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本研究の目的は，腫瘍切除による上顎欠損患者の発話の特徴と，顎義歯の効果を明らかにすることである．腫瘍切除後上顎欠損を生じ，顎義歯を作製した9名に，開鼻声，構音検査，ブローイング検査，発語明瞭度検査を実施した．顎義歯非装着時は，無声破裂子音は摩擦音へ，有声子音は通鼻音へ異聴される傾向が見られた．この理由として，上顎欠損により呼気鼻漏出量が増加し，安定した構音位置が確保されないことが考えられた．開鼻声，構音，ブローイング検査時の呼気鼻漏出量，発語明瞭度とも顎義歯装着により改善された．顎義歯装着後，構音が改善されたことから，顎義歯が上顎欠損症例の構音障害の治療に効果があることが示された．

There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (35 degrees C) apparently via direct heat-evoked channel gating. beta-NAD(+)- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25 degrees C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca(2+) and insulin release, which is K(ATP) channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca(2+) entry into pancreatic beta-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.

The capsaicin receptor, TRPV1 (VR1), is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. Extracellular Ca 2+ -dependent desensitization of TRPV1 observed in patch–clamp experiments when using both heterologous expression systems and native sensory ganglia is thought to be one mechanism underlying the paradoxical effectiveness of capsaicin as an analgesic therapy. Here, we show that the Ca 2+ -binding protein calmodulin binds to a 35-aa segment in the C terminus of TRPV1, and that disruption of the calmodulin-binding segment prevents TRPV1 desensitization. Compounds that interfere with the 35-aa segment could therefore prove useful in the treatment of pain.

mDia, one of the target proteins of the GTPase Rho, is known to be involved in cytoskeletal reorganization and cytokinesis. Here, we report that mDia enters the nucleus and binds to the transcription factor, Pax6. In cultured non-neuronal cells, overexpression of mDia with Pax6 causes redistribution of some Pax6 molecules from the nucleus to the cytosol and decreases Pax6 transcriptional activity. Because Pax6 functions in the early central nervous system morphogenesis, we also examined the effects of mDia on endogenous Pax6 localization and neurite extension in cerebellar granule cells. Here too, Pax6 was partially mislocalized to the cytosol, and its expression level was decreased by mDia overexpression. In addition, mDia overexpression in these cells led to increased neurite branching and length. These results strongly suggest that mDia influences Pax6-induced transcriptional activity and axonal pathfinding in a way opposite from ROCK (Rho kinase) and that it may act via Pax6 to modulate early neuronal development.

The capsaicin receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that ATP, one of the inflammatory mediators, potentiates the VR1 currents evoked by capsaicin or protons and reduces the temperature threshold for activation of VR1 through metabotropic P2Y(1) receptors in a protein Kinase C (PKC)-dependent pathway, suggesting the phosphorylation of VR1 by PKC. In this study, direct phosphorylation of VR1 upon application of phorbol 12-myristate 13-acetate (PMA) was proven biochemically in cells expressing VR1. An in vitro kinase assay using glutathione S-transferase fusion proteins with cytoplasmic segments of VR1 showed that both the first intracellular loop and carboxyl terminus of VR1 were phosphorylated by PKCepsilon. Patch clamp analysis of the point mutants where Ser or Thr residues were replaced with Ala in the total 16 putative phosphorylation sites showed that two Ser residues, Ser(502) and Ser(800) were involved in the potentiation of the capsaicin-evoked currents by either PMA or ATP. In the cells expressing S502A/S800A double mutant, the temperature threshold for activation was not reduced upon PMA treatment. The two sites would be promising targets for the development of substance modulating VR1 function, thereby reducing pain.

