Synthesis of enantiopure (5R)-hydroxyhexane-2-one with immobilised whole cells of Lactobacillus kefiri.

Whole-cell reduction of (2,5)-hexanedione to yield highly enantiopure (5R)-hydroxyhexane-2-one (enantiomeric excess >99%) with Lactobacillus kefiri DSM 20587 was investigated. Cell immobilisation with sodium cellulose sulphate was chosen as the most suitable encapsulation matrix, giving an immobilisation yield of 40%. Despite the lowered biocatalytic activity from cell immobilisation, the bioreduction process was vastly improved with the help of reaction engineering techniques (batch to a plug flow reactor set-up). High selectivity (95%) and space-time yield (87 g L(-1) day(-1)) were achieved in the plug flow reactor. The biocatalyst remained active (68% residual activity) after 6 days of operation.

Cloning, sequence analysis, and heterologous expression of the gene encoding a (S)-specific alcohol dehydrogenase from Rhodococcus erythropolis DSM 43297.

The gene encoding an (S)-specific NAD-dependent alcohol dehydrogenase (RE-ADH) was isolated from the genomic DNA of Rhodococcus erythropolis DSM 43297. The nucleotide sequence of 1,047 bp, coding for 348 amino acids, was cloned in Escherichia coli cells and successfully expressed. The subunit molecular mass as deduced from the amino acid sequence was determined to be 36.026 kDa. The recombinant enzyme exhibited high thermostability, which facilitated its purification by heat treatment, followed by two column-chromatography steps. RE-ADH shows high similarity to several zinc-containing medium-chain alcohol dehydrogenases. All zinc ligands seem to be conserved except one of the catalytic zinc ligands, where Cys is probably substituted by Asp. A similarity of 84% with a phenylacetaldehyde reductase from Corynebacterium sp. ST-10 was determined. Biochemical properties such as thermostability and substrate specificity of the two enzymes were compared.

Diastereoselective synthesis of optically active (2 R,5 R)-hexanediol.

Diastereoselective reduction of diketones with Lactobacillus kefir DSM 20587 was examined. The reduction of both oxo-functions proceeded highly diastereoselectively. (2 R,5 R)-Hexanediol 3 was produced starting from (2,5)-hexanedione 1 in quantitative yields with enantiomeric excess >99% and diastereomeric excess >99%. The reaction conditions were optimized: maximum yield of (2 R,5 R)-hexanediol was reached at pH 6, 30 degrees C and with equal amounts of substrate and cosubstrate. The applicability of the system in fed-batch experiments was demonstrated. The feed specific biomass concentration required to reach maximal yield and selectivity in fed-batch mode was determined.

Sorption behaviour of Am on marl groundwater colloids.

The sorption of americium on marl colloids is studied in a comprehensive way. In Na-HCO3 groundwater conditions, americium in the concentration range 3 x 10(-11)-3 x 10(10) M sorbs onto groundwater colloids. In these tests, the colloid concentration is rather high, i.e. Ccoll 1.7+/-1.3 mg l(-1) for sizes 505000 nm or Ncoll (1.7+/-0.1) x 10(11) l(-1) for sizes larger than 100 nm. These colloids are mainly chlorite and smectite/illite with an average size smaller than 500 nm. For the groundwater (pH 8.6, [Na+]-[HCO3]-2 x 10(-2) M), Kd values in the range 1-3 x 10(6)mlg(-1) are measured. These values are compared with experimental and modelled values reported earlier in the literature.

Biocatalytic reduction of beta,delta-diketo esters: a highly stereoselective approach to all four stereoisomers of a chlorinated beta,delta-dihydroxy hexanoate.

