Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.     

An extension of the moment closure method

The moment closure method was recently shown in N?sell (Theor. Popul. Biol. 63 (2) (2003a) 159) to give asymptotic approximations of the first few cumulants for the stochastic logistic model. A slight extension of the method is introduced. It is shown to be robust with regard to the specific distributional assumption that is used to achieve moment closure. The phenomenon of spurious solutions is shown to be related to the domain of attraction of the non-spurious critical point.     

Heparin and calcium ions dramatically enhance antithrombin reactivity with factor IXa by generating new interaction exosites

Blood coagulation factor IXa has been presumed to be regulated by the serpin, antithrombin, and its polysaccharide activator, heparin, but it has not been clear whether factor IXa is inhibited by the serpin with a specificity comparable to that for thrombin and factor Xa or what determinants govern this specificity. Here we show that antithrombin is essentially unreactive with factor IXa in the absence of heparin (k(ass) approximately 10 M(-1) s(-1)) but undergoes a remarkable approximately 1 million-fold enhancement in reactivity with this proteinase to the physiologically relevant range (k(ass) approximately 10(7) M(-1) s(-1)) when activated by heparin in the presence of physiologic levels of calcium. This rate enhancement is shown to derive from three sources: (i) allosteric activation of antithrombin by a sequence-specific heparin pentasaccharide (300-500-fold), (ii) allosteric activation of factor IXa by calcium ions (4-8-fold), and (iii) heparin bridging of antithrombin and factor IXa augmented by calcium ions (130-1000-fold depending on heparin chain length). Mutagenesis of P6-P3' reactive loop residues of antithrombin further reveals that the reactivity of the unactivated inhibitor is principally determined by the P1 Arg residue, whereas exosites outside the loop which are present on the activated serpin and on heparin are responsible for heparin enhancement of this reactivity. These results together with our previous findings demonstrate that exosites are responsible for the unusual specificity of antithrombin and heparin for three clotting proteases with quite distinct substrate specificities.     

American foulbrood and African honey bees (Hymenoptera: Apidae)

We have taken samples of honey from individual beekeepers (N = 64), and of domestic (N = 35) and imported honey (N = 15) retailed in supermarkets in several sub-Saharan countries and cultivated these samples for Paenibacillus larvae subsp. larvae Heyndrickx et al. causing American foulbrood in honey bee colonies. The results are compared with samples of similar backgrounds and treated the same way but collected in Sweden (N = 35). No P. larvae subsp. larvae spores were found in any honey produced in Africa south of the Sahara although honey imported into this region frequently contains the pathogen. Swedish honey frequently contains P. larvae subsp. larvae spores although the general level of visibly infected bee colonies is low (roughly 0.5%). The results suggest that large parts of Africa may be free from American foulbrood. Behavioral studies (hygienic behavior) on Apis mellifera subsp. scutellata Lepeletier in Zimbabwe suggest that hygienic behavior of African bees could influence the apparent low level, or even absence of American foulbrood in large parts of Africa.     

Importance of lysine 125 for heparin binding and activation of antithrombin

The anticoagulant sulfated polysaccharide, heparin, binds to the plasma coagulation proteinase inhibitor, antithrombin, and activates it by a conformational change that results in a greatly increased rate of inhibition of target proteinases. Lys125 of antithrombin has previously been implicated in this binding by chemical modification and site-directed mutagenesis and by the crystal structure of a complex between antithrombin and a pentasaccharide constituting the antithrombin-binding region of heparin. Replacement of Lys125 with Met or Gln in this work reduced the affinity of antithrombin for full-length heparin or the pentasaccharide by 150-600-fold at I = 0.15, corresponding to a loss of 25-33% of the total binding energy. The affinity decrease was due both to disruption of approximately three ionic interactions, indicating that Lys125 and two other basic residues of antithrombin act cooperatively in binding to heparin, and to weakened nonionic interactions. The mutations caused a 10-17-fold decrease in the affinity of the initial, weak binding step of the two-step mechanism of heparin binding to antithrombin. They also increased the reverse rate constant of the second, conformational change step by 10-50-fold. Lys125 is thus a major heparin-binding residue of antithrombin, contributing an amount of binding energy comparable to that of Arg129, but less energy than Lys114. It is the first residue identified so far that has a critical role in the initial recognition of heparin by antithrombin, but also appreciably stabilizes the heparin-induced activated state of the inhibitor. These effects are exerted by interactions of Lys125 with the nonreducing end of the heparin pentasaccharide.     

