Biomedical subjects

E Furuya

Publications and source records attributed to E Furuya.

At least 19 recordsLinked to original sources

PubMed2001

Alternative splicing of novel exons of rat heart-type fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase gene.

Three C-terminal sequences (VSIPVV, VCKWT, and LTLLS) of rat heart-type fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase have been reported. To elucidate the mechanism of generating the heterogeneity of the enzyme, we carried out reverse transcriptase PCR using three pairs of specific primers. The existence of mRNAs encoding VSIPVV and LTLLS was confirmed but not that of VCKWT. In addition to these cDNAs, we found four novel mRNAs that encode the C-termini of the enzyme. The genomic sequence reveals that the heterogeneity is generated by alternative splicing of exon 15 and four novel exons (exon 16a-d).

Alternative Splicing↗

PubMed2000

[Coping with stress in the elderly].

In terms of adaptability, unlike inanimate objects, living organisms exist in a dynamic balance between "wear and tear" and "repair and recovery". We regarded 17-hydroxycorticosteroids (17-OHCS) as a compound related to tissue "wear and tear" (Hans Selye) and sought for a compound related to tissue "repair and recovery". This led us to the discovery of 17-ketosteroid sulfates (17-KS-S) in urine. Elderly persons, unlike young adults, show low levels in 17-KS-S and little diurnal changes. In an elderly person 17-KS-S can decrease on significant life events (e.g. a spouse's death) and remain at low levels for a long time. Elderly persons with frailty need to improve their lifestyle (meals, exercise, rest, sleep, etc.) qualitatively and quantitatively to adapt themselves to stress adequately. Increased 17-KS-S levels were to be related to improvement in lifestyle.

17-Ketosteroids↗

PubMed2000

Accumulation of cAMP evoked by acetylcholine stimulation in rat submandibular acinar cells: observation of exocytosis, fluid secretion and [Ca2+]i.

The effects of cAMP accumulation evoked by acetylcholine (ACh) stimulation were studied in rat submandibular acinar cells by observing the exocytotic events, swelling of intercellular canaliculi (IC) and intracellular Ca2+ concentration ([Ca2+]i), which were monitored using an optical microscope. ACh stimulation evoked transient increases followed by sustained increases in the frequency of exocytotic events and IC swelling, while isoproterenol (isoprenaline; IPR) stimulation evoked sustained increases in these parameters. BAPTA treatment reduced the frequency of exocytotic events evoked by 5 microM ACh in the absence of extracellular Ca2+, and further addition of Rp-cAMPS or H-89 (protein kinase A (PKA) inhibitors) eliminated the remaining ACh-evoked responses (50 %). Addition of PKA inhibitors in the presence of extracellular Ca2+ reduced the frequency of exocytotic events evoked by 500 microM ACh in non-BAPTA-loaded cells. However, IC swelling evoked by 5 microM ACh was not affected by addition of PKA inhibitors, and was eliminated in BAPTA-loaded cells perfused with Ca2+-free solution. These results indicate that the IC swelling is regulated by [Ca2+]i and the frequency of exocytotic events is regulated by both [Ca2+]i and [cAMP]i during ACh stimulation. Addition of H-89 inhibited the capacitative Ca2+ entry into ACh-stimulated acinar cells. Biochemical analysis revealed that ACh stimulation increased the cAMP content in perfused submandibular glands. These results indicate that ACh stimulates the accumulation of cAMP in submandibular acinar cells and that this accumulation of cAMP modulates Ca2+-regulated exocytosis.

Acetylcholine↗

PubMed1999

Tissue-specific alternative splicing of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase.

We have reported the occurrence of eight splice variants of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase (RB2K). In the present study, we quantified these splice variants in various tissues using a RNAse protection assay and found a tissue-specific pattern of alternative splicing of the RB2K gene. Splice variants containing exon F were specifically expressed in brain. Moreover, exons D and E were spliced in brain, skeletal muscle and heart. Consequently, eight, six, four and two splice variants were expressed in brain, skeletal muscle, heart and liver plus testis, respectively. These results suggest that distinct RB2K isoforms could be involved in regulation of glycolysis in a tissue-specific manner.

Alternative Splicing↗

PubMed1999

A combined analysis of regional energy metabolism and immunohistochemical ischemic damage in the gerbil brain.

