Validation of a decision support system for the cytodiagnosis of fine needle aspirates of the breast using a prospectively collected dataset from multiple observers in a working clinical environment

We have used a 692 case dataset, collected retrospectively by a single observer, to develop decision support systems for the cytodiagnosis of fine needle aspirates of breast lesions. In this study, we use a 322 case dataset that was prospectively collected by multiple observers in a working clinical environment to test two predictive systems, using logistic regression and the multilayer perceptron (MLP) type of neural network. Ten observed features and the patient age were used as input features. The systems were developed using a training set and test set from the single observer dataset and then applied to the multiple observer dataset. For the independent test cases from the single observer dataset, with a threshold set for no false positives on the training set, logistic regression produced a sensitivity of 82% (95% confidence interval 73-91) and a predictive value of a positive result (PV +) of 98% (95-99), the values for the MLP were 79% (69-89) and 100%, respectively. However the performance on the prospective multiple observer dataset was much worse, with a sensitivity of 72% (65-80), and PV + of 97% (94-99) for logistic regression and 67% (60-75) and 91% (85-97) for the MLP. These results suggest that there is considerable interobserver variability for the defined features and that this system is unsuitable for further development in the clinical environment unless this problem can be overcome.     

Hypothalamic-pituitary-adrenal responses to short-duration high-intensity cycle exercise

beta-Endorphin (beta-EP), adrenocorticotropin (ACTH), and cortisol plasma concentrations were examined before and after maximal exercise at four intensities [36, 55, 73, and 100% of maximal leg power (MLP)] by means of a computerized cycle ergometer. All intensities were greater than those eliciting peak O2 uptake for the individual subjects. Blood samples were collected at rest, immediately after exercise, and at 5 and 15 min postexercise. Significant (P less than 0.05) increases were observed at 36% MLP for beta-EP and ACTH immediately after exercise and at 5 and 15 min postexercise. Plasma cortisol increased at 36% MLP at 15 min postexercise. Blood lactate significantly increased at all postexercise collection points for exercise intensities of 36, 55, and 73% MLP and at 5 min postexercise for 100% MLP. beta-EP concentrations at 36% MLP were significantly correlated (r = 0.75) with capillary density (mm-2), and cortisol concentrations at 36% MLP were significantly correlated (r = 0.89) with percentage of type II muscle fibers. No other significant relationships were observed. These data show that brief, high-intensity exercise up to maximal power production results in a nonlinear response pattern in peripheral blood hormone concentrations. Furthermore, blood lactate levels do not appear to be related to hypothalamic-pituitary-adrenal hormone plasma concentrations at high exercise intensities.     

Molecular Evolution of Miraculin-Like Proteins in Soybean Kunitz Super-Family

Miraculin-like proteins (MLPs) belong to soybean Kunitz super-family and have been characterized from many plant families like Rutaceae, Solanaceae, Rubiaceae, etc. Many of them possess trypsin inhibitory activity and are involved in plant defense. MLPs exhibit significant sequence identity (~30-95%) to native miraculin protein, also belonging to Kunitz super-family compared with a typical Kunitz family member (~30%). The sequence and structure-function comparison of MLPs with that of a classical Kunitz inhibitor have demonstrated that MLPs have evolved to form a distinct group within Kunitz super-family. Sequence analysis of new genes along with available MLP sequences in the literature revealed three major groups for these proteins. A significant feature of Rutaceae MLP type 2 sequences is the presence of phosphorylation motif. Subtle changes are seen in putative reactive loop residues among different MLPs suggesting altered specificities to specific proteases. In phylogenetic analysis, Rutaceae MLP type 1 and type 2 proteins clustered together on separate branches, whereas native miraculin along with other MLPs formed distinct clusters. Site-specific positive Darwinian selection was observed at many sites in both the groups of Rutaceae MLP sequences with most of the residues undergoing positive selection located in loop regions. The results demonstrate the sequence and thereby the structure-function divergence of MLPs as a distinct group within soybean Kunitz super-family due to biotic and abiotic stresses of local environment.     

Melphalan, a second chemical for which specific-locus mutation induction in the mouse is maximum in early spermatids.

