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Chiral cyclic β-hydroxy ketones represent key motifs in the production of natural products of biological interest. Although the molecules are structurally simple, they require cumbersome synthetic steps to get access to them and their synthesis remains a challenge in organic chemistry. In this report, we describe a straightforward approach to enantiomerically enriched (R)- and (S)-3-hydroxycyclopentanone 2a, (R)- and (S)-3-hydroxycyclohexanone 2b, and (R)- and (S)-3-hydroxycycloheptanone 2c involving a transesterification resolution of the racemates using whole cells of marine microorganisms as catalysts and vinyl acetate the acyl donor and solvent. Twenty-six strains from a wide collection of isolates from marine sediments were screened, and seven strains were found to markedly catalyze the resolution in an asymmetric fashion. Using the strain Serratia sp., (R)-2a was isolated in 27% yield with 92% ee and (S)-2a in 65% yield with 43% ee, corresponding to an E-value of 37; (R)-2b was isolated in 25% yield with 91% ee and (S)-2b in 67% yield with 39% ee, corresponding to an E-value of 40; and (R)-2c was isolated in 30% yield with 96% ee and (S)-2c in 63% yield with 63% ee, corresponding to an E-value of 75.

The first enantioselective synthesis was the selective addition of cyanide to benzaldehyde catalysed by a hydroxynitrile lyase (HNL). Since then these enzymes have been developed into a reliable tool in organic synthesis. HNLs to prepare either the (R)- or the (S)-enantiomer of the desired cyanohydrin are available and a wide variety of reaction conditions can be applied. As a result of this, numerous applications of these enzymes in organic synthesis have been described. Here the examples of the last decade are summarised, the enzyme catalysed step is discussed and the follow-up chemistry is shown. This proves HNLs to be part of main stream organic synthesis. Additionally the newest approaches via immobilisation and reaction engineering are introduced.

Water addition to carbon-carbon double bonds using hydratases is attracting great interest in biochemistry. Most of the known hydratases are involved in primary metabolism and to a lesser extent in secondary metabolism. New hydratases have recently been added to the toolbox, both from natural sources or artificial metalloenzymes. In order to comprehensively understand how the hydratases are able to catalyse the water addition to carbon-carbon double bonds, this review will highlight the mechanistic and stereochemical studies of the enzymatic water addition to carbon-carbon double bonds, focusing on the syn/anti-addition and stereochemistry of the reaction., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The enantioselective Michael addition using water as both nucleophile and solvent has to date proved beyond the ability of synthetic chemists. Herein, the direct, enantioselective Michael addition of water in water to prepare important β-hydroxy carbonyl compounds using whole cells of Rhodococcus strains is described. Good yields and excellent enantioselectivities were achieved with this method. Deuterium labeling studies demonstrate that a Michael hydratase catalyzes the water addition exclusively with anti-stereochemistry., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal-dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg(-1) at pH 6 with (R)-mandelonitrile, and synthesized (R)-mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether., (© 2013 FEBS.)

Rhodococcus rhodochrous ATCC 17895 possesses an array of mono- and dioxygenases, as well as hydratases, which makes it an interesting organism for biocatalysis. R. rhodochrous is a Gram-positive aerobic bacterium with a rod-like morphology. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 6,869,887 bp long genome contains 6,609 protein-coding genes and 53 RNA genes. Based on small subunit rRNA analysis, the strain is more likely to be a strain of Rhodococcus erythropolis rather than Rhodococcus rhodochrous.

Hydroxynitrile lyases are a versatile group of enzymes that are applied both in the laboratory and on an industrial scale. What makes them particularly interesting is that to date five structurally unrelated categories of hydroxynitrile lyases have been discovered. Given their great importance they have often been immobilised utilising many different methodologies. Therefore the hydroxynitrile lyases are ideally suited to compare different immobilisation methods and their dependence on the structural features of the enzyme in question, since the activity is the same in all cases. This review examines all the different immobilisation methods applied to hydroxynitrile lyases and draws conclusions on the effect of the approach.

The base is a key factor in aldol reactions in organic media, determining the selectivity. Here, we describe a tetrahedral phenylboronate salt as a mild non-nucleophilic base that is able to catalyse the aldol reaction and significantly decrease the formation of undesired elimination products.

