Gain-of-function screen identifies a role of the Sec61alpha translocon in Drosophila postmitotic neurotoxicity

To elucidate the intrinsic mechanisms of neurotoxicity induction, including those underlying neural cell death and neurodegeneration, we developed a gain-of-function screen for gene products causing neural cell loss. To identify novel genes with a cell-death-related function in neurons, we screened 4,964 Drosophila GS lines, in which one or two genes from much of the Drosophila genome can be overexpressed. Approximately 0.68% of the GS lines produced phenotypes involving a loss of postmitotic neurons. Of these, we identified and characterized the endd2 gene, which encodes the Drosophila ortholog of Sec61alpha (DSec61alpha), an endoplasmic reticulum protein with protein translocation activity. Ectopic expression of DSec61alpha caused neural cell death accompanied by the accumulation of ubiquitinated proteins, which was mediated by DSec61alpha's translocon activity. This supported our previous observation that the DSec61alpha translocon contributes to expanded polyglutamine-mediated neuronal toxicity, which is also associated with ubiquitinated protein accumulation. These data suggest that the translocon may be a novel component of neural cell death and degeneration pathways. Our approach can be used to identify potential neurotoxic factors within the whole genome, which will increase our understanding of the molecular mechanisms of various types of cell death, including those associated with human neurodegenerative diseases.     

Transcriptional induction of Smurf2 ubiquitin ligase by TGF-beta

Smad ubiquitination regulatory factor 2 (Smurf2), a ubiquitin ligase for Smads, plays critical roles in the regulation of transforming growth factor-beta (TGF-beta)-Smad signaling via ubiquitin-dependent degradation of Smad2 and Smad7. We found that TGF-beta stimulates Smurf2 expression. TGF-beta activated the Smurf2 promoter in a TGF-beta responsive cell lines, whereas IL-1alpha, PDGF and epidermal growth factor did not. TGF-beta-mediated Smurf2 promoter activation was inhibited by Smad7 or an activin receptor-like kinase 5 inhibitor but not by dominant negative Smad or disruption of Smad-binding elements in the promoter. Moreover, inhibition of the phosphatidil inositol 3 kinase (PI3K)/Akt pathway suppressed TGF-beta-mediated Smurf2 induction. These results suggest that TGF-beta stimulates Smurf2 expression by Smad-independent pathway such as PI3K/Akt pathway via TGF-beta receptor.     

Differential effect of IL-4 and IL-13 on the expression of recombination-activating genes in mature B cells from human peripheral blood

We examined the expression of recombination-activating genes (RAG-1 and RAG-2) and activation-induced cytidine deaminase (AID) by mature human blood B cells stimulated with anti-CD40 in the presence of IL-4 or IL-13. IL-4 was an effective cofactor for RAG-1 and RAG-2 expression, whereas IL-13 was not. In addition, IL-4-dependent RAG expression combined with AID and IgE expression allowed predominant expression of newly rearranged lambda light chains on IgE+ cells generated from kappa+ cells. Although the magnitudes of IL-4- and IL-13-dependent AID and IgE expression were related to expression levels of binding subunits of the IL-4 and IL-13 receptors, IL-13 was ineffective for light chain replacement in the induced IgE+ cells due to the failure in RAG expression. Our studies using mature blood B cells indicate that IL-4-responsive cells, unlike IL-13-responsive cells, undergo lambda gene rearrangement leading to replacement in parallel with RAG expression and suggest that this replacement may contribute to the regulation of affinity maturation of IgE antibodies.     

Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.     

Development of novel yeast cell surface display system for homo-oligomeric protein by coexpression of native and anchored subunits

Streptavidin derived from Streptomyces avidinii was displayed on the cell surface of the yeast Saccharomyces cerevisiae by cell-surface engineering using two types of plasmid for the expression of a native subunit and an anchored subunit fused with the C-terminus of 318 amino acids of Flo1p containing a glycosylphosphatidylinositol anchor attachment signal. The displayed streptavidin had the binding ability for biotinylated compounds. This was confirmed by fluorescence microscopy after the adsorption of yeast cells displaying streptavidin and biotinylated fluorescein isothiocyanate. On the other hand, streptavidin produced by cells harboring only the plasmid for the expression of the anchored subunit showed a very low binding activity for biotinylated compounds. Cells displaying streptavidin may constitute novel whole-cell affinity adsorbents widely used for immunoassay and biosensing. This coexpression method will ensure that proteins, such as homo- and hetero-oligomeric proteins, are displayed on the cell surface in an active form.     

Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry

We developed an integrated platform consisting of machinery and software modules that can apply vast amounts of data generated by nanoflow LC-MS to differential protein expression analyses. Unlabeled protein samples were completely digested with modified trypsin and separated by low speed (200 nl/min) one-dimensional HPLC. Mass spectra were obtained every 1 s by using the survey mode of a hybrid Q-TOF mass spectrometer and displayed in a two-dimensional plane with m/z values along the x axis, and retention time was displayed along the y axis. The time jitter of nano-LC was adjusted using newly developed software based on a dynamic programming algorithm. The comprehensiveness (60,000-160,000 peaks above the predetermined threshold detectable in 60-microg cell protein samples), reproducibility (average coefficient of variance of 0.35-0.39 and correlation coefficient of over 0.92 between duplicates), and accurate quantification with a wide dynamic range (over 10(3)) of our platform warrant its application to various types of experimental and translational proteomics.     

