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We report a case of immunotactoid glomerulonephritis (ITG) in a patient with cold agglutinins. An 86-year-old Japanese male with a history of hypertension, dyslipidemia, and gastric malignancy presented to our hospital for the evaluation of proteinuria and hematuria. He had an elevated blood pressure of 200/77 mmHg and edema of the lower extremities. Initial blood test results revealed an impaired renal function (creatinine, 1.37 mg/dL) and hypoalbuminemia (albumin, 2.6 g/dL). His estimated daily urinary protein was 5.89 g/g creatinine, meeting the diagnostic criteria for nephrotic syndrome. The selectivity index for proteinuria indicated low selectivity (0.329). We conducted a renal biopsy to identify the cause of nephrotic syndrome. Immunofluorescence microscopy demonstrated positive staining of IgM, C4, and C1q. Electron microscopy exhibited mesangial expansion with inflammatory cells and a lobular structure, suggesting membranoproliferative glomerulonephritis. Subendothelial deposits containing microtubular structures with a diameter of approximately 30–200 nm were found, concurrent with the criteria for the diagnosis of ITG. Screening for lymphoproliferative diseases and immunological abnormalities revealed a positive direct Coombs test result and the presence of cold agglutinins. Paraproteinemia was absent. The similarities between cold agglutinin disease and ITG, including the production of autoantibodies and involvement of complement pathways, raise the possibility that cold agglutinins played a role in the development of ITG; however, we were unable to prove it due to difficulties in detecting cold agglutinins on renal histology. We discuss the possible implications for pathogenesis considering prior reports on nephrotic syndrome being potentially associated with cold agglutinins.

Dienogest (DNG), is an effective and widely used progestin used in the treatment of endometriosis, yet clinically, a subset of cases show resistance to DNG treatment. During a previous investigation on the effect of DNG of cytokines and growth factor production, we incidentally found that endometriotic cyst fluid did not demonstrate inhibitory effects to DNG in a subset of cases. To clarify the mechanisms of this resistance to DNG, we performed proteomics analysis to compare the protein expression between DNG-sensitive and resistant cases. Based upon our results, several proteins were extracted that relate to neutrophil granulocyte activation marker (myeloperoxidase, lactotransferrin), inflammation (azurocidin, neutrophil gelatinase-associated lipocalin, etc.), and others biological processes reflecting the clinical environment of the endometriotic cyst. Among these proteins, azurocidin (AZU) is perhaps most interesting one as azurocidin is a protease that cleaves insulin-like growth factor-1 (IGFBP-1) associated with clear cell carcinoma of the ovary. We propose that the proteins extracted in the present study warrant further investigation in their relationship to carcinogenesis of endometrioma.

Podocyte damage is a major pathological lesion leading to focal segmental glomerulosclerosis (FSGS). Podocytes damaged by cellular stress undergo hypertrophy to compensate for podocytopenia. It is known that cyclin-dependent kinase inhibitors induced by p53 ensure podocytes hypertrophy; however, its precise mechanism remains to be further investigated. In this study, we found that ubiquitin specific protease 40 (USP40) is a novel regulator of p53. Although USP40 knockout mice established in the present study revealed no abnormal kidney phenotype, intermediate filament Nestin was upregulated in the glomeruli, and was bound to and colocalized with USP40. We also found that USP40 deubiquitinated histidine triad nucleotide-binding protein 1 (HINT1), an inducer of p53. Gene knockdown experiments of USP40 in cultured podocytes revealed the reduction of HINT1 and p53 protein expression. Finally, in glomerular podocytes of mouse FSGS, upregulation of HINT1 occurred in advance of the proteinuria, which was followed by upregulation of USP40, p53 and Nestin. In conclusion, USP40 bound to Nestin deubiquitinates HINT1, and in consequence upregulates p53. These results provide additional insight into the pathological mechanism of podocyte hypertrophy in FSGS.

The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process.C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody.Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution.The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

The quality control and export of mRNA by RNA-binding proteins are necessary for the survival of malaria parasites, which have complex life cycles. Nuclear poly(A) binding protein 2 (NAB2), THO complex subunit 4 (THO4), nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2) and serine/arginine-rich splicing factor 1 (SR1) are involved in nuclear mRNA export in malaria parasites. However, their roles in asexual and sexual development, and in cellular localization, are not fully understood. In this study using the rodent malaria parasite, Plasmodium berghei, we found that NAB2 and SR1, but not THO4, NPL3 or GBP2, played essential roles in the asexual development of malaria parasites. By contrast, GBP2 but not NPL3 was involved in male and female gametocyte production. THO4 was involved in female gametocyte production, but had a lower impact than GBP2. In this study, we focused on GBP2 and NAB2, which play important roles in the sexual and asexual development of malaria parasites, respectively, and examined their cellular localization. GBP2 localized to both the nucleus and cytoplasm of malaria parasites. Using immunoprecipitation coupled to mass spectrometry (IP-MS), GBP2 interacted with the proteins ALBA4, DOZI, and CITH, which play roles in translational repression. IP-MS also revealed that phosphorylated adapter RNA export protein (PHAX) domain-containing protein, an adaptor protein for exportin-1, also interacted with GBP2, implying that mRNA export occurs via the PHAX domain-containing protein pathway in malaria parasites. Live-cell fluorescence imaging revealed that NAB2 localized at the nuclear periphery. Moreover, IP-MS indicated that NAB2 interacted with transportin. RNA immunoprecipitation coupled to RNA sequencing revealed that NAB2 bound directly to 143 mRNAs, including those encoding 40S and 60S ribosomal proteins. Our findings imply that malaria parasites use an evolutionarily ancient mechanism conserved throughout eukaryotic evolution.