By combining immunohistochemical technique with microassay methods, we analyzed regional energy metabolism in vulnerable and tolerant areas of gerbil brains during evolution of neuronal damage after bilateral common carotid artery occlusion for 10 min with subsequent reperfusion. Four animals were used for each reperfusion period. Based on the information from the immunohistochemical examination, we dissected out vulnerable and tolerant subregions of the hippocampus, cerebral cortex, and thalamus from freeze-dried 20-microm-thick sections, and measured the levels of creatine phosphate (P-Cr), adenine nucleotides, guanine nucleotides, and purine bodies by HPLC, and the levels of glucose, glycogen, and lactate by an enzyme-immobilized column method. There were no significant differences in the levels of metabolites between vulnerable and tolerant subregions of control brains. After reperfusion, both vulnerable and tolerant subregions recovered preischemic metabolic profiles by 2 days. Although the regional differences between vulnerable and tolerant subregions were minimal at each reperfusion period, there were delays in the recovery of P-Cr, ATP, and/or total adenine nucleotides in all vulnerable subregions. A decline of P-Cr, ATP, and GTP levels without change in %ATP, AMP, or purine bodies occurred after reperfusion for 3 days, coinciding with the development of immunohistochemical damage by the immunoreaction for microtubule-associated protein 1A. The results supported the notion that subtle but sustained impairment of energy metabolism caused by mitochondrial dysfunction in the early reperfusion period might trigger delayed neuronal death in vulnerable subregions.

Adenine Nucleotides↗

PubMed1999

Keloids have continuous high metabolic activity.

The adenosine triphosphate (ATP) content of keloids and scars resected from patients was demonstrated by high-performance liquid chromatography. The surface color of hypertrophic scars was red or pink and that of atrophic scars was white. The ATP content of red scars was (in mmol/g protein) 1.06 +/- 0.14, of pink scars 0.12 +/- 0.02, of white atrophic scars 0.19 +/- 0.06, and of keloids 1.06 +/- 0.19. The longer the elapsed time after the trauma, the lower the level of ATP in scar tissues (correlation coefficient = -0.506; p = 0.005 by Spearman's rank correlation). However, ATP levels in keloids were still high 10 years after the injury. Fibroblasts and fibrocytes in keloids and scars were counted in histologic preparations stained with hematoxylin and eosin. The average number of fibroblasts in a definite area (56 x 10(-4) mm at a magnification of x400) was 4.8 in keloids, 5.1 in red scars, 2.4 in pink scars, and 1.3 in white atrophic scars. The number of fibrocytes in the same area was 0.4 in keloids, 0.4 in red scars, 2.3 in pink scars, and 1.3 in white atrophic scars. These results indicate that keloids and red hypertrophic scars have higher ATP levels and contain more fibroblasts than pink or white scars, and they also suggest that the levels of ATP and the number of fibroblasts decrease when red hypertrophic scars change into atrophic scars. In keloids, ATP and fibroblasts seem to remain at high levels for a long time.

Adenosine Triphosphate↗

PubMed1998

[Stress and anticortisols--17-ketosteroid sulfate conjugate as a biomarker in tissue repair and recovery].

We attempted to determine compounds in human urine which, differing from cortisol (17-OHCS), show high values in healthy individuals, decrease with failing health, clearly decline with advancing disease and finally reach very low values in severe disease. We have eventually established that 17-ketosteroid sulfate conjugates (17-KS-S) are the compounds we were searching for. Hans Selye regarded stress as the rate of wear and tear and 17-OHCS as its indicator, but we considered that, differing from inanimate objects, living organisms consume energy to cope with stress and "repair" "wear and tear" of the tissue and "recover" its function. This concept led us to determine the organism's requirement for two mechanisms: "Wear and tear" which could be represented by the secretion of 17-OHCS (Selye), and "repair and recovery", which could be determined by the amounts of 17-KS-S derived from dehydroepiandrosterone (DHEA)-sulfate, a product of the adrenal cortex, which enhances neuronal survival, longterm memory, maintains the function of peripheral tissues, and stimulates immune system (17-KS mainly consists of 17-KS-S and 17-KS glucuronides 17-KS-G, the latter derived from cortisol, DHEA and testosterone). We hold that stress response is a series of biological process, beginning from CRH.ACTH and catecholamines, cortisol (17-OHCS), followed by insulin, acting as an anabolic agent and finally DHEA (17-KS-S) leads the tissues to repair by utilizing produced energy. Balanced changes of hormones, such as 17-KS-S, 17-OHCS, cortisol, catecholamines and insulin are seen in healthy individuals under healthy lifestyles and disruption of the balance brings apparent reduction in 17-KS-S. From this standpoint, we wish to develop research in interrelations between biologically antagonistic 17-KS-S and 17-OHCS, focusing particularly at 17-KS-S, which represents a contact point for mind and body, the healthy state of which is kept by appropriate sleeping and exercise programs on the basis of adequate food intake. The above three factors, 17-KS-S, 17-OHCS, and 17-KS-G, are expressed in creatinine ratio (mg/g creatinine).

