[Clinical management of ischemic heart diseases. Discussion].
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Aged↗
Biomedical subjects
H Murakami
Publications and source records attributed to H Murakami.
Cloning and expression study of the mouse tetrodotoxin-resistant voltage-gated sodium channel alpha subunit NaT/Scn11a.
We have cloned a tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel alpha subunit from a mouse cDNA library and designated it as NaT. It encodes 1765 amino acid residues and is virtually identical to that of Scn11a, which has been reported recently, except for 40 nt and 14 aa substitutions. The amino acid identity of NaT/Scn11a with rat NaN/SNS2 is 88%. NaT/Scn11a was mapped to mouse chromosome 9F3-F4 by fluorescence in situ hybridization (FISH). While rat NaN/SNS2 has been reported to be expressed specifically in the peripheral sensory neurons, NaT/Scn11a is expressed not only in the peripheral sensory neurons but also in the spinal cord, uterus, testis, ovary, placenta, and small intestine. NaT is detectable in mouse embryos 15 days postcoitus (p.c.), around the phase of organogenesis and gonadal differentiation. These findings demonstrate a unique distribution of NaT/Scn11a and suggest some of its roles in the above-mentioned processes.
Amino Acid Sequence↗
Multiple myeloma presenting high fever and high serum levels of lactic dehydrogenase, CRP, and interleukin-6.
Two myeloma patients presented high fever with no signs or data indicating infection at diagnosis or relapse. Both patients had plasmablastic myeloma, and serum levels of lactic dehydrogenase (LDH) and CRP were extremely high. Plasmablastic morphology, high LDH, and CRP were recognized as poor prognostic factors, indicating a fulminant phase of multiple myeloma. Interleukin-6 (IL-6) was only high in measured cytokines. We proposed that IL-6 caused high fever and induced the fulminant phase in these 2 cases.
C-Reactive Protein↗
Effects of L-lysine deficient diet on the hypothalamic interstitial norepinephrine and diet-induced thermogenesis in rats in vivo.
Rats readily recognize an amino acid deficient diet, presumably via central mechanisms that involve hypothalamic circuits. Presently, effects of the essential amino acid L-lysine deficiency on the ventromedial (VMH) and lateral (LH) hypothalamus norepinephrine (NE) release were evaluated in free moving rats. Microdialysis measurement was undertaken once in 48 h (12:00 noon-14:00) in rats that had free access to food and drink. Significant decline in the food intake and VMH NE release were found in rats fed L-lysine diet. No changes were identified in LH NE release. Additionally, no significant differences in diet-induced spatial thermogenesis between normal and L-lysine deficient non-stressed rats were found in vivo. The results suggested that the VMH NE release was specifically involved in the integration of signals about amino acid deficiency. However, the decrease in VMH NE was not translated into changes of thermogenic responses to diet.
Animals↗
Traumatic cardiac rupture with acute ascites.
A rare accumulation of serous ascites following traumatic cardiac rupture is reported for a 21-year-old man transferred to the emergency center of our hospital from a community hospital suffering from severe shock due to a motorcycle accident. Computed tomography scan showed moderate pericardial and intrapelvic fluid accumulation strongly suggesting cardiac and visceral injuries. An emergency sternotomy disclosed a rupture of the right atrial appendage, successfully closed with primary sutures. A laparotomy was done to aspirate moderate serous fluid, which was clear and not bloody. Edema of the retroperitoneal space and hepatic congestion were noted with no accompanying organ injury. Ascites pathogenesis is unknown but appeared to be related to portal venous congestion induced by cardiac tamponade combined with massive intravenous fluid infusion done to correct the patient's deteriorating hemodynamics.
Accidents, Traffic↗
Molecular design of novel metal-binding oligomeric human metallothioneins.