Orally supplemented Lactobacillus acidophilus strain L‐92 inhibits passive and active cutaneous anaphylaxis as well as 2,4‐dinitroflurobenzene and mite fecal antigen induced atopic dermatitis‐like skin lesions in mice

Oral supplementation of lactic acid bacteria is a potential approach to the prevention and manipulation of allergic diseases such as atopic dermatitis. Our previous report showed that heat‐killed Lactobacillus acidophilus strain L‐92 (L‐92) possessed anti‐allergic properties, although its physiological function in atopic dermatitis has largely remained undefined. To evaluate the anti‐allergic efficacy of L‐92, we used four experimental animal models with the major features of atopic dermatitis and compared the results to those of clinically active drugs. ICR mice were passively sensitized by anti‐dinitrophenyl mouse monoclonal IgE for passive cutaneous anaphylaxis (PCA), and BALB/c mice were actively sensitized by ovalbumin for active cutaneous anaphylaxis (ACA). Allergic reaction was induced by repeated exposure to 2,4‐dinitroflurobenzene (DNFB) and mite (Dermatophagoides farinae) fecal allergen, in BALB/c and NC/Nga mice, respectively. Orally administrated L‐92 significantly inhibited the vascular permeability increase in both PCA and ACA, and the elevation of ovalbumin‐specific IgE titer in ACA. Moreover, repeated applications of DNFB and mite fecal antigen onto the BALB/c and NC/Nga mouse ear, respectively, caused clinical symptoms similar to atopic dermatitis such as ear swelling, scratching behavior and elevation of total serum IgE levels that were also moderately suppressed by L‐92. In addition, L‐92 treated mice exhibited lower levels of mast cells, eosinophil infiltration and Th1/Th2 cytokine expression. Our results, therefore, suggest that oral administration of L‐92 might be useful for alleviating allergic symptoms.     

Divergent Evolution of Norovirus GII/4 by Genome Recombination from May 2006 to February 2009 in Japan

