Towards π-wires on a semiconductor surface: Benzyne on Si(001).

Towards the goal of covalently bound molecular wires on silicon, the adsorption of benzyne on Si(001) was studied by means of scanning tunneling microscopy (STM), X-ray photoelectron spectroscopy (XPS), ultraviolet photoelectron spectroscopy (UPS), and density functional calculations (DFT). The benzyne molecule is found to adsorb preferentially via the strained triple bond on one dimer of the Si(001) surface which results in an intact π system covalently bound to the surface. With increasing coverage, the molecules primarily adsorb along the dimer rows; on stepped surfaces, these molecular wires are all oriented in the same direction.

The potential of δ2Hn-alkanes and δ18Osugar for paleoclimate reconstruction - A regional calibration study for South Africa.

The hydrogen isotopic composition of leaf wax-derived n-alkanes (δ2Hn-alkanes) is a widely applied proxy for (paleo)climatic changes. It has been suggested that the coupling with the oxygen isotopic composition of hemicellulose-derived sugars (δ18Osugar) - an approach dubbed 'paleohygrometer' - might allow more robust and quantitative (paleo)hydrological reconstructions. However, the paleohygrometer remains to be evaluated and tested regionally. In this study, topsoil samples from South Africa, covering extensive environmental gradients, are analysed. δ2Hn-alkanes correlates significantly with the isotopic composition of precipitation (δ2Hp), whereas no significant correlation exists between δ18Osugar and δ18Op. The apparent fractionation (εapp) is the difference between δ2Hn-alkanes and δ2Hp (εapp 2H) and δ18Osugar and δ18Op (εapp 18O), respectively, and integrates i) isotopic enrichment due to soil water evaporation, ii) leaf (and xylem) water transpiration and iii) biosynthetic fractionation. We find no correlation of εapp 18O nor for εapp 2H with temperature, and no correlation of εapp 2H with potential evapotranspiration and an aridity index. By contrast, εapp 18O correlates significantly with both potential evapotranspiration and the aridity index. This highlights the strong effect of evapotranspirative enrichment on δ18Osugar. In study areas without plant predominance using Crassulacean Acid Metabolism (CAM), coupling δ18Osugar and δ2Hn-alkanes enables to reconstruct δ2Hp and δ18Op with an offset of Δδ2H = 6 ± 27‰ and Δδ18O = 0.8 ± 3.7‰, respectively, as well as relative humidity (RH) with an offset of ΔRH = 6 ± 17%. The paleohygrometer does, however, not work well for our study areas where CAM plants prevail (reconstructed δ18Op, δ2Hp and RH are off by 3.1‰, 27.2‰ and 31.7%). This probably reflects plant-specific (phenological) adaptations and/or post-photosynthetic exchange reactions related to CAM metabolism. Overall, our findings corroborate that δ2Hn-alkanes and δ18Osugar are valuable proxies, and the paleohygrometer is a promising approach for paleoclimate reconstructions in southern Africa.

Climate deteriorations and Neanderthal demise in interior Iberia.

Time and circumstances for the disappearance of Neanderthals and its relationship with the advent of Modern Humans are not yet sufficiently resolved, especially in case of the Iberian Peninsula. Reconstructing palaeoenvironmental conditions during the last glacial period is crucial to clarifying whether climate deteriorations or competition and contacts with Modern Humans played the pivotal role in driving Neanderthals to extinction. A high-resolution loess record from the Upper Tagus Basin in central Spain demonstrates that the Neanderthal abandonment of inner Iberian territories 42 kyr ago coincided with the evolvement of hostile environmental conditions, while archaeological evidence testifies that this desertion took place regardless of modern humans' activities. According to stratigraphic findings and stable isotope analyses, this period corresponded to the driest environmental conditions of the last glacial apart from an even drier period linked to Heinrich Stadial 3. Our results show that during Marine Isotope Stages (MIS) 4 and 2 climate deteriorations in interior Iberia temporally coincided with northern hemisphere cold periods (Heinrich stadials). Solely during the middle MIS 3, in a period surrounding 42 kyr ago, this relation seems not straightforward, which may demonstrate the complexity of terrestrial climate conditions during glacial periods.

Brain cholinesterases. Differentiation of target enzymes for toxic organophosphorus compounds.

