Premedication with midazolam in intellectually disabled dental patients: intramuscular or oral administration? A retrospective study.

BACKGROUND: The use of midazolam for dental care in patients with intellectual disability is poorly documented. The purpose of this study was to determine which method of premedication is more effective for these patients, 0.15 mg/kg of intramuscular midazolam or 0.3 mg/kg of oral midazolam. MATERIAL AND METHODS: This study was designed and implemented as a non-randomized retrospective study. The study population was composed of patients with intellectual disability who required dental treatment under ambulatory general anesthesia from August 2009 through April 2013. Patients were administered 0.15 mg/kg of midazolam intramuscularly (Group IM) or 0.3 mg/kg orally (Group PO). The predictor variable was the method of midazolam administration. The outcome variables measured were Observer's Assessment of Alertness/ Sedation (OAA/S) Scale scores, the level of cooperation when entering the operation room and for venous cannulation, post-anesthetic agitation and recovery time. RESULTS: Midazolam was administered intramuscularly in 23 patients and orally in 21 patients. More patients were successfully sedated with no resistance behavior during venous cannulation in Group PO than in Group IM (p=0.034). There were no differences in demographic data and other variables between the groups. CONCLUSIONS: The results of this study suggest that oral premedication with 0.3 mg/kg of midazolam is more effective than 0.15 mg/kg of midazolam administered intramuscularly, in terms of patient resistance to venous cannulation. If both oral and intramuscular routes of midazolam are acceptable in intellectually disabled patients, the oral route is recommended.

Both head extension and mouth opening impair the ability to swallow in the supine position.

Head position and mouth opening in the supine position may impair the ability to swallow. If this does occur, it would lead to retention of intra-oral fluids during dental treatment, which would lead to stimulation of the cough reflex. This study was conducted to investigate how head position and mouth opening affect swallowing ability. The water swallowing test was performed in 13 healthy adult subjects in the supine position. The subjects were asked to swallow 10 mL of water that was injected into the mouth in a single attempt. After swallowing, the residual intra-oral water was suctioned and its volume was measured. An electromyogram (EMG) of the suprahyoid (SH) muscles was also recorded during the test. The duration of SH muscle activity and peak amplitude of SH EMG were examined. The water swallowing test was performed under three head positions (neutral, extended and flexed) and four mouth opening patterns (interincisal distances of 0, 20, 30 and 40 mm). The wider the subject opened the mouth, the more the water remained in the mouth after swallowing. The residual volume of water was more in the extended position compared with that in the neutral and flexed positions. Peak amplitude of SH EMG decreased with mouth opening. Duration of SH muscle activity was longer in the extended position than in the neutral and flexed positions. Head extension and mouth opening can induce difficulty in swallowing in the supine position by extending the duration of SH muscle activity while reducing its intensity.

Role of activated protein C in facilitating basement membrane invasion by tumor cells.

The present study was undertaken to investigate the role of plasminogen activator inhibitor type 1 (PAI-1) and activated protein C (APC) in the regulation of tumor cell invasion. PAI-1 was purified in active form from conditioned medium of human umbilical vein endothelial cells under denaturing conditions (4 M guanidine-HCl). The purified inhibitor reacts with urokinase-type plasminogen activator (uPA) and APC. Two selected human lines, HOC-I (ovarian cancer cells) and SMT-ccl (choriocarcinoma cells), preferentially invaded through reconstituted basement membranes in an in vitro invasion assay using a modified Boyden chamber. The present study determined the efficacy of these two agents (PAI-1 and APC) used alone or in combination in inhibiting or facilitating tumor cell invasion. Active PAI-1 inhibited the tumor cell surface receptor-bound uPA activity. In an in vitro invasion assay, active PAI-1 reduced tumor cell invasive potential in a dose-dependent manner. When SMT-ccl cells saturated with uPA-PAI-1 complexes were treated with a 50-fold molar excess of APC, PAI-1-APC complex was demonstrated in conditioned medium, indicating that PAI-1 was dissociated from receptor-bound uPA on tumor cells and that tumor cell-associated uPA restored its enzymatic activity. Although APC alone had no effect on tumor cell invasion, the addition of APC to the cells saturated with uPA-PAI-1 complexes showed regeneration of tumor cell surface receptor-bound uPA activity and produced substantial and efficient invading effects. These data suggest that PAI-1 activity may be neutralized by APC or that APC may promote tumor cell invasion via inactivation of PAI-1 by formation of a stable PAI-1-APC complex. These observations suggest that APC may play a critical role in the initiation of a hematogenous metastatic process (extravasation step).

