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Biomedical subjects

M Araki

Publications and source records attributed to M Araki.

At least 55 records · Page 3Linked to original sources
PubMed1999

Assembly of lipoprotein (a) in transgenic rabbits expressing human apolipoprotein (a).

The study of human lipoprotein (a) [Lp(a)] has been hampered due to the lack of appropriate animal models since apolipoprotein (a) [apo(a)] is found only in primates and humans. In addition, human apo(a) in transgenic mice can not bind to murine apoB to form Lp(a) particles. In this study, we generated three independent transgenic rabbits expressing human apo(a) in their plasma at 1.8-4.5 mg/dl. In the plasma of transgenic rabbits, unlike the plasma of transgenic mice, about 80% of the apo(a) was covalently associated with rabbit apo-B and was contained in the fractions with density 1.02-1.10 g/ml, indicating the formation of Lp(a). These results suggest that transgenic rabbits expressing human apo(a) exhibit efficient assembly of Lp(a) and can be used as an animal model for the study of human Lp(a).

Animals↗
PubMed1999

Antioxidant function of the mitochondrial protein SP-22 in the cardiovascular system.

The mitochondrial protein SP-22 has recently been reported to be a member of the thioredoxin-dependent peroxide reductase family, suggesting that it may be one of the antioxidant systems in mitochondria, which are the major site of reactive oxygen intermediate generation. The aim of this study was to examine whether SP-22 is involved in mitochondrial antioxidant mechanisms and whether its expression is induced by oxidative stresses, particularly those in mitochondria. The expression of SP-22 protein was enhanced by about 1.5-4.6-fold when bovine aortic endothelial cells (BAEC) were exposed to various oxidative stresses, including mitochondrial respiratory inhibitors which increased the superoxide generation in BAEC mitochondria. The expression of SP-22 mRNA increased 2.0-3.5-fold with a peak at 3-6 h after exposure to Fe2+/dithiothreitol or a respiratory inhibitor, antimycin A. BAEC with an increased level of SP-22 protein caused by pretreatment with mild oxidative stress became tolerant to subsequent intense oxidative stress. On the other hand, BAEC that had been depleted of SP-22 with an antisense oligodeoxynucleotide against SP-22 mRNA became more labile to oxidative stress than control BAEC. The induction of SP-22 protein by oxidative stress in vivo was demonstrated in an experimental model of myocardial infarction in rat heart. These findings indicate that SP-22 functions as an antioxidant in mitochondria of the cardiovascular system.

Amino Acid Sequence↗
PubMed1999

The role of endothelin-converting enzyme-1 in the development of alpha1-adrenergic-stimulated hypertrophy in cultured neonatal rat cardiac myocytes.

BACKGROUND: Accumulating evidence suggests that the local synthesis of endothelin-1 (ET-1) plays a role in the development of heart failure in vivo. We investigated the role of endothelin-converting enzyme-1 (ECE-1), which mediates the conversion of big ET-1 to mature ET-1, in the development of alpha1-adrenergic-stimulated hypertrophy in cultured neonatal rat cardiac myocytes. METHODS AND RESULTS: Phenylephrine (PE) induced the expression of ET-1 in rat cardiac myocytes and accelerated the conversion of big ET-1 to ET-1. The ECE-1 mRNA levels were markedly increased 3 hours after PE stimulation (3.6-fold compared with saline stimulation, P<0.005). A specific ECE-1 antagonist, FR901533, inhibited the PE-stimulated increase in protein synthesis rate by 45% (P<0.05). As genetic markers for the hypertrophic response, FR901533 inhibited the PE-stimulated transcriptional activities of the 3.5-kb beta-myosin heavy chain promoter by 79% (P<0.01) but did not affect that of the 3.4-kb atrial natriuretic factor (ANF) promoter. In Bio14.6 Syrian cardiomyopathic hamsters, ventricular ET-1 and ANF mRNA levels did not correlate at 2 different stages. CONCLUSIONS: ET-1-independent pathways may mediate activation of the ANF gene program in ventricular myocytes both in vitro and in vivo. These results also indicate that the conversion of big ET-1 to ET-1 in rat cardiac myocytes is required for the development of alpha1-adrenergic-stimulated hypertrophy and beta-myosin heavy chain gene transcription.

