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Bromovalerylurea (BU), an acyl urea derivative, was originally developed as a hypnotic/sedative. We recently reported that BU at a dose of 50 mg/kg ameliorates sepsis, Parkinson's disease, and traumatic brain injury in Wistar rat models through its anti-inflammatory actions on microglia and macrophages. However, since BU was developed more than 100 years ago, its hypnotic mechanism and characteristics are poorly understood. Herein, we conducted an electroencephalogram (EEG) study and found that BU, when administered at a dose of more than 125 mg/kg but not at a dose of 50 mg/kg in Wistar rats, significantly increased non-rapid eye movement (NREM) sleep duration and dose-dependently decreased rapid eye movement (REM) sleep duration. This characteristic of sleep induced by BU is similar to the effect of compounds such as barbiturate, benzodiazepine, and z-drugs, all of which require γ-aminobutyric acid A receptors (GABA A R) for hypnotic/sedative activity. To investigate whether BU could potentiate GABA A ergic neurotransmission, we conducted a whole-cell patch-clamp recording from pyramidal neurons in rat cortical slices to detect spontaneous GABA A R-mediated inhibitory postsynaptic currents (IPSCs). We found that BU dose-dependently prolonged IPSCs. Importantly, the prolonged IPSCs were not attenuated by flumazenil, a benzodiazepine receptor antagonist, suggesting that modulation of IPSCs by BU is mediated by different mechanisms from that of benzodiazepine. Taken together, these data elucidate the basic characteristics of the hypnotic effects of BU and suggest that the enhancement of GABA A R-mediated Cl - flux may be a possible mechanism that contributes to its hypnotic/sedative activity., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in lipid and glucose metabolism. In this study, the function of PPARγ on lung development was investigated. Lung-specific Pparg conditional knockout mice (Pparg ΔLuEpC ) were developed using Cre-Lox system. Pparg ΔLuEpC mice showed abnormal lung development with enlarged airspaces and followed by increase of apoptotic cells at E14.5 to E18.5. Gene analysis revealed that expression of Pmaip1, a gene related to apoptosis, was significantly increased while expression of Retnla, a gene related to anti-apoptosis, was dramatically decreased in the fetal lung (E14.5) of Pparg ΔLuEpC mice. In addition, expression of Pthlh, a gene phenotypically expressed in the congenital cystic adenomatoid malformation (CCAM), was increased at E14.5 to E18.5 in the lung of Pparg ΔLuEpC mice. Cell culture studies revealed that PPARγ could bind to promoter region of Pthlh gene as a repressor in the immortalized mouse lung epithelial cell line MLE-15. Surprisingly, phenotypic changes in MLE-15-shPparg cells, stably transfected with shPparg plasmid, were similar to the Pparg ΔLuEpC mice model. In addition, MLE-15-shPparg cells were easily detached from the cultured plate when cold phosphate buffered saline was applied. Furthermore, expression of Cdh1, a gene related to cell adhesion, was significantly reduced in the MLE-15-shPparg cells. Taken together, PPARγ may play an important role in fetal lung development via alveolar cell-to-cell adhesion system., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We investigated the effects of antipsychotics on human induced pluripotent stem cell (hiPSC)-derived neural stem cell (NSC) differentiation. Induction of NSCs from hiPSCs was performed using PSC neural induction medium. Induced NSCs were subsequently cultured in neural differentiation medium containing antipsychotics. Cultured cells were subjected to neural differentiation marker analysis. As previously shown in rodent cells, antipsychotics promoted neural differentiation compared with vehicle treatment. Atypical antipsychotics appear to possess more differentiation induction potential than typical ones. Most NSCs do not express dopamine D2 receptor; however, our in vitro study indicates the clinical potential of antipsychotics could include effects independent of monoamine receptor expression in NSCs. Our study shows NSCs derived from hiPSCs provide opportunity to investigate the underlying direct effect of antipsychotics treatment on NSCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Rac1, a GTPase of the Rho subfamily, has a crucial role in cytoskeletal architecture, as well as the regulation of cell migration and growth. However, renal injury in mice with podocyte-specific deletion of Rac1 has yet to be elucidated fully due to conflicting findings. Herein, we identified a possible role for Rac1 in podocytes of streptozotocin- (STZ) induced diabetic mice. The urinary albumin/creatinine ratio (ACR) in the knockout (KO) group was significantly higher than that in the wild type (WT) group at any week of age. A more marked ACR increase was observed in STZ/KO group than STZ/WT group, although ACR did increase with weeks of age in both diabetic groups. The kidney sections from diabetic mice revealed a glomerular hypertrophy with mesangial expansion, but there was no appreciable difference in glomerular findings under a light microscope between STZ/WT and STZ/KO mice. However, an electron microscopy analysis revealed that regardless of the presence or absence of diabetes, both KO (KO and STZ/KO) groups had a higher rate of foot process effacement compared with both WT (WT and STZ/WT) groups. The expression levels of the slit diaphragm protein, podocin, was reduced with the induction of diabetes, and the levels in the STZ/KO group experienced a further reduction compared with the STZ/WT group. The number of WT1-positive cells in the STZ/KO group was more significantly decreased than that in the other three groups. In contrast, the numbers of cleaved caspase 3- and TUNEL-positive cells in the glomeruli of the STZ/KO group were more increased than those in the STZ/WT group. Thus, this study provides evidence that podocyte-specific deletion of Rac1 results in morphological alteration in podocytes, and that the induction of apoptosis or decreased expression of the slit diaphragm proteins by hyperglycemic stimuli are associated with the progression of diabetic nephropathy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Isocitrate dehydrogenase 2 (IDH2) mutations have been reported in gliomas, osteosarcomas, cartilaginous tumors, giant cell tumors of bone, and acute myeloid leukemias. Although IDH2 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate (α-KG) in mitochondria, mutated IDH2 proteins possess the ability to change α-KG into the oncometabolite R(-)-2-hydroxyglutarate (2-HG). To date, several monoclonal antibodies (mAbs) specific for IDH2 mutations have been established, such as KMab-1 against IDH2-R172K, MMab-1 against IDH2-R172M, and WMab-1 against