Determinants of Slow Walking Speed in Ambulatory Patients Undergoing Maintenance Hemodialysis

Walking ability is significantly lower in hemodialysis patients compared to healthy people. Decreased walking ability characterized by slow walking speed is associated with adverse clinical events, but determinants of decreased walking speed in hemodialysis patients are unknown. The purpose of this study was to identify factors associated with slow walking speed in ambulatory hemodialysis patients. Subjects were 122 outpatients (64 men, 58 women; mean age, 68 years) undergoing hemodialysis. Clinical characteristics including comorbidities, motor function (strength, flexibility, and balance), and maximum walking speed (MWS) were measured and compared across sex-specific tertiles of MWS. Univariate and multivariate logistic regression analyses were performed to examine whether clinical characteristics and motor function could discriminate between the lowest, middle, and highest tertiles of MWS. Significant and common factors that discriminated the lowest and highest tertiles of MWS from other categories were presence of cardiac disease (lowest: odds ratio [OR] = 3.33, 95% confidence interval [CI] = 1.26–8.83, P<0.05; highest: OR = 2.84, 95% CI = 1.18–6.84, P<0.05), leg strength (OR = 0.62, 95% CI = 0.40–0.95, P<0.05; OR = 0.57, 95% CI = 0.39–0.82, P<0.01), and standing balance (OR = 0.76, 95% CI = 0.63–0.92, P<0.01; OR = 0.81, 95% CI = 0.68–0.97, P<0.05). History of fracture (OR = 3.35, 95% CI = 1.08–10.38; P<0.05) was a significant factor only in the lowest tertile. Cardiac disease, history of fracture, decreased leg strength, and poor standing balance were independently associated with slow walking speed in ambulatory hemodialysis patients. These findings provide useful data for planning effective therapeutic regimens to prevent decreases in walking ability in ambulatory hemodialysis patients.     

Expression of bovine lung prostaglandin F synthase in Escherichia coli

The full-length bovine lung prostaglandin(PG) F synthase cDNA was constructed from partial cDNA clones and ligated into bacterial expression vector pUC8 to develop expression plasmid pUCPF1. This plasmid permitted the synthesis of bovine lung PGF synthase in Escherichia coli. The recombinant bacteria overproduced a 36-KDa protein that was recognized by anti-PGF synthase antibody, and the expressed protein was purified to apparent homogeneity. The expressed protein reduced not only carbonyl compounds including PGD2 and phenanthrenequinone but also PGH2; and the Km values for phenanthrenequinone, PGD2, and PGH2 of the expressed protein were 0.1, 100, and 8 microM, respectively, which are the same as those of the bovine lung PGF synthase. The protein produced PGF2 alpha from PGH2, and 9 alpha, 11 beta-PGF2 from PGD2 at different active sites. Moreover, the structure of the purified protein from Escherichia coli was essentially identical to that of the native enzyme in terms of C-terminal sequence, sulfhydryl groups, and CD spectra except that the nine amino acids provided by the lac Z' gene of the vector were fused to the N-terminus. These results indicate that the expressed protein is essentially identical to bovine lung PGF synthase. We confirmed that PGF synthase is a dual function enzyme catalyzing the reduction of PGH2 and PGD2 on a single enzyme and that it has one binding site for NADPH.     

A lung type prostaglandin F synthase is expressed in bovine liver: cDNA sequence and expression in E. coli.

Using the cDNA of bovine lung prostaglandin F synthase (EC 1.1.1.2) as a probe, we isolated a clone from a bovine liver cDNA library which differed in only eleven nucleotides from the probe. The corresponding protein contained three amino acid substitutions, including a leucine residue which is conserved throughout all aldo-keto reductases. We inserted the liver cDNA into expression vector pUC19 and expressed the recombinant liver enzyme in E.coli. The purified liver enzyme reduced prostaglandin H2 as well as prostaglandin D2 and various carbonyl compounds. The high relative activity against prostaglandin H2 in combination with a high Km value for prostaglandin D2 identified this liver enzyme as a lung type prostaglandin F synthase. However, the binding constant for NADPH of the liver enzyme was 3.5 fold higher than that of lung prostaglandin F synthase.     

