ATP consumption by uncoupled mitochondria activates sarcolemmal K(ATP) channels in cardiac myocytes

We tested whether close coupling exists between mitochondria and sarcolemma by monitoring whole cell ATP-sensitive K(+) (K(ATP)) current (I(K,ATP)) as an index of subsarcolemmal energy state during mitochondrial perturbation. In rabbit ventricular myocytes, either pinacidil or the mitochondrial uncoupler dinitrophenol (DNP), which rapidly switches mitochondria from net ATP synthesis to net ATP hydrolysis, had little immediate effect on I(K,ATP). In contrast, in the presence of pinacidil, exposure to 100 microM DNP rapidly activated I(K,ATP) with complex kinetics consisting of a quick rise [time constant of I(K,ATP) increase (tau) = 0.13 +/- 0.01 min], an early partial recovery (tau = 0.43 +/- 0.04 min), and then a more gradual increase. This DNP-induced activation of I(K,ATP) was reversible and accompanied by mitochondrial flavoprotein oxidation. The F(1)F(0)-ATPase inhibitor oligomycin abolished the DNP-induced activation of I(K,ATP). The initial rapid rise in I(K,ATP) was blunted by atractyloside (an adenine nucleotide translocator inhibitor), leaving only a slow increase (tau = 0.66 +/- 0.17 min, P < 0.01). 2,4-Dinitrofluorobenzene (a creatine kinase inhibitor) slowed both the rapid rise (tau = 0.20 +/- 0.01 min, P < 0.05) and the subsequent declining phase (tau = 0.88 +/- 0.19 min, P < 0.05). From single K(ATP) channel recordings, we excluded a direct effect of DNP on K(ATP) channels. Taken together, these results indicate that rapid changes in F(1)F(0)-ATPase function dramatically alter subsarcolemmal energy charge, as reported by pinacidil-primed K(ATP) channel activity, revealing cross-talk between mitochondria and sarcolemma. The effects of mitochondrial ATP hydrolysis on sarcolemmal K(ATP) channels can be rationalized by reversal of F(1)F(0)-ATPase and the facilitation of coupling by the creatine kinase system.     

Comparison of oligosaccharides derived from salivary mucin of Japanese secretor and non-secretor individuals of blood group type-A

Comparison of oligosaccharide components derived from salivary mucin was performed between secretor and non-secretor individuals. Salivary mucin was collected from four secretors and three non-secretors having blood group type-A. Compositional analysis showed that the contents of galactose and N-acetylglucosamine in the non-secretor were higher than those in the secretor. The O-linked oligosaccharides obtained by treatment with alkaline borohydride were separated by gel filtration using Sephadex G-50. The results indicated that the size of the type-A active oligosaccharides from the secretor was similar to or smaller than that of the non-secretor. Ion-exchange chromatography showed that the secretors had strong type-A activities in both the neutral and acidic fractions but the non-secretors showed type-A activity mainly in the neutral fraction. These results suggest that compositional differences in blood group substances exist between secretors and non-secretors.     

Heart rate variability during long truck driving work

We recorded ambulatory electrocardiograms of 6 long distance truck drivers during their work period in order to observe the affect of autonomic nervous function and symptoms while doing their work. We also recorded their work patterns every minute. The RR50 value and the LFP/HFP ratio were calculated every two minutes based on R-R interval data. RR50 was significantly higher during taking naps than during other periods of work shifts, while, the LFP/HFP ratio showed significantly lower during taking naps than during other periods of work shifts. RR50 in the morning was significantly higher than that in the afternoon. On the contrary, the LFP/HFP ratio showed opposite tendency. Only on the times of driving, RR50 was significantly higher in the morning than that in the afternoon. On the other hand, the LFP/HFP ratio showed an opposite tendency. These results show that the parasympathetic nervous activities were more dominant than sympathetic nervous activities in the morning during the subjects were doing long distance truck driving including midnight work. Driving while in high parasympathetic nervous activity levels may add to cardiovascular stress and lead to drowsiness. And this may result in disrupted attention. It is necessary to decrease work time and improve working conditions of truck drivers working long-hour shifts.     

