Sec22b Is a Negative Regulator of Phagocytosis in Macrophages

The endoplasmic reticulum (ER) is proposed to be a membrane donor for phagosome formation. In support of this, we have previously shown that the expression level of syntaxin 18, an ER-localized SNARE protein, correlates with phagocytosis activity. To obtain further insights into the involvement of the ER in phagocytosis we focused on Sec22b, another ER-localized SNARE protein that is also found on phagosomal membranes. In marked contrast to the effects of syntaxin 18, we report here that phagocytosis was nearly abolished in J774 macrophages stably expressing mVenus-tagged Sec22b, without affecting the cell surface expression of the Fc receptor or other membrane proteins related to phagocytosis. Conversely, the capacity of the parental J774 cells for phagocytosis was increased when endogenous Sec22b expression was suppressed. Domain analyses of Sec22b revealed that the R-SNARE motif, a selective domain for forming a SNARE complex with syntaxin18 and/or D12, was responsible for the inhibition of phagocytosis. These results strongly support the ER-mediated phagocytosis model and indicate that Sec22b is a negative regulator of phagocytosis in macrophages, most likely by regulating the level of free syntaxin 18 and/or D12 at the site of phagocytosis.     

IRF family proteins and type I interferon induction in dendritic cells

Dendritic cells （DC）, although a minor population in hematopoietic cells, produce type I interferons （IFN） and other cytokines and are essential for innate immunity. They are also potent antigen presenters and regulate adaptive immunity. Among DC subtypes plasmacytoid DC （pDC） produce the highest amounts of type I IFN. In addition, pro- and anti-inflammatory cytokines such as IL-12 and IL-10 are induced in DC in response to Toll like receptor （TLR） signaling and upon viral infection. Proteins in the IRF family control many aspects of DC activity. IRF-8 and IRF-4 are essential for DC development. They differentially control the development of four DC subsets. IRF-8^-/- mice are largely devoid of pDC and CD8α^＋ DC, while IRF-4^-/- mice lack CD4^＋ DC. IRF-8^-/-, IRF4^-/-, double knock-out mice have only few CD8α CD4^-DC that lack MHC Ⅱ. IRF proteins also control type Ⅰ IFN induction in DC. IRF-7, activated upon TLR signaling is required for IFN induction not only in pDC, but also in conventional DC （cDC） and non-DC cell types. IRF-3, although contributes to IFN induction in fibroblasts, is dispensable in IFN induction in DC. Our recent evidence reveals that type Ⅰ IFN induction in DC is critically dependent on IRF-8, which acts in the feedback phase of IFN gene induction in DC. Type Ⅰ IFN induction in pDC is mediated by MyD88 dependent signaling pathway, and differs from pathways employed in other cells, which mostly rely on TLR3 and RIG-Ⅰ family proteins. Other pro-inflammatory cytokines are produced in an IRF-5 dependent manner. However, IRF-5 is not required for IFN induction, suggesting the presence of separate mechanisms for induction of type Ⅰ IFN and other pro-inflammatory cytokines. IFN and other cytokines produced by activated DC in turn advance DC maturation and change the phenotype and function of DC. These processes are also likely to be governed by IRF family proteins.     

Characterization of katD, a kinesin-like protein gene specifically expressed in floral tissues of Arabidopsis thaliana

Kinesin and kinesin-like proteins (KLPs) are microtubule-based motor proteins that play important roles in organelle transport. Based on the homology to these proteins, a katD cDNA has now been isolated from a library prepared from flowers of Arabidopsis thaliana ecotype Columbia. Sequence analysis of the katD cDNA revealed an open reading frame of 2691bp [corrected], encoding a protein of 987 amino acids. Comparison of the nucleotide sequences of katD genomic and cDNA clones revealed the presence of 18 introns, 17 of which conform to the GU-AG rule. The central region of the KatD polypeptide exhibits substantial amino acid sequence homology to the motor domain of kinesin heavy chains, although the motor domain of KatD appears to be phylogenetically distant from those of other KLPs in plants. The amino-terminal region of KatD shares marked sequence similarity with the calponin homology domain, whereas the approximately 240-residue carboxyl-terminal region shows no significant homology to other known proteins. The predicted secondary structure of KatD revealed the lack of an alpha-helical coiled coil structure typical of kinesin heavy chains, suggesting that KatD may function as a monomeric motor. A recombinant truncated KatD protein containing the putative motor domain was shown both to bind to mammalian microtubules in a manner dependent on a non-hydrolyzable ATP analog, and to possess microtubule-dependent ATPase activity. Immunoblot and Northern blot analyses showed that both KatD protein and mRNA are expressed specifically in floral tissues. These results suggest that the structurally distinct KatD protein functions as a floral tissue-specific motor protein.     

