Design and evolution of new catalytic activity with an existing protein scaffold

The design of enzymes with new functions and properties has long been a goal in protein engineering. Here, we report a strategy to change the catalytic activity of an existing protein scaffold. This was achieved by simultaneous incorporation and adjustment of functional elements through insertion, deletion, and substitution of several active site loops, followed by point mutations to fine-tune the activity. Using this approach, we were able to introduce beta-lactamase activity into the alphabeta/betaalpha metallohydrolase scaffold of glyoxalase II. The resulting enzyme, evMBL8 (evolved metallo beta-lactamase 8), completely lost its original activity and, instead, catalyzed the hydrolysis of cefotaxime with a (kcat/Km)app of 1.8 x 10(2) (mole/liter)(-1) second(-1), thus increasing resistance to Escherichia coli growth on cefotaxime by a factor of about 100.     

An instrument measuring mechanical characteristics of the potato tuber

Summary A type of penetrometer is described in which the probe passes through raw or cooked material at a constant speed, the force required to maintain this speed being continuously recorded to an accuracy of about 3% of scale. Examples of results obtained with unpeeled boiled potatoes are illustrated.     

Response of limed lakes to episodic acid events in Southwestern Sweden

Occurrence of stratified acidic water is described for five forest lakes situated in the westcoast region of Sweden. Differentiation between two types of acid events is made related to origin; one is caused by heavy rains and the other associated with snowmelt during winter and spring. Acid events are due to incomplete mixing between lake and inflow water. Both mixing forces like wind and density differences are important factors regulating the actual stratification. As observed in the lakes studied, stratification therefore occurs during periods of ice-cover when wind-induced mixing is impeded and generally results in an acidic surface water of varying depth. However, in this study we also describe the stratification of acidic inflow water at the sediment-water interface. This type of inlayering is presumably less frequent as a complex set of conditions must be satisfied for its occurrence. Our study shows that acid events may cause temporal and spatial water chemistry changes even in lakes and streams with relatively high pH and buffer capacity. Thus, early biotic damages can be expected in neutral and limed (soft water) lakes.     

Effects of dietary anthocyanins on tocopherols and lipids in rats

The effects of dietary cyanidin-3-O-glucoside (C3G) and concentrates from blackcurrant [Ribes nigrum] (BC) and elderberry [Sambucus nigra] (EC) on plasma and tissue concentrations of alpha- (alpha-T) and gamma-tocopherol (gamma-T) and cholesterol, as well as the fatty acid composition of the liver lipids were investigated in growing, male rats of the Sprague-Dawley strain. Animals were fed semisynthetic diets supplemented with 2 g/kg C3G, BC, or EC for 4 weeks. Dietary anthocyanins did not affect feed intake, body weight, and organ weights. C3G elevated the concentrations of tocopherols in the liver and lungs (P < 0.05). Cholesterol levels in plasma and liver were not affected by any of the regimens. C3G and BC reduced the relative amount of saturated fatty acids in the liver (P < 0.05). BC also lowered the percentage of 22:6 + 24:0 and EC the ratio of 20:3/20:4 n-6 (P < 0.05). In conclusion, dietary C3G, BC, and EC appear to have little effect on cholesterol levels and the fatty acid pattern in the liver but seem to be capable of sparing vitamin E in healthy, growing rats.     

Results of the international aqueous mercury speciation intercomparison exercise

Twenty-seven laboratories from around the world agreed to participate in an intercomparison exercise for total Hg (Hg_t) and methyl Hg (MMHg) in pristine lake water. Unfiltered samples from a remote brown water lake in northern Wisconsin (USA) were collected into acid cleaned Teflon^® bottles using ultra-clean sample handling techniques. The samples were acidified in the field with 0.4% by volume of pre-analyzed HCl (12N; <5 pg Hg/mL), and sent to the primary reference laboratory (PRL) by overnight mail. Within one week of receipt, the samples were randomized, and 10% analyzed for Hgt and MMHg at the PRL to verify the homogeneity of the set. Each participating laboratory was then sent 3 randomly selected 1 L bottles, while the PRL retained 30, and the secondary reference laboratory (SRL) retained 12 samples. The participating laboratories were asked to analyze each bottle in triplicate for both Hgt and MMHg, reporting all QA data including blanks, spike recoveries, and detection limits. The PRL analyzed samples in triplicate at both the beginning and the end of the analytical window, to provide a controlled estimate of any changes in concentration or speciation over that time. Of the 23 laboratories that returned results, 18 utilized BrCl oxidation, gold trapping, and cold vapor atomic fluorescence (CVAFS) detection for Hg_t. Four laboratories reported similar techniques, varying either in detector (cold vapor atomic absorption), or wet chemistry. Only 16 laboratories reported MMHg results, with 15 using a variation of the aqueous phase ethylation, GC separation, and CVAFS detection technique. The results show good convergence between the participating labs for both Hg_t and MMHg. For Hgt 18 of 23 labs reported means within 20% of the consensus value and PRL results (1.27±0.18 ng/L and 1.27±0.14 ng/L respectively). For MMHg, 13 of the 16 labs reported results within 20% of either the consensus value (0.420±0.055 ng/L) or the PRL value (0.446±0.041 ng/L).     

