Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer.

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 microl of diluted nipple aspirate fluid over a 5-40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75-84% of samples from women with cancer but in only 0-9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer.

Thrombin-induced platelet aggregation is inhibited by the heptapeptide Leu271-Ala277 of domain 3 in the heavy chain of high molecular weight kininogen.

The ability of kininogens to modulate thrombin-induced aggregation of human platelets has been assigned to domain 3 (D3) in the common heavy chain coded for by exons 7, 8, and 9 of kininogen gene. We expressed each of the exons 7, 8, and 9, and various combinations as glutathione S-transferase fusion proteins in Escherichia coli. Each of the exon products 7 (Lys236-Gln292), 9 (Val293-Gly328), and 8 (Gln329-Met357), and their combinations were evaluated for the ability to inhibit thrombin induced platelet aggregation. Only products containing exon 7 inhibited platelet aggregation induced by thrombin with an IC50 of > 20 microM. A deletion mutant of exon 7 product, polypeptide 7A product (Lys236-Lys270) did not block thrombin-induced platelet aggregation, while 7B product (Thr255-Gln292) and 7C product (Leu271-Gln292) inhibited aggregation. These findings indicated that the inhibitory activity is localized to residues Leu271-Gln292. Peptides Phe279-Ile283 and Phe281-Gln292 did not block thrombin, and Asn275-Phe279 had only minimal inhibitory activity. A heptapeptide Leu271-Ala277 inhibited thrombin-induced aggregation of platelets with an IC50 of 65 microM. The effect is specific for the activation of platelets by thrombin but not ADP or collagen. No evidence for a thrombin-kininogen complex was found, and neither HK nor its derivatives directly inhibited thrombin activity. Knowledge of the critical sequence of kininogen should allow design of compounds that can modulate thrombin activation of platelets.

Contiguous binding and inhibitory sites on kininogens required for the inhibition of platelet calpain.

Both high molecular weight kininogen (HK) and low molecular weight kininogens (LK) are potent tight binding inhibitors of platelet calpain (Ki = 2 nM), but the molecular basis for the inhibitory function is not well delineated. The amino acid sequences of the calpain inhibitory domain 2 from human and rat HK were compared for homology with the noninhibitory domains from human and rat domain 3 and from domain 2 of rat T-kininogen, and two areas of nonconserved differences were detected. Computer three-dimensional models were constructed on a template built using the x-ray crystallographic data for cystatin, an evolutionary precursor of HK. Two nonconserved regions in the calpain inhibitory domains flank the highly conserved motif QVVAG to form a continuous surface for interaction with cysteine proteases. Three peptide sequences, components of the modeled surface, were chosen for synthesis from HK D-2: VHPISTQSPDLE (peptide 146-156, NH2-terminal), CTDNAYIDIQLRIASFSQNC (peptide 229-248, COOH-terminal), and CQRQVVAGLNFRIC (185-189, central) containing QVVAG. This last peptide differs from the natural sequence by substitutions of A185C and T195C. Peptides 185-198 and 229-248 were folded by air oxidation of their cysteine residues and then tested for their ability to inhibit calpain and papain. The folded peptide 229-248 inhibited calpain with an IC50 35 microM and unfolding reduced this effect. The folded peptide 185-198 did not inhibit calpain, but when preincubated with calpain, could block the inhibition by HK indicating a probable enzyme binding site. Peptide 146-157 did not inhibit calpain but could inhibit papain with an IC50 of 20 microM. We have thus defined separate binding and inhibitory sequences on HK which form a contiguous surface for thiol protease interactions.

Nonlinear viscoelastic properties of articular cartilage in shear.

The strain dependence of the intrinsic viscoelastic properties of the cartilage matrix in shear was investigated. Stress relaxation experiments were performed on bovine articular cartilage at shear strains ranging from approximately 3% to 16%. The tissue was found to exhibit nonlinear strain-dependent viscoelastic behavior, with the nonlinearity occurring primarily in the short-time transient during stress relaxation. In addition, the equilibrium stress was found to fit a quadratic relation with strain. This relationship was noted to be nearly linear with strain from 3% to 16%. The instantaneous stress was seen to be highly nonlinear, and followed a cubic relationship with applied shear strain. Fung's quasilinear theory can be used to describe the stress relaxation response over the range of strains examined when a nonlinear regression is performed to determine an "average" normalized relaxation function. Alternately, strain dependence can be incorporated into the model to describe and predict more accurately the strain-dependent stress relaxation response.