Norovirus GII/4 is a leading cause of acute viral gastroenteritis in humans. We examined here how the GII/4 virus evolves to generate and sustain new epidemics in humans, using 199 near-full-length GII/4 genome sequences and 11 genome segment clones from human stool specimens collected at 19 sites in Japan between May 2006 and February 2009. Phylogenetic studies demonstrated outbreaks of 7 monophyletic GII/4 subtypes, among which a single subtype, termed 2006b, had continually predominated. Phylogenetic-tree, bootscanning-plot, and informative-site analyses revealed that 4 of the 7 GII/4 subtypes were mosaics of recently prevalent GII/4 subtypes and 1 was made up of the GII/4 and GII/12 genotypes. Notably, single putative recombination breakpoints with the highest statistical significance were constantly located around the border of open reading frame 1 (ORF1) and ORF2 (P ≤ 0.000001), suggesting outgrowth of specific recombinant viruses in the outbreaks. The GII/4 subtypes had many unique amino acids at the time of their outbreaks, especially in the N-term, 3A-like, and capsid proteins. Unique amino acids in the capsids were preferentially positioned on the outer surface loops of the protruding P2 domain and more abundant in the dominant subtypes. These findings suggest that intersubtype genome recombination at the ORF1/2 boundary region is a common mechanism that realizes independent and concurrent changes on the virion surface and in viral replication proteins for the persistence of norovirus GII/4 in human populations.Norovirus (NoV) is a nonenveloped RNA virus that belongs to the family Caliciviridae and can cause acute gastroenteritis in humans. The NoV genome is a single-stranded, positive-sense, polyadenylated RNA that encodes three open reading frames, ORF1, ORF2, and ORF3 (68). ORF1 encodes a long polypeptide (∼200 kDa) that is cleaved in the cells by the viral proteinase (3C^pro) into six proteins (4). These proteins function in NoV replication in host cells (19). ORF2 encodes a viral capsid protein, VP1. The capsid gene evolved at a rate of 4.3 × 10⁻³ nucleotide substitutions/site/year (7), which is comparable to the substitution rates of the envelope and capsid genes of human immunodeficiency virus (30). The capsid protein of NoV consists of a shell (S) and two protruding (P) domains: P1 and P2 (47). The S domain is relatively conserved within the same genetic lineages of NoVs (38) and is responsible for the assembly of VP1 (6). The P1 subdomain is also relatively conserved (38) and has a role in enhancing the stability of virus particles (6). The P2 domain is positioned at the most exposed surface of the virus particle (47) and forms binding clefts for putative infection receptors, such as human histo-blood group antigens (HBGA) (8, 13, 14, 60). The P2 domain also contains epitopes for neutralizing antibodies (27, 33) and is consistently highly variable even within the same genetic lineage of NoVs (38). ORF3 encodes a VP2 protein that is suggested to be a minor structural component of virus particles (18) and to be responsible for the expression and stabilization of VP1 (5).Thus far, the NoVs found in nature are classified into five genogroups (GI to GV) and multiple genotypes on the basis of the phylogeny of capsid sequences (71). Among them, genogroup II genotype 4 (GII/4), which was present in humans in the mid-1970s (7), is now the leading cause of NoV-associated acute gastroenteritis in humans (54). The GII/4 is further subclassifiable into phylogenetically distinct subtypes (32, 38, 53). Notably, the emergence and spread of a new GII/4 subtype with multiple amino acid substitutions on the capsid surface are often associated with greater magnitudes of NoV epidemics (53, 54). In 2006 and 2007, a GII/4 subtype, termed 2006b, prevailed globally over preexisting GII/4 subtypes in association with increased numbers of nonbacterial acute gastroenteritis cases in many countries, including Japan (32, 38, 53). The 2006b subtype has multiple unique amino acid substitutions that occur most preferentially in the protruding subdomain of the capsid, the P2 subdomain (32, 38, 53). Together with information on human population immunity against NoV GII/4 subtypes (12, 32), it has been postulated that the accumulation of P2 mutations gives rise to antigenic drift and plays a key role in new epidemics of NoV GII/4 in humans (32, 38, 53).Genetic recombination is common in RNA viruses (67). In NoV, recombination was first suggested by the phylogenetic analysis of an NoV genome segment clone: a discordant branching order was noted with the trees of the 3D^pol and capsid coding regions (21). Subsequently, many studies have reported the phylogenetic discordance using sequences from various epidemic sites in different study periods (1, 10, 11, 16, 17, 22, 25, 40, 41, 44-46, 49, 51, 57, 63, 64, 66). These results suggest that genome recombination frequently occurs among distinct lineages of NoV variants in vivo. However, the studies were done primarily with direct sequencing data of the short genome portion, and information on the cloned genome segment or full-length genome sequences is very limited (21, 25). Therefore, we lack an overview of the structural and temporal dynamics of viral genomes during NoV epidemics, and it remains unclear whether NoV mosaicism plays a role in these events.To clarify these issues, we collected 199 near-full-length genome sequences of GII/4 from NoV outbreaks over three recent years in Japan, divided them into monophyletic subtypes, analyzed the temporal and geographical distribution of the subtypes, collected phylogenetic evidence for the viral genome mosaicism of the subtypes, identified putative recombination breakpoints in the genomes, and isolated mosaic genome segments from the stool specimens. We also performed computer-assisted sequence and structural analyses with the identified subtypes to address the relationship between the numbers of P2 domain mutations at the times of the outbreaks and the magnitudes of the epidemics. The obtained data suggest that intersubtype genome recombination at the ORF1/2 boundary region is common in the new GII/4 outbreaks and promotes the effective acquisition of mutation sets of heterogeneous capsid surface and viral replication proteins.     

Molecular identification of Ehrlichia species and 'Candidatus Neoehrlichia mikurensis' from ticks and wild rodents in Shizuoka and Nagano Prefectures, Japan

A total of 293 ticks and 111 wild rodents that were collected in Shizuoka and Nagano Prefectures, Japan, were examined for infection of Ehrlichia species and 'Candidatus Neoehrlichia mikurensis.' The 16S rDNA or the omp-1 gene of these bacterial DNAs were detected from the spleens of tick-inoculated mice (5 positive/total 29 mice) or from the spleens of wild rodents (25 positive/total 111 rodents) by PCR amplifi-cation. Sequencing of the 16S rDNA revealed Ehrlichia spp. from the 5 tick-inoculated mice and 8 wild rodents, and 'Candidatus N. mikurensis' from 17 wild rodents. The data suggest the presence of additional genetic variants, and potential vectors and/or reservoirs for these bacteria in central Japan.     

A protocol for the construction of microsatellite enriched genomic library

An improved protocol for constructing microsatellite-enriched libraries was developed. The procedure depends on digesting genomic DNA with a restriction enzyme that generates blunt-ends, and on ligating linkers that, when dimerized, create a restriction site for a different blunt-end producing restriction enzyme. Efficient ligation of linkers to the genomic DNA fragments is achieved by including restriction enzymes in the ligation reaction that eliminate unwanted ligation products. After ligation, the reaction mixture is subjected to subtractive hybridization without purification. DNA fragments containing microsatellites are captured by biotin-labeled oligonucleotide repeats and recovered using streptavidin-coated beads. The recovered fragments are amplified by PCR using the linker sequence as primer, and cloned directly into a plasmid vector. The linker has the sequence GTTT on the 5′ end, which promotes efficient adenylation of the 3′ ends of the PCR products. Consequently, the amplified fragments could be cloned into vectors without purification. This procedure enables efficient enrichment and cloning of microsatellite sequences, resulting in a library with a low level of redundancy.