[Molecular mechanism for monitoring chromosomal DNA replication].
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Chromosomes↗
Biomedical subjects
H Araki
Publications and source records attributed to H Araki.
Thermostabilization by proline substitution in an alkaline, liquefying alpha-amylase from Bacillus sp. strain KSM-1378.
alpha-Amylase (LAMY) from alkaliphilic Bacillus sp. strain KSM-1378 is a novel semi-alkaline enzyme which has 5-fold higher specific activity than that of a Bacillus licheniformis enzyme. The Arg124 in LAMY was replaced with proline by site-directed mutagenesis to increase thermostability of the enzyme. The wild-type and engineered LAMYs were very similar with respect to specific activity, kinetic values, pH-activity curve, and degree of inhibition by chelating reagents. Thermostability and structure stiffness of LAMYs as measured by fluorescence were increased by the proline substitution. The change of Arg124 to proline is assumed to stabilize the loop region involving amino acid residues from 122 to 134. This is the first report that thermostability of an alpha-amylase is improved by proline substitution.
Amino Acid Sequence↗
[Renal angiomyolipoma with marked extrarenal development: a case report].
A 28-year-old female complained of minimal fever elevation. Computed tomography (CT) revealed a left renal tumor of 10 cm in diameter. Ultrasonogram and CT, magnetic resonance imaging and angiography suggested a renal angiomyolipoma (AML) with marked extrarenal development. Partial nephrectomy was performed using a microwave tissue coagulater without clamping of the renal artery. The tumor weight was 800 g and the pathological diagnosis was AML. The management of large AML is reviewed in the literature. Nephron sparing surgery should be performed even in patients who have a larger tumor with extrarenal development.
Adult↗
[A case of bladder tumor producing granulocyte colony-stimulating factor].
A case of bladder tumor producing granulocyte colony-stimulating factor (G-CSF) is reported. A 78-year-old male presented with macroscopic hematuria. Cystoscopy demonstrated a large bladder tumor. Drip infusion pyelography and computerized tomography of the chest, abdomen and pelvis revealed right hydronephrosis, but did not reveal any metastasis. Total cystectomy and construction of bilateral cutaneous ureterostomy was performed. Histologically, the tumor consisted of sarcomatoid carcinoma with foci of transitional cell carcinoma (G3 > G2), showing marked infiltration of polymorphonuclear leukocytes. Immunohistochemical examination revealed a high concentration of G-CSF in the tumor specimen. Preoperatively, the white blood cell, count in the peripheral blood and serum G-CSF concentration were elevated, 45,400/mm3 and 73.4 pg/ml, respectively. After surgery these values became normal. This is the 19th reported case of G-CSF producing bladder tumor in Japan. The prognosis of the reported cases of G-CSF producing bladder tumor has been very poor. Fourteen of the reported 19 cases died within one year. This patient should be carefully followed up.
Aged↗
Improved thermostability of a Bacillus alpha-amylase by deletion of an arginine-glycine residue is caused by enhanced calcium binding.
alpha-Amylase from alkaliphilic Bacillus KSM-1378 (LAMY) is a novel semi-alkaline enzyme which has a high specific activity, a value 5-fold higher than that of a Bacillus licheniformis enzyme at alkaline pH. Thermostability of this enzyme could be improved by deletion of the Arg181-Gly182 residue by means of site-directed mutagenesis. The wild-type and engineered LAMYs were very similar with respect to specific activity, pH-activity curve, temperature-activity curve, susceptibility to inhibitors, and pattern of hydrolysis products from soluble starch and maltooligosaccharides. However, the engineered enzyme also acquired increased pH stability and resistance to sodium dodecyl sulfate and especially chelating reagents, such as ethylenediaminetetraacetate and ethyleneglycol-bis (beta-aminoethylether)tetraacetate. This is the first report that thermostability of alpha-amylase is improved by enhanced calcium binding to the enzyme molecule.
Amino Acid Sequence↗
Meiotic role of SWI6 in Saccharomyces cerevisiae.
