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Pulsed radiofrequency (PRF) therapy is one of the most common treatment options for neuropathic pain, albeit the underlying mechanism has not been hitherto elucidated. In this study, we investigated the efficacy and mechanism of PRF therapy on resiniferatoxin (RTX)-induced mechanical allodynia, which has been used as a model of postherpetic neuralgia (PHN). Adult male rats were intraperitoneally injected with a vehicle or RTX. Furthermore, PRF current was applied on a unilateral sciatic nerve in all RTX-treated rats. On both ipsilateral and contralateral sides, the paw mechanical withdrawal thresholds were examined and L4-6 dorsal root ganglia (DRG) were harvested. In the DRG of rats with RTX-induced mechanical allodynia, Na

Comprehensive pharmacology education in nursing based on the "Patient-oriented Pharmacology" is effective against the improvement of quality of pharmacotherapy and patient satisfaction. Two active learning programs of practical pharmacotherapy for nursing students have been performed in School of Nursing, University of Miyazaki; (1) pharmacotherapy role-play for interprofessional education (IPE) and (2) practical excise for Kampo medicine. Pharmacotherapy role-play for IPE was performed as joint lecture both medical students and nursing students. This pharmacotherapy role-play is named CaseCommunication based approach (CC approach), since it is studied through communication between physicians, nurses and patients based on cases presented beforehand. In the practical excise for Kampo medicine, nursing students studied Kampo medicines and tried to taste 9 frequently used Kampo medicines. These active-learning programs in nursing pharmacology education may be effective for better understanding of pharmacotherapy and patient's feeling, and improvement of students' motivation as a nurse.

Changes in the shape and size of the dendritic spines are critical for synaptic transmission. These morphological changes depend on dynamic assembly of the actin cytoskeleton and occur differently in various types of neurons. However, how the actin dynamics are regulated in a neuronal cell type-specific manner remains largely unknown. We show that Fhod3, a member of the formin family proteins that mediate F-actin assembly, controls the dendritic spine morphogenesis of specific subpopulations of cerebrocortical pyramidal neurons. Fhod3 is expressed specifically in excitatory pyramidal neurons within layers II/III and V of restricted areas of the mouse cerebral cortex. Immunohistochemical and biochemical analyses revealed the accumulation of Fhod3 in postsynaptic spines. Although targeted deletion of Fhod3 in the brain did not lead to any defects in the gross or histological appearance of the brain, the dendritic spines in pyramidal neurons within presumptive Fhod3-positive areas were morphologically abnormal. In primary cultures prepared from the Fhod3-depleted cortex, defects in spine morphology were only detected in Fhod3 promoter-active cells, a small population of pyramidal neurons, and not in Fhod3 promoter-negative pyramidal neurons. Thus, Fhod3 plays a crucial role in dendritic spine morphogenesis only in a specific population of pyramidal neurons in a cell type-specific manner.

Superoxide production by Nox1, a member of the Nox family NAPDH oxidases, requires expression of its regulatory soluble proteins Noxo1 (Nox organizer 1) and Noxa1 (Nox activator 1) and is markedly enhanced upon cell stimulation with phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC). The mechanism underlying PMA-induced enhancement of Nox1 activity, however, remains to be elucidated. Here we show that, in response to PMA, Noxo1 undergoes phosphorylation at multiple sites, which is inhibited by the PKC inhibitor GF109203X. Among them, Thr341 in Noxo1 is directly phosphorylated by PKC in vitro, and alanine substitution for this residue reduces not only PMA-induced Noxo1 phosphorylation but also PMA-dependent enhancement of Nox1-catalyzed superoxide production. Phosphorylation of Thr341 allows Noxo1 to sufficiently interact with Noxa1, an interaction that participates in Nox1 activation. Thus phosphorylation of Noxo1 at Thr341 appears to play a crucial role in PMA-elicited activation of Nox1, providing a molecular link between PKC-mediated signal transduction and Nox1-catalyzed superoxide production. Furthermore, Ser154 in Noxo1 is phosphorylated in both resting and PMA-stimulated cells, and the phosphorylation probably participates in a PMA-independent constitutive activity of Nox1. Ser154 may also be involved in protein kinase A (PKA) mediated regulation of Nox1; this serine is the major residue that is phosphorylated by PKA in vitro. Thus phosphorylation of Noxo1 at Thr341 and at Ser154 appears to regulate Nox1 activity in different manners. Structured digital abstract Noxo1 binds to p22phox by pull down (1, 2, 3) Noxo1 binds to Noxo1 by pull down (View interaction) Noxa1 binds to Noxo1 by pull down (1, 2, 3, 4, 5)