Adrenergic alpha-Agonists↗
PubMed1999

Clinical application of a blood pressure autoregulation system during hypotensive anesthesia.

A blood pressure autoregulation system based on the state-predictive control method was developed to minimize intraoperative blood loss and hence avoid blood transfusion. In this report, the system is further improved by incorporating fuzzy logic with a fail-safe function and an individual parameter-identifying function. The safety and stability of this system had been confirmed by preclinical experiments with dogs. Thereafter clinical application with 17 patients was conducted to maintain their mean arterial pressure at around 60 mmHg during major surgery. The use of this system resulted in decreased blood loss and more speedy and accurate surgery due to a clearer surgical field. Unwanted effects of hypotension were not observed clinically or in laboratory tests. This system is therefore safe, stable, and effective in reducing the blood loss during major surgery that otherwise might cause substantial blood loss.

Anesthesia↗
PubMed1999

Differentiation of pinopsin-immunoreactive cells in the developing quail pineal organ: an in-vivo and in-vitro immunohistochemical study.

The avian pineal organ contains several types of photoreceptors with different photopigments: rhodopsin, iodopsin, and pinopsin. We have previously examined the differentiation of both rhodopsin-like and iodopsin-like immunoreactive cells during pineal development in quail embryos to determine the onset of synthesis of specific proteins and their cellular localization. In the present study, we have performed pinopsin immunohistochemistry on in-vivo developing and in-vitro cultured pineal organs of quail embryos. The results were compared with those obtained with rhodopsin and iodopsin immunohistochemistry. In the developing pineal organs, pinopsin immunoreactivity was detected at embryonic day 8, i.e. five days earlier than rhodopsin-like and iodopsin-like immunoreactivities. It was localized exclusively in the protrusions extending into the lumen throughout development, whereas rhodopsin-like and iodopsin-like immunoreactivities were usually found both in cell bodies and processes. These differences were also observed under two different types of culture conditions (dissociated cell culture and organ culture) indicating that, in the avian pineal organ, the expression pattern of the pinopsin gene is basically different from those of the other two pineal photopigments. The present study suggests that pineal cells have a mechanism for the polarized transport of pinopsin molecules.

Animals↗
PubMed1999

Upregulated expression of cardiac endothelin-1 participates in myocardial cell growth in Bio14.6 Syrian cardiomyopathic hamsters.

OBJECTIVES: The purpose of this study is to investigate the role of endogenous endothelin-1 (ET-1) in myocardial growth in Bio 14.6 Syrian cardiomyopathic hamsters (Bio). BACKGROUND: While ET-1, as a growth-promoting peptide, has been implicated in the development of secondary cardiac hypertrophy, the role of endogenous ET-1 in cardiac growth in primary myocardial disease is unknown. METHODS: We measured left ventricular ET-1 levels by a specific sandwich enzyme-linked immunosorbent assay. Furthermore, we examined the chronic effect of T0201, an ET type A receptor-specific antagonist. RESULTS: The ET-1 levels in the left ventricles were 1.8-fold higher (p < 0.0005) at 20 weeks and 6.4-fold higher (p < 0.0001) at 35 weeks in the Bio compared to age-matched control F1B hamsters (F1B). The Bio ET-1 levels in the lungs exhibited an only 1.3-fold elevation at 35 weeks. Immunohistochemistry demonstrated the localization of ET-1 mainly in the cardiac myocytes. The treatment with T0201 significantly reduced the heart weight/body weight ratio in the Bio, but did not affect the heart weight/body weight ratio in the F1B. Histologically, T0201 reduced the myocyte diameter of Bio to a level similar with that of F1B. However, T0201 did not affect the extent of fibrosis in Bio or F1B. CONCLUSIONS: The ET-1 level in the heart of cardiomyopathic hamsters increases in stage-dependent and organ-specific manners. Though myocyte degeneration and subsequent replacement fibrosis do not require an ET-1 pathway, the accelerated synthesis of ET-1 in the heart may contribute to the pathological growth of remaining myocytes in this animal model.

Animals↗
PubMed1999

Activation pattern of Langerhans cells in the afferent and efferent phases of contact hypersensitivity.

