[Dry clinical chemistry--analysis of proteins and enzyme].
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Amylases↗
Biomedical subjects
Y Takagi
Publications and source records attributed to Y Takagi.
Characterization of elongin C functional domains required for interaction with elongin B and activation of elongin A.
The Elongin (SIII) complex stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates. The Elongin (SIII) complex is composed of a transcriptionally active A subunit, a chaperone-like B subunit, which promotes assembly and enhances stability of the Elongin (SIII) complex, and a regulatory C subunit, which (i) functions as a potent activator of Elongin A transcriptional activity, (ii) interacts specifically with Elongin B to form an isolable Elongin BC complex, and (iii) is bound and negatively regulated in vitro by the product of the von Hippel-Lindau tumor suppressor gene. As part of our effort to understand how Elongin C regulates the activity of the Elongin (SIII) complex, we are characterizing Elongin C functional domains. In this report, we identify Elongin C mutants that fall into multiple functional classes based on their abilities to bind Elongin B and to bind and activate Elongin A under our assay conditions. Characterization of these mutants suggests that Elongin C is composed of multiple overlapping regions that mediate functional interactions with Elongin A and B.
Amino Acid Sequence↗
Conserved neuron promoting activity in Drosophila and vertebrate laminin alpha1.
Drosophila S2 cells were transfected with constructs that code for two portions of the Drosophila laminin alpha chain. Construct recalphaL coded for domains III, I/II, and G of laminin alpha. Construct recalphaS coded for only the COOH-most 12% of the I/II domain and the G domain. The corresponding polypeptides were isolated and characterized from the culture media. The recalphaL chain partly formed disulfide-linked heterotrimers with the endogenously produced beta and gamma laminin chains. Like normal Drosophila laminin, a substrate coating of either recalphaL or recalphaS supported neuron differentiation and neurite extension of primary Drosophila embryo cell cultures. However, at the same low concentrations, only Drosophila laminin-1, but neither recalphaL nor recalphaS supported myogenesis in these cultures. Previously, an overlapping set of dodecapeptides that covered a region of the murine laminin alpha1 chain similar to recalphaS had been synthesized and tested for cell culture support properties (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). The Drosophila laminin alpha homologues of the six most active vertebrate dodecapeptides were now synthesized and tested as substrates for differentiation of primary Drosophila embryo cells. Peptides that contained either the Drosophila sequence SIKVGV or the murine homologue, SIKVAV, provided support for neurite extension.
Amino Acid Sequence↗
Somatic in vivo alterations of the DPC4 gene at 18q21 in human lung cancers.
The chromosome region 18q21 has been shown to be frequently deleted in lung cancers. Recent identification at this locus of DPC4, a gene whose inactivation has been suggested to play a role in pancreatic carcinogenesis, prompted as to examine whether it might also be altered in lung cancers. Two missense and 2-bp frameshift somatic mutations in DPC4 were detected among 42 lung cancer specimens taken directly from patients. DPC4 mutations, however, were not present in all lung cancers carrying l8q21 deletions. These findings suggest that DPC4 may play a role in a limited fraction of lung cancers and that another tumor suppressor gene may also exist in this chromosome region.
Base Sequence↗
A 5'-(trifluoromethyl)anthracycline glycoside: synthesis of antitumor-active 7-O-(2,6-dideoxy-6,6,6-trifluoro-alpha-L-lyxo-hexopyranosyl) adriamycinone.
7-O-(2,6-Dideoxy-6,6,6-trifluoro-alpha-L-lyxo-hexopyranosyl)adriam ycinone (3), whose substituent at C-5' is a lipophilic trifluoromethyl group, has been prepared by coupling of 3,4-di-O-acetyl-2,6-dideoxy-6,6,6-trifluoro-alpha-L-lyxo-hexopyran osyl bromide (20) with 14-O-(tert-butyldimethylsilyl)adriamycinone under the Koenigs-Knorr conditions. The key step in this synthesis was the C-trifluoromethylation of 5-O-acetyl-2,3-di-O-benzyl-4-deoxy-aldehydo-L-erythro-pentose (10), derived from D-lyxose in 10 steps, with (trifluoromethyl)trimethylsilane in the presence of tetrabutylammonium fluoride, whereupon 1,1,1-trifluoro-L-arabino-hexitol (11) was obtained along with its 2-epimer. The synthetic product 3 showed remarkable antitumor activity in vivo in a low dose range compared to the analogs including doxorubicin. The fact may be ascribed to the presence of a trifluoromethyl group at C-5', suggesting the importance of the group in view of biological activity.