Actin filament assembly in nonmuscle cells is regulated by the actin polymerization machinery, including the Arp2/3 complex and formins. However, little is known about the regulation of actin assembly in muscle cells, where straight actin filaments are organized into the contractile unit sarcomere. Here, we show that Fhod3, a myocardial formin that localizes to thin actin filaments in a striated pattern, regulates sarcomere organization in cardiomyocytes. RNA interference-mediated depletion of Fhod3 results in a marked reduction in filamentous actin and disruption of the sarcomeric structure. These defects are rescued by expression of wild-type Fhod3 but not by that of mutant proteins carrying amino acid substitution for conserved residues for actin assembly. These findings suggest that actin dynamics regulated by Fhod3 are critical for sarcomere organization in striated muscle cells.

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox–p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

Formin-family proteins, in the active state, form actin-based structures such as stress fibres. Their activation mechanisms, however, are largely unknown except that mDia and its closely related formins can be activated by direct binding of the small GTPase Rho or Cdc42. Here we show that the Rho-dependent protein kinase ROCK phosphorylates the C-terminal residues Ser1131, Ser1137, and Thr1141 of formin homology domain protein 1 (FHOD1), a major endothelial formin that is normally autoinhibited by intramolecular interaction between the N- and C-terminal regions. Phosphorylation of FHOD1 at the three residues fully disrupts the autoinhibitory interaction, which culminates in formation of stress fibres. We also demonstrate that, in vascular endothelial cells, thrombin, a vasoactive substance leading to Rho activation, elicits both FHOD1 phosphorylation and stress fibre formation in a ROCK-dependent manner, and that FHOD1 depletion by RNA interference impairs thrombin-induced stress fibre formation. Based on these findings we propose a novel mechanism for activation of formin-family proteins: ROCK, activated by G protein-coupled receptor ligands such as thrombin, directly phosphorylates FHOD1 at the C-terminal region, which renders this formin in the active form, leading to stress fibre formation.

Deliberate production of reactive oxygen species (ROS) are catalyzed by enzymes that belong to the NAD(P)H oxidase (Nox) family. The human genome contains seven members of the Nox family: the superoxide-producing enzymes Nox1 through Nox5 and the dual oxidases Duox1 and Duox2 that release hydrogen peroxide but not superoxide. Among them, the classical member gp91( phox )/Nox2 functions as the phagocyte NADPH oxidase, playing a crucial role in host defense. Although Nox2, heterodimerized with its membrane-spanning partner p22( phox ), is inactive in resting cells, during phagocytosis it forms an active complex with soluble regulatory proteins such as the organizer p47( phox ), the activator p67( phox ), and the small GTPase Rac. Here the authors describe how the novel superoxide-producing Nox oxidases (Nox1, 3, 4, and 5) with different functions are regulated by p22( phox ), the Nox organizers, the Nox activators, and Rac, and how their expression is controlled at the transcriptional level.

Activation of the non-phagocytic superoxide-producing NADPH oxidase Nox1, complexed with p22(phox) at the membrane, requires its regulatory soluble proteins Noxo1 and Noxa1. However, the role of the small GTPase Rac remained to be clarified. Here we show that Rac directly participates in Nox1 activation via interacting with Noxa1. Electropermeabilized HeLa cells, ectopically expressing Nox1, Noxo1, and Noxa1, produce superoxide in a GTP-dependent manner, which is abrogated by expression of a mutant Noxa1(R103E), defective in Rac binding. Superoxide production in Nox1-expressing HeLa and Caco-2 cells is decreased by depletion or sequestration of Rac; on the other hand, it is enhanced by expression of the constitutively active Rac1(Q61L), but not by that of a mutant Rac1 with the A27K substitution, deficient in binding to Noxa1. We also demonstrate that Nox1 activation requires membrane recruitment of Noxa1, which is normally mediated via Noxa1 binding to Noxo1, a protein tethered to the Nox1 partner p22(phox): the Noxa1-Noxo1 and Noxo1-p22(phox) interactions are both essential for Nox1 activity. Rac likely facilitates the membrane localization of Noxa1: although Noxa1(W436R), defective in Noxo1 binding, neither associates with the membrane nor activates Nox1, the effects of the W436R substitution are restored by expression of Rac1(Q61L). The Rac-Noxa1 interaction also serves at a step different from the Noxa1 localization, because the binding-defective Noxa1(R103E), albeit targeted to the membrane, does not support superoxide production by Nox1. Furthermore, a mutant Noxa1 carrying the substitution of Ala for Val-205 in the activation domain, which is expected to undergo a conformational change upon Rac binding, fully localizes to the membrane but fails to activate Nox1.