Cholinesterases in hen brain were characterized with respect to inhibition kinetics and substrate specificity. Three organophosphorus inhibitors were used: diethyl p-nitrophenyl phosphate (Paraoxon, E 600), di-isopropylphosphorofluoridate (DFP), and N,N'-di-isopropylphosphorodiamidic fluoride (Mipafox). The kinetics of irreversible cholinesterase inhibition were studied using two substrates, acetylthiocholine and butyrylthiocholine. The inhibition curves were analysed by the method of iterative elimination of exponential functions. Final classification of the different enzymes was done by combining two inhibitors in sequential inhibition expts. Six cholinesterases were shown to hydrolyse choline esters in hen brain, one was identified as acetylcholinesterase (EC 3.1.1.7) and one as cholinesterase (EC 3.1.1.8). Four enzymes can be classified as intermediate type cholinesterases according to their substrate specificity and to their inhibition constants. The possible role of different brain cholinesterases for the development of atypical symptoms following organophosphate intoxication is discussed.

Cholinesterases of heart muscle. Characterization of multiple enzymes using kinetics of irreversible organophosphorus inhibition.

Cholinesterases of porcine left ventricular heart muscle were characterized with respect to substrate specificity and inhibition kinetics with organophosphorus inhibitors N,N'-di-isopropyl-phosphorodiamidic fluoride (Mipafox), di-isopropylphosphorofluoridate (DFP), and diethyl p-nitro-phenyl phosphate (Paraoxon). Total myocardial choline ester hydrolysing activity (234 nmol/min/g wet wt with 1.5 mM acetylthiocholine, ASCh; 216 nmol/min/g with 30 mM butyrylthiocholine, BSCh) was irreversibly and covalently inhibited by a wide range of inhibitor concentrations and, using weighted least-squares non-linear curve fitting, residual activities as determined with four different substrates in each case were fitted to a sum of up to four exponential functions. Quality of curve fitting as assessed by the sum of squares reached its optimum on the basis of a three component model, thus, indicating the presence of three different enzymes taking part in choline ester hydrolysis. Final classification of heart muscle cholinesterases was obtained according to both substrate hydrolysis patterns with ASCh, BSCh, acetyl-beta-methylthiocholine and propionylthiocholine, and second-order rate constants for the reaction with organophosphorus inhibitors Mipafox, DFP, and Paraoxon. One choline ester-hydrolysing enzyme was identified as acetylcholinesterase (EC 3.1.1.7), and one as butyrylcholinesterase (EC 3.1.1.8). The third enzyme with relative resistance to organophosphorus inhibition was classified as atypical cholinesterase.

Influence of the organophosphorus compound DFP on inhibitory motor systems and esterase activity in the spinal cord of cats.

In high spinal cats, the acute time-dependent changes of both the activity of spinal reflex pathways and the activity of three different esterases (acetylcholinesterase, carboxylesterase and neurotoxicant target enzyme) in the spinal cord were investigated after intravenous application of the organophosphorus compound di-isopropyl phosphofluoridate (DFP). There is no general depression of spinal reflexes by DFP. While the recurrent inhibition is completely abolished for a long time and the reflexes to a flexor (PBSt) are depressed but with a shorter recovery time, the reflexes to an extensor (GS) are distinctly less depressed or even facilitated. Reflex pathways from skin afferents to motoneurones did not react in a uniform way to DFP, e.g. inhibitory nociceptive pathways were less affected than excitatory ones. Esterase activities were heavily depressed and recovered with different time courses. The acute DFP action cannot be explained by a uniform intoxication of all spinal functions but probably emerges from a differential action on different interneuronal systems.

Neurotoxic esterase: gel filtration and isoelectric focusing of carboxylesterases solubilized from hen brain.

Carboxylesterase activity (EC 3.1.1.1) of hen brain including neurotoxic esterases NTEA and NTEB is solubilized from lyophilized lipid-extracted brain material by the use of n-octylglucoside. The solubilized enzymes are subjected to free isoelectric focusing, six carboxyl - esterase activity peaks are obtained. By gel filtration on Sephacryl S-300 neurotoxic esterases are separated from carboxylesterase isoenzymes V and X. The molecular weight of the neurotoxic esterases is estimated to be 1.8 X 10(6).

Computerized analysis of covalent inhibition kinetics for identification of heart muscle cholinesterase and brain carboxylesterase isoenzymes. Design of differential inhibition assays.

The kinetics of time- and concentration-dependent covalent organophosphorus inhibition of carboxylesterase isoenzymes (EC 3.1.1.1) and cholinesterase isoenzymes (EC 3.1.1.7 and EC 3.1.1.8) were investigated using a wide range of organophosphate inhibitor concentrations (10(-10)-10(-3) mol/l) and different inhibition times. Computerized analysis of inhibition curves by weighted non-linear least-squares curve fitting was compared to graphic analysis by iterative elimination of exponential functions. Possible experimental errors due to inhibitor saturation kinetics and enzymatic organophosphate hydrolysis were thoroughly investigated. In mammalian heart muscle, three different cholinesterase isoenzymes were identified. High sensitivity and specificity of the classic differential inhibition test for carboxylesterase activity of hen brain neuropathy target esterase (NTE) could be confirmed independently with both methods of inhibition curve analysis.