IDH2-R172W. Although a multi-specific mAb MsMab-1 reacted with IDH2-R172G and IDH2-R172S, a mono-specific mAb against IDH2-R172S has not been established. In this study, we established a novel mAb SMab-2, which recognizes IDH2-R172S but not with wild type IDH2 in ELISA. Although SMab-2 reacted with both IDH1-R132S and IDH2-R172S expressed in Escherichia coli, it reacted with only IDH2-R172S expressed in U-2 OS osteosarcoma cells. Furthermore, SMab-2 recognized endogenous IDH2-R172S protein expressed in SW1353 chondrosarcoma cells in Western blot and immunocytochemical analyses. SMab-2 is expected to be useful for diagnosis of IDH2-R172S-bearing tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Previous studies have revealed that podocytes normally can be associated with a very high degree of autophagic activity, and that a lack of autophagic activity in podocytes is associated with susceptibility to disease and to late-onset glomerulosclerosis. In the present study, we conducted unilateral nephrectomy as a surgical model for acute nephron reduction. First, using GFP-LC3 transgenic mice to monitor autophagy, we found that glomerular autophagy could be transiently suppressed by surgery, but that it was restored quickly. To further explore the significance of podocyte autophagy after unilateral nephrectomy, we investigated podocyte-specific Atg7-deficient mice. The knockout mice exhibited no pathological phenotype compared with wild-type mice before nephrectomy. However, 1 day after nephrectomy, significantly higher levels of proteinuria and ultrastructural changes that included foot process effacement and a significant reduction in podocyte number were detected in mice harboring Atg7-deficient podocytes. Moreover, biochemical and immunohistochemical analyses showed a robust increase in polyubiquitin levels and ER stress markers in the glomeruli of the mice with autophagy-deficient podocytes. These results show the importance of the autophagic process in podocytes for maintaining a normal degree of filtration function during the adaptation to compensatory kidney hypertrophy following unilateral nephrectomy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Skeletal muscle is a primary organ that uses blood glucose. Insulin- and 5'AMP-activated protein kinase (AMPK)-regulated intracellular signaling pathways are known as major mechanisms that regulate muscle glucose transport. It has been reported that macrophage migration inhibitory factor (MIF) is secreted from adipose tissue and heart, and affects these two pathways. In this study, we examined whether MIF is a myokine that is secreted from skeletal muscles and affects muscle glucose transport induced by these two pathways. We found that MIF is expressed in several different types of skeletal muscle. Its secretion was also confirmed in C2C12 myotubes, a skeletal muscle cell line. Next, the extensor digitorum longus (EDL) and soleus muscles were isolated from mice and treated with recombinant MIF in an in vitro muscle incubation system. MIF itself did not have any effect on glucose transport in both types of muscles. However, glucose transport induced by a submaximal dose of insulin was diminished by co-incubation with MIF in the soleus muscle. MIF also diminished glucose transport induced by a maximal dose of 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), an AMPK activator, in the EDL muscle. These results suggest that MIF is a negative regulator of insulin- and AICAR-induced glucose transport in skeletal muscle. Since MIF secretion from C2C12 myotubes to the culture medium decreased during contraction evoked by electrical stimulations, MIF may be involved in the mechanisms underlying exercise-induced sensitization of glucose transport in skeletal muscle., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47(phox) and p67(phox) deficiency. The p47(phox) or p67(phox) protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Chondrocytes are known to express Sp7/Osterix (Osx) to varying degrees, but the role of Osx in chondrocytes is still unknown. In the current study, we investigated the role of the Osx gene using the clonal mouse embryonic cell line ATDC5, which retains the properties of the chondroprogenitor. ATDC5 cells express Osx; therefore, the effects of Osx gene silencing with shRNA lentiviral particles on chondrocyte marker gene expression and alkaline phosphatase (ALP) activity were investigated. At confluency, gene silencing down-regulated expression of the Sox trio (Sox5, 6, 9), Dlx5 and Alp mRNA, as well as ALP enzyme activity. Bone morphogenetic protein 2 (BMP2) is known to induce Osx gene expression in chondrocytes, and stimulation with BMP2 rescued Osx expression accompanied by up-regulation of Alp expression and ALP enzyme activity in a dose-dependent manner. To clarify the role of Osx in chondrocyte differentiation, cells induced to differentiate by 10μg/ml insulin for 21days were examined. Gene silencing inhibited the expression of the hypertrophic chondrocyte marker gene, type X collagen (Col X), and attenuated up-regulation of Dlx5 and Alp mRNA, as well as ALP enzyme activity. Taken together, these results suggest that Osx is involved in chondrogenic gene activation and is a positive regulator of the chondrocyte differentiation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Assembly of Amyloid beta (Abeta) peptides, in particular Abeta-42 is central to the formation of the amyloid plaques associated with neuro-pathologies such as Alzheimer's disease (AD). Molecular assembly of individual Abeta-42 species was observed using a simple fluorescence microscope. From the molecular movements (aka Brownian motion) of the individual peptide assemblies, we calculated a temporal evolution of the hydrodynamic radius (R(H)) of the peptide at physiological temperature and pH. The results clearly show a direct relationship between R(H) of Abeta-42 and incubation period, corresponding to the previously reported peptide's aggregation kinetics. The data correlates highly with in solution-based label-free electrochemical detection of the peptide's aggregation, and Abeta-42 deposited on a solid surface and analysed using atomic force microscopy (AFM). To the best of our knowledge, this is the first analysis and characterisation of Abeta aggregation based on capturing molecular trails of individual assemblies. The technique enables both real-time observation and a semi-quantitative distribution profile of the various stages of Abeta assembly, at microM peptide concentration. Our method is a promising candidate for real-time observation and analysis of the effect of other pathologically-relevant molecules such as metal ions on pathways to Abeta oligomerisation and aggregation. The method is also a promising screening tool for AD therapeutics that target Abeta assembly.

