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IMPORTANCE: Cell therapy is a promising treatment approach for stroke and other diseases. However, it is unknown whether MultiStem (HLCM051), a bone marrow–derived, allogeneic, multipotent adult progenitor cell product, has the potential to treat ischemic stroke. OBJECTIVE: To assess the efficacy and safety of MultiStem when administered within 18 to 36 hours of ischemic stroke onset. DESIGN, SETTING, AND PARTICIPANTS: The Treatment Evaluation of Acute Stroke Using Regenerative Cells (TREASURE) multicenter, double-blind, parallel-group, placebo-controlled phase 2/3 randomized clinical trial was conducted at 44 academic and clinical centers in Japan between November 15, 2017, and March 29, 2022. Inclusion criteria were age 20 years or older, presence of acute ischemic stroke (National Institutes of Health Stroke Scale [NIHSS] score of 8-20 at baseline), confirmed acute infarction involving the cerebral cortex and measuring more than 2 cm on the major axis (determined with diffusion-weighted magnetic resonance imaging), and a modified Rankin Scale (mRS) score of 0 or 1 before stroke onset. Data analysis was performed between May 9 and August 15, 2022. EXPOSURE: Patients were randomly assigned to either intravenous MultiStem in 1 single unit of 1.2 billion cells or intravenous placebo within 18 to 36 hours of ischemic stroke onset. MAIN OUTCOMES AND MEASURES: The primary end points were safety and excellent outcome at day 90, measured as a composite of a modified Rankin Scale (mRS) score of 1 or less, a NIHSS score of 1 or less, and a Barthel index score of 95 or greater. The secondary end points were excellent outcome at day 365, mRS score distribution at days 90 and 365, and mRS score of 0 to 1 and 0 to 2 at day 90. Statistical analysis of efficacy was performed using the Cochran-Mantel-Haenszel test. RESULTS: This study included 206 patients (104 received MultiStem and 102 received placebo). Their mean age was 76.5 (range, 35-95) years, and more than half of patients were men (112 [54.4%]). There were no between-group differences in primary and secondary end points. The proportion of excellent outcomes at day 90 did not differ significantly between the MultiStem and placebo groups (12 [11.5%] vs 10 [9.8%], P = .90; adjusted risk difference, 0.5% [95% CI, −7.3% to 8.3%]). The frequency of adverse events was similar between treatment groups. CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, intravenous administration of allogeneic cell therapy within 18 to 36 hours of ischemic stroke onset was safe but did not improve short-term outcomes. Further research is needed to determine whether MultiStem therapy for ischemic stroke has a beneficial effect in patients who meet specific criteria, as indicated by the exploratory analyses in this study. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02961504

To study the time-course of infectious asthma, we retrospectively examined [FEV.sub.1] from 5 days before to 10 days after the onset of illness in 31 asthmatic children (20 boys and [...]

Any polymers composed of racemic repeating units are obviously optically inactive and hence chiral functions, such as asymmetric catalysis, will not be expected at all. Contrary to such a preconceived notion, we report an unprecedented helical polymer-based highly enantioselective organocatalyst prepared by polymerization of a racemic monomer with no catalytic activity. Both the right- and left-handed helical poly(biarylylacetylene)s (PBAs) composed of dynamically racemic 2-arylpyridyl-N-oxide monomer units with N-oxide moieties located in the vicinity of the helical polymer backbone can be produced by noncovalent interaction with a chiral alcohol through deracemization of the biaryl pendants. The macromolecular helicity and the axial chirality induced in the PBAs are retained (“memorized”) after complete removal of the chiral alcohol. Accordingly, the helical PBAs with dual static memory of the helicity and axial chirality show remarkable enantioselectivity (86% ee) for the asymmetric allylation of benzaldehyde. The enantioselectivity is slightly lower than that (96% ee) of the homochiral PBAs prepared from the corresponding enantiopure (R)- and (S)-monomers, but is comparable to that (88% ee) of the helical PBA composed of nonracemic monomers of ca. 60% ee.