The formin homology protein mDia is a Rho GTPase effector protein that participates in stress fiber formation, cytokinesis, and transcriptional activation of the serum response factor. Although the function of another effector of Rho, Rho-associated kinase, is well established, relatively little is known about the functional mechanism and the downstream targets of mDia. Our recent report of a Rho-mDia-Src-tyrosine kinase pathway suggested an important role for mDia in cell adhesion turnover. We identified a new mDia-interacting protein which is expressed ubiquitously. The new protein mainly binds to the proline-rich region of mDia through its Src homology 3 domain and also binds to Grb2 through its proline-rich domain. The protein is localized at the cell periphery and membrane ruffles and co-localizes with mDia. Co-expression of vSrc and the mDia-interacting protein induces significant morphological changes at focal contacts and activation of vSrc. Furthermore, we found that the mDia-interacting protein plays an important role in stress fiber formation induced by active mDia1. Our results suggest that this new protein regulates actin polymerization and cell adhesion turnover in the downstream portion of the Rho-mDia pathway by interacting with Grb2 and Src.

The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg148, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2.1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K+ > Rb+ > NH4+ >> Na+) was preserved. The coexpression made the IRK1 channel more sensitive to extracellular block by Mg2+ and Ca2+, and turned this blockade from a voltage-independent to a -dependent process. The sensitivity of the mutant channels to Mg2+ was enhanced at higher pH and by an increased ratio of mutant:WT mRNA, suggesting that the charge on the Arg site controlled the sensitivity. The blocking rate of open channel blockers, such as Cs+ and Ba2+, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg2+ or Ca2+ and that to the barrier peak for block by Cs+ or Ba2+ suggest that Arg148 is located between the external blocking site for Mg2+ or Ca2+ and the deeper blocking site for Cs+ or Ba2+ in the IRK1 channel. It is concluded that Arg148 serves as a barrier to cationic blockers, keeping Mg2+ and Ca2+ out from the electric field of the membrane.

Using a specific monoclonal antibody (mAb) recognizing the rat interleukin-2 receptor (IL-2R) and immunocytochemical techniques, we examined the expression of IL-2R in rat anterior pituitary and adrenal cells in vitro. IL-2R immunoreactivity was seen in a variety types of cells in both the pituitary and adrenals. Under the condition to detect the IL-2R-like immunoreactivity, we found that overnight preincubation with IL-2 enhanced the immunostaining in these cells. Moreover, co-incubation of the cells with dexamethasone attenuated the effect of IL-2 on the IL-2R-like immunostaining. These findings indicate that IL-2R expression in the pituitary and adrenal cells is influenced by IL-2 and glucocorticoid, and support the hypothesis that IL-2 is an immunomediator affecting the pituitary and the adrenal function.

To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells.

To examine how people react to palatalized articulation, we used one cleft palate speech (CPS) sample of palatalized articulation that was purchased in Japan and one recorded sample of speech from a non-cleft palate individual. Study design The two speech samples were rated by 137 native listeners. Each participant rated the set of speech samples for 10 features using a 10-point scale. Alpha factor analysis was performed. Results Two factors were extracted from the entire set of features with alpha factor analysis. Conclusions Although native listeners could not distinguish between CPS and non-CPS using the psychometrical measurements applied in this study, this method of analyzing speech represents a useful technique for planning treatments in cleft disorder patients.

In the accompanying paper (Nemoto, Y., Namba, T., Teru-uchi, T., Ushikubi, F., Morii, N., and Narumiya, S. (1992) J. Biol. Chem. 267, 20916-20920), we have identified rhoA protein as the sole substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human blood platelets. Here we examined the role of rhoA protein in platelet functions. C3 exoenzyme added to washed platelets dose- and time-dependently ADP-ribosylated rhoA protein in situ in the cells. Concomitant with this modification, inhibition of thrombin-induced platelet aggregation was observed. This inhibition was not reversed by washing the treated platelets, but was not found when C3 exoenzyme was pretreated with mouse monoclonal anti-C3 exoenzyme antibody. C3 exoenzyme treatment did not affect thrombin-induced inositol 1,4,5-trisphosphate production. Secretion of preloaded [14C]serotonin was delayed by the enzyme treatment, but the extent of the secretion was not influenced. In addition, the enzyme treatment did not change the expression of the glycoprotein IIb-IIIa complex on the platelet surface. The enzyme treatment also suppressed platelet aggregation induced by phorbol myristate acetate. These results suggest that rhoA protein plays a role mainly in the aggregation process downstream from receptor-phospholipase C coupling. This, together with the previous finding that rhoA protein modulates stress fiber formation in cultured fibroblasts (Paterson, H. F., Self, A. J., Garrett, M. D., Just, I., Aktories, K., and Hall, A. (1990) J. Cell Biol. 111, 1001-1007), suggests that rhoA protein regulates the assembly of actin filaments and the avidity of the platelet integrin (glycoprotein IIb-IIIa) in the aggregation process.