Mutation of High-Affinity Methionine Permease Contributes to Selenomethionyl Protein Production in Saccharomyces cerevisiae

The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.Structural analyses of proteins have provided meaningful insights into the relationship between protein conformation and biological function. Different approaches, including X-ray crystallographic analysis, nuclear magnetic resonance (NMR) analysis, and electron microscopy analysis, are applicable to determine protein structures. Although the principal method for determining three-dimensional structures of purified proteins is X-ray crystallography, substantial efforts are required to determine protein structures using this method, such as the expression and purification of recombinant proteins, optimization of crystallization conditions, and solving phase problems. Recent advances in structural biology have resulted from the substitution of Met residues for selenomethionine (SeMet) for the phase determination of proteins, using single-wavelength anomalous dispersion (SAD) and multiwavelength anomalous dispersion (MAD) phasing methods (9, 22). In addition, the use of SeMet derivatives for solving phase problems is indispensable for high-throughput determination of protein structure for structural genomic studies that aim to understand biological phenomena in whole-cell systems at the atomic level (10, 26).The use of SeMet-incorporated proteins for X-ray crystallography was originally reported in the 1990s (9). At that time, the majority of tertiary structures were determined by SAD or MAD phasing using SeMet-containing crystals that were routinely prepared in Escherichia coli cells cultured with SeMet. However, it is considered more difficult to incorporate SeMet into proteins expressed in eukaryotic systems than in E. coli cells, and eukaryotic proteins which require posttranslational modification often fail to be expressed in E. coli cells. Therefore, the incorporation of SeMet into eukaryotic proteins is limited to those proteins that can be successfully expressed in E. coli. Although there are a few reports on the production of recombinant proteins labeled with SeMet in mammalian and insect cells, these reports emphasize mainly the practical use of the specified host cells and did not examine the mechanisms by which SeMet toxicity is overcome (1, 8). Yeast is an attractive host for the production of eukaryotic proteins of interest, as cells are capable of rapid growth under simple culture conditions and production of large amounts of recombinant proteins at low cost. In addition, the potential exists to minimize or eliminate SeMet toxicity through the isolation of a SeMet-resistant mutant of yeast.The first report of SeMet-resistant mutants in the budding yeast Saccharomyces cerevisiae suggested that the observed resistance of the eth10 and eth2 mutants was dependent upon the increase of intracellular Met concentrations as a result of enhanced sulfate assimilation during biosynthesis (6). Subsequent genetic and biochemical analyses identified that the eth10 and eth2 mutant cells possess a single, recessive mutation in the unlinked genes SAM1 and SAM2, which encode isomers of S-adenosylmethionine (AdoMet) synthetase (5). A recent study demonstrated that the deletion of both SAM1 and SAM2 confers increased SeMet resistance and allows the production of recombinant proteins with 95% of SeMet occupancy (18). In a different approach, Bockhorn et al. screened a collection of single-gene deletion mutants of S. cerevisiae for resistance to SeMet and demonstrated that a mutant lacking cystathionine γ-lyase activity (cys3Δ) showed the highest resistance to SeMet and has an ability to incorporate SeMet that is equal to or slightly higher than that of sam1Δ sam2Δ cells (2). However, the extracellular supply of expensive AdoMet or Cys, which are involved in a wide range of important biological phenomena, is required to support cellular growth of these mutants and thus limits their use in practical applications (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Metabolic pathways of sulfur compounds in S. cerevisiae. The main sulfur compounds are methionine, S-adenosylmethionine, and cysteine, which are involved in protein synthesis and sulfur metabolism regulation. The S-adenosylmethionine also participates in the methylation of nucleic acids, proteins, and lipids as a methyl group donor and in the biosynthesis of biotin and polyamines. Glutathione plays a pivotal role in redox homeostasis.Previously, we isolated a SeMet-resistant mutant of the methylotrophic yeast Pichia pastoris (SMR-94 strain) that also showed resistance to selenate (13). The mutant cells were able to produce recombinant human lysozyme containing a sufficient amount of SeMet to allow determination of its crystal structure by the SAD phasing method without the need for supplementation of AdoMet and Cys. However, the mutation sites of the P. pastoris SMR-94 strain responsible for SeMet resistance remain unclear because unlike S. cerevisiae, there is a lack of established genetic approaches and techniques for P. pastoris. Here, in an attempt to reveal the molecular basis for SeMet resistance and generate a suitable host for the production of SeMet derivatives of eukaryotic proteins, we isolated SeMet-resistant mutants of S. cerevisiae. Two obtained mutants (SRY5-3 and SRY5-7) were characterized genetically and biochemically. Furthermore, we examined the ability of these mutants to produce SeMet derivatives of epidermal growth factor (EGF) peptide.     