A stimuli-responsible reversible structural transformation is of key importance in biological systems. We now report a unique water-mediated reversible transformation among three discrete double-stranded dinuclear titanium(IV) achiral meso- and chiral rac-helicates linked by a mono(μ-oxo) or a bis(μ-hydroxo) bridge between the titanium ions through hydration/dehydration or its combination with a water-mediated dynamic cleavage/re-formation of the titanium-phenoxide (Ti–OPh) bonds. The bis(μ-hydroxo) bridged titanium(IV) meso-helicate prepared from two tetraphenol strands with titanium(IV) oxide was readily dehydrated in CD3CN containing a small amount of water upon heating, accompanied by Ti–OPh bond cleavage/re-formation catalyzed by water, resulting in the formation of the mono(μ-oxo)-bridged rac-helicate, which reverted back to the original bis(μ-hydroxo)-bridged meso-helicate upon hydration in aqueous CD3CN. These reversible transformations between the meso- and rac-helicates were also promoted in the presence of a catalytic amount of an acid, which remarkably accelerated the reactions at lower temperature. Interestingly, in anhydrous CD3CN, the bis(μ-hydroxo)-bridged meso-helicate was further slowly converted to a different helicate, while its meso-helicate framework was maintained, namely the mono(μ-oxo)-bridged meso-helicate, through dehydration upon heating and its mesoto mesotransformation was significantly accelerated in the presence of cryptand[2.2.1], which contributes to removing Na+ions coordinated to the helicate. Upon cooling, the backward mesoto mesotransformation took place via hydration. Hence, three different, discrete double-stranded chiral rac- and achiral meso-titanium(IV) helicates linked by a mono(μ-oxo) or a bis(μ-hydroxo) bridge were successfully generated in a controllable manner by a change in the water content of the reaction media.

Adequate surgical planning includes a precise understanding of patient-specific anatomy and is a necessity for neurosurgeons. Although the use of virtual reality (VR) technology is emerging in surgical planning and education, few studies have examined the effectiveness of immersive VR during surgical planning using a modern head-mounted display.

In the electrocatalytic CO2reduction reaction (CO2RR), tin-based catalysts are known for their high formate faradaic yield. However higher overpotentials are required to attain a high faradaic yield with high partial current density for formate. Here, we describe the increase in the electrocatalytic CO2RR activity of Sn nanoparticles decorated on nitrogen-doped carbon fibers (NCFs) by adding a small amount of Pd. Nitrogen-doped carbon fibers decorated with SnPd nanoparticles (Sn100−yPdy–NCF) of different Sn : Pd ratios were synthesized using the electrospinning method and their electrocatalytic CO2RR activity was studied. The Sn100−yPdy–NCF catalyst with 3 wt% (y= 3) Pd displayed superior activity for the CO2RR and attained a faradaic efficiency of 85%, whereas the NCF with Sn nanoparticles (Sn100–NCF) attained only 57% efficiency at the same potential. The surface electronic configuration, Tafel slope (79 mV dec−1) and bicarbonate reduction activity of the catalyst reveal that the combination of SnOx–PdO on the catalyst surface is responsible for the superior CO2RR activity.

Ultrasound-targeted microbubble destruction (UTMD) induces cellular inflow of drugs at low intensity, while high intensity eradicates tumor vessels. Since vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), VEGFR2-targeted microbubble (MB) might additionally increase the tissue specificity of drugs and thus improve antitumor effects. In addition, fixing the dual pulse intensity could maximize MB properties. This study evaluated the one-off (experiment 1) and cumulative (experiment 2) treatment effect of UTMD by regulating the dual pulse output applied to PDAC using VEGFR2-targeted MB.

The deracemization reaction of a racemic double-stranded spiroborate helicate containing a bisporphyrin unit in the middle assisted by optically-active guests efficiently proceeded even below room temperature in the presence of a catalytic amount of strong acids that triggered the partial B–O bond cleavage/reformation of the spiroborate groups, affording an optically-active helicate with a high enantioselectivity up to 93% ee.We have achieved the acid-catalyzed deracemization of a racemic double-stranded spiroborate helicate containing a bisporphyrin unit in the middle assisted by optically-active guests below room temperature, thus producing a highly enantio-enriched helicate with up to 93% ee.

ABSTRACTCell size control is one of the prerequisites for plant growth and development. Recently, a GRAS family transcription factor, SCARECROW-LIKE28 (SCL28), was identified as a critical regulator for both mitotic and postmitotic cell-size control. Here, we show that SCL28is specifically expressed in proliferating cells and exerts its function to delay G2 progression during mitotic cell cycle in Arabidopsis thaliana. Overexpression of SCL28provokes a significant enlargement of cells in various organs and tissues, such as leaves, flowers and seeds, to different extents depending on the type of cells. The increased cell size is most likely due to a delayed G2 progression and accelerated onset of endoreplication, an atypical cell cycle repeating DNA replication without cytokinesis or mitosis. Unlike DWARF AND LOW-TILLERING, a rice ortholog of SCL28, SCL28 may not have a role in brassinosteroid (BR) signaling because sensitivity against brassinazole, a BR biosynthesis inhibitor, was not dramatically altered in scl28mutant and SCL28-overexpressing plants. Collectively, our findings strengthen a recently proposed model of cell size control by SCL28 and suggest the presence of diversified evolutionary mechanisms for the regulation and action of SCL28.

