Effect of furosemide on urinary excretion of prostaglandin E in normal volunteers and pateints with essential hypertension

Urinary excretion of prostaglandin E was measured radioimmunologically in 19 healthy persons ( 15 men and 4 women ) and in 16 patients ( 10 men and 6 women ) with essential hypertension before and after the administration of furosemide. The excretion rates were increased from 26.3±3.0 to 64.5±11.3 ng/hr in the former and from 11.9±2.7 to 26.9±85 ng/hr in the latter. There was a significant difference between them, healthy subjects showing a greater increase than patients with essential hypertension.There was an obvious sexual difference in urinary excretion of prostaglandin. In men, greater increase in the excretion rates was found than in the women. Greater increases were also obtained in healthy men than in hypertensive men and in healthy women than in hypertensive women. The present results suggest that furosemide enhances urinary excretion of prostaglandin E by mechanisms which entails either an increase in prostaglandin synthesis or a decrease in renal metabolism.     

Purification and characterization of human testis aldose and aldehyde reductase

1. Aldose reductase and aldehyde reductase were purified to homogeneity from human testis. 2. The molecular weight of aldose reductase and aldehyde reductase were estimated to be 36,000 and 38,000 by SDS-PAGE, and the pI values of these enzymes were found to be 5.9 and 5.1 by chromatofocusing, respectively. 3. Aldose reductase had activity for aldo-sugars, whereas aldehyde reductase was virtually inactive for aldo-sugars. The Km values of aldose reductase for D-glucose, D-galactose and D-xylose were 57, 49 and 6.2 mM, respectively. Aldose reductase utilized both NADPH and NADH as coenzymes, whereas aldehyde reductase only NADPH. 4. Sulfate ion caused 3-fold activation of aldose reductase, but little for that of aldehyde reductase. 5. Sodium valproate inhibited significantly aldehyde reductase, but not aldose reductase. Aldose reductase was inhibited strongly by aldose reductase inhibitors being in clinical trials at concentrations of the order of 10(-7)-10(-9) M. Aldehyde reductase was also inhibited by these inhibitors, but its susceptibility was less than aldose reductase. 6. Reaction of aldose reductase with pyridoxal 5'-phosphate (PLP) resulted ca 2.5-fold activation, but aldehyde reductase did not cause the activation. PLP-treated aldose reductase has lost the susceptibility to aldose reductase inhibitor.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

Nucleotide sequence of the gene coding for four subunits of cytochrome c oxidase from the thermophilic bacterium PS3

The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing. Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 [Sone et al. (1988) J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct. PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and CO4 are much more similar to E. coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.     

Vacuolar targeting and posttranslational processing of the precursor to the sweet potato tuberous root storage protein in heterologous plant cells

Sporamin, the tuberous root storage protein of the sweet potato, which is localized in vacuoles, is synthesized as a prepro-precursor with an N-terminal sequence of amino acids that includes a signal peptide and an additional pro-segment of 16 amino acids. A full-length cDNA for sporamin was placed downstream of the 35 S promoter of cauliflower mosaic virus and introduced into tobacco and sunflower genomes by Ti plasmid-mediated transformation. A polypeptide of nearly the same size as mature sporamin from the sweet potato was detected in transformed calli of tobacco and sunflower, as well as in the leaves, stems, and roots of regenerated, transgenic tobacco plants. Amino acid sequence analysis of the nearly mature-sized form of sporamin from the transformed tobacco cells revealed that it is actually longer by three amino acids at its N terminus than authentic sporamin purified from the sweet potato. By pulse labeling of suspension-cultured tobacco cells with [35S]methionine, the pro-form of the precursor to sporamin, but not the prepro-precursor, was detected. The 35S-labeled proform was chased to the nearly mature-sized form via an intermediate form which is slightly larger than the nearly mature-sized form. Analysis by Edman degradation of the intermediate form that was labeled in vivo with [3H]histidine suggested that it is longer by two amino acids at its N terminus than the nearly mature-sized form of sporamin. These results suggest that at least two steps of posttranslational processing of the pro-form occurs sequentially in tobacco cells. The posttranslational processing of the pro-form of the precursor to sporamin was inhibited by monensin, suggesting that this step takes place in the acidic compartment, probably in the vacuole. All of the sporamin polypeptides synthesized in transformed tobacco cells were retained inside the cell and sporamin was localized in the vacuole, as judged from results of subcellular fractionation. These results indicate that sporamin is appropriately targeted to the vacuole in tobacco cells.     

Molecular cloning and expression of the rat beta 1-adrenergic receptor gene.

Using the sequence homology approach for cloning related genes within the G-protein-coupled receptor gene family, we have cloned the gene for the rat beta 1-adrenergic receptor (beta 1-AR). The rat beta 1-adrenergic receptor gene was isolated from a lambda EMBL3 rat genomic DNA library using the hamster beta 2-adrenergic receptor (beta 2-AR) coding sequence as a probe under low stringency hybridization conditions. The rat beta 1-AR gene encodes a protein of 466 amino acids that contains one consensus site for N-linked glycosylation (Asn-15) and three consensus sites for cAMP-dependent protein kinase phosphorylation (Ser-296, Ser-301, and Ser-401). The encoded rat beta 1-AR is 98 and 91% similar at the amino acid level with the human beta 1-AR in the transmembrane domains and in the overall sequence, respectively. Genomic Southern blot and gene dosage analyses indicate that the rat beta 1-AR gene is a single copy gene. The tissue distribution of the rat beta 1-AR mRNA was highest in the pineal gland with other brain regions and peripheral tissues, including the heart, expressing the mRNA at moderate levels. The bacteriophage clone containing the rat beta 1-AR gene with its natural promoter was co-transfected with the selectable marker (pRSVneo) conferring neomycin resistance into beta 1-AR-deficient mouse L cells. Analyses of the selected transfectant demonstrates efficient expression of the beta 1-AR gene and functional receptor. 125I-Labeled iodocyanopindolol bound transfectant membranes with an affinity of KD = 24 pm; the beta 1-AR-selective antagonist ICI 89,406 displaced iodocyanopindolol binding with a Ki approximately 140 times lower than that for the beta 2-AR-selective antagonist ICI 118,551. In addition, in the transfectant cell line, adenylylcyclase was stimulated by beta-adrenergic receptor agonists with the rank order of potency of isoproterenol greater than norepinephrine = epinephrine, consistent with properties expected of the beta 1-AR subtype.     

Differential effect of UV irradiation on induction of intragenic and intergenic recombination during commitment to meiosis in Saccharomyces cerevisiae

A comparison was made between the induction of intragenic and intergenic recombinations during meiosis in a wild-type diploid of Saccharomyces cerevisiae. Under non-irradiated normal conditions, production of both intragenic and intergenic recombinants greatly increased in the cells with commitment to meiosis. The susceptibility of cells to the induction of both the spontaneous intra- and intergenic recombinations in meiotic cells was similar. However, under condition of UV irradiation, there were striking differences between intra- and intergenic recombinations. Susceptibility to induction of intragenic recombination by UV irradiation was not enhanced at meiosis compared with mitosis, and was not altered through commitment to meiotic processes. In contrast, however, susceptibility to the induction of intergenic recombination by UV irradiation was enhanced markedly during commitment to meiosis compared with mitosis. Genetic analysis suggested that the enhanced susceptibility to recombination during meiosis is specifically concerned with reciprocal-type recombination (crossing-over) but not non-reciprocal-type recombination (gene conversion). Hence it is concluded that the meiotic process appears to be intimately concerned with the mechanism(s) of induction of recombination, especially reciprocal-type recombination.