To enhance the activity of transketolase towards nonphosphorylated substrates and enlarge the scope of its substrates, notably to long polyol aldehyde acceptors (D-ribose or D-glucose), a rational design-supported evolution strategy was applied. By using docking experiments, an in silico library, and iterative mutagenesis, libraries of single- and double-point mutants were designed and generated. A double-screening approach was implemented, coupling a preselection activity assay (HPLC method) and a selective assay (GC method) to find the best enzymes. Several mutants (R526N, R526Q, R526Q/S525T, R526K/S525T) showed improved activities towards nonphosphorylated substrates as the coupled products of lithium hydroxypyruvate (HPA) with glycolaldehyde (GO), D-ribose or D-glucose. These mutated enzymes were further characterised. They were shown to be up to four times more active than the wild-type (mutant R526Q/S525T) for nonphosphorylated substrates LiHPA/GO (V(m) /K(m) for LiHPA = 92.4 instead of 28.8×10(-3) min(-1) for the wild-type) and 2.6 times more active for substrates LiHPA/rib., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Different acidic ionic liquids (ILs; namely, 1-methylimidazolium tetrafluoroborate, 1-methylimidazolium trifluoroacetate, N-methyl-2-pyrrolidone hydrogen sulfate, N,N,N-trioctyl-n-butanesulfonic acid ammonium hydrogen sulfate, 1-methyl-3-(3-sulfobutyl)imidazolium hydrogen sulfate) and basic ILs (namely, 1,1,3,3-tetramethylguanidinium lactate and choline hydroxide) were tested as catalysts for the aldol reaction. The choline hydroxide catalysed reaction gave high yield (94.3%) and selectivity of the 4-hydroxy-4-phenylbutan-2-one after a short reaction time (15 min) at 0 °C. This article demonstrates the potential of choline hydroxide, which is a derivative of choline and a naturally occurring water-soluble essential nutrient, as a highly active and selective green catalyst., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The production of renewable chemicals is gaining attention over the past few years. The natural resources from which they can be derived in a sustainable way are most abundant in sugars, cellulose and hemicellulose. These highly functionalized molecules need to be de-functionalized in order to be feedstocks for the chemical industry. A fundamentally different approach to chemistry thus becomes necessary, since the traditionally employed oil-based chemicals normally lack functionality. This new chemical toolbox needs to be designed to guarantee the demands of future generations at a reasonable price. The surplus of functionality in sugars and glycerol consists of alcohol groups. To yield suitable renewable chemicals these natural products need to be defunctionalized by means of dehydroxylation. Here we review the possible approaches and evaluate them from a fundamental chemical aspect., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Water is the liquid of life. Nature has therefore evolved countless enzymes that catalyse the addition of water to C=C bonds, isolated or conjugated. These reactions are regio- and enantioselective, they are part of primary metabolism as well as the secondary metabolism. The enzymes that catalyse these reactions (hydratases or hydro-lyases) are applied industrially in selected cases. However, they are not generally used in the laboratory although they outperform all currently available catalytic chemical methodologies. This feature article highlights the potential that hydratases have for chemistry compared to the acid catalysed addition of water.

Bimetallic three-dimensional amorphous mesoporous materials, Al-Zr-TUD-1 materials, were synthesised by using a surfactant-free, one-pot procedure employing triethanolamine (TEA) as a complexing reagent. The amount of aluminium and zirconium was varied in order to study the effect of these metals on the Brønsted and Lewis acidity, as well as on the resulting catalytic activity of the material. The materials were characterised by various techniques, including elemental analysis, X-ray diffraction, high-resolution TEM, N(2) physisorption, temperature-programmed desorption (TPD) of NH(3), and (27) Al MAS NMR, XPS and FT-IR spectroscopy using pyridine and CO as probe molecules. Al-Zr-TUD-1 materials are mesoporous with surface areas ranging from 700-900 m(2) g(-1), an average pore size of around 4 nm and a pore volume of around 0.70 cm(3) g(-1). The synthesised Al-Zr-TUD-1 materials were tested as catalyst materials in the Lewis acid catalysed Meerwein-Ponndorf-Verley reduction of 4-tert-butylcyclohexanone, the intermolecular Prins synthesis of nopol and in the intramolecular Prins cyclisation of citronellal. Although Al-Zr-TUD-1 catalysts possess a lower amount of acid sites than their monometallic counterparts, according to TPD of NH(3), these materials outperformed those of the monometallic Al-TUD-1 as well as Zr-TUD-1 in the Prins cyclisation of citronellal. This proves the existence of synergistic properties of Al-Zr-TUD-1. Due to the intramolecular nature of the Prins cyclisation of citronellal, the hydrophilic surface of the catalyst as well as the presence of both Brønsted and Lewis acid sites synergy could be obtained with bimetallic Al-Zr-TUD-1. Besides spectroscopic investigation of the active sites of the catalyst material a thorough testing of the catalyst in different types of reactions is crucial in identifying its specific active sites., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Michael addition of water is a major challenge. Here an enzymatic approach is described. Interestingly, the enzyme (MhyADH) does not only catalyse the Michael addition of water but also the in situ oxidation of the product.

