Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair

Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.     

Detection of Spiroplasma from the mite Macrocheles sp. (Acari; Macrochelidae) ectoparasitic to the fly Drosophila hydei (Diptera; Drosophilidae): a possible route of horizontal transmission?

Some members of the genus Spiroplasma are vertically transmitted endosymbionts of insects. Among them, Spiroplasma sp. Dhd, a member of the Spiroplasma poulsonii clade, is highly prevalent among worldwide populations of Drosophila hydei. Here we found that 53 out of 3,763 wild-caught D. hydei (1.4 %) were ectoparasitized by the mite that belong to the genus Macrocheles. Many of the ectoparasitized flies (79 %) had a single mite, but some flies had up to five mites. Among 59 mites subjected to Spiroplasma-specific PCR, 15 individuals were found to be positive. Infection status of Spiroplasma in flies and the associated mites were incongruent. Partial nucleotide sequences of the Spiroplasma P58 gene suggest that some of the mites are infected with a Spiroplasma, which is identical or closely related to Spiroplasma sp. Dhd. This finding provides a potential route of horizontal Spiroplasma transmission between D. hydei individuals in natural populations. In addition, a Spiroplasma strain that does not form a monophyletic group with S. poulsonii was also found from a mite individual.     

Neurite Outgrowth of PC12 Cells by 4′-O-β-d-Glucopyranosyl-3′,4-Dimethoxychalcone from Brassica rapa L. ‘hidabeni’ was Enhanced by Pretreatment with p38MAPK Inhibitor

The cellular effects of eleven compounds including chalcone glycosides isolated from Brassica rapa L. ‘hidabeni’ and their synthetic derivatives were studied in rat pheochromocytoma PC12 cells. Of the compounds tested, 4′-O-β-d-glucopyranosyl-3′,4-dimethoxychalcone (A2) significantly increased the levels of the phosphorylated forms of extracellular signal-regulated kinases 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38MAPK), and stress-activated protein kinases/Jun amino-terminal kinases (JNK/SAPK), but it did not affect Akt. Nerve growth factor (NGF), a well-known neurotrophic factor, increased the levels of phosphorylated ERK1/2, JNK/SAPK, and Akt but not p38MAPK, which may mediate marked neurite outgrowth. Signals evoked by A2 shared common characteristics with those induced by NGF; therefore, we evaluated the neuritogenic activity of A2 and found it induced only weak neurite outgrowth. However, this effect was enhanced by pre-treatment with a p38MAPK inhibitor, suggesting that the phosphorylation of p38MAPK down-regulated neurite outgrowth. From the results of this study, it was found that A2 in combination with a p38MAPK inhibitor can induce NGF-like effects. Hence, a combination of chalcone glycosides containing A2 and a p38MAPK inhibitor increases the likelihood that chalcone glycosides could be put to practical use in the form of drugs or alternative medicines to maintain neural health.     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Accumulation of major histocompatibility complex class II+CD11c− non‐lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte‐dependent

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II⁺CD11c^? non‐T, non‐B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II⁺CD11c^? non‐T, non‐B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag‐2^?/? mice with adoptively transferred normal spleen cells indicated that these cells were non‐lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II⁺CD11c^? non‐T, non‐B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin‐6 in response to infected red blood cells, but had only a limited ability to activate antigen‐specific CD4⁺ T cells. This study revealed a novel interaction between MHC II⁺CD11c^? non‐lymphoid cells and lymphoid cells in the accumulations of these non‐lymphoid cells in the spleen during infection with P. yoelii.

     

Rice Resistance to Planthoppers and Leafhoppers

For over 50 years, host-plant resistance has been regarded as an efficient method to reduce yield losses to rice caused by delphacid and cicadelid hoppers. Already a number of resistant rice varieties have been developed and deployed throughout Asia. To date, over 70 hopper resistance genes have been identified in rice; however, less than 10 genes have been deliberately introduced to commercial rice varieties. Currently, due to recent brown planthopper (Nilaparvata lugens [Stål]) and whitebacked planthopper (Sogatella furcifera [Horvath]) outbreaks occurring at an unprecedented scale, researchers are working toward a second generation of resistant varieties using newly identified gene loci and applying new molecular breeding methods. This paper reviews advances in the identification of resistance genes and QTLs against hoppers in rice. It collates all published information on resistance loci and QTLs against the major rice planthoppers and leafhoppers and presents information on gene locations, genetic markers, differential varieties, and wild rice species as sources of resistance. The review indicates that, whereas progress in the identification of genes has been rapid, considerable tidying of the information is required, especially regarding gene nomenclature and resistance spectra. Furthermore, sound information on gene functioning is almost completely lacking. However, hopper responses to resistance mechanisms are likely to be similar because a single phenotyping technique has been applied by most national and international breeding programs during germplasm screening. The review classifies genes occurring at two chromosome regions associated with several identified resistance loci and highlights these (Chr4S: BphR-R and Chr12L: BphR-R) as general stress response regions. The review calls for a greater diversity of phenotyping methods to enhance the durability of resistant varieties developed using marker-aided selection and emphasizes a need to anticipate the development of virulent hopper populations in response to the field deployment of genes.     

Identification of a New Interaction Mode between the Src Homology 2 Domain of C-terminal Src Kinase (Csk) and Csk-binding Protein/Phosphoprotein Associated with Glycosphingolipid Microdomains

Proteins with Src homology 2 (SH2) domains play major roles in tyrosine kinase signaling. Structures of many SH2 domains have been studied, and the regions involved in their interactions with ligands have been elucidated. However, these analyses have been performed using short peptides consisting of phosphotyrosine followed by a few amino acids, which are described as the canonical recognition sites. Here, we report the solution structure of the SH2 domain of C-terminal Src kinase (Csk) in complex with a longer phosphopeptide from the Csk-binding protein (Cbp). This structure, together with biochemical experiments, revealed the existence of a novel binding region in addition to the canonical phosphotyrosine 314-binding site of Cbp. Mutational analysis of this second region in cells showed that both canonical and novel binding sites are required for tumor suppression through the Cbp-Csk interaction. Furthermore, the data indicate an allosteric connection between Cbp binding and Csk activation that arises from residues in the βB/βC loop of the SH2 domain.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).