Pneumomediastinum secondary to hyperemesis gravidarum during early pregnancy.
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Abortion, Induced↗
Biomedical subjects
M Yokoi
Publications and source records attributed to M Yokoi.
E2F regulates growth-dependent transcription of genes encoding both catalytic and regulatory subunits of mouse primase.
DNA polymerase alpha-primase is one of the principal enzymes involved in eukaryotic chromosomal DNA replication. Mouse DNA polymerase alpha-primase consists of four subunits with molecular masses of 180, 68, 54 and 46 kDa. Protein and mRNA expression levels of the four subunits are up-regulated in a coordinated manner in response to growth stimulation. We have previously analysed the transcription of the 180 kDa (p180) and 68 kDa (p68) subunits, which form the DNA polymerase catalytic complex, and found that growth-dependent regulation of transcription of the mouse p180 and p68 genes is mediated by a common factor, E2F, while the basal transcription of the genes is regulated by different transcription factors. We characterized the transcriptional regulation of the 54 kDa (p54) and 46 kDa (p46) subunits, which form the DNA primase catalytic complex. We isolated genomic clones spanning the 5'-flanking regions of the p54 and p46 genes and showed, using transient expression and gel mobility shift assays, that the basal transcription of p54 is controlled by Sp1 and GA-binding protein, as is the basal transcription of the p180 gene. The basal transcription of p46 is controlled by unknown factor(s) which were bound to the upstream sequence. The variant E2F sites close to the transcription initiation sites of the p54 and p46 genes had no basal promoter activity, but were essential for the growth-dependent transcription of both genes. The promoter regions of the four subunits of mouse DNA polymerase d-primase complex share several common features. The coordinated transcription of all four subunits in response to growth stimulation appears to be controlled by E2F.
3T3 Cells↗
The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase.
Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.
Amino Acid Sequence↗
Anthocyanins in flowers of genus Rosa, sections Cinnamomeae (=Rosa), Chinenses, Gallicanae and some modern garden roses.
Forty-four taxa of three sections (Cinnamomeae (=Rosa) 26, Chinenses 8 and Gallicanae 10) and eight modern garden roses in the genus Rosa were surveyed for their floral anthocyanins. Eleven anthocyanins: 3-glucosides and 3,5-diglucosides of cyanidin (Cy), pelargonidin (Pg) and peonidin (Pn), 3-rutinosides and 3-rho-coumaroylglucoside-5-glucosides of Cy and Pn, and Cy 3-sophoroside, were isolated from flowers of these taxa and identified by chemical and spectroscopic techniques. Four anthocyanins: Cy 3-rutinoside, Pn 3-rutinoside, Pn 3-rho-coumaroylglucoside-5-glucoside and Cy 3-sophoroside were found for the first time in Rosa flowers.Investigated sections of wild roses showed characteristic distribution of anthocyanins. Cy 3,5-diglucoside was the dominant anthocyanin detected in all three sections, but accumulation of Pn 3,5-diglucoside distinguished sections Cinnamomeae from other sections, and the occurrence of Cy 3-glucoside separates section Chinenses from others.Cy 3-sophoroside was detected in large amount in some taxa of section Cinnamomeae: e.g., R. moyesii and its related cultivars, and R. rugosa cv. Salmon Pink. The acylated Cy glycoside was found in all sections and also in some modern garden roses, while the acylated Pn glycoside was detected in the section Cinnamomeae, but not in sections Chinenses and Gallicanae. According to anthocyanin distribution patterns, eight groups were classified chemotaxonomically in genus Rosa.
Journal Article↗
Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase alpha is controlled by E2F, an Ets-related transcription factor, and Sp1.
We have isolated a genomic DNA fragment spanning the 5'-end of the gene encoding the catalytic subunit of mouse DNA polymerase alpha. The nucleotide sequence of the upstream region was G/C-rich and lacked a TATA box. Transient expression assays in cycling NIH 3T3 cells demonstrated that the GC box of 20 bp (at nucleotides -112/-93 with respect to the transcription initiation site) and the palindromic sequence of 14 bp (at nucleotides -71/-58) were essential for basal promoter activity. Electrophoretic mobility shift assays showed that Sp1 binds to the GC box. We also purified a protein capable of binding to the palindrome and identified it as GA-binding protein (GABP), an Ets- and Notch-related transcription factor. Transient expression assays in synchronized NIH 3T3 cells revealed that three variant E2F sites near the transcription initiation site (at nucleotides -23/-16, -1/+7 and +17/+29) had no basal promoter activity by themselves, but were essential for growth-dependent stimulation of the gene expression. These data indicate that E2F, GABP and Sp1 regulate the gene expression of this principal replication enzyme.
