Study of genotoxicity and antigenotoxicity of the Cotinus coggygria Scop. methanol extract by Drosophila melanogaster sex-linked recessive lethal test.

The genotoxic and antigenotoxic effects of Cotinus coggygria Scop. methanol extract was investigated using the Drosophila sex-linked recessive lethal (or SLRL) test. The results presented here show that the methanol extract of Cotinus coggygria in a concentration of 5% and artificial chemical agent ethyl methanesulfonate EMS (0.75 ppm) induce recessive lethal mutations on X-chromosome on Drosophila melanogaster in all broods (I, II and III). Post-treatment with lower concentration of the methanol extract of Cotinus coggygria (2%) was effective in reducing genotoxicity ofmutagen.

Differences between molecular mechanisms involved in the regulation of haptoglobin gene expression during the acute phase response and dietary restriction.

Haptoglobin is a glycoprotein involved in the acute phase response. Previously we reported that haptoglobin gene expression was up-regulated during dietary restriction in young female rats. The present study aimed at determining whether chronic dietary restriction affects haptoglobin blood levels through changing levels and/or activities of IL-6-related transcription factors STAT and C/EBP in the liver as is the case during the acute phase response. To this end, we compared a female Wistar rat model of 50% 6-week-long dietary restriction with the standard laboratory model for the acute phase response induced by turpentine administration. During the turpentine-induced acute phase response, the transitory 5.4-fold increase of rat haptoglobin expression was accompanied by a prominent rise of serum IL-6 concentration and the increased binding of STAT3 and 35kD C/EBPbeta/LAP transcription factors to the haptoglobin gene hormone-responsive element. Results obtained after immunoblotting and DNA affinity chromatography (using hormone-responsive element) suggest that the stable 1.7-fold increase of serum haptoglobin level during dietary restriction was the result of increased amounts and activities of constitutive transcription factors C/EBPalpha and STAT5b, and to a smaller extent of STAT3. When dietary restriction rats were administered turpentine, a 8.7-fold increase in haptoglobin expression was followed by a considerable increase in the amount and hormone-responsive element binding activity of STAT3 but not 35kD C/EBPbeta/LAP. We concluded that haptoglobin gene up-regulation during chronic dietary restriction was regulated by different mechanisms than during the acute phase response, and that it depended on the amount(s) and activit(ies) of transcription factor(s) that characterize low-grade inflammatory conditions.

Molecular characterization of hsp90 isoforms in colorectal cancer cells and its association with tumour progression.

A key role of hsp90 in the activity of various oncogenic proteins and pathways is currently of intense interest. To clarify the molecular basis of biological behaviour of colorectal cancers we analysed the expression characteristics of hsp90 in cytosolic, nuclear and plasma membranous fractions of cancer cells. As determined by Western blot assay all hsp90 isoforms studied, alpha (84 kDa), beta (86 kDa) and hsp90N (75 kDa), were up-regulated and differentially expressed in various stages of colorectal carcinoma. The inducible hsp90alpha isoform is a component of invasive phenotype of cancer cells thus pointing to the importance of hsp90alpha for metastasis generation. The expression of hsp90beta is definitely higher in poorly-differentiated carcinomas than in well-differentiated cancers, suggesting an involvement of hsp90beta in the inhibition of cancer cell differentiation. Especially, the expression of cytosolic hsp90N isoform in malignant cells points to the possibility that induction or overexpression of hsp90N might be causally related to tumour formation. Hsp90N is the plasma-membrane-associated protein in poorly-differentiated colorectal cancers with metastasis. This suggests that the expression of hsp90N is elevated with progressive dedifferentiation often associated with advanced cancer stages. Hsp90 was exclusively localized in the invasive front in a majority of metastatic cancers as visualized by immunohistochemical study. Consistent with these facts, the frequent expression of hsp90alpha and hsp90N on the surface of colorectal cancer cells may enable hsp90 to act as a mediator of metastasis generation. The above results indicate more complex roles for hsp90 in colorectal tumourigenesis. In this way, the hsp90 would be at the crossroads of both signalling and cell migration events.

Acute-phase related binding ability of p53 for the hormone response element of the haptoglobin gene in adult rats.