Purification and characterization of poly(ADP-ribose) glycohydrolase. Different modes of action on large and small poly(ADP-ribose)

Poly(ADP-ribose) glycohydrolase was purified approximately 74,000-fold to apparent homogeneity from calf thymus with a yield of 3.2%. The enzyme was a monomeric protein of Mr = 59,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The action of glycohydrolase on poly(ADP-ribose) was exoglycosidic in the direction of adenosine terminus----ribose terminus; radioactive ADP-ribose monomers were immediately produced from evenly labeled poly(ADP-ribose), but not from the polymer labeled selectively at the ribose terminus. The enzymatic degradation of large poly(ADP-ribose) (greater than 20 ADP-ribose residues) proceeded in a biphasic as well as bimodal manner. In the early and rapid phase, the enzyme degraded part of large polymers successively, leaving the remainder completely intact, and accumulated ADP-ribose monomers and small polymers of the size less than half of original polymers, indicating that the enzyme action was processive up to a certain extent. In the late and 20-fold slower phase, by contrast, the enzyme degraded the accumulated small polymers gradually and evenly, i.e. in a nonprocessive manner. The Km for large polymers was approximately 100-fold lower than that for small polymers. Similar rates and processivities were observed with large and small polymers bound to various proteins. These results suggested that the glycohydrolase may regulate differentially the levels of large and small poly(ADP-ribose) in the cell.     

6R-L-erythro-5,6,7,8-tetrahydrobiopterin as a regulator of dopamine and serotonin biosynthesis in the rat brain

6R-L-Erythro-tetrahydrobiopterin (6R-BH4), the natural isomer of tetrahydrobiopterin, was synthesized from 7,8-dihydrobiopterin using dihydrofolate reductase. The effects of intracerebroventricular injection of 6R-BH4 on the biosyntheses of neurotransmitter monoamines in the rat brain were investigated by measuring accumulation of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) after the inhibition of aromatic L-amino acid decarboxylase by NSD 1015 and the contents of metabolites of dopamine (DA) and 5-hydroxytryptamine (5-HT). The formation of DOPA and 5-HTP increased after the injection, reached a maximum level at about 1 h, then leveled off and reached a plateau over 2 h up to 8 h. The formation of DOPA and 5-HTP increased dose-dependently in the whole brain after the injection of 6R-BH4, and reached a plateau when the dose was more than 300 micrograms/rat. The enhancement was 100 and 70% for DOPA and 5-HTP, respectively. The formation of DOPA and 5-HTP increased in four brain regions, but the degree of stimulation was different among the brain regions. The contents of DA and 5-HT metabolites increased after the injection of 6R-BH4 in all brain regions tested, especially in the diencephalon and brain stem. The contents of DA and 5-HT increased slightly after the injection of 6R-BH4. These results suggest that 6R-BH4 concentration may be submaximal within DA and 5-HT neurons, and that an increase in 6R-BH4 in the brain enhances the biosyntheses of DA and 5-HT.     

Purification and characterization of rat brain prostaglandin D synthetase

Prostaglandin D synthetase was purified 2,600-fold from rat brain to apparent homogeneity, as judged by polyacrylamide gel electrophoresis and ultracentrifugation. The purified enzyme was a monomeric protein with a molecular weight of 27,000 +/- 1,000. The pI value, sedimentation coefficient, and partial specific volume were 4.6, 4.1 s, and 0.73 ml/g, respectively. The enzyme was stable between pH 4 and 11 at the temperature lower than 25 degrees C and resistant to a heat treatment under alkaline conditions (pH 8-11). About 50% of the activity was detected after a heat treatment at 100 degrees C for 5 min at pH 10. However, the enzyme was readily inactivated by the isomerase reaction of prostaglandin H2 to prostaglandin D2. The enzyme required sulfhydryl compounds such as dithiothreitol, glutathione, beta-mercaptoethanol, cysteine, and cysteamine for the reaction, but stoichiometric oxidation of these sulfhydryl compounds was not observed. The optimum pH, Km value for prostaglandin H2, and the turnover number were 9.5, 14 microM, and 170 min-1, respectively. The antibody was raised against the purified enzyme in a rabbit, which showed only one positive band in immunoblotting after gel electrophoresis of crude extracts of the brain at the same position as that of the purified enzyme. More than 90% of the prostaglandin D synthetase activity in the brain was absorbed by an excess amount of the antibody, indicating that our preparation is a major component of the enzyme responsible for the biosynthesis of prostaglandin D2 in the brain.     

Developmental changes in the activities of prostaglandin synthesizing enzymes in the digestive and immune systems of rat

The developmental changes of prostaglandin (PG) synthesizing enzymes in the digestive system (stomach and small intestine) and the immune system (spleen and thymus) of rats were investigated. In all the digestive organs, the predominant PG produced from PGH2 changed at around 2 weeks after birth to another PG. Further, the predominant activities of PG synthesizing enzymes were different organ by organ in the digestive system. In the case of the immune system, only the activity of PGD2 synthesizing enzyme displayed a significant increase during development and the activities of other PG synthesizing enzymes remained insignificant throughout the development. These results suggest that PGs may play important roles during the development of each organ.     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

A new species of Oithona (Copepoda, Cyclopoida) from the neritic waters of Australia

A new species of Oithona predominating in the neritic watersof Australia is described. This species resembles three otherspecies of the genus, but has several unique characters in thefamily Oithonidae. The morphological and geographical relationshipsof this species with allied species are discussed.     

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).