β-hemoglobinopathies are the most common monogenic disorders worldwide, and are defined based on whether patients have quantitative (β-thalassemia) or qualitative (Sickle Cell Disease (SCD)) defects in β-globin synthesis. Unfortunately, there are limited treatment options for β-hemoglobinopathies, and the majority of patients continue to develop life-threatening complications from their diseases. An alternative β-like subunit, γ-globin, has been shown to inhibit pathogenic hemoglobin polymerization in SCD and to functionally replace β-globin in β-thalassemia. The discovery of novel strategies to upregulate γ-globin expression may lead to effective therapies for β-hemoglobinopathies.

Erythropoietin (EPO) is a plasma glycoprotein that binds bone marrow erythroid progenitors, stimulating their proliferation and differentiation. EPO is secreted into the circulation by hepatocytes and specialized kidney peritubular fibroblasts, which contribute ~20% and ~80% of the total plasma EPO level, respectively. EPO production is induced during hypoxia, due to enhanced stability of the transcription factor hypoxia-inducible factor (HIF). However, increased HIF activity results in enhanced expression of angiogenesis and oncogenesis promoting genes, raising concerns about clinical strategies that promote EPO production via increasing HIF levels.

Red blood cell (RBC) development is regulated by a few external signaling molecules which act to influence the ongoing intracellular processes responsible for producing erythrocytes. While much is known about the signaling molecules important for blood production, comparatively little is known about the genes required for successfully generating erythrocytes. Several genes have already been identified as essential for RBC development through loss-of-function studies in the mouse or in human erythroid cells in vitro. However, these studies have been limited in scope to genes that are already suspected or known to influence RBC production. Since there are ~10,000 genes expressed in erythroid cells, it is unclear how many of these genes are functionally required for erythroid development.

Shinya Kondo, MD(*) and Masaki Ito, MD. Division of Respiratory Disease, Tokyo Metropolitan Children's Hospital. PURPOSE: Most asthmatic children clinically outgrow their asthma around puberty despite lack of normalization of [...]

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Organonitriles (RCN) were selectively converted into the corresponding diborylamines (RCH2N(Bpin)2) in reactions with pinacolborane (pinBH) in the presence of a catalytic amount of the iron–indium complex [Fe(CH3CN)6][cis-Fe(CO)4(InCl3)2]. The catalytic reaction mechanism was tentatively proposed. In addition, this catalytic system was found to be applicable for the synthesis of borylsilylamine in high yield when organonitrile was treated with hydroborane and hydrosilane simultaneously.

BACKGROUND:Whether additional indirect bypasses effectively contribute to revascularization in combined procedures remains unclear in patients with moyamoya disease.OBJECTIVE:To evaluate the longitudinal changes associated with combined procedures while following up pediatric and adult patients long term and to assess whether any other clinical factors or hemodynamic parameters affected these changes to determine an optimal surgical strategy.METHODS:We studied 58 hemispheres in 43 adults and 39 hemispheres in 26 children who underwent combined revascularization for moyamoya disease. To evaluate bypass development, we assessed the sizes of the superficial temporal artery and middle meningeal artery using magnetic resonance angiography. Multivariate analysis determined the effects of multiple variables on bypass development.RESULTS:Indirect bypass (middle meningeal artery) development occurred in 95% and 78% of the pediatric and adult hemispheres, respectively. Of these, dual development of direct and indirect bypasses occurred in 54% of the pediatric hemispheres and in 47% of the adult hemispheres. Reciprocal superficial temporal artery regression occurred in 28% of the hemispheres during the transition from the postoperative acute phase to the chronic phase during indirect bypass development. Good indirect bypass development was associated with adult hemispheres at Suzuki stage 4 or greater (odds ratio, 7.4; 95% confidence interval, 1.4-39.4; P= .02). Disease onset type and preoperative hemodynamic parameters were not considered predictors for the development of surgical revascularization.CONCLUSION:Simultaneous direct and indirect bypass development was most frequently observed, regardless of patient age and hemodynamic status. Applying indirect bypass as an adjunct to direct bypass could maximize revascularization in adults and children.

