial cell line, A549. Also, rGrA stimulated release of a neutrophil chemoattractant, interleukin-8, from the cells, via a mechanism involving microtubule disruption, probably resulting from reduction of cell adhesion to culture dishes. These findings suggest that GrA might be involved in the pathogenesis of certain lung diseases characterized by loss of alveolar wall structures, neutrophil accumulation, and chronic inflammation.     

Prolyl-isomerase Pin1 accumulates in lewy bodies of parkinson disease and facilitates formation of alpha-synuclein inclusions

Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of alpha-synuclein. Although certain post-translational modifications of alpha-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of alpha-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50-60% of the LBs that show an intense halo pattern resembling that of alpha-synuclein. By utilizing a cellular model of alpha-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of alpha-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of alpha-synuclein. Finally, we show that Pin1 binds synphilin-1, an alpha-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with alpha-synuclein, thus likely facilitating the formation of alpha-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for alpha-synuclein aggregation and in the resultant Lewy body formations in PD.     

A critical role of LFA-1 in the development of Th17 cells and induction of experimental autoimmune encephalomyelytis

Snu13p is a Saccharomyces cerevisiae protein essential for pre-messenger RNA splicing and pre-ribosomal RNA processing. Snu13p binds U4 snRNA of the spliceosome and box C/D snoRNAs of the pre-ribosomal RNA processing machinery to induce assembly of each ribonucleoprotein complex. Here, we present structural and biochemical analysis of Snu13p. The crystal structure of Snu13p reveals a region of the protein which could be important for protein interaction during ribonucleoprotein assembly. Using the structure of Snu13p we have designed the first temperature-sensitive mutants in Snu13p, L67W and I102A. Wild-type and mutant Snu13p proteins were assayed for binding to U4 snRNA and U3 snoRNA. Both temperature-sensitive mutants displayed significantly reduced RNA binding compared to wild-type protein. As the temperature-sensitive mutations are not in the known RNA binding region of Snu13p this indicates that these mutants indirectly influence the RNA binding properties of Snu13p. This work provides insight into Snu13p function during ribonucleoprotein assembly.     

Long-term persistence of both functional and non-functional alleles at the leukocyte immunoglobulin-like receptor A3 (LILRA3) locus suggests balancing selection

The leukocyte immunoglobulin-like receptor (LILR) family consists of 13 loci, and a number of variations have been identified in these genes. Some polymorphisms of the LILR genes are reported to be associated with susceptibility to diseases such as rheumatoid arthritis and multiple sclerosis. LILRA3, one of the LILR genes, exhibits a presence or absence variation due to a 6.7-kb deletion in various populations. In this study, variation screening of the LILRA3 gene revealed high allele frequency of the 6.7-kb LILRA3 deletion (71%) in Japanese, in contrast to the frequency reported for the other populations. In addition, we identified a splice acceptor mutation in intron 1 with allele frequency of 19%, resulting in three alternatively spliced isoforms. Surprisingly, all of these isoforms were found to contain premature termination codons (PTCs) in the exon 3. Taken together, approximately 80% of Japanese lack functional LILRA3 alleles. The maximum likelihood coalescent analysis suggested that two major lineages, functional alleles and PTC-containing alleles, have been maintained for 2.75 million years in humans. These results prompted us to hypothesize that balancing selection had maintained both the functional and non-functional alleles at the LILRA3 locus. This hypothesis is consistent with the observation that the 6.7-kb LILRA3 deletion is detected worldwide in the presence of functional LILRA3 alleles.Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB236941, AB236942, AB236943, AB236944, AB236945, AB236946, AB236947, AB236948, AB236949, AB236950.     

Granzyme A and thrombin differentially promote the release of interleukin-8 from alveolar epithelial A549 cells

Some of extracellular serine proteases with trypsin-like specificity of cleavage have been known to increase the release of inflammatory mediators from various cell types. For instance, two well-known trypsin-like serine proteases circulating in blood, granzyme A (GrA) and thrombin, have been found to promote interleukin (IL)-8 release from an alveolar epithelial A549 cell line. However, the mechanisms by which the proteases promote IL-8 release from the cells are not fully understood. In the present study, using A549 cells we found that (1) thrombin promoted IL-8 release from the cells via a mechanism partially involving activation of protease-activated receptor-1, a G-protein coupled receptor, whereas a recombinant form of GrA (rGrA) did it via a mechanism that does not involve the receptor activation; that (2) unlike rGrA, thrombin did not cause detachment and microtubule disruption of the cells; and that (3) the release of IL-8 induced by rGrA was inhibited in the presence of taxol, a microtubule-stabilizing reagent, whereas that induced by thrombin was not. These findings suggest that rGrA and thrombin promote the release of IL-8 from A549 cells through distinct mechanisms.