A novel and automatic mammographic texture resemblance marker is an independent risk factor for breast cancer.

OBJECTIVE: We investigated whether breast cancer is predicted by a breast cancer risk mammographic texture resemblance (MTR) marker. METHODS: A previously published case-control study included 495 women of which 245 were diagnosed with breast cancer. In baseline mammograms, 2-4 years prior to diagnosis, the following mammographic parameters were analysed for relation to breast cancer risk: (C) categorical parenchymal pattern scores; (R) radiologist's percentage density, (P) computer-based percentage density; (H) computer-based breast cancer risk MTR marker; (E) computer-based hormone replacement treatment MTR marker; and (A) an aggregate of P and H. RESULTS: Density scores, C, R, and P correlated (tau=0.3-0.6); no other pair of scores showed large (tau>0.2) correlation. For the parameters, the odds ratios of future incidence of breast cancer comparing highest to lowest categories (146 and 106 subject respectively) were C: 2.4(1.4-4.2), R: 2.4(1.4-4.1), P: 2.5(1.5-4.2), E: non-significant, H: 4.2(2.4-7.2), and A: 5.6(3.2-9.8). The AUC analysis showed a similarly increasing pattern (C: 0.58±0.02, R: 0.57±0.03, P: 0.60±0.03, H: 0.63±0.02, A: 0.66±0.02). The AUC of the aggregate marker (A) surpasses others significantly except H. HRT-MTR (E) did not significantly identify future cancers or correlate with any other marker. CONCLUSIONS: Breast cancer risk MTR marker was independent of density scores and more predictive of risk. The hormone replacement treatment MTR marker did not identify patients at risk.

Parallel assessment of the impact of different hormone replacement therapies on breast density by radiologist- and computer-based analyses of mammograms.

OBJECTIVES: First, to compare the impact of nasally and orally dosed estradiol on breast density; second, to investigate the utility of computer-based automated approaches to the assessment of breast density with reference to traditional methods. METHODS: Digitized images from two 2-year, randomized, placebo-controlled trials formed the basis of the present post hoc analysis. Active treatments were 1 mg estradiol continuously combined with 0.125 mg trimegestone (oral hormone replacement therapy, HRT) or low-dose (150 or 300 microg estradiol) nasal estradiol cyclically combined with 200 mg micronized progesterone (nasal HRT). The effects on breast density were assessed by a radiologist, providing the BI-RADS score and the interactive threshold, and by a computer-based approach, providing the measure of stripiness and the HRT-effect specific measure of breast density. RESULTS: In the oral HRT trial, active treatment induced a significant increase in breast density, which was consistent in all methods used (all p < 0.05). In contrast, none of the methods detected significant changes in women receiving nasal HRT. The sensitivity of automated methods to discriminate HRT- from placebo-treated women was equal or better than the sensitivity of methods performed by the radiologist. CONCLUSIONS: The markedly different pharmacokinetic profile of nasal estrogen seems to be associated with better breast safety. Automated computer-based analysis of digitized mammograms provides a sensitive measure of changes in breast density induced by hormones and could serve as a useful tool in future clinical trials.

Filter learning: application to suppression of bony structures from chest radiographs.

A novel framework for image filtering based on regression is presented. Regression is a supervised technique from pattern recognition theory in which a mapping from a number of input variables (features) to a continuous output variable is learned from a set of examples from which both input and output are known. We apply regression on a pixel level. A new, substantially different, image is estimated from an input image by computing a number of filtered input images (feature images) and mapping these to the desired output for every pixel in the image. The essential difference between conventional image filters and the proposed regression filter is that the latter filter is learned from training data. The total scheme consists of preprocessing, feature computation, feature extraction by a novel dimensionality reduction scheme designed specifically for regression, regression by k-nearest neighbor averaging, and (optionally) iterative application of the algorithm. The framework is applied to estimate the bone and soft-tissue components from standard frontal chest radiographs. As training material, radiographs with known soft-tissue and bone components, obtained by dual energy imaging, are used. The results show that good correlation with the true soft-tissue images can be obtained and that the scheme can be applied to images from a different source with good results. We show that bone structures are effectively enhanced and suppressed and that in most soft-tissue images local contrast of ribs decreases more than contrast between pulmonary nodules and their surrounding, making them relatively more pronounced.

Bi-substrate analogue ligands for affinity chromatography of protein kinases.

Novel affinity ligands, consisting of ATP-resembling part coupled with specificity determining peptide fragment, were proposed for purification of protein kinases. Following this approach affinity sorbents based on two closely similar ligands AdoC-Aoc-Arg4-Lys and AdoC-Aoc-Arg4-NH(CH2)6NH2, where AdoC stands for adenosine-5'-carboxylic acid and Aoc for amino-octanoic acid, were synthesized and tested for purification of recombinant protein kinase A catalytic subunit directly from crude cell extract. Elution of the enzyme with MgATP as well as L-arginine yielded homogeneous protein kinase A preparation in a single purification step. Also protein kinase A from pig heart homogenate was selectively isolated using MgATP as eluting agent. Protein kinase with acidic specificity determinant (CK2) as well as other proteins possessing nucleotide binding site (L-type pyruvate kinase) or sites for wide variety of different ligands (bovine serum albumin) did not bind to the column, pointing to high selectivity of the bi-functional binding mode of the affinity ligand.

Calcium-dependent protein kinase from maize seedlings activated by phospholipids.

