Multiple forms of the glucocorticoid receptor steroid binding protein identified by affinity labeling and high-resolution two-dimensional electrophoresis

Potential charge heterogeneity within the glucocorticoid binding protein (GBP) of the glucocorticoid receptor was examined by a combination of affinity labeling, immunopurification, and high-resolution two-dimensional (2D) gel electrophoresis. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of [3H]dexamethasone 21-mesylate ([3H]DM) labeled cytosol identified a major, competable, component of Mr approximately equal to 92 000 (92K). This component was recognized by anti-human glucocorticoid receptor antibodies but not by nonimmune serum, indicating that the 92K component was the reduced denatured GBP. Examination of [3H]DM-labeled GBP by conventional 2D electrophoresis utilizing equilibrium isoelectric focusing in the first dimension failed to resolve the 92K GBP into discrete isoelectric components. This behavior was not representative of other, nonspecifically [3H]DM-labeled proteins or proteins in general. Nonequilibrium pH gradient electrophoresis (NEPHGE) was therefore employed to achieve separation in the first dimension. Immunopurified, [3H]DM-labeled GBP subjected to NEPHGE reached isoelectric equilibrium after 6 h of electrophoresis at 400 V. A single, broad peak of radioactivity was identified at pH approximately equal to 6.3. Second-dimension analysis of the NEPHGE-separated GBP by SDS-PAGE resolved this peak into two discrete, 92K, isoforms of apparent pI = 5.7 and 6.0-6.5. The GBP charge heterogeneity was confirmed by NEPHGE 2D analysis of [3H]DM-labeled GBP prepared directly from crude cytosol. Two isoforms indistinguishable from those observed in immunopurified samples were identified. An additional, more acidic, isoform (apparent pI approximately equal to 5.2) was also identified. Thus, there are at least two, and perhaps three, isoforms of the GBP. These data therefore suggest that there is significant charge heterogeneity in the GBP of the glucocorticoid receptor.     

Low content of protein S29 in ribosomes of human lung cancer cell line a549: detected by two-dimensional electrophoresis

This study revealed that the content of protein S29 in ribosomes of cancer cell line A549 was distinctly low (equivalent to about 30% of that of 2BS). The conclusion was acquired based on the ratios of spot volume of ribosomal protein S29 to that of several other ribosomal proteins (S29/L37a, S29/L38, S29/S27 and S29/S28) in the same gel plate. The possible biological roles of ribosomal protein S29 in malignant transformation and translation regulation are briefly discussed.     

Purification of Gc-2 globulin from human serum by displacement chromatography: a model for the isolation of marker proteins identified by two-dimensional electrophoresis

A protein that was initially known only as a minor spot in two-dimensional electrophoresis patterns of serum obtained from certain psoriasis patients, particularly those with a pustular component to their disease, has been purified by two stages of ion-exchange displacement chromatography on DEAE-Sephacel at different pH levels, followed by elution chromatography on hydroxylapatite. The purification was followed by examining the column fractions directly by two-dimensional gel electrophoresis. The capacity of the displacement system, which utilized carboxymethyldextrans as displacers, was very high; 6 ml of dialyzed serum applied to a 7-ml column in the initial stage resulted in a very substantial enrichment of the target protein. The second displacement stage yielded a highly purified product, contaminated only by A-1 lipoprotein. The latter was removed by hydroxylapatite chromatography. The purified protein was subsequently identified as Gc-2 globulin, a vitamin D-binding protein, by immunological procedures. The results demonstrate the effectiveness of ion-exchange displacement chromatography in focusing resolving power on the relatively narrow range of affinities represented by the target protein and its immediate neighbors in a chromatogram, as well as the applicability of the system to the isolation of a protein known only by its position in a two-dimensional electrophoretic pattern.     