Animals↗
The alternative exons 1 of the mouse aromatase cytochrome P-450 gene.
Aromatase cDNA clones were isolated from cDNA libraries of mouse hypothalamus, amygdala and ovary. Analysis of the nucleotide sequences of the 5' regions of the obtained cDNAs suggested that the mouse aromatase gene is tissue-specifically regulated by alternative exons 1. There were obvious differences between the 5' regions of the brain and ovary aromatase cDNAs, but no difference was found between the sequences of the hypothalamus and amygdala ones. We further isolated a mouse genomic DNA clone containing brain- and ovary-specific exons 1. The brain specific exons 1 and their promoters were highly homologous in the human and mouse aromatase genes. In contrast there were several differences in the sequences among the promoter regions of the ovary-specific exons 1 of the mouse, human and rat aromatase genes, significant homology between their sequences was also observed. The present results demonstrate that expression of the mouse aromatase gene is also tissue-specifically regulated through the use of alternative exons 1 and promoters, as reported for man.
Alternative Splicing↗
Influence of glutamine-supplemented parenteral nutrition on intestinal amino acid metabolism in rats after small bowel resection.
Glutamine (Gln)-supplemented total parenteral nutrition (TPN) has been shown to improve mucosal adaptation after massive small bowel resection (SBR); however, its influences on intestinal amino acid metabolism remain unknown. In this study, intestinal amino acid flux, circulating plasma aminogram, mucosal glutaminase activity and protein, and DNA content were measured 7 days after massive SBR in rats receiving either standard (Std) or Gln-supplemented TPN. Sham-operated rats and rats fed chow after enterectomy served as controls. The uptake of Gln and the release of citrulline (Cit) by the remaining intestine was significantly decreased, with reduced mucosal glutaminase activity after SBR in the Chow and Std-TPN groups. Glutamine supplementation resulted in significantly increased gut Gln uptake compared with Std-TPN (P < 0.01). Mucosal glutaminase activity, mucosal protein, and DNA content was also increased by Gln; however, the gut release of Cit remained unchanged (P > 0.05). The subsequent decrease in circulating arginine (Arg) in the Gln-TPN group compared with the Std-TPN group (P < 0.05) was attributed to an insufficient exogenous supply. These findings show that Gln-supplemented TPN improves mucosal growth and gut Gln uptake after SBR. However, the intestinal production of Cit, which remained low in both TPN groups, may lead to an insufficiency of endogenous Arg synthesis. Thus, both Gln and Arg may be essential amino acids after SBR.
Amino Acids↗
Heated oyster shell-seaweed calcium (AAA Ca) on osteoporosis.
A randomized, prospective, double-blind test was carried out to compare the effects of heated oyster shell-seaweed calcium (AAA Ca), calcium carbonate, and placebo in 58 elderly, hospitalized women with the mean age of 80 divided into three groups. Group A received 900 mg/day Ca as AAA Ca, Group B 900 mg/day Ca as CaCO3, and Group C placebo besides regular hospital diet containing approximately 600 mg Ca/day for 24 months. From the 25th to the 30th month, all groups were given AAA Ca. Lumbar spine and radial bone mineral density (BMD) were measured at 3-month intervals. Urinary Ca/Cr and serum alkaline phosphatase, intact and midportion serum parathyroid hormone (PTH), and calcitonin were also measured at intervals. From the 6th to the 24th month of the study, the ratio of lumbar spine BMD (L2-L4 by DPX, Lunar) to the basal pretest value was consistently and significantly higher in Group A than Group C but not higher in Group B than in Group C. PTH, measured 12 months after the beginning of the study, was lower in Group A than in Group C, but no significant difference was found between Groups B and C. At 3 months after the placebo was switched to AAA Ca in Group C, serum PTH was significantly decreased from the level during placebo supplement. Morning urine Ca/Cr decreased in Groups A after 18 months and in B after 12 months, but not in C. Serum alkaline phosphatase decreased in Group A significantly compared with Group C, but not in Group B. AAA Ca appears to be effective for increasing BMD in elderly subjects.