The transcript levels of DNA replication genes and some recombination genes in Saccharomyces cerevisiae fluctuate and peak at the G1/S boundary in the mitotic cell cycle. This fluctuation is regulated by MCB (Mlu I cell cycle box) elements which are bound by the DSC1/MBF1 complex consisting of Swi6 and Mbp1. It is also known that some of the MCB-regulated genes are induced by treatment with DNA damaging agents and in meiosis. In this report, the function of SWI6 in meiosis was investigated. Delta swi6 cells underwent sporulation as did wild-type cells. However, the deletion mutant cells showed reduced spore viability and lower frequency of recombination. The transcript levels of the recombination genes RAD51 and RAD54 , which have MCB elements, were reduced in Delta swi6 cells. The transcript levels of SWI6 itself were also induced and declined in meiosis. Furthermore, an increased dosage of SWI6 enhanced the transcript level of the RAD51 gene and also the recombination frequency in meiosis. These results suggest that SWI6 enhances the expression level of the recombination genes in meiosis in a dosage-dependent manner, which results in an effect on the frequency of meiotic recombination.
DNA Helicases↗
Age-related decrease in centrally-mediated pressor response to clonidine in conscious rats.
An age-related change in pressor response to intracerebroventricular (i.c.v.) injection of clonidine in conscious rats was investigated. In 15-week-old rats, clonidine (2 to 20 micrograms) caused a dose-dependent pressor response concomitant with decrease in heart rate. The depressor response to i.c.v. clonidine at lower doses (2 and 5 micrograms) but not higher doses (10 and 20 micrograms) was observed. The pressor response to clonidine in 20- and 30-week-old rats was markedly diminished, and the depressor response appeared at any of the doses injected. However, no age-related change in heart rate responses was observed. These results suggest that the centrally mediated pressor response to clonidine is age-related and masks the depressor response.
Adrenergic alpha-Agonists↗
HL-004, the ACAT inhibitor, prevents the progression of atherosclerosis in cholesterol-fed rabbits.
HL-004, N-(2,6-diisopropylphenyl) tetradecylthioacetamide, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was evaluated concerning the possible prevention of hyperlipidemia and atherosclerosis in 1% cholesterol-fed rabbits. HL-004 (0.2, 5 and 25 mg/kg) was orally administered once a day for 12 weeks. HL-004 inhibited the rise of total serum cholesterol at a dose of 5 mg/kg and over. In the thoracic aorta, HL-004 at the doses of 5 mg/kg and 25 mg/kg reduced the total cholesterol content by 56.3% and 84.2% compared with control, and decreased ACAT activity, dose-dependently. HL-004 also attenuated the development of aortic lesions. The area of atherosclerotic lesions was reduced by 30.3% with 5 mg/kg of HL-004 and 100% with 25 mg/kg. In this study, we suggest that the main reason for HL-004 preventing the progression of atherosclerosis is its hypocholesterolemic effect due to the inhibition of cholesterol absorption in the intestine.
Acetanilides↗
The ACAT inhibitor HL-004 inhibits cholesterol absorption and lowers serum cholesterol in rats.
1. HL-004 decreased absorption of cholesteryl ester from the intestine into the lymph in a rat lymph-fistula model. 2. HL-004 reduced serum cholesterol level in acute cholesterol-fed rats. 3. HL-004 simultaneously decreased hepatic cholesteryl ester content and increased free cholesterol in cholesterol-fed rats. 4. These findings suggest that: (1) the hypocholesterolemic effect of HL-004 is principally due to inhibition of cholesterol absorption via inhibition of ACAT in the intestine; and (2) changes in hepatic cholesterol metabolism due to direct inhibition by HL-004 of hepatic ACAT may account in part for reduction of serum cholesterol level by HL-004.
Absorption↗
N-glycan structures of a recombinant mouse soluble Fcgamma receptor II.
N-glycans of a recombinant mouse soluble Fcgamma receptor II (sFcgammaRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226:139-46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was alpha-(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and alpha-(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry. Carbohydrate sequence [see text]
Animals↗
Involvement of calcitonin gene-related peptide (CGRP) receptors in insulin-induced vasodilatation in mesenteric resistance blood vessels of rats.