3T3 Cells↗
Cloning and characterization of the 5'-upstream sequence governing the cell cycle-dependent transcription of mouse DNA polymerase alpha 68 kDa subunit gene.
We have isolated the genomic DNA fragment spanning the 5-end and the first four exons encoding the 68 kDa subunit (p68) of the mouse DNA polymerase alpha-primase complex [corrected]. The p68 promoter region lacks TATA and CAAT boxes, but contains a GC-rich sequence, two palindrome sequences and two putative E2F-binding sites [corrected]. A series of transient expression assays using a luciferase reporter gene indicated that a region from nucleotide position -89 to -30 (-89/-30) with respect to the transcription initiation site is crucial for basal transcription of the p68 gene in proliferating NIH 3T3 cells. In particular, part of the GC-rich sequence (-57/-46) and the palindrome (-81/-62) elements were necessary for promoter activity, both of which share homology with the E-box sequence. Gel mobility shift assays using NIH 3T3 nuclear extracts revealed that the upstream stimulatory factor, known as an E-box-binding protein, binds to these sites. Moreover, we observed binding of E2F to two sites near the transcription initiation site (-11/-3 and +9/+16). A transient luciferase expression assay using synchronized NIH 3T3 cells in G(0)phase revealed that these E2F sites are essential for transcription induction of the p68 gene after serum stimulation, but are dispensable for basal transcription. These results indicate that growth-dependent regulation of transcription of the mouse p68 and p180 genes is mediated by a common factor, E2F; however, basal transcription of the genes, interestingly, is regulated by different transcription factors.
3T3 Cells↗
The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA.
The xeroderma pigmentosum group C protein complex XPC-HR23B was first isolated as a factor that complemented nucleotide excision repair defects of XP-C cell extracts in vitro. Recent studies have revealed that this protein complex plays an important role in the early steps of global genome nucleotide excision repair, especially in damage recognition, open complex formation, and repair protein complex formation. However, the precise function of XPC-HR23B in global genome repair is still unclear. Here we demonstrate that XPC-HR23B interacts with general transcription factor IIH (TFIIH) both in vivo and in vitro. This interaction is thought to be mediated through the specific affinity of XPC for the TFIIH subunits XPB and/or p62, which are essential for both basal transcription and nucleotide excision repair. Interestingly, association of TFIIH with DNA was observed in both wild-type and XP-A cell extracts but not in XP-C cell extracts, and XPC-HR23B could restore the association of TFIIH with DNA in XP-C cell extracts. Moreover, we found that XPC-HR23B was necessary for efficient association of TFIIH with damaged DNA in cell-free extracts. We conclude that the XPC-HR23B protein complex plays a crucial role in the recruitment of TFIIH to damaged DNA in global genome repair.
Cell Line↗
Xeroderma pigmentosum variant: from a human genetic disorder to a novel DNA polymerase.
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DNA Damage↗
[Preoperative complications and intraoperative hemodynamic changes in very old patients with femoral neck fractures].
We compared preoperative complications and intraoperative hemodynamic changes in very old patients, 85 years or older, and those with elderly patients aged 70-84 for hip fracture repair. Spinal anesthesia with 0.25 or 0.5% of bupivacaine was performed except for the patients with dementia and/or deformity of the spinal column. The incidence of cardiac disease and anemia was higher in very old patients than in elder patients, and its odds ratios were 2.29 and 3.10, respectively. There is no difference in intraoperative hemodynamic changes between the two groups. Two patients of very old groups had severe intraoperative complications, heart failure and grave arrhythmia, but other patients underwent the operation without severe complication. In conclusion, even in very old patients with hip fracture, spinal anesthesia was performed safely unless patients had serious diseases preoperatively.
Aged↗
Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome.
hHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that specifically complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro. Although a small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo. Here we demonstrate that the human NER factor hHR23B as well as another human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit of the human 26 S proteasome using the yeast two-hybrid system. Furthermore, hHR23 proteins were detected with S5a at the position where 26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100 extracts. Intriguingly, hHR23B showed the inhibitory effect on the degradation of (125)I-lysozyme in the rabbit reticulocyte lysate. hHR23 proteins thus appear to associate with 26 S proteasome in vivo. From co-precipitation experiments using several series of deletion mutants, we defined the domains in hHR23B and S5a that mediate this interaction. From these results, we propose that part of hHR23 proteins are involved in the proteolytic pathway in cells.
Amino Acid Sequence↗
The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta.