Effects of Obesity and Weight Loss on Cardiac Function and Valvular Performance

KARASON, KRISTJAN, INGEMAR WALLENTIN, BO LARSSON, LARS SJOSTROM. Effects of obesity and weight loss on cardiac function and valvular performance. Obes Res. 1998;6:422–429. Objective : To study the consequences of long-standing obesity on myocardial function and valvular performance and to determine the effects of weight loss on these cardiovascular features. Research Methods and Procedures : We included 41 patients with obesity referred for weight-reducing gastroplasty, 31 patients with obesity who received dietary recommendations, and 43 lean subjects. Body weight and blood pressure were measured, and cardiac function and valvular performance were estimated echocardiographically. Left ventricular ejection fraction was used to assess systolic heart function, and the ratio of transmitral early to atrial (E/A) peak flow velocity was used as an estimate of diastolic filling. All three study groups were investigated at baseline, and the two groups with obesity were re-examined at 1-year follow-up. Results : Patients with obesity had higher blood pressure, greater cardiac output, lower ejection fraction, and reduced E/A ratio, compared with lean subjects (p<0.01). Surgical treatment of obesity led to significant decreases in body weight, whereas body weight remained unchanged in the group treated with dietary recommendations (p<0.001). In the weight loss group, blood pressure and cardiac output decreased and the E/A ratio increased (p<0.001). Left ventricular ejection fraction tended to increase in the weight loss group and decrease in the obese control group p<0.01). No significant valvular disease was observed in any of the subjects with obesity at baseline or after weight loss. Discussion : We conclude that weight reduction in subjects with obesity is associated with improvements in left ventricular diastolic filling and has favorable effects on left ventricular ejection fraction. Neither obesity nor weight loss seem to promote valvular heart disease.     

Continuous control of water activity during biocatalysis in organic media

Summary This paper describes a newly developed technique to adjust and control the water activity in enzymatic reactions in organic media. A saturated salt solution of known water activity is circulated inside a silicone tube, submerged into the reaction medium. The circulating solution can both absorb and release water. Water activity control during lipase catalyzed esterification was demonstrated with diisopropyl ether as solvent.     

25-Hydroxylation of vitamin D3 by a reconstituted system from rat liver microsomes.

Using isotope dilution—mass fragmentography as assay technique, it was shown that highly purified preparations of cytochrome P-450 from rat liver microsomes catalyzed 25-hydroxylation of vitamin D₃ when combined with NADPH-cytochrome P-450 reductase and a phospholipid. The rate of conversion was approximately linear with the amount of cytochrome P-450, and was considerably higher than the rate of conversion obtained with crude liver microsomes. The possibility is discussed that the microsomal fraction contains inhibitors of 25-hydroxylase activity, which may be of regulatory importance in vitamin D₃ metabolism.     

An improved histofluorescence procedure for freeze-dried paraffin-embedded tissue based on combined formaldehyde-glyoxylic acid perfusion with high magnesium content and acid pH

Summary A technique is described for highly sensitive and precise visualization of central catecholamine systems in paraffin sections of freeze-dried tissue. The procedure is based on perfusion of the animal with a solution containing formaldehyde and/or glyoxylic acid, in the presence of a very high magnesium content (40 g MgSO₄/150 ml solution) and acid pH. The perfused tissue is rapidly frozen, freeze-dried, treated with formaldehyde vapours (at +80°C for 1h), embedded in paraffin in vacuo, and finally sectioned.The present technique has a sensitivity for the dopamine- and noradrenaline-containing systems that is comparable with that of the glyoxylic acid-Vibratome technique, which utilizes fresh, glyoxylic acid-perfused tissue. Thus, the preterminal axon pathways become fluorescent throughout their full extent and the several new terminal systems, discovered with the glyoxylic acid-Vibratome method, are well demonstrable. The method is also highly useful for the study of the cell bodies and their dendritic processes. The catacholamine fibre systems are visualized without any signs of diffusion and with a richness in detail. In animals pretreated with l-tryptophan and MAO-inhibitor the technique is also useful for studies on central indolamine-containing systems.