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.

An anti-c-Fms antibody inhibits orthodontic tooth movement.

Orthodontic force induces osteoclastogenesis in vivo. It has recently been reported that administration of an antibody against the macrophage-colony-stimulating factor (M-CSF) receptor c-Fms blocks osteoclastogenesis and bone erosion induced by tumor necrosis factor-alpha (TNF-alpha) administration. This study aimed to examine the effect of an anti-c-Fms antibody on mechanical loading-induced osteoclastogenesis and osteolysis in an orthodontic tooth movement model in mice. Using TNF receptor 1- and 2-deficient mice, we showed that orthodontic tooth movement was mediated by TNF-alpha. We injected anti-c-Fms antibody daily into a local site, for 12 days, during mechanical loading. The anti-c-Fms antibody significantly inhibited orthodontic tooth movement, markedly reduced the number of osteoclasts in vivo, and inhibited TNF-alpha-induced osteoclastogenesis in vitro. These findings suggest that M-CSF plays an important role in mechanical loading-induced osteoclastogenesis and bone resorption during orthodontic tooth movement mediated by TNF-alpha.

An uncommon cleft subtype of unilateral cleft lip and palate.

The finding that the vomer plays a crucial role in maxillary growth suggests that the bilateral cleft configuration of unilateral cleft lip and palate (UCLP), in which the vomer is detached from the non-cleft-side secondary hard palate, negatively influences palatal development, and this hypothesis was tested. Sixty persons with complete UCLP, including those with the vomer detached from (n = 30, b-UCLP) and attached to (n = 30, u-UCLP) the secondary hard palate, were analyzed morphologically, with the use of cast models taken at 10 days, 3 mos, and 12 mos of age. The anterio-posterior palatal length at 12 mos of age in those with b-UCLP was significantly shorter than that in those with u-UCLP, by 8.7% (p < 0.05). In addition, palatal width development in the first year in those with b-UCLP was also significantly retarded. These results suggest that the uncommon bilateral cleft subtype in UCLP should be included in the cleft classification.

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.

Negative regulation of catalase gene expression in hepatoma cells.

For an understanding of the molecular basis of the marked decrease in catalase activity of various tumor cells, expression of the catalase gene was studied in rat and human hepatoma cell lines and in rat liver, which was used as a control with high activity. RNA blot hybridization profiles and run-on assays indicated that the decrease in catalase activity was due to depression of catalase gene transcription. Chloramphenicol acetyltransferase (CAT) assays for the fragments with various lengths of the 5'-flanking region (up to -4.5 kb from the ATG codon) of the catalase gene revealed the presence of several cis-acting elements involved in the negative regulation of transcription. The most-upstream element with the strongest activity (-3504 to -3364 bp), when linked to the catalase promoter region (-126 bp) of the CAT construct and subjected to an in vitro transcription assay, did not yield transcripts in experiments with the hepatoma nuclear extract, whereas the unlinked template did yield transcripts. A gel shift competition assay using hepatoma nuclear extract showed the core sequence of the silencer element to be 5'-TGGGGGGAG-3'. A homology search found that the same core sequence was also present in 5'-flanking regions of the albumin gene and of some other liver enzyme genes, the expression of which has been reported to be down regulated in some hepatoma cells. Southwestern (DNA-protein) analysis demonstrated that an approximately 35-kDa nuclear protein bound to the silencer element was present in hepatoma cells but not in rat liver cells.

Therapeutic vaccination based on side population cells transduced by the granulocyte-macrophage colony-stimulating factor gene elicits potent antitumor immunity.

Among cancer immunotherapies, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccine (GVAX) therapies appear promising and have been shown to be safe and effective in multiple clinical trials. However, the antitumor efficacies of GVAX therapy alone are in some cases limited. Here we showed that GVAX therapy targeting cancer stem cells (CSCs) substantially suppressed tumor development in syngeneic immunocompetent mice recapitulating normal immune systems. CSCs were isolated as side population (SP) cells from 4T1 murine breast carcinoma cell line and transduced with GM-CSF gene delivered by non-transmissible Sendai virus (4T1-SP/GM). Impaired tumorigenicity of subcutaneously injected 4T1-SP/GM depended on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Mice therapeutically vaccinated with irradiated 4T1-SP/GM cells had markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with those treated with irradiated cells of non-transduced 4T1-SP cells or non-SP (4T1-NSP/GM) cells. Tumor suppression was accompanied by the robust accumulation of mature dendritic cells at vaccination sites and T-helper type 1-skewed systemic cellular immunity. Our results suggested that CSC cell-based GVAX immunotherapy might be clinically useful for inducing potent tumor-specific antitumor immunity.

Stereospecific removal of the 16 alpha-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol from pregnenolone.