We examined the effects of dexamethasone (Dex), a synthetic glucocorticoid, on the formation of mineralized bone nodules and the gene expressions of the late osteoblastic markers, bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN) in mature osteoblast ROS17/2.8 cells. Treatment of ROS17/2.8 cells with Dex resulted in the induction of mineralization accompanied with increasing BSP and OC expressions. Previous reports have demonstrated that BSP and OC expressions are regulated by Runx2. Then, we hypothesized that Dex might promote osteoblastic differentiation and mineralization on ROS17/2.8 by Runx2. In this study, no effect was observed in mRNA and protein expression of Runx2. However, the transcriptional activity of Runx2 was enhanced by Dex treatment. Furthermore, the Dex-induced BSP and OC expressions decreased after the transfection of Runx2 small-interfering RNAs (siRNAs). These results suggested that the enhancement of Runx2 transcriptional activity by Dex treatment may be followed by the activation of osteoblast marker genes, such as BSP and OC to thereby produce a bone-specific matrix that subsequently becomes mineralized.

The Fox family of transcription factors is expressed in various organs and tissues during development, and is involved in a variety of developmental and cellular differentiation processes. Foxc2 mRNA is strongly expressed in mesoderm-derived tissues in the embryo, but the molecular mechanism of Foxc2-induced cellular differentiation and the physiological role of Foxc2 are unclear. In mouse myoblast C2C12 cells, over-expression of Foxc2 increased the expression of desmin, the muscle-specific member of the intermediate filament family of proteins, and induced the synthesis of myotubes. Transient expression of Foxc2 increased MyoD mRNA and protein levels, as assessed by real-time PCR and Western blot, respectively. Chromatin immunoprecipitation (ChIP) analysis showed that Foxc2 does not bind to the promoter region of the MyoD gene, which indicated that Foxc2 does not directly activate MyoD. In contrast to reports that Foxc2 regulates the production of basement membrane components in endothelial cells, we found no evidence of Foxc2-mediated regulation of Collagen type IV alpha 1 (Col4a1) or Col4a2 in myoblast cells. Taken together, these results indicate that Foxc2 plays an important role in the development of the mesenchyme through the regulation of MyoD gene expression.