Melphalan (MLP), a bifunctional alkylating agent structurally related to the highly mutagenic chemical chlorambucil (CHL), was found to induce high frequencies of specific-locus mutations in postspermatogonial germ cells of the mouse, and to be one of only a few chemicals that is also mutagenic in spermatogonial stem cells. Productivity patterns following MLP exposures resembled those that had been found for CHL. Mutation rates in successive male germ-cell stages were measured at three MLP-exposure levels in a total of 95,375 offspring. While the induced (experimental minus historical-control) mutation rate is relatively low in stem-cell spermatogonia (1.2 x 10(-5) per locus at a weighted-mean exposure of 7.3 mg/kg), it is about 5 times higher in poststem-cell stages overall, and peaks at 26.7 x 10(-5) per locus in early spermatids at a weighted-mean exposure of only 5.7 mg/kg. This "type-2 pattern" of mutation yield (Russell et al., 1990), i.e., peak sensitivity in early spermatids, has heretofore been found for only one other chemical, CHL. Mutation-rate data earlier reported for CHL (Russell et al., 1989) were augmented in the present study for comparison with MLP-induced rates. Because of the greater toxicity of MLP, average exposures used for this chemical were only about one-half of those for CHL. When MLP and CHL mutation rates are extrapolated to equimolar doses, they appear very similar for poststem-cell stages overall. However, in the case of CHL, a somewhat higher proportion of the mutations is induced in early spermatids than in the case of MLP.     

A protein whose binding to Na,K-ATPase is regulated by ouabain

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.     

Effects of high-intensity cycle exercise on sympathoadrenal-medullary response patterns

Plasma proenkephalin peptide F immunoreactivity and catecholamines were examined on separate days in nine healthy males before and after maximal exercise to exhaustion at four intensities [36, 55, 73, and 100% of maximal leg power (MLP)] by use of a computerized cycle ergometer. The mean duration of 36, 55, 73, and 100% MLP was 3.31, 0.781, 0.270, and 0.1 min, respectively. All intensities were greater than those eliciting peak O2 uptake for the individual subjects. Blood samples were obtained before, immediately after exercise, and 5 and 15 min after exercise. Significant (P less than 0.05) increases in plasma peptide F immunoreactivity (i.e., from mean resting value of 0.18 to 0.43 pmol/ml) were observed immediately after exercise at 36% MLP. Significant increases in plasma epinephrine were observed immediately after exercise at 36% MLP (i.e., from mean resting value of 2.22 to 3.11 pmol/ml) and 55% MLP (i.e., from mean resting value of 1.67 to 2.98 pmol/ml) and 15 min after exercise at 100% MLP (i.e., from mean resting value of 1.92 to 3.88 pmol/ml). Significant increases for plasma norepinephrine were observed immediately after exercise (36, 55, 73, and 100% MLP), 5 min after exercise (36, 55, and 73% MLP), and 15 min after exercise (36% MLP). Increases in whole blood lactate were observed at all points after exercise for 36, 55, and 73% MLP and 5 min after exercise for 100% MLP. These data show that brief high-intensity exercise results in differential response patterns of catecholamines and proenkephalin peptide F immunoreactivity.     

Prenatal diet determines susceptibility to cardiac ischaemia-reperfusion injury following treatment with diethylmaleic acid and N-acetylcysteine

Fetal undernutrition programmes increased risk of developing cardiovascular disease in adult life. We hypothesized that prenatal protein restriction would impair recovery in post-ischaemic cardiac function in adult offspring through antioxidant-mediated processes. Pregnant Wistar rats were fed control or maternal low protein diets (MLP) throughout gestation. The offspring of these rats were treated with either saline, N-acetylcysteine (NAC), diethylmaleate (DEM), or both NAC and DEM to manipulate glutathione status at 6 months of age. Hearts were rapidly excised and retro-perfused (Langendorff) to assess isolated cardiac function before (baseline), and during 30 min global ischaemia and 60 min reperfusion. Hearts from adult rats exposed to a MLP diet in utero suffered greater cardiac dysfunction than those from controls following 30 min ischaemia. Left ventricular developed pressure (LVDP) was significantly reduced upon early reperfusion (p<0.042) in MLP rats compared to controls. NAC pre-treatment had no effect on LVDP of hearts from control animal hearts but improved the revival of MLP hearts to the same level as controls. DEM treatment did not affect control hearts but significantly reduced recovery of LVDP of MLP hearts during early (p<0.008) and late reperfusion (0.035). Combined NAC and DEM treatment had no effect on LVDP between control and MLP fed offspring. Prenatal protein restriction throughout pregnancy increases the susceptibility of the adult rat heart to suffer a functional deficit following ischaemia-reperfusion injury. Pharmacologically improving antioxidant status prevented this injury. A nutritionally-imbalanced developmental environment may increase susceptibility to coronary heart disease through the programming of myocardial glutathione metabolism.     

Identification of sequence-specific DNA-binding factors by label transfer: application to the adenovirus-2 major late promoter.

A method of affinity labelling proteins specifically associated with DNA target sequences is proposed. The method utilizes covalent UV-crosslinking of proteins to highly labelled DNA (e.g. in crude cell or nuclear extracts) followed by degradation of the DNA to short oligonucleotides. Proteins selectively labelled by attached residual oligonucleotides are readily amenable to molecular mass determination. Using this approach, we have characterized a HeLa polypeptide specifically bound to a short segment of the adenovirus-2 major late promoter (Ad2 MLP). A molecular mass value (approximately 51 kD) and precise location of the crosslinking site(s) of the protein within the MLP (-55 with respect to the cap site) were determined.     