[16 alpha-2H]Pregnenolone was synthesized by catalytic deuteriation of 3 beta-hydroxy-5,16-pregnadien-20-one followed by base-catalyzed back exchange of the 17 alpha-2H atom, and [16 beta-2H]pregnenolone by catalytic hydrogenation of 3 beta-hydroxy-5,16-[16-2H]pregnadien-20-one, which had been synthesized from [16,16-2H]dehydroepiandrosterone. The labelled pregnenolones were incubated separately with the microsomal fraction of boar testis. The metabolites were analyzed by gas chromatography-mass spectrometry, and the isotope compositions of the following six metabolites were determined: 17-hydroxypregnenolone, dehydroepiandrosterone, 5-androstene-3 beta,17 alpha-diol, 5-androstene-3 beta,17 beta-diol,16 alpha-hydroxypregnenolone and 5,16-androstadien-3 beta-ol. The first four metabolites derived either from [16 alpha-2H]- or from [16 beta-2H]pregnenolone showed essentially the same isotope compositions as those of their respective precursors. The 16 alpha-hydroxypregnenolone and the 5,16-androstadien-3 beta-ol biosynthesized from [16 alpha-2H]pregnenolone lost the 2H label, while the same metabolites biosynthesized from [16 beta-2H]pregnenolone retained the albel. The result shows that the 16 alpha-hydrogen is stereospecifically removed with the retention of the 16 beta-hydrogen in the biosynthesis of 5,16-androstadien-3 beta-ol.

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system.

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.

Conjugation types of 7 beta-hydroxylated bile acids detected in healthy human urine.

Bile acids extracted from the urine of a healthy volunteer who excreted 7 beta-hydroxylated bile acids were fractionated to nonamidated, glycine-conjugated, taurine-conjugated, and sulfated bile acid fractions. The chemical conjugation types of the 7 beta-hydroxylated bile acids were then determined by treatment with several enzymes and by capillary column gas chromatography. Large amounts of 3 alpha,7 beta,12 alpha-trihydroxycholanoic acid were present as nonamidated and nonconjugated bile acids, while 3 beta,7 beta-dihydroxycholanoic acid formed nonamidated bile acid N-acetylglucosaminide. In addition, ursodeoxycholic acid formed both glycine-conjugated bile acid and glycine-conjugated bile acid N-acetylglucosaminide. Bile acid N-acetylglucosaminides were hydrolyzed by solvolysis.

Studies on the phosphorylation of a M(r) 25,000 protein, a putative protein phosphatase 2A modulator, by casein kinase I, and analysis of multiple endogenous phosphates.

A M(r) 25,000 protein, which was isolated from the cytosolic fraction of Xenopus laevis oocytes, is a newly identified substrate for casein kinase II and protein kinase C [Hashimoto et al. (1995) J. Biochem. 118, 453-460], and was recently shown to have the ability to modulate protein phosphatase 2A activity [Hashimoto et al. (1996) J. Biochem. 119, 626-632]. Acid phosphatase treatment of the protein shifted its electrophoretic mobility from 25 to 20 kDa on SDS-PAGE. The content of alkali-labile phosphate bound covalently to the protein was 53 mol per mol of M(r) 25,000 protein. Amino acid composition analysis revealed that there are 50 serine residues and 6 threonine residues per mol of this protein. Therefore, this M(r) 25,000 protein seems to be highly phosphorylated in vivo. The M(r) 25,000 protein, once partially dephosphorylated by acid phosphatase, served as an efficient substrate for casein kinase I and casein kinase II. When entirely dephosphorylated, the M(r) 25,000 protein was used as a substrate, the rate of phosphorylation with both casein kinases being decreased. This behavior of casein kinases toward the M(r) 25,000 protein reflects the possible mechanism of multisite phosphorylation in which the introduction of a phosphate group facilitates sequential phosphorylation.

Analysis of bile acids in urine specimens from healthy humans: determination of several bile acids with beta-hydroxyl and carbonyl groups.

Urinary bile acids of 39 healthy male undergraduates were analyzed by capillary gas chromatography and capillary gas chromatography-mass spectrometry. 3 alpha-Hydroxy-12-oxo-5 beta-cholanoic acid, 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid, 3 beta, 7 alpha-dihydroxy-5 beta-cholanoic acid, and 3 alpha, 7 alpha, 12 beta-trihydroxy-5 beta-cholanoic acid, in addition to known bile acids, were identified and then quantified. The major part of the urinary bile acids was occupied by secondary bile acids. Every 7 beta-hydroxylated bile acid species was found in more than 80% of the subjects. The bile acid detected in the largest amount was 3 alpha-hydroxy-12-oxo-5 beta-cholanoic acid. The metabolites of cholic acid were quantitatively more predominant than those of chenodeoxycholic acid. These results indicate that bile acids with beta-hydroxyl and carbonyl groups at the C-3,7 and/or 12 positions are usual bile acids usually found in the urine of healthy humans. It is concluded that the occurrence of these bile acids is an effect of the intestinal bacterial flora and living conditions.