There exists a significant difference in pre-tRNA preference among prokaryotic tRNase Zs. This is an enigma, because pre-tRNAs should form the common L-shaped structure and tRNase Zs should form the common structure based on the alphabeta/betaalpha-fold. To address this issue, we examined six different eubacterial and archaeal tRNase Zs including two newly isolated tRNase Zs for cleavage of 18 different pre-tRNA substrates. Two Thermotoga maritima, one Thermus thermophilus, one Bacillus subtilis, one Thermoplasma acidophilum, and one Pyrobaculum aerophilum enzymes were tested. To our surprise, the newly isolated proteins T. maritima and T. thermophilus showed the weak tRNase Z activity, even though their primary amino acid sequences are, on the whole, quite different from those of the typical tRNase Zs. We confirmed that substrate recognition ability is quite different among those tRNase Zs. In addition, we found that the optimal conditions as a whole differ significantly among the enzymes. From these results, we provided several clues to solve the enigma by showing the potential importance of the 74th-76th nucleotide sequence of pre-tRNA, the flexible arm length of tRNase Z, the divalent metal ion species, and the histidine corresponding His222 in T. maritima tRNase Z.

The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.

Phytochelatins (PCs) are well known as the heavy metal-detoxifying peptides in higher plants, eukaryotic algae, fungi, and nematode. In contrast, neither PCs nor PC synthase genes have ever been identified in any prokaryotes. The genome sequences for the cyanobacterium Nostoc sp. PCC 7120 were recently completed and allowed us to identify a gene encoding a PC synthase-like protein, termed alr0975. The predicted product of alr0975 contains the conserved N-terminal domain but not the variable C-terminal domain found in eukaryotic PC synthases. The recombinant alr0975 protein strongly catalyzed the first step of PC synthesis, in which glutathione (GSH) is converted to gamma-glutamylcysteine (gamma-EC), although the protein only weakly catalyzed the second step of PC synthesis, namely the transfer of gamma-EC moiety to an acceptor GSH molecule to form PC(2). These results suggest alr0975 protein may be a more primitive form of the PC synthases found in eukaryotes.

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.

The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein (Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit beta-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation.

The YlALK1 gene, which encodes cytochrome P450ALK1, plays a primary role in the assimilation of n-decane by yeast Yarrowia lipolytica and is inducible by n-decane at the transcriptional level. Deletion analysis of the YlALK1 promoter revealed that a 95-bp region on the YlALK1 promoter (from the position -400 to -304 upstream of the ATG codon) is essential for the induction by n-decane and we named this region ARR1 (alkane-responsive region). ARR1 was found to be made up of two different elements, ARE1 (alkane-responsive element 1; from -394 to -371) and ARE2 (from -325 to -305). By electrophoretic mobility shift assay, we found that the respective elements gave specific shift bands with the extracts from Y. lipolytica cells grown on n-alkane, but not much evidently from the cells grown on glycerol or glucose. This suggests that proteins that specifically bind to these elements are present and their binding or synthesis is dependent on n-alkane.

LAP1s (lamina-associated polypeptide 1s) are type 2 integral membrane proteins with a single membrane-spanning region of the inner nuclear membrane. We report here on the cloning of the full-length cDNA of human LAP1B (huLAP1B) that encodes 584 amino acids. The sequence homology between the predicted rat LAP1B and huLAP1B was found to be 73.6%. A topological analysis was carried out by transiently expressing N-terminal GFP fused deletion mutants of huLAP1B in cells. The transmembrane (TM) domain (aa 346-368) is required for the localization of the nuclear and endoplasmic reticulum membrane and that the TM domain and the C-terminal half of the nucleoplasmic domain (aa 190-331) are sufficient for the proper localization of LAP1B. In contrast, the well-conserved lumenal domain of the nuclear membrane is not required for its topological function. Biochemical analysis showed that huLAP1B is retained within the nucleus via interactions of the nucleoplasmic portion with nuclear components., ((c) 2002 Elsevier Science (USA).)

Cycloheximide (CYH) resistance in Candida maltosa is dependent on the induction of a ribosomal protein, Q-type L41, the 56th residue of which is glutamine, not proline as in ordinary P-type L41. We found that a 38-kDa protein in a wild-type C. maltosa ribosomal fraction became undetectable upon CYH treatment but detectable again with the establishment of CYH resistance by the induction of Q-type L41. We cloned a gene coding for this protein and named it RAY38 (ribosome-associated protein of yeast). Ray38p is a homolog of a purine motif triple-helical DNA-binding protein, Stm1p, and has a putative RNA-binding motif RGG. The ribosome-associated Ray38p was phosphorylated at serine and threonine residues, and Ray38p that was dissociated from ribosome by CYH treatment was highly phosphorylated in threonine residues. A ray38 null mutant recovered faster from CYH-caused growth stasis than the wild-type strain, suggesting that the dissociation of Ray38p from ribosome facilitates the induction of CYH resistance in C. maltosa., (Copyright 2001 Academic Press.)