Water concentration affects both the enantioselectivity and activity of enzymes in dry organic media. Its influence has been investigated using the hydrocyanation of benzaldehyde catalyzed by hydroxynitrile lyase cross-linked enzyme aggregate (MeHNL-CLEA) as a model reaction. The enzyme displayed higher enantioselectivity at higher water concentration, thus suggesting a positive effect of enzyme flexibility on selectivity. The activity increased on reducing the solvent water content, but drastic dehydration of the enzyme resulted in a reversible loss of activity.

The three-dimensional sponge-like mesoporous material TUD-1 is straightforward to prepare. Its synthesis can readily be modified to introduce metals into the framework of TUD-1, imparting many different catalytic activities. M-TUD-1 catalysts have proven to be very active, unlimited by diffusion and very stable. By combining two metals into one TUD-1 catalyst, synergy between Lewis and Brønsted acid sites could be induced; incorporation of zeolites similarly gave rise to synergy. In addition to successful applications in redox-, acid- and photo-catalysis TUD-1 proved to be an excellent carrier material for catalysts, enabling new applications. TUD-1 was used as a contrast agent and drug delivery system, indicating that this material is but at the beginning of its potential applications.

Enzymes are versatile catalysts in the laboratory and on an industrial scale. To broaden their applicability in the laboratory and to ensure their (re)use in manufacturing the stability of enzymes can often require improvement. Immobilisation can address the issue of enzymatic instability. Immobilisation can also help to enable the employment of enzymes in different solvents, at extremes of pH and temperature and exceptionally high substrate concentrations. At the same time substrate-specificity, enantioselectivity and reactivity can be modified. However, most often the molecular and physical-chemical bases of these phenomena have not been elucidated yet. This tutorial review focuses on the understanding of enzyme immobilisation.

Synergy between Brønsted acid sites and Lewis acid sites in mesoporous Al-Zr-TUD-1 was demonstrated to exist in Brønsted acid catalysed reactions, but not in Lewis acid catalysed reactions.

A three-dimensional, mesoporous, silicate containing zirconium, Zr-TUD-1, was synthesized by a direct hydrothermal treatment method with triethanolamine as a complexing and templating reagent to ensure that zirconium was incorporated as isolated atoms. The mesoporosity of Zr-TUD-1 was confirmed by X-ray diffraction (XRD), N(2) sorption and high-resolution transmission electron micrograph (HR-TEM) studies. The nature and strength of the Lewis acid sites present in Zr-TUD-1 were evaluated by FTIR studies of pyridine adsorption and temperature-programmed desorption of ammonia. FTIR, X-ray photoelectron spectroscopic (XPS) and UV/Vis spectroscopic studies showed that, at Si/Zr ratios of 25 and higher, all the zirconium was tetrahedrally incorporated into the mesoporous framework, while at low Si/Zr ratios, a small part of the zirconium was present as ZrO(2) nanoparticles. Zr-TUD-1 is a Lewis acidic, stable and recyclable catalyst for the Meerwein-Ponndorf-Verley (MPV) reaction and for the Prins reaction.

Fatty acid desaturases catalyze the introduction of double bonds at specific positions of an acyl chain and are categorized according to their substrate specificity and regioselectivity. The current understanding of membrane-bound desaturases is based on mutant studies, biochemical topology analysis, and the comparison of related enzymes with divergent functionality. Because structural information is lacking, the principles of membrane-bound desaturase specificity are still not understood despite of substantial research efforts. Here we compare two membrane-bound fatty acid desaturases from Aspergillus nidulans: a strictly monofunctional oleoyl-Delta12 desaturase and a processive bifunctional oleoyl-Delta12/linoleoyl-omega3 desaturase. The high similarities in the primary sequences of the enzymes provide an ideal starting point for the systematic analysis of factors determining substrate specificity and bifunctionality. Based on the most current topology models, both desaturases were divided into nine domains, and the domains of the monofunctional Delta12 desaturase were systematically exchanged for their respective corresponding matches of the bifunctional sister enzyme. Catalytic capacities of hybrid enzymes were tested by heterologous expression in yeast, followed by biochemical characterization of the resulting fatty acid patterns. The individual exchange of two domains of a length of 18 or 49 amino acids each resulted in bifunctional Delta12/omega3 activity of the previously monofunctional parental enzyme. Sufficient determinants of fatty acid desaturase substrate specificity and bifunctionality could, thus, be narrowed down to a membrane-peripheral region close to the catalytic site defined by conserved histidine-rich motifs in the topology model.

Catalytic cascade reactions increase atom efficiency and help to reduce waste. Therefore they have recently attracted considerable attention. When performing them we have experienced that the immobilisation of the catalyst and the nature of the support are crucial factors. The carriers can play various roles from facilitating filtration and compartmentalisation to specifically binding unwanted compounds in the reaction media. In this Feature Article we give an overview of our work on such carrier dependent cascade reactions.

