Detection of mitochondrial dysfunction by EPR technique in mouse model of dilated cardiomyopathy

Tgalphaq44 mice with targeted overexpression of activated Galphaq protein in cardiomyocytes mimic many of the phenotypic characteristics of dilated cardiomyopathy in humans. However, it is not known whether the phenotype of Tgalphaq44 mice would also involve dysfunction of cardiac mitochondria. The aim of the present work was to examine changes in EPR signals of semiquinones and iron in Fe-S clusters, as compared to classical biochemical indices of mitochondrial function in hearts from Tgalphaq44 mice in relation to the progression of heart failure. Tgalphaq44 mice at the age of 14 months displayed pulmonary congestion, increased heart/body ratio and impairment of cardiac function as measured in vivo by MRI. However, in hearts from Tgalphaq44 mice already at the age of 10 months EPR signals of semiquinones, as well as cyt c oxidase activity were decreased, suggesting alterations in mitochondrial electron flow. Furthermore, in 14-months old Tgalphaq44 mice loss of iron in Fe-S clusters, impaired citrate synthase activity, and altered mitochondrial ultrastructure were observed, supporting mitochondrial dysfunction in Tgalphaq44 mice. In conclusion, the assessment of semiquinones content and Fe(III) analysis by EPR represents a rational approach to detect dysfunction of cardiac mitochondria. Decreased contents of semiquinones detected by EPR and a parallel decrease in cyt c oxidase activity occurs before hemodynamic decompensation of heart failure in Tgalphaq44 mice suggesting that alterations in function of cardiac mitochondria contribute to the development of the overt heart failure in this model.     

Immunocytochemical evaluation of metallothionein (MT) expression in myoepithelial cells of ductal mammary carcinoma and its relation to survival time: analysis of 7-year course of the disease

Myoepithelial cells participate in the development of mammary glands and have been suggested to play a role in the biology of mammary cancer. Recent studies demonstrated in the cells expression and overexpression of metallothionein (MT): a low molecular weight, cystein-rich protein which participates, i.a., in the multidrug resistance to cvtostatic drugs. The present study was aimed at examining the relation between results of immunocytochemical (ICC) detection of MT expression in myoepithelial cells, present in structures of a ductal mammary carcinoma, and survival of the patients. In sections originating from 43 patients with ductal mammary carcinoma ICC reactions were performed to detect MT and to confirm the presence of myoepithelial cells (using antibodies to smooth muscle actin). Survival of the patients was also determined in the course of 7-year observations. Statistical analysis using the Coxe's model did not detect relations between MT expression intensity, in the myoepithelium on one hand and patient survival on the other (chi2=0.003 p=0.96).     

Stromal myofibroblasts in breast cancer: relations between their occurrence, tumor grade and expression of some tumour markers

It is suggested that tumour stromal myofibroblasts exert an unfavourable effect on the biology of breast cancer. We are aware of only a single study which examined relationships between manifestation of myofibroblasts in the stroma of breast cancer and clinicopathological data of the patients. The present study was aimed at estimation of the effect exerted by myofibroblasts present in the tumour stroma on principal pathological parameters and on expression of Ki67, P53 and HER-2 proteins in the group of the most frequent breast cancers, the ductal cancers. In paraffin sections of 60 ductal breast cancers (20 cases in G1, 20 in G2 and 20 in G3), immunohistochemical reactions were performed to detect expression of smooth muscle actin (SMA) in order to visualize myofibroblasts, Ki67, P53 and HER-2. The studies demonstrated that the most numerous myofibroblasts were present in G3 cases and they were the least frequent in G1 cases (P = 0.02). Positive correlations were observed between the presence of myofibroblasts in tumour stroma and expression of Ki67 and HER-2 in breast cancer cells in the entire group (P < 0.001 and P = 0.001, respectively), in G2 cases (P = 0.003 and P = 0.03) and in G3 cases (P = 0.01 and P = 0.03). Considering that the higher grade, Ki67 and HER-2 are thought to represent unfavourable prognostic factors, the elevated content of myofibroblasts in tumour stroma is probably typical for cases with worse prognosis.     

The N-terminal part of recombinant human tear lipocalin/von Ebner's gland protein confers cysteine proteinase inhibition depending on the presence of the entire cystatin-like sequence motifs.

Human Tear Lipocalin/von Ebner's gland protein (TL) is a member of the lipocalin superfamily. The protein is secreted by a number of serous glands and tissues and is overproduced under conditions of stress, infection and inflammation. In addition to its typical affinity for lipophilic ligands it was recently found to be able to inhibit cysteine proteinases [van't Hof et al., J. Biol. Chem. 272 (1997), 1837-1841], probably due to the presence of amino acid motifs resembling the papain binding domains of family 2 cystatins. In this work we have used a recombinant protein to confirm the results obtained with native TL. The inhibitory activity of the recombinant protein against papain was dependent on the ratio of papain and TL. At higher papain concentrations, the N-terminal sequence of TL was cleaved off by the protease, indicating that it can act in an inhibitor- or a substrate-like mode. This behaviour resembles that observed with certain chicken cystatin mutants. Using a recombinant TL mutant we found that the two Leu residues (Leu4-Leu5) contained within the first cystatin-like motif are absolutely essential for the inhibitory activity. These results were supported by experiments using a recombinant form of the corresponding pig von Ebner's gland protein (VEGp). This protein, which does not possess a fully conserved first cystatin-like motif, is unable to inhibit papain.     

