Comparison of osseous healing after sagittal split ramus osteotomy and intraoral vertical ramus osteotomy.

The sagittal split ramus osteotomy (SSRO) is generally associated with greater postoperative stability than the intraoral vertical ramus osteotomy (IVRO); however, it entails a risk of inferior alveolar nerve damage. In contrast, IVRO has the disadvantages of slow postoperative osseous healing and projection of the antegonial notch, but inferior alveolar nerve damage is believed to be less likely. The purposes of this study were to compare the osseous healing processes associated with SSRO and IVRO and to investigate changes in mandibular width after IVRO in 29 patients undergoing mandibular setback. On computed tomography images, osseous healing was similar in patients undergoing SSRO and IVRO at 1year after surgery. Projection of the antegonial notch occurred after IVRO, but returned to the preoperative state within 1year. The results of the study indicate that IVRO is equivalent to SSRO with regard to both bone healing and morphological recovery of the mandible.

Mechanism of motor coordination of masseter and temporalis muscles for increased masticatory efficiency in mice.

The demand for the use of mice as animal models for elucidating the pathophysiologies and pathogeneses of oral motor disorders has been increasing in recent years, as more and more kinds of genetically modified mice that express functional disorders of the stomatognathic system become available. However, the fundamental characteristics of mouse jaw movements during mastication have yet to be fully elucidated. The purpose of this study was to investigate the roles of the masseter and temporalis muscles, and the mechanisms of motor coordination of these muscles for increasing masticatory efficiency in the closing phase in mice. Twenty-two male Jcl:ICR mice were divided into control (n = 8), masseter-hypofunction (n = 7) and temporalis-hypofunction groups (n = 7). Botulinum neurotoxin type A (BoNT/A) was used to induce muscle hypofunction. The masticatory movement path in the horizontal direction during the occlusal phase became unstable after BoNT/A injection into the masseter muscle. BoNT/A injection into the temporalis muscle decreased antero-posterior excursion of the late-closing phase corresponding to the power phase of the chewing cycle. These results suggest that the masseter plays an important role in stabilizing the grinding path, where the food bolus is ground by sliding the posterior teeth from back to front during the occlusal phase. The temporalis plays a major role in retracting the mandible more posteriorly in the early phase of closing, extending the grinding path. Masticatory efficiency is thus increased based on the coordination of activities by the masseter and temporalis muscles.

Affinity imaging mass spectrometry (AIMS): high-throughput screening for specific small molecule interactions with frozen tissue sections.

A novel screening system, using affinity imaging mass spectrometry (AIMS), has been developed to identify protein aggregates or organ structures in unfixed human tissue. Frozen tissue sections are positioned on small (millimetre-scale) stainless steel chips and incubated with an extensive library of small molecules. Candidate molecules showing specific affinity for the tissue section are identified by imaging mass spectrometry (IMS). As an example application, we screened over a thousand compounds against Alzheimer's disease (AD) brain tissue and identified several compounds with high affinity for AD brain sections containing tau deposits compared to age-matched controls. It should also be possible to use AIMS to isolate chemical compounds with affinity for tissue structures or components that have been extensively modified by events such as oxidation, phosphorylation, acetylation, aggregation, racemization or truncation, for example, due to aging. It may also be applicable to biomarker screening programs.

Effects of feeding a soft diet and subsequent rehabilitation on the development of the masticatory function.

It has been suggested that feeding a soft diet could possibly inhibit normal development of the masticatory function. However, the consequences of such changes in the alimentary habits have yet to be fully clarified. Therefore, the aim of this study was to determine whether a soft diet prevents the development of masticatory function and whether a critical period for programming the masticatory system exists. To examine these hypotheses, we used a three-dimensional jaw-movement tracking device and jaw muscle electromyography (EMG) to analyse masticatory function changes in mice. Jcl:ICR mice were divided into three groups, with the normal group fed a hard diet, the hypofunctional group fed a soft diet, and the rehabilitation group first fed a soft diet that was then changed to a hard diet. Our results showed that the excursion and duration of late-closing phase (occlusal phase) of the chewing cycle and EMG activity in the masseter muscle were not only reduced in the hypofunctional but also in the rehabilitation group as compared with the normal group. These results suggest that optimisation of the chewing pattern and acquisition of appropriate masticatory function are impeded by feeding a soft diet during the animal's growth period and that no catch-up effect of the masticatory function is observed when there is a prolonged period of time prior to changing the diet from soft to hard. In conclusion, masticatory function can only be fully developed through a learning process such as exposure to chewing various kinds of foods with different food textures.

