Changes In Cytoplasmic Proteins Research Articles

A Simple Method to Study Host-Pathogen Interaction in Sesamum Indicum

Differential activation of early defense responses like increased activity of phenylalanine ammonia-lyase (PAL), peroxidases (PO), and pathogenesis proteins (PR proteins) predisposes the host plant resistance against pathogens. Therefore, in the present study interaction between this defenses related compounds and pathogen (Macrophomina phaseolina) was used as new strategy to enhance sesamum defense responses against root rot disease. There was a marked increase in these biochemical compounds in pathogen treated sesamum plants after pathogen attack. The investigation was carried after infection, which shows increase in activities of all defense related proteins to varying degrees of infection. Interestingly, there are significant changes in cytoplasmic proteins under pathogen inoculation. In the control plants, 8 major bands and 3 minor bands are obtained. In contrast, in the inoculated plants 8 major and 4 minor bands are obtained, which however, are of different Rf- values than the control plants. The differences in the protein are more pronounced in the molecular mass range of 15 to 36 kda and 40 to 59 kda. Individual protein bands obtained through SDS-PAGE were further quantified by scion image programme. This programme gives the peak area values of the corresponding band. The peak area denotes the densities of protein bands on the gel. A comparison of the densities of the protein bands in control and inoculated plants amply demonstrated that exposure to pathogen not only results in higher protein amounts but synthesis of a few new proteins, possibly PR-proteins. The biochemical approach described in this paper should provide the basis for further efforts concentrating on genetic regulation of sesamum in the form of transcript accumulation for expression of disease resistance proteins.

Reperfusion-triggered stress protein response in the myocardium is blocked by post-conditioning. Systems biology pathway analysis highlights the key role of the canonical aryl-hydrocarbon receptor pathway

Ischaemic post-conditioning (IPost-Co) exerts cardioprotection by diminishing ischaemia/reperfusion injury. Yet, the mechanisms involved in such protection remain largely unknown. We have investigated the effects of IPost-Co in cardiac cells and in heart performance using molecular, proteomic and functional approaches. Pigs underwent 1.5 h mid-left anterior descending balloon occlusion and then were sacrificed without reperfusion (ischaemia; n= 7), subjected to 2.5 h of cardiac reperfusion and sacrificed (n= 5); or subjected to IPost-Co before reperfusion and sacrificed 0.5 h (n= 4) and 2.5 h (n= 5) afterwards. A sham-operated group was included (n= 4). Ischaemic and non-ischaemic myocardium was obtained for molecular/histological analysis. Proteomic analysis was performed by two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization-time-of-flight identification. Potential protein networks involved were identified by bioinformatics and Ingenuity Pathway Analysis (IPA). Cardiac function was assessed by echocardiography. IPost-Co diminished (up to 2.5 h) reperfusion-induced apoptosis of both the intrinsic and extrinsic pathways whereas it did not affect reperfusion-induced Akt/mammalian target of rapamycin (mTOR)/P70S6K activation. Proteomic studies showed that IPost-Co reverted 43% of cardiac cytoplasmic protein changes observed during ischaemia and ischaemia + reperfusion. Systems biology assessment revealed significant changes in the aryl-hydrocarbon receptor (AhR) pathway (cell damage related). Bioinformatic data were confirmed since the expression of HSP90, AhR, ANRT, and β-tubulin (involved in AhR-signalling transduction) were accordingly modified after IPost-Co. IPost-Co rescued 52% of the left ventricle-at-risk compared with reperfusion alone and resulted in a ≈30% relative improvement in left ventricular ejection fraction (P <0.05). IPost-Co improves cardiac function post-myocardial infarction and reduces reperfusion-induced cell damage by down-regulation of the AhR-signalling transduction pathway ultimately leading to infarct size reduction.