1. The vascular effect of insulin in the mesenteric resistance blood vessel and the role of calcitonin generelated peptide (CGRP)-receptor in insulin-induced vascular responsiveness were investigated in rats. 2. The mesenteric vascular beds isolated from Wistar rats were perfused with Krebs solution, and perfusion pressure was measured with a pressure transducer. In preparations contracted by perfusion with Krebs solution containing methoxamine in the presence of guanethidine, the perfusion of insulin (from 0.1 to 3000 nM) caused a concentration-dependent decrease in perfusion pressure due to vasodilatation. The pD2 value and maximum relaxation (%) were 6.94+/-0.22 and 43.9+/-5.2, respectively. 3. This vasodilator response to insulin was unaffected by 100 nM propranolol (beta-adrenoceptor antagonist) plus 100 nM atropine (muscarinic cholinoceptor antagonist), 100 microM L-NG-nitroarginine (nitric oxide synthase inhibitor), 1 microM ouabain (Na+-K+ ATPase inhibitor), or 1 microM glibenclamide (ATP sensitive K+-channel inhibitor). 4. In preparations without endothelium, perfusion of insulin produced a marked vasodilatation. The pD2 value and maximum relaxation (%) were 7.62+/-0.21 and 81.0+/-4.6, respectively, significantly greater than in preparations with intact endothelium. 5. The vasodilator responses to insulin in the preparations without endothelium were significantly inhibited by CGRP[8 37], a CGRP receptor antagonist, whereas pretreatment with capsaisin, a toxin for CGRP-containing nerves, did not affect insulin-induced vasodilatation. 6. These results suggest that insulin induces non-adrenergic, non-cholinergic and endothelium-independent vasodilatation, which is partially mediated by CGRP receptors.
Animals↗
DNA polymerase epsilon encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe.
BACKGROUND: DNA polymerase II (PolII), the homologue of mammalian DNA polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA polymerase epsilon. It has been shown that DNA polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. RESULTS: We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA polymerase epsilon (Pol epsilon). The predicted amino acid sequence of Pol epsilon is highly homologous to that of S. cerevisiae PolII and human Pol epsilon. Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. CONCLUSION: These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.
Amino Acid Sequence↗
Fatal cardiac beta2-microglobulin amyloidosis in patients on long-term hemodialysis.
We report two long-term hemodialysis patients who developed severe congestive heart failure attributable to cardiac heavy amyloid deposition. Both patients became hypotensive during dialysis sessions, gradually making it difficult to continue hemodialysis, and they died of congestive heart failure. At autopsy, left ventricle walls in each case contained diffuse extensive deposits of amyloid. The distribution of amyloid was not localized to vessel walls but was widely disseminated throughout the left ventricle walls and replaced myocardial muscle fibers. Immunohistochemical examination showed positive staining for anti-human beta2-microglobulin antibody. We conclude that cardiac dialysis-related amyloidosis should also be considered in long-term hemodialysis patients with congestive heart failure as a life-threatening complication.
Amyloidosis↗
Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication.
The DPB11 gene, which genetically interacts with DNA polymerase II (epsilon), one of three replicative DNA polymerases, is required for DNA replication and the S phase checkpoint in Saccharomyces cerevisiae. To identify factors interacting with Dbp11, we have isolated sld (synthetically lethal with dpb11-1) mutations which fall into six complementation groups (sld1 to -6). In this study, we characterized SLD2, encoding an essential 52-kDa protein. High-copy SLD2 suppressed the thermosensitive growth defect caused by dpb11-1. Conversely, high-copy DPB11 suppressed the temperature-sensitive growth defect caused by sld2-6. The interaction between Sld2 and Dpb11 was detected in a two-hybrid assay. This interaction was evident at 25 degreesC but not at 34 degreesC when Sld2-6 or Dpb11-1 replaced its wild-type protein. No interaction between Sld2-6 and Dpb11-1 could be detected even at 25 degreesC. Immunoprecipitation experiments confirmed that Dpb11 physically interacts with Sld2. sld2-6 cells were defective in DNA replication at the restrictive temperature, as were dpb11-1 cells. Further, in dpb11-1 and sld2-6 cells, the bubble-shaped replication intermediates formed in the region of the autonomously replicating sequence reduced quickly after a temperature shift. These results strongly suggest the involvement of the Dpb11-Sld2 complex in a step close to the initiation of DNA replication.
Amino Acid Sequence↗
The RFC2 gene, encoding the third-largest subunit of the replication factor C complex, is required for an S-phase checkpoint in Saccharomyces cerevisiae.