Association between hydration volume and symptoms in terminally ill cancer patients with abdominal malignancies.

BACKGROUND: To explore the association between hydration volume and symptoms during the last 3 weeks of life in terminally ill cancer patients. PATIENTS AND METHODS: This was a multicenter, prospective, observational study of 226 consecutive terminally ill patients with abdominal malignancies. Primary responsible physicians and nurses evaluated the severity of membranous dehydration (dehydration score calculated from three physical findings), peripheral edema (edema score calculated from seven physical findings), ascites and pleural effusion (rated as physically undetectable to symptomatic), bronchial secretion, hyperactive delirium (Memorial Delirium Assessment Scale), communication capacity (Communication Capacity Scale), agitation (Agitation Distress Scale), myoclonus and bedsores. RESULTS: Patients were classified into two groups: the hydration group (n=59) who received 1 l or more of artificial hydration per day, 1 and 3 weeks before death, and the non-hydration group (n=167). The percentage of patients with deterioration in dehydration score in the final 3 weeks was significantly higher in the non-hydration group than the hydration group (35% versus 14%; P=0.002), while the percentages of patients whose symptom scores for edema, ascites and pleural effusion increased were significantly higher in the hydration group than the non-hydration group (44% versus 29%, P=0.039; 29% versus 8.4%, P <0.001; 15% versus 5.4%, P=0.016; respectively). After controlling for multiple covariates and treatment settings, the association between hydration group and dehydration/ascites score was statistically significant. Subgroup analysis of patients with peritoneal metastases identified statistically significant interaction between hydration group and dehydration/pleural effusion score. There were no significant differences in the degree of bronchial secretion, hyperactive delirium, communication capacity, agitation, myoclonus or bedsores. CONCLUSIONS: Artificial hydration therapy could alleviate membranous dehydration signs, but could worsen peripheral edema, ascites and pleural effusions. It is suggested that the potential benefits of artificial hydration therapy should be balanced with the risk of worsening fluid retention symptoms. Further clinical studies are strongly needed to identify the effects of artificial hydration therapy on overall patient well-being, and an individualized treatment and close monitoring of dehydration and fluid retention symptoms is strongly recommended.

Purification and characterization of a monoacylglycerol lipase from Pseudomonas sp. LP7315.

A monoacylglycerol lipase (MGL) was purified from Pseudomonas sp. LP7315 by ammonium sulfate precipitation, anion-exchange chromatography, and preparative electrophoresis. The purified enzyme was homogeneous on SDS-PAGE with a molecular mass of 59 kDa. Its hydrolytic activity was confirmed to be specific for monoglycerides: the enzyme did not hydrolyze di- and triglycerides. MGL was found to be stable even after 1-h incubation at 65 degrees C. The optimum pH for monopalmitin hydrolysis was approximately 8. The hydrolytic activity depended not only on temperature and pH but also on the type of monoglyceride used. MGL also catalyzed monoglyceride synthesis at 65 degrees C in a solvent-free two-phase system, in which fatty acid droplets were dispersed in the glycerol phase with a low water content. The synthetic reaction proceeded at a constant rate for approximately 24 h and approximately reached an equilibrium after 48 h of reaction. The initial rate and equilibrium yield of the synthetic reaction depended on the type of fatty acid used as the substrate.

Analysis of monoglyceride synthetic reaction in a solvent-free two-phase system catalyzed by a monoacylglycerol lipase from Pseudomonas sp. LP7315.