Human mitochondrial DNA takes on a large protein-DNA complex called a nucleoid or mitochromosome. Mitochondrial transcription factor A (TFAM) is a major component of the complex. During an attempt to search for proteins associated with the TFAM-containing complex by a proteomic method, we found one protein that has not been considered to be mitochondrial: PDIP38. PDIP38 was initially identified as a binding protein to nuclear DNA polymerase delta. PDIP38 is almost exclusively recovered from the mitochondrial fraction of human HeLa cells. PDIP38 is completely cleaved when TritonX-100-solubilized mitochondria are treated with proteinase K, but not when mitoplasts devoid of outer membranes are treated, indicating that PDIP38 is located in the mitochondrial matrix. TFAM and mitochondrial single-stranded DNA binding protein (mtSSB) are co-immunoprecipitated with PDIP38 by anti-PDIP38 antibodies. On the other hand, only the latter is crosslinked to PDIP38 when mitochondria are treated with a crosslinker, formaldehyde. In addition to mtSSB, 60 kDa heat shock protein and a Lon protease homolog, both of which have single-stranded DNA binding activity, are also crosslinked. PDIP38 associates with the nucleoid components and could be involved in the metabolism of mitochondrial DNA.

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91(phox), tightly complexed with p22(phox), and its activation requires the association with p47(phox), p67(phox), and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91(phox)-related enzymes of the NAD(P)H oxidase (Nox) family--seven homologues identified in humans--deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.

Formins constitute a family of eukaryotic proteins that are considered to function as a cytoskeleton organizer to regulate morphogenesis, cell polarity and cytokinesis. Fhos is a recently identified mammalian formin, which contains the conserved domains FH (formin homology) 1 and FH2 in the middle region and the Dia-autoregulatory domain (DAD) in the C-terminus. The role of Fhos in the regulation of cytoskeleton, however, has remained unknown. Here we show that Fhos, in an active form, induces the formation of actin stress fibers and localizes to the actin-based structure. Fhos appears to normally exist in a closed inactive form via an intramolecular interaction between the N-terminal region and the C-terminal DAD. Both FH1 and FH2 domains are required for the induction of the stress fiber formation. However, the N-terminal region of Fhos is required for the targeting of this protein to stress fibers, which is probably mediated via its F-actin-binding activity. We also show that Fhos occurs as a homotypic complex in cells. The self-association of Fhos seems to be mediated via the FH2 domain: the domains bind to each other in a direct manner. Thus, the mammalian formin Fhos, which directly binds to F-actin via the N-terminal region, forms a homotypic complex via the FH2 domain to organize actin cytoskeleton.

Protein–phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47 phox , a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47 phox with phosphoinositides. Although the isolated phox homology (PX) domain of p47 phox can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47 phox is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47 phox to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47 phox , because protein kinase C treatment of the wild-type p47 phox but not of a mutant protein with the S303/304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47 phox translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47 phox nor lipid-binding-defective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47 phox enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47 phox from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase.

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline‐rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47 phox , binds to the C‐terminal SH3 domain from p67 phox . We applied the NMR cross‐saturation method to locate the interaction sites for the non‐PxxP peptides on their cognate SH3 domains from p67 phox , Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP‐binding site, whereas those of p67 phox and Pex13p SH3s are located in different surface regions. The non‐PxxP peptide from p47 phox binds to the p67 phox SH3 more tightly when it extends to the N‐terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non‐PxxP peptide segment interacted with the p67 phox SH3 in a compact helix–turn–helix structure (PDB entry 1K4U).

We have cloned human cDNA encoding a novel protein of 782 amino acids that contains the lipase consensus sequence Gly-Xaa-Ser-Xaa-Gly and several stretches surrounding the motif, which are homologous to those of the catalytic domain of cytosolic calcium-independent phospholipase A 2 (iPLA 2 ). When expressed in COS-7 cells, the protein predominantly exists in the membrane fraction and exhibits a phospholipase A 2 activity in a calcium-independent manner. The transcript of the membrane-bound iPLA 2 gene is ubiquitously observed as a single band of approximately 3.3 kb on Northern blot, with the most abundant expression in the skeletal muscle and heart. By a search of the database, we have also identified its putative C. elegans homologue, which shows 47% identity with that of human in the iPLA 2 catalytic region. Thus the novel type of iPLA 2 is evolutionarily well conserved, suggestive of its biological significance.

