An investigation of heparinase immobilization

A systematic investigation of the parameters that affect the efficiency of immobilizing heparinase onto cyanogen bromide activated crosslinked 8% agarose beads was conducted. Two experimental measures, the “fraction bound” and the “fraction retained,” were used to monitor the coupling efficiency. The fraction bound is the portion of the total initial enzyme that is bound to the agarose gel. The fraction retained is the fraction of bound enzyme that is active. The product of the two measures indicates the coupling efficiency. The activity of the immobilized heparinase was measured under conditions free of both internal and external mass transfer limitations, and thus, the fraction retained represents the true immobilized enzyme activity. Increasing the degree of activation of the beads results in an increase in the fraction bound, the fraction retained, and consequently, the coupling efficiency. As the ratio of enzyme solution to gel volume increases from 1.5 to 2.2, the fraction bound remains constant but the fraction retained decreases (heparinase concentration; 0.15 mg/mL and degree of activation; 9.5 μmol of cyanate esters/g of gel). At volume ratios greater than 2.2, both the fraction bound and the fraction retained decline continuously. Changing the heparinase concentration in the coupling solution changes the coupling efficiency in a manner similar to that of the volume ratio change. When heparin is added during the coupling process, the fraction bound declines as the heparin concentration increases, whereas the fraction retained increases up to a heparin concentration of 12 mg/mL and decreases thereafter. When arginine, lysine, and glycine are used to block the unreacted cyanate ester groups after the coupling process, the immobilized heparinase shows different pH optima of 6.5, 6.9, and 7.2, respectively. Based upon these findings, a protocol to optimize heparinase immobilization is developed.     

On the 6-exo-trig ring closure of substituted 5-hexen-1-oxyl radicals

Substituted 5-hexen-1-oxyl radicals have been generated from N-(5-hexen-1-oxy)-5-(p-methoxyphenyl)-4-methylthiazole-2(3H)-thiones under tin-free conditions and have been successfully applied as reactive intermediates in a mechanistic study on the formation of bromomethyl-substituted tetrahydropyrans via 6-exo-trig selective cyclizations.     

Raman and cathodoluminescence spectroscopic investigations on Permian fossil wood from Chemnitz--a contribution to the study of the permineralisation process

Samples of different three-dimensionally preserved fossil plants (Medullosa sp., Dadoxylon sp., Calamodendron striatum, Psaronius sp.) from the Lower Permian petrified forest of Chemnitz were examined with regard to their chemical composition and structural order. Raman spectroscopy and cathodoluminescence microscopy are shown to be powerful tools for such investigations. Silicified wood from Chemnitz-Hilbersdorf generally shows yellow cathodoluminescence (CL) of the cell walls and only weak yellow-brownish CL of the cell lumina. By time-resolved cathodoluminescence spectroscopy, a secondary mineralisation of hydrothermal origin was recognized. The latter is shown by short-lived blue CL at the cell walls extinguishing the yellow signal. Therefore, after the primary silicification step a secondary mineralisation step initiated by hydrothermal processes, seems to have taken place at probably slightly higher temperatures. The resulting silica matrix consists of phanerocrystalline and microcrystalline alpha-quartz as well as microcrystalline moganite, both partially associated with iron oxides. Dadoxylon sp. is a prominent example for parallel permineralisation by alpha-quartz and fluorspar, which is outstanding for the Chemnitz Petrified Forest. CL on this samples shows parallel silicification and fluoritisation, followed by infiltration of iron oxides. Permineralised samples show very low percentage of original organic remains. The seed fern Medullosa, for example, shows dispersed carbon, which is mainly restricted to the centres of the typical star-shaped vascular bundles. Raman spectroscopy revealed that these carbonaceous particles are of an anthracite structure. For experimental confirmation coal samples of different rank, especially anthracite from different geological times and localities, were studied by means of Raman spectroscopy. The remaining pith of the vascular bundles is white-coloured and consists of alpha-quartz and moganite, whereas surrounding tracheides exhibit white and reddish coloured parts. The reddish parts, mainly found in the rays, additionally contain alpha-Fe2O3 and Fe3O4 among the SiO2 polymorphs of alpha-quartz and moganite. Sometimes iron oxides could have dominated permineralisation processes as the peak intensities of distinct parts of the samples suggest.     