Kinetics of monoglyceride synthesis catalyzed by a monoacylglycerol lipase (MGL) isolated from Pseudomonas sp. LP7315 was studied at 65 degrees C in a solvent-free two-phase system, in which fatty acid droplets were dispersed in a glycerol phase containing a small amount of water. The initial rate of the synthetic reaction depended on several factors: the amounts of fatty acid and glycerol, and the concentration of MGL in the glycerol phase. To analyze the effects of these factors, a kinetic model was developed based on the assumption that the adsorption equilibrium of MGL molecules at the interface between the two phases is the crucial factor for the synthetic reaction. The model was found to yield good approximations of the initial synthetic rate under various reaction conditions. The analysis suggests that the adsorption behavior of MGL onto the interface had a large effect on the initial rate of the monoglyceride synthesis.

Hypoxic induction of prolyl 4-hydroxylase alpha (I) in cultured cells.

Accumulated evidence indicates that hypoxia activates collagen synthesis in tissues. To explore the molecular mechanism of activation, we screened genes that are up-regulated or down-regulated by hypoxia. Fibroblasts isolated from fetal rat lung were cultured under hypoxia. Differential display technique showed that the mRNA level of prolyl 4-hydroxylase (PH) alpha(I), an active subunit that catalyzes the oxygen-dependent hydroxylation of proline residue in procollagen, increased 2-3-fold after an 8-h exposure to hypoxia. This elevated level was maintained over 40 h and returned to the basal level after reoxygenation. The transcription rate, protein level, and hydroxyproline content (an indicator of the prolyl hydroxylation) were all elevated by hypoxic culture. Analysis of the promotor region of PHalpha(I) gene indicated that a motif similar to hypoxia-responsive element (HRE) of hypoxia-inducible genes such as erythropoietin, was identified within a 120-base pair sequence upstream of the transcription start site. Luciferase reporter assay and mutational analysis showed that a site similar to the HRE in this motif is functionally essential to hypoxic response. Electrophoretic mobility shift assay revealed that hypoxia-inducible factor-1 was stimulated and bound to the PHalpha(I) HRE upon hypoxic challenge. Our results indicate that PHalpha(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.

New estrogenic antagonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore.

We have designed and synthesized estrogen antagonists bearing dicarba-closo-dodecaborane (carborane) as a hydrophobic pharmacophore based on the structure of 1-(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane, a potent estrogen agonist that we reported previously. Compounds with a long alkyl chain bearing an amide moiety on the carborane skeleton (6, 7) showed estrogen antagonistic activity in a luciferase reporter gene assay using COS-1 cells transfected with a rat ER alpha-expression plasmid and as an appropriate reporter plasmid.

Changes in lung-specific molecular expression during differentiation of hamster embryonic M3E3/C3 cell line.

To clarify the differentiation mechanisms of bronchiolar epithelial cells, changes at the transcription level of epithelial cell-specific proteins were examined using M3E3/C3, a cell line derived from hamster fetal lung. During a 9-day incubation period with 24 microg/mL of retinol, the cells became attached to each other and formed large bud-like structures which could be detected with periodic acid-Schiff staining. During the incubation period, the mRNA level of surfactant-associated protein-B significantly increased 2.6- and 5.4-fold higher than cells incubated without retinol on days 3 and 9, respectively. The Clara cell-specific secretory protein mRNA level also increased and peaked at 5.1-fold (P < 0.05) on day 5 compared with control cells. In contrast, mRNA for surfactant-associated protein-C, an alveolar type II cell-specific protein, decreased. Moreover, the expression of the gene for hepatocyte nuclear factor 3alpha, a putative transactivating factor for lung-related genes, was up-regulated resulting in consistently higher levels (2.4- to 5.6-fold) compared with controls, while those for transmembrane-type mucin-1 and glyceraldehyde-3-phosphate dehydrogenase were constantly expressed during the incubation. The present study confirms that at the gene transcription level M3E3/C3 cells differentiate into Clara-like cells with mucus granules in the presence of retinol.

Protein kinase C modulators bearing dicarba-CLOSO-dodecaborane as a hydrophobic pharmacophore.