Cellular and functional defects in a mouse model of heart failure

Heart failure and dilated cardiomyopathy develop in mice that lack the muscle LIM protein (MLP) gene (MLP(-/-)). The character and extent of the heart failure that occurs in MLP(-/-) mice were investigated using echocardiography and in vivo pressure-volume (P-V) loop measurements. P-V loop data were obtained with a new method for mice (sonomicrometry) using two pairs of orthogonal piezoelectric crystals implanted in the endocardial wall. Sonomicrometry revealed right-shifted P-V loops in MLP(-/-) mice, depressed systolic contractility, and additional evidence of heart failure. Cellular changes in MLP(-/-) mice were examined in isolated single cells using patch-clamp and confocal Ca(2+) concentration ([Ca(2+)]) imaging techniques. This cellular investigation revealed unchanged Ca(2+) currents and Ca(2+) spark characteristics but decreased intracellular [Ca(2+)] transients and contractile responses and a defect in excitation-contraction coupling. Normal cellular and whole heart function was restored in MLP(-/-) mice that express a cardiac-targeted transgene, which blocks the function of beta-adrenergic receptor (beta-AR) kinase-1 (betaARK1). These data suggest that, despite the persistent stimulus to develop heart failure in MLP(-/-) mice (i.e., loss of the structural protein MLP), downregulation and desensitization of the beta-ARs may play a pivotal role in the pathogenesis. Furthermore, this work suggests that the inhibition of betaARK1 action may prove an effective therapy for heart failure.     

Synergistic up-regulation of muscle LIM protein expression in C2C12 and NIH3T3 cells by myogenin and MEF2C

Although the role of muscle LIM protein (MLP, also known as CRP3), a LIM-only protein of LIM domain-containing protein family, is well-characterized, the mechanism by which the MLP gene expresses remains unclear. Herein, we demonstrate that myogenin and myocyte enhancer factor 2C (MEF2C) cooperate in activating the MLP gene in myogenesis. RT-PCR, real-time PCR and Western blotting showed that overexpression of myogenin or myogenin plus MEF2C led to induction of the MLP gene in differentiating C2C12 and NIH3T3 fibroblasts. By contrary, knocking-down of myogenin by RNA interference (RNAi) suppressed MLP expression in differentiating C2C12. Deletion and reporter enzyme assay revealed that the promoter activity was determined largely by the region extending from −260 to −173, which containing three E-box (CANNTG motif) candidates. Site-directed mutagenesis demonstrated that the E-box at position −186 to −180 was crucial for activating the promoter by myogenin. Furthermore, MEF2C could enhance myogenin-mediated activation of the promoter. In addition, chromatin immunoprecipitation (ChIP) and re-ChIP showed that myogenin and MEF2C were associated with the activated MLP promoter. Together, these results suggest that myogenin and MEF2C cooperate in the MLP gene activation. The linking of the MLP gene activation with myogenin and MEF2C may facilitate myogenin-mediated differentiation of striated muscle.     

Interaction of the adenovirus IVa2 protein with viral packaging sequences

We have demonstrated previously that the adenovirus L1 52/55-kDa protein binds to the viral IVa2 protein in infected cells. The significance of this interaction was unclear, however, based on the known functions of these two proteins: the 52/55-kDa protein is required for viral DNA packaging, while the IVa2 protein is a transactivator of the major late promoter (MLP). In this report, we have attempted to elucidate a role for each of the two proteins in the other's known function. There is no apparent effect of the 52/55-kDa protein on the interaction of the IVa2 protein with the MLP. Surprisingly, however, we found that the IVa2 protein can interact with the adenoviral packaging signal and that this interaction involves DNA sequences that have previously been demonstrated to be required for packaging.     

Purification and characterization of a unique alkaline elastase from Micrococcus luteus

Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.     

Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo.

We have constructed recombinant adenoviruses (Ad), with functional or defective E1a genes, which harbor either the hepatitis B (HB) virus s gene encoding the HB surface antigen, as well as the pre-S2 epitopes, or the bacterial gene encoding chloramphenicol acetyltransferase (CAT) under control of the Ad major late promoter (MLP). The recombinant viruses defective for E1a (Ad.MLP.S2 and Ad.CAT), which can be efficiently propagated only on 293 cells that complement this defect, and the nondefective (Ad.MLP.S2.E1A) recombinant were used to infect a wide spectrum of cells of different origin. The yields of HBs and CAT proteins obtained with these different recombinant viruses demonstrate no real advantage to using nondefective vectors, whatever the cell type infected. The injection into chimpanzees of Ad.MLP.S2 does not elicit the production of antibodies, but can immunologically prime the animals, resulting in a partial protection against HBV challenge.