Langerhans cells are MHC class II (Ia) positive antigen-presenting cells that play a crucial role in the induction of contact hypersensitivity (CHS). The topical application of a hapten modifies the cell surface moieties of Langerhans cells, and activates Langerhans cells to increase their size and Ia intensity. The haptenated and activated Langerhans cells emigrate from the epidermis and thus the in situ density of Langerhans cells usually decreases during 24-48 h after the hapten application in CHS. To determine whether the early activation pattern of Langerhans cells is different between the afferent phase and the efferent phase of CHS, we compared the density and morphologic changes of Langerhans cells in CHS to trinitrochlorobenzene using nonsensitized and sensitized mice. We found that the application of a hapten induces more significant enlargement of Langerhans cell size in the afferent phase than in the efferent phase, whereas the reduction of Langerhans cell density is more marked in the efferent than in the afferent phase of CHS. Moreover, topical immunosuppressive drugs inhibit the in situ activation of Langerhans cells.

Animals↗
PubMed1999

Extracranial metastasis of anaplastic ganglioglioma through a ventriculoperitoneal shunt: a case report.

Extracranial metastasis of an intracranial anaplastic ganglioglioma through a ventriculoperitoneal shunt is reported. A 53-year-old woman was treated by surgery, radiation and chemotherapy and died 2 years later. At autopsy, multiple metastatic lesions were found in the spinal cord, and the abdominal and pleural cavities. Histologically, all the metastatic lesions were composed of atypical cells which resembled primitive glial elements found in intracranial anaplastic ganglioglioma, suggesting that anaplastic glial elements have a metastatic potential. Extracranial metastasis of ganglioglioma is a rare occurrence; however, the spread of glial elements through the shunt further suggests that caution is required in therapy and indicates a need for protective filters in the shunt system.

Biomarkers, Tumor↗
PubMed1999

17beta-estradiol induces protein thiol/disulfide oxidoreductases and protects cultured bovine aortic endothelial cells from oxidative stress.

OBJECTIVE: To examine whether or not estrogens induced the expression of protein thiol/disulfide oxidoreductases such as protein disulfide isomerase (PDI), thioredoxin (Trx), Trx reductase, and glutaredoxin (Grx) in vascular endothelial cells. METHODS: The regenerative effects of the protein thiol/disulfide oxidoreductases, PDI, Trx and Grx, on oxidatively damaged proteins were assayed using H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reporter enzyme. The induction of protein thiol/disulfide oxidoreductases and the accumulation of protein adducts generated by lipid peroxidation were examined by Western blotting in estrogen-treated bovine aortic endothelial cells (BAECs). RESULTS: Reduced PDI, Trx and Grx regenerated the H2O2-inactivated GAPDH in vitro. The levels of these protein disulfide oxidoreductases in BAECs were increased by pretreatment with 0.01-10 micromol/l 17beta-estradiol, the largest increase (about fourfold of the control) being found for PDI. Other sex hormones such as progesterone and testosterone did not affect the contents of these oxidoreductases in BAECs. 4-Hydroxy-2-nonenal (HNE)-protein adducts, which are generated by lipid peroxidation, were accumulated in BAECs exposed to paraquat, whereas the pretreatment of BAECs with 17beta-estradiol suppressed their accumulation. CONCLUSIONS: The estrogen-mediated induction of the protein thiol/disulfide oxidoreductases such as PDI, Trx, Trx reductase and Grx suggested a possible involvement of these oxidoreductases in the antioxidant protection of estrogen observed in the vascular system.

Animals↗
PubMed1999

[Virological response to interferon therapy in patients dually infected with TT virus and hepatitis C virus].

In 30 chronic hepatitis C patients co-infected with TTV, TTV DNA in the sera immediately after cessation of IFN and 6 months after the end of IFN was examined. Sustained loss of TTV DNA was observed in 12 of 30 (40.0%) patients. In 7 of 30 (23.3%) patients, TTV DNA re-appeared 6 months after becoming undetectable at the end of IFN therapy. In the remaining 11 patients (36.7%), TTV DNA remained detectable during the entire follow-up period. The ALT values correlated only with the presence of HCV RNA regardless of the effect of IFN on TTV replication. This study indicates that IFN therapy is effective against TTV.

Adult↗
PubMed1999

[The detection of apoptosis cells by TUNEL stain in peripheral blood smears].