Fast atom bombardment tandem mass spectrometric analysis of phospholipids in Drosophila melanogaster.

Direct determination of the phospholipid components in adult Drosophila melanogaster was carried out by using fast atom bombardment tandem mass spectrometry (FAB-MS/MS) of both the positive and negative ions. Approximately 50 molecular species were detected, including phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylinositol (PI). Eight PE, one PC and three PS molecular species were identified. Some variations with age and a few differences among the D. melanogaster strains in the PE and PC molecular species were found. There was a difference in the fatty acid structure of a 741 Da PE molecular species between the wild-types and a mutant strain (EthAR201) which requires a higher concentration of diethylether for anesthesia than the wild-types; in the mutant sn-1-oleoyl-2-linoleoyl (18:1/18:2) but in the wild-types sn-1-linoleoyl-2-oleoyl (18:2/18:1) were speculated. This suggests that this technique will be useful for the screening of phospholipid molecular species mutation.

TRH axon terminals in synapsis with GRF neurons in the arcuate nucleus of the rat hypothalamus as revealed by double labeling immunocytochemistry.

Synaptic input to neurons containing growth hormone-releasing factor (GRF) by axon terminals containing thyrotropin-releasing hormone (TRH) in the arcuate nucleus (AN) of the rat hypothalamus was examined using a method combining pre-embedding peroxidase-anti-peroxidase for GRF with postembedding immunocolloidal gold staining for TRH. The TRH-like immunoreactive axon terminals were found to make synaptic contact with GRF-like immunoreactive neurons with unlabeled axon terminals. From these findings, TRH-containing neurons in the hypothalamic AN of the rat may be considered to innervate GRF neurons, to regulate GRF secretion or to have some other functions via synapses.

Formation of 5,16-androstadien-3 beta-ol from pregnenolone in human testicular microsomes.

A microsomal fraction of testicular tissue from a patient with prostatic carcinoma was incubated with [4-14C]pregnenolone in the presence of an NADPH-generating system for different periods of time. The metabolites were separated by Sephadex LH-20 column chromatography and then identified by thin-layer chromatography, radio-gas chromatography, and crystallization studies. Pregnenolone was converted to a major metabolite, 5-androstene-3 beta,17 beta-diol via 17-hydroxypregnenolone and then dehydroepiandrosterone. Another major metabolite was 5,16-androstadien-3 beta-ol, which increased with the time of incubation and accumulated in the incubation medium. After 120 min of incubation, 34.6% of the precursor was converted to 5-androstene-3 beta,17 beta-diol and 15.1% to 5,16-androstadien-3 beta-ol. In addition to the above-mentioned steroids, 16 alpha-hydroxypregnenolone, 5-pregnene-3 beta,20 alpha-diol, and 5-androstene-3 beta,17 alpha-diol were identified as minor metabolites of pregnenolone. From these results it was concluded that human testicular microsomes possess enzymic activities for the synthesis of 5,16-androstadien-3 beta-ol, as well as androgens from pregnenolone.

Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry.

A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with 2H2O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using [2H]diethylene glycol and [2H]hydrazine hydrate afforded [11,11,12,12,23,23(-2)H]lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-[11,11,12,12(-2)H]pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-[11,11,12,12(-2)H] progesterone (XIV), consisting of 0.3% 2H0-, 1.1% 2H1-, 8.6% 2H2-, 37.1% 2H3-, 52.1% 2H4-, and 0.8% 2H5-species.

Effect of docetaxel with cisplatin or vinorelbine on lung cancer cell lines.

Docetaxel shows substantial activity against lung cancer. To find the optimal drug combination for docetaxel, we evaluated the effects of cisplatin, etoposide, mitomycin C, irinotecan, vindesine, and vinorelbine using three human lung cancer cell lines, ABC-1, EBC-1, and SBC-3. Drug cytotoxicity was determined by MTT assay. Tumor cells were incubated for 96 hours in the presence of docetaxel and each of the test drugs stated above. The combined drug interaction was evaluated by median-effect plot analysis and improved IC50-isobologram analysis. Both methods showed strong antagonism (subadditive or protective effect) between docetaxel and etoposide when tested on ABC-1 and EBC-1 cells. Docetaxel and cisplatin displayed additive effects on all cell lines tested, when evaluated by improved IC50-isobologram analysis. The combination of docetaxel and vinorelbine exerted synergistic effect on the growth inhibition of SBC-3 cells, which showed a wide range of fractional cytotoxicity when analyzed by median-effect plot and supraadditive when analyzed by improved IC50-isobologram. These observations suggest a possibility that docetaxel can be used in combination with vinorelbine or cisplatin in the treatment of lung cancer.