Inhibition of in vitro ovarian cancer cell invasion by modulation of urokinase-type plasminogen activator and cathepsin B.

HOC-I ovarian cancer cells express the single-chain form of the urokinase-type plasminogen activator (uPA) and cathepsin B (cath B) on their cell surface. The significance of the expression of cell surface uPA/cath B activity to the invasive potential was examined by preincubating with uPA/cath B-modulating agents in in vitro invasion assay. The anti-uPA monoclonal antibody 394 effectively inhibited invasion in a dose-dependent manner. On the contrary, anti-cath B antibody did not affect the invasive potential of the cells. E-64, a specific inhibitor for cysteine proteases, blocked invasion as effectively as monoclonal antibody 394. The data reveal that the uPA and cysteine proteases contribute significantly to the invasive capacity of the cells. We suggest that the cysteine proteases facilitate the action of uPA, possibly by activating proenzyme uPA produced by cancer cells. Evidence for the role of a cathepsin-uPA activation cascade in HOC-I cell invasion is provided.

Increased expression of cyclooxygenase-2 protein in 4-nitroquinoline-1-oxide-induced rat tongue carcinomas and chemopreventive efficacy of a specific inhibitor, nimesulide.

Expression of cyclooxygenase (COX)-2 protein in 4-nitroquinoline-1-oxide (4-NQO)-induced rat tongue lesions and the postinitiation chemopreventive potential of a selective COX-2 inhibitor, nimesulide (NIM), were examined in Fischer 344 male rats. NIM was administered in the diet at doses of 150, 300, and 600 ppm for 14 weeks after treatment with 25-35 ppm 4-NQO in the drinking water for 12 weeks. Western blot analysis revealed COX-2 protein to be barely expressed in the normal tongue epithelia, whereas it was increased approximately 6-fold in squamous cell carcinomas (SCCs). Immunohistochemically, COX-2 protein was diffusely present in SCCs and dysplasia but expressed only in basal cells in hyperplasia and papillomas. In basal cells of normal epithelia, it was also occasionally weakly stained. NIM dose-dependently decreased at doses of 150 and 300 ppm, the incidences of SCCs to 4 of 12 (33.3%) and 1 of 13 (7.7%) and their multiplicity to 0.33+/-0.49 and 0.08+/-0.28 per rat, respectively, as compared with 4-NQO alone group values of 9 of 11 (81.8%) and 1.00+/-0.77. A lesser decrease was observed with 600 ppm, the values being 5 of 12 (41.7%) and 0.50+/-0.67. NIM did not significantly affect the development of hyperplasias, dysplasias, and papillomas. These results clearly indicate chemopreventive potential of a selective COX-2 inhibitor against the postinitiation development of SCCs in rat tongue carcinogenesis.

Effects of membrane-associated cathepsin B on the activation of receptor-bound prourokinase and subsequent invasion of reconstituted basement membranes.

The present study was undertaken to assess the role of membrane-associated cathepsin B as an activator of receptor-bound single-chain urokinase-type plasminogen activator (pro-uPA) and to determine the importance of receptor-bound uPA activity in the destruction of extracellular matrix by tumor cells with subsequent invasion through basement membranes. Ovarian cancer HOC-I cells express pro-uPA/HMW-uPA and cathepsin B on their surface. uPAs are bound to a specific surface receptor, about 30% of which is saturated. 60% of the receptor-bound uPA is pro-uPA. No reduction in the specific binding of biotinylated DFP-HMW-uPA was observed when cells were cultivated in the presence of E-64, a cysteine proteinase inhibitor, for 24 h. Inhibition of cell-surface cathepsin B activity was associated with a decrease in cell-bound uPA activity to undetectable levels, and > 95% of the membrane-associated uPA was pro-uPA in cells cultivated with E-64. This suggested that receptor-bound pro-uPA cannot be converted to HMW-uPA in the absence of enzymatically-active cathepsin B. The significance of the expression of cell-surface uPA activity regarding invasive potential was examined in an in-vitro Matrigel invasion assay. Decreased cell-surface uPA activity was associated with a decrease in invasive potential. These data support our hypothesis that membrane-associated cathepsin B may be important for the conversion of pro-uPA to HMW-uPA and that receptor-bound uPA activity constitutes an efficient mechanism which contributes to tumor cell invasion. As HOC-I cells produce both uPA and cathepsin B, the implications of tumor-cell-derived pro-uPA activation by cellular proteinase cathepsin B should be considered.