Genes for dimeric and tetrameric human metallothionein (hMT) were designed and successfully overexpressed in Escherichia coli to generate functional oligomeric hMTs. An hMT synthesized with prokaryotic codons, a linker encoding a gly-gly-gly tripeptide, and Met-deficient hMT-II was ligated to create a dimeric hMT, from which a tetrameric hMT was then constructed. The increased molecular size of the constructs resulted in improved stability and productivity in E. coli. The oligomeric proteins formed inclusion bodies which were dissolved with dithiothreitol, and the purified apometallothioneins were reconstituted with Cd or Zn ions in a reducing condition. The oligomeric hMT proteins incubated with Cd ions showed a typical Cd-thiolate absorbance peak at 245-255 nm. The dimeric and tetrameric hMT proteins exhibited both Cd and Zn binding activities that were respectively two and four times higher than those of the hMT-II monomer protein. These novel oligomeric hMTs may be useful in bioremediation for heavy metals.
Amino Acid Sequence↗
Plasma oxysterols and tocopherol in patients with diabetes mellitus and hyperlipidemia.
The plasma levels of free oxysterols (7-ketocholesterol; 7alpha-hydroxy-, 7beta-hydroxy-, 25-hydroxy-, and 27-hydroxycholesterol; and 5alpha,6alpha-epoxycholestanol) in patients with diabetes mellitus and hypercholesterolemia were determined using gas chromatography-mass spectrometry with selective ion monitoring. We studied 39 patients with diabetes mellitus, 20 nondiabetic patients with hypercholesterolemia, and 37 normal controls. Plasma cholesterol levels in diabetic and hypercholesterolemic patients showed no statistical difference. Plasma 7-ketocholesterol was significantly higher in patients with diabetes (31.6+/-2.8 ng/mL) or hypercholesterolemia (52.3+/-5.9) than in the control group (22.4+/-1.2). The increased plasma cholesterol can be regarded as an oxidation substrate for the oxidant stress and the higher absolute levels of oxysterols in hypercholesterolemic plasma compared with the control plasma. This difference disappeared when 7-ketocholesterol was expressed in proportion to total cholesterol. The oxidizability of plasma cholesterol was evaluated by comparing the increased ratio of 7-ketocholesterol after CuSO4 oxidation to the ratio before. We demonstrated that the patients with diabetes showed increased oxidizability (77.5%) compared with the control (36.6%) or hyperlipemic group (45.3%), which is likely due to the lower amounts of alpha-tocopherol in the diabetics. Measurement of oxysterols may serve as a marker for in vivo oxidized lipoproteins in diabetes and hyperlipemia.
Cholesterol↗
Production of transgenic pigs expressing human DAF (CD55) regulated by the porcine MCP gene promoter.
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Animals↗
Porcine MCP gene promoter directs high level expression of human DAF (CD55) in transgenic mice.
Porcine membrane cofactor protein (pMCP), a complement regulatory protein, is widely expressed in various tissues. Particularly, it is highly expressed on vascular endothelium. The objective of this study was to investigate whether the pMCP gene promoter can induce efficient expression of a human complement regulatory protein, decay-accelerating factor (DAF; CD55) in transgenic mice. Two fragments of the 5'-flanking region of pMCP gene (0.9 kb and 5.4 kb) connected with human DAF minigene (0.9/hDAF and 5.4/hDAF) were used to produce transgenic mice. The expression of hDAF in heart, liver, kidney, lung, pancreas, brain and testis of the transgenic mice was examined by immunohistochemical analysis. The vascular endothelia and the nerves in all organs examined were intensely stained. The staining pattern in these tissues was similar in all transgenic mice examined regardless of the length of the promoters. The surface expression levels of hDAF on peripheral red blood cells and splenocytes from a mouse carrying 5.4/hDAF hemizygously was twice the level of expression on corresponding human cells. The red blood cells and splenocytes from the transgenic mice exhibited resistance to lysis by human serum in a manner dependent upon expressed hDAF level. The hearts from the transgenic mice functioned for a significantly longer time than those from normal mice under perfusion with human serum in the Langendorff perfusion system. These results demonstrated that the pMCP gene promoter is a good candidate of the regulatory element in the transgene to produce transgenic animals for xenotransplantation.
