Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis.

Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.     

Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy

Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.     

Improved Cassava Starch by Antisense Inhibition of Granule-bound Starch Synthase I

Cassava is a poor man's crop which is mainly grown as a subsistence crop in many developing countries. Its commercial use was first as animal feed (also known as tapioca), but has shifted since the late sixties to a source of native starch. The availability of native starches, which on the one hand do not require substantial chemical derivatisation and on the other hand have improved properties, would make cassava also for small farmers a potentially attractive cash crop. Since breeding is difficult in this polyploid, vegetatively propagated, crop a transgenic approach would be ideal to improve certain characteristics. We have created a cassava genotype producing amylose-free starch by genetic modification. The absence of amylose increased the clarity and stability of gels made with the transgenic starch, without requiring treatment with environment-unfriendly chemicals such as epoxides (propylene oxide, ethylene oxide) and acetic anhydride, which are normally used to improve stability. The amylose-free starch showed no changes in particle size distribution, chain length distribution or phosphorous content when compared to amylose-containing starch, but the granule melting temperature was increased by almost 2°C. Furthermore, the amylose-free cassava starch shows enhanced clarity and stability properties. These improved functionalities are desired in technical applications in paper and textile manufacturing, but also in the food industry for the production of sauces, dairy products and noodles.     

Custom chemical microarray production and affinity fingerprinting for the S1 pocket of factor VIIa

The goal of this study was to explore the applicability of surface plasmon resonance (SPR)-based fragment screening to identify compounds that bind to factor VIIa (FVIIa). Based on pharmacophore models virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa and immobilized these compounds on microarrays. In affinity fingerprinting experiments, a number of compounds were identified to be specifically interacting with FVIIa and shown to fall into four structural classes. The results demonstrate that the chemical microarray technology platform using SPR detection generates unique chemobiological information that is useful for de novo discovery and lead development and allows the detection of weak interactions with ligands of low molecular weight.     

Quantifying BSE control by calculating the basic reproduction ratio R 0 for the infection among cattle

The safety of using meat and bone meal (MBM) in mammal feed was studied in view of BSE, by quantifying the risk of BSE transmission through different infection routes. This risk is embodied in the basic reproduction ratio R ₀ of the infection, i.e. the average number of new infections induced by one initial infection. Only when R ₀ is below 1, will the disease die out with certainty and the population will become free from BSE. Unfortunately this is a slow process due to the slow progression of the disease. We calculate R ₀ explicitly from basic ingredients taking several different transmission routes into account. Several of the basic ingredients are functions of age or of infection-age. We also calculate the exponential growth rate r in terms of the same basic ingredients. Next we quantify the ingredients from available data and compute the effects on R ₀ of various scenarios for controlling BSE, with examples for the UK and the Netherlands. This revised version was published online in November 2003. A correction was made to formula 10 of this paper, indicies were previously printed in an incorrect order and an extraneous element has been removed.     

Quantification of left ventricular mechanical dyssynchrony by conductance catheter in heart failure patients

Mechanical dyssynchrony is an important codeterminant of cardiac dysfunction in heart failure. Treatment, either medical, surgical, or by pacing, may improve cardiac function partly by improving mechanical synchrony. Consequently, the quantification of ventricular mechanical (dys)synchrony may have important diagnostic and prognostic value and may help to determine optimal therapy. Therefore, we introduced new indexes to quantify temporal and spatial aspects of mechanical dyssynchrony derived from online segmental conductance catheter signals obtained during diagnostic cardiac catheterization. To test the feasibility and usefulness of our approach, we determined cardiac function and left ventricular mechanical dyssynchrony by the conductance catheter in heart failure patients with intraventricular conduction delay (n = 12) and in patients with coronary artery disease (n = 6) and relatively preserved left ventricular function. The heart failure patients showed depressed systolic and diastolic function. However, the most marked hemodynamic differences between the groups were found for mechanical dyssynchrony, indicating a high sensitivity and specificity of the new indexes. Comparison of conductance catheter-derived indexes with septal-to-lateral dyssynchrony derived by tissue-Doppler velocity imaging showed highly significant correlations. The proposed indexes provide additional, new, and quantitative information on temporal and spatial aspects of mechanical dyssynchrony. They may refine diagnosis of cardiac dysfunction and evaluation of interventions, and ultimately help to select optimal therapy.