Organophosphate inhibition of human heart muscle cholinesterase isoenzymes.

The rate of acetylcholine hydrolysis of mammalian heart muscle influences cardiac responses to vagal innervation. We characterized cholinesterases of human left ventricular heart muscle with respect to both substrate specificity and irreversible inhibition kinetics with the organophosphorus inhibitor N,N'-di-isopropylphosphorodiamidic fluoride (mipafox). Specimens were obtained postmortem from three men and four women (61 +/- 5 years) with no history of cardiovascular disease. Myocardial choline ester hydrolyzing activity was determined with acetylthiocholine (ASCh; 1.25 mM), acetyl-beta-methylthiocholine (AbetaMSCh; 2.0 mM), and butyrylthiocholine (BSCh; 30 mM). After irreversible and covalent inhibition (60 min; 25 degrees C) with a wide range of mipafox concentrations (50 nM-5 mM), residual choline ester hydrolyzing activities were fitted to a sum of up to five exponentials using weighted least-squares non-linear curve fitting. In each ease, quality of curve fitting reached its optimum on the basis of a four component model. Final classification of heart muscle cholinesterases was achieved according to substrate hydrolysis patterns (nmol/min per g wet weight) and to second-order organophosphate inhibition rate constants k2 (1/mol per min); one choline ester hydrolyzing enzyme was identified as acetylcholinesterase (AChE; k2/mipafox = 6.1 (+/- 0.8) x 10(2)), and one as butyrylcholinesterase (BChE; k2/mipafox = 5.3 (+/- 1.1) x 10(3)). An enzyme exhibiting both ChE-like substrate specificity and relative resistance to mipafox inhibition (k2/mipafox = 5.2 (+/- 1.0) x 10(-1)) was classified as atypical cholinesterase.

Paraoxonase polymorphism in rabbits.

Paraoxonase in serum and liver of rabbits and cattle was investigated. In serum the two substrates paraoxon and phenylacetate are exclusively hydrolyzed by alpha-lipoprotein-bound paraoxonase. In rabbit liver paraoxon is hydrolyzed only by paraoxonase, while phenylacetate is hydrolyzed by paraoxonase (20%) and additionally by an organophosphate sensitive carboxylesterase (B-Esterase), which is responsible for 80% of total liver phenylacetate hydrolysis. Phenyl acetate hydrolysis by B-Esterase of rabbit liver was shown to be inhibited by paraoxon and by mipafox covalently in a time and concentration dependent manner. Rabbit serum exhibits by far the highest serum paraoxonase activity (2.6 +/- 0.66 U/ml) of all vertebrate species tested up to now, while rabbit liver contains only 0.5 +/- 0.2 U/g fresh weight. In cattle extremely high paraoxonase activity is found in liver (2.8 U/g), while bovine serum contains only 0.2 U/g. The paraoxonase activity ratio (hydrolysis rate paraoxon: phenylacetate x 1000) in cattle does not show interindividual variation (activity ratio 4.0 +/- 0.4, correlation coefficient 0.996, P < 0.001). In contrast, the paraoxon/phenylacetate hydrolysis ratio of rabbit paraoxonase in serum as well as in liver does vary considerably between individuals. In cross-bred rabbits paraoxonase activity ratios from three to ten are found. In a strain of pure-bred New Zealand White rabbits three polymorphic serum paraoxonase phenotypes could be clearly differentiated by the activity ratio. By analogy with the human paraoxonase polymorphism, the rabbit paraoxonase isotypes were classified as paraoxonase A (activity ratio 3.8-4.3), AB (ratio 5.5-6.0) and B (ratio 7.3-8.6). The corresponding frequencies of the three isotypes were 40, 35 and 25%.

Lipoproteins and hydrolysis of organophosphorus compounds.

Paraoxonase of human and animal sera was shown to be a structural part of high density lipoproteins (HDL) by immunoprecipitation, heparin- or polyethyleneglycol fractionation, ultracentrifugation and gel chromatography. Frequency distribution of paraoxonase activity in human sera is trimodal. Human individuals, with respect to paraoxon detoxication, can be distinguished into low and high detoxicators using ratios of phenylacetate and paraoxon hydrolysis as well as activation with ethanolamine and sodium chloride. With conversion of alpha-lipoprotein subtype HDL3 to HDL2, specific activities of paraoxonase and arylesterase are increasing about 3.5-fold in low detoxicator individuals and 1.9-fold in high detoxicators, indicating that more than 90% of HDL2 particle-bound paraoxonase and arylesterase activity are incorporated during the HDL conversion process. HDL cholesterol concentrations in individual sera were shown to be positively correlated to both serum paraoxonase and arylesterase activities.