Ab initio density functional theory molecular dynamics simulations of the solvated states of the hydroxyl radical and hydroxide ion are performed using the Becke-Lee-Yang-Parr (BLYP) exchange-correlation functional (Becke, A. D. Phys. Rev. A 1988, 38, 3098. Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785). The structures of the solvation shells of the two species are examined. It is found that the OH radical forms a relatively well-defined solvation complex with four neighboring water molecules. Three of these molecules are hydrogen bonded to the OH, while the fourth is hemibonded via a three-electron two-centered bond between the oxygen atoms of the OH and water. The activity and the diffusion mechanism of the OH radical in water is discussed in comparison with the OH- ion. Although the results are partially influenced by the tendency of the BLYP density functional to overestimate hemibonded structure, the present simulations suggest that the widely accepted picture of rapid diffusion of OH radical in water through hydrogen exchange reaction may need to be reconsidered.

The enantioselective synthesis of C-C bonds is often the pivotal step of a synthesis. Nature has made a variety of versatile enzymes available that catalyse this type of reaction very selectively under mild conditions. Cyanohydrins, acyloins (alpha-hydroxy ketones), alpha-hydroxy acids and aldols (beta-hydroxy ketones) are very efficiently synthesised enantioselectively with the aid of C-C bond forming enzymes, which we discuss in this tutorial review. In the case of the alpha-hydroxy acids the applications of nitrilases in a synthetic dkr even allows a disconnection that has no enantioselective chemical equivalent.

A practical racemisation-epimerisation method for chiral secondary alcohols has been developed. Meerwein-Ponndorf-Verley-Oppenauer catalysts such as neodymium(III) isopropoxide are able to racemise these alcohols with retention of other stereocentres in the molecule. This is particularly useful for the recycling of the undesired products of kinetic resolutions of alcohols. By combination of such a racemisation with an acylation using isopropenyl or ethoxyvinyl esters as acyl donors, a fast straightforward recycling of starting materials may be achieved. The combined epimerisation and acylation process is demonstrated for the steroid estradiol methyl ether.

Commonly, hydrolase-catalysed reactions are used for kinetic resolutions of alcohols, amines and acids. Only a few applications in total syntheses have been described. Most of these examples are for the synthesis of amides and peptides. Here, the synthesis of all the different types of activated acids that can be applied in hydrolase-catalysed acylations is described. The application of these activated acids for both the formation of esters and amides is discussed and the different reactions are compared with the equivalent chemical reactions.

We have used directed two-component self-assembly to "pattern" organic monolayers on the nanometer scale at the liquid/solid interface. The ability of the scanning tunneling microscope to investigate structural details in these adlayers was used to gain insight into the two-component two-dimensional phase behavior. The components are symmetrically alkylated bisurea derivatives (R1-urea-spacer-urea-R2; R1, R2=alkyl, spacer=alkyl or bisthiophene). The bisthiophene unit acts as a marker and its bisurea derivative (T2) is a component in all the mixtures investigated. By varying the position of the hydrogen-bond forming urea groups along the molecule and the length of the alkyl chains of the other components, the effect of 1) hydrogen bonding, 2) molecule length, 3) odd-even effects, and 4) shape complementarity on the two-dimensional phase behavior was investigated. Insight into the effect of these parameters leads to the control of the two-dimensional patterning: from randomly intermixed systems to phase separation.

Critical care testing in a point of care (POC) setting can be very demanding for the laboratory. Lack of continuous monitoring of the normal functioning of POC instruments leads to late response to technical problems, and frequently results in more comprehensive interventions and reduced instrument availability. As most POC instruments lack adequate data transfer capabilities, manual data entry into the medical records with a high error rate is a component of POC programs. To reduce this data handling drawback of POC testing, our hospital opted for the replacement of the existing analysers with network-ready, expandable analysers (ABL700), linked to an integrated management system (RADIANCE). This new set-up enabled us to have continuous instrument control and maximal data management. The implementation of the integrated management system was well accepted by the operators. The network connectivity has led to real time technical support while reducing the workload by automating data collection.

We evaluated a direct assay for the determination of LDL-cholesterol (LDL-C) L-Type assay, Wako Pure Chemicals in two laboratories. This assay is applicable to most random access clinical chemistry analyzers, allowing full automation. Between-run coefficient of variation (NCCLS EP5) varied between 1.29% and 3.13% and thus met the National Cholesterol Education Program (NCEP) goal. The assay was considered linear over a physiologically relevant range of LDL-C, 2.22 to 7.04 mmol/l (NCCLS EP6). Method comparison yielded identical results at both evaluation sites for LDL-C when assayed with the direct method. LDL-C results obtained with the homogeneous method under investigation (y) differed significantly from values from density-gradient ultracentrifugation (x) according to Chung (y = 0.87x + 0.43 mmol/l, s(yx) = 0.38 mmol/l, r = 0.91). With the latter method as a reference method, mean bias was 3.16% meeting the NCEP criteria. Diagnostic performance was excellent at a clinically relevant cut-off level of 3.37 mmol/l. Results of the direct method (y) and the commonly used Friedewald formula (x) were highly correlated (s(yx) = 0.22 mmol/l, r = 0.97), but both slope and intercept differed significantly from one and zero respectively (y = 0.90x + 0.37 mmol/l). Bilirubin, hemolysis and ascorbate did not interfere; triglycerides did not cause clinically relevant interference below 11.3 mmol/l. The direct method we investigated is user-friendly and provides an improvement in the determination of LDL-C in routine laboratories.

