Delineation of selective cyclic GMP-dependent protein kinase Ialpha substrate and inhibitor peptides based on combinatorial peptide libraries on paper.

Peptide libraries on cellulose paper have proven to be valuable tools for the a priori determination of substrate specificities of cyclic AMP- and cyclic GMP-dependent protein kinases (cAMP-kinase and cGMP-kinase) on the basis of octa-peptide sequences. Here, we report the extension of our peptide library screens to 12-mer and 14-mer peptide sequences, resulting in highly cGMP-kinase Ialpha selective peptides. The sequences TQAKRKKSLAMA-amide and TQAKRKKSLAMFLR-amide, with Km values for cGMP-kinase Ialpha of 0.7 and 0.26 microM and Vmax values of 11.5 and 10.9 micromol/min/mg, respectively, display a high specificity for this enzyme. Furthermore, replacing the phosphate acceptor residue serine with alanine in TQAKRKKSLAMA-amide resulted in the highly cGMP-kinase Ialpha selective inhibitor peptide TQAKRKKALAMA-amide, with inhibitor constants for cGMP-kinase Ialpha and cAMP-kinase of 7.5 microM and 750 microM, respectively. Selective cGMP-kinase inhibitors have the potential to play an important role in the elucidation of the distinct cellular functions of cGMP-kinase separate from those activated by cAMP-kinases, and, therefore, may play an important role as pharmaceutical targets. Molecular docking experiments of the most cGMP-kinase selective sequences on a molecular model of the catalytic domain of cGMP-kinase Ialpha suggest that they adopt unique conformations, which differ significantly from those observed for the cAMP-kinase-specific inhibitor PKI(5-24). Our results suggest that despite their structural similarities, cAMP-kinase and cGMP-kinase use distinct peptide substrate and inhibitor conformations, which could account for their unique substrate specificities. These findings are further supported by cAMP- and cGMP-kinase-selective inhibitor analogs with (D)-Ala residues at the inhibitory positions.

It takes acid, rather than ice, to freeze glucose.

Plasma glucose levels provide the cornerstone of diabetes evaluation. Unfortunately, glucose levels drop in vitro due to glycolysis. Guidelines provide suitable conditions which minimize glycolysis, such as immediate centrifugation or the use of ice/water slurry storage containers. For obvious practical reasons, most laboratories use blood collection tubes containing glycolysis inhibitors. We describe the effect of a variety of commonly used blood collection tubes on in vitro stability of glucose. Furthermore, we looked at the validity of the assumption that glycolytic activity is minimal when blood is kept in an ice/water slurry. Sodium fluoride alone does not reduce in vitro glycolysis in the first 120 minutes after phlebotomy. Addition of citrate almost completely prevented in vitro glycolysis, but showed a positive bias (0.2 mmol/l) compared to control. This is partly due to a small drop in glucose level in control blood, drawn according to the current guidelines. This drop occurs within 15 minutes, in which glycolysis has been described to be minimal and acceptable. NaF-EDTA-citrate based test tubes provide the best pre-analytical condition available. Furthermore, glucose levels are not stable in heparinized blood placed in an ice/water slurry. We strongly advise the use of NaF-EDTA-citrate based test tubes in diabetes research.

Determination of the calibration factor of polysulphone film UV dosemeters for terrestrial solar radiation.

Polysulphone film is used as a personal UV dosemeter in dermatological or epidemiological studies. The relative efficiency of this detector does not exactly match the action spectrum as proposed by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) and to which the UV dose and exposure limits refer. Therefore, the calibration of the dosemeter depends on the spetrum. In the present paper the variation of the calibration factor for terrestrial solar UV spectra is analysed on the basis of a two year observation period at a site near Munich. Germany. A detailed error estimation is included. It is shown that the variation of the calibration factor within this class of spectra is the main contribution to the total uncertainty of the dose determination, which can be up to 40%. The shape of the spectrum of terrestrial solar UV radiation is mainly determined by the total ozone column and the solar elevation angle. It is shown how the calibration depends on these two parameters and how this additional information can help to reduce the measurement error to a residual uncertainty of 17%. Exposure studies of terrestrial solar UV radiation using polysulphone film as a dosemeter would gain in accuracy if total ozone column values at the study's site could be measured or taken from satellite or weather service data. The interpretation of the magnitude of the dose uncertainty depends on the further use of these data.

Purification and characterization of two mannan-binding lectins from mouse serum.

Mannan-binding lectin (MBL) is a serum protein that activates the complement system after binding to glycoconjugates found on the surface of microorganisms. By molecular cloning two forms of MBL have been identified in the mouse (mMBL-A and mMBL-C), but only mMBL-A has been purified and characterized at the protein level. MBL-C has been termed the liver form of MBL. The present report describes the purification and characterization of mMBL-A and mMBL-C from serum. The two forms of mMBL could be separated both by ion-exchange and carbohydrate-affinity chromatography. The initial identification by immunochemical technique was confirmed by N-terminal amino-acid sequencing. Both proteins give bands corresponding to polypeptide chains of 28 kDa on SDS-PAGE in the reduced state, but mMBL-A migrated more rapidly than mMBL-C in acid/urea-PAGE, in accordance with the calculated pIs. Both forms mediated activation of complement component C4 in mannan-coated microtiter wells. MBL-A showed a higher affinity for d -glucose and alpha-methyl-d -glucose then did MBL-C. Serum concentrations of mMBL-A in laboratory strains and wild mice were found to vary from 5 to 80 microg/ml, with wild mice tending to show higher levels than laboratory strains.