Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.     

Building-site camps and extended work hours: A two-week monitoring of self-reported physical exertion, fatigue, and daytime sleepiness

Large-scale construction work often requires people to work longer daily hours and more than the ordinary five days in a row. In order to minimize transportation times and optimize the use of personnel, workers are sometimes asked to live in temporary building-site camps in the proximity of the work site. However, little is known about the biological and psychological effects of this experience. The objective of the present study was to investigate whether exposure to long work hours and extended workweeks while living in building-site camps in between work shifts was associated with a build-up of increased complaints of poor sleep, daytime sleepiness, physical exertion, and fatigue across a two-week work cycle. Two groups of construction workers were examined. The camp group of 13 participants (mean age: 42+/-11 S.D. yrs) lived in building-site camps and worked extended hours (between 07:00 and 18:00 h) and extended workweeks (six days in a row, one day off, five days in a row, nine days off). The home group of 16 participants (mean age 40+/-9 yrs) worked ordinary hours between 07:00 and 15:00 h and returned home after each workday. Self-ratings of daytime sleepiness (Karolinska Sleepiness Scale), physical exertion (Borg CR-10), and mood were obtained six or seven times daily during two workweeks. Fatigue ratings were obtained once daily in the evening, and ratings of sleep disturbances were obtained once daily in the morning with the Karolinska Sleep Diary. Data were evaluated in a repeated measures design. The results showed that both groups reported a similar level of daytime sleepiness, physical exertion, and mood across workdays and time points within a workday (all three-way interactions had p>0.898). Although the home group reported earlier wake-up times, the pattern of sleep disturbance ratings across the workdays did not differ between the groups. Both groups reported few sleep disturbances and good mood. However, the camp group reported higher physical exertion already at the start of work and showed a more gentle increase in ratings during the work shift and a smaller decline between the end of work and bedtime. The camp group also reported higher fatigue scores than the home group. However, none of the groups showed signs of increasing ratings in the progress of the two workweeks. For both groups, the ratings of daytime sleepiness formed a U-shaped pattern, with the highest scores at awakening and at bedtime. Yet, the camp group reported higher daytime sleepiness than the home group at lunch break and at the second break in the afternoon. In conclusion, there were no signs of fatigue build-up or accumulation of daytime sleepiness, physical exertion, or sleep disturbances in either group. Despite the fact that the camp group showed some signs of having trouble in recuperating in between work shifts, as indicated by the higher physical exertion ratings at the start of work, higher fatigue scores, and higher daytime sleepiness, the results constitute no real foundation for altering the camp group's current work schedule and living arrangements.     

The Human Red Cell Voltage-regulated Cation Channel. The Interplay with the Chloride Conductance,the Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> Channel and the Ca<Superscript>2+</Superscript> Pump

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K⁺ channels. We report here the cloning of a K⁺ channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K⁺ channel characteristics. The amino-acid sequence of the eel K⁺ channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.     

Evolution of ant-cultivar specialization and cultivar switching in Apterostigma fungus-growing ants

Almost all of the more than 200 species of fungus-growing ants (Formicidae: Attini) cultivate litter-decomposing fungi in the family Lepiotaceae (Basidiomycota: Agaricales). The single exception to this rule is a subgroup of ant species within the lower attine genus Apterostigma, which cultivate pterulaceous fungi distantly related to the Lepiotaceae. Comparison of cultivar and ant phylogenies suggests that a switch from lepiotaceous to pterulaceous fungiculture occurred only once in the history of the fungus-growing ants. This unique switch occurred after the origin of the genus Apterostigma, such that the basal Apterostigma lineages retained the ancestral attine condition of lepiotaceous fungiculture, and none of the Apterostigma lineages in the monophyletic group of pterulaceous fungiculturists are known to have reverted back to lepiotaceous fungiculture. The origin of pterulaceous fungiculture in attine ants may have involved a unique transition from the ancestral cultivation of litter-decomposing lepiotaceous fungi to the cultivation of wood-decomposing pterulaceous fungi. Phylogenetic analyses further indicate that distantly related Apterostigma ant species sometimes cultivate the same cultivar lineage, indicating evolutionarily frequent, and possibly ongoing, exchanges of fungal cultivars between Apterostigma ant species. The pterulaceous cultivars form two sister clades, and different Apterostigma ant lineages are invariably associated with, and thus specialized on, only one of the two cultivar clades. However, within clades Apterostigma ant species are able to switch between fungi. This pattern of broad specialization by attine ants on defined cultivar clades, coupled with flexible switching between fungi within cultivar clades, is also found in other attine lineages and appears to be a general phenomenon of fungicultural evolution in all fungus-growing ants.     