17-Hydroxycorticosteroids↗

PubMed1998

[17-KS sulfate as a biomarker in psychosocial stress].

We found an association between low levels of 17-Ketosteroid Sulfates (17-KS-S) in subjects under psychosocial stress. Stress associated with overwork and lack of sleep resulted in decreased levels of 17-KS-S and an increase in 17-Hydroxycorticosteroids (17-OHCS) levels. Alleviation of stress condition was associated with a restoration of the ratio of 17-KS-S/17-OHCS resulting from a slow increase in 17-KS-S and a decrease in 17-OHCS. In subjects with severe mental stress the ratio of 17-KS-S/17-OHCS showed markedly reduced values with a transient marked increase in 17-OHCS. There was a decrease in the levels of 17-KS-S in depressives, which was more pronounced during severe depression. There was no significant difference in 17-OHCS levels. In a cohort experiencing bereavement, there was a reduction in the levels of 17-KS-S, which remained low for about 50 days. With time, the level of 17-KS-S returned to the basal level established prior to spousal death. In this case, there was no significant change in the levels of 17-OHCS. Persistent severe stress over several months in a physician, led to a Herpes Zoster outbreak and Arrhythmia, resulting in hospital admission. The levels of 17-KS-S gradually decreased over this time reaching 1/3 of the normal baseline value, while 17-OHCS levels remained within normal ranges. These results suggest that measurement of 17-KS-S is indispensable for current research on psychosocial stress.

17-Hydroxycorticosteroids↗

PubMed1998

[17-ketosteroid sulfates and its application to kampo-medicine].

Traditional oriental medicine tells us that everything is interrelated and consists of combinations of opposing factors, "Yin-Yang". So we need to evaluate the normal state and pathologic condition from the perspective of dynamic hormone balance between cortisol and DHEA which are opposing and physiologically very important. Cortisol has a more functional side and is considered Yang, while DHEA has a more material side and is considered Yin. We tried to explain the concepts of KAMPO which include SHO (Yin-Yang. Excess-Deficiency), the 6 Stages of Disease and Deficiency of the Kidney, by measuring urinary 17-OHCS and 17-KS-S, metabolites of cortisol and DHEA. We think our effort may develop a good objective indicator of KAMPO's diagnosis and be useful for selecting of treatment.

17-Hydroxycorticosteroids↗

PubMed1998

[Significance of measuring urinary 17-ketosteroid sulfates at the work-place].

We examined availability of urinary 17-KS-S to evaluate work and job strain by presented the results in two field research and one experiment. It became clear that urinary S/OH was a comprehensive parameter of thermal strain, under the combined load condition of temperature and exercise, and as an additional factor, under the various load conditions of air velocity. Significant changes of urinary S/OH before and after the rest break and holiday suggested that the findings reflected fatigue conditions. This suggestion was strongly supported by the finding that the values of urinary S/OH on the first day-off after a mid-night shift and those 1 to 3 days later were not able to recover to the value before the night shift. A ratio between urinary 17-KS-S and 17-OHCS, S/OH, suggests a method of evaluating the degree of strain and the condition of fatigue and over-fatigue by measuring these parameters and thus clarifying the work conditions and work-place environment.

17-Hydroxycorticosteroids↗

PubMed1997

Novel isoforms of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase are generated by tissue-specific alternative splicing.

Fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, which is the most potent activator of glycolysis. We have shown previously the occurrence of a partial cDNA (RB7) encoding the catalytic core domain of a novel brain-type isozyme. To elucidate the full-length sequence of RB7 cDNA, we have carried out cDNA cloning using the 3'- and 5'-rapid amplification cDNA ends method and have isolated eight isoforms from rat brain. The cDNA sequences encoding the 5'-untranslated region, the amino-terminal domain, and the catalytic core domain were identical among all the isoforms. However, heterogeneity of the carboxyl-terminus was found by sequence analysis. This heterogeneity was shown not to have resulted from transcription of multiple genes, as Southern blot and genomic sequence analysis revealed that the gene was a single copy in the rat genome. It is likely that these transcripts represent splice variants of the gene. High-level expression of the gene was also observed by northern blot analysis in skeletal muscle. However, the pattern of alternative splicing was different from that of brain, and only four isoforms were detected in skeletal muscle.

Alternative Splicing↗

PubMed1997

Distribution of fiber types determined by in situ hybridization of myosin heavy chain mRNA and enzyme histochemistry in rat skeletal muscles.

