Phosphoinositide-dependent regulation of VAN3 ARF-GAP localization and activity essential for vascular tissue continuity in plants

ACAP-type ARF GTPase activating proteins (ARF-GAPs) regulate multiple cellular processes, including endocytosis, secretion, phagocytosis, cell adhesion and cell migration. However, the regulation of ACAP functions by other cellular proteins is poorly understood. We have reported previously that a plant ACAP, VAN3, plays a pivotal role in plant venation continuity. Here, we report on newly identified VAN3 regulators: the CVP2 (cotyledon vascular pattern 2) 5 PTase, which is considered to degrade IP3 and also to produce PtdIns(4) P from PtdIns(4,5) P-2; and a PH domain-containing protein, VAB (VAN3 binding protein). Combinational mutations of both CVP2 and its closest homologue CVL1 (CVP2 like 1) phenocopied the strong allele of van3 mutants, showing severe vascular continuity. The phenotype of double mutants between van3, cvp2 and vab suggested that VAN3, CVP2 and VAB function in vascular pattern formation in the same pathway. Localization analysis revealed that both CVP2 and VAB colocalize with VAN3 in the trans-Golgi network (TGN), supporting their functions in the same pathway. The subcellular localization of VAN3 was dependent on its PH domain, and mislocalization of VAN3 was induced in cvp2 or vab mutants. These results suggest that CVP2 and VAB cooperatively regulate the subcellular localization of VAN3 through the interaction between its PH domain and phosphoinositides and/or inositol phosphates. In addition, PtdIns(4) P, to which VAN3 binds preferentially, enhanced the ARF-GAP activity of VAN3, whereas IP3 inhibited it. These results suggest the existence of PtdIns(4) P and/or IP3-dependent subcellular targeting and regulation of VAN3 ACAP activity that governs plant vascular tissue continuity

Acid-denatured small heat shock protein HdeA from Escherichia coli forms reversible fibrils with an atypical secondary structure

The periplasmic small heat shock protein HdeA from Escherichia coli is inactive under normal growth conditions (at pH 7) and activated only when E. coli cells are subjected to a sudden decrease in pH, converting HdeA into an acid-denatured active state. Here, using in vitro fibrillation assays, transmission EM, atomic-force microscopy, and CD analyses, we found that when HdeA is active as a molecular chaperone, it is also capable of forming inactive aggregates that, at first glance, resemble amyloid fibrils. We noted that the molecular chaperone activity of HdeA takes precedence over fibrillogenesis under acidic conditions, as the presence of denatured substrate protein was sufficient to suppress HdeA fibril formation. Further experiments suggested that the secondary structure of HdeA fibrils deviates somewhat from typical amyloid fibrils and contains α-helices. Strikingly, HdeA fibrils that formed at pH 2 were immediately resolubilized by a simple shift to pH 7 and from there could regain molecular chaperone activity upon a return to pH 1. HdeA, therefore, provides an unusual example of a “reversible” form of protein fibrillation with an atypical secondary structure composition. The competition between active assistance of denatured polypeptides (its “molecular chaperone” activity) and the formation of inactive fibrillary deposits (its “fibrillogenic” activity) provides a unique opportunity to probe the relationship among protein function, structure, and aggregation in detail

Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana

PIN-FORMED (PIN) proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture

Probing the Functional Mechanism of Escherichia coli GroEL Using Circular Permutation

Background: The Escherichia coli chaperonin GroEL subunit consists of three domains linked via two hinge regions, and each domain is responsible for a specific role in the functional mechanism. Here, we have used circular permutation to study the structural and functional characteristics of the GroEL subunit. Methodology/Principal Findings: Three soluble, partially active mutants with polypeptide ends relocated into various positions of the apical domain of GroEL were isolated and studied. The basic functional hallmarks of GroEL (ATPase and chaperoning activities) were retained in all three mutants. Certain functional characteristics, such as basal ATPase activity and ATPase inhibition by the cochaperonin GroES, differed in the mutants while at the same time, the ability to facilitate the refolding of rhodanese was roughly equal. Stopped-flow fluorescence experiments using a fluorescent variant of the circularly permuted GroEL CP376 revealed that a specific kinetic transition that reflects movements of the apical domain was missing in this mutant. This mutant also displayed several characteristics that suggested that the apical domains were behaving in an uncoordinated fashion. Conclusions/Significance: The loss of apical domain coordination and a concomitant decrease in functional ability highlights the importance of certain conformational signals that are relayed through domain interlinks in GroEL. W

Immunotherapy of transitional cell carcinoma of urinary bladder

Karcinom prijelaznog epitela mokraćnog mjehura najčešći je karcinom mokraćnog sustava. Klinički razlikujemo dva oblika ove bolesti: mišićno-neinvazivni i mišićno-invazivni oblik. Temeljna klinička odrednica mišićno-neinvazivnih tumora su recidivne novotvorinske promjene; 60 – 90 % mišićno-neinvazivnih tumora recidivirat će ako se liječe samo transuretralnom resekcijom (TUR). Upravo stoga se nakon TUR-a u pacijenata u kojih postoji visoki rizik od ponovne pojave ili progresije bolesti provodi intravezikalna imunoterapija BCGom (bacillus Calmette-Guerin). BCG predstavlja živi atenuirani soj Mycobacterium bovis. U primjeni BCG-a intravezikalne terapije razlikujemo indukcijsku terapiju i terapiju održavanja. Intravezikalna aplikacija BCG-a uzrokuje masivan ulazak upalnih stanica u stijenku mokraćnog mjehura te produkciju citokina detektibilnih u sluznici mjehura i u urinu, što dovodi do imunog odgovora protiv tumora. Činjenica je kako BCG uzrokuje dugotrajan i dugodjelujući imuni odgovor. Do eradikacije tumorskih stanica dolazi zbog celularnih fokusa u stijenci mjehura, a kao direktan antitumorski efektorski mehanizam navodi se direktna antitumorska aktivnost IFN (interferon) i citotoksičnost NK (engl. natural killer) stanica.Bladder cancer is the most common cancer in urinary system. There are two clinical aspects of this disease: muscle non-invasive and muscle invasive disease. Basic characteristic of non-muscle invasive disease is tumor recurrence; 60-90 % of tumors will recurr if treated by transurethral resection (TUR) only. That is the reason why patients in whom exists the risk of recurrence or progression undergo intravesical bacillus Calmette-Guerin (BCG) immunotherapy. BCG represents live attenuated Mycobacterium bovis. There are two phases in BCG therapy: induction and maintenance therapy. Intravesical BCG application causes massive agregation of immune cells in bladder wall and producton of cytokins which causes cytotoxic tumor response. Direct effector mechanism is achieved by IFN (interferon) and NK (natural killer) cell cytotoxicity