The size and position of a hydrophobic moiety on a benzolactam skeleton, which reproduces the active conformation and biological activity of teleocidins, play an important role in the appearance of the activity. We have designed and synthesized benzolactams bearing dicarba-closo-dodecaborane. These compounds showed potent binding affinity to protein kinase C, providing a further example of the application of carborane as the hydrophobic pharmacophore of biologically active molecules.

Urocortin in human placenta and maternal plasma.

Plasma immunoreactive (IR-) urocortin (Ucn) and corticotropin-releasing factor (CRF) levels in pregnant women were measured by their specific radioimmunoassays after extraction. Although plasma IR-CRF levels were increased in pregnant women as compared to men and non-pregnant women, there was no difference of plasma IR-Ucn levels among groups. Ucn mRNA was detected in cytotrophoblasts and syncytiotrophoblasts by in situ hybridization. A reverse-phase high-performance liquid chromatography (HPLC) showed the major peak of IR-Ucn in placenta and plasma that had similar chromatographic mobility to synthetic Ucn1-40. These data suggest that Ucn is produced and processed into the same form of synthetic Ucn in placenta, but not secreted into maternal blood.

Serum thrombopoietin levels in patients with chronic hepatitis and liver cirrhosis.

OBJECTIVE: Thrombocytopenia is a common manifestation of cirrhosis. The aim of this study was to examine the relationship between serum thrombopoietin concentrations, circulating platelet levels, and the stage of hepatic fibrosis in patients with chronic viral hepatitis. METHODS: The study included 48 patients with chronic viral hepatitis (14 with stage 1 fibrosis; five with stage 2 fibrosis; three with stage 3 fibrosis; 26 with cirrhosis) and 30 healthy volunteers. Serum thrombopoietin levels were measured using an enzyme-linked immunosorbent assay. Spleen size, platelet counts, and prothrombin time were measured. RESULTS: Thrombopoietin levels of patients with fibrosis stage 1 (2.50 +/- 1.60 fmol/ml) or stage 2 (1.89 +/- 0.65) were significantly higher than those in patients with cirrhosis (1.21 +/- 0.55) or healthy volunteers (1.26 +/- 0.74). Mean platelet counts of patients with cirrhosis (8.0 +/- 4.6 x 10(4)/microl) were significantly lower than those with fibrosis stage 1 (18.6 +/- 3.9) or stage 2 (16.0 +/- 5.8), or healthy volunteers (24.5 +/- 7.3). Patients with cirrhosis had larger spleens (30.9 +/- 18.4 cm2) than those with fibrosis stage 1 (18.2 +/- 6.4). Platelet counts showed a significant inverse relationship to spleen size (p = -0.51, p < 0.0005) and a significant positive relationship with thrombopoietin levels (p = 0.34, p < 0.02). Thrombopoietin levels were significantly correlated to prothrombin time (p = 0.45, p < 0.005). CONCLUSIONS: Serum thrombopoietin levels are elevated in patients with an early stage of chronic viral hepatitis. As the disease progresses from mild fibrosis to cirrhosis, decreased production of thrombopoietin may contribute to the further development of thrombocytopenia in cirrhosis.

OPC-13013, a cyclic nucleotide phosphodiesterase type III, inhibitor, inhibits cell proliferation and transdifferentiation of cultured rat hepatic stellate cells.