The DNA hexamer d(CACGPG), in which dP is the ambivalent pyrimidine nucleoside analogue 2'-deoxy-beta-d-ribofuranosyl-(6H,8H-3, 4-dihydropyrimido[4,5-c][1,2]oxazin-7-one), crystallises as a left-handed Z-DNA duplex. X-ray analysis at 1.5 A shows that both P. A base-pairs are of the wobble type. This result appears inconsistent with other evidence from hybridisation and NMR studies of P-containing oligonucleotides, which suggests that, while P can form stable base-pairs with either A or G, thymine-like properties are more pronounced. Thermal denaturation experiments over a range of solution pH values indicate that protonation of the P.A base-pairs is unlikely to be responsible for the anomalous behaviour. No specific crystal packing effects can be identfied as an explanation, and it is concluded that base stacking and other interactions between nucleotide residues in Z-DNA are responsible., (Copyright 1998 Academic Press.)

Hydroquinone is a well known reagent used in the treatment of pigmentation disorders. The instability of the quinones and the required active concentration make topical treatment rather difficult. We tested the efficacy of an ascorbate-phytohydroquinone complex that inhibits the synthesis of melanin and promotes the degradation of the existing melanin. Lentigo senile lesions were evaluated before and after 1 month of treatment. Objective skin color evaluation was performed instrumentally. After one month of treatment, a clear depigmentation of the macules was measured. None of the volunteers reported any side effects from the prolonged treatment with the hydroquinone containing product.

The preservative properties of thyme essential oil (3%) with a known composition were evaluated in two types of final formulations, suitable for use as pharmaceutical or cosmetic vehicles, by means of a standard challenge test proposed by the latest European Pharmacopoeia. The required preservation efficacy criteria were satisfied against the bacterial strains, against the yeast in one of the formulations, but not against the mould strain involved in this study. Interactions between the essential oil compounds and other factors present in the final formulation might have influenced the activity of this essential oil, leading to an incomplete satisfaction of the criteria.

As indicated by in vitro experiments the penetration of irritants through the skin is significantly influenced by the temperature of the solution. In vivo experiments, demonstrated equally a significant influence of temperature in surfactant-induced skin irritation. In order to evaluate the irritant potential of detergent solutions under normal user conditions, we used the hand/forearm immersion test. We compared 2 detergents with different anionic character in a repetitive immersion protocol (30 min immersion on 4 consecutive days). The solutions were tested at 2 temperatures (37 degrees C and 40 degrees C). The irritation was quantified by assessment of the stratum corneum barrier function (transepidermal water loss), skin redness (a* colour parameter) and skin dryness (capacitance method). Both detergents affected the integrity of the skin in a significant way. The anionic content as well as the temperature of the solutions were found to be determinative for the irritant potential, with a stronger response for higher anionic content and temperature, respectively.

Haptoglobin (Hp) is a haemoglobin-binding acute phase protein with three genetic types: Hp 1-1, Hp 2-1, Hp 2-2. We investigated 45 patients during the first 48 hours of acute myocardial infarction, and studied determinant factors and clinical correlates. Upon hospital admission, serum haptoglobin concentration was increased (1.95 +/- 0.94 g/l, mean +/- SD, P < 0.001) versus the reference population (0.97 +/- 0.46 g/l, n = 107), independent of haptoglobin type: 1.84 +/- 0.64 g/l (Hp 1-1, n = 11) (P < 0.01), 1.98 +/- 0.79 g/l (Hp 2-1, n = 25) (P < 0.001), 1.98 +/- 1.58 g/l (Hp 2-2, n = 9) (P < 0.001). Moreover, during the first hours of hospitalization, a temporal lowering of haptoglobin was observed suggesting acute haemolysis, independent of the haptoglobin type. Minimal serum haptoglobin was reached 9.6 +/- 5.8 hours after admission. The amplitude of the haptoglobin decrease correlated with initial serum haptoglobin (r = 0.78) and was more pronounced (P < 0.05) in men (0.53 +/- 0.57 g/l) than in women (0.18 +/- 0.17 g/l). Decrease of serum haptoglobin did not correlate with infarct size (based on creatine kinase-MB release). Out of the other acute phase proteins measured upon admission, only C-reactive protein was significantly increased (P < 0.05). During the next 36 hours, haptoglobin increased as a result of the acute phase response to myocardial injury. Our findings suggest that acute myocardial infarction is also preceded by an acute phase response, characterized by an initial high haptoglobin and followed by a temporal haptoglobin decrease due to haemolysis.