We analyzed fiber types in rat skeletal muscles using a novel combination of in situ hybridization of myosin heavy chain (MyHC) mRNA, and enzyme histochemistry for succinate dehydrogenase (SD), which displayed metabolic properties. The fiber types were classified into the four major subtypes of I(beta/slow), IIA, IIX and IIB, and their intermediate types coexpressed two MyHC mRNAs: I and IIA, IIA and IIX, or IIX and IIB. The distribution of fiber types differed markedly in each skeletal muscle. The superficial region of limb muscles was composed mainly of fast-twitch fibers with oxidative-glycolytic and glycolytic activities, such as type IIX and type IIB. In contrast, the deep region was composed almost exclusively of type I and type IIA fibers both with oxidative activity. In this region, type IIA/IIX hybrid fibers were noted more frequently than type I/IIA and IIX/IIB hybrid fibers. In axial muscles, slow-twitch fibers and fast-twitch fibers composed predominantly of type IIB were distributed dispersively. The diaphragm and masseter showed a high proportion of type IIX and type IIB, respectively, to adapt to tissue-specific functional requirements and more frequently contained type IIX/IIB hybrid fibers than other observed muscles.

Animals↗

PubMed1996

Improvement of rat liver graft function by insulin administration to donor.

BACKGROUND & AIMS: The nutritional status of a donor is considered to be an important factor affecting organ viability. The purpose of the present study was to examine hepatic energy status in connection with posttransplantation liver function. METHODS: The following five groups of donor rat livers were prepared and stored in University of Wisconsin solution at 4 degrees C: fasted group, rats fasted for 24 hours; fed group, rats provided standard laboratory chow; fed plus insulin group, fed rats treated with insulin before harvest; fasted plus insulin group, fasted rats treated with insulin before harvest; and fed plus glucagon group, fed rats pretreated with glucagon. RESULTS: Hepatic levels of adenosine triphosphate and total adenine nucleotides were maintained in the decreasing order of the fed plus insulin, fed, fasted plus insulin, fasted, and fed plus glucagon groups during preservation. Lactate production rate and fructose 2,6-bisphosphate level increased in the order of the fed plus insulin, fed, fed plus glucagon, fasted, and fasted plus insulin groups. Liver function after transplantation evaluated by the bile flow rate and enzyme leakage was well restored in the fed plus insulin group. CONCLUSIONS: Insulin administration to nutritionally well-supported livers before harvest improved energy metabolism during preservation and liver function after transplantation.

Adenosine Triphosphate↗

PubMed1996

Insulin pretreatment protects the liver from ischemic damage during Pringle's maneuver.

BACKGROUND: Although maintenance of adenosine triphosphate (ATP) levels is important to restore liver functions during anoxia, ATP production by oxidative phosphorylation is inhibited during Pringle's maneuver, and only a little ATP can be supplied by glycolysis. The glycolytic activity of the liver is controlled by the nutritional condition and hormones. Enhancement of glycolytic activity by insulin may increase ATP production and thus may protect the liver from ischemia. METHODS: Rats were divided into three groups: fasted group, food was withheld for 24 hours; fed group, food was provided ad libitum; and insulin group, fed rats were administered insulin (12 units/kg during a 30-minute period) before portal triad clamping (PTC) was performed. After laparotomy was performed, PTC was performed for 30 minutes. The fructose 2,6-bisphosphate (F-2,6-BP) level, the hepatic levels of lactate and ATP, the bile flow rate, the plasma levels of aspartate transaminase and lactate dehydrogenase, and the indocyanine green clearance were measured at appropriate times. RESULTS: The hepatic F-2,6-BP levels before PTC in the fasted, fed, and insulin groups were 6.2 +/- 3.8, 55.6 +/- 10.6, and 122.2 +/- 31.3 nmol/gm dry weight liver, respectively. The glycolytic activity of the insulin group before PTC was significantly enhanced compared with that of the other groups. Lactate was more rapidly accumulated in livers of the insulin group during PTC than in those of the other groups. The ATP level and energy charge during PTC of the insulin group were higher than those of the other groups. The bile flow rate and indocyanine green clearance after PTC were restored in the order of the insulin, fed, and fasted groups. CONCLUSIONS: Insulin administration before PTC increased the hepatic F-2,6-BP content and enhanced glycolytic activity. Insulin pretreatment combined with feeding improved the hepatic energy metabolism during PTC and restored the liver functions after PTC. Insulin has protective effects on the liver during PTC.

Adenine Nucleotides↗

PubMed1996

Cloning of cDNA encoding for a novel isozyme of fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase from human placenta.

