A Facile Fluorometric Assay of Orotate Phosphoribosyltransferase Activity Using a Selective Fluorogenic Reaction for Orotic Acid.

Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells. The technique utilizes 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent, which produces selective fluorescence for orotic acid. To perform the OPRT reaction, orotic acid was added to HeLa cell lysate, and a portion of the enzyme reaction mixture was heated at 80 °C for 4 min in the presence of 4-TFMBAO under basic conditions. The resulting fluorescence was measured using a spectrofluorometer, which reflects the consumption of orotic acid by the OPRT. After optimization of the reaction conditions, the OPRT activity was successfully determined in 15 min of enzyme reaction time without further procedures such as purification of OPRT or deproteination for the analysis. The activity obtained was compatible with the value measured by the radiometric method with [3H]-5-FU as the substrate. The present method provides a reliable and facile measurement of OPRT activity and could be useful for a variety of research fields targeting pyrimidine metabolism.

Cerebral infarction by paradoxical gas embolism detected after laparoscopic partial hepatectomy with an insufflation management system: a case report.

BACKGROUND: Laparoscopic surgery has reduced surgical morbidity and postoperative duration of hospital stay. Gas embolism is commonly known as a risk factor for all laparoscopic procedures. We report a case of severe cerebral infarction presumably caused by paradoxical CO2 embolism in laparoscopic partial hepatectomy with an insufflation management system. CASE PRESENTATION: A male in his 60 s was diagnosed with recurrence of liver metastasis in the right hepatic lobe after laparoscopic lower anterior resection for rectal cancer. We performed laparoscopic partial hepatectomy with an AirSeal® under 10 mmHg of intra-abdominal pressure. During the surgery, the patient's end-tidal CO2 and percutaneous oxygen saturation dropped from approximately 40-20 mmHg and 100-90%, respectively, while the heart rate increased from 60 to 120 beats/min; his blood pressure remained stable. Postoperatively, the patient developed right hemiplegia and aphasia. Brain magnetic resonance imaging showed cerebral infarction in the broad area of the left cerebral cortex. Thereafter, transesophageal echocardiography revealed a patent foramen ovale, suggesting cerebral infarction due to paradoxical gas embolism. CONCLUSIONS: A patent foramen ovale is found in approximately 15-20% of healthy individuals. While gas embolism is a rare complication of laparoscopic surgery, cerebral infarction must be considered a possible complication even if the intra-abdominal pressure is constant under 10 mmHg with an insufflation management system.

Cell-autonomous signal transduction in the Xenopus egg Wnt/ß-catenin pathway.

Wnt proteins are thought to bind to their receptors on the cell surfaces of neighboring cells. Wnt8 likely substitutes for the dorsal determinants in Xenopus embryos to dorsalize early embryos via the Wnt/ß-catenin pathway. Here, we show that Wnt8 can dorsalize Xenopus embryos working cell autonomously. Wnt8 mRNA was injected into a cleavage-stage blastomere, and the subcellular distribution of Wnt8 protein was analyzed. Wnt8 protein was predominantly found in the endoplasmic reticulum (ER) and resided at the periphery of the cells; however, this protein was restricted to the mRNA-injected cellular region as shown by lineage tracing. A mutant Wnt8 that contained an ER retention signal (Wnt8-KDEL) could dorsalize Xenopus embryos. Finally, Wnt8-induced dorsalization occurred only in cells injected with Wnt8 mRNA. These experiments suggest that the Wnt8 protein acts within the cell, likely in the ER or on the cell surface in an autocrine manner for dorsalization.

Maternal overprotection score of the Parental Bonding Instrument predicts the outcome of cognitive behavior therapy by trainees for depression.

AIMS: The purpose of this study was to predict the outcome of cognitive behavior therapy (CBT) by trainees for major depressive disorder (MDD) based on the Parental Bonding Instrument (PBI). The hypothesis was that the higher level of care and/or lower level of overprotection score would predict a favorable outcome of CBT by trainees. METHODS: The subjects were all outpatients with MDD treated with CBT as a training case. All the subjects were asked to fill out the Japanese version of the PBI before commencing the course of psychotherapy. The difference between the first and the last Beck Depression Inventory (BDI) score was used to represent the improvement of the intensity of depression by CBT. In order to predict improvement (the difference of the BDI scores) as the objective variable, multiple regression analysis was performed using maternal overprotection score and baseline BDI score as the explanatory variables. RESULTS: The multiple regression model was significant (P = 0.0026) and partial regression coefficient for the maternal overprotection score and the baseline BDI was -0.73 (P = 0.0046) and 0.88 (P = 0.0092), respectively. Therefore, when a patient's maternal overprotection score of the PBI was lower, a better outcome of CBT was expected. CONCLUSION: The hypothesis was partially supported. This result would be useful in determining indications for CBT by trainees for patients with MDD.

[A case of advanced gastric cancer with peritoneal dissemination successfully treated with S-1 and intraperitoneal docetaxel administration].

A 76-year-old man was admitted to our hospital for the treatment of remnant gastric cancer. Laparotomy revealed massive lymph node metastasis, direct invasion of the transverse colon, and peritoneal dissemination. Partial resection of remnant stomach with transverse colon and intraperitoneal infuser port implantation were performed. After surgery, he underwent chemotherapy with docetaxel(DOC)administered intraperitoneally, and S-1. CT scan showed no tumors, and the patient was judged to be a complete response(CR)without serious adverse events. We switched DOC to intravenous injection because of port damage, and grade 3 adverse events appeared frequently until the chemotherapy was stopped. It has been 30 months since we stopped the chemotherapy, and the patient is still alive with no evidence of tumor recurrence 48 months after surgery.

Effects of unilateral voluntary movement on motor imagery of the contralateral limb.

OBJECTIVE: To investigate whether unilateral voluntary movement affects voluntary drive of motor imagery for the contralateral limb. METHODS: The subjects were asked to maintain the left index-finger movements with different directions (abduction and adduction, 5% of maximum voluntary contraction; MVC) and with different force levels (10% and 25% MVC). Under these conditions, transcranial magnetic stimulation was applied over the left motor cortex to record motor evoked potential (MEP) from the right first dorsal interosseous muscle with or without motor imagery of the right index-finger abduction. RESULTS: Voluntary movement of the left index finger with isodirection, but not mirrored direction, to the imagined movement reduced the MEP enhancement induced by motor imagery. MEP was gradually increased depending on increment of the force level on the left side, while the motor imagery-induced MEP enhancement was consequently reduced. CONCLUSIONS: Enhancement of the motor cortex excitability driven by motor imagery of the contralateral limb is interfered with by isodirection and forceful movement of the ipsilateral limb, which may be due to an increase in the transcallosal inhibitory effects. SIGNIFICANCE: Using motor imagery as a therapeutic tool, the voluntary movements on the other side of the body should be taken into account.

Spinal neuronal mechanisms explaining the modulation of soleus H-reflexes during sustained passive rotation of the hip joint.

OBJECTIVES: The aim of this study was to investigate the mechanism of modulation of soleus H-reflexes during sustained passive rotation of the hip joint. METHODS: Healthy men participated as subjects in this study. In the supine position, soleus maximum H-reflexes were recorded during rotation of the hip joint. To examine the roles of muscle spindles, we recorded H-reflexes while applying vibration to muscles around the hip joint. Furthermore, to assess the modulation of pre- and postsynaptic inputs to soleus motoneurons, H-reflexes were conditioned by common peroneal nerve stimulation or transcranial magnetic stimulation (TMS). RESULTS: The ratio of the maximum H-reflex to the maximum M-response was significantly reduced in externally rotated positions of the hip joint compared to neutral position. In these positions, presynaptic inhibition, assessed by D1 inhibition, was significantly increased. H-reflexes were reduced during vibration, irrespective of muscles involved and hip position. Facilitation of H-reflex induced by conditioning-TMS showed insignificant differences across all hip positions. CONCLUSIONS: These findings suggest that Ia afferents induced by rotation of the hip joint presumably inhibit soleus H-reflexes presynaptically. SIGNIFICANCE: Based on the results, therapists could efficiently modulate the spasticity of the lower legs of hemiplegic patients during rotation of the hip joint.

Influence of negative cognition on the parental bonding instrument (PBI) in patients with major depression.

The aim of this study was to examine the effects of negative cognition on PBI score before and after treatment for depression. Forty major depressive disorder outpatients were assessed with the PBI scale and Structured Interview Guide for Hamilton Rating Scale for Depression (SIGH-D) at the time of the first medical examination (baseline) and 8 weeks later. The SIGH-D scores decreased by about 50% from baseline to 8 weeks, but there was no significant change in the PBI scores of the depressed outpatients from baseline to 8 weeks. Analysis of covariance with the SIGH-D scores as covariate was conducted for PBI scores between baseline and 8 weeks to remove effects of MDD. No significant differences were found on any of the PBI scales. Even though the therapeutic values on the SIGH-D of the depressed patients indicated that depressive symptoms were reduced by about 50%, depression level did not influence the PBI scores. This study provides evidence for the stability of parental representations throughout treatment, as measured by the PBI.

Further evidence for excitability changes in human primary motor cortex during ipsilateral voluntary contractions.

The present study aimed to further investigate whether the intracortical neural circuits within the primary motor cortex (M1) are modulated during ipsilateral voluntary finger movements. Single- and paired-pulse (interstimulus intervals, ISIs; 3 ms and 12 ms) transcranial magnetic stimulations of the left M1 were applied to elicit motor evoked potential (MEP) in the right first dorsal interosseous (Rt-FDI) muscle during voluntary contractions (10% and 30% maximum voluntary contraction) of the left FDI (Lt-FDI) muscle. F-waves of Rt-FDI muscle were recorded under these left index-finger conditions for ensuring that the excitability changes occur at the supraspinal level. MEPs were also recorded during motor imagery of the left index-finger abduction instead of overt movement. The results showed that, in single-pulse transcranial magnetic stimulation (TMS) paradigm, MEPs in Rt-FDI muscle were markedly enhanced during voluntary contractions of Lt-FDI muscle compared with the complete resting state. In paired-pulse TMS paradigm, the short intracortical inhibition was significantly reduced in proportion to increments of the ipsilateral muscle contraction, whereas the intracortical facilitation had no change. F-wave of Rt-FDI muscle was unchanged under these conditions, while MEP in Rt-FDI muscle was also enhanced during motor imagery of the left index-finger abduction. Based on the present results, it is suggested that the intracortical inhibitory neural circuits may be modulated in the transition from rest to activity of the ipsilateral homonymous muscle. The excitability changes in M1 might be induced by overflows of voluntary drive given to the ipsilateral limb, probably via the transcallosal pathway.

A Wnt3a variant participates in chick apical ectodermal ridge formation: distinct biological activities of Wnt3a splice variants in chick limb development.

Wnt/beta-catenin signaling is involved in the formation of the apical ectodermal ridge (AER) during vertebrate limb development. Although Wnt3a is a potent ligand for chick AER formation, whether chick Wnt3a can induce Fgf8 expression in chick embryos is unclear and the Wnt ligand involved in chick AER formation remains unknown. Here, we examined whether another Wnt3a isoform is expressed in the AER, and whether Wnt3 contributes to AER formation in chick as well as mouse embryos. We found that chick Wnt3 was not expressed in the presumptive limb ectoderm at the early stages of AER formation. Using 5'-rapid amplification of cDNA ends, we isolated another chick Wnt3a transcript. This novel variant, Wnt3a variant 2, induced Fgf8 in the limb ectoderm and activated the beta-catenin pathway in vivo and in vitro. These data showed that Wnt3a variant 2 is an active form of chick Wnt3a that regulates chick AER formation.

Psychosomatic analysis of atopic dermatitis using a psychological test.

In patients with atopic dermatitis (AD), psychosomatic factors are important elements in treating the condition. In this study, we surveyed 51 outpatients with AD who consulted the Department of Dermatology of Fujita Health University Hospital using a questionnaire involving present illness/treatment history regarding AD to analyze psychosomatic factors. The severity of AD was evaluated using the severity classification described by Yoshiike et al. Four psychological tests were used to examine depression, anxiety, personality, and upbringing experiences during childhood. Beck Depression Inventory (BDI) was used as a scale for depression, Self-rating Anxiety Scale (SAS) as a scale for anxiety, the Temperament and Character Inventory (TCI) as a scale for the personality tendency, and the Parental Bonding Instrument (PBI) as a scale for upbringing experiences during childhood. The BDI and SAS scores were high in the severe AD group. Among patients with the same grade of AD, the BDI and SAS scores were higher in the low IgE RIST group. In the patients with AD, the BDI scores were significantly higher than those in the healthy controls (P<0.05). In clinical practice, the treatment of AD should include psychosomatic approaches.

Wnt10a is involved in AER formation during chick limb development.

The apical ectodermal ridge (AER) is indispensable for vertebrate limb development and requires Wnt/beta-catenin signaling for induction and maintenance. We report identification and involvement of Wnt10a in AER formation during chick limb development. Chicken Wnt10a has 82% identity with mouse Wnt10a in the amino acid sequence. The Wnt10a gene was expressed broadly in the surface ectoderm from as early as stage 10. By stage 15, the expression was restricted to the surface ectoderm overlying the lateral plate mesoderm. Wnt10a expression became intensified in the presumptive limb ectoderm during AER formation, and subsequently intense expression signals persisted in the AER. Wnt10a misexpression led to ectopic Fgf8 expression in the developing limb ectoderm and induced translocation of beta-catenin in chick embryo fibroblasts. These results suggest that Wnt10a is involved in AER formation in the chick limb bud through the Wnt/beta-catenin signaling pathway.

Excessive portal flow causes graft failure in extremely small-for-size liver transplantation in pigs.

AIM: To evaluate the effects of a portocaval shunt on the decrease of excessive portal flow for the prevention of sinusoidal microcirculatory injury in extremely small-for-size liver transplantation in pigs. METHODS: The right lateral lobe of pigs, i.e. the 25% of the liver, was transplanted orthotopically. The pigs were divided into two groups: graft without portocaval shunt (n = 11) and graft with portocaval shunt (n = 11). Survival rate, portal flow, hepatic arterial flow, and histological findings were investigated. RESULTS: In the group without portocaval shunt, all pigs except one died of liver dysfunction within 24 h after transplantation. In the group with portocaval shunt, eight pigs survived for more than 4 d. The portal flow volumes before and after transplantation in the group without portocaval shunt were 118.2+/-26.9 mL/min/100 g liver tissue and 270.5+/-72.9 mL/min/100 g liver tissue, respectively. On the other hand, in the group with portocaval shunt, those volumes were 124.2+/-27.8 mL/min/100 g liver tissue and 42.7+/-32.3 mL/min/100 g liver tissue, respectively (P<0.01). As for histological findings in the group without portocaval shunt, destruction of the sinusoidal lining and bleeding in the peri-portal areas were observed after reperfusion, but these findings were not recognized in the group with portocaval shunt. CONCLUSION: These results suggest that excessive portal flow is attributed to post transplant liver dysfunction after extreme small-for-size liver transplantation caused by sinusoidal microcirculatory injury.

Evaluating the state dependency of the Temperament and Character Inventory dimensions in patients with major depression: a methodological contribution.

BACKGROUND: Little is known as to whether or not the seven personality dimensions of Cloninger's theory, particularly the three character dimensions newly included in the theory, are independent of the states of depression. METHODS: One hundred and eight patients with major depression filled out the Temperament and Character Inventory (TCI) before and after a 16-week antidepressant treatment. RESULTS: The level of depression, as assessed by the Hamilton Rating Scale for Depression, was correlated positively to the harm avoidance score and negatively to the self-directedness and cooperativeness scores. During the treatment, the scores on these three dimensions significantly changed toward normal values in treatment-responders, but were stable in treatment-nonresponders. The changes in these dimensions were significantly explained by the change in the depression severity during treatment. Scores on novelty seeking, reward dependence, persistence, and self-transcendence were not correlated significantly to the level of depression and did not change significantly during the treatment in either treatment-responders or nonresponders. LIMITATIONS: The changes in the TCI scores during treatment in this study may reflect a non-specific tendency for the scores to change on retest. CONCLUSIONS: The results suggest that a depressive state can significantly affect assessments of harm avoidance, self-directedness, and cooperativeness in major depression. The administration of the TCI during a depressive episode may elevate the HA score, and may lower the SD and C scores. These findings highlight the importance of considering the state of depression before drawing conclusions about the TCI personality traits, when a patient with major depression is still experiencing a depressive episode.