Xeroderma pigmentosum variant (XP-V) is an inherited disorder which is associated with increased incidence of sunlight-induced skin cancers. Unlike other xeroderma pigmentosum cells (belonging to groups XP-A to XP-G), XP-V cells carry out normal nucleotide-excision repair processes but are defective in their replication of ultraviolet-damaged DNA. It has been suspected for some time that the XPV gene encodes a protein that is involved in trans-lesion DNA synthesis, but the gene product has never been isolated. Using an improved cell-free assay for trans-lesion DNA synthesis, we have recently isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in XP-V cell extracts. Here we show that this polymerase is a human homologue of the yeast Rad30 protein, recently identified as DNA polymerase eta. This polymerase and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. We found that all XP-V cells examined carry mutations in their DNA polymerase eta gene. Recombinant human DNA polymerase eta corrects the inability of XP-V cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA. Together, these results indicate that DNA polymerase eta could be the XPV gene product.
Alleles↗
Metabotropic glutamate receptor subtype 7 ablation causes deficit in fear response and conditioned taste aversion.
Metabotropic glutamate receptors (mGluRs) consist of eight different subtypes and exert their effects on second messengers and ion channels via G-proteins. The function of individual mGluR subtypes in the CNS, however, largely remains to be clarified. We examined the fear response of freezing after electric shock in wild-type and mGluR7(-/-) knockout littermates. Wild-type mice displayed freezing immediately after and 1 d after footshock. In comparison, mGluR7(-/-) knockout mice showed significantly reduced levels in both immediate postshock and delayed freezing responses. However, the knockout mice exhibited no abnormalities in pain sensitivity and locomotor activity. To further examine amygdala-dependent behavior, we performed conditioned taste aversion (CTA) experiments. In wild-type mice, the administration of saccharin followed by intraperitoneal injection of the malaise-inducing agent LiCl resulted in an association between saccharin and LiCl. This association caused strong CTA toward saccharin. In contrast, mGluR7(-/-) knockout mice failed to associate between the taste and the negative reinforcer in CTA experiments. Again, the knockout mice showed no abnormalities in taste preference and in the sensitivity to LiCl toxicity. These results indicate that mGluR7 deficiency causes an impairment of two distinct amygdala-dependent behavioral paradigms. Immunohistochemical and immunoelectron-microscopic analyses showed that mGluR7 is highly expressed in amygdala and preferentially localized at the presynaptic axon terminals of glutamatergic neurons. Together, these findings strongly suggest that mGluR7 is involved in neural processes subserving amygdala-dependent averse responses.
Animals↗
Oxidative stress and haematological changes in immobilized rats.
Immobilization stress induces formation of reactive oxygen species (ROS) and leads to the oxidative injury in various tissues. In this study, the effects of immobilization stress on peripheral blood cells distribution, plasma level of thiobarbituric acid reactive substances (TBARS), and activities of antioxidant enzymes in erythrocytes were investigated in male Fischer rats. A significant increase in plasma TBARS was observed during and after the stress. Dramatic increases of neutrophils and monocytes imply that ROS formation resulted from their activation. Furthermore, the antioxidant activities of catalase and superoxide dismutase (SOD) in erythrocytes were dramatically increased during and after the stress, while a large fall in erythrocyte number was observed. These findings suggest that the activation of immune cells can be a source of the immobilization-induced ROS production, and that antioxidant enzymes in erythrocytes play an important role in preventing the ROS-induced injuries.
Animals↗
Effect of positive end-expiratory pressure on left and right ventricular diastolic filling assessed by transoesophageal Doppler echocardiography.
The effect of positive end-expiratory pressure (PEEP) on left and right ventricular diastolic filling dynamics was assessed by transmitral and transtricuspid flow patterns. Using transoesophageal Doppler echocardiography in fourteen ASA physical status 1 female patients, the following measurements were performed at baseline (0 cm H2O PEEP) and at 5, 10, 15, and 20 cm H2O PEEP: 1. peak velocity of early filling (peak E velocity), 2. peak velocity of atrial contraction (peak A velocity), 3. the ratio of the peak E to A velocity (peak E/A velocity ratio), 4. isovolumic relaxation time (IRT), 5. acceleration half-time (AHT), 6. deceleration half-time (DHT) of early filling, and 7. end-diastolic and end-systolic areas of both ventricles. Increasing PEEP progressively deceased peak E velocity of both ventricles. In contrast, peak A velocity did not change and the peak E/A velocity ratio decreased significantly with PEEP. IRT and AHTs remained unchanged, but DHTs of both ventricles increased following PEEP. End-diastolic and end-systolic areas of both ventricles decreased gradually and significantly with PEEP. It is concluded that PEEP was associated with decreased preload as well as reduced compliance of both ventricles, which was considered to contribute to the changes in diastolic ventricular filling.