Prostaglandin E2(PGE2) and prostaglandin I2(PGI2) are major inflammatory mediators that play important roles in pain sensation and hyperalgesia. The role of their receptors (EP and IP, respectively) in inflammation has been well documented, although the EP receptor subtypes involved in this process and the underlying cellular mechanisms remain to be elucidated. The capsaicin receptor TRPV1 is a nonselective cation channel expressed in sensory neurons and activated by various noxious stimuli. TRPV1 has been reported to be critical for inflammatory pain mediated through PKA- and PKC-dependent pathways. PGE2or PGI2increased or sensitized TRPV1 responses through EP1or IP receptors, respectively predominantly in a PKC-dependent manner in both HEK293 cells expressing TRPV1 and mouse DRG neurons. In the presence of PGE2or PGI2, the temperature threshold for TRPV1 activation was reduced below 35°C, so that temperatures near body temperature are sufficient to activate TRPV1. A PKA-dependent pathway was also involved in the potentiation of TRPV1 through EP4and IP receptors upon exposure to PGE2and PGI2, respectively. Both PGE2-induced thermal hyperalgesia and inflammatory nociceptive responses were diminished in TRPV1-deficient mice and EP1-deficient mice. IP receptor involvement was also demonstrated using TRPV1-deficient mice and IP-deficient mice. Thus, the potentiation or sensitization of TRPV1 activity through EP1or IP activation might be one important mechanism underlying the peripheral nociceptive actions of PGE2or PGI2.

The ubiquitously expressed Na–H exchanger, NHE1, acts downstream of RhoA in a pathway regulating focal adhesion and actin stress fiber formation. p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA‐induced assembly of focal adhesions and stress fibers. Here, stress fiber formation induced by p160ROCK was inhibited by the addition of a specific NHE1 inhibitor, ethylisopropylamiloride, in CCL39 fibroblasts, and was absent in PS120 mutant fibroblasts lacking NHE1. In CCL39 cells, NHE1 activity was stimulated by expression of mutationally active p160ROCK, but not by mutationally active protein kinase N, another RhoA target kinase. Expression of a dominant interfering p160ROCK inhibited RhoA‐, but not Cdc42‐ or Rac‐activation of NEH1. In addition, the p160ROCK‐specific inhibitor Y‐27632 inhibited increases in NHE1 activity in response to RhoA, and to lysophosphatidic acid (LPA), which stimulates RhoA, and it also inhibited LPA‐increased phosphorylation of NHE1. A C‐terminal truncation of NHE1 abolished both LPA‐induced phosphorylation and activation of the exchanger. Furthermore, mutationally active p160ROCK phosphorylated an NHE1 C‐terminal fusion protein in vitro , and this was inhibited in the presence of Y‐27632. Phosphopeptide maps indicated that identical residues in NHE1 were phosphorylated by p160ROCK in vivo and in vitro . These findings identify p160ROCK as an upstream, possibly direct, activator of NHE1, and suggest that NHE1 activity and phosphorylation are necessary for actin stress fiber asssembly induced by p160ROCK.

Guinea pig ventricular myocytes exhibit a Cl − -selective current regulated by the cAMP-dependent pathway. We have investigated the distribution of cAMP-activated Cl − channel current density and cystic fibrosis transmembrane-conductance regulator (CFTR) mRNA in three regions of the guinea pig heart: the atrium, and the epicardium and endocardium of the free wall of the left ventricle. The regional differences in the Cl − current density were investigated in enzymatically isolated myocytes using the whole-cell patch-clamp technique. Forskolin (1 μmol/L) activated Cl − -selective currents in all ventricular myocytes and 21% of atrial myocytes examined. The conductance density, estimated as the outward chord conductance normalized to cell capacitance, was greatest in epicardial myocytes (79.8±8.4 pS/pF, n=21) and significantly lower in endocardial (59.8±9.5 pS/pF, n=22) and atrial (10.9±5.0 pS/pF, n=38) myocytes. The regional differences in CFTR mRNA expression levels were investigated by competitive reverse-transcribed polymerase chain reaction. The regional distribution of the mRNA levels was similar to that of the Cl − conductance density, ie, highest in the epicardium (23 230±1840 molecules/μg total RNA, n=3), significantly lower in endocardium (10 610±780 molecules/μg total RNA, n=3), and lowest in atrium (1450±290 molecules/μg total RNA, n=3). The data indicate that regional differences in CFTR mRNA expression in the guinea pig heart are responsible, at least in part, for the regional differences in cAMP-activated Cl − current density.