New compound heterozygous mutation in the CYP17 gene in a 46,XY girl with 17 alpha-hydroxylase/17,20-lyase deficiency

BACKGROUND: 17alpha-Hydroxylase/17,20-lyase deficiency is caused by a defect of P450c17 which catalyzes both 17alpha-hydroxylase and 17,20-lyase reactions in adrenal glands and gonads. RESULTS: In the present study, we analyzed the CYP17 gene in a Japanese patient with 17alpha-hydroxylase/17,20-lyase deficiency. The patient was a phenotypic girl and referred to us for right-sided inguinal hernia at the age of 4 years. Biopsy of the herniated gonad showed testicular tissue. The karyotype was 46,XY. At 6 years of age, hypertension was clearly recognized and the patient was diagnosed as having 17alpha-hydroxylase/17,20-lyase deficiency based on the clinical and laboratory findings. Analysis of the CYP17 gene revealed a compound heterozygous mutation. One mutation was an undescribed single nucleotide deletion at codon 247 in exon 4 (CTT to CT: 247delT) and the other was a missense mutation resulting in a substitution of His to Leu at codon 373 in exon 6 (CAC to CTC: H373L), which has been previously shown to abolish both 17alpha-hydroxylase and 17,20-lyase activities. The functional expression study of the 247delT mutant showed that this 247delT mutation completely eliminates both 17alpha-hydroxylase and 17,20-lyase activities. CONCLUSIONS: Together, these results indicate that the patient is a compound heterozygote for the mutation of the CYP17 gene (247delT and H373L) and that these mutations inactivate both 17alpha-hydroxylase and 17,20-lyase activities and give rise to clinically manifest 17alpha-hydroxylase/17,20-lyase deficiency.     

Effects of PEMF on a murine osteosarcoma cell line: drug-resistant (P-glycoprotein-positive) and non-resistant cells

After pulsed exposure of Dunn osteosarcoma cells (nonresistant cells) to Adriamycin (ADR) at increasing concentrations and single-cell cloning of surviving cells, ADR-resistant cells were obtained. These resistant cells expressed P-glycoprotein and had resistance more than 10 times that of their nonresistant parent cells. Compared to the nonresistant cells not exposed to pulsing electromagnetic fields (PEMF) in ADR-free medium, their growth rates at ADR concentrations of 0.01 and 0.02 micrograms/ml, which were below IC50, were 83.0% and 61.8%, respectively. On the other hand, in the nonresistant cells exposed to PEMF (repetition frequency, 10 Hz; rise time, 25 microsec, peak magnetic field intensity, 0.4-0.8 mT), the growth rate was 111.9% in ADR-free medium, 95.5% at an ADR concentration of 0.01 micrograms/ml, and 92.2% at an ADR concentration of 0.02 micrograms/ml. This promotion of growth by PEMF is considered to be a result of mobilization of cells in the non-proliferative period of the cell cycle due to exposure to PEMF. However, at ADR concentrations above the IC50, the growth rate tended to decrease in the cells not exposed to PEMF. This may be caused by an increase in cells sensitive to ADR resulting from mobilization of cells in the non-proliferative period to the cell cycle. The growth rate in the resistant cells exposed to PEMF was significantly lower than that in the non-exposed resistant cells at all ADR concentrations, including ADR-free culture (P相似文献   

Differential electron flow around photosystem I by two C(4)-photosynthetic-cell-specific ferredoxins

In the C(4) plant maize (Zea mays L.), two ferredoxin isoproteins, Fd I and Fd II, are expressed specifically in mesophyll and bundle-sheath cells, respectively. cDNAs for these ferredoxins were introduced separately into the cyanobacterium Plectonema boryanum with a disrupted endogenous ferredoxin gene, yielding TM202 and KM2-9 strains expressing Fd I and Fd II. The growth of TM202 was retarded under high light (130 micromol/m(2)/s), whereas KM2-9 grew at a normal rate but exhibited a nitrogen-deficient phenotype. Measurement of photosynthetic O(2) evolution revealed that the reducing power was not efficiently partitioned into nitrogen assimilation in KM2-9. After starvation of the cells in darkness, the P700 oxidation level under far-red illumination increased significantly in TM202. However, it remained low in KM2-9, indicating an active cyclic electron flow. In accordance with this, the cellular ratio of ATP/ADP increased and that of NADPH/NADP(+) decreased in KM2-9 as compared with TM202. These results demonstrated that the two cell type-specific ferredoxins differentially modulate electron flow around photosystem I.     

