Inhibition of Klenow DNA polymerase and poly(A)-specific ribonuclease by aminoglycosides

Aminoglycosides are known to bind and perturb the function of catalytic RNA. Here we show that they also are potent inhibitors of protein-based catalysis using Escherichia coli Klenow polymerase (pol) and mammalian poly(A)-specific ribonuclease (PARN) as model enzymes. The inhibition was pH dependent and released in a competitive manner by Mg2+. Kinetic analysis showed that neomycin B behaved as a mixed noncompetitive inhibitor. Iron-mediated hydroxyl radical cleavage was used to show that neomycin B interfered with metal-ion binding in the active sites of both enzymes. Our analysis suggests a mechanism of inhibition where the aminoglycoside binds in the active site of the enzyme and thereby displaces catalytically important divalent metal ions. The potential causes of aminoglycoside toxicity and the usage of aminoglycosides to probe, characterize, and perturb metalloenzymes are discussed.     

Improved partitioning in aqueous two-phase system of tyrosine-tagged recombinant lactate dehydrogenase

The partitioning of Bacillus stearothermophilus lactate dehydrogenase (LDH) in an aqueous two-phase system was studied. Particularly, the influence of tyrosine tags on the partitioning was evaluated. The hydrophobic effect, caused by the addition of tyrosine residues, was determined in a system based on dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer (EO30PO70). Five different LDH variants were constructed with N-terminal tags containing tyrosines (Y3 and Y6), tyrosines and prolines (Y3P2 and Y6P2), and only prolines (P2). LDH fused with tags containing tyrosines increased the partitioning coefficient, and the more tyrosines added to the protein, the larger improvement in partitioning. When prolines were added between the tyrosine-rich tag and the protein, a further increased partitioning was obtained. The enhanced partitioning was attributed to the rigid structure of the proline, which in turn led to an increase in the exposure of the tag to the surroundings. The best tyrosine tag, Y6P2, increased the partition coefficient four times and additionally, a higher thermostability was observed.     

Selective extraction of lecithin:cholesterol acyltransferase (EC 2.3.1.43) from human plasma

A method for the rapid extraction of lecithin:cholesterol acyltransferase (LCAT) from human plasma or serum has been developed. The method is based on direct treatment of acidified plasma of fully conserved enzyme activity, with the strong ion exchanger Q-Sepharose, which under the experimental conditions bound all LCAT but only about 10% of the total protein content of the plasma, no albumin and essentially no lipoproteins. This corresponds to a 10-fold purification. Only traces of apolipoprotein A-I remained in the quantitatively desorbed LCAT preparation which, however, contained a residual fraction of apolipoprotein D and acidic plasma proteins.The present one-step procedure for extraction of LCAT in high yields from human plasma represents a simple and efficient alternative to the first step in previously described methods for preparation of the enzyme to homogeneity.     

Effects of exposure to 17alpha-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish (Danio rerio)

The effect of acute stress on plasma beta-corticosterone (B), testosterone (T) and estradiol-17beta (E2) concentrations in juvenile alligators collected from sites with varying sediment contaminants was examined in this study. Dramatic increases in plasma B concentrations were observed in alligators from all of the sites after 2 h of capture although females from the intermediate contaminant site exhibited a significantly lower percentage increase in B than females from the other two sites. Males from the site with the highest contaminant levels exhibited elevated initial B concentrations relative to the other sites. This pattern was not observed after 2 h of restraint. Females from the highest contaminant site exhibited depressed initial T when compared to the other sites although this pattern was not observed after 2 h of restraint. Neither E2 nor T decreased after 2 h in females, whereas T concentrations decreased in all males over the same time period. The variance associated with these endpoints was also examined to determine whether it could serve as a more sensitive marker for perturbations of the endocrine system and stress response. Females from the higher and intermediate contaminant sites exhibited the lowest and highest standard errors (respectively) associated with 2 h plasma B concentrations with no differences among mean concentrations suggesting a perturbation of the stress response in these animals that was not detected by examining the means. We concluded that the environmental contaminants could be acting as stressors, leading to the observed differences.     