Neutrophil peptides (NPs, defensins), which consist of approximately 30 amino acids with a highly conserved backbone of six Cys residues, possess several biological activities, such as antimicrobial, antiviral, cytotoxic, and anti-adrenocorticotropin (corticostatic) activity in vitro. In the rabbit, six NPs, i.e., NP-1, -2, -3A, -3B, -4, and -5, have been isolated, among them, NP-3A has the most potent corticostatic activity, which involves the inhibition of adrenocorticotropin-stimulated corticosterone synthesis in adrenal cells. Using a sensitive radioimmunoassay for NP-3A and reverse-phase HPLC, we found a novel component in rabbit tissue extracts with NP-3 A-like immuno-reactivity. We further purified and characterized the component and found two novel peptides. One of these peptides, designated NP-6, has an amino acid sequence identical to that of NP-3A except for three amino acids, i.e. Leu11, Phe13, and Gln15, in the central portion of the sequence. The other has a sequence corresponding to that of NP-6 except that the N-terminal Gly was deleted and is thus named des-G1-NP-6. Using chemically synthesized NP-6 and des-G1-NP-6, we measured their putative corticostatic activities by a dispersed rat adrenal cell bioassay. NP-6 showed corticostatic activity comparable to that of NP-3 A while des-G1-NP-6 showed weak activity. These findings are also compatible with the notion that the N-terminal Gly, but not the central portion, is important for the anti-adrenocorticotropin activity of the NPs.

Botulinum C3 exoenzyme specifically ADP-ribosylates a group of ras-related small molecular weight GTP-binding proteins, rho, and inhibits their biological activity. Using this enzyme, we examined the function of rho in PMA-induced activation of lymphocyte function-associated antigen-1 (LFA-1) in a B lymphoblastoid cell line, JY. Northern blot analysis revealed that among the three rho genes, rhoA mRNA was predominantly expressed in JY cells. Consistently, only one [32P]ADP-ribosylated band was found when the lysate of the cells was subjected to ADP ribosylation by C3 exoenzyme. When the cells were cultured with C3 exoenzyme, this substrate was ADP-ribosylated in situ in a time- and concentration-dependent manner. Concomitant with this ADP ribosylation, PMA-induced LFA-1/intercellular adhesion molecule (ICAM)-1-dependent aggregation of JY cells was inhibited. This inhibition was blocked by prior treatment of the enzyme with an anti-C3 monoclonal antibody, and overcome by stimulation with higher concentrations of PMA. The C3 exoenzyme-induced inhibition was not affected by shaking of the cell suspension, while inhibition of aggregation by cytochalasin B was abolished by this procedure, suggesting that the inhibitory effect of the C3 exoenzyme treatment was not due to decrease in cell motility. The C3 exoenzyme treatment affected neither protein phosphorylation in JY cells before and after PMA stimulation, nor affected surface expression of LFA-1 and ICAM-1. These results suggest that rhoA protein works downstream of protein kinase C activation linking PMA stimulation to LFA-1 activation and aggregation in JY cells.

Therapeutic applications of hypothalamic hypophysiotropic hormones were discussed. Pulsatile administration of gonadotropin-releasing hormone (Gn RH) is effective to maintain menstrual cycles and ovulation in female patients with hypothalamic gonadal dysfunction and testicular function in male patients. On the other hand, continuous administration of Gn-RH or its analog is able to suppress gonadal function and thereby gonadal hormonedependent activities effectively. Application of this medical gonadectomy includes treatments for excessive menstrual bleeding, endometriosis, prostatic cancer and benign prostatic hypertrophy. Thyrotropin-releasing hormone has been applied in patients with spinocerebellar degeneration or some kinds of consciousness disturbance and somatostatin analog has benn tried to treat acromegaly. Corticotropin-releasing hormone and growth hormone-releasing hormone have also therapeutic potentials. We believe that many-sided research trials, which include development of new potent analogs, new effective drug delivery systems and further elucidation of physiology and pathophysiology about the functions of the hypothalamic hormones, are inevitable to materialize therapeutic potentials of these hormones.