Urinary trypsin inhibitor (UTI) and fragments derived from UTI by limited proteolysis efficiently inhibit tumor cell invasion.

We investigated the effects of purified human urinary trypsin inhibitor (UTI) and fragments derived from UTI by proteolysis on the invasive potential of ovarian cancer cells (HOC-I) and gestational choriocarcinoma cells (SMT-ccl) using an in vitro reconstituted basement membrane invasion assay. These cells express cell-associated plasmin and functional uPA receptors that are partially occupied by ligands. SMT-ccl cells, which express threefold higher levels of cell-associated plasmin activity than HOC-I cells, showed approximately twofold increase in their invasive potential. For the invasion assay, HOC-I cells were primed with exogenous plasminogen, but SMT-ccl cells were not. Human leukocyte elastase (HLE)-digested UTI (22 kDa fragment; UTI-22) inhibited plasmin practically with the same strength as native UTI. Trypsin-digested UTI (20 kDa fragment; UTI-20), however, did not inhibit plasmin significantly. Treatment of cells with UTI or UTI-22 reduced the incidence of tumor cell invasive capacity, whereas the inhibitory effect of UTI-20 was not remarkable. The inhibitory effect on tumor cell invasion was dose-dependent and non-toxic; moreover, it was not mediated by inhibition of the tumor cell chemotactic response or of cell attachment to matrigel. These results indicate that inhibition of the proteolytic enzyme plasmin specifically reduced the invasive capacity of tumor cells in vitro.

Idebenone and vinpocetine augment long-term potentiation in hippocampal slices in the guinea pig.

The effects of idebenone and vinpocetine which reportedly prevent impairment of learning and memory were studied in vitro, on the long-term potentiation of the population spike in the pyramidal layer of CA3 region of slices of hippocampus in the guinea pig. Idebenone (10(-9) M-10(-6) M) or vinpocetine (10(-7) M-10(-6) M) significantly augmented long-term potentiation in the mossy fibre-CA3 pyramidal cell system, without any significant changes in population spikes in the absence of tetanic stimulation. These results suggest that both drugs have direct actions on the hippocampal neurones to augment long-term potentiation at fairly small concentrations. Further, when the two drugs were applied together, the augmenting effects were additive.

Different drug-susceptibilities of long-term potentiation in three input systems to the CA3 region of the guinea pig hippocampus in vitro.

The susceptibilities to several drugs of long-term potentiations in the three input systems (mossy, commissural/associational and fimbrial fibres) to CA3 pyramidal neurones were investigated in hippocampal slices from the guinea pig. D-2-Amino-5-phosphonovalerate (D-APV), a selective antagonist at N-methyl-D-aspartate (NMDA) receptors, blocked the long-term potentiations in the commissural/associational fibre- and fimbrial fibre-CA3 systems, but did not significantly affect that in the mossy fibre-CA3 system. The latter was suppressed by kynurenate, a non-selective glutamate receptor antagonist. On the other hand, naloxone, an opioid antagonist, inhibited and bifemelane, which improves metabolism in brain and has an anti-amnesic action, augmented long-term potentiation in mossy fibre-CA3 system but did not influence those in commissural/associational fibre- and fimbrial fibre-CA3 systems. These findings suggest that the mechanisms, relevant to production of long-term potentiation in the mossy fibre-CA3 system, are different from those in the commissural/associational fibre- and fimbrial fibre-CA3 systems. N-Methyl-D-aspartate receptors are involved in the latter systems, while non-NMDA receptors for L-glutamate and opioid receptors are involved in the former. Further, the mossy fibre-CA3 system is more susceptible to a drug, having an anti-amnesic action, than are the other two systems.

Establishment of a rat thyroid carcinoma cell line in vitro demonstrating high DNA synthesis in response to insulin-like growth factor I.