Introduction:Functional neuroimaging studies have reported increased activation in the contralesional cortex in both experimental and clinical settings of stroke. However, the dynamics of activation and neuronal subtypes involved is unclear. In this study we performed a time course analysis of neuronal activation and its subtypes in the contralesional cortex during post-stroke recovery using neuronal activity marker and cell type-specific markers.Methods:C57BL6 male mice (11-13 weeks old) underwent transient middle cerebral artery occlusion (MCAO, 30 minutes). Infarct was visualized by T2-weighted MRI and/or histology. Time course study used brains collected at PD1, 3, 7, 15, 21 and 28 and were processed for immunostaining using activity-dependent marker Early growth response 1 (Egr1). Egr1 positive cells were quantitatively assessed in the primary motor cortex (M1), pre-motor cortex (M2), and somatosensory cortex (SS) regions in the contralesional hemispheres. Cell type markers for NeuN (mature neurons), CaMKII and SATB2 (excitatory neurons), Parvalbumin (GABAergic neurons) and GFAP (astrocytes) were co-stained with Egr1 to identify its cell type specificity.Results:In the contralesional cortex, Egr1 predominantly co-localized with NeuN, indicating mature neurons. After stroke, contralesional cortical activity of Egr1 is detected at PD1-3 and peaks at PD7-15. Activity of Egr1 in contralesional cortex persisted at PD21-28 but begins to decrease in some regions. In-depth analysis of specific regions indicate that these changes occurred at contralesional M1, M2 and SS. Cell type co-localization analysis showed that Egr1 expressing cells are mostly colocalized with excitatory markers SATB2 and CaMKII but not inhibitory neuronal marker Parvalbumin, suggesting activated neurons in these contralesional cortical regions are mostly excitatory.Conclusion:Our results show that Egr1 expression is increased in excitatory neurons in the contralesional cortex after stroke, particularly in somatosensory and primary motor cortex. These data reveal dynamic changes of excitatory neuronal activity in the contralesional cortex post-stroke and highlight its involvement in post-stroke recovery.

Spherical powders via gas atomization etc. are recommended for use in SLM, however, the spherical powders are expensive and able to make in limited types of metal materials. Using non-spherical powder in SLM are capable of applying SLM to cheap part production and diversify powder materials for SLM. In this paper, to study the feasibility of using Ti6Al4V milled powder via hydride-dehydride method for SLM, characteristics of powders, powder bed performances, and SLM fabricated samples made from gas atomization powder and milled powder were analyzed and compared. The milled powder which added a fumed silica as a lubricant were also analyzed. In the results, flow and packing characteristics of the milled powder are lower than the gas atomized one. The characteristics of the milled powder are improved by adding fumed silica as lubricant, and had comparable characteristics to the gas atomized one. Microstructure of the SLM fabricated samples has a comparable size and morphology regardless of powders. Hardness of the SLM fabricated sample made from milled powder are larger than that of it made from gas atomized powder, owing to solid solution hardening by oxygen.

Plant MYB3R transcription factors, homologous to Myb oncoproteins, regulate the genes expressed at G2 and M phases in the cell cycle. Recent studies showed that MYB3Rs constitute multiprotein complexes that may correspond to animal complexes known as DREAM or dREAM. Discovery of the putative homologous complex in plants uncovered their significant varieties in structure, function, dynamics, and heterogeneity, providing insight into conserved and diversified aspects of cell cycle-regulated gene transcription.

The mechanisms underlying frontal lobe dysfunction in moyamoya disease (MMD) are unknown. We aimed to determine whether chronic ischemia induces subtle microstructural brain changes in adult MMD and evaluated the association of changes with neuropsychological performance.

We have demonstrated in vitro motility assay of microtubules on a kinesin-coated substrate by using adenosine triphosphate (ATP) generated from microsphere gels containing thylakoid membranes. Upon photoirradiation, the gels generated ATP, which was utilized for performing gliding motions of microtubules. This work may facilitate the development of a localized ATP generation system in the in vitro motility assay, which consequently would widen the applications of biomolecular motors in nanotechnology.A new ATP generation system has been developed by encapsulating thylakoid membranes of spinach leaves into alginate based microsphere gels. Upon photoirradiation the gels produce ATP which can be used for in vitro motility assay of biomolecular motors.

ABSTRACTWe estimated the efficacy of the current single administration of peramivir on the basis of peramivir pharmacokinetics in the upper respiratory tract (URT) and determined the predictive peramivir concentration-time curve to assess its efficacy against viruses with decreased susceptibility to neuraminidase inhibitors. Serum, nasal swab, or aspiration samples were collected from 28 patients treated with 10 mg/kg body weight peramivir. The sequential influenza viral RNA load and susceptibility after peramivir administration were measured using a quantitative real-time reverse transcription-PCR and neuraminidase inhibition assay. The peramivir concentrations in the serum and URT after a single administration at 10 mg/kg were measured, and the predictive blood and URT peramivir concentration-time curves were determined to assess various administration regimens against resistant variants. The peramivir concentration decreased to <0.1% of the maximum concentration of drug in serum (Cmax) at 24 h after administration. Rapid elimination of peramivir from the URT by 48 h after administration may contribute to an increase in the influenza A viral load after day 3 but not to a decrease in the influenza B viral load, despite the absence of a decrease in the susceptibility to peramivir. A longer maintenance of a high level of peramivir in the URT is expected by divided administration rather than once-daily administration. When no clinical improvement is observed in patients with normal susceptibility influenza A and B, peramivir readministration should be considered. In severe cases caused by resistant variants, better inhibitory effectiveness and less frequent adverse events are expected by divided administration rather than once-daily administration with an increased dosage.