Crystal structures of 3-isopropylmalate dehydrogenases with mutations at the C-terminus: crystallographic analyses of structure-stability relationships

Thermal stability of the Thermus thermophilus isopropylmalate dehydrogenase enzyme was substantially lost upon the deletion of three residues from the C-terminus. However, the stability was partly recovered by the addition of two, four and seven amino acid residues (called HD177, HD708 and HD711, respectively) to the C-terminal region of the truncated enzyme. Three structures of these mutant enzymes were determined by an X-ray diffraction method. All protein crystals belong to space group P2(1) and their structures were solved by a standard molecular replacement method where the original dimer structure of the A172L mutant was used as a search model. Thermal stability of these mutant enzymes is discussed based on the 3D structure with special attention to the width of the active-site groove and the minor groove, distortion of beta-sheet pillar structure and size of cavity in the domain-domain interface around the C-terminus. Our previous studies revealed that the thermal stability of isopropylmalate dehydrogenase increases when the active-site cleft is closed (the closed form). In the present study it is shown that the active-site cleft can be regulated by open-close movement of the minor groove located at the opposite side to the active-site groove on the same subunit, through a paperclip-like motion.     

Steroid 5alpha-reductase inhibitory activity and hair regrowth effects of an extract from Boehmeria nipononivea

The acetone extract of Boehmeria nipononivea showed both potent 5alpha-reductase inhibitory activity and hair regrowth promotion effects on mice. 5alpha-Reductase inhibitory activity-guided fractionation led to six active fatty acids: alpha-linolenic, linoleic, palmitic, elaidic, oleic and stearic acids. The extract of B. nipononivea, and alphalinolenic, elaidic and stearic acids exhibited a hair regrowth effect.     

Cloning and expression of the Momordica charantia trypsin inhibitor II gene in silkworm by using a baculovirus vector

MCTI-II (Momordica charantia trypsin inhibitor II) isolated from bitter gourd (Momordica charantia LINN.) seeds is one of the serine protease inhibitors of the squash family. We cloned cDNA that encodes MCTI-II and constructed an expression system for MCTI-II by using a baculovirus vector. The recombinant baculovirus was inoculated to early fifth-instar larvae of the silkworm (strain: Shunrei x Shougetsu). Four days after infection, the hemolymph of silkworm larvae was collected and the recombinant protein was purified. Two kinds of expressed MCTI-II protein were obtained. An amino acid sequence analysis of the two proteins indicates that both were similar to the authentic inhibitor, except for the addition of a tripeptide derived from the vector at the N-terminus. One of the two inhibitors (MCTI-II A) resulted in a single PTH-amino acid in each Edman degradation cycle, while the other (MCTI-II B) resulted in two PTH-amino acids, suggesting the occurrence of cleavage of the reactive site. The inhibitory activities of MCTI-II expressed toward trypsin are examined in terms of the Ki value, these being 6.4 x 10(-10)M for MCTI-II A and 5.2 x 10(-10) M for MCTI-II B.     

Chromosomal and extrachromosomal synthesis of exfoliative toxin from Staphylococcus hyicus

Evidence for the existence of two molecular species of exfoliative toxin (ET) synthesized by Staphylococcus hyicus (SHET) under chromosomal and plasmid control is presented. Serological evidence that these molecular species of toxins are distinct from each other is given. The molecular weights of SHET from plasmidless strain P-1 (SHETA) and from plasmid-carrying strains P-10 and P-23 (SHETB) were almost equal. Both of the serotypes of SHET exhibited exfoliation in 1-day-old chickens. The plasmid-cured (P(-)) substrains (P-23C1 and P-23C2) of S. hyicus P-23 did not cause exfoliation in 1-day-old chickens, whereas P(-) substrains (P-10C1 and P-10C2) of strain P-10 caused exfoliation, but they decreased their exfoliative activity. These findings suggest that SHETB was synthesized along with SHETA by strain P-10, whereas the P-23 strain synthesized SHETB alone. The plasmid-carrying strain (P-23) as well as the plasmidless strain (P-1) exhibited the typical clinical signs of exudative epidermitis in pigs. However, plasmid-cured (P(-)) substrains of P-23 (P23C1 and P23C2) did not exhibit the typical clinical signs of exudative epidermitis. These findings suggest that SHETA is synthesized under chromosomal control and SHETB is synthesized under plasmid control and that SHET-producing strains can be divided into three groups: SHETA-producing strains, SHETB-producing strains, and strains producing both toxins.