Characterization of the Ah receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin: use of chemical crosslinking and a monoclonal antibody directed against a 59-kDa protein associated with steroid receptors

The Ah receptor regulates induction of cytochrome P450IA1 (aryl hydrocarbon hydroxylase) by "3-methylcholanthrene-type" compounds and mediates the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons. Hepatic Ah receptor from untreated rodents is localized in the cytosol and has an apparent molecular mass of 250 to 300 kDa. This large form can be dissociated into a smaller ligand-binding subunit upon exposure to high ionic strength. The Ah receptor displays many structural similarities to the receptors for steroid hormones. Two non-ligand-binding proteins have been identified to be associated with the cytosolic forms of the steroid hormone receptors. The first is a 90-kDa heat shock protein (hsp 90); the second is a 59-kDa protein (p59) of unknown function. The cytosolic Ah receptor ligand-binding subunit previously has been shown to be associated with hsp 90. In the present study, we used a monoclonal antibody, KN 382/EC1, generated against the 59-kDa protein which is associated with rabbit steroid receptors to determine if p59 also is a component of the large cytosolic Ah receptor complex. Cytosolic forms of rabbit progesterone receptor, glucocorticoid receptor, and Ah receptor were analyzed by velocity sedimentation on sucrose gradients under low-ionic-strength conditions and in the presence of molybdate. Progesterone receptor from rabbit uterine cytosol and glucocorticoid receptor from rabbit liver each had a sedimentation coefficient of approximately 9 S. In the presence of KN 382/EC1 antibody the progesterone receptor and the glucocorticoid receptor both underwent a shift in sedimentation to a value of approximately 11 S. The increase in sedimentation velocity is an indication that the receptor-protein complexes are interacting with the antibody. Under low-ionic-strength conditions the Ah receptors from rabbit uterine cytosol and liver cytosol had a sedimentation coefficient of approximately 9 S. However, in contrast to the steroid receptors, the Ah receptor showed no change in its sedimentation properties in either tissue in the presence of KN 382/EC1, indicating that the antibody is not interacting with the Ah receptor. Multimeric Ah receptor complexes that were chemically crosslinked still did not show any interaction with KN 382/EC1. These data indicate that the 59-kDa protein either is not associated with the Ah receptor or is present in an altered form which the antibody cannot recognize.     

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase.

The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide-dependent kinases, calcium/calmodulin-dependent kinases, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.     

Factors associated with an individual's decision to withdraw from genetic testing for breast and ovarian cancer susceptibility: implications for counseling

Our study aimed to examine why individuals withdraw from genetic testing for breast and ovarian cancer susceptibility. We explored the characteristics of 334 individuals from high-risk breast and ovarian cancer families who declined genetic testing for BRCA1/2 mutations, when, and why they did so. Individuals who declined genetic testing were older, and a greater proportion had never developed breast or ovarian cancer. Fifty one per cent (51.1%) of individuals withdrew after the first genetic counseling session. Most of those who declined were afraid of the psychological effects of genetic testing (36.3%). The next most-cited explanations concerned logistic problems such as a limited ability to travel, lack of time, personal issues, advanced age, or health problems (21.7%). The third category included individuals who did not see any advantage in being tested (14.5%). Insurability was a concern (5.9%), mainly for men. Surprisingly, confidentiality was not a frequently reported issue (1.3%). Sixty eight per cent (68%) of individuals belonging to a family in which at least one individual has been tested withdrew after the presence of a deleterious BRCA1/2 mutation in a relative was disclosed, compared to 42% after the disclosure of a nonconclusive test result in at least one relative. Concern about the psychological effects of the result was still one of the major reasons. Several factors may influence an individual's decision to decline genetic testing; a greater understanding of these issues may help health professionals to better meet the needs and concerns of individuals from high-risk families, thus possibly improving their health outcomes.     

Detection of the associated state of membrane proteins by polyacrylamide gradient gel electrophoresis with non-denaturing detergents Application to band 3 protein from erythrocyte membranes

Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes.     