We have cloned the cDNA encoding human PICK1 (protein interacting with C kinase 1), a PDZ domain-containing protein of 415 amino acids, and also identified the Drosophila homologue by search of the databank. Northern blot analysis shows a single mRNA of about 2.0 kb ubiquitously expressed in human tissues. Although PICK1 proteins harbor a region homologous to arfaptin1 and arfaptin2, two proteins that bind to the ARF (ADP-ribosylation factor), this region of PICK1 does not interact with ARFs in the yeast two-hybrid system. On the other hand, the PDZ domain of PICK1 is capable of interacting with constitutively active, GTP-bound forms of ARF1 and ARF3, but neither with those of ARF5/6 nor with the GDP-bound ARFs. The PICK1–ARF interaction is abrogated by introduction of mutations in the PDZ domain or by deletion of the extreme C-terminus of ARF1. Thus, PICK1 specifically interacts with ARF1/3 in the GTP-bound state, suggesting that PICK1 participates in ARF1/3-mediated cellular processes.

Interleukin (IL)-4 plays an important role in IgE synthesis in B cells and in Th2 differentiation in T cells. IL-4 conducts its biological activities through binding to the IL-4 receptor (IL-4R) on the surface of target cells. IL-4R are thought to be composed of the IL-4R alpha chain (IL-4R alpha) and either the IL-2R gamma chain or the IL-13R alpha chain. We have previously shown that the membrane-proximal portion in the cytoplasmic domain of the human IL-4R alpha (hIL-4R alpha) is critical for proliferation, generation of germline epsilon transcript, and activation of STAT6, based on analyses of truncated hIL-4R alphas. In this study, we found that p47phox, an activator of the phagocyte NADPH oxidase, binds to this portion by the two-hybrid system. Furthermore, we observed the association of p47phox with the hIL-4R alpha in B cells derived from a normal donor. These results suggest that p47phox is involved in the signal transduction of IL-4 in B cells. However, activation of STAT6, CD23 expression, and IgE synthesis induced by IL-4 were not affected in p47phox-deficient patients, which raises the possibility that p47phox may be important in other signaling activities as well in B cells.

Dilated cardiomyopathy (DCM) is characterized by a dilated left ventricular cavity with systolic dysfunction manifested by heart failure. It has been revealed that mutations in genes for cytoskeleton or sarcomere proteins cause DCM. However, the disease-causing mutations can be found only in far less than half of patients with a family history, indicating that there should be other disease genes for DCM. Formin homology 2 domain containing 3 (FHOD3) is a sarcomeric protein expressed in the heart that plays an essential role in sarcomere organization during myofibrillogenesis. The purpose of this study was to explore a possible novel disease gene for DCM.We analyzed 48 Japanese familial DCM patients for mutations in FHOD3, and a missense variant, Tyr1249Asn, which was predicted to modify the 3D structure and damage protein function, was found in a case with adult-onset DCM. Functional studies revealed that the DCM-associated mutation significantly reduced the ability to induce actin dynamics-dependent activation of serum response factor, although no remarkable change in the cellular localization was induced in neonatal rat cardiomyocytes transfected with a mutant construct of FHOD3.The DCM-associated FHOD3 variant may cause DCM by interfering with actin filament assembly.

The formin family proteins play pivotal roles in actin filament assembly via the FH2 domain. The mammalian formin Fhod3 is highly expressed in the heart, and its mRNA in the adult heart contains exons 11, 12, and 25, which are absent from non-muscle Fhod3 isoforms. In cultured neonatal cardiomyocytes, Fhod3 localizes to the middle of the sarcomere and appears to function in its organization, although it is suggested that Fhod3 localizes differently in the adult heart. Here we show, using immunohistochemical analysis with three different antibodies, each recognizing distinct regions of Fhod3, that Fhod3 localizes as two closely spaced bands in middle of the sarcomere in both embryonic and adult hearts. The bands are adjacent to the M-line that crosslinks thick myosin filaments at the center of a sarcomere but distant from the Z-line that forms the boundary of the sarcomere, which localization is the same as that observed in cultured cardiomyocytes. Detailed immunohistochemical and immuno-electron microscopic analyses reveal that Fhod3 localizes not at the pointed ends of thin actin filaments but to a more peripheral zone, where thin filaments overlap with thick myosin filaments. We also demonstrate that the embryonic heart of mice specifically expresses the Fhod3 mRNA isoform harboring the three alternative exons, and that the characteristic localization of Fhod3 in the sarcomere does not require a region encoded by exon 25, in contrast to an essential role of exons 11 and 12. Furthermore, the exon 25-encoded region appears to be dispensable for actin-organizing activities both in vivo and in vitro, albeit it is inserted in the catalytic FH2 domain.