Aged↗
Risk factors for stroke in patients undergoing coronary artery bypass grafting.
OBJECTIVE: To determine predictors of stroke in patients undergoing first-time coronary bypass grafting, we prospectively collected data on 1631 consecutive patients. METHODS: Patients with a history of stroke and/or central nervous system symptoms (n = 134) and/or carotid bruits (n = 95) underwent carotid Doppler evaluation. Stenosis greater than 70% was considered significant. Patients with symptomatic disease or asymptomatic bilateral disease were referred for combined coronary bypass and carotid endarterectomy (n = 21). Patients with neurologic symptoms after the operation were assessed by a neurologist and underwent a computed tomographic scan. Events were classified as reversible transient ischemic attack, reversible ischemic neurologic deficit, or irreversible stroke. RESULTS: There were 19 strokes (1.2%) and 20 deaths (1.2%) in this series. In patients with carotid screening, risk of stroke increased with severity of carotid disease and ranged from 0% in patients without stenosis, to 3.2% (1/31) in those with greater than 70% stenosis, and to 27.3% (6/22) in those with carotid occlusion. By stepwise logistic regression analysis six variables were identified as risk factors for stroke. The most important predictor was carotid occlusion with or without contralateral stenosis (odds ratio = 28, 95% confidence interval (8,105). In this group, four of five strokes occurred on the occluded side. Other risk factors were presence of ascending aortic disease at the time of surgery (odds ratio = 12.8, confidence interval 3,48), perioperative myocardial infarction (odds ratio = 8.2, confidence interval 2,33), poor left ventricular function (odds ratio = 4.6, confidence interval 1,19), peripheral vascular disease (odds ratio = 3.2, confidence interval 1,9), and age > 60 years (odds ratio = 2.9, confidence interval 0.8,11). CONCLUSION: We conclude that risk factors for perioperative stroke in patients undergoing coronary artery bypass grafting are multiple. Carotid scanning in patients with neurologic symptoms or carotid bruits can identify patients at increased risk. Patients with carotid occlusion are at high risk for stroke on the occluded side.
Adult↗
Neurotransmitter-mediated regulation of brain aromatase: protein kinase C- and G-dependent induction.
Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by alpha 1-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the alpha 1-agonist phenylephrine than in control neurons, whereas prazosin, an alpha 1-antagonist, suppressed this increase, and ligands for alpha 2- or beta-adrenergic receptors did not exert any influence. The profile of alpha 1-adrenergic receptor subtypes during actual development in vivo suggested that the alpha 1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyrylcyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via alpha 1 (possibly alpha 1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. beta-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.
Adrenergic alpha-1 Receptor Agonists↗
Somatic alterations of the DPC4 gene in human colorectal cancers in vivo.
BACKGROUND & AIMS: The chromosome region 18q21 has been shown to be frequently deleted in colorectal cancers, and such frequent allelic loss is a hallmark of the presence of a tumor-suppressor gene. The DPC4 gene, which is located at 18q21, has been identified as a tumor-suppressor gene from examination of pancreatic cancers. The aim of the present study was to determine if it might also be altered in colorectal cancers. METHODS: Mutation analyses of the DPC4 gene were performed on complementary DNA samples from 31 primary colorectal cancer specimens using a combination of polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. RESULTS: Four missense mutations producing amino acid substitutions and a somatic 12-base pair deletion in the coding region of the DPC4 gene were detected in the 31 cancers (16%; 5 of 31). CONCLUSIONS: The DPC4 gene may play a role as a tumor-suppressor gene in a fraction of colorectal cancers; however, while allelic loss at 18q21 is very often seen in colorectal cancers, only a minority show DPC4 mutations, suggesting that there might be another tumor-suppressor gene in this chromosome region.
Adult↗
Alanyl-glutamine dipeptide-supplemented parenteral nutrition improves intestinal metabolism and prevents increased permeability in rats.