A calcium- and phospholipid-dependent protein kinase of apparent molecular mass 54 kDa (designated ZmCPKp54) was partially purified from etiolated maize seedlings. Activity of ZmCPKp54 is stimulated by phosphatidylserine and phosphatidylinositol, but is not essentially affected by diolein and phorbol esters. The enzyme cross-reacts with polyclonal antibodies against the calmodulin like-domain of the calcium-dependent protein kinase, but not with antibodies against catalytic or regulatory domains of protein kinase C. ZmCPKp54 is not able to phosphorylate the specific substrates of protein kinase C (MARCKS peptide and protein kinase C substrate peptide derived from pseudosubstrate sequence) and its activity is not inhibited by specific PKC inhibitors (bisindolylmaleimide, protein kinase C pseudosubstrate inhibitory peptide). The substrate specificity and sensitivity to the inhibitors of the maize enzyme resembles calcium-dependent protein kinase. The biochemical and immunological properties indicate that ZmCPKp54 belongs to the calcium-dependent protein kinase family.

Peptide phosphorylation by calcium-dependent protein kinase from maize seedlings.

Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS15VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cbeta (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L-5X-4R-3X-2X-1SX+1R+2Z+3R+4, where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA.

Kinetic study of the inhibition of CK2 by heparin fragments of different length.

The structure-activity relationships for the inhibition of protein kinase CK2 by heparin were investigated using purified heparin fragments of different length, varying from 4 to 24 oligosaccharide sugar units. The inhibitory potency was shown to decrease concomitant with the shortening of the heparin fragment length. The fragment of 24 oligosaccharide sugar units was the most potent inhibitor with a K(i) value of 22 nM which is close to the K(i) value for the commercial heparin mixture available. Shortening of the heparin from 24 to 12 sugar units had a moderate influence on the inhibitory potency causing an increase in K(i) values up to 151 nM while fragments shorter than 12 sugar units showed a more drastic increase in K(i) values reaching up to micromolar range. The mode of inhibition was studied in respect to the protein substrate beta-casein and it was shown to be competitive for the long as well as for the short heparin fragments. In contrast, the inhibition mode in respect to a synthetic peptide substrate RRRADDSDDDDD was found to be hyperbolic partial non-competitive mixed-type. Such a kinetic model suggests that heparin binds to a site on CK2 which does not overlap with the peptide substrate binding site and that a productive enzyme complex exists where both heparin and peptide substrate are simultaneously bound. This is in contrast to the competitive inhibition model of the phosphorylation of protein substrate beta-casein where the binding of the protein substrate and inhibitor was mutually exclusive.

Adenosine-5'-carboxylic acid peptidyl derivatives as inhibitors of protein kinases.

A new class of protein kinase bisubstrate-analog inhibitors was designed on the basis of adenosine-5'-carboxylic acid derivatives, where a short peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. The potency and selectivity of these inhibitors were adjusted by relevant combination of these structural fragments, resembling the structure of the bisubstrate complex of the peptide phosphorylation reaction.

Contribution of endopeptidase 3.4.24.15 to central neurotensin inactivation.

The tridecapeptide, neurotensin elicits naloxone-insensitive analgesia after its intracebroventricular administration in mice. We used this central pharmacological effect to assess the putative contribution of the endopeptidase 3.4.24.15 to central inactivation of the peptide. By means of combinatorial chemistry, we previously designed the first potent endopeptidase 3.4.24.15 inhibitor. This agent, Z-(L,D)Phe psi(PO2CH2)(L,D)Ala-Lys-Met (phosphodiepryl 21), is shown here to behave as a fully specific endopeptidase 3.4.24.15 inhibitor, as demonstrated by the absence of effect on a series of other exo- and endopeptidases belonging to various classes of proteolytic activities present in murine brain membranes. Furthermore, central administration of phosphodiepryl 21 drastically prolongs the forepaw licking latency of mice tested on the hot plate and injected with sub-maximally active doses of neurotensin. Altogether, our results demonstrated that, in addition to endopeptidase 3.4.24.16, endopeptidase 3.4.24.15 likely contributes to the physiological termination of the neurotensinergic message in murine brain.

Effect of a novel selective and potent phosphinic peptide inhibitor of endopeptidase 3.4.24.16 on neurotensin-induced analgesia and neuronal inactivation.

1. We have examined a series of novel phosphinic peptides as putative potent and selective inhibitors of endopeptidase 3.4.24.16. 2. The most selective inhibitor, Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 displayed a Ki value of 12 nM towards endopeptidase 3.4.24.16 and was 5540 fold less potent on its related peptidase endopeptidase 3.4.24.15. Furthermore, this inhibitor was 12.5 less potent on angiotensin-converting enzyme and was unable to block endopeptidase 3.4.24.11, aminopeptidases B and M, dipeptidylaminopeptidase IV and proline endopeptidase. 3. The effect of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2, in vitro and in vivo, on neurotensin metabolism in the central nervous system was examined. 4. Pro-Phe-psi(PO2CHH2)-Leu-Pro-NH2 dose-dependently inhibited the formation of neurotensin 1-10 and concomittantly protected neurotensin from degradation by primary cultured neurones from mouse embryos. 5. Intracerebroventricular administration of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 significantly potentiated the neurotensin-induced antinociception of mice in the hot plate test. 6. Altogether, our study has established Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 as a fully selective and highly potent inhibitor of endopeptidase 3.4.24.16 and demonstrates, for the first time, the contribution of this enzyme in the central metabolism of neurotensin.