A recently introduced automated nephelometric immunoassay involving shell/core particles for determination of alpha-foetoprotein in serum and amniotic fluid was evaluated with the Behring Nephelometer analyser II. Method stability was good: reconstituted reagents and calibration curve were stable for at least one week. The intra-assay CV varied between 2.3% and 4.0%. The inter-assay CV varied between 3.5% and 4.6%. Samples with alpha-foetoprotein concentrations up to 273000 micrograms/l were analysed without high-dose "hook" effect after automatic dilution. No significant interference from haemoglobin, bilirubin, rheumatoid factors, or human anti-mouse antibodies was detected up to concentrations of 0.15 mmol/l haemoglobin, 268 mumol/l bilirubin, 470 int, units/l rheumatoid factor and a titre of 1/1000 human anti-mouse antibodies. Interference due to triacylglycerols depended on the size of triacylglycerol containing particles: for VLDL, interference did not occur up to triacylglycerol levels of 6.0 mmol/l, for chylomicrons interference was already noted at triacylglycerol levels of 1.0 mmol/l. Correlation with a commercial RIA (Kabi Pharmacia) was excellent both for serum (n = 65) and amniotic fluid (n = 100). The effect of the molecular variation of the carbohydrate moiety of alpha-foetoprotein on the test results was studied using concanavalin A affinity chromatography. The detection of both concanavalin A-reactive and concanavalin A-non-reactive alpha-foetoprotein was equivalent by both methods. Multimeric forms of alpha-foetoprotein were prepared by gel permeation chromatography. The effect of autopolymerization of alpha-foetoprotein on the nephelometric determination of alpha-foetoprotein was negligible. We conclude that latex-enhanced immunonephelometry is a rapid, practical, and reliable method for measuring alpha-foetoprotein in serum and amniotic fluid.

In an earlier study [Smith, Davies, Dodson & Moore (1995). Biochemistry, 34, 415-425] the crystal structure of the d(TGATCA)-nogalamycin complex was determined to 1.8 A and refined with PROLSQ to R = 19.5% against 4767 reflections with F> 1sigma(F). A low-temperature crystallographic study on this complex has now been performed. Native data collection at liquid-nitrogen temperature (120 K) improved the resolution to 1.4 A. The structure has now been refined against these new diffraction data in the resolution range 8-1.4 A using NUCLSQ, PROLSQ, SHELXL93 and X-PLOR, in order to determine to what extent the resulting DNA conformation and associated solvent structure would differ and to examine the suitability of these programs for the refinement of oligonucleotide structures. With the advent of more DNA-protein structure determinations, it is of interest to see how well the protein-refinement packages, PROLSQ and X-PLOR, and the small-molecule program, SHELXL93, are able to accommodate DNA. Comparisons are made between the dictionaries, weights and restraints used and the final models obtained from each program. Although the final R values, using all data in the resolution range 8.0-1.4 A, from PROLSQ (22.8%), SHELXL93 (R1 =21.7% after isotropic refinement) and X-PLOR (24.4%) are higher than the R value from the NUCLSQ refinement (21.2%), the root-mean-square deviations between the four final models are very small. Using this high-quality 8.0-1.4 A data set neither the dictionary nor the refinement program leave an imprint on the final fully refined complex. Likewise, the helical parameters and backbone conformation including sugar-puckering modes are not influenced by the refinement procedure used. Although a different number of water molecules is found in each refinement, varying from 62 (X-PLOR) to 86 (NUCLSQ), the first hydration sphere is well conserved in all four models.

More than 50 years of noninvasive studies of epidermal and sebaceous lipids has revealed a vast amount of information concerning secretion and regulation of SSLs. The first techniques developed (solvent extraction and cigarette paper) required very long experimental procedures to obtain valuable parameters; however, a great part of the knowledge on SSL production and regulation was obtained by these pioneers. Because of the difficulties encountered in these multistep procedures, applicability was restricted to a limited number of research centers. The sampling procedures in the newly developed techniques are significantly reduced and are followed by a fast and accurate evaluation. Moreover, the new sampling procedures permit us to study other aspects of sebum secretion, as demonstrated with the Sebutape method. When using photometric techniques an estimate of the SSL can be obtained within minutes (casual level measurements). After such a screening procedure, more standardized parameters can be accurately determined. Whatever the parameter under investigation, strictly controlled experimental procedures are required. Control of temperature and relative humidity of the experimental room, of the acclimatization periods, and of the volunteers during the collection periods is of capital importance. The studies discussed in this article clearly demonstrate the applicability of the new techniques not only for research and cosmetic purposes but also for clinical use.