A stereoselective chemoenzymatic synthesis of all four stereoisomers of tert-butyl 6-chloro-3,5-dihydroxy-hexanoate (6a) is presented. The key step of the sequence is a highly regio- and enantioselective single-site reduction of tert-butyl 6-chloro-3,5-dioxohexanoate (1a) by two enantiocomplementary biocatalysts. Alcohol dehydrogenase from Lactobacillus brevis (recLBADH) afforded a 72% yield of enantiopure tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate [(S)-2a]. The enantiomer (R)-2a was prepared with 90-94% ee by Baker's yeast reduction in a biphasic system (50% yield). Both biotransformations were performed on a gram scale. The beta-keto group of the enantiomeric delta-hydroxy-beta-keto esters 2a thus obtained was reduced by syn- and anti-selective borohydride reductions. Permutation of the reduction methods yielded all four stereoisomers of the crystalline target compound 6a (> or = 99.3% ee, dr > or = 205:1), which is a versatile 1,3-diol building block. recLBADH accepts a variety of beta,delta-diketo esters as was determined in a photometric assay. tert-Butyl 3,5-dioxo-hexanoate (1b) and tert-butyl 3,5-dioxo-heptanoate (1c) were reduced on a preparative scale as well to afford the corresponding delta-hydroxy-beta-keto esters (R)-2b and (R)-2c with 99.4% ee and 98.1% ee, respectively.

Crystallization and preliminary characterization of crystals of R-alcohol dehydrogenase from Lactobacillus brevis.

The R-specific alcohol dehydrogenase (RADH) from Lactobacillus brevis is a valuable catalyst for the production of chiral alcohols that can be used as synthons in asymmetric syntheses. RADH is a homotetramer with 222 symmetry and a molecular mass of 107 kDa. The recombinant enzyme has been expressed in Escherichia coli, purified to homogeneity and crystallized. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 56.5, b = 85.1, c = 115.4 A, and diffract X-rays to at least 1.8 A resolution. The calculated crystal packing parameter V(M) = 2.59 A(3) Da(-1), corresponding to a solvent content of 52.5% and suggesting that one RADH monomer is contained in the asymmetric unit. The RADH tetramer lies on a special position with its molecular dyads coinciding with the crystallographic twofold axes and with its centre of mass on the origin of the unit cell.

Sex differences in 'climacteric symptoms' with increasing age? A hypothesis-generating analysis of cross-sectional population surveys.

Are the established clinical symptoms of the female climacteric really specific for women? We were interested to compare the prevalence of these symptoms across age and sex. In the absence of longitudinal (cohort) studies, we tried to find cross-sectional, population-based studies with a broad age span to find a preliminary answer. We obtained six surveys that contained information for at least some of the symptoms. We re-analyzed these databases, which included over 8000 males and 8000 females, in a pooled fashion. Sixteen symptoms of predominantly somatic, psychological and urogenital origin were identified and the age-dependent increase of the relative frequencies was compared between genders. A general tendency for women to report more symptoms was confirmed; however, no significant differences were found between genders regarding the time trend of these symptoms. The analyses suggest that males experience a similar symptomatology in the same age span as women. One may thus be tempted to conclude that aging males experience similar phenomena to women during their menopausal transition. This should be confirmed in a specifically designed study.

Large-scale applications of NAD(P)-dependent oxidoreductases: recent developments.

NAD(P)-dependent dehydrogenases are useful catalysts for the synthesis of chiral compounds. Many active and stable enzymes are available that (with high enantioselectivity) reduce ketones or keto acids to chiral alcohols, hydroxy acids or amino acids. For economic reasons, these reactions need coupling to the simultaneous regeneration of NAD(P)H. For preparative applications, three components have to be combined: an appropriate enzyme, an efficient coenzyme-regenerating step and a suitable reaction-engineering technique.

Assisted reproductive technology: the state of the ART.

At least one in ten couples of reproductive age is affected by infertility. Tubal disease, ovulatory defects, endometrosis and abnormal sperm physiology are the most common causes of failure to conceive. Many of these disorders can be treated successfully with surgery, ovulation induction or intrauterine insemination, but in selected cases, or where there is long-standing intractable infertility, assisted reproductive technology (ART) becomes the treatment of choice. We provide an overview of the techniques for assisted reproduction, including in vitro fertilization, gamete intrafallopian transfer and other related procedures. Indications for treatment, patient evaluation and advances in reproductive technology including embryo cryopreservation, micromanipulation and donor gametes are also reviewed.

