Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides.

A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed.     

Chemical synthesis of new trypsin, chymotrypsin and elastase inhibitors by amino-acid substitutions in a trypsin inhibitor from squash seeds (CMTI III)

Five new analogues of squash trypsin inhibitor CMTI III were obtained by the solid-phase method, exhibiting antitrypsin, antichymotrypsin or antielastase activity. Modification in the reactive site region changes dramatically the specificity, whereas substitution in non-contact positions facilitates the refolding of the reduced form of the peptides and does not have a significant influence on the association equilibrium constants of the inhibitor analogues with cognate enzymes.     

Synthesis of an elastase inhibitor by monosubstitution of arginine-5 with valine at the reactive site in a trypsin inhibitor from squash seeds (CMTI III)

Using the solid-phase procedure an analog of trypsin inhibitor CMTI III containing Val5 instead of Arg5 in position P1, was synthesized. The substitution in only this one position P1 increased the affinity of synthetic inhibitor to porcine pancreatic elastase and human leukocyte elastase by the factor of 10(3) and 10(7), respectively.     

Linkage analysis and allelic imbalance in human breast cancer kindreds using microsatellite markers from the short arm of chromosome 3

Eight Icelandic breast cancer kindreds were subjected to linkage analyses with respect to 28 microsatellite loci dispersed along the short arm of chromosome 3. Breast tumors derived from these kindreds were concurrently scored for allelic imbalance with ten of the markers. Linkage to most markers could be excluded on the basis of negative LOD scores and haplotype analyses, although some moderately positive LOD scores resulted. A high frequency of imbalance in the familial tumors was seen with two of the markers in comparison with results obtained from sporadic material. The highest frequency (68%) of imbalance was detected with the marker D3S1217, which is located on 3p14.2-p14.1. Imbalance at the D3S1211 locus, which is more telomeric (3p24.2-p22), was not significantly elevated in the familial tumors. We suggest that the genetic defect responsible for breast cancer susceptibility in these families either promotes instability in the 3p14.2-p14.1 region or enhances the selective advantage of such changes.     

Separation of basic,hydrophilic peptides by reversed-phase ion-pair chromatography: II. Analytical applications with particular reference to phosphoserine peptides

Phosphoserine peptides, obtained by phosphorylation of synthetic precursors with cyclic AMP-dependent protein kinase, can be efficiently separated from the corresponding non-phosphorylated peptides and from each other by ion-pair high-performance liquid chromatography. All experiments were performed under isocratic conditions on a C₁₈ column, using phosphate buffers with pH 3.2–4.5, n-hexane sulfonic acid as counter ion, and ethanol as organic modifier.     

Phosphopeptide substrates of a phosphoprotein phosphatase from rat liver

The substrate specificity of a preparation of phosphoprotein phosphatase (Mr = 32 000) from rat liver was investigated. Phosphopeptides based on the structure Leu-Arg-Arg-Ala-Ser(P)-Val-Ala-Glx-Leu and Ala-Arg-Thr-Lys-Arg-Ser-Gly-Ser(P)-Val-Tyr-Glu-Pro-Leu-Lys were used. These phosphopeptides correspond to the phosphorylation sites of rat liver pyruvate kinase (type L) and the beta subunit of rabbit muscle phosphorylase b kinase, respectively. A decrease in the apparent Km values and a concomitant increase in Vmax values was observed when the number of amino acyl residues after the phosphoseryl residue in the respective phosphopeptides were increased from 2 to 4, 5, or 6. Most of the phosphopeptides investigated generally showed apparent Km values higher than the values obtained with phosphopyruvate kinase. Ala-Ser(P)-Val-Ala and Gly-Ser(P)-Val-Tyr appeared to be the shortest phosphopeptides that could be dephosphorylated rapidly. These findings support the hypothesis that a small part of the phosphoprotein may be sufficient to fulfill the minimal requirements for its dephosphorylation.     

Quantifying Cancer Absolute Risk and Cancer Mortality in the Presence of Competing Events after a Myotonic Dystrophy Diagnosis

Recent studies show that patients with myotonic dystrophy (DM) have an increased risk of specific malignancies, but estimates of absolute cancer risk accounting for competing events are lacking. Using the Swedish Patient Registry, we identified 1,081 patients with an inpatient and/or outpatient diagnosis of DM between 1987 and 2007. Date and cause of death and date of cancer diagnosis were extracted from the Swedish Cause of Death and Cancer Registries. We calculated non-parametric estimates of absolute cancer risk and cancer mortality accounting for the high non-cancer competing mortality associated with DM. Absolute cancer risk after DM diagnosis was 1.6% (95% CI=0.4-4%), 5% (95% CI=3-9%) and 9% (95% CI=6-13%) at ages 40, 50 and 60 years, respectively. Females had a higher absolute risk of all cancers combined than males: 9% (95% CI=4-14), and 13% (95% CI=9-20) vs. 2% (95%CI= 0.7-6) and 4% (95%CI=2-8) by ages 50 and 60 years, respectively) and developed cancer at younger ages (median age =51 years, range=22-74 vs. 57, range=43-84, respectively, p=0.02). Cancer deaths accounted for 10% of all deaths, with an absolute cancer mortality risk of 2% (95%CI=1-4.5%), 4% (95%CI=2-6%), and 6% (95%CI=4-9%) by ages 50, 60, and 70 years, respectively. No gender difference in cancer-specific mortality was observed (p=0.6). In conclusion, cancer significantly contributes to morbidity and mortality in DM patients, even after accounting for high competing DM mortality from non-neoplastic causes. It is important to apply population-appropriate, validated cancer screening strategies in DM patients.     

Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers

Permeability coefficients across monolayers of the human colon carcinoma cell line Caco-2, cultured on permeable supports, are commonly used to predict the absorption of orally administered drugs and other xenobiotics. This protocol describes our method for the cultivation, characterization and determination of permeability coefficients of xenobiotics (which are, typically, drug-like compounds) in the Caco-2 model. A few modifications that have been introduced over the years are incorporated in the protocol. The method can be used to trace the permeability of a test compound in two directions, from the apical to the basolateral side or vice versa, and both passive and active transport processes can be studied. The permeability assay can be completed within one working day, provided that the Caco-2 monolayers have been cultured and differentiated on the permeable supports 3 weeks in advance.