[Age related decrease of high density lipoproteins (HDL) in women after menopause. Quantification of HDL with genetically determined HDL arylesterase in women with healthy coronary vessels and in women with angiographically verified coronary heart disease]. / Zur altersabhängigen Verminderung von Lipoproteinen hoher Dichte (HDL) bei Frauen nach der Menopause. Quantifizierung von HDL über die genetisch determinierte HDL-Arylesterase bei koronargesunden Frauen und bei Frauen mit angiographisch gesicherter koronarer Herzkrankheit.

BACKGROUND: The decline in the concentration of high density lipoproteins (HDL) observed in postmenopausal women is thought to contribute to the increasing incidence of coronary artery disease (CAD) after menopause. Human serum arylesterase (EC 3.1.1.2) is exclusively associated with HDL. We therefore investigated possible differences in the decline of HDL-levels and of HDL-subfractions HDL2 and HDL3 between postmenopausal women without and with angiographically documented CAD. PATIENTS AND METHODS: HDL-, HDL2-and-HDL3- concentrations were studied in postmenopausal women with angiographically documented CAD (n = 24; 51 to 72 years mean: 62 years) and compared to HDL-parameters of women without CAD (n = 22; 51 to 81 years, mean: 58 years). Arylesterase activities of HDL2-and HDL3-subfractions and HDL2-cholesterol concentrations were determined after differential precipitation with polyethylene glycol (4.7 mM PEG). Phenotyping of HDL-arylesterase was achieved in CAD patients and in women without CAD after determining hydrolysis of arylesterase substrates paraoxon (PO) and phenylacetate (PA) by calculating paraoxonase/arylesterase activity ratios R (R = [PO]/[PA] x 1000): phenotype A (n = 26) with R < 2.5, phenotype AB (n = 16) with 5.0 < R < 10.7, and phenotype B (n = 4) with R > 13.5. RESULTS: In postmenopausal women with documented CAD, as compared to women without CAD, HDL-cholesterol (55 +/- 3 mg/dl vs. 69 +/- 3 mg/dl HDL2-arylesterase (25 +/- 1 kU/l vs. 33 +/- 2 kU/l), and HDL3-arylesterase (89 +/- 4 kU/l vs. 106 +/- 5 kU/I) were found to be significantly reduced. Analysis of the correlation of lipid parameters and age revealed in CAD patients, but not in postmenopausal women without CAD, a significant increase of total cholesterol (r = 0.42), and significant reductions of both HDL2-arylesterase (r = -0.47) and HDL3-arylesterase (r = 0.74) with increasing age. In contrast, HDL-cholesterol (r = -0.14) and HDL2-cholesterol (r = -0.06) of CAD patients showed only slight and non-significant reductions with age. Since HDL3-arylesterase was found to be age-dependently reduced in women without CAD (r = 0.17), HDL2-arylesterase of postmenopausal women, among all lipid parameters showed the most pronounced differences between women without CAD and CAD patients. The age-dependent decrease of HDL2-arylesterase in postmenopausal women with CAD does not result from an increased frequency of B-allele carriers in the subgroup of CAD patients with an age above the median (64 years). CONCLUSION: Genetically determined serum HDL-arylesterase is well suited to quantify HDL in postmenopausal women without and with CAD. HDL2-arylesterase of postmenopausal women should be evaluated as a screening parameter for both primary and secondary CAD prevention.

Preparation of two neurotoxic esterases from the chick central nervous system.

A standard procedure for lipid-extraction of lyophilized hen brain material is described. Nine carboxylesterase isoenzymes (EC 3.1.1.1) are identified in lipid-extracted lyophilized material (LELM) using kinetic analysis of organophosphate inhibition. Total phenyl valerate (PV) hydrolysing carboxylesterase activity in LELM is 43.3 U X g-1. Two carboxylesterase isoenzymes of LELM are classified as neurotoxic esterases (NTEA and NTEB). Using n-octylglucoside 51% of the water-insoluble neurotoxic esterase activity from LELM are solubilized. Six carboxylesterase isoenzymes including NTEA (6.5 U X 1(-1] and NTEB (4.2 U X 1(-1] are present in the solubilized preparation. Throughout purification and separation steps carboxylesterase isoenzymes are identified by their rate constants for the reaction with organophosphorus inhibitors.

Maintenance of hemostasis during exodontia in two hemophiliacs with factor VIII inhibitors.

This paper reviews hemophilia A with emphasis on the 10% of afflicted patients who have inhibitors to factor VIII. Two patients with the latter problem are presented, and a management program is described and illustrated.