We previously established transplantable rat thyroid carcinoma cell lines in vivo from primary thyroid tumors induced by N-bis-(2-hydroxypropyl)nitrosamine (DHPN). In the present study, an insulin-like growth factor I (IGF-I)-responsive cell line (TRTC-G1-C-A4) in culture was derived from one (well differentiated papillary type) of these carcinoma cell lines G1. TRTC-G1-C-A4 cells were found to exhibit specific saturable binding of IGF-I with a Kd of 1.16 nM at approximately 43.6 fmol/10(5) cells. Inclusion of IGF-I (10 and 50 ng/ml) in the culture medium resulted in a significant increase of [3H]thymidine incorporation and marked cell proliferation. IGF-II (10 ng/ml) and insulin (1 microgram) produced no such effects. The molecular weight of IGF-I receptors on the cell membrane was determined by Western blotting analysis, a single band of binding proteins with a molecular weight of 125 kDa being evident under non-reducing conditions. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that the TRTC-G1-C-A4 cells contained IGF-I receptor mRNA with a sequence corresponding to that determined from rat uterus. These results demonstrate that the IGF-I receptor can be expressed in a thyroid carcinoma with an important contribution to cell growth.

Inhibition of neuronal nitric oxide synthase activity by 3-[2-[4-(3-chloro-2-methylphenyl)- 1-piperazinyl]ethyl]-5, 6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel neuroprotective agent, in vitro and in cultured neuroblastoma cells in situ.

DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5, 6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel calmodulin (CaM) antagonist, possesses neuroprotective activity. In the current study, we examined the effects of DY-9760e on nitric oxide synthase (NOS) activities in vitro and on calcium ionophore-induced NO production in situ. DY-9760e inhibited both neuronal NOS and endothelial NOS activities without affecting inducible NOS activity. It also inhibited purified neuronal NOS activity with a potency similar to that seen for purified CaM kinase II activity in vitro. Furthermore, DY-9760e significantly inhibited Ca(2+) ionophore (A23187)-induced NO production in mouse N1E-115 neuroblastoma cells, at a concentration of less than 1 microM. In contrast, no apparent inhibitory effect on Ca(2+)/CaM-dependent protein kinase II activity was observed in cultured hippocampal neurons up to 5 microM. These results suggest that the inhibitory effect of DY-9760e on CaM-dependent NOS activities underlies neuroprotective effects of the agent.

Annexin V inhibits phosphatidylserine-induced intrauterine growth restriction in mice.

To investigate the role of phosphatidylserine (PS) derived from the activated platelets in placental circulation, we established an artificial PS-induced intrauterine growth restriction (IUGR) model in mice and examined whether annexin V, a PS-binding anticoagulant protein, could prevent the development of IUGR in the animal experiments. The ICR mice were injected in the tail vein on days 8, 11 and 14 of pregnancy with filtered PS/phosphatidylcholine (PC) microvesicles (<0.3 microm in maximal diameter). The microvesicles have a procoagulant activity which was inhibited by annexin V in a dose-dependent fashion. The mice were killed on day 18 of pregnancy and the placental and fetal weights were measured. The placental tissue specimens were examined microscopically. PS/PC vesicles induced significant reductions in fetal weights compared with PC vesicles alone. The placental tissue revealed severe congestion with fibrin depositions although the lung and kidney tissue specimens showed minimal histological changes. PS/PC microvesicles with recombinant annexin V showed no significant reductions in fetal weights in mice with PS/PC vesicles alone. These results suggest that PS from the activated platelets induces IUGR by enhancing the coagulation cascade in the placental circulation and that annexin V from the villous trophoblast prevents the development of IUGR.

Epigenetic alterations in methylation in oral squamous cell carcinoma cell lines detected by two-dimensional gel electrophoresis.

It has been described that DNA alterations in human oral squamous cell carcinomas were successfully demonstrated as 6 spots being commonly reduced in more than 50% of 10 lesions using the restriction landmark genomic scanning (RLGS) method. In the present study, the question of whether the DNA alterations involve not only genetic but also epigenetic change was examined with the use of cell lines Ca9-22, HO-1-u-1, HSC-2 and KB and treatment with a demethylating agent 5-aza-2'-deoxycytidine (5-AzaCDR). Intensities of two of the reduced spots were amplified in the cell lines by the 5-AzaCDR treatment, showing that they were due to altered DNA methylation. Our study provides clear evidence that epigenetic changes, like the methylation are related to carcinogenesis in human oral squamous cell epithelium.