We have determined the fixative conditions for TUNEL stain in peripheral blood smears. Murine IL-3 dependent B cell line (BAF-3) was used as positive control. The intensity of fixing solutions effected on the positivity/negativity for TUNEL stain. Alive cells showed positivity for TUNEL stain (false positive), when 70% methyl alcohol, acetone or carnoy fluid was used. In contrast to the strong fixation by high concentrated glutaraldehyde and the phosphate buffer, denatured cells showed to be negative (false negative). The fixation by 0.4% glutaraldehyde and 70% methyl alcohol for 15 min at room temperature, followed by the treatment by 20 micrograms/ml proteinase K for 15 min at 37 degrees C was most suitable for TUNEL stain to detect the apoptotic cells in peripheral blood smears. The frequency of TUNEL positive cells increased significantly in peripheral blood cells treated by apoptosis induced reagents, or infected by CMV. This is a new approach for the techniques to detect the apoptotic cells in peripheral blood smears.

Apoptosis↗
PubMed1999

Exchangeable gene trap using the Cre/mutated lox system.

The gene trap technique is a powerful approach for characterizing and mutating genes involved in mouse development. However, one shortcoming of gene trapping is the relative inability to induce subtle mutations. This problem can be overcome by introducing a knock-in system into the gene trap strategy. Here, we have constructed a new gene trap vector, pU-Hachi, employing the Cre-mutated lox system (Araki et al., 1997), in which a pair of mutant lox, lox71 and lox66, was used to promote targeted integrative reaction by Cre recombinase. The pU-Hachi carries splicing acceptor (SA)-lox71-internal ribosomal entry site (IRES)-beta-geo-pA-loxP-pA-pUC. By using this vector, we can carry out random insertional mutagenesis as the first step, and then we can replace the beta-geo gene with any gene of interest through Cre-mediated integration. We have isolated 109 trap clones electroporated with pU-Hachi, and analyzed their integration patterns by Southern blotting to select those carrying a single copy of the trap vector. By use of some of these clones, we have succeeded in exchanging the reporter gene at high efficiency, ranging between 20-80%. This integration system is also quite useful for plasmid rescue to recover flanking genomic sequences, because a plasmid vector sequence can be introduced even when the pUC sequence of the trap vector is lost through integration into the genome. Thus, this method, termed exchangeable gene trapping, has many advantages as the trapped clones can be utilized to express genes with any type of mutation.

Animals↗
PubMed1998

Blockade of ATP-sensitive K+ channels attenuates preconditioning effect on myocardial metabolism in swine: myocardial metabolism and ATP-sensitive K+ channels.

OBJECTIVE: We investigated if blockade of ATP-sensitive K+ channels (KATP) abolishes the protective effect of ischemic preconditioning (IP) on myocardial metabolism and ischemia-induced reactive hyperemia (RH) in pigs. METHODS: IP was elicited by a single cycle of 5 min occlusion and 5 min reperfusion of coronary artery, followed by 15 min of test ischemia and 120 min of reperfusion. Vehicle or the ATP-sensitive K+ channels (KATP) blocker, glibenclamide (3 or 6 mg/kg; G3 or G6) was administered before IP (groups; IP, G3+IP, G6+IP). As respective controls, the same treatment was performed in groups without IP (groups; C, G3, G6). Tissue levels of ATP, creatine phosphate (CP) and intracellular pH (pHi) in the area at risk were measured by 31P-nuclear magnetic resonance spectroscopy. RH after 5 min of preconditioning ischemia was assessed by regional myocardial blood flow. RESULTS: ATP and pHi were preserved after 15 min of ischemia in the IP group [C/IP; ATP=57+/-4/76+/-10% of baseline, pHi=6.18+/-0.08/6.66+/-0.03, P<0.05, C vs. IP]. Both doses of glibenclamide completely abolished the ATP sparing effect of IP. The high dose completely abolished pHi preservation (G6+IP=6.33+/-0.06), while the low dose showed only a partial effect (G3+IP=6.48+/-0.03). Glibenclamide did not adversely affect myocardial metabolism in groups without IP. Glibenclamide attenuated RH after 5 min of ischemia by 30% in both subendocardium and subepicardium. CONCLUSIONS: Blockade of KATP abolished the preconditioning effect on myocardial metabolism, and partially attenuated post-ischemic reactive hyperemia in pigs. These results indicate that KATP activation might be involved in the mechanisms of these phenomena, reactive hyperemia is not sufficient to induce IP protection.