One hundred and sixteen samples for toxicological analysis (78 urines and 38 stomach contents) were analysed both with the Bio-Rad REMEDi Drug Profiling System and with a set of immunoassays and standard chromatographic procedures (HPLC, GC/NPD and GC/MS). As a screening method, REMEDi performed only moderately well, compared with immunoassays. Amphetamines and tricyclic antidepressants were detected by REMEDi in 80% and 68%, respectively, of the positive immunoassay results. Positive immunoassay screening tests were more often confirmed by the conventional chromatographic methods (80%) than by REMEDi (35%). The highest confirmation score for REMEDi was observed in the amphetamine group (9 of 12), while low scores were obtained for barbiturates (1 of 7) and benzodiazepines (13 of 75). However, in these samples REMEDi detected 53 additional compounds, which were neither reactive in the immunoassay screening tests, nor detected by the standard chromatographic procedures. These results clearly demonstrate that REMEDi can be a useful complementary technique in the clinical toxicology laboratory, since it expands the range of drugs covered by the immunoassays and provides a rapid, preliminary report in emergency situations.

The Arabidopsis thaliana 2 S albumins are examples of vacuolar proteins which undergo intensive posttranslational processing. An in vitro processing assay to screen for processing enzymes present in seeds was developed using an in vitro synthesized 2 S albumin precursor as the substrate. A protease was characterized which cleaved the substrate into two fragments with molecular weights (as determined from their migration distance on SDS-polyacrylamide gel) corresponding to those of the small and large subunits of Arabidopsis 2 S albumin. The pH optimum of this protease activity, its inhibition by pepstatin A, and partial sequence data led to the conclusion that the protease under study was an aspartic proteinase. Synthetic peptides representing two 2 S albumin propeptides allowed the determination of the in vitro cleavage sites, and suggested that the protease activity is capable, in vitro, of cleaving the amino-terminal propeptide as well as the internal propeptide linking the two subunits. Alterations of the amino acids in and around the cleavage sites, made to study the specificity of the protease activity, suggest that both structural and sequence determinants are important in cleavage site recognition.

Procedures are described for the quantitative, semi-preparative isolation of individual cyanobacterial heterocyst-type glycolipids (HGs) by reverse-phase HPLC (RP-HPLC) and the modifications to conventional techniques necessary to prevent significant HG losses during sample preparation. Total lipids are obtained from cultures of nitrogen-fixing cyanobacteria by triplicate extraction with 200 packed-cell volumes of chloroform/methanol (1/1, v/v), filtered, and redissolved in chloroform for loading onto a short disposable column of acid-washed silica. After removal of neutral lipids, pigments, and the majority of monogalactosyldiacylglycerol with chloroform and chloroform/acetone, HGs are eluted along with other complex lipids in methanol. The complex lipids are then fractionated by TLC and the HGs isolated as two classes of differing mobilities. The individual components of each class are then resolved by isocratic elution with methanol/water (91/9, v/v) from a C18 RP-HPLC column with refractive index detection. Samples of up to approximately 1.0 mg lipid can be completely separated and the major components isolated in high purity from a single run. Structural studies on the major HG of Nostoc azollae show it to be the glycosylated hexacosane-1,3,25-triol found by others in Anabaena cylindrica.

Procedures are described for the rapid and quantitative analysis of cyanobacterial heterocyst-type glycolipids (HGs) by normal-phase HPLC of their per-O-benzoylated derivatives. Total lipids are obtained from 1 ml of nitrogen-fixing cyanobacterial culture by triplicate extraction with chloroform/methanol, 1/1 (v/v), and the HGs are isolated from other complex lipids by preparative silica gel TLC. A C18 solid-phase extraction cartridge is used to ensure quantitative salt-free recovery of the HGs, and the purified glycolipids are then rendered uv-absorbing by a per-O-benzoylation derivatization reaction for which optimal conditions have been established. Derivatives are analyzed within 12 min on a 3-microns silica HPLC column using a linear gradient of 2-propanol in n-hexane and uv monitoring at 230 nm. The reaction product was also used to determine the relative proportions of the glucosyl and galactosyl epimers of individual members of this class of glycolipid.