Effects of several cerebroprotective drugs on NMDA channel function: evaluation using Xenopus oocytes and [3H]MK-801 binding.

The effects of several cerebroprotective and nootropic drugs on the function of excitatory amino acid (EAA) receptor subtypes expressed in Xenopus oocytes after injection of rodent brain poly(A)+ mRNA were investigated. The oocyte response to N-methyl-D-aspartate (NMDA) in the presence of glycine (Gly) was inhibited dose-dependently by bifemelane, indeloxazine, vinpocetine and vincamine while no effect was observed by idebenone, Ca hopantenate, aniracetam or piracetam. Bifemelane, indeloxazine and vinpocetine suppressed the maximum response of NMDA and Gly without affecting their EC50 values. Unlike Mg2+, they did not affect the current-voltage relationship of the NMDA response below 0 mV. On the non-NMDA-type responses of the injected oocytes to kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and quisqualate (QA), no significant effects were observed by these drugs at 100 microM. On the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) to brain membranes, the estimated IC50 values were 88 microM for bifemelane, 102 microM for indeloxazine, and 115 microM for vinpocetine. The dissociation rate of [3H]MK-801 was significantly slowed by Zn2+ and vinpocetine, but not affected by bifemelane or indeloxazine. The Kd value for [3H]MK-801 binding was increased by bifemelane and indeloxazine while Bmax was unchanged. These results suggest that the inhibition of NMDA channels by vinpocetine shows a similarity to the action of Zn2+ which closes the gate of the NMDA channel. In contrast, bifemelane and indeloxazine may affect the phencyclidine (PCP)-site in the open channels and inhibit NMDA function.

DY-9760e, a novel calmodulin antagonist, reduces brain damage induced by transient focal cerebral ischemia.

Perturbations in Ca2+ homeostasis have been proposed to lead to neuronal damage after cerebral ischemia. DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1- (4-imidazolylethyl)-1H-indazole dihydrochloride 3.5 hydrate) is a novel calmodulin antagonist. In this study, we examined the effects of DY-9760e on brain damage in rats subjected to transient (1 h) focal cerebral ischemia. DY-9760e (0.25-1.00 mg kg(-1) h(-1)) was intravenously infused for 6 h, starting 1 h after middle cerebral artery occlusion. Treatment with DY-9760e 0.25, 0.50 and 1.00 mg kg(-1) h(-1) reduced infarct volume by 30, 42 (P < 0.05), and 60% (P < 0.05), respectively. Furthermore, the effect of DY-9760e on ischemia-induced fodrin breakdown was examined in the same model. Pronounced fodrin breakdown was observed in the cerebral cortex and striatum at 24 h after ischemia. DY-9760e caused potent suppression of fodrin breakdown in the cerebral cortex at the same doses as those that had a protective action against cerebral infarction. From these results DY-9760e may have a therapeutic effect against cerebral ischemic damage in the acute stage.

DY-9760e, a novel calmodulin antagonist with cytoprotective action.

We report the pharmacological characterization and cytoprotective effect of DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-( 4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel antagonist of calmodulin. DY-9760e inhibited calmodulin-dependent enzymes, including calmodulin-dependent protein kinase II and IV, calcineurin, [corrected] calmodulin-dependent phosphodiesterase and myosin light chain kinase with Ki values of 1.4, 12, 2.0, 3.8 and 133 microM, respectively. These antagonistic effects of DY-9760e were more potent than those of W-7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, another calmodulin antagonist. This compound showed little or no effect on calmodulin-independent enzymes, such as protein kinase A and C and calpain I and II. Analysis of the hydrophobic interaction of DY-9760e with calmodulin by using 2-p-toluidinylnaphthalene-6-sulfonate and 9-anthroylcholine revealed that, like W-7, DY-9760e bound to the hydrophobic regions of calmodulin. The [14C]DY-9760e binding assay indicated that DY-9760e bound to calmodulin at one class of binding site. Finally, DY-9760e substantially protected N1E-115 neuroblastoma cells from cytotoxicity induced by the Ca2+ ionophore, A23187. These results indicate that DY-9760e, a novel calmodulin antagonist, possesses a cytoprotective action and suggest that calmodulin plays a critical role in mediating some of the biochemical events leading to cell death following Ca2+ overload.