Activated hepatic stellate cells (HSC; lipocytes; Ito cells) proliferate and are responsible for extracellular matrix synthesis during hepatic fibrogenesis. During activation, HSC undergo transdifferentiation into myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA). Adenosine 3', 5'-cyclic monophosphate (cyclic AMP) is an ubiquitous intracellular signaling molecule, and is upregulated by the activation of adenylate cyclase and downregulated via hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). Recently, increased intracellular cyclic AMP has been shown to inhibit HSC activation. The aim of the current study was to determine the effects of inhibition of PDEs on cell proliferation and transdifferentiation in cultured rat HSC. Cell proliferation was determined by [3H]thymidine incorporation, and Western blot analysis was performed for detection of alpha-SMA, a phenotypic marker of transdifferentiation into myofibroblast. When the cells were exposed to 3-isobutyl-1-methylxanthine (IBMX; 50-1000 microM), a nonselective PDE inhibitor, serum-stimulated [3H]thymidine incorporation was suppressed in a dose-dependent manner with a maximum inhibition of 66% at a concentration of 500 microM OPC-13013 (1-60 microM), a selective PDE III isoenzyme inhibitor, induced a dose-dependent inhibitory effect on serum-stimulated DNA synthesis that reached a maximum inhibition of 95% at a concentration of 60 microM, while neither 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX), a PDE I isoenzyme inhibitor, nor Ro-20-1724, a PDE IV isoenzyme inhibitor, had an inhibitory effect. Western blot analysis revealed that IBMX or OPC-13013 decreased alpha-SMA expression, while other selective PDE isoenzyme inhibitors did not have a suppressive effect. IBMX, OPC-13013 or Ro-20-1724, but not 8-MMX augmented forskolin-induced increase in intracellular cyclic AMP levels although cyclic AMP levels were not affected by treatment with any of these PDE inhibitors alone. These data indicate that inhibition of PDEs, especially PDE III isoenzyme, can produce an inhibitory effect on HSC activation. The PDE III isoenzyme may contribute to the regulation of HSC activation during fibrogenesis. In addition, OPC-13013 may have the potential to inhibit initiation and progression of hepatic fibrosis by interfering with HSC activation.

Urocortin expression in the human central nervous system.

OBJECTIVE AND STUDY DESIGN: Urocortin is a recently identified neuropeptide of the corticotrophin-releasing factor (CRF) family in the mammalian brain and has been demonstrated to stimulate ACTH secretion from pituitary cells, but its expression in human brain tissue including the hypothalamus has not been examined. In this study, we first examined urocortin expression in the hypothalamus (20 cases) and pituitary stalks (17 cases) of human brain obtained from autopsy using immunohistochemistry and mRNA in situ hybridization. RESULTS: Neither urocortin immunoreactivity nor mRNA hybridization signals were detected in the hypothalami and pituitary stalks while CRF immunoreactivity was detected in the paraventricular nuclei of the hypothalami in 10/20 cases and in nerve fibres of the stalks in 17/17 cases. These results indicate that urocortin does not act on the hypothalamo-pituitary-adrenal axis, at least not in the same manner as CRF in humans. We then examined urocortin expression in various portions of the brain in 7 cases. Both urocortin immunoreactivity and mRNA hybridization were detected in Purkinje cells of the cerebellum and anterior horn cells of the spinal cord in specimens examined. Urocortin expression was, however, variably seen in superior olivary nuclei (two out of six cases examined) and in the Edingar-Westphal nuclei (one out of three cases examined). CONCLUSIONS: The distribution of urocortin in the human central nervous system suggests that urocortin may work as a neurotransmitter like other neuropeptides in the human.

Involvement of Arg-328, Arg-334 and Arg-342 of DnaA protein in the functional interaction with acidic phospholipids.

We reported previously that three basic amino acids (Arg-360, Arg-364 and Lys-372) of DnaA protein are essential for its functional interaction with cardiolipin. In this study, we examined the effect of mutation of some basic amino acids in a potential amphipathic helix (from Lys-327 to Ile-345) of DnaA protein on this interaction. ATP binding to the mutant DnaA protein, in which Arg-328, Arg-334 and Arg-342 were changed to acidic amino acids, was less inhibited by cardiolipin than that of the wild-type protein, as was the case for mutant DnaA protein with mutations of Arg-360, Arg-364 and Lys-372. A mutant DnaA protein with mutations of all six basic amino acids showed the most resistance to the inhibition of ATP binding by cardiolipin. These results suggest that Arg-328, Arg-334 and Arg-342, like Arg-360, Arg-364 and Lys-372, are also involved in the functional interaction between DnaA protein and acidic phospholipids.

Mechanism involved in synergistic adrenocorticotropin response to activating protein kinase-A and -C in rat anterior pituitary cells.