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The membrane-integrated protein gp91(phox) serves as the catalytic core, because it contains a complete electron-transporting apparatus from NADPH to molecular oxygen for superoxide production. Activation of gp91(phox) requires the cytosolic proteins p67(phox), p47(phox), and Rac (a small GTPase). p67(phox), comprising 526 amino acids, moves upon cell stimulation to the membrane together with p47(phox) and there interacts with Rac; these processes are prerequisite for gp91(phox) activation. Here we show that a region of p67(phox) (amino acids 190-200) C-terminal to the Rac-binding domain is evolutionarily well conserved and participates in oxidase activation at a later stage in conjunction with an activation domain. Alanine substitution for Tyr-198, Leu-199, or Val-204 abrogates the ability of p67(phox) to support superoxide production by gp91(phox)-based oxidase as well as its related oxidases Nox1 and Nox3; the activation also involves other invariant residues such as Leu-193, Asp-197, and Gly-200. Intriguingly, replacement of Gln-192 by alanine or that of Tyr-198 by phenylalanine or tryptophan rather enhances superoxide production by gp91(phox)-based oxidase, suggesting a tuning role for these residues. Furthermore, the Y198A/V204A or L199A/V204A substitution leads to not only a complete loss of the activity of the reconstituted oxidase system but also a significant decrease in p67(phox) interaction with the gp91(phox) NADPH-binding domain, although these mutations affect neither the protein integrity nor the Rac binding activity. Thus the extended activation domain of p67(phox) (amino acids 190-210) containing the D(Y/F)LGK motif plays an essential role in oxidase activation probably by interacting with gp91(phox).

Reactive oxygen species (ROS) have been attracting attention as mediators of various cell-signaling pathways. Nox-family NADPH oxidases have proven to be a major source of ROS production in various cell types and have crucial roles in various physiological and pathological processes. In this study, we show that Nox4, a member of Nox family, is prominently expressed in various neuroepithelial tumors by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical studies. We quantified Nox4 mRNA expression by real-time PCR in tumor specimens from 58 patients with astrocytomas and found that the expression levels of Nox4 mRNA in glioblastomas (WHO grade IV) were significantly higher than those in other astrocytomas (WHO grade II and III). In addition, we show that specific knockdown of Nox4 expression by RNA interference results in cell-growth inhibition and enhances induction of apoptosis by chemotherapeutic agents, such as cisplatin, in cultured glioma cell lines. Based on these observations, enhanced expression of Nox4 appears to be involved in cell proliferation and survival in glioma cells.

NADPH oxidase organizer 1 (Noxo1), harboring a PX domain, two SH3 domains, and a proline-rich region (PRR), participates in activation of superoxide-producing Nox-family NADPH oxidases. Here, we show that Noxo1 supports superoxide production in a cell-free system for gp91phox/Nox2 activation by arachidonic acid. This lipid enhances an SH3-mediated binding of Noxo1 to p22phox, a protein complexed with Nox oxidases; the binding is known to be required for Nox activation. We also demonstrate that the bis-SH3 domain directly interacts with the Noxo1 PRR. The interaction appears to prevent the bis-SH3 domain and PRR from binding to their target proteins; disruption of the intramolecular interaction facilitates Noxo1 binding to p22phox and also allows the PRR to associate with the Nox activator Noxa1, which association is crucial for Nox activation as well. These findings suggest that Nox activation involves a conformational change leading to disruption of the bis-SH3–PRR interaction in Noxo1.