To study the cellular mechanisms of interleukin-1 (IL-1) in the pituitary corticotroph, we studied the properties of IL-1 receptors on a mouse pituitary ACTH-producing cell line, AtT-20. Scatchard plot analysis revealed a single type of receptor with a Kd (dissociation constant) of 93 pM, and 482 binding sites/cell. [125I]IL-1 alpha binding competed with IL-1 alpha and IL-1 beta in an equimolar fashion. A 24 h pre-incubation with either CRH, epinephrine or nor-epinephrine increased the [125I]IL-1 alpha binding sites in the AtT-20 cells and conversely, a similar pre-incubation with either dexamethasone or tumour necrosis factor-alpha (TNF alpha) decreased them without affecting the affinity of the receptors in either case.

腎奇形およびgrowth hormone(以下GH)分泌障害を伴ったKallmann症候群を報告する. 18才,男性.二次性徴遅延,低身長,肥満を主訴に入院.嗅覚低下,停留睾丸,右腎低形成,左重複腎盂重複尿管,慢性糸球体腎炎を伴っていた.内分泌検査上,血漿LH, FSH,テストステロンは低値で, clomiphene試験, LH-RH試験に対し, LH, FSHは無反応であったが, LH-RH連続負荷試験により反応性の改善が認められ,視床下部性性機能低下症の所見を呈していた. GHの基礎値も低値で,インスリン負荷およびアルギニン負荷に対し無反応であったが, GRF試験ではわずかな反応を認めた. Kallman症候群に合併したGH分泌障害,肥満に関し,視床下部障害に起因する可能性もあり,考察を加えた.

We examined effects of sodium valproate, a gamma amino butyric acid (GABA)-transaminase inhibitor, on the secretion of immunoreactive (IR)-ACTH and IR-beta-endorphin/LPH from cultured rat anterior pituitary cells to determine whether sodium valproate has a direct action on the secretion of ACTH and its related peptides from the cultured rat anterior pituitary gland. During the 3 h incubation, the basal secretion of IR-ACTH and IR-beta-endorphin/LPH decreased to 50.8% and 58.3%, respectively, of the control concentration after adding 10(-7) M sodium valproate into the incubation media and to 67.7% and 69.3%, respectively, of the control levels with 10(-8) M sodium valproate. However, sodium valproate at a concentration of 10(-6) M or 10(-9) M did not affect the basal concentration of IR-ACTH and IR-beta-endorphin/LPH. Sodium valproate at a concentration of 10(-7) M significantly attenuated the stimulated release of IR-ACTH and IR-beta-endorphin/LPH by 10(-9) or 10(-10) M of ovine corticotrophin releasing factor. These results indicate that sodium valproate could directly effect rat anterior pituitary cells to suppress both basal and stimulated release of proopiomelanocortin derived peptides and this supports the hypothesis that sodium valproate has a direct effect at the pituitary corticotroph in reducing plasma ACTH.

We have examined the role of the mouse Diaphanous-related formin (DRF) Rho GTPase binding proteins, mDia1 and mDia2, in cell regulation. The DRFs are required for cytokinesis, stress fiber formation, and transcriptional activation of the serum response factor (SRF). 'Activated' mDia1 and mDia2 variants, lacking their GTPase binding domains, cooperated with Rho-kinase or ROCK to form stress fibers but independently activated SRF. Src tyrosine kinase associated and co-localized with the DRFs in endosomes and in mid-bodies of dividing cells. Inhibition of Src also blocked cytokinesis, SRF induction by activated DRFs, and cooperative stress fiber formation with active ROCK. Our results show that the DRF proteins couple Rho and Src during signaling and the regulation of actin dynamics.