Expression and regulation of delta5-desaturase,delta6-desaturase,stearoyl-coenzyme A (CoA) desaturase 1, and stearoyl-CoA desaturase 2 in rat testis

In mammalian cells, essential polyunsaturated fatty acids (PUFAs) are converted to longer PUFAs by alternating steps of elongation and desaturation. In contrast to other PUFA-rich tissues, the testis is continuously drained of these fatty acids as spermatozoa are transported to the epididymis. Alteration of the germ cell lipid profile from spermatogonia to condensing spermatids and mature spermatozoa has been described, but the male gonadal gene expression of the desaturases, responsible for the PUFA-metabolism, is still not established. The focus of this study was to characterize the expression and regulation of stearoyl-CoA desaturase 1 (SCD1), stearoyl-CoA desaturase 2 (SCD2), and Delta5- and Delta6-desaturase in rat testis. Desaturase gene expression was detected in testis, epididymis, and separated cells from seminiferous tubulus using Northern blot analysis. For the first time, SCD1 and SCD2 expression is demonstrated in rat testis and epididymis, both SCDs are expressed in epididymis, while testis mainly contains SCD2. Examination of the testicular distribution of Delta5- and Delta6-desaturase and SCD1 and SCD2 shows that all four desaturases seem to be localized in the Sertoli cells, with far lower expression in germ cells. In light of earlier published results showing that germ cells are richer in PUFAs than Sertoli cells, this strengthens the hypothesis of a lipid transport from the Sertoli cells to the germ cells. As opposed to what is shown in liver, Delta5- and Delta6-desaturase mRNA levels in Sertoli cells are up-regulated by dexamethasone. Furthermore, dexamethasone induces SCD2 mRNA. Insulin also up-regulates these three genes in the Sertoli cell, while SCD1 mRNA is down-regulated by both insulin and dexamethasone. Delta5- and Delta6-desaturase, SCD1, and SCD2 are all up-regulated by FSH. A similar up-regulation of the desaturases is observed when treating Sertoli cells with (Bu)2cAMP, indicating that the desaturase up-regulation observed with FSH treatment results from elevated levels of cAMP. Finally, testosterone has no influence on the desaturase gene expression. Thus, FSH seems to be a key regulator of the desaturase expression in the Sertoli cell.     

A new type of dihydroorotate dehydrogenase,type 1S,from the thermoacidophilic archaeon<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

