Amidase activity and thermal stability of human thrombin

Previous studies of amidase activity of human α-thrombin have yielded variable results and the decrease of this activity as a function of time and temperature has never been quantified. As this protease is an efficient tool in biochemistry and biotechnology thanks to its extreme selectivity, amidase activity and stability of thrombin were investigated with the synthetic substrate Tos-Gly-Pro-Arg-pNa. Enzyme activity as a function of temperature showed an optimum peak at 45‡C. The pH dependence of the activity showed a maximum around 9.5. The addition of NaCl promoted an increase of the activity. Stability of thrombin decreased rapidly when increasing the temperature from 25–45‡C and when diluting the enyzme. The presence of glycerol and ethylene glycol promoted a small increase of thrombin half life, whereas polyethylene glycol had a more pronounced positive effect even at very low concentrations.     

Parallel supported synthesis of polyamine-imidazole conjugates

[reaction: see text] A small collection of nine polyamine-imidazole conjugates, potentially acting as RNases A mimics, has been synthesized on SynPhase lanterns using amino alcohols and diamines as building blocks. Couplings were performed via S(N)2 alkylation of methanesulfonates with amines. The final introduction of N-4-nitrobenzyloxycarbonyldiamines allowed easy purification of the cleaved compounds.     

Reaction of 2,5‐dimethylpyrroles with quinones. Synthesis of new pyrrolylquinones

The reaction of 1,4‐naphthoquinone ( 1 ) with 2,5‐dimethylpyrroles ( 7a‐7d ) gives only 3‐(1,4‐naphtho‐quinonyl)‐2,5‐dimethylpyrroles. Extending the reaction to other quinones: 5‐hydroxy‐1,4‐naphthoquinone ( 2 ), 1,2‐naphthoquinone ( 3 ), quinoline‐5,8‐dione ( 4 ) and quinoxaline‐5,8‐dione ( 5 ), of which nothing was known, allows the synthesis of new pyrrolylquinones.     

The electronic structure of a diarsaallene –As=C=As– and a phosphaarsaallene –P=C=As–: UV photoelectron spectroscopy and theoretical studies

The electronic properties of a diarsaallene ArAs=C=AsAr and a phosphaarsaallene ArP=C=AsAr (Ar: 2,4,6-tri-tert-butylphenyl) have been investigated by using UV photoelectron spectroscopy and by density functional calculations on model compounds [(CH₃)₂C₆H₃Pn=C=AsC₆H₃(CH₃)₂, Pn: As, P]. Moreover, a comparison of the geometrical and electronic structures of the parent heteroallenes with those of the arsaethene H₂C=AsH and phosphaethene H₂C=PH has also been undertaken in order to determine the magnitude of the interaction between the π bond and the pnictogen lone pair n_Pn.     

Rapid, accurate and precise quantitative drug analysis: comparing liquid chromatography tandem mass spectrometry and chip-based nanoelectrospray ionisation mass spectrometry

We have developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) system capable of achieving better than 2% accuracy, routinely over a wide concentration range of 1800 ng mL-1. We demonstrate that the necessary high precision, high accuracy and rapid analysis can be achieved using LC-MS/MS technology. Automated nanoelectrospray ionisation tandem mass spectrometry (nanoESI-MS/MS) technology can be employed to eliminate the chromatographic step completely. In this paper, nanoESI-MS/MS is evaluated and compared directly with LC-MS/MS for the quantitative analysis of two-test analytes, amitriptyline (ATT) and 5-methoxytryptamine (5-MTT), in aqueous/organic mixture. Calibration curves were found to be linear over a wide concentration range of 1800 ng mL-1 for both analytes using LC-MS/MS. Using nanoESI-MS/MS ATT gave a linear response while 5-MTT gave a non-linear response using nanoESI-MS/MS over the same concentration range as in LC-MS/MS. Accuracy and precision values of quality control samples (QCs) at four concentration levels were analysed in replicates of six at each level using 5-MTT and ATT as test analytes for both techniques. The LC-MS/MS system was capable of achieving accuracy levels of 99.50101.96% for ATT and 100.17100.40% for 5-MTT. Accuracy levels using nanoESI-MS/MS were not comparable to LC-MS/MS, they ranged from 90.09100.18% for ATT and 95.95113.55% for 5-MTT. The precision values obtained for nanoESI-MS/MS were in good agreement with those obtained by LC-MS/MS.     

Symmetry breaking in biological systems. From molecules to tissues

Chirality of biomolecules is one of the major characteristics of living systems. Molecular asymmetry can be detected in the earliest prokaryotes and stereoselective synthesis of proteins and polysaccharides was recognized since Pasteur’s time. More recently much attention was paid to the molecular origin of asymmetric development of organs. This asymmetry can be followed up to the cellular and tissue levels. We compared the periorbital wrinkle pattern (crow’s feet) around the left and right eyes, as well as the microdepressional skin surface relief on the left and right arms. The pattern of these reliefs was shown to change with age in a typical manner. Apparently this age-dependent modification of the skin surface micro-relief as well as wrinkle formation is largely determined by the progressive modifications of the dermal extracellular matrix (ECM) and especially of the elastic fiber network as the result of the oriented (vectorial) biosynthetic activity of dermal fibroblasts. These cells do not form a homogeneous population as shown by their proliferation pattern. The reaction of these cells to receptor-mediated stimuli—by growth factors, cytokines, and hormones also exhibit microheterogeneity. Cell–cell interactions, especially between the keratinocyte layers of the epidermis and dermal fibroblasts might also play an important role in the regulation of cell-patterning. These mechanisms might well explain the asymmetry detected by our semi-automated image analysis program on left–right microdepressionary skin surface relief, as well as periorbital wrinkle formation.