Characterization of the cardiac holotroponin complex reconstituted from native cardiac troponin T and recombinant I and C.

Cardiac troponin I (cTnI), the inhibitory subunit of cardiac troponin (cTn), is phosphorylated by the cAMP-dependent protein kinase A at two adjacently located serine residues within the heart-specific N-terminal elongation. Four different phosphorylation states can be formed. To investigate each monophosphorylated form cTnI mutants, in which each of the two serine residues is replaced by an alanine, were generated. These mutants, as well as the wild-type cardiac troponin I (cTnI-WT) have been expressed in Escherichia coli, purified and characterized by isoelectric focusing, MS and CD-spectroscopy. Monophosphorylation induces conformational changes within cTnI that are different from those induced by bisphosphorylation. Functionality was assessed by measuring the calcium dependence of myosin S1 binding to thin filaments containing reconstituted native, wild-type and mutant cTn complexes. In all cases a functional holotroponin complex was obtained. Upon bisphosphorylation of cTnI-WT the pCa curve was shifted to the right to the same extent as that observed with bisphosphosphorylated native cTnI. However, the absolute values for the midpoints were higher when recombinant cTn subunits were used for reconstitution. Reconstitution itself changed the calcium affinity of cTnC: pCa50-values were higher than those obtained with the native cardiac holotroponin complex. Apparently only bisphosphorylation of cTnI influences the calcium sensitivity of the thin filament, thus monophosphorylation has a function different from that of bisphosphorylation; this function has not yet been identified.     

Evaluation of a direct spectrophotometric method for the rapid determination of ascorbate and dehydroascorbate in blood using ascorbate oxidase

The application of a technique for the direct spectrophotometric determination of ascorbate and dehydroascorbate in blood samples is described. The reliability of the test system and various disturbing factors are assessed. It is concluded that the technique possesses a high specificity and that interferences by various biochemicals and metabolic intermediates are low. The lowest ascorbate concentration detectable in plasma by this assay is estimated to be about 1 mumol/l.     

Native fluorescence of platelets from patients with occlusive arterial disease

Healthy platelets exhibit a fluorescence band with a peak at 475 nm if excited at 360 nm. This peak increases first with the progression of occlusive arterial disease (OAD) followed by a decrease at an advanced stage. Concomitantly, a new fluorescence band at 445 nm will appear, which increases steadily with the progression of OAD. These findings can be explained by the oxidation of NADH (fluorescence at 475 nm) to NAD (445 nm) and support, thus, the assumption that oxidative processes are involved in the formation of OAD.     

Structure-activity relationship of a series of non peptidic RGD integrin antagonists targeting α5β1: part 2

Potent antagonists of the integrin α(5)β(1), which are RGD mimetics built from tyrosine are described. This paper describes the optimization of in vitro potency obtained by variation of two parts of the molecule, the central aromatic core and the amide moiety.