In recent years, brown adipose tissue (BAT), which has a high heat-producing capacity, has been confirmed to exist even in adults, and it has become a focal point for the prevention and the improvement of obesity and lifestyle-related diseases. However, the influences of obesity and physical activity (PA) on the fluid factors secreted from BAT (brown adipokines) are not well understood. In this study, therefore, we focused on brown adipokines and investigated the effects of obesity and PA. The abnormal expressions of gene fluid factors such as galectin-3 (Lgals3) and Lgals3 binding protein (Lgals3bp), whose proteins are secreted from HB2 brown adipocytes, were observed in the interscapular BAT of obese mice fed a high-fat diet for 4 months. PA attenuated the abnormalities in the expressions of these genes. Furthermore, although the gene expressions of factors related to brown adipocyte differentiation such as peroxisome proliferator-activated receptor gamma coactivator 1-α were also down-regulated in the BAT of the obese mice, PA suppressed the down-regulation of these factors. On the other hand, lipogenesis was increased more in HB2 cells overexpressing Lgals3 compared with that in control cells, and the overexpression of Lgals3bp decreased the mitochondrial mass. These results indicate that PA attenuates the obesity-induced dysregulated expression of brown adipokines and suggests that Lgals3 and Lgals3bp are involved in brown adipocyte differentiation.

Urate is the final oxidation product of purine metabolism in humans. We have recently reported that the paracellular route is the major urate transport pathway across the blood-placental barrier. In this study, the mechanism of urate paracellular transport was investigated in several epithelial cell lines including Madin-Darby canine kidney (MDCK) type I, Lilly Laboratories cell-porcine kidney 1 (LLC-PK1) and Caco-2 cells. Very little urate passed through MDCK and LLC-PK1 cell layers. In contrast, one of the Caco-2 cell lines was found to be urate-permeable. This urate paracellular movement across Caco-2 cell layer was not inhibited by the urate transporter inhibitor benzbromarone but was partially inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride transport. Detection and quantification of claudin proteins that are important for paracellular transport of ions were performed by LC/MS. Claudins 1, 3, 4, 6, 7 and 12 were detected in urate-permeable cell lines, BeWo cells and Caco-2 cells. We compared claudin expression patterns in urate-permeable and urate-non-permeable Caco-2 cells by LC/MS and found that claudin 12 had a higher expression level in urate-permeable Caco-2 cells. Overexpression of these claudins in MDCK cells did not increase urate paracellular transport. Although there were differences in claudin expression pattern between urate-permeable and non-permeable cells, increased expression of single claudin alone did not explain paracellular permeability of urate.

Mastitis is an inflammation of the mammary gland in the breast and is typically due to bacterial infection. In malaria-endemic areas, mastitis with accompanying fever can be challenging to differentiate from malaria. At the same time, it is unclear whether malaria infection is directly involved in the development of mastitis. In the present study, whether mastitis develops during infection with malaria parasites was investigated using a rodent malaria model with Plasmodium berghei (P. berghei; Pb) ANKA. The course of parasitemia in postpartum mice infected with Pb ANKA was similar to the course in infected virgin mice. However, infected postpartum mice died earlier than did infected virgin mice. In addition, the weight of pups from mice infected with Pb ANKA was significantly reduced compared with pups from uninfected mice. The macroscopic and histological analyses showed apparent changes, such as destruction of the alveolus wall and extensive presence of leukocytes, in mammary gland tissue in mice infected during the postpartum period. The findings suggest that women during the postpartum period are more vulnerable to complications when infected with malaria parasites, particularly women who do not acquire protective immunity against malaria parasites. Based on the proteomic analysis, IFN-γ signaling pathway-related proteins in mammary gland tissue of the infected postpartum mice were increased. Our results indicate that inflammation induced by IFN-γ, a proinflammatory cytokine, may contribute to negative histological changes in mammary gland tissue of postpartum mice infected with Pb ANKA. In IFN-γ receptor 1-deficient (IFNGR1-KO) mice, the histological changes in mammary gland tissue of the infected postpartum wild-type mice were improved to almost normal mammary gland structure. Furthermore, weight loss in pups delivered by infected IFNGR1-KO postpartum mice was not observed. Taken together, these findings indicate that inflammation induced by IFN-γ is associated with development of mastitis in postpartum mice infected with Pb ANKA. The present study results may increase our understanding of how disease aggravation occurs during postpartum malaria.