Effects of compressed hydrocarbon gases on the growth activity of Saccharomyces cerevisiae

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane < ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

Induction of tumor-specific acquired immunity against already established tumors by selective stimulation of innate DEC-205+ dendritic cells

Two major distinct subsets of dendritic cells (DCs) are arranged to regulate our immune responses in vivo; 33D1⁺ and DEC-205⁺ DCs. Using anti-33D1-specific monoclonal antibody, 33D1⁺ DCs were successfully depleted from C57BL/6 mice. When 33D1⁺ DC-depleted mice were stimulated with LPS, serum IL-12, but not IL-10 secretion that may be mediated by the remaining DEC-205⁺ DCs was markedly enhanced, which may induce Th1 dominancy upon TLR signaling. The 33D1⁺ DC-depleted mice, implanted with syngeneic Hepa1-6 hepatoma or B16-F10 melanoma cells into the dermis, showed apparent inhibition of already established tumor growth in vivo when they were subcutaneously (sc) injected once or twice with LPS after tumor implantation. Moreover, the development of lung metastasis of B16-F10 melanoma cells injected intravenously was also suppressed when 33D1⁺ DC-deleted mice were stimulated twice with LPS in a similar manner, in which the actual cell number of NK1.1⁺CD3⁻ NK cells in lung tissues was markedly increased. Furthermore, intraperitoneal (ip) administration of a very small amount of melphalan (l-phenylalanine mustard; l-PAM) (0.25 mg/kg) in LPS-stimulated 33D1⁺ DC-deleted mice helped to induce H-2K^b-restricted epitope-specific CD8⁺ cytotoxic T lymphocytes (CTLs) among tumor-infiltrating lymphocytes against already established syngeneic E.G7-OVA lymphoma. These findings indicate the importance and effectiveness of selective targeting of a specific subset of DCs, such as DEC-205⁺ DCs alone or with a very small amount of anticancer drugs to activate both CD8⁺ CTLs and NK effectors without externally added tumor antigen stimulation in vivo and provide a new direction for tumor immunotherapy.     

Detection of Sapovirus in oysters

SaV sequences which are either genetically identical or similar were detected from oysters, feces from gastroenteritis patients, and domestic wastewater samples in geographically close areas. This is the first report of the detection of SaV in oysters which meet the legal requirements for raw consumption in Japan.     

New Vector System for Random,Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome

We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D₃ hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.The actinobacteria or actinomycetes are a group of Gram-positive bacteria with a high G+C content. Many species of actinobacteria are well known as attractive hosts for the production of biologically active compounds since they can easily utilize cheap complex industrial media and possess excellent secretion capacities. This group includes several antibiotic producers (12, 15) and manufacturers of enzymes (5), amino acids (11), and heterologous proteins (3); hence, they are of high industrial, pharmacological, and commercial interest. Among the genera in this group are Corynebacterium, Mycobacterium, Streptomyces, Nocardia, and Rhodococcus.While a few Rhodococcus species are pathogenic, most are benign and have been found to thrive in a broad range of environments, including soil, water, and eukaryotic cells. Rhodococcus is an experimentally advantageous system due to its relatively fast growth rate and simple developmental cycle. Rhodococcus erythropolis can grow at temperatures ranging from 4 to 35°C (41), which enables the investigation of protein production over a wide range of temperatures (24). Strains of Rhodococcus have important applications due to their ability to bioconvert cheap starting material into more valuable compounds (23) and to metabolize harmful environmental pollutants such as toluene, naphthalene, herbicides, and polychlorinated biphenyls (PCBs) (6, 17). This genetic and catabolic diversity of Rhodococcus is the result of not only its large bacterial chromosome but also the presence of large linear plasmids (37). To date, 43 species of Rhodococcus (7; reference periodically updated at http://www.bacterio.net.) have been recognized (http://www.bacterio.cict.fr/qr/rhodococcus.html). However, Rhodococcus is not yet fully characterized.Various genetic tools have been established for the genetic manipulation of Rhodococcus. These include the development of efficient transformation techniques using electroporation (33); construction of expression vectors for protein production (24, 25); and development of shuttle vectors using cryptic, antibiotic-resistant, and temperature-sensitive plasmids (16, 18, 19, 22) derived from Rhodococcus strains as well as the generation of random mutagenesis using transposons.Several transposon mutagenesis systems have been reported for Rhodococcus species (1, 8, 20, 21). These systems can generate a single copy of insertion into the host cell genome. To date, no efficient tool is available for the creation of random multiple gene disruptions in a single step. Previous researches on the creation of multiple integrations in a genome have been based on site-directed mutagenesis or gene disruption in sequential steps, which requires different antibiotic markers for mutant selection (9, 28, 31, 36, 39, 40).We recently established the transposon-based vector system pTNR that can efficiently generate a random mutagenesis library by transposition in various Rhodococcus species (30). Inside the Rhodococcus cell, pTNR is unstable due to the lack of a replication origin for Rhodococcus. The expression of the transposase (IstA) and its helper protein (IstB) in pTNR is regulated under the influence of the constitutive promoter, P_nit (25). Once pTNR is electroporated into Rhodococcus cells, transposition occurs, whereby IstA and IstB are simultaneously expressed and initiate the integration of a single copy of the transposable-marker DNA into the host cell genome while the rest of the plasmid itself is lost. The transposable-marker DNA of pTNR locates between the two inverted repeats, IR1 and IR2, and encodes a replication origin for Escherichia coli and a kanamycin resistance gene, enabling easy identification of the insertion site via plasmid rescue from the genome (30).pTNR was further modified to be used for protein expression through insertion of the protein expression cassette into the host cell genome (29). Currently, four variants of pTNR vectors are available, each with a different antibiotic resistance marker gene. Two or more variants of pTNR can be used for creating double or multiple integrations in sequential steps or even in one step if the variants are cotransposed in combinations. Nonetheless, the incidence of achieving double or multiple integrations from the cotransposed pTNR variants is very low; hence, the statistical chance of inactivating multiple genes within a definite metabolic pathway or bioprocess by such a method is extremely low. To knock out these functionally related genes, an effective method to produce huge numbers of mutations with random insertions at multiple loci is required. To that end, the present study aimed to develop an efficient genome engineering system for random integration of multiple DNA copies into the Rhodococcus genome in a single step.     

Regulation of hepatic branched-chain α-keto acid dehydrogenase kinase in a rat model for type 2 diabetes mellitus at different stages of the disease

Branched-chain α-keto acid dehydrogenase (BCKDH) kinase (BDK) is responsible for the regulation of BCKDH complex, which is the rate-limiting enzyme in the catabolism of branched-chain amino acids (BCAAs). In the present study, we investigated the expression and activity of hepatic BDK in spontaneous type 2 diabetes using hyperinsulinemic Zucker diabetic fatty rats aged 9 weeks and hyperglycemic, but not hyperinsulinemic rats aged 18 weeks. The abundance of hepatic BDK mRNA and total BDK protein did not correlate with changes in serum insulin concentrations. On the other hand, the amount of BDK bound to the complex and its kinase activity were correlated with alterations in serum insulin levels, suggesting that hyperinsulinemia upregulates hepatic BDK. The activity of BDK inversely corresponded with the BCKDH complex activity, which was suppressed in hyperinsulinemic rats. These results suggest that insulin regulates BCAA catabolism in type 2 diabetic rats by modulating the hepatic BDK activity.