Sex-specific post-translational regulation of the gamete fusogen GCS1 in the isogamous volvocine alga Gonium pectorale

Male and female, generally defined based on differences in gamete size and motility, likely have multiple independent origins, appearing to have evolved from isogamous organisms in various eukaryotic lineages. Recent studies of the gamete fusogen GCS1/HAP2 indicate that this protein is deeply conserved across eukaryotes, and its exclusive and/or functional expression generally resides in males or in male homologues. However, little is known regarding the conserved or primitive molecular traits of males and females within eukaryotes. Here, using morphologically indistinguishable isogametes of the colonial volvocine Gonium pectorale, we demonstrated that GCS1 is differently regulated between the sexes. G. pectorale GCS1 molecules in one sex (homologous to “male”) are transported from the gamete cytoplasm to the protruded fusion site, whereas those of the other sex (“females”) are quickly degraded within the cytoplasm upon gamete activation. This molecular trait difference might be conserved across various eukaryotic lineages and may represent male and female prototypes originating from a common eukaryotic ancestor

Serum amyloid A-induced IL-6 production by rheumatoid synoviocytes

AbstractIn this study, we investigated the role of serum amyloid A protein (SAA) in the production of interleukin-6 (IL-6) using rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). Recombinant SAA stimulation induced the production of pro-inflammatory cytokine, IL-6, from RA-FLS. The signaling events induced by SAA included the activation of the mitogen-activated protein kineases, p38 and JNK1/2 and the activation of nuclear factor-kappa B (NF-κB). Inhibitor studies have shown SAA-induced IL-6 production to be down-regulated by NF-κB inhibition and partially inhibited by p38 or JNK inhibitors. Our findings demonstrate that SAA is a significant inducer of IL-6, which is critically involved in RA pathogenesis

Structural insights into the HBV receptor and bile acid transporter NTCP

B型肝炎ウイルスの受容体“胆汁酸輸送体”の立体構造を解明. 京都大学プレスリリース. 2022-05-18.Roughly 250 million people are infected with hepatitis B virus (HBV) worldwide, and perhaps 15 million also carry the satellite virus HDV, which confers even greater risk of severe liver disease. Almost ten years ago the HBV receptor was identified as NTCP (sodium taurocholate co-transporting polypeptide), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large (L) protein. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria, and these models with ten transmembrane helices are believed to resemble strongly both NTCP and ASBT. Using cryo-electron microscopy we have solved the structure of NTCP bound to an antibody, clearly showing the transporter has no equivalent to the first transmembrane helix of other SLC10 models, leaving the N-terminus exposed on the extracellular face. Comparison of the different structures indicates a common mechanism of bile acid transport, but the NTCP structure also displays a pocket formed by residues known to interact with preS1, presenting new and enticing opportunities for structure-based drug design

ショクブツ ニ オケル メンブレン トラフィック ノ ビジュアル バイオロジー

京都大学0048新制・課程博士博士(生命科学)甲第11747号生博第34号新制||生||7(附属図書館)23390UT51-2005-D496京都大学大学院生命科学研究科統合生命科学専攻(主査)教授 竹安 邦夫, 教授 佐藤 文彦, 教授 佐邊 壽孝学位規則第4条第1項該当Doctor of Life ScienceKyoto UniversityDA

ユーザー ト スクリーン ディスプレイ ノ インタラクション ニオケル シクミ ト デザイン ニ カンスル ケンキュウ : ケイケン ノ カシカ ヒョウゲン オ ダイザイ トシテ

本研究は，ユーザと｢製品及びコンピュータのスクリーンディスプレイ(以下ディスプレイとする)｣とのインタラクションの問題について着目する。研究目的は，インタラクションを｢出来事(event)｣として捉え，｢ディスプレイ上の表現が原因で生まれる出来事の仕組み｣について明かにし，それをもとに出来事のデザイン方法について検討することである。筑波大学博士 (感性科学) 学位論文・平成17年3月25日授与 (乙第2112号)付: DVD-R(1枚)標題紙，目次 -- 用語の定義 -- 論文要旨 -- 序論：研究の背景と目的 -- 第１章：出来事に関する理論的展開　 -- 第２章：映画表現の仕組みとオーディエンスの視点（事例研究１） -- 第３章：インタラクティブ表現作品の仕組みとオーディエンスの作品理解（事例研究２） -- 第４章：インタラクティブ表現作品の仕組み（事例研究３） -- 第５章：身体表現から生まれるビジュアル表現作品の仕組み（事例研究４） -- 第６章：身体表現から描く人工物と人のインタラクションの仕組み（事例研究５） -- 結論：出来事をデザインすること -- 参考文献 -- 謝辞 -- 付録資