Microtubule (MT)–kinesin, a biomolecular motor system, is a promising candidate for construction of artificial biomachines for a variety of nanotechnology applications. An active self-organization (AcSO) method involving a specific streptavidin (St)–biotin (Bt) interaction has been developed to assemble MTs into a highly ordered structure by exploiting their motility on a kinesin-coated surface. Dynein is another biomolecular motor that moves along the MTs in the opposite direction from kinesin. Dynein has not yet been used to demonstrate the AcSO of MTs. In this study, we report the first successful demonstration of the AcSO of MTs on a dynein-coated surface to produce ring-shaped MT assemblies similar to those of kinesin. We found that ring-shaped MT assemblies obtained on dynein showed equal clockwise and counterclockwise rotational motion. This work will enrich the building blocks for designing future oriented motor protein-based artificial devices.

Hydrogenation of chlorosilane was achieved in acetonitrile using NaBH4, a safe and easy-to-handle reagent. This reaction converted Si–Cl portion(s) in organosilanes into Si–H portion(s) without hydrogenation of cyano, chloro, and aldehyde groups on an alkyl substituent of the Si reagents. In addition, the Si–Cl/Si–H exchange reaction was applicable to dichlorodisilane without Si–Si bond cleavage.Hydrogenation of chlorosilane was achieved in acetonitrile using NaBH4being safe and easy to handle. This reaction converted Si–Cl portion(s) in organosilanes into Si–H portion(s) without hydrogenation of cyano, chloro, and aldehyde groups on an alkyl substituent of the Si reagents. In addition, the Si–Cl/Si–H exchange reaction was applicable to dichlorodisilane without Si–Si bond cleavage.

Introduction:Moyamoya disease (MMD) is a rare cerebrovascular disorder with unknown etiology. Although vascular smooth muscle cells (VSMCs) are known to be involved in the histopathology of MMD, VSMCs have not been biologically characterized in contrast to well-characterized endothelial cells (ECs).Methods:VSMCs were differentiated from neural crest stem cells (NCSCs) using induced pluripotent stem (iPS) cell lines established from peripheral blood mononuclear cells of healthy control subjects and MMD patients with RNF213founder polymorphism (n=3, each). Cellular proliferation, migration, contractile, and array-based transcriptome profiles of VSMCs were compared between the groups. The transcriptome profile of VSMCs was also compared with ECs using the same iPS cell lines and analysis platform.Results:Homogeneous spindle-shaped cells were differentiated from NCSCs. These cells exhibited similar expression of myosin heavy chain-11, calponin, smooth muscle protein 22α, and α-smooth muscle actin in both groups, and real-time PCR analysis further demonstrated no expression of CD31 and CD34, thereby suggesting the features of differentiated VSMCs.No significant differences in the proliferation, migration, and contractile profiles of VSMCs were observed between the two groups. Transcriptome analysis demonstrated similar gene expression profile of VSMCs in MMD and control subjects, resulting in six differentially expressed genes (DEGs, 4 upregulated and 2 downregulated genes in MMD), whereas the iPS cell-derived ECs showed distinct transcriptome profile in MMD patients as compared to control subjects with 120 DEGs (78 upregulated and 42 downregulated genes in MMD).Conclusions:This study showed similar biological and transcriptome features in VSMCs, in contrast to ECs with distinct transcriptome profile between MMD patients and control subjects. Thus, ECs, rather than VSMCs, may play a major role in the pathophysiology of MMD.

Ewing sarcoma family of tumors (ESFT) comprise common sarcomas in children and young adults. Such tumors usually occur in the medullary cavity of long bones and directly invade cortical bone and circumferential soft tissue, including periosteum. Periosteal ESFT is extremely rare and only a small numbers of cases, including cases not confirmed by molecular detection of chimeric fusion gene, have been reported. We describe herein a case of periosteal ESFT of the femur in a 13-year-old boy harboring EWS-FLI1 chimeric fusion gene transcripts detected by reverse transcription-polymerase chain reaction analysis. This report may provide an opportunity for further evaluation of the EWS-FLI1 type 1 fusion gene and detection of prognostic significance in periosteal ESFT.

To evaluate the effects of dementia on denture‐handling ability, we measured the amount of time that elderly subjects spent inserting and removing dentures. Elderly subjects with dementia tended to take more time inserting and removing dentures than those who were not demented. Only elderly patients who were severely demented needed signifi‐cantly more time removing partial dentures than they did removing complete dentures. There was no significant difference between the time spent inserting dentures and removing dentures. The greater amount of time required to inseri or remove dentures for subjects with dementia was due to dis‐orientation. The results indicate that elderly people with mild to moderate dementia are able to insert and remove their dentures without help.