The dietary hydroxycinnamate caffeic acid and its conjugate chlorogenic acid increase vitamin e and cholesterol concentrations in Sprague-Dawley rats

Vegetarian diets are correlated with a reduced risk of developing cardiovascular disease and comprise a great variety of bioactive compounds, including hydroxycinnamic acid derivatives. Therefore, this study aimed to identify dietary hydroxycinnamic acid derivatives that may alter two important factors related to the development of cardiovascular disease, namely, tocopherol (T) and cholesterol (C) concentrations in the body. The effects of caffeic acid (CA), chlorogenic acid (CGA), and ferulic acid (FA) on alpha-T, gamma-T, and C levels in blood plasma, liver, and lungs were investigated after these compounds had been fed to rats for 4 weeks at concentrations of 2 g/kg in semisynthetic diets. None of the regimens affected weight gain, feed intake, or absolute weights of livers and lungs, although CA increased the liver weight relative to the body weight (P < 0.05). CA- and CGA-fed animals showed a tendency toward sparing vitamin E in all tissues, but statistical significance was obtained only for gamma-T in the liver of CA-fed animals (P < 0.005) and for alpha-T in the lungs of CGA-treated rats (P < 0.05). CGA supplementation reduced concentrations of lipids in the lung tissue (P < 0.05). CA and CGA elevated the concentrations of C in liver tissue and lipids to a similar extent, but only CA decreased the ratio of high-density lipoprotein C to total C in blood plasma (P < 0.05 for all effects). Animals eating FA showed T and C values comparable to those in the control group. In conclusion, this study demonstrates that dietary caffeic and chlorogenic acid may elevate tocopherols and cholesterol in vivo.     

Gamma-ray flares from the Crab Nebula

A young and energetic pulsar powers the well-known Crab Nebula. Here, we describe two separate gamma-ray (photon energy greater than 100 mega-electron volts) flares from this source detected by the Large Area Telescope on board the Fermi Gamma-ray Space Telescope. The first flare occurred in February 2009 and lasted approximately 16 days. The second flare was detected in September 2010 and lasted approximately 4 days. During these outbursts, the gamma-ray flux from the nebula increased by factors of four and six, respectively. The brevity of the flares implies that the gamma rays were emitted via synchrotron radiation from peta-electron-volt (10(15) electron volts) electrons in a region smaller than 1.4 × 10(-2) parsecs. These are the highest-energy particles that can be associated with a discrete astronomical source, and they pose challenges to particle acceleration theory.     

Canine superficial bacterial folliculitis: Current understanding of its etiology,diagnosis and treatment

Superficial bacterial folliculitis (SBF) is more common in the dog than other mammalian species. Until recently, a successful outcome in cases of canine SBF was possible by administering a potentiated amoxicillin, a first generation cephalosporin or a potentiated sulfonamide. Unfortunately, this predictable susceptibility has changed, because methicillin resistant Staphylococcus pseudintermedius (MRSP) and Staphylococcus aureus (MRSA) are becoming more prevalent in canine SBF cases. The increasing frequency of multidrug resistance complicates the selection of antimicrobial therapy. Antimicrobial agents that were once rarely used in cases of canine SBF, such as amikacin, rifampicin and chloramphenicol, are becoming the drugs of choice, based on bacterial culture and susceptibility testing. Furthermore, changes in antimicrobial susceptibility have helped to re-emphasize the importance of a multimodal approach to treatment of the disease, including topical therapy. Due to the increasing frequency of identification of highly resistant Staphylococcus spp., topical antimicrobial therapy, including the use of diluted sodium hypochlorite (bleach), is becoming necessary to successfully treat some cases of canine SBF. Other important antiseptics that can be used include chlorhexidine, benzoyl peroxide, ethyl lactate, triclosan and boric acid/acetic acid. This review discusses the diagnostic and therapeutic management of canine SBF, with a special emphasis on treating methicillin resistant staphylococcal infections.     

Genetic correlations between production with disease resistance and fertility in dairy cattle and consequences for total merit selection

Twelve years of data from progeny‐test results of 1486 Swedish Red and White (SRB) and 756 Swedish Black and White (SLB) AI bulls were analysed to provide estimates of genetic correlations between yield of protein and three health‐ and fertility traits. For both breeds, the correlations were unfavourable (r_G= —0.13 to —0.37). The effects of negative genetic correlations (compared to a situation with genetic correlations with zero values) on the b‐values in the total merit index of bulls were rather small but the effects on the estimated genetic gain were large. The effect of not including health and fertility traits in the index, although included in the breeding goal, resulted in a 9–10% reduced accuracy of estimated breeding values for total merit and thus a corresponding loss in total gain.