Dysfunctional mitochondria are observed in β-cells of diabetic patients, which are eventually removed by autophagy. Vesicle-associated membrane protein (VAMP)7, a vesicular SNARE protein, regulates autophagosome formation to maintain mitochondrial homeostasis and control insulin secretion in pancreatic β-cells. However, its molecular mechanism is largely unknown. In this study, we investigated the molecular mechanism of VAMP7-dependent autophagosome formation using VAMP7-deficient β-cells and β-cell–derived Min6 cells. VAMP7 localized in autophagy-related (Atg)9a–resident vesicles of recycling endosomes (REs), which contributed to autophagosome formation, and it interacted with Hrb, Syntaxin16, and SNAP-47. Hrb recruited VAMP7 and Atg9a from the plasma membrane to REs. Syntaxin16 and SNAP-47 mediated autophagosome formation at a step later than the proper localization of VAMP7 to Atg9a-resident vesicles. Knockdown of Hrb, Syntaxin16, and SNAP-47 resulted in defective autophagosome formation, accumulation of dysfunctional mitochondria, and impairment of glucose-stimulated insulin secretion. Our data indicate that VAMP7 and Atg9a are initially recruited to REs to organize VAMP7 and Atg9a-resident vesicles in an Hrb-dependent manner. Additionally, VAMP7 forms a SNARE complex with Syntaxin16 and SNAP-47, which may cause fusions of Atg9a-resident vesicles during autophagosome formation. Thus, VAMP7 participates in autophagosome formation by supporting Atg9a functions that contribute to maintenance of mitochondrial quality.

The affinity for K+ of silkworm nerve Na+/K+-ATPase is markedly lower than that of mammalian Na+/K+-ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K+ affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na+/K+-ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na+/K+-ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na+/K+-ATPase. Na+/K+-ATPase expressed in the cultured cells showed a low affinity for K+ and a high affinity for Na+, characteristic of the silkworm nerve Na+/K+-ATPase. These results suggest that the β subunit is responsible for the affinity for K+ of Na+/K+-ATPase.

The function of actin is regulated by various posttranslational modifications. We have previously shown that in the kidneys of nonobese type 2 diabetes model Goto-Kakizaki rats, increased O-GlcNAcylation of β-actin protein is observed. It has also been reported that both O-GlcNAcylation and phosphorylation occur on Ser199of β-actin. However, their roles are not known. To elucidate their roles in diabetic nephropathy, we examined the rat kidney for changes in O-GlcNAcylation of Ser199(gS199)-actin and in the phosphorylation of Ser199(pS199)-actin. Both gS199- and pS199-actin molecules had an apparent molecular weight of 40 kDa and were localized as nonfilamentous actin in both the cytoplasm and nucleus. Compared with the normal kidney, the immunostaining intensity of gS199-actin increased in podocytes of the glomeruli and in proximal tubules of the diabetic kidney, whereas that of pS199-actin did not change in podocytes but decreased in proximal tubules. We confirmed that the same results could be observed in the glomeruli of the human diabetic kidney. In podocytes of glomeruli cultured in the presence of the O-GlcNAcase inhibitor Thiamet G, increased O-GlcNAcylation was accompanied by a concomitant decrease in the amount of filamentous actin and in morphological changes. Our present results demonstrate that dysregulation of O-GlcNAcylation and phosphorylation of Ser199occurred in diabetes, which may contribute partially to the causes of the morphological changes in the glomeruli and tubules. gS199- and pS199-actin will thus be useful for the pathological evaluation of diabetic nephropathy.

Glucocorticoids (GCs) potently induce T-cell apoptosis in a GC receptor (GR)-dependent manner and are used to control lymphocyte function in clinical practice. However, its downstream pathways remain controversial. Here, we showed that GC-induced transcript 1 (GLCCI1) is a novel downstream molecule of the GC-GR cascade that acts as an antiapoptotic mediator in thymic T cells. GLCCI1 was highly phosphorylated and colocalized with microtubules in GLCCI1-transfected human embryonic kidney QBI293A cells. GR-dependent up-regulation of GLCCI1 was associated with GC-induced proapoptotic events in a cultured thymocyte cell line. However, GLCCI1 knockdown in a thymocyte cell line led to apoptosis. Consistently, transgenic mice overexpressing human GLCCI1 displayed enlarged thymi that consisted of larger numbers of thymocytes. Further molecular characterization showed that GLCCI1 bound to both dynein light chain LC8-type 1 (LC8) and its functional kinase, p21-protein activated kinase 1 (PAK1), thereby inhibiting the kinase activity of PAK1 toward LC8 phosphorylation, a crucial event in apoptotic signaling. GLCCI1 induction facilitated LC8 dimer formation and reduced Bim expression. Thus, GLCCI1 is a candidate factor involved in apoptosis regulation of thymic T cells.-Kiuchi, Z., Nishibori, Y., Kutsuna, S., Kotani, M., Hada, I., Kimura, T., Fukutomi, T., Fukuhara, D., Ito-Nitta, N., Kudo, A., Takata, T., Ishigaki, Y., Tomosugi, N., Tanaka, H., Matsushima, S., Ogasawara, S., Hirayama, Y., Takematsu, H., Yan, K. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.