Taenia solium: a two-dimensional Western blotting method combined with the use of an EST-library for the identification of immunogenic proteins recognized by sera from neurocysticercosis patients

Commercial antigens used to diagnose human neurocysticercosis (NCC) are obtained from either a soluble parasite extract or a parasite-derived glycoprotein fraction. The aim of the present study was to identify antigenic proteins as potential diagnostic candidates in this context. Soluble immunogenic proteins from Taenia solium cysticerci were identified by two-dimensional electrophoresis Western blotting using human sera from Nicaragua confirmed to be positive for NCC by computer tomography. Six antigenic proteins were identified and sequenced by liquid chromatography–mass spectrometry. Among these immunogenic proteins, a novel sequence was found and named Tsol-p27. To determine the antigenicity of Tsol-p27, the previously reported antigen TsolHSP36 and the new Tsol-p27 were expressed as recombinant proteins and evaluated serologically. Immunoblotting demonstrated that Tsol-p27 was recognized by sera from 13 NCC-positive humans, whereas TsolHSP36 was identified by only two of those 13 positive sera. None of the antigens were recognized by negative control sera. Despite the limited number of serum samples evaluated in this study, the results indicate that Tsol-p27 might be a suitable candidate for diagnosis of human NCC.     

Expression of the insulin-like growth factor-I receptor in primary breast cancer and lymph node metastases: correlations with estrogen receptors alpha and beta.

Numerous laboratory studies and some epidemiological data have suggested the involvement of the insulin-like growth factor-I receptor (IGF-IR) in breast cancer development and progression. However, data on IGF-IR expression in human tissues, including breast cancer sections, are limited and often inconsistent. We therefore examined by immunohistochemistry the expression of IGF-IR in primary tumors and breast cancer metastases to lymph nodes, and correlated IGF-IR positivity with estrogen receptor (ER) status and selected clinicopathological features. We found that 1) IGF-IR was expressed in primary tumors as well as in lymph node metastases, but the expression in primary tumors was more frequent (56 % vs. 44.4 %); 2) IGF-IR expression in primary tumors was associated with negative node status (p < 0.033); 3) in node-negative primary tumors, IGF-IR positively correlated with ERbeta (p < 0.008; r = 0.538), but not with ERalpha, tumor size or grade; 4) both IGF-IR-positive and IGF-IR-negative primary tumors were found to produce IGF-IR-positive as well as IGF-IR-negative metastases; 5) in metastases, IGF-IR expression did not associate with ERalpha, ERbeta or any of the studied pathobiological markers. The results suggest that IGF-IR could become a viable pharmaceutical target in breast cancer therapy, but such therapy should be based on IGF-IR assessment in primary tumor and metastasis in each potential patient.     

Markers of breast cancer stromal fibroblasts in the primary tumour site associated with lymph node metastasis: a systematic review including our case series

CAFs (cancer-associated fibroblasts), the most abundant cell type in breast cancer stroma, produce a plethora of chemokines, growth factors and ECM (extracellular matrix) proteins, that may contribute to dissemination and metastasis. Axillary nodes are the first metastatic site in breast cancer; however, to the present date, there is no consensus of which specific proteins, synthesized by CAFs, might be related with lymph node involvement. The purpose of this study was to perform a systematic review of CAF biomarkers associated with the presence of regional metastasis. PubMed was searched using the words: ‘breast cancer’ and ‘lymph node’ and fibroblast or stroma or microenvironment. After exclusions, eight studies evaluating biomarkers immunoexpression in CAFs and lymph node status were selected. Biomarkers evaluated in these studies may be divided in two groups, according to their ontology: extracellular matrix components [MMP13 (matrix metalloproteinase 13), TIMP2 (tissue inhibitor of metalloproteinases-2), THBS1 (thrombospondin 1), LGALS1 (lectin, galactoside-binding, soluble, 1)] and response to wounding [PDPN (podoplanin), PLAU (plasminogen activator, urokinase), PLAUR (plasminogen activator, urokinase receptor), CAV1 (caveolin 1), THBS1, LGALS1]. A positive expression of MMP13 and LGALS1 in CAFs was associated with enhanced OR (odds ratio) for regional metastasis. Contrariwise, CAV1 positive staining of fibroblasts was associated with decreased OR for nodal involvement. Expression of MMP13, PDPN and CAV1 was further tested in a new series of 65 samples of invasive ductal breast carcinomas by immunohistochemistry and no association between biomarkers expression in CAFs and nodal status was found. It was suggested that breast cancer subtypes may differentially affect CAFs behaviour. It would be interesting to evaluate the prognostic significance of these biomarkers in CAFs from different tumour types.     