Time dependent changes in cytoplasmic proteins of the right ventricle during prolonged pressure overload

In many forms of congenital heart disease, the right ventricle (RV) is subject to abnormal loading conditions resulting in RV hypertrophy and remodeling. We determined the alterations in RV cytoplasmic proteomic phenotype that occur during prolonged periods of RV pressure overload. We performed a differential proteomic profiling study on RV hypertrophy using an animal model of various durations of pulmonary artery banding (PAB) in parallel with hemodynamic characterization. This hemodynamic evaluation showed that after 6, 12 and 20 weeks of PAB, the RV is in a compensated state of hypertrophy. Overall, the majority of protein changes were metabolism related indicating a shift towards the glycolytic pathway at the expense of β-oxidation in the RV of the PAB animals. The changes in proteins related to the glycolytic pathway, exemplified by enolase and creatine kinase B-chain, tended to precede changes in β-oxidation. In parallel, increases in stress chaperones, exemplified by several phosphorylated HSP-27 species, are present from the 6 week time point, whereas increases in antioxidant proteins, exemplified by peroxiredoxin 2 and 6, appear to be restricted to the 12 week time point. The p38 MAPK signal transduction pathway appears not to be activated. Observed protein changes are likely part of a protective mechanism against the development of RV failure.

Selective depletion of cyclin-dependent kinases is associated with Fas-mediated apoptosis in human leukemia T-cell lines

The Fas/Apo-1 molecule is a member of tumor necrosis factor/nerve growth factor (TNF/NGF) receptor family and is able to induce apoptosis in various type of malignant cells, including most of the human leukemia T-cells. We previously demonstrated that the Fas-resistant variants may exist in highly Fas-sensitive human leukemia T-cell lines. The surface expression of Fas antigen was unchanged in the variant cells, suggesting the requirement of the cytoplasmic mechanism to exert apoptosis. In the present study, we examined the changes in cytoplasmic proteins of the Fas-sensitive and Fas-resistant cells after stimulation with anti-Fas antibody, 2D 1. In Western blotting analysis, we found that the stimulation of Fas-sensitive cells with 2D 1, but not resistant variants, induced a repression of cyclin-dependent kinases (cdks), p34cdc2 and p33cdk2, along with apoptosis. There was no alteration in the expression of bcl-2, HSP70, HSP90, and cyclin proteins examined. This observation seemed specific to Fas-mediated apoptosis, because calcium ionophore A23187 or sodium azide failed to repress the expression of cdks. These results indicate that the specific depletion of cdks, most likely due to proteolysis, may play a role in Fas-mediated apoptosis.

Regulation of gene expression by tumor promoters. II. Control of cell shape and developmental programs for macrophages and granulocytes in human myeloid leukemic cells

AbstractRegulation of the developmental programs for macrophages and granulocytes has been analysed, using two‐dimensional gel electrophorels of the cytoplasmic protein changes, in a human myeloid leukemic cell line (HL60) that can be induced to differentiate to macrophages by the macrophage and granulocyte‐inducing (MGI) protein and the tumor promoter 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), and to granulocytes by dimethylsulfoxide (DMSO). Studies on the protein changes induced by the different inducers showed a similar developmental program for macrophage differentiation induced by MGI or TPA, which differed from the beginning from the granulocyte program induced by DMSO. Comparison with normal cells from human peripheral blood has shown that the developmental programs induced in the HL60 cells are relevant to the normal programs of differentiation for these two cell types. Unlike MGI and DMSO, TPA induces rapid attachment of the HL60 cells to the surface of the Petri dish. Combined treatment with TPA and DMSO showed cell attachment, extensive spreading of the cells, the regulation of specific proteins and expression of the macrophage program. The addition of TPA to cells induced for the granulocyte program by DMSO resulted in cell attachment and spreading and a switchover from the granulocyte to the macrophage program. The results indicate that cells in suspension can express either the macrophage or granulocyte program depending on the inducer, and that changes in cell shape associated with cell attachment induced by TPA can regulate specific proteins and restrict the developmental program to macrophages. It is suggested that the in vivo environment of cells in relation to the possibilities for cell adhesion, which can be regulated by tumor promotors, may play a major role in determining the developmental program of myeloid and other cell types.

Constitutive gene expression in myeloid leukemia and cell competence for induction of differentiation by the steroid dexamethasone.