The superoxide‐producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40 phox associates via the PB1 domain with the essential oxidase activator p67 phox , and is considered to function by recruiting p67 phox to phagosomes; in this process, the PX domain of p40 phox binds to phosphatidylinositol 3‐phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P‐binding activity of p40 phox is normally inhibited by the PB1 domain both in vivo and in vitro . The crystal structure of the full‐length p40 phox reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40 phox PB1 domain for the PX domain localizes on the opposite side of that for the p67 phox PB1 domain, and thus the PB1‐mediated PX regulation occurs without preventing the PB1–PB1 association with p67 phox .

Activation of the superoxide-producing NADPH oxidase Nox1 requires both the organizer protein Noxo1 and the activator protein Noxa1. Here we describe an alternative splicing form of Noxo1, Noxo1gamma, which is expressed in the testis and fetal brain. The Noxo1gamma protein contains an additional five amino acids in the N-terminal PX domain, a phosphoinositide-binding module; the domain plays an essential role in supporting superoxide production by NADPH oxidase (Nox) family oxidases including Nox1, gp91(phox)/Nox2, and Nox3, as shown in this study. The PX domain isolated from Noxo1gamma shows a lower affinity for phosphoinositides than that from the classical splicing form Noxo1beta. Consistent with this, in resting cells, Noxo1gamma is poorly localized to the membrane, and thus less effective in activating Nox1 than Noxo1beta, which is constitutively present at the membrane. On the other hand, cell stimulation with phorbol 12-myristate 13-acetate (PMA), an activator of Nox1-3, facilitates membrane translocation of Noxo1gamma; as a result, Noxo1gamma is equivalent to Noxo1beta in Nox1 activation in PMA-stimulated cells. The effect of the five-amino-acid insertion in the Noxo1 PX domain appears to depend on the type of Nox; in activation of gp91(phox)/Nox2, Noxo1gamma is less active than Noxo1beta even in the presence of PMA, whereas Noxo1gamma and Noxo1beta support the superoxide-producing activity of Nox3 to the same extent in a manner independent of cell stimulation.

The membrane-integrated protein gp91phox functions as the catalytic center of the superoxide-producing phagocyte NADPH oxidase. Recent studies have identified homologs of gp91phox in nonphagocytic cells, which constitute the NADPH oxidase (Nox) family. Activation of the Nox oxidases leads to production of reactive oxygen species (ROS), thereby participating in a variety of biological events, such as host defense, hormone biosynthesis, and signal transduction. The activity of the Nox enzymes is regulated by various proteins, including the small GTPase Rac; regulatory mechanisms differ dependent on the type of the Nox proteins. For example, an oxidase activator (p47phox or Noxo1) and an oxidase activator (p67phox or Noxa1) are absolutely required for superoxide production by gp91phox and Nox1, but not by Nox3. Rac, albeit probably dispensable to the Nox3 activity, plays an essential role in activation of gp91phox. Thus, functional reconstitution of Nox systems is crucial for the study of Nox regulation. Here we describe a basic method for the reconstitution of Nox systems by expression of oxidase proteins in transfectable cells.

The phagocyte NADPH oxidase plays a crucial role in host defense against microbial infections by generating reactive oxygen species. It is a multisubunit enzyme composed of membrane-bound flavocytochrome b558 as well as cytosolic components, including p47phox, which is essential for assembly of the complex. When phagocytes are activated, the cytosolic components of the NADPH oxidase translocate to flavocytochrome b558 due to binding of the tandem Src homology 3 (SH3) domains of p47phox to a proline-rich region in p22phox, a subunit of flavocytochrome b558. Using NMR titration, we first identified the proline-rich region of p22phox that is essential for binding to the tandem SH3 domains of p47phox. We subsequently determined the solution structure of the p47phox tandem SH3 domains complexed with the proline-rich peptide of p22phox using NMR spectroscopy. In contrast to the intertwined dimer reported for the crystal state, the solution structure is a monomer. The central region of the p22phox peptide forms a polyproline type II helix that is sandwiched by the N- and C-terminal SH3 domains, as was observed in the crystal structure, whereas the C-terminal region of the peptide takes on a short alpha-helical conformation that provides an additional binding site with the N-terminal SH3 domain. Thus, the C-terminal alpha-helical region of the p22phox peptide increases the binding affinity for the tandem SH3 domains of p47phox more than 10-fold.

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response.

Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47(phox) and p67(phox), each harboring two SH3 domains. p67(phox) interacts with p47(phox) via simultaneous binding of the p67(phox) C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360-369 and its C-terminally flanking region of p47(phox); the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47(phox)-p67(phox) interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47(phox) extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation.