Dihydroorotate dehydrogenase (DHOD) (EC 1.3.3.1) from the thermoacidophilic archaeon Sulfolobus solfataricus P2 (DSM 1617) was partially purified 3,158-fold, characterized, and the encoding genes identified. Based on enzymological as well as phylogenetic methods, dihydroorotate dehydrogenase from S. solfataricus (DHODS) represents a new type of DHOD, type 1S. Furthermore, it is unable to use any of the (type-specific) natural electron acceptors employed by all other presently known DHODs. DHODS shows optimal activity at 70 degrees C in the pH range 7-8.5. It is capable of using ferricyanide, 2,6-dichlorophenolindophenol (DCIP), Q(0), and molecular oxygen as electron acceptor. Kinetic studies employing ferricyanide indicate a two-site ping-pong mechanism with K(M) values of 44.2+/-1.9 microM for the substrate dihydroorotate and 344+/-21 microM for the electron acceptor ferricyanide, as well as competitive product inhibition with a K(i) of 23.7+/-3.4 microM for the product orotate (OA). The specific activity, as determined from a partially purified sample, is approximately 20 micromol mg(-1) min(-1). DHODS is a heteromeric enzyme comprising a catalytic subunit encoded by pyrD (291 aa; MW=31.1 kDa) and an electron acceptor subunit (208 aa; MW=23.6 kDa), encoded by orf1. DHODS employs a serine as catalytic base, which is unique for a cytosolic DHOD. To our knowledge, this work represents not only the first study on an archaeal DHOD but the first on a nonmesophilic DHOD as well.     

Preoperative Acute Normovolaemic Hemodilution (ANH) in combination with Hypotensive Epidural Anaesthesia (HEA) during knee arthroplasty surgery. No effect on transfusion rate. A randomized controlled trial [ISRCTN87597684]

BACKGROUND: Hypotensive epidural anaesthesia (HEA) combines a high epidural anaesthesia, performing a sympathetic blockade, with low-dose iv-infusion of epinephrine to stabilize circulation in the conscious patient. Mean artery blood pressure is reduced to 45-50 mmHg and hereby a reduced blood loss. In this study we have combined HEA with preoperative acute normovolaemic hemodilution (ANH) in attempt to further reduce the blood loss and need for blood transfusion in total knee arthroplasty surgery (TKR). METHODS: Twenty-eight patients scheduled for TKR are randomised to ANH or no hemodilution (non-ANH). Both groups are anaesthetized with HEA. ANH is established with predonation of 20 % of the total blood volume, and replacement with equal volume of HAES 6 %. Blood re-transfusion is completed within 6 h. RESULTS: A mean of 877 ml blood was predonated (19.7 % of the total blood volume). Blood loss was, except from the intraoperative loss, significantly higher in ANH group. The total loss was 1306 mL (ANH) vs. 1026 mL (non-ANH), p < 0.05. Except from the first hour postoperatively, hematocrit was identical in between groups postoperatively. The amount of blood transfusion was identical 386 ml (ANH) vs. 343 ml (non-ANH) (ns). 50 % went through surgery without receiving blood (ANH) vs. 58 % (non-ANH). No renal, neurological or cardiopulmonary complications were registered. CONCLUSIONS: These data suggest no benefits in combining HEA and ANH in TKR surgery. Probably because of the reduced viscosity of the blood after ANH, there is an increased postoperative blood loss. The need for homologous blood transfusion was identical.     

Characterization of Laccases and Peroxidases from Wood-Rotting Fungi (Family Coprinaceae)

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60°C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.     

Purification and characterization of bovine mannan-binding protein

Bovine mannan-binding protein (bMBP) was observed in serum byits Ca²⁺ -dependent binding to mannan and by an M_rof 28 kDaunder reducing conditions on sodium dodecyl sulphate—polyacrylamidegel electrophoresis (SDS—PAGE). The lectin was isolatedby precipitation with polyethyl-eneglycol (PEG), affinity chromatographyon mannan—Sepharose eluted with EDTA, and absorption onSepharose 4B rabbit anti-bovine Ig to remove anti-mannan antibodies.Fractions containing the lectin were reapplied to mannan—Sepharoseand eluted first with N-acetyl-D-glucosamine (GlcNAc) to removeconglutinin, and then with mannose to elute the 28 kDa lectin.Further purification was achieved by ion-exchange chromatographyon Mono-Q and by man-nose-gradient elution from a mannan-Sepharosecolumn. SDS—PAGE of the purified lectin showed three highmolecular weight bands under non-reducing conditions. The reducedprotein gave a single band of 28 kDa. On gel permeation chromatographyunder non-dissociating conditions, the protein emerged at avolume corresponding to M_r