Abstract Evidence is emerging that the E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S transition in plants. Here, we show that in the tobacco proliferating cell nuclear antigen (PCNA), whose transcript is specifically expressed at G1/S phase, the two E2F binding sites are synergistically responsible for transcriptional activation at G1/S phase in synchronized tobacco BY-2 cells transformed with promoter constructs fused to a reporter gene. In addition, we have isolated the tobacco DP cDNA (NtDP) and showed that significant activation of the reporter gene was observed in transient expression assays by concomitantly transfecting with plasmids expressing NtE2F and NtDP. This transcriptional activation was repressed by co-transfection with a plasmid expressing NtRBR1; in vitro pull-down assays also revealed that NtRBR1 binds directly to NtE2F, thereby potentially blocking the transcriptional activation of NtE2F. Importantly, this repressor activity was cancelled when NtRBR1 was further co-transfected with a plasmid expressing cyclin D but not with cyclin A or cyclin B. These results are discussed with respect to the repression activity of NtRBR1 on the NtE2F/NtDP complex.

Summary: (S)-9-chloro-5-[p-aminomethyl-o-(carboxymethoxy)phenylcarbamoylmethyl]-6,7-dihydro-1H,5H-pyrido[1,2,3-de]quinoxaline-2,3-dion hydrochloride trihydrate (SM-18400) was given intravenously to rats and dogs and its pharmacokinetics was investigated. By LC/MS/MS analysis, the major metabolite in the rat serum was identified asN-acetylated SM-18400 (SM-NAc). In rats,AUC ratio of SM-NAc to SM-18400 was approximately 50%. However, 71% of the dose was excreted as unchanged SM-18400 and only 9.8% as SM-NAc in the urine and bile, indicating that the contribution ofN-acetylation clearance (CL NAc) to the total clearance (CL tot) is limited to 10–30% in rats. No SM-NAc or other metabolites were detected in the dog serum, urine or bile. Thein vitro intrinsic clearance (CL int, ml/min/mg cytosolic protein) ofN-acetyltransferase (NAT) activities of dog liver cytosol towards SM-18400 and hepaticN-acetylation clearance (CL NAc, ml/min/kg body weight) estimated by well-stirred model were both only 5% of the respective rat value, well reflecting the relativein vivo CL NAc/CL tot ratios.CL int values for human live cytosol samples (n=4) and estimatedCL NAc were all less than 18% and 7% of the rat, respectively. Based on these results, we concluded that theCL NAc/CL tot of human would be small enough to avoid major inter-individual variance in SM-18400 pharmacokinetics due toN-acetylation polymorphism. In addition, even a human liver cytosol sample lacking polymorphic NAT2 activity as determined by sulfamethazine (SMZ)N-acetylation analysis, proved capable of acetylating SM-18400, suggesting that NAT2 is not the major enzyme responsible forN-acetylation of SM-18400 in human. This fact would also reduce the risk ofN-acetylation polymorphism playing a role in clinical use of this drug.

Cytokinesis is the last essential step in the distribution of genetic information to daughter cells and partition of the cytoplasm. In plant cells, various proteins have been found in the phragmoplast, which corresponds to the cytokinetic apparatus, and in the cell plate, which corresponds to a new cross wall, but our understanding of the functions of these proteins in cytokinesis remains incomplete. Reverse genetic analysis of NPK1 MAPKKK (nucleus– and phragmoplast–localized protein kinase 1 mitogen–activated protein kinase kinase kinase) and investigations of factors that might be functionally related to NPK1 have helped to clarify new aspects of the mechanisms of cytokinesis in plant cells. In this review, we summarize the evidence for the involvement of NPK1 in cytokinesis. We also describe the characteristics of a kinesin–like protein and the homologue of a mitogen–activated protein kinase that we identified recently, and we discuss possible relationships among these proteins in cytokinesis.

O: -methyltransferase, and cinnnamyl alcohol dehydrogenase were localized to differentiating xylem. These enzymes are particularly abundant during secondary wall formation. Immunolabeling was observed on polysomes and in the cytosol of the cells during secondary wall formation, indicating that these enzymes are synthesized in the polysomes and released in the cytosol. The synthesis of monolignols might occur in the cytosol. Immunolabeling of anionic peroxidase was also localized to the differentiating xylem, particularly during secondary wall formation. The labeling, however, was observed in the rough endoplasmic reticulum (r-ER), the Golgi apparatus, and the plasma membrane, indicating that peroxidase is synthesized in the r-ER, transported to the Golgi apparatus, and localized on the plasma membrane by fusion of the Golgi vesicles to the membrane.