Modern medical management of endometriosis.

The modern medical management of endometriosis has changed considerably since the first attempts were made to control this disease hormonally over four decades ago. Currently, there are multiple choices for the clinician and patient, including oral contraceptives, danazol, GnRH agonist analogues, and gestrinone. Several advances have been made in the use of GnRH agonists in preventing some of the untoward effects of prolonged hypoestrogenism. These add-back regimens provide the best therapy available today for prolonged medical control of endometriotic symptoms. The antiprogesterones (RU-486) hold promise for the future, but are still in the investigational stage of development.

Simulation of measurements of uptake of 123I-iodide in the thyroid of fetal chimpanzees.

The quantitative assessment of the uptake of 123I-iodide (123I-) in the fetal thyroid in vivo was simulated in phantom measurements. First, the relationship between the depth of the fetal thyroid phantom and the two-peak ratio, the ratio of the counts in the gamma-ray and the X-ray energy windows of the registered spectrum, was determined. Subsequently, the attenuation of the gamma-ray signal in relationship to the depth of the fetal thyroid phantom was determined. Finally, the relationship between the two-peak ratio and the attenuation of the gamma-ray signal was deduced. For a reliable correlation, the signal recorded from the radioactivity surrounding the fetal thyroid phantom has to be subtracted from that obtained with the fetal thyroid phantom present. A correction curve was generated to be applied to the in vivo measurements. It is concluded that with this method determination of uptake of 123I- in the fetal thyroid is feasible.

Protection of the maternal and fetal thyroid from radioactive contamination by the administration of stable iodide during pregnancy. An experimental evaluation in chimpanzees.

The safety and efficacy of the administration of stable iodide to protect the fetal thyroid from exposure to radioactive iodide were investigated in chimpanzees in weeks 19 to 21 of pregnancy. The mean 24-h uptake of iodide in the fetal thyroid, determined with 123I-, was 1.8%. Administration of stable potassium iodide (KI), 0.65, 1.95 or 6.5 mg per kg body weight, 1 h before tracer injection reduced the fetal uptake satisfactorily. Only the higher doses were effective after 20 h. Excess iodide may impair a child's thyroid status. However, adverse effects were not found during the 11 days the animals ingested these doses. Tracer concentrations in the amniotic fluid were 30- to 130-fold lower than in the urine. The dose to the fetus from radioactivity in the maternal bladder was estimated by computer simulation. The potential increment of the risk from this dose during the ingestion of stable iodide is smaller than the reduction of risk achieved by inhibiting the uptake of radioactive iodide by the fetal thyroid. The conclusion of the experiments is that stable iodide can be used safely and effectively to protect the fetal thyroid against contamination with radioactive iodine.

Protection of the infant thyroid from radioactive contamination by the administration of stable iodide. An experimental evaluation in chimpanzees.

Protection of the thyroid from radioactive contamination by the administration of stable iodide was investigated in chimpanzees aged 2 to 98 weeks. The uptake of iodide in the thyroid was measured with 123I-. The animals were subjected to a control measurement first, and subsequently the thyroid uptake of 123I- was determined twice; once at the start and once at the end of 11 days of ingestion of 0.5, 1.5 or 5.0 mg of stable iodide per kg body weight per day. The three doses of iodide reduced the control thyroid iodide uptake of 10 to 30% to lower than 1% when ingested 1 h before exposure to the tracer and to 2-4% when ingested 20 h before exposure. In the latter experiments 0.5 mg iodide/kg was less effective than doses of 1.5 mg/kg or higher. The physiological state of the thyroid of control infant chimpanzees does not differ from that of human infants. Incidentally, an increased level of TSH was found during the ingestion of iodide, but with unaltered thyroxine levels. Therefore, it is concluded that a daily dose of 1.5 mg stable iodide/kg body weight and higher offers optimal protection of the thyroid against exposure to radioactive iodine in infants and that, when used during 10 days, it leaves the thyroid unaffected.