Replication factor C (RF-C), an auxiliary factor for DNA polymerases delta and epsilon, is a multiprotein complex consisting of five different polypeptides. It recognizes a primer on a template DNA, binds to a primer terminus, and helps load proliferating cell nuclear antigen onto the DNA template. The RFC2 gene encodes the third-largest subunit of the RF-C complex. To elucidate the role of this subunit in DNA metabolism, we isolated a thermosensitive mutation (rfc2-1) in the RFC2 gene. It was shown that mutant cells having the rfc2-1 mutation exhibit (i) temperature-sensitive cell growth; (ii) defects in the integrity of chromosomal DNA at restrictive temperatures; (iii) progression through cell cycle without definitive terminal morphology and rapid loss of cell viability at restrictive temperatures; (iv) sensitivity to hydroxyurea, methyl methanesulfonate, and UV light; and (v) increased rate of spontaneous mitotic recombination and chromosome loss. These phenotypes of the mutant suggest that the RFC2 gene product is required not only for chromosomal DNA replication but also for a cell cycle checkpoint. It was also shown that the rfc2-1 mutation is synthetically lethal with either the cdc44-1 or rfc5-1 mutation and that the restrictive temperature of rfc2-1 mutant cells can be lowered by combining either with the cdc2-2 or pol2-11 mutation. Finally, it was shown that the temperature-sensitive cell growth phenotype and checkpoint defect of the rfc2-1 mutation can be suppressed by a multicopy plasmid containing the RFC5 gene. These results suggest that the RFC2 gene product interacts with the CDC44/RFC1 and RFC5 gene products in the RF-C complex and with both DNA polymerases delta and epsilon during chromosomal DNA replication.
Amino Acid Sequence↗
Hyperexpression of the gene for a Bacillus alpha-amylase in Bacillus subtilis cells: enzymatic properties and crystallization of the recombinant enzyme.
We have constructed a new excretion vector, pHSP64, to develop a hyperexcretion system for Bacillus subtilis [Sumitomo et al., Biosci. Biotech. Biochem., 59, 2172-2175 (1995)]. The structural gene for a novel liquefying semi-alkaline alpha-amylase from the alkaliphilic Bacillus sp. KSM-1378 was amplified by PCR. It was cloned into a SalI-SmaI site of pHSP64 and the recombinant plasmid obtained was introduced into B. subtilis. The transformed B. subtilis hyperproduced the alpha-amylase activity extracellularly, corresponding to approximately 1.0 g (5 x 10(6) units) per liter of an optimized liquid culture. The recombinant enzyme was purified to homogeneity by a simple purification procedure with very high yield. No significant differences in physiochemical and catalytic properties were observed between the recombinant enzyme and the native enzyme produced by Bacillus sp. KSM-1378. The enzymatic properties of the recombinant enzyme were further examined with respect to the responses to various metal ions. The recombinant enzyme could easily be crystallized at room temperature within one day in a buffered solution of 10% (w/v) ammonium sulfate (pH 6.5).
Amino Acid Sequence↗
Steady-state serum concentrations of carbamazepine and valproic acid in obese and lean patients with epilepsy.
Steady-state serum concentrations of carbamazepine (CBZ) and valproic acid (VPA) were investigated in normal weight (body mass index; BMI 20 to 25), lean (smaller than 20 BMI) and moderately obese subjects (greater than 25 BMI) who received either 400 mg/day of CBZ or 800 mg/day of VPA. The CBZ serum concentration in lean subjects was significantly higher than that in normal weight subjects. However, no significant differences in VPA serum concentration were found between the three groups. The CBZ serum concentration decreased with increases in total body weight, and the VPA serum concentration decreased with increases in ideal body weight. However, both serum concentrations were not correlated with BMI. These results suggest that VPA doses should be calculated using ideal body weight and that degree of obesity may affect CBZ serum concentration rather than VPA serum concentration.
Adolescent↗
Repeated mazindol and methamphetamine administration produces cross-sensitization to stereotyped behavior induced by these agents in rats.
The cross-sensitization to stereotyped behavior between mazindol (MZD) and methamphetamine (MAP) was investigated in rats. MZD (5 and 10 mg/kg/day, p.o.), MAP (5 and 10 mg/kg/day, p.o.) and saline (1 ml/kg, p.o.) were administered once daily for a week. Challenge with MZD (10 mg/kg, p.o.) on the 8th day caused markedly stereotyped behavior in MAP-pretreated group compared with the saline-pretreated control group. MAP (10 mg/kg, p.o.)-induced stereotyped behavior on the 8th day was also greater in MZD-pretreated group rather than the saline-pretreated control group. These results suggest that repeated MZD and MAP administration cross-sensitizes to their stereotype-producing effects.
Animals↗