Activation of protein kinase C (PKC) stimulates adrenocorticotropin (ACTH) release synergistically in the presence of corticotropin releasing factor (CRF). We examined the effect of a cyclic nucleotide-specific phosphodiesterase inhibitor, 1-isoamyl-3-isobutylxanthine (IIX), on arginine vasopressin (AVP)-induced ACTH release and intracellular cAMP accumulation in normal rat anterior pituitary cells. IIX alone elevated intracellular cAMP accumulation. IIX potentiated AVP-induced ACTH release synergistically without further increase in cAMP accumulation, suggesting that synergistic ACTH release has an alternative mechanism other than the synergistic elevation of intracellular cAMP accumulation which has been reported. Phorbol 12-myristate-13-acetate (PMA) also induced synergistic ACTH release when incubated with IIX. IIX had no additional effect on ACTH response when incubated with maximal dose of CRF, forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Moreover, the combination of PMA and 8-Br-cAMP produced synergistic ACTH response. In conclusion, the synergistic ACTH release from rat pituitary corticotrophs occurs at least in the presence of directly activating events of PKC and PKA as well as PKC-induced inhibition of phosphodiesterase activity.

Corticotropin-releasing factor but not urocortin is involved in adrenalectomy-induced adrenocorticotropin release.

Urocortin, a new corticotropin-releasing factor (CRF)-related peptide, has been reported to have the ability to bind to CRF receptors and to stimulate adrenocorticotropin (ACTH) secretion from the rat anterior pituitary in vivo and in vitro. In this study, we examined the effect of intravenous administration of urocortin-antiserum to investigate the role of endogenous urocortin on ACTH secretion from rat anterior pituitary after adrenalectomy. Male Sprague-Dawley rats, which were maintained in a conscious and undisturbed condition, were administered non-immunized rabbit serum (NRS), CRF-antiserum or urocortin-antiserum at a volume of 1 ml/kg b.w. 15 min before the injection of secretagogues. Synthetic rat urocortin (2 microg/kg B.W.) increased plasma ACTH concentrations by about sixfold the basal concentration. The pretreatment with urocortin-antiserum but not CRF-antiserum abolished the urocortin-induced increase in plasma ACTH concentrations. In adrenalectomized rats, plasma ACTH concentrations were markedly increased at basal conditions, and rapidly reduced after the administration of CRF-antiserum. By contrast, administration of urocortin-antiserum did not alter ACTH secretion induced by adrenalectomy. Our results suggest that endogenous urocortin is unlikely to be involved in ACTH release in adrenalectomized rats.

Estrogenic antagonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore.

Dicarba-closo-dodecaboranes (carboranes), which have spherical geometry and hydrophobicity, are applicable as a hydrophobic pharmacophore of biologically active molecules. We have designed and synthesized estrogenic antagonists based on the structure of the potent agonist 1-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-d odecaborane, which we have developed. The compounds showed potent antagonistic activity in luciferase reporter gene assay using COS-1 cells transfected with rat ER alpha-expression plasmid and an appropriate reporter plasmid.

Site-directed mutational analysis for the membrane binding of DnaA protein. Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, interacts with acidic phospholipids, such as cardiolipin, and its activity seems to be regulated by membrane binding in cells. In this study we introduced site-directed mutations at the positions of hydrophobic or basic amino acids which are conserved among various bacteria species and which are located in the putative membrane-binding region of DnaA protein (from Asp357 to Val374). All mutant DnaA proteins showed much the same ATP and ADP binding activity as that of the wild-type protein. The release of ATP bound to the mutant DnaA protein, in which three hydrophobic amino acids were mutated to hydrophilic ones, was stimulated by cardiolipin, as in the case of the wild-type protein. On the other hand, the release of ATP bound to another mutant DnaA protein, in which three basic amino acids were mutated to acidic ones, was not stimulated by cardiolipin. These results suggest not only that the region is a membrane-binding domain of DnaA protein but also that these basic amino acids are important for the binding and the ionic interaction between the basic amino acids and acidic residues of cardiolipin and is involved in the interaction between DnaA protein and cardiolipin.