New alcohol dehydrogenases for the synthesis of chiral compounds.

The enantioselective reduction of carbonyl groups is of interest for the production of various chiral compounds such as hydroxy acids, amino acids, hydroxy esters, or alcohols. Such products have high economic value and are most interesting as additives for food and feed or as building blocks for organic synthesis. Enzymatic reactions or biotransformations with whole cells (growing or resting) for this purpose are described. Although conversions with whole cells are advantageous with respect to saving expensive isolation of the desired enzymes, the products often lack high enantiomeric excess and the process results in low time-space-yield. For the synthesis of chiral alcohols, only lab-scale syntheses with commercially available alcohol dehydrogenases have been described yet. However, most of these enzymes are of limited use for technical applications because they lack substrate specificity, stability (yeast ADH) or enantioselectivity (Thermoanaerobium brockii ADH). Furthermore, all enzymes so far described are forming (S)-alcohols. Quite recently, we found and characterized several new bacterial alcohol dehydrogenases, which are suited for the preparation of chiral alcohols as well as for hydroxy esters in technical scale. Remarkably, of all these novel ADHs the (R)-specific enzymes were found in strains of the genus Lactobacillus. Meanwhile, these new enzymes were characterized extensively. Protein data (amino acid sequence, bound cations) confirm that these catalysts are novel enzymes. (R)-specific as well as (S)-specific ADHs accept a broad variety of ketones and ketoesters as substrates. The applicability of alcohol dehydrogenases for chiral syntheses as an example for the technical use of coenzyme-dependent enzymes is demonstrated and discussed in this contribution. In particular NAD-dependent enzymes coupled with the coenzyme regeneration by formate dehydrogenase proved to be economically feasible for the production of fine chemicals.

Screening and characterization of new enzymes for biosensing and analytics.

The development of new or improved analytical methods requires new enzymes. Screening techniques utilizing enrichment cultures and rapid assay methods supported by automated or miniaturized methods are useful tools to detect new enzyme producers. Notably, oxidoreductases are well suited for analytical purposes. The NAD(P)- and oxygen-independent quinoprotein dehydrogenases with a covalently bound redox cofactor can be used advantageously for the development of biosensors. Examples are given of selective enrichment methods used in screening for useful enzyme-producing microorganisms. Enrichment under chemostatic conditions proved to be successful because enzymes with a remarkably high affinity against the analyte could be obtained. This is demonstrated by the screening of a trimethylamine-converting enzyme. The frequently observed high selectivity of these enzymes against the substrate is demonstrated in a few examples. In exploitation of these new oxidoreductases, new analytical methods were developed which are useful for the detection and during monitoring of phenylketonuria (PKU) or maple syrup urine disease (MSUD).

Ovulation induction in the estrogenized anovulatory patient.

Ovulation induction is one of the greatest success stories in reproductive endocrinology. With appropriate therapy in properly f1p4cted patients, the conception and live birth-rates in treated patients are almost indistinguishable from normal. In the estrogenized woman there are many different techniques to reverse the condition of chronic anovulation. With clomiphene citrate, up to 80% of patients will ovulate, and approximately half will conceive. In women who fail clomiphene therapy, injectable gonadotropins are usually successful in inducing ovulation. New protocols for administering these powerful agents have minimized the risk of ovarian hyperstimulation and multiple pregnancy. When medical therapy fails to result in successful ovulatory cycles, surgical treatments can be considered. Laparoscopic ovarian ablation has been shown to be effective treatment. Whether this less invasive surgery results in fewer adhesions than conventional ovarian wedge resection remains to be proven. By carefully considering each patient and individualizing treatment based on a full knowledge of alternatives, ovulation induction remains one of the most challenging and rewarding treatments in reproductive endocrinology.