Nanoinjection as a tool to mimic vertical transmission of Flavobacterium psychrophilum in rainbow trout Oncorhynchus mykiss

Newly fertilised eggs of rainbow trout Oncorhynchus mykiss were nanoinjected with Flavobacterium psychrophilum in order to mimic vertical transmission. Two bacterial isolates with different elastin-degrading capacity were used. All infected groups (10, 100 and 1000 colony forming units egg(-1)) showed significantly higher cumulative mortalities than the control groups at the end of the experiment, 70 d post-hatching. The total mortalities in the control groups were below 2.5%. In the high-dose groups, 95 to 100% of the eggs died during the eyed stage. In the intermediate group infected with the elastin-negative isolate, the major mortality occurred during the eyed stage of the egg, with a total cumulative mortality of 83% at the end of the experiment. In the intermediate group infected with the elastin-positive isolate, a total mortality of 63% was recorded. In this group, diseased fry showed clinical signs of disease and morphological changes similar to those described in connection with rainbow trout fry syndrome (RTFS) shortly after the beginning of feeding. In the low dose groups, the mortality in the elastin-negative group was 14% and in the elastin-positive group 11%. The bacterium was isolated from dead eggs and fry in infected groups and demonstrated in internal organs of dead and moribund fry by immunohistochemistry. The nanoinjection method used in this study may be a useful method to study pathogens, like F. psychrophilum, that can be vertically transmitted.     

Long-term history of land-use and vegetation at Ire,an agriculturally marginal area in Blekinge,south Sweden

Pollen records from a small lake and a small peatland at Ire in northern Blekinge, Sweden, reveal that until a.d. 300 there was a mixed deciduous type of woodland dominated by Quercus, Tilia and Corylus, with Betula, Pinus, Populus, Fraxinus, Ulmus and Acer as important constituents. The first, but weak, signs of human influence on the vegetation are detected around 2300 b.c. At this time, the area was probably used for woodland grazing. The regional expansion of Fagus occurred around 100 b.c.–a.d. 200, and later, at about a.d. 500, woods dominated by Fagus were common in the area. Around a.d. 600–700 an isolated farmstead may have been established in the area, as single pollen grains of Secale (rye) were found. This farmstead may represent the first permanent settlement in the area. A more widespread opening of the vegetation occurred around a.d. 1000, probably as an effect of a more pronounced use of the area, and an expansion of settlements in the region. The openness of the area seems to have peaked around a.d. 1400–1800, and during this period the vegetation was highly fragmented with small stands of woodland, intensively grazed pastures, and arable fields where Secale and Triticum were mainly cultivated. Around a.d. 1600 Fagus pollen percentages sharply decrease, most probably caused by a deliberate and selective felling of beech trees for the production of potash, which consumed enormous amounts of wood. Picea seems to have been established around a.d. 1600, but it did not become a regional dominant until the first part of the 20th century, when land use decreased and it became favoured by forestry.     

Two-electron electrochemical oxidation of quercetin and kaempferol changes only the flavonoid C-ring

Bulk electrolysis of the antioxidant flavonoids quercetin and kaempferol in acetonitrile both yield a single oxidation product in two-electron processes. The oxidation products are more polar than their parent compounds, with an increased molecular weight of 16 g/mol, and were identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone and 2-(4-hydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone for quercetin and kaempferol, respectively. Two-electron oxidation of the parent flavonoid is suggested to yield a 3,4-flavandione with unchanged substitution pattern in the A- and B-ring, which may rearrange to form the substituted 3(2H)-benzofuranone through the chalcan-trione ring-chain tautomer. The acidity of the 3-OH group is suggested to determine the fate of the flavonoid phenoxyl radical, originally formed by one-electron oxidation, as no well-defined oxidation product of luteolin (lacking the 3-OH group) could be isolated despite rather similar half-peak potentials: E_P/2 = 0.97 V, 0.98 V and 1.17 V vs. NHE for quercetin, kaempferol and luteolin, respectively, as measured by cyclic voltammetry in acetonitrile.