Fhos1 is a mammalian formin-family protein, and functions as an organizer of the actin microfilament. Here we have cloned human and mouse cDNAs for a novel Fhos homolog, designated Fhos2. The messages for Fhos2 are expressed in the heart, kidney, and brain, where the Fhos1 mRNAs are not abundant. Two splice variants of Fhos2 exist in a tissue-specific manner; the longer variant Fhos2L is the major form in the heart, whereas the kidney and brain predominantly express Fhos2S that encodes a shorter protein. Over-expression of an active form of the two Fhos2 variants, as well as that of Fhos1, induces the formation of actin stress fibers in HeLa cells, suggesting that Fhos2 acts as an actin-organizing protein. Biochemical analysis using rat cardiomyoblastic H9c2 (2-1) cells reveals that endogenous Fhos2 is enriched in the intermediate filament fraction. Consistent with this, Fhos2 localizes to the nestin intermediate filament but not to other cytoskeletons, as demonstrated by staining of H9c2 (2-1) cells with anti-Fhos2 antibodies. Furthermore, Fhos2 is present in nestin-expressing neuroepithelial cells of the fetal rat brain. Thus, Fhos2 not only has the actin-organizing activity but also associates with nestin, which may imply a Fhos2-mediated link between the nestin intermediate filament and actin microfilament.

Nox3, a member of the superoxide-producing NADPH oxidase (Nox) family, participates in otoconia formation in mouse inner ears, which is required for perception of balance and gravity. The activity of other Nox enzymes such as gp91(phox)/Nox2 and Nox1 is known to absolutely require both an organizer protein (p47(phox) or Noxo1) andanactivatorprotein (p67(phox) or Noxa1); for the p47(phox)-dependent activation of these oxidases, treatment of cells with stimulants such as phorbol 12-myristate 13-acetate is also indispensable. Here we show that ectopic expression of Nox3 in various types of cells leads to phorbol 12-myristate 13-acetate-independent constitutive production of a substantial amount of superoxide under the conditions where gp91(phox) and Nox1 fail to generate superoxide, i.e. in the absence of the oxidase organizers and activators. Nox3 likely forms a functional complex with p22(phox); Nox3 physically interacts with and stabilizes p22(phox), and the Nox3-dependent superoxide production is totally dependent on p22(phox). The organizers p47(phox) and Noxo1 are capable of enhancing the superoxide production by Nox3 in the absence of the activators, and the enhancement requires the interaction of the organizers with p22(phox), further indicating a link between Nox3 and p22(phox). The p47(phox)-enhanced Nox3 activity is further facilitated by p67(phox) or Noxa1, whereas the activators cancel the Noxo1-induced enhancement. On the other hand, the small GTPase Rac, essential for the gp91(phox) activity, is likely dispensable to the Nox3 system. Thus Nox3 functions together with p22(phox) as an enzyme constitutively producing superoxide, which can be distinctly regulated by combinatorial use of the organizers and activators.

A complex of atypical PKC and Par6 is a common regulator for cell polarity-related processes, which is an essential clue to evolutionary conserved cell polarity regulation. Here, we determined the crystal structure of the complex of PKCiota and Par6alpha PB1 domains to a resolution of 1.5 A. Both PB1 domains adopt a ubiquitin fold. PKCiota PB1 presents an OPR, PC, and AID (OPCA) motif, 28 amino acid residues with acidic and hydrophobic residues, which interacts with the conserved lysine residue of Par6alpha PB1 in a front and back manner. On the interface, several salt bridges are formed including the conserved acidic residues on the OPCA motif of PKCiota PB1 and the conserved lysine residue on the Par6alpha PB1. Structural comparison of the PKCiota and Par6alpha PB1 complex with the p40phox and p67phox PB1 domain complex, subunits of neutrophil NADPH oxidase, reveals that the specific interaction is achieved by tilting the interface so that the insertion or extension in the sequence is engaged in the specificity determinant. The PB1 domain develops the interaction surface on the ubiquitin fold to increase the versatility of molecular interaction.

In this study, we identify the extent of deformation that causes cell lysis using a simple technique where a drop of cell suspension is compressed by two flat plates. The viability of human prostatic adenocarcinoma PC-3 cells in solutions of various concentrations of NaCl is determined as a function of the gap size between the plates. The viability declines with decreasing gap size in the following order: 700 mM >150 mM >75 mM NaCl. This is considered to be due to the difference in cell size, which is caused by the osmotic volume change before deformation; cell diameter becomes smaller in a solution of higher NaCl concentration, which appears to increase the survival ratio in a given gap size. The deformation-induced decrease in cell viability is correlated with the cell surface strain, which is dependent on the increase in surface area, irrespective of NaCl concentration. In addition, the treatment of cells with cytochalasin D results in the disappearance of cortical actin filaments and a marked drop in the viability, indicating that cell lysis is closely related to the deformation of the cytoskeleton.