SK32 mutant cells, which were isolated as peroxisome-deficient Chinese hamster ovary (CHO) cells by an advantage of a visible peroxisome form of green fluorescent protein (GFP), were found to suffer from a functional loss of PEX5 gene encoding for PTS1R. The sequence analysis of cDNA indicated that PEX5 gene encoded for the two isoforms composed of 603 amino acids (PTS1RS) and 640 amino acids (PTS1RL). The mutation changed glycine to arginine at amino acid position 343 of PTS1RL (corresponding to the position 306 of PTS1RS) in SK32 cells. The mutant cells exhibited a temperature-sensitive (TS) phenotype on the peroxisomal localizations of the recombinant GFP and urate oxidase appending a genuine peroxisome targeting signal 1 (PTS1), a tripeptide of Ser-Lys-Leu (SKL) at the C-terminus, but did not on that of catalase harboring a divergent PTS1, Lys-Ala-Asn-Leu (KANL) sequence. 3-ketoacyl-CoA thiolase (hereafter referred to as thiolase), which harbors an extension sequence (PTS2) at the N-terminus, never appeared to be affected on the peroxisomal localization in the mutant cells. When thiolase was examined on the molecular size in the mutant cells, the enzyme existed as the larger precursor form in the peroxisomes at 37°C and a considerable part (almost half) was converted to the mature size at 30°C. These results indicate that the amino acid substitution, Gly306Arg in PTS1RS and/or Gly343Arg in PTSRL, gives rise to TS phenotype on the peroxisomal translocation of PTS1 proteins and the maturation of PTS2 protein.

Branched‐chain α‐keto acid dehydrogenase (BCKDH), a multienzyme complex, plays a key role in branched‐chain amino acid catabolism. However, it remains unclear whether expression of each subunit is coordinately regulated in plants, which should be important for the efficient assembly of subunits into a functional multienzyme complex. We show that the transcripts from the ArabidopsisE1α subunit gene accumulated in dark‐adapted leaves and in sugar‐starved suspension cells. These results are complementary to our previous report that the transcripts for the E1β and E2 subunit genes accumulated in sugar‐starved cells. Expression of the E1α gene is likely to be regulated by hexokinase‐mediated sugar signaling, indicating that sugar plays a regulatory role in the coordinated expression of BCKDH subunit genes. Furthermore, Leu and its metabolite α‐ketoisocaproate have synergistic effects on the enhanced expression of BCKDH subunit genes under sugar starvation. We hence suggest that branched‐chain amino acids activate their own degradation pathway in sugar‐starved cells through co‐induction of each subunit gene of BCKDH.

We present a unique case of a 9-month-old infant with a left adrenal neuroblastoma with sarcoid reaction, detected by mass screening. There was no clinical evidence indicating systemic sarcoidosis or pulmonary mycobacterial infection. Histological examination of the resected adrenal tumor revealed many noncaseating epithelioid granulomas with lymphocytic infiltrate, composed of epithelioid cells and few giant cells, arising in tumor parenchyma and fibrovascular stroma. Most of the lymphocytes in the granulomas were CD3- or CD45RO-positive T cells, with fewer being CD20-positive B cells. The lymphocytes in the epithelioid granulomas expressed CD4 or CD8, but not CD56 and CD57. CD4-positive cells were observed more within the granulomas (internal area) than in the surrounding area (external area) of the same granulomas, while most of the CD8-positive cells were seen consistently at the outer margin of the granulomas (marginal zone). CD45RA-positive T cells were observed predominantly in the external area. The results of immunostaining demonstrated that lymphocytes in granulomas of this case showed the same distribution pattern as that seen in systemic sarcoidosis. Although the sarcoid reaction is a phenomenon known to be associated with the region of cancer, granuloma within the primary neuroblastoma is extremely rare. The sarcoid reaction in the present case of neuroblastoma may be associated with a delayed-type hypersensitivity reaction, and its significance and relevance still remain obscure.

Cyclins control the transition between the phases of the eukaryotic cell cycle as regulatory subunits of the cyclin-dependent kinases (CDKs). Phase-specific activation of the CDK is in part regulated by phase-specific expression of their cyclin component. In most eukaryotic cells including higher plant, B-type cyclin genes are expressed specifically at G2/M phase during the cell cycle. Promoters from yeast, plant and animal B-type cyclin genes are all activated in a cell cycle-regulated manner. In yeast, a transcription factor, Mcm1, in cooperation with an uncloned factor SFF, regulates the cell cycle-dependent promoter activation of mitotic B-type cyclin genes, CLB1and CLB2.Activity of the human cyclin B1 promoter is regulated by a complex mechanism involving multiple cis-acting elements, none of which are sufficient for G2/M-specific promoter activation. In contrast, plants employ a simple mechanism for cell cycle-regulated promoter activation of B-type cyclin genes. Plant B-type cyclin gene promoters contain a common cis-acting element, called the MSA element, which is necessary and sufficient for the phase-specific promoter activation. MSA-like sequences are also found in the promoters of G2/M-specific genes encoding kinesin-like proteins, suggesting that a defined set of G2/M-specific genes are co-regulated by a common MSA-mediated mechanism in plants. Thus, the molecular mechanisms regulating B-type cyclin gene expression are evolutionarily divergent, and the MSA-mediated mechanism seems to be specific to plants. The consensus sequence of the MSA element resembles the binding sites of animal Myb transcription factors. A set of our data suggest the possibility that plant Myb may have unexpected roles in G2/M by inducing B-type cyclin genes, together with other cell cycle-related genes in plants.