Urate is the final oxidation product of purine metabolism in humans. We have recently reported that the paracellular route is the major urate transport pathway across the blood-placental barrier. In this study, the mechanism of urate paracellular transport was investigated in several epithelial cell lines including Madin-Darby canine kidney (MDCK) type I, Lilly Laboratories cell-porcine kidney 1 (LLC-PK1) and Caco-2 cells. Very little urate passed through MDCK and LLC-PK1 cell layers. In contrast, one of the Caco-2 cell lines was found to be urate-permeable. This urate paracellular movement across Caco-2 cell layer was not inhibited by the urate transporter inhibitor benzbromarone but was partially inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS), which inhibits chloride transport. Detection and quantification of claudin proteins that are important for paracellular transport of ions were performed by LC/MS. Claudins 1, 3, 4, 6, 7 and 12 were detected in urate-permeable cell lines, BeWo cells and Caco-2 cells. We compared claudin expression patterns in urate-permeable and urate-non-permeable Caco-2 cells by LC/MS and found that claudin 12 had a higher expression level in urate-permeable Caco-2 cells. Overexpression of these claudins in MDCK cells did not increase urate paracellular transport. Although there were differences in claudin expression pattern between urate-permeable and non-permeable cells, increased expression of single claudin alone did not explain paracellular permeability of urate.

Ezrin is an actin binding protein which cross-links membrane proteins with cytoskeleton directly or indirectly via PDZ domain-containing scaffold proteins. It is mainly expressed at the brush border membrane (BBM) of gastrointestinal tracts, and is involved in the construction of microvilli structure and the functional expression of membrane protein complexes at the cell surface. To precisely study the roles of ezrin on the expression of membrane proteins at the cell surface, here we prepared the BBM fractions of ileums from the wild-type and ezrin-knockdown (Vil2(kd/kd)) mice, analyzed them by mass spectrometry, and compared their proteomic patterns. Totally 313 proteins were identified in the BBM fractions. Several transport proteins, cytoskeleton-associated proteins, and trafficking proteins were up- or down-regulated in the BBM fraction of the ileum in the Vil2(kd/kd) mice. Among them, the expressions of i) Na(+)/H(+) exchanger regulatory factor 1 (a PDZ domain-containing scaffold protein), ii) sodium monocarboxylate transporter 1, which contains a PDZ domain-binding motif at their carboxy-terminal, and iii) chloride intracellular channel protein 5 were down-regulated at the BBM fraction of the ileum in the Vil2(kd/kd) mice, suggesting that ezrin is involved in their expression in the BBM.

G-strand binding protein 2 (GBP2) is a Ser/Arg-rich (SR) protein involved in mRNA surveillance and nuclear mRNA quality control in yeast. However, the roles of GBP2 in virulence and sexual development in Plasmodium parasites are unclear, although GBP2 is involved in the asexual development of Plasmodium berghei, the rodent malaria parasite. In this study, we investigated the role of GBP2 in virulence and sexual development of P. berghei using gbp2-deleted P. berghei (Δgbp2 parasites). Then, to identify factors affected by gbp2 deletion, we performed a comparative proteomic analysis of the Δgbp2 parasites. We found that GBP2 was not associated with the development of experimental cerebral malaria during infection with P. berghei, but asexual development of the parasite was delayed with deletion of gbp2. However, the development of P. berghei gametocytes was significantly reduced with deletion of gbp2. Comparative proteomic analysis revealed that the levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in Δgbp2 parasites were significantly higher than those in wild-type (WT) parasites, suggesting that biosynthesis of purine nucleotides may be involved in function of GBP2. Therefore, we investigated the effect of purine starvation on the sexual development and proteome. In nt1-deleted P. berghei (Δnt1 parasites), the production of male and female gametocytes was significantly reduced compared to that in WT parasites. Moreover, we found that protein levels of GBP2 in Δnt1 parasites were markedly lower than in WT parasites. These findings suggest that GBP2 is primarily involved in the sexual development of malaria parasites, and its function may be suppressed by purine starvation.

Sodium-coupled monocarboxylate transporters SMCT1 (SLC5A8) and SMCT2 (SLC5A12) mediate the high- and low-affinity transport of lactate in the kidney, but their regulatory mechanism is still unknown. Since these two transporters have the PDZ-motif at their C-terminus, the function of SMCTs may be modulated by a protein–protein interaction. To investigate the binding partner(s) of SMCTs in the kidney, we performed yeast two-hybrid (Y2H) screenings of a human kidney cDNA library with the C-terminus of SMCT1 (SMCT1-CT) and SMCT2 (SMCT2-CT) as bait. PDZK1 was identified as a partner for SMCTs. PDZK1 coexpression in SMCT1-expressing HEK293 cells enhanced their nicotinate transport activity. PDZK1, SMCT1, and URAT1 in vitro assembled into a single tri-molecular complex and their colocalization was confirmed in the renal proximal tubule in vivo by immunohistochemistry. These results indicate that the SMCT1-PDZK1 interaction thus plays an important role in both lactate handling as well as urate reabsorption in the human kidney.