Regulation of the cytoplasmic protein changes during myeloid cell differentiation has been analyzed with two-dimensional gel electrophoresis and differentiation-defective cell mutants. The cells studied include a clone of myeloid leukemia cells (clone 11) that can be induced to differentiate to macrophages by the protein inducer MGI and the steroid dexamethasone (Dex) and mutant clones that were inducible for differentiation to macrophages by MGI but not by Dex. The mutants were not defective in the specific binding of [3H]Dex to cytoplasmic receptors or in the transport and nuclear binding of the receptor--steroid complex. The protein patterns in the mutants showed both specific constitutive protein changes and nonresponding proteins. Twenty-one percent of the Dex-induced protein changes and 2% of the MGI-induced protein changes in clone 11 were constitutively expressed in the mutants. In addition, 28% of the proteins that responded to Dex in clone 11 did not respond to Dex in the mutants, whereas only 4% of the proteins that responded to MGI in clone 11 did not respond to MGI. The higher percentage of constitutive changes was thus associated with a larger defect in induction. The proteins with an abnormal response to Dex still showed a normal response to MGI, and the constitutive changes and nonresponding proteins were different for the two inducers. It is suggested that specific constitutive protein changes expressed by the mutants produced an asynchrony in the developmental program, resulting in a defective response to Dex and to MGI, and that this may apply to other inducers and developmental programs.

Protein fractionation, nucleic acids, and enzymatic activity of cytoplasmic extracts from the intestinal mucosa of sheep

A method was designed to separate and study the cytoplasmic proteins of the intestinal mucosa of sheep. Specimens of mucosa from healthy sheep were extracted and analyzed for protein and nucleic acid content, and for selected enzyme activities. The proteins of this extract were separated by DEAE-cellulose chromatography. Samples of ulcerated mucosa were also extracted and analyzed for protein and nucleic acid content. Extracts from healthy mucosa contained more protein (71 ± 7 versus 52 ± 8 mg/ml) and less nucleic acid (8.5 ± 3 versus 14 ± 3 mg/ml) than extracts from ulcerated mucosa. The enzyme assay on healthy extracts showed that protease, amylase, alkaline phosphatase, transaminase, and lactase activity were present. Fractionation of the extracted proteins on DEAE-cellulose, using a Tris-HCl buffer (0.05 m, pH 9.0) and a sodium chloride gradient of 0.0–0.04 m, yielded 10 major protein fractions, of which 6 were eluted at less than 0.1 m NaCl. This system is useful for investigating changes in cytoplasmic proteins caused by drugs and diseases affecting the intestinal mucosa.

Regulation of actin and other proteins in the differentiation of myeloid leukemic cells

The regulation of cytoplasmic proteins in mutants of mouse myeloid leukemic cells, differing in their competence to be induced to differentiate by the normal macrophage- and granulocyte-inducing protein (MGI) and the steroid inducer dexamethasone, was analyzed using SDS-polyacrylamide gel electrophoresis of 35S-methionine-labeled proteins. Before induction, no consistent differences in the pattern of cytoplasmic proteins were found between clones with different capabilities to differentiate. Four MGI +D + clones, which are induced by MGI for Fc and C3 rosettes, the synthesis and secretion of lysozyme, and the formation of mature macrophages and granulocytes, all showed the same nine prominent changes in cytoplasmic proteins after induction. Five of these changes were either an increase or a decrease in proteins present in uninduced cells; four proteins appeared to be newly synthesized. One of the proteins that increased after induction was identified as actin. The pattern of cytoplasmic proteins from MGI-induced MGI +D + clones more closely resembled that of normal peritoneal macrophages and granulocytes than the pattern of the uninduced clones. The relationship of these protein changes to cell differentiation was further substantiated by the finding that MGI +D − cells, which can be induced by MGI for Fc and C3 rosettes and lysozyme, but not for mature cells, showed only four cytoplasmic protein changes which were quantitatively less than those found for MGI +D + clones. An MGI −D − clone which was not inducible for any differentiation-associated properties by MGI showed no alteration in protein synthesis. Thus in all the clones studied, there was a correlation between the number and extent of protein changes and the degree of MGI-induced differentiation. In MGI +D + clones, some of the differentiation-associated properties induced by MGI can be induced by the steroid hormone dexamethasone. Of the nine protein changes induced by MGI, six were also induced by dexamethasone, and no changes were induced by dexamethasone which were not also induced by MGI. These results, which were also shown by two-dimensional polyacrylamide gel electrophoresis, indicate that in cells which can respond to both MGI and dexamethasone, the proteins induced by dexamethasone were a subset of those induced by MGI.

Increased glycolytic metabolism in cardiac hypertrophy and congestive failure

Increased glycolytic metabolism in cardiac hypertrophy and congestive failure