Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.     

Expressions of receptor gene for hepatocyte growth factor in kidney after unilateral nephrectomy and renal injury.

The renal expressions of the receptor gene (c-met) for hepatocyte growth factor (HGF) were examined in unilateral nephrectomy (UNX), renal ischemia or folic acid administration. The levels of c-met mRNA were increased rapidly in all rat models at 6h after the operations. On the other hand, the expression of c-met mRNA in a kidney cell line (MDCK cells) was down-regulated for 8 h after HGF addition, indicating that c-met mRNA induction in rat models may be independent of the stimulated production of HGF. The stimulated expression of c-met in these models suggest that HGF may play an important role in renal hypertrophy after UNX and regeneration after ischemic or nephrotoxic injury.     

Regulation mechanisms of intracellular pH of Xenopus laevis oocyte.

Intracellular pH values (pHi) of Xenopus oocytes were optically measured using a fluorescent dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The oocytes were loaded with dye by incubation with a membrane-permeable form (BCECF-AM). Mean pHi of the oocytes in pH 7.6 solution was 7.69. Increasing ambient pCO2 rapidly decreased pHi and estimated buffering power was 23.8 mM/pH unit. Changing ambient HCO3- from 5 to 30 mM did not alter pHi. After incubation in a Na(+)-free solution, Na+ addition to the bath rapidly increased pHi and this response was blocked by amiloride (ED50 2 microM). The addition of NH4Cl to the bath caused an initial transient increase in PHi followed by a secondary decrease. The secondary decrease was greatly inhibited by a histidine specific reagent, diethylpyrocarbonate. It was also slightly inhibited by ouabain, Ba2+ and furosemide, but not by amiloride. These data suggest that (1), fluorescence technique is applicable to PHi measurements of Xenopus oocytes; (2), Xenopus oocytes have an amiloride sensitive Na+/H(+)-exchange, and permeabilities to CO2, NH3, and NH+4. These observation may be useful in studying the relationship between pHi and oocytes development, and the expression of acid/base transporters in Xenopus oocytes.     

Lithium chloride-tolerant character of the heterobasidiomycetous yeastRhodotorula glutinis

The heterobasidiomycetous yeastRhodotorula glutinis was able to grow in medium containing a high concentration of LiCl. This character ofR. glutinis was presumed to be attributable to its ability to incorporate [¹⁴C]-adenine and [¹⁴C]-leucine into nucleic acids and proteins, respectively, in the presence of LiCl. Intracellular levels of Li⁺ and Cl^– ions, production and accumulation of glycerol as an osmoregulator, and respiration in the LiCl-stressed condition were almost the same in the tolerant yeastR. glutinis and the sensitive yeastRhodosporidium sphaerocarpum.     

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA ⁺ gene were grown at 30° C and then transferred to 42° C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB ⁺ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42° C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Ischemic changes in myocardial ionic contents of the isolated perfused rat hearts as studied by NMR

Using³¹P-,²³Na- and³⁹K-NMR, we assessed ischemic changes in high energy phosphates and ion contents of isolated perfused rat hearts continuously and systematically. To discriminate intra- and extracellular Na⁺, a shift reagent (Dy(TTHA)^3–) was used in²³Na-NMR study. In³⁹K-NMR study, the extracellular K⁺ signal was suppressed by inversion recovery pulse sequence in order to obtain intracellular K⁺ signal without using shift reagnets. During the early period of ischemia, increases in intracellular Na⁺ and inorganic phosphate (Pi) were observed in addition to the well-documented decreases in creatine phosphate and ATP and a fall of intracellular pH, suggesting an augmented operation of Na⁺–H⁺ exchange triggered by a fall of the intracellular pH resulted from breakdown of ATP. At around 15 min of ischemia, a second larger increase in intracellular Na⁺ and a decrease in intracellular K⁺ were observed in association with a second increase in Pi. This was accompnanied by an abrupt rise of the ventricular end-diastolic pressure. As there was a depletion of ATP at this time, the increase in intracellular Na⁺ and associated decrease in intracellular K⁺ may be explained by inhibition of the Na⁺–K⁺ ATPase due to the depletion of ATP. A longer observation with³¹P-NMR revealed a second phosphate peak (at lower magnetic field to ordinary Pi peak) which increased its intensity as ischemic time lengthened. The pH of this 2nd peak changed in parallel with the changes in pH of the bathing solution, indicating the appearance of a compartment whose hydrogen concentration is in equilibrium with that of the external compartment. Thus, the peak could be used as an index of irreversible membrane damage of the myocardium.     

Analysis of the thromboxane/prostacyclin balance in human urine by gas chromatography/selected ion monitoring: abnormalities in diabetics

We microanalyzed 2,3-dinor-6-keto-prostaglandin F_1α (2,3-dinor-6-keto-PGF_1α 1) and 11-dehydrothromboxane B₂ (11-dehydro-TXB₂, 2) in human urine. Samples containing a [²H₄]-analogue as an internal standard were extracted by chromatography using Sep Pak tC18 and silica gel. The compounds were then analysed by means of the lactone ring opening reaction and dimethylisopropylsilylation. The conversion of 1 to 1-methyl ester (ME)-propylamide (PA)-9,12,15-dimethylisopropylsilyl (DMIPS) ether derivative and of 2 to 1-ME-6-methoxime (MO)-9,12,15-tris-DMIPS ether derivative was followed by gas chromatography/selected ion monitoring (GC/SIM). Interfering substances from the urine matrix were eliminated during GC/SIM analysis using a DB-5 column. We were able to detect 1 (222–1031 pg/mg creatinine) and 2 (18–155 pg/mg creatinine) in human urine. Furthermore, the thromboxane/prostacyclin (IX/PGI) ratio in the urine of diabetics was higher than that of healthy volunteers. This method can be used to determine the TX/PGI balance in human urine.     

Sex-related spatial kin structure in a spring population of grey-sided voles Clethrionomys rufocanus as revealed by mitochondrial and microsatellite DNA analyses

Polymerase chain reaction-directed mitochondria (mt) and microsatellite DNA analyses were performed to examine the kin structure in a spring population of grey-sided voles Clethrionomys rufocanus in Hokkaido, Japan. The spatial distribution of 81 voles in a trapping grid (about 1 ha) was estimated by using the catch-mark-release method. DNA samples were extracted from the toes clipped for individual identification. Maternal lineages of voles were unequivocally determined by the mtDNA haplotypes, as identified by nucleotide sequencing of the control region. Relatedness between individuals was estimated based on the genotype and allele frequencies at several microsatellite loci. Although the distribution of voles was uniform within the grid, neighbouring females were frequently from the same maternal lineage. Relatedness values between females correlated negatively with geographical distances. Combination of the two molecular markers revealed four clusters of closely related, matrilineal females in the population, whereas no such cluster was apparent in males. The present study first demonstrated a sex-related spatial kin structure in a natural population of the grey-sided vole.