Plasmodium berghei (Pb) XAT, a rodent malaria parasite, is an irradiation-attenuated variant derived from the lethal strain Pb NK65. Differences in genome sequence, protein structure and function between Pb XAT and Pb NK65 are currently unknown. In this study, to investigate genetic alterations in Pb XAT, we performed comparative genomics and proteomics analyses of nonlethal and lethal strains of Pb. We found mutations, such as a deletion mutation in rhoptry-associated protein (rap) 1, and deletion of rap2/3 and skeleton-binding protein 1 (sbp1), in Pb XAT. RAP1 is required for targeting of RAP2 to the rhoptries. However, the contribution of RAP2/3 to the lethality of Plasmodium is unclear. Therefore, we generated RAP1- and RAP2/3-deficient mutants of Pb ANKA, a reference strain of P. berghei. Furthermore, we investigated the effect of RAP1 and RAP2/3 deficiency on the outcome of infection. The parasitemia in mice infected with RAP1-deficient parasites was increased compared to that in control parasite-infected mice during the early phase of infection. However, mice infected with RAP1-deficient parasites survived longer than did control parasite-infected mice. Moreover, mice infected with RAP2/3-deficient parasites showed low levels of parasitemia and ultimately recovered from the infection The aim of this study was to investigate the effect of RAP2/3 expression on the outcome of infection with Pb XAT using a RAP2/3-expressing Pb XAT. Results showed that complementation of RAP2/3 expression in Pb XAT partially restored virulence. Our findings suggest that RAP1 and RAP2/3 contribute to virulence and a decrease in their expression explains the loss of virulence of the Pb XAT strain.

The affinity for K

Helicobacter pylori is one of the most common causes of bacterial infection in humans, and it forms biofilms on human gastric mucosal epithelium as well as on in vitro abiotic surfaces. Bacterial biofilm is critical not only for environmental survival but also for successful infection. We previously demonstrated that strain TK1402, which was isolated from a Japanese patient with duodenal and gastric ulcers, has high biofilm-forming ability in vitro relative to other strains. In addition, we showed that outer membrane vesicles (OMV) play an important role in biofilm formation. The aim of this study was to analyze which protein(s) in the OMV contributes to biofilm formation in TK1402. We obtained a spontaneous mutant strain derived from TK1402 lacking biofilm-forming ability. The protein profiles of the OMV were compared between this mutant strain and the wild type, and it was found that AlpB, an outer membrane protein in the OMV of the mutant strain, was markedly decreased compared to that of the wild type. Restoration of TK1402 alpB to the mutant strain fully recovered the ability to form biofilm. However, restoration with alpB from other strains demonstrated incomplete recovery of biofilm-forming ability. We therefore inferred that the variable region of AlpB (amino acid positions 121 to 146) was involved in TK1402 biofilm formation. In addition, diversification of the AlpB sequence was shown to affect the ability to adhere to AGS cells. These results demonstrate a new insight into the molecular mechanisms of host colonization by H. pylori . IMPORTANCE Bacterial biofilm is critical not only for environmental survival but also for successful infection. The mechanism of Helicobacter pylori adherence to host cells mediated by cell surface adhesins has been the focus of many studies, but little is known regarding factors involved in H. pylori biofilm formation. Our study demonstrated that AlpB plays an important role in biofilm formation and that this property depends upon the specific sequence of alpB . This in turn was shown to be important in the ability to adhere to gastric cells. We anticipate that these results will provide new insight into the molecular mechanisms of H. pylori colonization.

Bordetella pertussis, the causative agent of whooping cough, is highly adapted to cause human infection. The production of virulence factors, such as adhesins and toxins, is just part of an array of mechanisms by which B. pertussis causes infection. The stringent response is a global bacterial response to nutritional limitation that is mediated by the accumulation of cellular ppGpp and pppGpp [termed together as (p)ppGpp]. Here, we demonstrate that production of (p)ppGpp was controlled by RelA and SpoT proteins in B. pertussis, and that mutation-induced loss of both proteins together caused deficiencies in (p)ppGpp production. The (p)ppGpp-deficient mutants also exhibited defects in growth regulation, decreases in viability under nutritionally limited conditions, increases in susceptibility to oxidative stress and defects in biofilm formation. Analysis of the secreted proteins and the respective transcripts showed that lack of (p)ppGpp led to decreased expression of fim3 and bsp22, which encode a fimbrial subunit and the self-polymerizing type III secretion system tip protein, respectively. Moreover, electron microscopic analysis also indicated that (p)ppGpp regulated the formation of filamentous structures. Most virulence genes - including fim3 and bsp22 - were expressed in the Bvg(+) phase during which the BvgAS two-component system was activated. Although fim3 and bsp22 were downregulated in a (p)ppGpp-deficient mutant, normal expression of fhaB, cyaA and ptxA persisted. Lack of coherence between virulence gene expression and (p)ppGpp production indicated that (p)ppGpp did not modulate the Bvg phase. Taken together, our data indicate that (p)ppGpp may govern an as-yet-unrecognized system that influences B. pertussis pathogenicity.