Using cDNA subtraction screening, we identified five Saccharomyces cerevisiae genes whose expressions is up-regulated when culture temperature was down-shifted from 30 to 10 degrees C. Among these LOT (low temperature-responsive) genes, three (LOT1, LOT2, and LOT3) were identical to FBA1, RPL2B, and NOP1, encoding a fructose biphosphate aldolase, a ribosomal protein L2B, and a nucleolar protein for rRNA processing, respectively. No functions were assigned for LOT5 and LOT6, which are identical to YKL183w and YLR011w, respectively. Northern hybridization analysis revealed that these genes are not uniformly regulated in response to the change of growth temperature. In addition, all the LOT genes, except for LOT1/FBA1, were induced by a low concentration of cycloheximide. The data indicate that multiple mechanisms, including translational functionality may be involved in the regulation of LOT gene expression in yeast.

Yarrowia lipolytica is a yeast which can utilize n-alkane as a sole carbon source. We isolated a Y. lipolytica peroxisomal acetoacetyl-CoA thiolase gene, PAT1, by complementation of a mutant that cannot utilize n-decane as a sole carbon source. We found that the putative PAT1 product had conserved features of peroxisomal acetoacetyl-CoA thiolase. We showed that the PAT1 disruptant was not able to grow on n-decane, and that n-decane-inducible acetoacetyl-CoA thiolase activity largely depended on PAT1. The original mutant carried a mutation involving the replacement of Gly382 with Glu. This mutation inactivated the ability of PAT1 to complement the defective n-decane utilization of the disruptant. These results indicate that PAT1 encodes peroxisomal acetoacetyl-CoA thiolase and is essential for n-decane utilization in Y. lipolytica., (Copyright 2001 Academic Press.)

Thermococcus kodakaraensis KOD1 produces two kinds of chaperonin subunits, CpkA and CpkB. To monitor the expression levels of CpkA and CpkB, anti-CpkA and anti-CpkB antisera were obtained by using synthesized peptides as the haptens. These haptens were prepared based on the carboxyl terminus regions of CpkA and CpkB, which show clear differences in amino acid sequence. Immunoblotting analysis using obtained antisera revealed that the expression levels of CpkA and CpkB changed depending on the cultivation temperature. When cells were grown at 95 degrees C, intracellular amount of CpkA was low, while CpkB was expressed at extremely high level in KOD1. In the case of 70 degrees C cultivation, CpkA existed as the major chaperonin in the cell, whereas CpkB existed as the minor one. Temperature-shift experiments showed that the expression of CpkB was induced by the up-shift and reduced by the down-shift of temperature. In contrast, the expression of CpkA was reduced by the up-shift and induced by the down-shift of temperature. Furthermore, native PAGE and immunoprecipitation experiments revealed that the stoichiometrical ratio of CpkA and CpkB in chaperonin complex changed according to growth temperature., (Copyright 2001 Academic Press.)

The in vitro heat effect on protein characteristics of thermostable enzyme was examined using a cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from the hyperthermophilic archaeon Thermococcus sp. B1001 as a model protein. The recombinant form of CGTase was obtained as an inclusion body from Escherichia coli cells harboring a plasmid which carried the B1001 CGTase gene (cgtA). CGTase was solubilized by 6 M urea, refolded, purified to homogeneity, and heat treated at 80 degrees C for 20 min. Enzyme characteristics were examined compared with those of unheated CGTase. Cyclization activity was increased by in vitro heat treatment, while hydrolysis activity was decreased. The heated and unheated CGTases were analyzed for structures by circular dichroism (CD). The near- and far-UV CD spectra indicated that the structure of unheated CGTase with low cyclization activity was different from that of heated CGTase with high activity. Differential scanning calorimetry of unheated CGTase showed two absorption peaks at 87 and 106 degrees C with increasing temperature. After heat treatment, the minor peak at 87 degrees C disappeared, suggesting that heat-dependent structural conversion occurred in CGTase. These results indicate that the thermal environment plays an important role for the protein folding process of thermostable CGTase., (Copyright 1999 Academic Press.)