Enzyme engineering aspects of biocatalysis: cofactor regeneration as example.

Reaction engineering is an important tool in the case of cofactor depending enzyme-catalyzed reactions. It allows the establishment of conditions resulting in lower product specific cofactor costs as compared with product-specific enzyme costs. This is shown for the stereospecific reduction of carbonyl compounds yielding chiral amino acids and alcohols. In continuous processes, cofactor costs can be reduced if the cofactor can be retained within the bioreactor or recycled into it after separation of the product. In case of readily water-soluble substrates it is even possible to recycle the cofactor during a single pass through a continuously operated reactor more than 4000 times because normally very low cofactor concentrations are sufficient to saturate the enzymes involved. L-tert-Leucine has been produced by reductive amination with a space-time yield of up to 366 g L(-1) d(-1) in a single continuously operated enzyme membrane reactor and a two-stage cascade. Total turnover number of the cofactor NAD(+) increased to 4230. (S)-1-Phenyl-2-propanol was obtained by reduction of the corresponding ketone in an membrane reactor with integrated extraction of the product. A new alcohol dehydrogenase from Rhodococcus erythropolis was used. A space-time yield of 63 g L(-1) d(-1) and a total turnover number of 1350 have been reached. L-Leucine has been produced using polymer-enlarged NADH. The total turnover number was 80,000 at a space-time yield of 214 g L(-1) d(-1).

Enzymatic method for determination of branched-chain amino acid aminotransferase activity.

A spectrophotometric assay for the determination of branched-chain L-amino acid aminotransferase activity is described. It is based on the transamination of L-leucine in the presence of 2-oxoglutarate yielding 4-methyl-2-oxopentanoate. The rate of formation of the branched-chain 2-oxo acid is specifically monitored in a coupled enzymatic reaction using NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei ssp. pseudoplantarum as coupling enzyme by measuring the decrease in NADH absorbance at 334 nm. Optimized assay conditions are provided as evaluated for the (iso)enzyme from rat heart.

Reduction of radiation exposure while maintaining high-quality fluoroscopic images during interventional cardiology using novel x-ray tube technology with extra beam filtering.

BACKGROUND: Radiographic technology plays an integral role in interventional cardiology. The number of interventions continues to increase, and the associated radiation exposure to patients and personnel is of major concern. This study was undertaken to determine whether a newly developed x-ray tube deploying grid-switched pulsed fluoroscopy and extra beam filtering can achieve a reduction in radiation exposure while maintaining fluoroscopic images of high quality. METHODS AND RESULTS: Three fluoroscopic techniques were compared: continuous fluoroscopy, pulsed fluoroscopy, and a newly developed high-output pulsed fluoroscopy with extra filtering. To ascertain differences in the quality of images and to determine differences in patient entrance and investigator radiation exposure, the radiated volume curve was measured to determine the required high voltage levels (kVpeak) for different object sizes for each fluoroscopic mode. The fluoroscopic data of 124 patient procedures were combined. The data were analyzed for radiographic projections, image intensifier field size, and x-ray tube kilovoltage levels (kVpeak). On the basis of this analysis, a reference procedure was constructed. The reference procedure was tested on a phantom or dummy patient by all three fluoroscopic modes. The phantom was so designed that the kilovoltage requirements for each projection were comparable to those needed for the average patient. Radiation exposure of the operator and patient was measured during each mode. The patient entrance dose was measured in air, and the operator dose was measured by 18 dosimeters on a dummy operator. Pulsed compared with continuous fluoroscopy could be performed with improved image quality at lower kilovoltages. The patient entrance dose was reduced by 21% and the operator dose by 54%. High-output pulsed fluoroscopy with extra beam filtering compared with continuous fluoroscopy improved the image quality, lowered the kilovoltage requirements, and reduced the patient entrance dose by 55% and the operator dose by 69%. CONCLUSIONS: High-output pulsed fluoroscopy with a grid-switched tube and extra filtering improves the image quality and significantly reduces both the operator dose and patient dose.