OBJECTIVE: The authors determined the effects of alanyl-glutamine-supplemented total parenteral nutrition (TPN) on mucosal metabolism, integrity, and permeability of the small intestine in rats. METHODS: Male Sprague-Dawley rats were randomized to receive TPN supplemented with a conventional amino acids mixture (STD group) or the same solution supplemented with alanyl-glutamine; both solutions were isocaloric and isonitrogenous. On the seventh day of TPN, D-xylose and fluorescein isothiocyanate (FITC)-dextran were administered orally. One hour later, superior mesenteric vein (SMV) D-xylose and plasma FITC-dextran concentration were measured. Intestinal blood flow and calculated intestinal substrates flux were measured with ultrasonic transit time flowmetery. RESULTS: Plasma FITC-dextran increased significantly in the STD group. Intestinal blood flow and SMV D-xylose concentration did not differ between the groups. Mucosa weight, villus height, mucosal wall thickness, mucosal protein, and DNA and RNA content in jejunal mucosa were significantly increased in the alanyl-glutamine group. Jejunal mucosal glutaminase activity and net intestinal uptake of glutamine (glutamine flux) were significantly higher in the alanyl-glutamine group as compared with the STD group. CONCLUSION: Addition of alanyl-glutamine dipeptide to the TPN solution improves intestinal glutamine metabolism and prevents mucosal atrophy and deterioration of permeability.
Animals↗
In vitro increase of aromatase mRNA in diencephalic neurons.
This study was designed to (1) examine the ability of fetal diencephalic neurons cultured in vitro to express aromatase mRNA and (2) evaluate the involvement of several environmental factors which may regulate the development and differentiation of the neurons in the central nervous system. Brain cells from fetal mice at various developmental stages were cultured as tissue slices and as primary monolayer cells, and the expression levels of aromatase mRNA in the cultured cells were measured by a quantitative reverse transcription-polymerase chain reaction method using an internal standard. On cultured slices of the diencephalic region from fetal mice on embryonic day 12 (E12), E13, or E15 on collagen-coated membranes, the expression level of the mRNA continued to increase for the initial 2-3 days as that in vivo. Time-dependent increase of aromatase mRNA was also observed for 3 days in E13 neuronal cells dissociated with papain and cultured on poly L-Lys-coated dishes in serum-free medium. However, no significant time-dependent increase of the aromatase mRNA level was observed in E10 or E11 brain cells cultured by either method. These findings suggest that the developmental increase of aromatase mRNA in diencephalic neurons is an endogenous characteristic and probably genetically determined after E12.
Animals↗
Clinical evaluation of bone metabolism after renal transplantation to support the theory to perform 1,25(OH)2D3 pulse therapy before transplantation.
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Adult↗
Prognostic significance of abnormal p53 accumulation in primary, resected non-small-cell lung cancers.
PURPOSE: This study was conducted to evaluate the prognostic significance of p53 abnormalities in primary, resected non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Methodologic validation of immunohistologic detection of p53 abnormalities in routine pathology sections was assessed using 31 lung cancer specimens for which p53 gene status was known from our previous molecular biologic studies. Applying the optimized cutoff value, we evaluated the prognostic significance of p53 abnormalities in an independent cohort of 208 NSCLC patients with complete follow-up data, whose resections were consecutively performed between January 1984 and December 1988. RESULTS: Immunohistologic detection of p53 abnormalities appeared to be reliable and showed approximately 90% concordance with the p53 gene status. Using the selected cutoff value of 10%, 46% of 208 NSCLCs showed p53 abnormalities. There was no relationship between p53 abnormalities and clinical outcome in the entire cohort, which represented all histologic subtypes of NSCLC (P = .58). Based on the reasoning that the influence of p53 abnormalities may have been obscured by distinct biologic roles depending on histologic subtypes, we also separately analyzed subsets of patients with adenocarcinomas (n = 100) and with squamous cell carcinomas (n = 88) and found that it may be a useful prognosticator only in adenocarcinoma patients (P = .04). CONCLUSION: p53 abnormalities are not a significant prognostic factor in primary, resected NSCLC when all histologic subtypes are combined, but may be a useful prognosticator for adenocarcinomas. Additional studies are warranted for further evaluation, specifically of adenocarcinomas.
Adenocarcinoma↗
Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function.
Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive agent used to treat osteoporosis. However, the mechanism of its action on osteoclasts and their precursor cells is not well understood. To determine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+]i) in osteoclasts and their precursors and measured specific binding of 3H-labeled ipriflavone. Highly purified chicken osteoclast precursors, which spontaneously differentiate into multinucleated osteoclasts in 3-6 days, were loaded with fura-2, and the subcellular [Ca2+]i distribution was monitored by videoimaging. Ipriflavone induced a rapid increase in [Ca2+]i followed by a sustained elevation [EC50 = 5 x 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basal levels, by 10(-6) M ipriflavone, sustained phase]. The responses were the same in differentiated chicken osteoclasts and isolated rabbit osteoclasts. An influx of extracellular Ca2+ is likely to be responsible for the ipriflavone-induced change in [Ca2+]i because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+]i levels were detected adjacent to the cell membrane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipine (10(-7)M) and verapamil (10(-7)M) had no effects on ipriflavone-induced [Ca2+]i responses. [3H]Ipriflavone binding studies indicated the presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (Kd), 7.60 x 10(-8)M, 2.67 x 10(-6)M] and in mature osteoclasts (Kd, 4.98 x 10(-8)M, 3.70 x 10(-6)M). Specific ipriflavone binding was not displaced by various modulators of avian osteoclast function, such as estradiol (10(-8)M) or retinoic acid (10(-6)M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The fusion of osteoclast precursor cells was significantly inhibited by ipriflavone, which led to dose-dependent inhibition of bone resorption and tartrate-resistant acid phosphatase activity. Novel specific ipriflavone receptors that are coupled to Ca2+ influx were demonstrated in osteoclasts and their precursor cells. These ipriflavone receptors may provide a mechanism to regulate osteoclast differentiation and function.
Acid Phosphatase↗
Presence of alternatively spliced transcripts of aromatase gene in human breast cancer.
The expression of aromatase (estrogen synthetase) is tissue specifically regulated through the alternative use of multiple exons 1 and promotors. We have determined the amounts of aromatase messenger ribonucleic acid (mRNA) and which type of multiple exons 1 of the human aromatase gene is used in breast tissues of 49 patients with breast cancer by reverse transcription-PCR analysis. The aromatase mRNA levels in these breast cancer tissues (4.53 +/- 0.66 x 10(-3) attomoles/micrograms RNA) were significantly (P < 0.01) higher than those in 16 nonmalignant breast tissues (1.73 +/- 0.40 x 10(-3) amol/micrograms RNA). Aromatase mRNA in all nonmalignant breast tissue were transcribed from skin fibroblast/fetal liver-specific exon 1 (exon 1b) of the gene. In 23 breast cancer tissues, the utilization of multiple exons 1 in the aromatase mRNA was the same as that in nonmalignant breast tissues, whereas in the other 26 cases, it changed from exon 1b to ovary-specific exon 1 (exon 1c). Such switching of tissue-specific exons 1 may affect strict regulation of the tissue-specific expression of aromatase, leading to abnormal expression of the aromatase. The consequent overproduction of local estrogen might promote carcinogenesis or the proliferation of breast cancers.
Adult↗
Protective effect of astragali radix by oral administration against Japanese encephalitis virus infection in mice.
We investigated the protective effect of Astragali Radix (AR) by oral administration against Japanese encephalitis virus (JEV) infection in mice, the pharmacological effects of AR extracts (AE) in different origin, and the chemical composition of the AEs. A protective effect was demonstrated in all four AEs used, however, the effective grade for each one was different. In the control group, an increase of hemagglutination inhibition (HI) antibody titer was observed in all mice surviving 25 d after JEV inoculation. However, the increase of HI antibody titer was not observed in some animals administered an AE. In the control group, the rate of HI antibody positive mice was 90% 3 d after JEV inoculation, while the four groups which received the AE had a 30-60% positive rate. In mice which received the AE, the peritoneal exudate cell (PEC) numbers increased significantly compared to the control group. The predominant cell population of PECs in mice receiving the AE was macrophages, and in the PEC, the active oxygen (AO) production was high. From these results, we propose that the protective effect of AE by oral administration is based on a non-specific mechanism during the early stage of infection, before shifting to antibody production, and that macrophages play an important role in this resistance to JEV infection, e.g., by inducing the production of AO. In the chemical composition of each AE, carbohydrate was the major component.
Animals↗