Interaction between transcription factor p53 and the hormone response element (HRE) of the haptoglobin (Hp) gene in adult rat liver was studied. We detected a sequence homologous to the p53 consensus DNA-binding site in the regulatory promoter element of the Hp gene. DNA-affinity chromatography, followed by Western immunoblot analysis with an antibody to p53 indicated that components of the nuclear extract possessed the same antigen determinants as p53. While p53 was identified in both control and acute-phase (AP) samples, DNA-binding affinity for the Hp gene HRE was detected only in the nuclear extract prepared from rats undergoing the AP response. Whether either as an inducible or as a constitutive transcription factor, p53 could be involved in the transcriptional regulation of the Hp gene in adult rat liver.

Expression of C/EBP delta in rat liver during development and the acute-phase response.

Using Western analysis, C/EBP delta was established in the nuclear extract and nuclear matrix throughout rat liver development and in the adult. During the acute-phase response (APR), C/EBP delta increased in the nuclear extract but remained unchanged in the nuclear matrix of fetal and postnatal rats, whereas it increased in both the nuclear extract and nuclear matrix of the adult. The solubility partitioning of gene regulatory proteins in the nucleus is important for their functioning (Uskokovic et al. 2002). The obtained different solubility partitioning profiles of C/EBP delta suggest that its activity is regulated by different mechanisms during development and in the adult.

The effect of O-glcnac glycosylation of rat liver nucleoproteins on their acute phase-dependent binding ability to the hormone responsive element of the haptoglobin gene.

We examined whether the transcriptional activation of the rat haptoglobin (Hp) gene during the acute phase (AP) response reflects the O-linked N-acetylglucosamine (O-GlcNAc) status of liver nucleoproteins (NPs) and their binding for the hormone responsive element (HRE). After deglycosylation with N-acetylglucosaminidase of the O-GlcNAc glycoproteins obtained by WGA, affinity chromatography and South-Western analysis, it was observed that only increased HRE binding ability of p64/p70 in control and p51 obtained from turpentine-treated rats can be directly attributed to the presence of O-GlcNAc residues. Therefore, expression of the rat Hp gene could be controlled by this modification of certain trans-acting NPs.

Acute-phase-dependent binding affinity of C/EBPbeta from the nuclear extract and nuclear matrix towards the hormone response element of the alpha2-macroglobulin gene in rat hepatocytes.

Interactions of nuclear extract and nuclear matrix proteins from rat hepatocytes with the hormone response element of the alpha2-macroglobulin gene were studied. By Western and South-Western blot analysis we have shown the presence of C/EBPbeta in the examined nuclear fractions as well as its increased binding affinity to the examined gene sequence during the acute-phase response. The results suggest that both nuclear protein fractions could participate in the transcriptional regulation of the alpha2-macroglobulin gene in the rat hepatocytes.

C/EBPalpha and C/EBPbeta are persistently associated with the rat liver nuclear matrix throughout development and the acute phase response.

The partitioning of C/EBPalpha and C/EBPbeta on the nuclear matrix structure was examined during the different transcriptional activities accompanying liver development and the acute phase (AP) response. The presence of C/EBPalpha and C/EBPbeta was established on the nuclear matrix. Their relative concentrations on the matrix always reflected the developmental stage- and AP-related fluctuations observed in the nuclear extract. Thus, they progressively increased as development proceeded, whereas during the AP response, C/EBPalpha decreased and C/EBPbeta increased. In addition, the levels of both transcription factors were always notably higher in the nuclear matrix than in the extracts. We conclude that the observed changes and overall enrichment of the nuclear matrix with regulatory proteins is a reflection of the importance of such interactions for the in vivo functioning of C/EBP proteins.

Correlation between acute phase-related expression of C/EBPbeta and transcriptional regulation of the haptoglobin gene during rat liver development.

The hepatic 35 kD nucleoprotein participates in transcriptional regulation of the haptoglobin gene during the development of rat liver. The same molecular mass and trans-acting role of the above protein as the active isoform of C/EBPbeta transcription factor, made homology between them possible. We could detect C/EBPbeta in control hepatic nuclear extracts not earlier than in the second week after the birth. However, the acute-phase reaction induced the expression of 35 kD-C/EBPbeta at day 7 of postnatal development suggesting that the above trans-active nucleoprotein displays the structural and functional characteristics of C/EBPbeta isoform from that day on.

The DNA binding affinity of rat liver nucleoproteins to the alpha 1-acid glycoprotein gene.

Transcriptional regulation and binding interactions between soluble nucleoproteins and the distal regulatory element (DRE) of the rat alpha 1-acid glycoprotein (alpha 1-AGP) gene were examined in the liver of rats during the acute-phase response. Our results show that the elevation of the alpha1-AGP gene transcription activity in acute phase liver relies basically on an increase in the binding-affinity of the constitutive soluble nucleoproteins with molecular masses 35 and 45 kD, enhancing their capability to bind to the distal regulatory element (DRE) of alpha-AGP gene. On the basis of in vitro phosphorylation/dephosphorylation experiments we discuss that the role of these proteins during alpha 1-AGP transcription may be dependent on their behavior as phosphoproteins. The 35kD nucleoprotein that displayed an acute-phase inducible affinity to bind DRE was identified as a C/EBP beta isoform.

Structural and functional homology between the 29 kD rat liver nucleoprotein and the high mobility group 1 protein.

A 29 kD soluble rat liver nucleoprotein (p29) has increased binding affinity for the hormone responsive element (RE) of the rat haptoglobin (Hp) gene during the acute-phase reaction. In this work the possibility of its structural and functional homology to the high mobility group 1 (HMG1) nonhistone protein constituent of chromatin was examined. The results of two-dimensional gel electrophoresis, Southwestern and Western immunoblot analyses, showed that p29 and HMG1 are homologous protein species. On the basis of in vitro and in vivo phosphorylation/dephosphorylation experiments, we discuss the modulatory role of phosphate groups in view of the structure and function of p29.

The DNA binding affinity of rat liver nucleoproteins to the regulatory elements of the haptoglobin and alpha 2-macroglobulin genes.

Transcriptional regulation and binding interactions between soluble nucleoproteins and the hormone response elements (REs) of the rat haptoglobin (Hp) and alpha 2-macroglobulin (alpha 2MG) genes was examined in the livers of rats during the acute-phase reaction. Our results demonstrate that the elevation of the Hp and alpha 2MG genes' transcription rates in acute-phase liver relies essentially on an increase in the binding-affinity of pre-existing soluble nucleoproteins, enhancing their capability to bind the examined cis-regulatory elements. The 35kD nucleoprotein that displayed an acute-phase inducible affinity to bind hormone REs of rat Hp and alpha 2MG genes, was identified as a C/EBP beta isoform.

Acute phase-dependent changes in the binding of rat liver nucleoproteins to the cytokine response element of the rat haptoglobin gene.

Hormones released during the acute phase reaction promote the transcriptional activation of the haptoglobin (Hp) gene and a consequent increase of Hp protein synthesis in the liver. The mechanisms underlying the alterations of basal transcription rates of eukaryotic genes are assumed to result from modulations of the binding affinities between nucleoproteins and specific DNA sequences in the enhancer and promoter elements. In order to characterize the changes in the interaction of nucleoproteins with the promoter that accompany the induction of the Hp gene, nuclear extracts from normal and inflamed livers were probed with hormone responsive element (HRE) of the rat Hp gene by gel mobility shift and Southwestern assays. Each of the three cis-acting sequences of the HRE, elements A, B, and C, recognized a distinct set of proteins. Together they conferred an additional level of specificity to the protein binding sites of the entire ABC-element. These sites were recognized by proteins in liver nuclear extracts isolated from both control and treated rats. The differences in the gel shift and Southwestern patterns of the corresponding DNA-protein complexes suggested that transcriptional activation of the Hp gene relied on changes in the concentrations and/or functional modifications of preexisting proteins rather than on the induction of new trans-acting factors.

Inflammation of the dam has different effects on the binding of maternal and fetal rat liver nucleoproteins to the rat haptoglobin gene promoter.

Transcriptional regulation of binding interactions between nucleoproteins and the hormone response element (RE) of the rat haptoglobin (Hp) gene was investigated in adult and fetal livers of rats exposed to inflammation on day 19 of pregnancy. Nuclear extracts from the embryonal liver displayed a barely detectable binding-affinity for hormone RE, but in extracts from the adult liver it was noticeable. The acute phase reaction of the mother promoted an increase of Hp gene expression in both adult and fetal livers, relying on stage-specific changes in hormone RE binding activities of nucleoplasmic proteins. The results indicated that the elevation of Hp gene expression in fetal liver to the steady basal level found in adults required the induction of new trans-acting proteins, whereas an overexpression of this gene in adult acute phase liver relied essentially on an increase in the binding-affinity of the preexisting hormone RE binding proteins.

Pretreatment with alpha 2-macroglobulin leads to recovery of rats exposed to a lethal scald.

The effect of alpha 2-macroglobulin (alpha 2M) administration on the survival rate of lethally injured rats and molecular mechanisms regulating its hepatic expression after sublethal and lethal scalding were examined. Transcriptional activity of nuclei for the alpha 2M gene increased after a sublethal 20 per cent TBSA scald reaching a maximal three-fold increase by 12 h, whereas concentrations of the corresponding mRNA and protein attained the maximal nine- and 18-fold enhancements by 24 h, respectively. After the second, lethal scald, the plasma alpha 2M level increased during the first few hours, then dropped rapidly below the control value although the abundance of its mRNA was several fold enhanced. This anomaly was ascribed to inhibition of the alpha 2M mRNA translation caused by the second scald-induced disturbance of the haemodynamic equilibria. Eighty per cent of rats receiving alpha 2M prior to rescalding survived the second injury. Their recovery proceeded in parallel with normalization of the plasma volume and reactivation of the process of acute phase protein synthesis in the liver. A functional link between these events is discussed.

The expression of liver acute-phase protein genes during rat development and in response to inflammation of the dam.

The hepatic expression of albumin (Al) and plasma acute phase protein genes (APP) was examined during the development of rat liver and in response to inflammation of the dam. Throughout the 10- to 20-day gestation period the level of alpha 2-macroglobulin (alpha 2M) mRNA in fetal liver exceeded twice that of the adult liver. The concentrations of the other APP and Al mRNAs were 10-30% of those of the adult liver between days 10 and 13 of gestation, then increased to values which ranged from 40% for haptoglobin (Hp) to 80% for Al and alpha 1-acid glycoprotein (AGP) mRNAs on day 19 of gestation. The transition of fetuses to an extrauterine environment was followed by a temporary overexpression of the Hp gene and an increase of the fibrinogen (Fb), AGP and thiostatin (TST) mRNAs to adult levels. Fetal liver responded to inflammation of the mother by a transcriptional induction of all of the investigated APP genes, except for the Fb gene whose level of expression remained unchanged. The pattern of individual APP genes expression in maternal and fetal livers was similar and characteristic for the acute phase reaction.

Testosterone metabolism and formation of cytosol 5alpha-dihydrotestosterone-receptor complex in the rat prostate in vitro: Effects of lindane and malathion.

The effects of gamma-1,2,3,4,5,6-hexachlorocyclohexane (lindane) and O,O-dimethyl-S-(1,2-dicarbethoxyethyl)phosphorodithioate (malathion) on testosterone metabolism in rat prostate tissue in vitro was investigated. The influence of lindane on the formation of 5alpha-androstan-17beta-ol-3-one (5alpha-DHT)-receptor complex in the rat prostatic cytosol has also been examined. Lindane and malathion inhibited the formation of 5alpha-DHT, and lindane caused a decrease in the formation of 5alpha-androstane-3,17-dione (androstanedione) in the rat prostate tissue. A mixture of lindane and malathion exerted a synergistic effect on testosterone metabolism in the rat prostate, as indicated by lower 5alpha-DHT and higher 5alpha-androstane-3alpha,17beta-diol formation. The presence of lindane significantly inhibited 5alpha-DHT-receptor complex formation in the rat prostatic cytosol, and this inhibition was of a fully noncompetitive type.

Acute-phase response to scalding: changes in serum properties and acute-phase protein concentrations.

The time course of changes in the concentrations of individual acute-phase proteins (APRs) in the plasma was examined in the model of rats exposed to a single (sublethal) and repeated (fatal) scalding. These data were interrelated with the rate of survival, plasma volume, corticosterone level, and immunosuppressive potency of the serum. The infliction of a sublethal scalding composing 20% body surface area resulted in an initial 50% reduction of the plasma volume and a severalfold increase of the corticosterone level and immunosuppressive activity of the serum. A subsequent normalisation of these deviations proceeded in parallel with an increased rate of APR synthesis. An early infliction of a second scald was fatal. It led to a 60% reduction of the plasma volume, a lack of plasma APRs, and an additional enhancement of the immunosuppressive activity of serum.