We previously found by using yeast, Candida maltosa, that cycloheximide (CYH) sensitivity of ribosomes is dependent on the 56th amino acid residues of a ribosomal protein, L413 (proline in sensitive and glutamine in resistant ribosomes). We also revealed that in this yeast, which has both L41-P type and L41-Q type genes, the expression of the latter type genes is induced by the addition of CYH in the medium to make the cells inducibly resistant to CYH. In this paper, we analyzed the promoter region of L41-Q2a, one of the CYH-inducible L41-Q type genes and found two elements required for the induction of expression: one was a GCRE (Gcn4p-responsive element of Saccharomyces cerevisiae)-like element and the other was a GT-rich element. This promoter region was also required for its expression under some other growth inhibitory conditions. Furthermore, it was suggested that Q-type ribosomes synthesized under these conditions are more resistant to these inhibitory conditions., (Copyright 1999 Academic Press.)

Two histone genes, hpkA and hpkB, from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 strain were cloned, sequenced, and expressed in Escherichia coli cells. Both hpkA and hpkB genes encoded a protein of 67 amino acids, however they possessed the different molecular weight (HpkA, 7,378:HpkB, 7,167). Deduced amino acid sequences of HpkA and HpkB were homologous to other archaeal histones and eucaryal core histones (H2A, H4). Gel mobility shift assays by purified proteins demonstrated that HpkB possessed higher affinity to DNA and more extensive ability to compact DNA than HpkA. HpkB prevented double stranded DNA from thermal denaturation in less amount than HpkA in vitro. In order to investigate intracellular contents of HpkA and HpkB in KOD1 cells, immunoblot analysis was performed by using anti-HpkA antisera obtained from immunized BALB/c mice, showing that HpkA was less abundantly expressed than HpkB in KOD1 cells. These results suggest that HpkB plays a major role to protect double stranded DNA from thermal denaturation in vivo., (Copyright 1999 Academic Press.)

In higher eukaryotic cells, it is generally thought that nuclear pore complex disassembles at the beginning of mitosis and reassembles at the end. Using a monoclonal antibody that recognizes nuclear pore antigens, we found that, at mitosis of mammalian somatic Hela cells, the nuclear pore complex disassembles into at least three subcomplexes, termed subcomplexes A, B and C (molecular mass; 2 MDa<, approximately 660 kDa, and approximately 440 kDa, respectively). The direct partial amino acid sequencing of the components of these subcomplexes indicates that the A subcomplex contains CAN/Nup214/p250 and p62 and the B subcomplex also contains p62, indicating that p62 is contained in two different subcomplexes. Subcomplex C was shown to consist of Nup98 and human RAE1, a human homolog of yeast Rae1p/Gle2p. Since Nup98 and Rae1p/Gle2p have been reported to be involved in mRNA export, this suggests that some of the mitotic subcomplex may be formed by functionally related proteins., (Copyright 1999 Academic Press.)

The CYP52A subfamily of the alkane-assimilating yeast Candida maltosa consists of six structurally related isoforms. Four of them (CYP52A3, 4, 5, and 9) are strongly induced by alkanes and play an important role for the conversion of various alkanes and fatty acids. Taking advantage of a homologous overexpression system, we found in the present study that both of the two other CYP52A forms, CYP52A10 and CYP52A11, represent specialists for the hydroxylation of lauric acid suggesting their preference for short-chain fatty acids. At the same time, they hydroxylated palmitic acid only moderately and failed to convert hexadecane. Based on the now completed knowledge about the principal substrate specificities of all members of the CYP52A subfamily of C. maltosa, it became apparent that evolutionarily more distantly related P450 forms developed either to alkane or to fatty acid hydroxylases, whereas P450 forms which retained the ability to convert both types of substrates were also found to be evolutionarily related to both alkane and fatty acid hydroxylases., (Copyright 1998 Academic Press.)

Intersubunit ion pairs are considered to be involved for maintaining a stable structure of the glutamate dehydrogenase (GDH) from hyperthermophiles. In order to demonstrate an effect of intersubunit ion pairs on the structural stability, two kinds of mutation (T138E, Thr at position 138 was replaced by Glu; E158Q, Glu at position 158 was replaced by Gln) which add and remove ion pairs, respectively, were introduced into Pk-gdhA gene encoding GDH from Pyrococcus kodakaraensis KOD1. Addition of one ion pair (Pk-GDHA-T138E) increased the optimum temperature and thermostability. In contrast, Pk-GDH-E158Q showed lower optimum temperature and less thermostability than wild type GDH. Structure analysis of GDHs was performed by circular dichroism (CD) and indicated that all recombinant enzymes (Pk-GDH, Pk-GDH-T138E, Pk-GDH-E158Q) possess different structures from that of natural GDH. Upon heat treatment (60 degrees C, 2 h), the structures of Pk-GDH and Pk-GDH-T138E were converted to another form close to the natural structure. However, no structural conversion by heat treatment was observed in Pk-GDH-E158Q. These results indicate that intersubunit ion pairs play an important role in forming thermostable structure of Pk-GDH.