Animals↗
Involvement of angiotensin II and endothelin-1 in the development of submandibular gland hypertrophy in response to isoproterenol in rats.
We investigated whether angiotensin II (Ang II) and endothelin-1(ET-1) are involved in submandibular hypertrophy in response to repeated treatment with isoproterenol (ISO) in rats. The immunoreactive Ang II (IRAng II) and immunoreactive ET-1 (IRET-1) contents of ISO-induced hypertrophy were significantly higher than those of control glands. Treatment of isolated gland tissues with ISO (1 microM) or dobutamine (1 microM) caused significant increases in the IRAng II and IRET- 1 contents of the glands compared with controls. These increases were suppressed by pretreatment with enalapril (3 microM) or captopril (3 microM). Treatment with Ang II (10 microM) also caused an increase in IRET-1 content. Our findings suggest that Ang II and ET-1 are involved in the submandibular gland hypertrophy that develops in rats repeatedly treated with ISO, and that these biologically active peptides may act as growth factors. They also imply that the tissue renin-angiotensin system and Ang II specific receptors are present in the submandibular glands.
Adrenergic beta-Agonists↗
Association of a polymorphism at the 5'-region of the angiotensin II type 1 receptor with hypertension.
Molecular variants of individual components of the renin-angiotensin system are thought to contribute to inherited predisposition towards essential hypertension. Using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequence analysis, we identified seven polymorphisms in the 5'-flanking region of the angiotensin II type 1 receptor (AGTR1/AT 1 ) gene. We conducted a case-control study in a sample from the Japanese population to determine whether polymorphic markers in the 5'-flanking region of the AT 1 gene were associated with essential hypertension. The study compared 149 hypertensive subjects to 156 normotensive control subjects. A significantly higher frequency of the AT 1 (-535)*T allele was observed in hypertensive subjects. Evidence was obtained that the AT 1 (-535)*T allele showed a synergistic effect on risk of hypertension with angiotensin I converting enzyme D allele (ACE*D).
5' Untranslated Regions↗
Site-directed incorporation of fluorescent nonnatural amino acids into streptavidin for highly sensitive detection of biotin.
Fluorescent nonnatural amino acids were incorporated into specific positions of streptavidin. The positions of the nonnatural amino acids were directed by a CGGG/CCCG four-base codon/anticodon pair. The nonnatural mutants with a single 2-anthrylalanine at the 22nd, 43rd, 54th, and 120th positions, respectively, were found to bind biotin, indicating that the mutants retained active conformation. The fluorescence intensities of the anthryl groups were relatively insensitive to the positions and the biotin binding when excited at 265 nm. When the anthryl group at the 120th position was excited through energy transfer from tryptophan units, the fluorescence intensity markedly decreased with biotin binding, because of a suppression of the energy transfer. Amino acids carrying 7-methoxycoumarine fluorophore were also incorporated at the 120th position. Their fluorescence quantum yields were very sensitive to the biotin binding. The high sensitivity of the coumarine-labeled streptavidin exemplifies potential applications of fluorescent nonnatural mutants for detecting specific molecules at very low concentrations.
Amino Acids↗
Characteristics of choline transport across the blood-brain barrier in mice: correlation with in vitro data.
PURPOSE: We examined the functional properties of choline transport across the blood-brain barrier (BBB) in mice. We compared the kinetic parameters and transport properties with those found in our in vitro uptake experiments using mouse brain capillary endothelial cells (MBEC4). METHODS: The permeability coefficient-surface area product (PS) values of [3H]choline at the BBB were estimated by means of an in situ brain perfusion technique in mice. RESULTS: [3H]Choline uptake was well described by a two-component model: a saturable component and a nonsaturable linear component. The [3H]choline uptake was independent of pH and Na+, but was significantly decreased by the replacement of Na+ with K+. Various basic drugs, including substrates and inhibitors of the organic cation transporter, significantly inhibited the [3H]choline uptake. These in situ (in vivo) results corresponded well to the in vitro results and suggest that the choline transporter at the BBB is a member of the organic cation transporter (OCT) family. CONCLUSION: The choline transport mechanism at the BBB is retained in MBEC4.