Adenosine Triphosphate↗
PubMed1998

Cardiac endothelin-1 plays a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats.

BACKGROUND: To investigate whether endogenous ET-1 participates in an adaptive process of left ventricular hypertrophy (LVH) or a maladaptive process from LVH to congestive heart failure (CHF), we used a Dahl salt-sensitive (DS) rat model, in which systemic hypertension caused compensated concentric LVH at the age of 11 weeks followed by marked LV dilatation and global hypokinesis at the age of 17 weeks. METHODS AND RESULTS: By specific sandwich enzyme immunoassay, serum and myocardial ET-1 levels at the LVH stage were not elevated compared with age-matched Dahl salt-resistant (DR) rats, despite the marked increase of LV/body weight ratio (LV/BW). However, at the CHF stage, serum and LV ET-1 levels increased by 3. 8-fold and 5.4-fold, respectively. LV ET-1 contents had close relationships with the fractional shortening (r=0.763) and the systolic wall stress (r=0.858) measured by in vivo transthoracic echocardiography. Immunohistochemistry demonstrated that the remarkably increased ET-1 in LV is located mainly in cardiomyocytes. By competitive reverse transcriptase-polymerase chain reaction, LV prepro-ET-1 mRNA levels increased by 4.1-fold in CHF rats. We randomized 11-week-old LVH rats to chronic treatment with the endothelin receptor antagonist bosentan (Bos, 100 mg. kg-1. d-1, n=14), the alpha1-receptor antagonist doxazosin (Dox, 1 mg. kg-1. d-1, n=12), or vehicle (Cont, n=14). Bos treatment did not alter the LV geometry and function at 15 weeks; however, it attenuated the decrease of LV fractional shortening by 51% (P<0.01) without reducing the LV/BW at 17 weeks. Conversely, Dox, which decreased the blood pressure to the same extent as Bos, did not affect the progression of LV dysfunction. Bos (93%; P<0.0001 versus Cont) but not Dox (42%; P=0.8465 versus Cont) ameliorated the survival rate at 17 weeks (Cont; 36%). CONCLUSIONS: The accelerated myocardial synthesis of ET-1 contributes directly to LV contractile dysfunction during the transition from LVH to CHF. Unelevated levels of LV ET-1 at the established LVH stage and lack of effects on LV mass by chronic bosentan treatment suggest that myocardial growth is mediated through alternative pathways. These studies indicate that chronic ET antagonism may provide an additional strategy for heart failure therapy in humans.

Adaptation, Physiological↗
PubMed1998

Allosteric regulation of a ribozyme activity through ligand-induced conformational change.

An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and aflavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN). Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k cat of Rz3 increases by approximately 10-fold upon addition of saturating amounts of FMN. To determine the rate constants( K m4and k cat), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k cat value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.

Allosteric Regulation↗
PubMed1998

Analysis of the murine Hoxa-9 cDNA: an alternatively spliced transcript encodes a truncated protein lacking the homeodomain.

Hoxa-9 is one of the homeo box (Hox) genes exhibiting similarity to the Drosophila Abdominal B gene. So far, only partial nucleotide sequences have been reported for mouse Hoxa-9 cDNA (Rubin et al., (1987) Mol. Cell. Biol. 7, 3836-3841). Here, we have determined the nucleotide sequence of the 5'-region of mouse Hoxa-9 cDNA and its genomic structure. Mouse Hoxa-9 cDNA contains a complete ORF encoding a protein of 271aa exhibiting 96.7% identity to its human counterpart. Interestingly, an alternatively spliced transcript (Hoxa-9T) was identified by RT-PCR. Sequence analysis revealed that 173bp within the Hoxa-9 ORF was missing from the Hoxa-9T cDNA. This additional splicing would potentially result in a frameshift, leading to the production of a truncated protein lacking the homeobox. Northern blot analysis revealed that the probe containing the homeodomain hybridized to two major transcripts (2.5 and 1.9kb) in the trunk region of 12.5 dpc embryos, and adult kidney and large intestine. On the other hand, the probe containing the additional intron detected only 2.5kb transcript in the same tissues, indicating that 1.9kb transcript corresponds to Hoxa-9T mRNA. We have also determined the transcriptional start site of Hoxa-9T.

Alternative Splicing↗