Propagation of embryonic stem (ES) cells with an undifferentiated pluripotential phenotype depends on leukemia inhibitory factor (LIF). The LIF receptor complex is composed of a heterodimer of LIF receptor alpha (LIFR alpha) and gp130. To activate LIFR signaling pathways independently from endogenous ones, we constructed chimeric receptors by linking the extracellular domain of human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha or beta (hGMR alpha or beta) to the transmembrane and cytoplasmic regions of either mouse LIFR alpha or gp130. hGMR alpha-mLIFR/hGMR beta-mgp130 or hGMR alpha-mgp130/hGMR beta-mgp130, but not hGMR alpha-mLIFR/hGMR beta-mLIFR, preserved the self-renewal activity in A3 ES cells. All of these chimeric receptors were phosphorylated after hGM-CSF stimulation without phosphorylation of endogenous gp130. Phosphorylation of the signal transducer and activator of transcription 3 through chimeric receptors correlated with the undifferentiated phenotype. Therefore, these chimeric receptors prove useful to analyze mechanisms of the self-renewal of ES cells.

Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus sp. KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in Escherichia coli. Determination of the molecular weight of these enzymes by SDS-PAGE and gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified as both monomeric and hexameric forms. Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active. Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and that circular dichroism spectra of these enzymes are distinctly different from each other. These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II). Upon heat treatment (80 degrees C, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6-fold. Likewise, upon heat treatment (70 degrees C for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated with the hexameric form. These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH.

A csmA gene that encodes chitin synthase with a myosin motor-like domain was isolated from the filamentous fungus Aspergillus nidulans. Initially, we obtained the csmA as a homolog of the Aspergillus fumigatus chsE-partial fragment. A large open reading frame encoding a polypeptide of 1,852 a.a. was identified by determining the cDNA sequences. The chitin synthase conserved region was situated at the C-terminus and classified into class V as reported previously. On the other hand, the N-terminal region showed significant similarity to myosin motors and could not be classified into any types of myosins identified so far. Thus, it is suggested that this is the first report of unconventional myosin fused to a metabolic enzyme. The finding of this new type of chitin synthase gene suggests that localization of chitin synthesis may be guided by association with cytoskeletal structures.

Starfish oocyte maturation was blocked by the addition of 100 microM MG115, a potent proteasome inhibitor, whereas no inhibition was observed by membrane permeable cysteine protease inhibitor, E-64-d. The inhibition by MG115 was diminished by adding at a time corresponding to the half time required for germinal vesicle breakdown. Potent inhibition of germinal vesicle breakdown was also observed by microinjection of anti-proteasome-a-subunit antibodies. The antibody-injected oocytes failed to activate pre-maturation promoting factor (pre-MPF), since the dephosphorylation of phospho-Tyr15 in cdc2 kinase was not observed even in the presence of 1-methyadenine, a maturation-inducing hormone. These results indicate that the proteasome triggers the activation of pre-MPF via the dephosphorylation of cdc2 kinase in the signal transduction pathway in response to the hormonal stimulus during starfish oocyte maturation.

In Drosophila, Serrate plays an important role to the wing margin formation. A putative secretory protein, Fringe, is indispensable for the wing margin formation inducing Serrate and other genes. Recently, Xenopus homologues of Fringe were identified and one of them, lunatic Fringe (X-lFng), was demonstrated to be involved in mesoderm induction. We have identified two chick Fringe homologous genes by reverse transcription-polymerase chain reaction and cDNA library screening. One of them, C-Fringe 1, showed sequence similarity to X-lFng. In situ hybridization study of C-Fringe 1 has demonstrated its expression in the developing nervous system and in the presomitic mesoderm. The hindbrain and spinal cord showed the distinct stripe pattern expression which was complementary to that of C-Serrate, indicating the correlation between them in vertebrate.

The n-alkane-assimilating yeast Candida maltosa contains several structurally related cytochromes P450 (P450) encoded by the CYP52 multigene family, which are inducible by various long-chain hydrocarbons and fatty acids and which are responsible for the initial hydroxylation steps in the metabolism of these substrates. In the present work, the four major n-alkane-inducible C. maltosa P450 forms; CYP52A3, CYP52A4, CYP52A5, and CYP52A9, were enzymatically characterized, taking advantage of heterologous P450/reductase coexpression in Saccharomyces cerevisiae. Testing various alkanes and fatty acids, distinct preferences of the individual P450 forms concerning substrate class and chain length were detected, thus providing new insight into the functional diversity of the C. maltosa CYP52 family. Moreover, the results obtained emphasize these structurally related enzymes as a powerful tool for future studies on P450 structure-function relationships.