Adult↗
Favorable life-style modification and attenuation of cardiovascular risk factors.
In order to develop an effective counseling system for prevention of cardiovascular diseases, the association of a favorably changed life-style with improved risk factors was examined. Participants were 7,321 office workers aged 30-69 years from in and around Nagoya city. The age-adjusted odds ratio (OR) and its 95% confidence interval (CI) were calculated to assess the likelihood of risk factor improvement by favorable life-style modifications during a 3-year period. Those who began to eat breakfast and increased their vegetable intake normalized their previously abnormal diastolic blood pressure with more than twice the likelihood (adjusted OR [95% CI] 2.89 [1.29-6.46] and 2.60 [1.18-5.75], respectively). 'Began to eat breakfast' was also significantly associated with normalized total cholesterol (TC) (1.84, [1.05-3.21]). 'Stopped eating till full' significantly normalized the body mass index (2.03; [1.25-3.28]), uric acid (1.65; [1.07-2.52]) and TC (1.43; [1.04-1.97]). Those who started regular exercise significantly normalized their high-density lipoprotein-cholesterol (HDL-C) abnormality with 1.69-times the likelihood (1.69; [1.24-2.29]) and those who began to walk briskly also improved their TC abnormality (1.85; [1.19-2.89]). HDL-C was normalized with 2.55-times the likelihood in those who quit smoking (2.55; [1.68-3.86]). Because favorable life-style modifications can attenuate abnormal cardiovascular risk factors, then proper advice on specific risk factors should be routinely given at each health check-up in order to prevent the onset of cardiovascular diseases in subsequent years.
Adult↗
Preparation and application of anti-idiotypic antibody against anti-gibberellin A4 antibody.
A monoclonal anti-idiotypic antibody was raised against anti-gibberellin A4 (GA4) antibody, which recognizes biologically active gibberellins such as GA1 and GA4 specifically. Amino acid sequences of variable regions of both anti-GA4 and anti-idiotypic antibodies were analyzed. By using the property of the anti-idiotypic antibody to compete with GA1/4 in binding to the anti-GA4 antibody, we successfully applied the anti-idiotypic antibody to ELISA as a tracer for measuring GA1/4. The single-chain Fv (scFv) gene of the anti-idiotypic antibody was constructed, and scFv expressed in E. coli showed binding activity to anti-GA4 antibody. These results suggest the possible application of anti-idiotypic antibody as a handy and stable source of an enzymatic tracer for ELISA by production of fusion protein of the scFv and an appropriate enzyme.
Amino Acid Sequence↗
Ablation of cerebellar Golgi cells disrupts synaptic integration involving GABA inhibition and NMDA receptor activation in motor coordination.
The role of inhibitory Golgi cells in cerebellar function was investigated by selectively ablating Golgi cells expressing human interleukin-2 receptor alpha subunit in transgenic mice, using the immunotoxin-mediated cell targeting technique. Golgi cell disruption caused severe acute motor disorders. These mice showed gradual recovery but retained a continuing inability to perform compound movements. Optical and electrical recordings combined with immunocytological analysis indicated that elimination of Golgi cells not only reduces GABA-mediated inhibition but also attenuates functional NMDA receptors in granule cells. These results demonstrate that synaptic integration involving both GABA inhibition and NMDA receptor activation is essential for compound motor coordination. Furthermore, this integration can adapt after Golgi cell elimination so as not to evoke overexcitation by the reduction of NMDA receptors.
Animals↗
The second-largest subunit of the mouse DNA polymerase alpha-primase complex facilitates both production and nuclear translocation of the catalytic subunit of DNA polymerase alpha.
DNA polymerase alpha-primase is a replication enzyme necessary for DNA replication in all eukaryotes examined so far. Mouse DNA polymerase alpha is made up of four subunits, the largest of which is the catalytic subunit with a molecular mass of 180 kDa (p180). This subunit exists as a tight complex with the second-largest subunit (p68), whose physiological role has remained unclear up until now. We set out to characterize these subunits individually or in combination by using a cDNA expression system in cultured mammalian cells. Coexpression of p68 markedly increased the protein level of p180, with the result that ectopically generated DNA polymerase activity was dramatically increased. Immunofluorescence analysis showed that while either singly expressed p180 or p68 was localized in the cytoplasm, cotransfection of both subunits resulted in colocalization in the nucleus. We identified a putative nuclear localization signal for p180 (residues 1419 to 1437) and found that interaction with p68 is essential for p180 to translocate into the nucleus. These results indicate that association of p180 with p68 is important for both protein synthesis of p180 and translocation into the nucleus, implying that p68 plays a pivotal role in the newly synthesized DNA polymerase alpha complex.
3T3 Cells↗