Isoforms of a cuticular protein from larvae of the meal beetle, Tenebrio molitor, studied by mass spectrometry in combination with Edman degradation and two-dimensional polyacrylamide gel electrophoresis.

Simultaneous sequencing, using a combination of mass spectrometry and Edman degradation, of three approximately 15-kDa variants of a cuticular protein extracted from the meal beetle Tenebrio molitor larva is demonstrated. The information obtained by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) time-course monitoring of enzymatic digests was found essential to identify the differences among the three variants and for alignment of the peptides in the sequence. To determine whether each individual insect larva contains all three protein variants, proteins extracted from single animals were separated by two-dimensional gel electrophoresis, electroeluted from the gel spots, and analyzed by MALDI MS. Molecular weights of the proteins present in each sample could be obtained, and mass spectrometric mapping of the peptides after digestion with trypsin gave additional information. The protein isoforms were found to be allelic variants.     

Direct activation of human resting T cells by IL 2: the role of an IL 2 receptor distinct from the Tac protein

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.     

Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein.

We have demonstrated and partially characterized the genetic control and pheromonal regulation of a soluble activity, produced only by mating-type a cells, that inhibits the action of the alpha mating pheromone, alpha-factor, on mating-type a cells. This activity was found to be associated with a heat-stable protein and to be secreted by MATa BAR1, mat alpha 2 BAR1, and mat alpha 1 mat alpha 2 BAR1 strains, but not by MAT alpha BAR1, MATa/MAT alpha BAR1, mat alpha 1 BAR1, or MATa barl strains, demonstrating that it is under the control of both the MAT alpha 2 and the BAR1 genes. Secretion of this activity was also found to be stimulated to as much as five times the basal level by exposure of the cells to alpha-factor. This stimulation was maximal after 6 h at a pheromone concentration of approximately 2 U/ml. An assay for this activity was developed by using a refined, quantitative assay for alpha-factor. The pheromone activity of samples added to wells in an agar plate was related to the size of the halo of growth inhibition produced in a lawn of mutant cells that are abnormally sensitive. The alpha-factor-inhibiting activity was related to a reduction of the halo size when active samples were added to the lawn. Although the assay for alpha-factor was found to be relatively insensitive to pH over a range of several units, the alpha-factor-inhibiting activity displayed a sharp pH optimum at approximately 6.5. The properties of this activity have important implications concerning the role of the BAR1 gene product in recovery of mating-type a cells from cell division arrest by alpha-factor.     

Partial characterization of an unusual 185 kDa protein synthesized by dermal fibroblasts from patients with Marfan syndrome: identification of the protein as type IV collagen

Production of an unusual collagenous protein was observed in culture of dermal fibroblasts from four patients with Marfan syndrome. The apparent molecular weight of the protein was about 185 kDa after reduction with 2-mercaptoethanol and 175 kDa after limited pepsin treatment. The 185 kDa protein was susceptible to the bacterial collagenase but resistant to the animal collagenase. Immunoprecipitation revealed the specific interaction of the pepsin-treated 175 kDa collagenous protein with monoclonal and polyclonal antibodies to human type IV collagen. From the patterns of CNBr peptide mapping the 185 kDa band was identified as alpha 1 (IV) chain. Type IV collagen in the skin is generally considered to be of non-fibroblastic origin. However, in "diseased" condition, dermal fibroblasts might produce type IV collagen. The clinical manifestation in relation to production of type IV collagen by cultured skin fibroblasts from Marfan patients is discussed.