To identify specific inhibitors of the human secretary pathway Ca(2+)-ATPase 2 (hSPCA2), a recombinant hSPCA2 was expressed in Saccharomyces cerevisiae, and purified by Co(2+)-chelating chromatography. The isolated hSPCA2 catalyzed ATP hydrolysis in the presence of Ca(2+) ions. The Ca(2+) dissociation constant for ATPase activation was 25 nM. hSPCA2 activity was inhibited by thapsigargin, 2,2'-methylenebis(6-tert-butyl-p-cresol), and 4-octylphenol in the low-micromolar concentration range. Unexpectedly, the organic solvent wash from standard laboratory polypropylene microtubes showed strong inhibitory potency toward hSPCA2 activity. The extract was found to comprise mainly primary fatty acid amides (PFAAs) by NMR analysis. Individual PFAAs, especially oleamide and linoleamide, almost completely inhibited hSPCA2 activity with IC50 values of 7.5 μM and 3.8 μM, respectively.

Ezrin is an actin binding protein which cross-links membrane proteins with cytoskeleton directly or indirectly via PDZ domain-containing scaffold proteins. It is mainly expressed at the brush border membrane (BBM) of gastrointestinal tracts, and is involved in the construction of microvilli structure and the functional expression of membrane protein complexes at the cell surface. To precisely study the roles of ezrin on the expression of membrane proteins at the cell surface, here we prepared the BBM fractions of ileums from the wild-type and ezrin-knockdown (Vil2(kd/kd)) mice, analyzed them by mass spectrometry, and compared their proteomic patterns. Totally 313 proteins were identified in the BBM fractions. Several transport proteins, cytoskeleton-associated proteins, and trafficking proteins were up- or down-regulated in the BBM fraction of the ileum in the Vil2(kd/kd) mice. Among them, the expressions of i) Na(+)/H(+) exchanger regulatory factor 1 (a PDZ domain-containing scaffold protein), ii) sodium monocarboxylate transporter 1, which contains a PDZ domain-binding motif at their carboxy-terminal, and iii) chloride intracellular channel protein 5 were down-regulated at the BBM fraction of the ileum in the Vil2(kd/kd) mice, suggesting that ezrin is involved in their expression in the BBM.

Urate (uric acid) is the final product of purine metabolism, and its antioxidant capacity has drawn attention recently for its protective role against oxidative stress. However, hyperuricemia has a known association with onset of illnesses such as gout and cardiovascular diseases. Renal urate transport mechanisms are known to be major determinants of serum urate levels, but the molecular mechanisms involved have not yet been fully elucidated. Molecular identification of a kidney-specific urate transporter SLC22A12 (URAT1) by our research group in 2002 marked the start of a subsequent compilation of information on several different molecules contributing to urate transport by the kidneys. In addition, recent genome-wide association (GWA) studies have contributed to the detection of novel candidate genes related to uric acid metabolism such as SLC2A9 (GLUT9/URATv1), ABCG2 (BCRP), SLC17A1 (NPT1), SLC17A3 (NPT4) and PDZK1. Furthermore, use of urate transporter gene-modified mice for Slc22a12 and Slc2a9 may provide clues for understanding the physiological role of each transporter. Results of recent studies on urate transport with emphasis on the relation to serum urate disorders are described in this report.

Serum proteins/peptides reflect physiological or pathological states in humans and are an attractive target for the discovery of disease biomarkers. However, the existence of high-abundance proteins and the large dynamic range of serum proteins/peptides make any quantitative analysis of low-abundance proteins/peptides challenging. Furthermore, analyses of peptides, including the cleaved fragments of proteins, are difficult because of carrier protein binding. Here, we developed a differential solubilization (DS) method to extract low-molecular-weight proteins/peptides in serum with good reproducibility and yield as compared to typical peptide-extraction methods such as organic solvent precipitation and ultrafiltration. Using the DS method combined with reverse-phase HPLC fractionation followed by MALDI-TOF-MS, we performed high-quality comparative analyses of more than 1500 peptides from 1 microL of serum samples, including low-abundance peptides in the subnanomolar range and containing many peptides bound to carrier proteins such as albumin. We applied this method and successfully discovered four new biomarker candidates of colon cancer, none of which have previously been observed in serum and one of which is a fragment of the protein zyxin that possibly originated from tumor cells. Our results indicate that serum peptide analyses based on the DS method should greatly contribute to the discovery of novel low-abundance biomarkers.

We analyzed the functional properties of five single nucleotide polymorphisms (SNPs) in organic cation transporter OCT3 gene (SLC22A3) resulting in the amino acid changes with a transient expression system. Three SNPs (A116S, T400I, and A439V) exhibited reduced uptake of both [ 3H]histamine and [3H]MPP+, although their protein expressions were detected in the plasma membrane of transfected cells. This study suggests that the OCT3 variants will contribute to interindividual variations leading to the differences in cationic drug disposition as well as certain disease processes such as hypertension, allergic diseases, and neuropsychiatric diseases by the clearance of endogenous organic cations such as biogenic amines. Keywords:: organic cation transporter, single nucleotide polymorphism, histamine