Algorithms↗
Improvement of extrathymic T cell type of large granular lymphocyte (LGL) leukemia by cyclosporin A: the serum level of Fas ligand is a marker of LGL leukemia activity.
We report a case of gammadelta T-cell-type large granular lymphocyte (LGL) leukemia (CD3 +,CD8 +, CD57 +,TCR gammadelta+), which was accompanied by pure red cell aplasia, neutropenia and thrombocytosis. Southern blotting analysis of the T-cell receptor beta gene showed the germline configuration, but clonal TCR J gamma rearrangements were identified. These granular lymphocytes demonstrated non-major histocompatibility complex-restricted cytotoxicitity. The serum-soluble FasL (sFasL) concentration of this patient was very high, whereas the serum levels of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), interleukin-1 beta (IL-1beta), interleukin-2 (IL-2) and thrombopoietin were normal. After treatment with cyclosporin A, anemia and thrombocytosis were improved, and LGL and the elevated sFasL concentration decreased. These observations suggested that FasL may have played a role in the establishment of the clinical symptoms of this patient and could be useful as an indicator of disease activity.
CD3 Complex↗
Pharmacokinetic analysis of felodipine-grapefruit juice interaction based on an irreversible enzyme inhibition model.
UNLABELLED: Aims Ingestion of grapefruit juice (GFJ) alters the pharmacokinetics of various orally administered drugs. Quantitative evaluation of this GFJ-drug interaction is required for the proper clinical management of patients. Methods Using felodipine as a model drug, we constructed a pharmacokinetic model based on irreversible inhibition of intestinal cytochrome P450 3A4 (CYP3A4) by GFJ. We fitted previously published data [5, 6] for felodipine ER (extended release formulation) to the ratio of CLGI,int before and after grapefruit juice ingestion by nonlinear least-squares regression analysis to estimate the reaction rate constant between GFJ and CYP3A4 (K) and the elimination rate constant of CYP3A4 (k ). RESULTS: The model gave a turnover rate of CYP3A4 of 0.0849 h-1, corresponding to a half-life of 8.16 h, in agreement with reported values. The AUC-time profiles of felodipine ER in the case of different amounts and schedules of GFJ ingestion were simulated using the parameter values estimated from the model. CONCLUSIONS: The modelling leads to the important conclusion that GFJ-felodipine interaction increases with increasing frequency and amount of GFJ ingestion, and that an interval of 2-3 days between GFJ intake and felodipine administration is necessary if GFJ-felodipine interaction is to be avoided.
Adult↗
The effect of dietary restriction and menstrual cycle on excess post-exercise oxygen consumption (EPOC) in young women.
The purpose of this study was to evaluate the effect of acute dietary restriction on excess post-exercise oxygen consumption (EPOC) in young women at two different phases of the menstrual cycle. Five young sedentary women (age 21-22 years) participated in this study. Each subject visited the laboratory eight times for measurement of EPOC. They performed cycle ergometer exercise for 60 min at a work rate corresponding to approximately 70% of VO2max under each four different conditions (i.e. standard diet/follicular phase (SF), standard diet/luteal phase (SL), restricted diet/follicular phase (RF) and restricted diet/luteal phase (RL)). The exercise was performed in the morning and VO2 was measured for the last 15 min of each hour for 7 h after the exercise. As a control, VO2 was also measured with an identical time schedule under the same four conditions but without exercise. EPOC was calculated as the difference of the VO2-time integral for 7 h between the exercise and control trial days in each of the four conditions (i.e. SL, SF, RL and RF). The diet was precisely controlled during 2 days (i.e. the test day and the day preceding it). The standard diet was 1600 kcal day-1 and the restricted diet was half of the standard diet. A two-way (dietary and menstrual cycle factors) ANOVA indicated that EPOC was significantly affected only by the dietary factor. The dietary restriction decreased EPOC compared to the standard dietary condition (SF 8.6 +/- 2.1, RF 5.3 +/- 1.6, SL 8.9 +/- 4.8, RL 4.0 +/- 1.2 l). These data indicate that for young sedentary women, EPOC is significantly lowered by prior acute dietary restriction but is not influenced by different phases of the menstrual cycle.