The NADPH oxidase 1 (Nox1) is a gp91 phox homologue preferentially expressed in the colon. We have established primary cultures of guinea pig large intestinal epithelial cells giving 90% purity of surface mucous cells. These cells spontaneously released superoxide anion (O 2 − ) of 160 nmol/mg protein/h and expressed the Nox1 , p22 phox , p67 phox , and Rac1 mRNAs, but not the gp91 phox , Nox4 , p47 phox , p40 phox , and Rac2 mRNAs. They also expressed novel homologues of p47 phox and p67 phox (p41 nox and p51 nox , respectively). Human colon cancer cell lines (T84 and Caco2 cells) expressed the Nox1 , p22 phox , p51 nox , and Rac1 mRNAs, but not the other NADPH component mRNAs, and secreted only small amounts of O 2 − ( p41 nox and p51 nox cDNAs in T84 cells enhanced PMA-stimulated O 2 − release 5-fold. Treatment of the transfected T84 cells with recombinant flagellin (rFliC) from Salmonella enteritidis further augmented the O 2 − release in association with the induction of Nox1 protein. The enhanced O 2 − production by cotransfection of p41 nox and p51 nox vectors further augmented the rFliC-stimulated IL-8 release from T84 cells. T84 cells expressed the Toll-like receptor 5, and rFliC rapidly phosphorylated TGF-β-activated kinase 1 and TGF-β-activated kinase 1-binding protein 1. A potent inhibitor for NF-κB (pyrrolidine dithiocarbamate) significantly blocked the rFliC-primed increase in O 2 − production and induction of Nox1 protein. These results suggest that p41 nox and p51 nox are involved in the Nox1 activation in surface mucous cells of the colon, and besides that, epithelial cells discern pathogenicities among bacteria to appropriately operate Nox1 for the host defense.

The gene encoding interferon (IFN)-gamma, IFNG, is known as one of the candidate susceptibility genes for type 1 diabetes. In addition, cytokines, including IFN-gamma, play important roles in the pathogenesis of type 1 diabetes. Therefore, we focused on the Th1-specific T-box transcription factor gene (T-bet), which contributes to the induction of the hallmark Th1 cytokine, IFN-gamma. We first screened for polymorphisms in the T-bet gene and detected two microsatellite repeat polymorphisms located in intron 1 and the 3'- flanking region, and two single nucleotide polymorphisms, including a His33Gln substitution within the coding region. By association studies, the Gln-positive phenotype and (CA)14 allele in 3'-flanking region of T-bet were found to be associated with type 1 diabetes in the Japanese population. Furthermore, Gln33 T-bet showed a significantly higher transcriptional activity of the IFNG gene via a dual luciferase reporter assay. Our study suggests the first evidence of an association between type 1 diabetes and polymorphisms in the T-bet gene, and that variation in T-bet transcriptional activity may play a role in the development of type 1 diabetes, possibly through the effect on IFN-gamma production in Th1 cells.

The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

The cell polarity protein PAR3, conserved from the nematode to the vertebrate, forms a complex with PAR6 and atypical protein kinase C (aPKC), and the protein complex occurs at the tight junctions in mammalian epithelial cells. Here we have cloned human cDNA for a novel PAR3 homologue, designated PAR3beta, whose messages are present in a variety of tissues and most abundantly expressed in the adult and fetal kidneys. The encoded protein of 1,205 amino acids contains a region homologous to the aPKC-binding domain of PAR3alpha, another human homologue previously identified, and three PDZ domains; the first PDZ domain of PAR3alpha is considered to interact with PAR6. Unexpectedly, in contrast to other PAR3s found in various species, PAR3beta is incapable of binding to any isotypes of PAR6 or aPKC. Nevertheless PAR3beta, expressed intrinsically or extrinsically, localizes to the tight junctions, indicating that the localization does not require the ternary complex formation.

The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox), the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking. Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: p40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P(2). In addition, the Bem1p PX domain also interacts with Ptdlns(4)P. When the p40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched. Furthermore, a mutant p40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes. Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes.