Expression analysis of zinc-metabolizing enzymes in the saliva as a new method of evaluating zinc content in the body: two case reports and a review of the literature.

BACKGROUND: The activity level of alkaline phosphatase, a zinc-requiring enzyme in the serum, is used to indicate zinc nutritional status; however, it does not correlate with serum zinc levels or subjective symptoms of taste disorder in many cases. Hence, this study focused on the total activity of alkaline phosphatase, a zinc-requiring enzyme. The total alkaline phosphatasa activity level in the saliva was measured before and after zinc supplementation, and the results were compared with serum zinc levels. CASE PRESENTATION: This study included patients with hypozincemia, specifically a patient with zinc-deficient taste disorder (patient 1: a 69-year-old Japanese woman) and a patient with glossodynia with zinc deficiency (patient 2: an 82-year-old Japanese woman). Saliva samples were collected, and blood tests were performed before and after zinc supplementation. Subjective symptoms and serum zinc levels were simultaneously evaluated. Zinc supplementation was performed using zinc acetate hydrate or Polaprezinc. CONCLUSIONS: Total alkaline phosphatase activity levels were found to be associated with serum zinc levels and subjective symptoms. A further study with a higher number of patients is necessary to confirm whether total alkaline phosphatase activity levels more accurately reflect the amounts of zinc in the body than serum zinc levels.

Metabolism of testosterone and progesterone by cytochrome P450 2C19 allelic variants.

CYP2C19 is a member of the human microsomal cytochrome P450 (CYP). Significant variation in CYP2C19 levels and activity can be attributed to polymorphisms in this gene. Wildtype CYP2C19 and 13 mutants (CYP2C19.1B, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.11, CYP2C19.13, CYP2C19.16, CYP2C19.19, CYP2C19.23, CYP2C19.30, and CYP2C19.33) were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Hydroxylase activity toward testosterone and progesterone was also examined. Ten CYP2C19 variants showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra. CYP2C19.11 and CYP2C19.23 showed higher testosterone 11α, 16α-/17- and progesterone 6ß-,21-,16α-/17α-hydroxylase activities than CYP2C19.1B. CYP2C19.6, CYP2C19.16, CYP2C19.19, and CYP2C19.30 showed lower activity than CYP2C19.1B. CYP2C19.9, CYP2C19.10. CYP2C19.13, and CYP2C19.33 showed different hydroxylation activities than CYP2C19.1B. These results indicated that CYP2C19 variants have very different substrate specificities for testosterone and progesterone.

Direct cleavage during the first mitosis is a sign of abnormal fertilization in cattle.

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.

Impact of the COVID-19 pandemic on patients with mental health problems and the differences among diagnostic categories.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has resulted in a total upending of our daily lives. While anxiety and depression were frequently reported among the general population, the pandemic's impact on patients with mental health problems remains unknown. METHODS: A cross-sectional questionnaire survey involving 1,166 patients was conducted at one psychiatric hospital and one mental health clinic. RESULTS: Symptom deterioration was reported in 23% to 34% of the patients and 9% to 20% reported increase in drug dosage. No significant differences were reported in these items among diagnostic categories. Patients with F3 (mood disorders) reported more psychological stress during the pandemic's beginning and during the emergency. Patients with F2 (schizophrenia, schizotypal, and delusional disorders) did online shopping and meetings less frequently, and reported poorer adherence of 3C's, while mask management was stricter in patients with F4 (neurotic, stress-related, and somatoform disorders). Symptom deterioration was significantly associated with increase in drug dosage, new physical symptoms, anxiety unrelated to COVID-19, stress at the beginning of pandemic, stress during the 'state of emergency', poor adaptability to environmental change, daily life changes, decrease in sleeping time, and decrease in time spent outside. CONCLUSION: One third of patients reported symptom deterioration during the pandemic, which was associated with stress and daily life changes. Patients with good adaptability to environmental changes might resilient against symptom deterioration. Providing continuous support to help patients manage their daily life in this COVID-19 era may minimize the risk of symptom deterioration.

Abnormal cleavage is involved in the self-correction of bovine preimplantation embryos.

Chromosome instability leading to aneuploidy during early cleavage is well known in humans and cattle. Partial compaction (PC), which occurs only in some blastomeres, is suggested as a self-correction mechanism through which human embryos avoid aneuploid mosaicism. Partially compacted embryos show abnormal cleavages more frequently during early development; however, the mechanism by which blastomeres are excluded has not been elucidated. Here, we confirmed PC in approximately half of the tested bovine embryos, similar to that in human embryos. DNA sequencing of single-cell and intact embryos revealed that the morulae that excluded some blastomeres had euploidy, but many of the excluded blastomeres had aneuploidy. Time-lapse imaging of zygotes without the zona pellucida revealed that the excluded blastomeres underwent reverse and direct cleavages, which are abnormal cleavages, more frequently than the blastomeres involved in compaction. These results suggest the potential role of abnormal cleavage in the self-correction mechanism during the development of mammalian preimplantation embryos.

Morphokinetic analysis of pronuclei using time-lapse cinematography in bovine zygotes.

The morphokinetics of pronuclei (PN) are considered crucial factors affecting embryogenesis in mammals. Whereas, since bovine zygotes contain a large number of cytosolic lipid droplets, detailed observation of PN has not been performed. In this study, we visualized PN using time-lapse cinematography (TLC) with light microscopy for the first time in delipidated bovine zygotes. The proportions of 0 PN, 1PN, 2PN, and multi-PN in delipidated bovine zygotes were 10.1%, 6.5%, 72.7%, and 10.8%, respectively. Abnormal fertilization, including 1 PN and multi-PN, was observed in 15.6% of blastocysts. The times from IVF to PN appearance, PN fading, and first cleavage in 2 PN bovine zygotes that developed into blastocysts were 10.4, 25.5, and 27.6 h, respectively, which were similar to PN morphokinetics in humans. The 2 PN zygotes showed that the prolonged time from IVF to the appearance of PN and from the fading of PN to the first cleavage negatively affected blastocyst formation. The time from appearance to fading of PN in multi-PN zygotes that developed into blastocysts was longer than that in multi-PN zygotes that did not develop into blastocysts. Besides, among zygotes that developed into blastocysts, the time from appearance to fading of PN in multi-PN zygotes was longer than that in 2 PN and 1 PN zygotes. These results suggest that PN morphokinetic abnormalities are associated with subsequent embryonic development. Observation of PN in bovine zygotes by using non-invasive visible light TLC by delipidation could be a powerful tool to clarify the relationship between PN morphokinetics and developmental competence.

Build-up functionalization of anti-EGFR × anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats.

Designing non-natural antibody formats is a practical method for developing highly functional next-generation antibody drugs, particularly for improving the therapeutic efficacy of cancer treatments. One approach is constructing bispecific antibodies (bsAbs). We previously reported a functional humanized bispecific diabody (bsDb) that targeted epidermal growth factor receptor and CD3 (hEx3-Db). We enhanced its cytotoxicity by constructing an Fc fusion protein and rearranging order of the V domain. In this study, we created an additional functional bsAb, by integrating the molecular formats of bsAb and high-affinity mutants previously isolated by phage display in the form of Fv. Introducing the high-affinity mutations into bsDbs successfully increased their affinities and enhanced their cytotoxicity in vitro and in vivo. However, there were some limitations to affinity maturation of bsDb by integrating high-affinity Fv mutants, particularly in Fc-fused bsDb with intrinsic high affinity, because of their bivalency. The tetramers fractionated from the bsDb mutant exhibited the highest in vitro growth inhibition among the small bsAbs and was comparable to the in vivo anti-tumor effects of Fc-fused bsDbs. This molecule shows cost-efficient bacterial production and high therapeutic potential.

Time-course colony tracking analysis for evaluating induced pluripotent stem cell culture processes.

Increasing the yield and maintaining a high quality of induced pluripotent stem cells (iPSCs) is necessary for manufacturing iPSCs at the industrial scale. However, because iPSCs are delicate, it is important to evaluate their quality during processing. To examine the status of cultured iPSCs non-invasively, morphology-based iPSC colony evaluation may be an efficient technology for cellular status monitoring and analysis. In this study, we examined the effectiveness of time-course colony tracking analysis for evaluating the iPSC culture process. Particularly, we obtained detailed time-course data to evaluate the effect of the pipetting technique on cell dissociation before seeding. Although the pipetting process causes severe shear stress to cells, which affects their quality, these effects have not been quantitatively analyzed because of their complex and uncontrollable parameters. By analyzing the heterogeneity and time-course responses of individual colonies, our colony tracking analysis revealed a critically damaged population caused by pipetting stress which could not be detected in conventional bulk analysis. Moreover, by comprehensively analyzing colony tracking data, which links the time-course morphology and marker staining results with each colony, we found that colony morphology is only highly correlated with the undifferentiated marker in the final stage, with a lower correlation in the early stages. Thus, colony tracking analysis provides a way to quantify cellular morphological information when evaluating complex iPSC manufacturing processes.

In-process evaluation of culture errors using morphology-based image analysis.

INTRODUCTION: Advancing industrial-scale manufacture of cells as therapeutic products is an example of the wide applications of regenerative medicine. However, one bottleneck in establishing stable and efficient cell manufacture is quality control. Owing to the lack of effective in-process measurement technology, analyzing the time-consuming and complex cell culture process that essentially determines cellular quality is difficult and only performed by manual microscopic observation. Our group has been applying advanced image-processing and machine-learning modeling techniques to construct prediction models that support quality evaluations during cell culture. In this study, as a model of errors during the cell culture process, intentional errors were compared to the standard culture and analyzed based only on the time-course morphological information of the cells. METHODS: Twenty-one lots of human mesenchymal stem cells (MSCs), including both bone-marrow-derived MSCs and adipose-derived MSCs, were cultured under 5 conditions (one standard and 4 types of intentional errors, such as clear failure of handlings and machinery malfunctions). Using time-course microscopic images, cell morphological profiles were quantitatively measured and utilized for visualization and prediction modeling. For visualization, modified principal component analysis (PCA) was used. For prediction modeling, linear regression analysis and the MT method were applied. RESULTS: By modified PCA visualization, the differences in cellular lots and culture conditions were illustrated as traits on a morphological transition line plot and found to be effective descriptors for discriminating the deviated samples in a real-time manner. In prediction modeling, both the cell growth rate and error condition discrimination showed high accuracy (>80%), which required only 2 days of culture. Moreover, we demonstrated the applicability of different concepts of machine learning using the MT method, which is effective for manufacture processes that mostly collect standard data but not a large amount of failure data. CONCLUSIONS: Morphological information that can be quantitatively acquired during cell culture has great potential as an in-process measurement tool for quality control in cell manufacturing processes.

Metabolism of steroids by cytochrome P450 2C9 variants.

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and ten mutants were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward steroids were examined. CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.48 and CYP2C9.52 had higher testosterone 6ß-hydroxylation than CYP2C9.1. CYP2C9.4 showed higher progesterone 6ß-hydroxylation activity than CYP2C9.1. CYP2C9.28 and CYP2C9.48 showed higher progesterone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.48 showed higher progesterone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.2, CYP2C9.3, CYP2C9.16 and CYP2C9.30 had higher estrone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.3 had higher estrone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.39 and CYP2C9.57 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.39 and CYP2C9.57 was not changed, but CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.30, CYP2C9.48 and CYP2C9.52 showed different hydroxylation activities toward steroids compared with CYP2C9.1.

Visualization of morphological categories of colonies for monitoring of effect on induced pluripotent stem cell culture status.

From the recent advances, there are growing expectations toward the mass production of induced pluripotent stem cells (iPSCs) for varieties of applications. For such type of industrial cell manufacturing, the technology which can stabilize the production efficiency is strongly required. Since the present iPSC culture is covered by delicate manual operations, there are still quality differences in produced cells from same culture protocols. To monitor the culture process of iPSCs with the quantified data to evaluate the culture status, we here introduce image-based visualization method of morphological diversity of iPSC colonies. We have set three types of experiments to evaluate the influential factors in iPSC culture technique that may disturb the undifferentiation status of iPSC colonies: (Exp. 1) technical differences in passage skills, (Exp. 2) technical differences in feeder cell preparation, and (Exp. 3) technical differences in maintenance skills (medium exchange frequency with the combination of manual removal of morphologically irregular colonies). By measuring the all existing colonies from real-time microscopic images, the heterogenous change of colony morphologies in the culture vessel was visualized. By such visualization with morphologically categorized Manhattan chart, the difference between technical skills could be compared for evaluating appropriate cell processing.

Parametric analysis of colony morphology of non-labelled live human pluripotent stem cells for cell quality control.

Given the difficulties inherent in maintaining human pluripotent stem cells (hPSCs) in a healthy state, hPSCs should be routinely characterized using several established standard criteria during expansion for research or therapeutic purposes. hPSC colony morphology is typically considered an important criterion, but it is not evaluated quantitatively. Thus, we designed an unbiased method to evaluate hPSC colony morphology. This method involves a combination of automated non-labelled live-cell imaging and the implementation of morphological colony analysis algorithms with multiple parameters. To validate the utility of the quantitative evaluation method, a parent cell line exhibiting typical embryonic stem cell (ESC)-like morphology and an aberrant hPSC subclone demonstrating unusual colony morphology were used as models. According to statistical colony classification based on morphological parameters, colonies containing readily discernible areas of differentiation constituted a major classification cluster and were distinguishable from typical ESC-like colonies; similar results were obtained via classification based on global gene expression profiles. Thus, the morphological features of hPSC colonies are closely associated with cellular characteristics. Our quantitative evaluation method provides a biological definition of 'hPSC colony morphology', permits the non-invasive monitoring of hPSC conditions and is particularly useful for detecting variations in hPSC heterogeneity.

[The Interaction of Low-dose Droperidol, Propofol, and Sevoflurane on QTc Prolongation].

BACKGROUND: Droperidol is an effective antiemetic, but its use is limited because of the warning of drug-induced QT prolongation. Some reports showed that low-dose droperidol does not significantly probing QT interval. This study was aimed to determine the effect of low-dose droperidol (1.25 and 2.5 mg) on QTc interval, and the interaction among droperidol, propofol and sevoflurane. METHODS: Patients received either 1.25 mg (group L : n = 25) or 2.5 mg (group H : n = 25) droperidol, and fentanyl (3 µg x kg(-1)) was administered 2.5 min later. One minute after fentanyl administration, anesthesia was induced using propofol (1.5 mg x kg(-1)) and vecuronium. One minute after propofol administration, sevoflurane (3%) was started. Tracheal intubation was performed 3 min after propofol administration, and then sevoflurane was reduced to 1%. RESULTS: Compared to baseline, the QTc interval in group L was unchanged by droperidol. In group H, the QTc interval was significantly prolonged after droperidol injection, but recovered after propofol injection. After tracheal intubation, QTc interval was significantly prolonged in both groups. CONCLUSIONS: Droperidol's effect on QTc prolongation was shown at the dose of 2.5 mg but not 1.25 mg. This prolongation effect was offset by propofol, and was unchanged by sevoflurane.

Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)-variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH-VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.

Comparative efficacy of levobupivacaine and ropivacaine for epidural block in outpatients with degenerative spinal disease.

BACKGROUND: Levobupivacaine has less toxic potential on both the cardiovascular and central nervous system and has been widely used for postoperative epidural analgesia in surgical patients. However, there are few reports on the efficacy of epidural levobupivacaine in outpatients with lumbosacral radiculopathy. This study was carried out to evaluate the comparative efficacy of levobupivacaine and ropivacaine for epidural block in outpatients with degenerative spinal disease and sciatica. OBJECTIVE: We studied 32 patients (19 men and 13 women) with degenerative spinal disease and sciatica. STUDY DESIGN: The study was performed in a prospective, randomized, double blind, and crossover fashion. SETTING: Treatment room for outpatients. METHODS: The epidural block was produced with a caudal approach (0.125% levobupivacaine or 0.2% ropivacaine, 15 mL). The upper level of analgesia, lumbosacral pain, motor blockade, and hemodynamic changes were evaluated by pin prick, visual analogue scale (VAS), Bromage scale, and arterial blood pressure and heart rate at 15, 30, 60, and 90 minutes after epidural block, respectively. The recovery time to mobilization, ambulation, and spontaneous micturition were measured. RESULTS: There were no significant differences (P < 0.05) in the upper level of analgesia, VAS, and Bromage scale between 0.125% levobupivacaine and 0.2% ropivacaine throughout the time course. There were no significant differences in the recovery times to mobilization, ambulation, and spontaneous micturition between 0.125% levobupivacaine and 0.2% ropivacaine. There were no significant differences in arterial blood pressure and heart rate between the 2 trials throughout the time course. CONCLUSION: The results showed that 0.125% levobupivacaine and 0.2% ropivacaine for epidural block by a caudal approach provide similar lumbosacral pain relief, hemodynamic effects, and the degree and the recovery of motor blockade in outpatients with degenerative spinal disease and sciatica.

Mitochondrial DNA reduced by hypoxic conditions in three-dimensional (3D) spheroid cell cultures.

Three-dimensional (3D) cell culture reflects many of the important properties of solid tumors, such as the inadequate diffusion of oxygen that results in hypoxia. To understand the mitochondrial states in cancer, we performed comparisons of the levels of mitochondrial DNA (mtDNA), fusion- and fission-related mitochondrial messenger RNA (mRNA), and mitochondrial protein expression between monolayer (2D)- and 3D-cultured cancer cells. The mtDNA levels were observed to be significantly lower in the 3D cells compared with the monolayer cells. In contrast, the differences in expression of the mitochondrial fusion- and fission-related mRNAs and mitochondrial proteins between 2D- and 3D-cultured cancer cells were not significant, as shown by real-time PCR and immunoblot analysis. Therefore, although mtDNA levels decrease as a whole during 3D culture, this does not appear to affect the fusion and fission of individual mitochondria. Indeed, the factors regulating mitochondrial dynamics during 3D cell culture remain unclear. This study provides the basis for future, more detailed studies on the regulation of mtDNA.

Label-free morphology-based prediction of multiple differentiation potentials of human mesenchymal stem cells for early evaluation of intact cells.

Precise quantification of cellular potential of stem cells, such as human bone marrow-derived mesenchymal stem cells (hBMSCs), is important for achieving stable and effective outcomes in clinical stem cell therapy. Here, we report a method for image-based prediction of the multiple differentiation potentials of hBMSCs. This method has four major advantages: (1) the cells used for potential prediction are fully intact, and therefore directly usable for clinical applications; (2) predictions of potentials are generated before differentiation cultures are initiated; (3) prediction of multiple potentials can be provided simultaneously for each sample; and (4) predictions of potentials yield quantitative values that correlate strongly with the experimental data. Our results show that the collapse of hBMSC differentiation potentials, triggered by in vitro expansion, can be quantitatively predicted far in advance by predicting multiple potentials, multi-lineage differentiation potentials (osteogenic, adipogenic, and chondrogenic) and population doubling potential using morphological features apparent during the first 4 days of expansion culture. In order to understand how such morphological features can be effective for advance predictions, we measured gene-expression profiles of the same early undifferentiated cells. Both senescence-related genes (p16 and p21) and cytoskeleton-related genes (PTK2, CD146, and CD49) already correlated to the decrease of potentials at this stage. To objectively compare the performance of morphology and gene expression for such early prediction, we tested a range of models using various combinations of features. Such comparison of predictive performances revealed that morphological features performed better overall than gene-expression profiles, balancing the predictive accuracy with the effort required for model construction. This benchmark list of various prediction models not only identifies the best morphological feature conversion method for objective potential prediction, but should also allow clinicians to choose the most practical morphology-based prediction method for their own purposes.

A new subfamily of polyphosphate kinase 2 (class III PPK2) catalyzes both nucleoside monophosphate phosphorylation and nucleoside diphosphate phosphorylation.

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by "high-energy" phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.

[The factors that require short acting beta-blockers during general anesthesia using remifentanil].

BACKGROUND: Short acting beta-blockers (SBB) have been utilized effectively to prevent adverse cardiac events perioperatively. After recent introduction of remifentanil in Japan, applications of SBB could have been changed because of its intense analgesic and negative chronotrophic effects. Thus, we evaluated the factors that require SBB during general anesthesia using remifentanil. MATERIALS AND METHODS: Total of 1,631 patients who had general anesthesia with remifentanil were enrolled. Groups were divided by the use of SBB. Using logistic multivariable analysis, the factors significantly increasing the chance of using SBB were evaluated including patients' characteristics, surgical procedures, and anesthetic methods. A P value < 0.05 was considered as statistical significance. RESULTS: One hundred thirty one patients received SBB perioperatively, 94 of them received only when awake and 34 of them received during remifentanil anesthesia. Emergency operation and preoperative ECG abnormalities were significant factors requiring SBB during anesthesia using remifentanil (OR; 3.0, 4.9 respectively). CONCLUSIONS: Even with use of remifentanil there are the patients, such as those under emergency operation or with ECG abnormalities who require SBB perioperatively.

The interaction of antiemetic dose of droperidol with propofol on QT interval during anesthetic induction.

PURPOSE: We investigated the effect of low-dose droperidol on heart rate-corrected QT (QTc) interval and interaction with propofol. METHODS: Seventy-two patients undergoing upper limb surgery were included in this study. Patients were randomly allocated to one of three groups: group S (n = 24), which received 1 ml saline; group D1 (n = 24), which received 1.25 mg droperidol; or group D2 (n = 24), which received 2.5 mg droperidol. One minute later, fentanyl (3 µg/kg) was administered. Two minutes after fentanyl administration, anesthesia was induced using propofol (1.5 mg/kg) and vecronium. Tracheal intubation was performed 3 min after the administration of propofol. Heart rate, mean arterial pressure, bispectral index, and QTc interval were recorded at the following time points: immediately before the droperidol injection (baseline); 3 min after the saline or droperidol injection; 3 min after the propofol injection; and 2 min after tracheal intubation. RESULTS: Compared to baseline, the QTc interval in group S and group D1 was significantly shorter after propofol injection, but recovered after tracheal intubation. In group D2, the QTc interval was significantly prolonged after droperidol injection, but recovered after propofol injection, and was significantly prolonged after tracheal intubation. CONCLUSIONS: We found that saline or 1.25 mg droperidol did not prolong QTc interval, whereas 2.5 mg droperidol prolonged the QTc interval significantly, and that propofol injection counteracted the prolongation of the QTc interval induced by 2.5 mg droperidol.