Candida maltosa can assimilate n-alkane as a sole carbon source and cytochromes P450ALK (P450ALK) are critical for the first oxidation step. Four major P450ALKs that are encoded by genes ALK1, ALK2, ALK3 and ALK5 are induced by n-alkane and repressed by glucose at the transcriptional level. In the present work, we found that all these four genes but ALK5 are transcriptionally activated in response to a peroxisome proliferator, clofibrate. This is the first report on the peroxisome proliferator responsive gene expression in lower eucaryotes.

It has been shown that the protein serine/threonine phosphatase type 2A alpha catalytic subunit (PP2A alpha) has a mutation consisting of a glycine substitution for cysteine at 269 in the okadaic acid resistant variant of CHO cells, and that the mutant protein is resistant to okadaic acid (Shima, H. et al. Proc. Natl. Acad. Sci. USA 91, 9267-9271, 1994). In this study we analyzed okadaic acid responsive sequences in PP2A alpha by introducing a mutation at around codon 269 of the cDNA strand. The recombinant mutant PP2A alpha proteins Y265F, C266G, Y267G and C269Y were resistant to okadaic acid, with IC50s of 10, 24, 20 and 10 nM, respectively, as opposed to the recombinant wild PP2A alpha protein which had an IC50 of 0.24 nM. This observation suggests that not only cysteine at 269, but also other YCY amino acids, between 265-267, are necessary for the high sensitivity of PP2A to okadaic acid.

The nucleotide sequence of the upstream region of the bphC gene from Pseudomonas sp. strain KKS102 was determined. Four genes were found in this region. Deduced amino acid sequences of the first, second, third and fourth genes showed significant homology with a large subunit of iron-sulfur protein, a small subunit of iron-sulfur protein, ferredoxin and dihydrodiol dehydrogenase, respectively, from other bacteria which degrade biphenyl/polychlorinated biphenyls, toluene and benzene. E. coli, in which the four genes, bphC and the gene for ferredoxin reductase from benzene degrading bacterium were expressed, was able to produce meta-cleavage compounds from chlorinated biphenyls. These results show that these gene products are functional in both biphenyl and polychlorinated biphenyls degradation.

We have identified a stem-and-loop region between the promoter and open reading frame of the Bacillus subtilis senS gene containing a 9 base sequence (TTTAGATAA) identical to a sequence found in a similar regulatory element of the inducible pC194 cat gene. A high copy number of this pC194 regulatory region repressed the expression of senS, indicating that the stem-and-loop region of the cat gene titrated a positive factor necessary for senS expression. These results suggest that factors with similar binding requirements control senS and cat expression. In addition, senS was found to be autoregulated.

Filamin, an actin cross-linker protein, has been shown to exist in platelet. The role of this protein in the platelet has remained unclear. In this report, we show that filamin inhibits the actin-activated Mg2+ -ATPase activity of platelet myosin. The activation caused by platelet actin is inhibited by 50% at the molar ratio of filamin to actin of 1/50. Platelet tropomyosin, which we showed to enhance the ATPase activity, does not abolish the effect of filamin. The results support the view that filamin stabilizes the actin network in the resting platelet.

Caldesmon, a calmodulin and actin binding protein, has been shown to exist in platelet. In this report, it is shown that caldesmon specifically inhibits the effect of tropomyosin to enhance the actomyosin ATPase activity in platelet. Platelet tropomyosin enhances the MgATPase activity of platelet actomyosin. This effect is abolished by platelet caldesmon. In the absence of tropomyosin, however, caldesmon has no effect on the ATPase activity. The inhibition is not due to displacement of the binding of tropomyosin to F-actin by caldesmon. The result indicates that caldesmon is the specific inhibitor of tropomyosin in resting platelet.

Endothelin is a recently-discovered vasoconstrictor peptide which is produced by endothelium and acts on vascular smooth muscle cells. At present its actions on other organs or cells are unknown. We studied the effect of endothelin on renin release in a dynamic superfusion system of dispersed rat juxtaglomerular (JG) cells. Endothelin in concentrations of 10(-11) M or more inhibited renin release dose-dependently and this inhibitory action vanished in the absence of extracellular Ca. It is suggested that endothelin is an inhibitory regulator of renin secretion from JG cells and its action is Ca-dependent.

The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer. A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin. About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current. The shifts indicated the gating of ion-permeable channels in the patches. The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events. The significance of these findings is discussed in relation to the mechanism of action of the toxin.

Effects of gelsolin on the actomyosin system in platelet have been studied. MgATPase activity of platelet actomyosin is enhanced up to two folds by 200 nM of platelet gelsolin in the presence, but not in the absence of Ca ion. The half maximum enhancement is observed at the concentration of Ca2+ around 10(-5) M. The effect of gelsolin to enhance the ATPase activity of actomyosin is potentiated by tropomyosin, which is a Ca2+-insensitive actomyosin enhancer. The results indicate that gelsolin may control the activity of actomyosin system in platelets.