SK24 and PT54 mutant cells, which are peroxisome-deficient Chinese hamster ovary (CHO) cells isolated using peroxisomal forms of green fluorescent protein (GFP), were found to be defective in the PEX2 gene. The nucleotide sequences of PEX2 cDNA from the mutant cells were determined to identify mutation sites in the mutant cells. The mutation in SK24 cells changed cysteine to tyrosine at amino acid position 258, which is a component of the RING finger (C3HC4) motif in the carboxyl terminus of the protein. PT54 cells contained a nonsense mutation in the codon for glutamine at position 101, resulting in premature termination. The immunocytochemical analyses revealed distinct phenotypes between mutant cells defective in the PEX2 gene. Both mutant cells exhibited cytosolic mislocalizations on catalase and urate oxidase containing PTS1. On the other hand, on 3-ketoacyl-CoA thiolase containing PTS2, PT54 cells exhibited cytosolic mislocalization, but SK24 cells exhibited peroxisomal localization. When wild-type or mutant-type PEX2 cDNA was transfected into both mutant cells, the stable transformants restored the phenotype in accordance with the transfected cDNA. These observations indicate that an amino acid substitution, cysteine-258 to tyrosine, in the RING finger motif of PEX2 protein, whose function is required for peroxisomal localizations of both PTS1- and PTS2-containing proteins, results in a complete defect in the PTS1 pathway but not in the PTS2 pathway.

The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1′-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.

Branched-chain α-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1β and E2 subunits of BCKDH, respectively, fromArabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines, were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti‐annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis‐negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression.

Background: Tyrosine kinase inhibitor (TKI) has been used in pediatric chronic myeloid leukemia (CML) for more than 10 years, but only a few prospective clinical studies have been conducted in pediatric patients with CML due to their rarity. We conducted the JPLSG CML-08 study to determine the efficacy and tolerability of TKIs in children and adolescents with newly diagnosed CML in chronic phase (CML-CP).

Idiopathic hyperammonemia (IHA) has been described as a complication of intensive chemotherapy for the treatment of hematologic malignancy but has subsequently been found in patients undergoing bone marrow transplantation and in those with solid tumors treated with 5-fluorouracil. Although IHA is a rare complication, it is sometimes associated with high mortality in hematologic malignancies. Here we report the case of a 15-year-old boy in whom hyperammonemia developed during the initial treatment with prednisolone for newly diagnosed acute lymphoblastic leukemia and who survived after early detection and oral lactulose therapy. To the best of our knowledge, this is the first report of IHA that was not induced by intensive chemotherapy, stem cell transplantation, or asparaginase therapy in a patient with newly diagnosed leukemia, but developed during an initial treatment with a steroid. Early detection of IHA by measuring the plasma ammonia level in patients with neurological symptoms may improve the outcome.

The transcription factor E2F regulates the expression of genes involved in the progression of G1/S transition and DNA replication in mammalian cells. We cloned and characterized a cDNA (NtE2F) corresponding to a E2F homolog of tobacco (Nicotiana tabacum). The transcription of NtE2Fwas induced as cells progressed from G1 to the S phase and expressed much earlier than that of the proliferating cell nuclear antigen (PCNA) gene. We demonstrated that NtE2F can interact with the tobacco retinoblastoma (Rb)‐related protein in a yeast two‐hybrid assay. To further characterize NtE2F, the trans‐activation activity of NtE2F was examined by using a transient assay in the tobacco Bright Yellow‐2 (BY‐2) cells with NtE2F fused to the DNA‐binding domain of the yeast transcriptional activator GAL4. NtE2F activated the transcription of the β‐glucuronidase (GUS) reporter gene driven by a cauliflower mosaic virus (CaMV) 35S core promoter containing the GAL4‐binding sequence. This is the first report of the identification of a functionally equivalent E2F‐like gene in plants.

Summary: This review paper describes the importance of synchronous cell cultures as experimental systems for investigations of mechanisms of the cell cycle of higher plants, and various methods of synchronization are discussed. The efficient synchronization methods were double phosphate starvation in Catharanthus roseus cells and aphidicolin treatment in tobacco cells. Using these systems, cell cycle-dependent genes were isolated and characterized. One of them, cyc07, was investigated in detail and the possible function of cyc07 is discussed as an example of genes involved in the progression of the cell cycle of higher plants. Finally, a perspective of investigations of the cell cycle of higher plant cells is discussed.