Two independent cDNA clones encoding fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase were isolated from a human placental cDNA library. The deduced amino acid sequences showed that one of the clones, 2K-1, was almost identical to the rat testis isozyme and the other, 2K-3, was different from any known isozymes expressed in mammalian tissues. The results of Southern blot analysis suggested that clones 2K-1 and 2K-3 were encoded as single copy genes and located in different parts of the genome. Since open reading frames of the cDNA clones were not complete, we obtained the 5'-end of the clone 2K-3 cDNA using the 5'-rapid amplification of cDNA end method. The entire cDNA (HP; 1,756 bp) had a coding capacity of 519 amino acids (M(r) = 59,410), and putative phosphorylation sites for protein kinases A and C on the C terminus. Northern blot analysis using a fragment of the HP as a probe showed that a major band of 5.4 kb, significantly different in size from known isozyme mRNAs such as liver (2.1 kb), muscle (1.9 kb), heart (4.0 kb), and testis (2.0 kb), was present in poly(A)+RNA preparations of human first trimester and term placentae. These results strongly suggested that this 5.4 kb mRNA codes a novel isozyme of fructose 6-phosphate,2-kinase/fructose 2,6-bisphosphatase.

Amino Acid Sequence↗

PubMed1995

Beneficial effect of fructose-1,6-bisphosphate on mitochondrial function during ischemia-reperfusion of rat liver.

BACKGROUND/AIMS: Several groups have reported that administration of fructose-1,6-bisphosphate (FBP) reduces ischemic injury. The aim of this study was to determine the protective effect of FBP on the impairment of mitochondrial oxidative phosphorylation by ischemia-reperfusion injury in the rat liver. METHODS: The respiratory control ratio (RCR) and the adenine nucleotide content of mitochondria isolated from ischemic and reperfused livers with or without FBP treatment were measured. RESULTS: In FBP-treated livers, the cellular adenosine triphosphate level was restored to more than 50% of normal after 120 minutes of reperfusion following 120 minutes of ischemia, whereas that of control livers only reached 15% of normal. The RCR and the adenine nucleotide content of mitochondria isolated from FBP-treated livers were significantly higher than those of mitochondria from control livers after ischemia and reperfusion. FBP strongly suppressed the formation of lipid peroxides during reperfusion. In vitamin E-deficient rats, the RCR decreased markedly during reperfusion, but FBP protected the mitochondria against reperfusion injury. CONCLUSIONS: FBP has a protective effect against ischemia-reperfusion injury on the liver and especially preserves the oxidative phosphorylation capacity of hepatic mitochondria.

Adenosine Triphosphate↗

PubMed1994

Cloning of cDNAs for fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase from frog skeletal muscle and liver, and their expression in skeletal muscle.

Frog (Rana catesbeiana) skeletal muscle (M-type) and liver (L-type) cDNAs of fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase were isolated from lambda gt10 phage cDNA library. The full-length L-type cDNA (1829 bp) encodes a 469 amino acids subunit (M(r) 54,800), while the M-type cDNA (1792 bp) encodes 455 amino acids (M(r) 52,901). The amino acid sequence of the M-type isozyme is identical to that of the L-type isozyme except for the N-terminus. The N-terminal 30 amino acids of the L-type isozyme are replaced by an unique sequence of 16 amino acids in the M-type isozyme. Both L- and M-type cDNAs were detected also in a lambda gt10 phage library of skeletal muscle. Relative amount of the M- and L-type mRNAs is skeletal muscle was determined by the reverse transcription-polymerase chain reaction method. The M/L mRNA ratio in frog skeletal muscle shows seasonal variations, being 0.56/1 in early summer and 5.3/1 in winter. These results suggest that there is a seasonal change in the isozyme composition and that the glycolysis in frog skeletal muscle may be regulated by type of the isozyme synthesized.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and tissue specific expression of fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase of rat brain.

A cDNA clone (3591 base pairs) encoding an isozyme of Fructose 6-phosphate, 2-kinase:Fructose 2,6-bisphosphatase was isolated from a rat brain cDNA library. The 5' sequence of this clone (1241 base pairs) was identical to that of the heart type isozyme cDNA (except for 7 base pair mismatches), but the 3' nucleotide sequence (2024 base pairs) was completely different. Its deduced amino acid sequence showed that the enzyme lacked a regulatory domain which contained phosphorylation sites for protein kinase A and C. The results of Northern blotting and polymerase chain reaction demonstrated that the mRNA, 7.4 kilobases long, was expressed also in heart, testis, liver, and skeletal muscle.

Alternative Splicing↗