In this study, we have elucidated that propionate, one of the short chain fatty acids (SCFAs), is the transport substrate for murine organic anion transporter 2 (mOat2), which is expressed in the kidneys and the liver. When expressed in Xenopus oocytes, mOat2-mediated [3H]PGE2transport was inhibited by three- to five-carbon SCFAs (C3 to C5). Among the SCFAs tested, propionate (3-carbon SCFA) was transported by mOat2 in a time-dependent manner. Since propionate is a potent glucogenic compound, Oat2 may be involved in the regulation of cellular metabolism through the transport of these metabolites in the kidneys and the liver. Keywords:: propionate, short chain fatty acid, organic anion transporter

Bordetella pertussis is a bacterium that is considered to be highly adapted to humans, and it has not been isolated from the environment. As this bacterium does not utilize sugars, the abundant supply of glutamate in Stainer Scholte (SS) medium enables B. pertussis to grow efficiently in liquid culture in vitro , and as such, SS medium is a popular choice for laboratory experiments. However, the concentration of glutamate in the in vivo niche of B. pertussis is quite low. We investigated the bacterial response to low concentrations of glutamate to elucidate bacterial physiology via the expression of the type 3 secretion system (T3SS), and we discuss its relationship to the Bvg mode in which the two-component regulator of pathogenesis (BvgAS) is activated. Glutamate limitation induced the expression of both the T3SS apparatus and effector genes at the transcriptional level. (p)ppGpp, a modulator of the stringent response, was necessary for maximum expression of the T3SS genes. These observations indicate that the expression of the T3SS is managed by nutrient starvation. In addition, the autoaggregation ability was high in the absence of glutamate and no autoaggregation was observed in glutamate-replete medium. Taken together, glutamate-limited conditions in Bvg + mode elicit the high expression of T3SS genes in B. pertussis and promotes its sessile form. IMPORTANCE Bordetella pertussis is a highly contagious pathogen that causes respiratory infectious disease. In spite of the increasing use of vaccination, the number of patients with pertussis is increasing. The proteins produced in vivo often are different from the protein profile under laboratory conditions; therefore, the development of conditions reflecting the host environment is important to understand native bacterial behavior. In the present study, we examined the effect of glutamate limitation, as its concentration in vivo is much lower than that in the culture medium currently used for B. pertussis experiments. As predicted, the T3SS was induced by glutamate limitation. These results are suggestive of the importance of regulation by nutrient conditions and in the pathogenicity of B. pertussis .

We developed a simple method for purifying peptides from crude extracts of animal tissues. Peptides free of high-molecular-mass (HMM) proteins were prepared by differential solubilization of proteins and peptides followed by tricine-SDS-PAGE. Proteins and peptides in a tissue extract were co-precipitated by slowly dropping the extracts into acetone at −40°C. The precipitates were dissolved in 2 mM DTT and separated by centrifugation. The peptide-enriched supernatant was resolved by tricine-SDS-PAGE and portions of the lanes below 10 kDa were excised. Peptides were extracted from the gel pieces in 40% acetonitrile and fractionated by two-step reversed-phase HPLC. We evaluated the method by extracting peptides from the livers and kidneys of rats and diabetic model mice. The HPLC elution profiles of peptide mixtures were reproducible, peptide recovery from tricine-SDS-PAGE gels was high and we identified one HPLC-fractionated peptide by amino-acid sequencing. The present method should serve as a good foundation for future peptidomics.

Recently, there has been increased research on millimeter-wave regions due to mobile communication. In addition to the characteristics of high-frequency carrier, an even larger-capacity communication becomes possible by making the communication circuits and the antennas broadband. This paper describes a high-frequency broadband printed antenna necessary for a monolithic small and lightweight transceiver capable of a large-capacity communication. As a broadband antenna, a two-arm spiral antenna was designed and tested at 90 GHz. At frequencies from 89 GHz through 99 GHz, the antenna pattern and the return loss were measured. In addition, Hallen's integral equation for a linear antenna with an arbitrary shape was analyzed numerically by means of the moment method so that the difference between the theory and the measurement was verified. Agreement of both is excellent. Hence, the fundamental data have been collected for realization of broadband active integrated antennas which combine the monolithic circuit and the planar antenna.

Urate is the final metabolite of purine in humans. Renal urate handling is clinically important because under-reabsorption or underexcretion causes hypouricemia or hyperuricemia, respectively. We have identified a urate-anion exchanger, URAT1, localized at the apical side and a voltage-driven urate efflux transporter, URATv1, expressed at the basolateral side of the renal proximal tubules. URAT1 and URATv1 are vital to renal urate reabsorption because the experimental data have illustrated that functional loss of these transporter proteins affords hypouricemia. While mutations affording enhanced function via these transporter proteins on urate handling is unknown, we have constructed kidney-specific transgenic (Tg) mice for URAT1 or URATv1 to investigate this problem. In our study, each transgene was under the control of the mouse URAT1 promoter so that transgene expression was directed to the kidney. Plasma urate concentrations in URAT1 and URATv1 Tg mice were not significantly different from that in wild-type (WT) mice. Urate excretion in URAT1 Tg mice was similar to that in WT mice, while URATv1 Tg mice excreted more urate compared with WT. Our results suggest that hyperfunctioning URATv1 in the kidney can lead to increased urate reabsorption and may contribute to the development of hyperuricemia.