Adult↗
Localization and characterization of antigen-presenting dendritic cells in the gastric mucosa of murine and human autoimmune gastritis.
BACKGROUND: Activated CD4+ T cells and inflammatory cytokines are implicated in the pathogenesis of experimental autoimmune gastritis. However, there is a paucity of information about the cells that induce them. Antigen-presenting dendritic cells (DCs) play a cardinal role in the formation and survival of activated lymphocytes. MATERIALS AND METHODS: Autoimmune gastritis was induced in neonatal BALB/c mice by thymectomy. DCs were detected in situ in the gastric mucosa from thymectomized mice and in patients with autoimmune gastritis, by immunohistochemistry and immunoelectron microscopy. The expression of MHC class II and CD86 antigens on DCs in the gastric mucosa and spleen was evaluated in dual-colour flow cytometry. RESULTS: DCs were detected in the gastric mucosa of mice with autoimmune gastritis, and the number of DCs increased as the levels of gastritis became more severe as time passed following thymectomy. Flow cytometric analyses revealed that more than 60% of the DCs in the gastric mucosa had a mature phenotype (expressed MHC class II and/or CD86 antigens) both at 4 and 16 weeks after thymectomy. Activated and mature DCs were localized in the gastric mucosa from patients with autoimmune gastritis. CONCLUSIONS: This is the first report on the localization and phenotypes of DCs in the gastric mucosa of autoimmune gastritis. The presence of mature DCs in the gastric mucosa of murine and human autoimmune gastritis, in spite of their absence in the gastric mucosa of normal mice, suggests that mature DCs play a role in the pathogenesis of autoimmune gastritis.
Animals↗
IF-LCM: laser capture microdissection of immunofluorescently defined cells for mRNA analysis rapid communication.
BACKGROUND: The next phase of the molecular revolution will bring functional genomics down to the level of individual cells in a tissue. Laser capture microdissection (LCM) coupled with reverse transcription-polymerase chain reaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless, targeting of specific cells may be difficult using routine morphologic stains. Immunohistochemistry can identify cells with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify specific tissue cells for LCM without mRNA loss. We report on a rapid immunofluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene expression. METHODS: A LCM microscope was outfitted for epifluorescence and light level video microscopy. Heat filters were added to shield the image intensifier from the laser. Frozen sections were fluorescently labeled by a rapid one minute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secondary antibody. Fluorescently labeled thick ascending limb (TAL) cells were detected by low light level video microscopy, captured by LCM, and mRNA was analyzed by RT-PCR for basic amino acid transporter, Tamm-Horsfall protein, and aquaporin-2. RESULTS: The immunofluorescently identified TAL could be cleanly microdissected without contamination from surrounding tubules. The recovery of RNA following rapid immunofluorescence staining was similar to that obtained following hematoxylin and eosin staining, as assessed by RT-PCR for malate dehydrogenase. CONCLUSIONS: We conclude that the new apparatus and method for the immunofluorescent labeling of tissue cells targeted for LCM can isolate pure populations of targeted cells from a sea of surrounding cells with highly acceptable preservation of mRNA. Since the TAL is minimally injured following ischemia, identification of the different responses between TAL and surrounding tissue in damaged kidneys may provide new therapeutic targets or agents for the treatment of acute renal failure.
Acridine Orange↗