Theoretical treatment of first-order reversible reactions occurring in a chromatographic reactor, on the basis of consecutive reactions

Summary Analytical peak-shape equations were derived for first-order reversible reactions occurring in a chromatographic reactor by treating the reversible reactions as consecutive reactions with alternating products. The results of the analytical peak-shape equations were compared with those from a numerical solution of the partial differential equation system modeling the chromatographic reactor. For small to medium conversions the correspondence was found to be sufficiently close to enable substitution of the numerical solution in fitting procedures for the determination of rate constants. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Direct identification of all oncogenic mutants in KRAS exon 1 by cycling temperature capillary electrophoresis

Over the past few decades, advances in genetics and molecular biology have revolutionized our understanding of cancer initiation and progression. Molecular progression models outlining genetic events have been developed for many solid tumors, including colon cancer. Previous reports in the literature have shown a relationship between different KRAS mutations and prognosis and response to medical treatment in colon cancer patients. Furthermore, the presence of a mutated KRAS has been correlated with different clinicopathological variables including age and gender of patients and tumor location. To our knowledge, few institutions screen for KRAS mutations on regular basis in colon cancer patients despite such evidence that knowledge of KRAS exon 1 status is informative. Here, we report on a mutation analysis method adapted to a 96-capillary electrophoresis instrument that allows identification of all 12 oncogenic mutations in KRAS exon 1 under denaturing conditions. To determine the optimal parameters, a series of DNA constructs generated by site-directed mutagenesis was analyzed and the migration times of all mutant peaks were measured. A classification tree was then made based on the differences in migration time between the mutants and an internal standard. A randomized series of 500 samples constructed with mutagenesis as well as 60 blind samples from sporadic colon carcinomas was analyzed to test the method. No wild-type samples were scored as mutants and all mutants were correctly identified. Post polymerase chain reaction (PCR) analysis time of 96 samples was performed within 40 min.     

2‐(4‐Hydroxy­phenyl)‐4,4‐dimethyl‐2‐oxazoline: X‐ray and density functional theory study

In the crystal structure of the title compound, C₁₁H₁₃NO₂, there are strong inter­molecular O—H⋯N hydrogen bonds which, together with weak intra­molecular C—H⋯O hydrogen bonds, lead to the formation of infinite chains of mol­ecules, held together by weak inter­molecular C—H⋯O hydrogen bonds. A theoretical investigation of the hydrogen bonding, based on density functional theory (DFT) employing periodic boundary conditions, is in agreement with the experimental data. The cluster approach shows that the influence of the crystal field and of hydrogen‐bond formation are responsible for the deformation of the 2‐oxazoline ring, which is not planar and adopts a ⁴T₃ (^C3T_C2) conformation.     

Enhancing the effectiveness of virtual screening by fusing nearest neighbor lists: a comparison of similarity coefficients

This paper evaluates the effectiveness of various similarity coefficients for 2D similarity searching when multiple bioactive target structures are available. Similarity searches using several different activity classes within the MDL Drug Data Report and the Dictionary of Natural Products databases are performed using BCI 2D fingerprints. Using data fusion techniques to combine the resulting nearest neighbor lists we obtain group recall results which, in many cases, are a considerable improvement on standard average recall values obtained for individual structures. It is shown that the degree of improvement can be related to the structural diversity of the activity class that is searched for, the best results being found for the most diverse groups. The group recall of active compounds using subsets of the class is also investigated: for highly self-similar activity classes, the group recall improvement saturates well before the full activity class size is reached. A rough correlation is found between the relative improvement using the group recall and the square of the number of unique compounds available in all of the merged lists. The Tanimoto coefficient is found unambiguously to be the best coefficient to use for the recovery of active compounds using multiple targets. Furthermore, when using the Tanimoto coefficient, the "MAX" fusion rule is found to be more effective than the "SUM" rule for the combination of similarity searches from multiple targets. The use of group recall can lead to improved enrichment in database searches and virtual screening.     

Application of flow-injection analysis in the on-line monitoring of sugars,lactic acid,protein and biomass during lactic acid fermentations

A laboratory system for the on-line monitoring of important lactic acid fermentation variables is described. The system contains flow-injection analysers for glucose, lactose, galactose, lactate and protein and a continuous-flow analyser for the biomass concentration. The sugar and lactate analysers are based on enzymatic reactions involving oxidases followed by chemiluminescence detection of the hydrogen peroxide formed. The protein analyser is based on the biuret reaction. The system has been used to monitor many fermentation experiments, and some results are presented as examples.