Orotate is a precursor of pyrimidine synthesis. The kidney uses exogenous orotate for the synthesis of uridine diphosphosugars, which are used in the glycosylation of collagen in glomerular and tubular basement membranes. Orotate uptake occurs in the liver and kidney, but its molecular mechanism is largely unknown. Since orotate has been shown to be a substrate of the renal urate/anion exchanger in brush border membrane vesicle studies, we investigated whether human URAT1 (hURAT1) mediates the transport of orotate using HEK293 cells expressing hURAT1 (HEK-hURAT1). hURAT1 mediated a time- and dose-dependent uptake of orotate (K m 5.2 μM). hURAT1-mediated [3H]orotate transport was inhibited strongly by non-labeled (cold) orotate and the uricouric agent benzbromarone, and moderately inhibited by urate, nicotinate, and another uricouric agent, probenecid. This is the first report demonstrating that hURAT1 mediates the transport of orotate. hURAT1 may function as one of the entrance pathways in renal proximal tubular cells.

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Salicylic acid derivatives are the most prescribed analgesic-antipyretic and anti-inflammatory agents. It is well known that salicylate has a paradoxical effect on renal urate excretion. At low doses (5 - 10 mg/dL serum), renal urate excretion is decreased, whereas at high doses (15 mg/dL serum), renal urate excretion is increased. Since the molecular identification of the renal apical urate/anion exchanger URAT1, it has been suggested that this protein is responsible for the paradoxical effect because of cis-inhibition of salicylate (1 mM) on urate uptake by URAT1-expressing oocytes. The purpose of this study was to examine whether or not URAT1 is responsible for the paradoxical effect of salicylate. In URAT1-stably expressing HEK293 (HEK293-URAT1) cells, salicylate inhibited [14C] urate uptake dose-dependently (IC50, 23.9 microM). URAT1 mediated the time-dependent uptake of [3H] salicylate in these cells. [3H] Salicylate uptake via URAT1 was inhibited by non-labelled urate and salicylate, and the uricosuric agent, benzbromarone. In the URAT1-expressing oocytes, we observed the time- and concentration-dependent transport of salicylate (Km : 25.3 microM). Moreover, non-labelled salicylate injected into the URATI-expressing oocytes stimulated [14C] urate uptake. These results suggest that the "paradoxical effect" of salicylate can be explained by two modes of salicylate interaction with URAT1 : (1) acting as an exchange substrate to facilitate urate reabsorption, and (2) acting as an inhibitor for urate reabsorption.

Mycotoxins are secondary metabolites of moulds that which exert adverse effects in humans and animals. It is known that direct cellular toxicity is often associated with increased cellular accumulation of toxic compounds, and membrane transport may be the first fundamental stage in the development of the cytotoxicity. To elucidate the entry pathway for mycotoxins into cells, we have investigated the interactions of human and rat organic anion transporters (hOATs/rOats) and human organic cation transporters (hOCTs) with mycotoxins using cells stably expressing hOATs/rOats/hOCTs. The mycotoxins tested were aflatoxin B1, α-zearalenol, citrinin, citrioveridine, cyclopiazonic acid, fumonisin B1, gliotoxin, patulin, penicillic acid, rubratoxin B, and zearalenone. These mycotoxins inhibited organic anion uptake mediated by hOAT1-4, and organic cation uptake mediated by hOCT1-2. By comparing the IC50values of mycotoxins for hOATs, it was found that hOAT1 and hOAT3 exhibited higher affinity interactions with mycotoxins than hOAT2 and hOAT4. There was no interspecies difference between humans and rats for the interactions of OATs with mycotoxins except that of OAT3 with rubratoxin B. Finally, we observed that hOAT1-4 and hOCT1-2 mediated the uptake of aflatoxin B1. In conclusion, hOATs and hOCTs interacted with various mycotoxins. Considering the localization of hOATs/rOats and hOCTs, it was suggested that these transporters were the possible entrance pathway for mycotoxins in kidney and liver, leading to the induction of adverse effects in humans and rats. Keywords:: organic anion transporter, organic cation transporter, SLC22, mycotoxin, aflatoxin

Nicotinate has been used as a lipid-lowering agent producing beneficial changes in serum lipids for atherosclerosis regression. The purpose of this study is to elucidate the molecular mechanisms underlying the transport of nicotinate by human organic anion transporters (OATs) of SLC22 family stably expressed in a mouse cell line derived from renal proximal tubule S2 segment. Among OATs tested (hOAT1-4), hOAT1 and hOAT2 mediated a time- and concentration-dependent uptake of nicotinate. We focused on the study of hOAT2 because it showed higher activity in nicotinate transport compared with hOAT1. hOAT2-mediated nicotinate uptake showed structural specificity, and was inhibited by some organic anions such as indocyanine green, glibenclamide, and probenecid and some NSAIDs (salicylic acid, diclofenac, ketoprofen, indomethacine, and ibuprofen). the identification of nicotinate transporter will contribute to understanding the molecular basis of such a drug-drug interaction as salicylate-induced increase in serum nicotinate level.