Interspecies differences in the population of multiple glutamate receptors expressed in Xenopus oocytes after injection of guinea pig, mouse and rat forebrain mRNAs.

Expression of multiple glutamatergic responses was compared in Xenopus oocytes several days after injection of poly(A)+ RNAs from guinea pig, mouse and rat forebrains. No difference either in the characteristics of each response or in the amplitude of N-methyl-D-aspartate response was observed between the oocytes injected with mRNA from different species. However, the average magnitudes of kainate (KA) and oscillatory quisqualate (QA) responses produced by guinea pig brain mRNA were 1/2 and 1/6 times, respectively, of those by rat brain mRNA. These results may reflect that the guinea pig forebrain contains smaller amounts of ionotropic KA and metabotropic QA receptors than the rat forebrain.

Effect of food size on the movement of the mandibular first molars and condyles during deliberate unilateral mastication in humans.

To date, the effect of food size on the movement of the mandibular first molars and condyles during chewing has not been fully examined due to methodological problems. The purpose of the present study was to examine the previously unknown effect of food size on masticatory jaw movement. Using a face bow, light-emitting diodes, and optical cameras, we recorded, in 16 young adults with good occlusion, mandibular movement for the first 10 strokes during the unilateral chewing of similarly shaped hard gummy jellies weighing 5 g and 10 g, respectively. The chewing cycle time for the 10-g jelly was significantly longer than that for the 5-g jelly. The jaw-closing and -opening maximum velocities, gapes at the maximum velocities, and maximum gape were significantly faster and larger when 10-g gummy jellies were chewed, compared with results with 5-g jellies, at the mandibular first molar on the chewing side and the condyle on the non-chewing side. With the exception of the velocity, similar tendencies were observed at the molar on the non-chewing side. However, such significant differences were not detected at the condyle on the chewing side. The mandibular first molar on the chewing side was that most affected by food size, and the mean value of the maximum gape coincided approximately with the height of each jelly. These results suggest that humans chew hard coherent food such that the mandibular teeth that come into contact with the food open to a height equivalent to that of the food bolus, and that the changes in movement of the other parts of the mandible are minimized, ensuring efficient mastication.

Motion of the human mandibular condyle during mastication.

It has been reported that loading to the mandible during closing movement makes the condylar path move more in the superior direction than that during the free closing movement. In this study, the hypothesis was tested that the displacement of the condyle on the chewing side is greater in the direction of the mandibular fossa than that on the non-chewing side. Using a six-degrees-of-freedom jaw movement recording system, we recorded condylar motion in 12 healthy adults without TMD, during the chewing of a large hard gummy jelly. The maximum displacements at the condyle on the chewing side from the maximum intercuspation (CO) position were significantly larger in the superior and medial directions at the initial stage and in the posterior direction at all stages (0.5 mm, 0.5 mm, and 0.6 mm, respectively) than those on the non-chewing side (0.0 mm, 0.1 mm, and 0.1 mm, respectively). This suggests that, in healthy adults, the condyles at CO are located in a position such that excessive load is not applied to the temporomandibular joint when there are the aforementioned displacements.

Purification and properties of urease from the leaf of mulberry, Morus alba.

Urease was purified from leaves of mulberry (Morus alba, L.) by ammonium sulfate fractionation, acetone fractionation and sequential column chromatography including Q-Sepharose HP, Phenyl-Sepharose HP, Superdex 200 HR and Mono Q. The enzyme was purified 5700-fold to apparent homogeneity with a recovery of 3.6%. The molecular mass of the enzyme was determined to be 90.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and 175 kDa by gel filtration, indicating that the enzyme was a homodimer. In the western blot analysis, 90.5 kDa subunit of the mulberry leaf urease cross-reacted with antiserum raised against jack bean seed urease. The N-terminal sequence of the first 20 residues of the enzyme revealed that it has a high similarity (80-90%) to ureases from other plant sources, suggesting that the mulberry leaf urease is closely related to other plant ureases. However, the mulberry leaf enzyme showed an optimum pH for activity of 9.0, while the optimum pH of most ureases isolated from plants and bacterial is neutral. In addition, the K(m) value for urea was 0.16 mM, which is lower than those of ureases from other sources. It is also proposed that urease activity ingested by browsing silkworm releases ammonia that is subsequently used in silkworm protein synthesis.