Several gas chromatographic columns were evaluated for the determination of methylmercury in aqueous solution. The goal of the study was to further decrease the detection limit of the recently developed method of head space gas chromatography with microwave-induced plasma detection (HS-GC-MIP) for the determination of methylmercury in biological samples. The columns were first evaluated using gas chromatography with electron-capture detection (ECD). At the same time, the column efficiencies for the determination of ethyl- and phenylmercury were also studied. Of the packed columns the stationary phase used previously in HS-GC-MIP, AT-1000, yielded the best results. Better results were obtained with two wide-bore thick-film fused-silica open tubular (FSOT) columns, one of which was suitable for aqueous injections (Superox-FA) and the other for benzene or toluene (RSL-300). With these FSOT columns, absolute detection limits at the sub-picogram level were reached. A new HS-GC-MIP system was then constructed, which was adapted for the use of FSOT columns. As more sensitive measurements were obtained with a Superox-FA FSOT column than with an AT-1000 packed column using the GC-ECD system in the first part of this study, the FSOT column was evaluated in this HS-GC-MIP system for the determination of methylmercury in real tissue samples. It was demonstrated that the use of an FSOT column gives only a small decrease in the detection limit compared with a packed column; reconditioning of the FSOT column is, however, a disadvantage in routine measurements.

The 1H NMR spectrum obtained with the alpha- and beta-anomers of D-[1-2H]fructose 6-phosphate generated from D-glucose 6-phosphate sequentially exposed in D2O to phosphoglucoisomerase, phosphofructokinase and fructose-1,6-diphosphatase differed from that recorded when the deuterated ketohexose phosphate was produced from D-mannose 6-phosphate sequentially exposed in D2O to phosphomannoisomerase, phosphofructokinase and fructose-1,6-diphosphatase. The identification of the 2 isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy provides a new tool to assess the relative extent of interconversion of hexose phosphates in the reactions catalyzed by phosphoglucoisomerase and phosphomannoisomerase, respectively.

About 40 new 2',3'-dideoxynucleoside analogues with different basic structures have been synthetized with the aim of finding more potent and selective inhibitors of HIV and the associated pathogenicity. Some new synthesis techniques were developed and existing ones were used for the preparation of these analogues. Among the 3'-fluorinated 2',3'-dideoxynucleosides several potent inhibitors of HIV were found and especially 3'-fluoro-2',3'-dideoxy-5-chloro-uridine (81) is endowed with a high selectivity index, comparable to the selectivity displayed by 3'-azido-3'-deoxythymidine (AZT). Because of its substantially reduced toxicity profile compared to AZT, this compound deserves further evaluation as a possible alternative for the treatment of AIDS. Likewise, the 5-chlorinated cytidine analogues 102 and 103 should be further examined because of their lack of toxicity in vitro.

Sampling, storing, sample pretreatment, and experimental conditions for selenium (Se) determination in human serum, plasma, and whole blood by X-ray emission spectrometric (XRS) methods are described. Concentration levels in these biological fluids, found by this technique, are discussed and compared to values found by other techniques for the same healthy population group in the same area. XRS analysis of blood from patients with various pathological conditions is reviewed, with special attention to the relation of Se with the concentration level of other essential or nonessential trace elements.

Epidemiological studies from Norway and England suggest that there is a relation between the frequency of Alzheimer's disease and the concentration of aluminum in the drinking water. A recent Dutch study suggests further that the role of aluminum from tooth pastes may be even more important than that from drinking water. For this reason we determined in samples from the Belgian market which tooth pastes contained aluminium oxyhydroxide as abrasive. This happened to be the case in 5 out of 27 brands. It is not know to us which part of the market these tooth pastes represent.

The reduction of Hg(II) at a glassy-carbon electrode in various electrolytes has been studied by rotating ring-disc voltammetry. Reduction proceeds directly to metallic mercury in a single 2-electron step. However, at the foot of the wave, and only during the first reduction sweep after pretreatment of the electrode surface, a small amount of Hg(I) species is detected at the ring. The appearance of an Hg(I) intermediate is most pronounced in sulphuric acid solution. The reduction of Hg(II) is found to proceed irreversibly and to be of first order. At sufficiently negative potentials the reduction is convective-diffusion controlled. Stripping voltammetric experiments indicate that the dissolution of mercury gives Hg(II) in complexing electrolytes. In non-complexing electrolytes the initially formed Hg(II) reacts with mercury atoms on the electrode surface to give Hg(I). During electrodissolution, two stripping peaks may be observed as a result of underpotential adsorption of mercury on glassy carbon. The difference in peak potential between the adsorption (mono) layer peak and the bulk mercury peak has been related to the difference in work functions of the deposit (mercury) and substrate (carbon). A rotating glassy-carbon electrode has been used for the anodic stripping determination of mercury. When an appropriate amount of a cation such as cadmium(II) or copper(II) is added to the test solution, mercury down to 2 x 10(-9)M (0.4 ng ml ) can be determined in acidified thiocyanate electrolyte with a relative standard deviation of about 22%.

It is shown that the mutual interference between the chlorides of Au, Pt and Pd in AAS using aqueous solutions nebulized into an air-acetylene flame, can be eliminated by using the dithizonates of these metals. This releasing action is ascribed to the formation of noble metal dithizonates. It is further shown that complex formation with dithizone favours the atomization of Au and that such an effect should be considered as due to complexation rather than to some bulk effect of the organic matrix.