No association between MTHFR C677T polymorphism and completed suicide

MTHFR C677T polymorphism (rs1801133) was associated with numerous psychiatric conditions but no prior study investigated whether it predisposes to completed suicide. We typed rs1801133 in 692 suicide victims and 3257 controls representative of a Polish adult population (the WOBASZ cohort). Although we had a power of 0.8 to detect (at alpha 0.05) an allelic OR = 1.19, we did not find significant difference among suicides vs. controls in the prevalence of the MTHFR 677T allele (OR = 1.02, p = 0.759) or the TT genotype (OR = 1.01, p = 0.926). Since among controls we found an association between TT and depression defined by Beck Depression Inventory (BDI, OR = 1.61, p = 0.049) we also compared suicides with controls without signs of depression (BDI ≤ 11) but found no association (OR = 1.0, p = 0.976). Analyses within suicides showed trends (not significant after Bonferroni correction) for correlations between the dose of the T allele and age at death among males and blood ethanol concentration among females, who committed suicide under the influence of alcohol. We conclude that MTHFR C677T polymorphism is not a risk factor for completed suicide. The sex-specific trends for correlations between rs1801133 and age at death, and blood ethanol concentration should be studied further.     

Complement component C8gamma is expressed in human fetal and adult kidney independent of C8alpha

Human complement component C8gamma is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily. So far, it has been found exclusively in plasma, covalently linked to C8alpha by disulfide bridging. We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8gamma. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8gamma is not dependent on C8alpha expression, since we could not detect C8alpha expression in kidney. Thus its physiological function is not restricted to a specific action in association with complement components. As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8gamma, we have expressed the encoding cDNA in Escherichia coli. To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm.     

Elevated metallothionein (MT) expression in invasive ductal breast cancers predicts tamoxifen resistance

Elevated expression of the low molecular weight metallothionein (MT) proteins can be found typically in breast cancer cases with less favourable prognosis. The MT gene has been described to be potentially down-regulated by estrogen receptor alpha. The present study is aimed at examining the predictive value of MT expression for results of tamoxifen treatment in breast cancer in relation to steroid receptor status. Sixty patients with primary invasive ductal breast cancers with post-operative tamoxifen treatment were enrolled in the study. In paraffin sections of the studied tumours immmunohistochemical reactions were performed using antibodies directed against MT, estrogen receptors (ER) and progesterone receptors (PgR). Results of the immunohistochemical reactions and of clinical observations were analysed using multivariate progression analysis based on the Cox proportional hazard model. Elevated MT expression was demonstrated to be typical for cases with documented relapse of the disease (P<0.001) or terminated by death (P=0.03). Decreased ER expression was found to be typical for cases of a higher grade (P=0.02) and cases terminated by death (P=0.006). The multivariate analysis showed that elevated MT expression was characteristic for cases with shorter overall survival time (P=0.04). The data showed that MT carried an independent, and also independent from ER status, unfavourable predictive value as far as results of tamoxifen treatment were concerned.     

Correlation of Ki-67 and MCM-2 proliferative marker expression with grade of histological malignancy (G) in ductal breast cancers

The study aimed at examining a relationship between expression of Ki-67 antigen and minichromosome maintenance 2 protein (MCM-2) and a grade of histological malignancy G in ductal breast cancers. The function of widely used marker of proliferation Ki-67 is still not clear. In contrast, the MCM-2 protein is well known to play an important role in controlling the cell cycle. Both proteins represent small protein molecules, which manifest nuclear expression only during cell division of normal and neoplastic cells. Their expression is noted in several malignant tumours. These studies were conducted on 56 archival paraffin blocks of ductal breast cancers. Immunohistochemical reactions were performed using monoclonal Ki-67- and MCM-2-specific antibodies. Statistical analysis demonstrated a positive correlation between expressions of two proteins (r=0.6; p<0.05). The most intense expression of these two markers was demonstrated in G3 grade cancers. Statistical analysis showed more pronounced expression of Ki-67 antigen in G3 grade cancers as compared to cancers of G1 and G2 grades (p<0.001) and, in the case of MCM-2 protein, a more pronounced expression in G3 grade cancers, as compared to those of G1 (p<0.05) or G2 grade (p<0.01). The results obtained in our study suggest that MCM-2 could be used as a marker of proliferation in breast carcinomas.