Aromatic sulphonate ion-selective electrode membrane with crystal violet as ion-exchange site.

Four triphenylmethane derivatives (cations) and a high molecular-weight quaternary ammonium ion were used as the ion-exchange site in the liquid membranes of electrodes responsive to aromatic sulphonate ions, such as benzenesulphonate and alpha-naphthalenesulphonate. The nitrobenzene or 1,2-dichloroethane membrane containing the Crystal Violet-aromatic sulphonate pair had good sensitivity, showing an approximately Nernstian response down to 10(-4)M sulphonate. The potential of the Crystal Violet membrane was independent of pH variation from 2.5 to 12. Chloride and sulphate ions in the aqueous sample solution did not affect the electrode potential. 1,3,6-Naphthalenetrisulphonate exerted essentially no influence on the potential of the alpha-naphthalenesulphonate electrode. The interference of the nitrate ion was relatively large. The conductivity and association of the solute species in the membrane were estimated.

Effects of the kallikrein-kinin system on phasic coronary vasospasm in dogs.

It is well known that kinins are liberated from kininogen in blood during angina attack to maintain blood flow in coronary artery. We examined the effects of bradykinin, one of kinins, on the coronary artery other than vasodilation. The isolated canine coronary artery ring was suspended in gassed (95% O2, 5% CO2) Krebs-Henseleit buffer at 37 degrees C in vitro. The experimental phasic contraction of coronary artery was induced by 6 x 10(-4)M of 3,4-diaminopyridine which decreases K conductance (Y. Uchida, Jpn. Circ. J: 49, 128, 1985). The effect of bradykinin and other substances on the cycle length of contraction (CL), the peak tension of contraction phase (PT) and the tension during relaxation phase (RT) were observed. The phasic contraction was eliminated by 10(-7)M nifedipine and 10(-6)M diltiazem which block voltage dependent Ca channels. These Ca blockers reduced PT, but slightly increased CL, and weakly reduced RT. The phasic contraction was also eliminated by 10(-6)M bradykinin. However, bradykinin, unlike Ca blockers, did not reduce PT, but markedly prolonged CL and decreased RT significantly. This inhibition mode was very similar to those of nicorandil which increases K conductance. These data suggest that bradykinin plays a protective role in coronary vasospasm, and this antivasospasm effect may be mediated through the increase in K conductance.

High-dose etoposide treatment for CNS involvement in a patient with primary non-Hodgkin's lymphoma of the breast.

A 46-year-old man was referred to us for treatment of non-Hodgkin's lymphoma (NHL; diffuse large immunoblastic B cell type), which had initially developed in the breast. He was treated with five courses of chemotherapy with CHOP (cyclophosphamide, adriamycin, vincristine, and prednisolone) and achieved a complete response. One year later, he noticed a gait disturbance. Magnetic resonance imaging (MRI) of the brain showed multiple nodules. A few abnormal cells were found in the cerebrospinal fluid (CSF). He was treated with high-dose etoposide (1,350 mg/ m2/course). After two courses, both the multiple nodular lesions in the brain and the abnormal cells in the CSF were resolved. High-dose etoposide is effective for CNS involvement by NHL.

Cardiorespiratory responses during cycle ergometer exercise with different ramp slope increments in patients with chronic obstructive pulmonary disease.

OBJECTIVE: The ramp exercise test has been widely used to evaluate cardiopulmonary responses to an incremental exercise load. This study was performed to clarify whether different slopes of the ramp exercise test influence exercise tolerance, exercise limiting factors, and respiratory pattern in patients with chronic obstructive pulmonary disease (COPD). SUBJECTS AND METHODS: We applied three different slopes (5 W/min, 10 W/min and 20 W/min) of the ramp exercise test in 9 patients with COPD and evaluated cardiopulmonary responses. RESULTS: There were no significant differences in peak oxygen uptake, anaerobic threshold (AT), minute ventilation, heart rate, arterial oxygen saturation, expired tidal volume, or respiratory rate at the maximal load among the three different ramp exercises tested. AT could be determined in six of nine patients (67%) at the slope of 5 W/min, in 8/9 (89%) at the slope of 10 W/min, and in 9/9 (100%) at the slope of 20 W/min. CONCLUSION: The findings suggest that the ramp slope does not affect exercise tolerance, exercise limiting factors, or respiratory patterns and each of these ramp slopes is useful for the evaluation of COPD. Ramp slopes of 10 W/ min or 20 W/min should be appropriate for the determination of AT.