Loss of p16 in recurrent malignant mixed müllerian tumors of the uterus.

Uterine malignant mixed müllerian tumors (MMMTs) are rare and highly aggressive malignancies with poor clinical prognoses. We examined for differences in the oncoprotein profiles of primary versus recurrent MMMTs. Five cases of recurrent uterine MMMT were examined by paraffin immunohistochemistry for the expression of p53, p16, P-cadherin, and Cerb-B2. P16, p53, and P-cadherin were each expressed in 100%, 80%, and 60% of the primary cases, respectively. Three cases expressed all three oncoproteins. All five cases were negative for Cerb-B2. No difference in antigen expression was seen in the epithelial versus sarcomatous components. Primary and recurrent tumors were concordant for p53, P-cadherin, and Cerb-B2. However, three cases of recurrent tumors were negative for p16 expression. P53, p16, and P-cadherin are common tumor suppressor genes expressed in uterine MMMT. Interestingly, p16 protein expression was lost in some cases of MMMTs when they recurred. This suggests that the oncoprotein and possibly genetic profile of p16 changes over time. We did not observe any difference in antigen expression between areas of epithelial or sarcomatous differentiation, which would support a single pluripotential malignant clone in the histogenesis of these tumors.

Oncoprotein profiles of primary peritoneal malignant mixed müllerian tumors.

Primary peritoneal malignant mixed müllerian tumors (MMMTs) are extremely rare and highly aggressive malignancies associated with poor clinical prognoses. We present a clinicopathologic review of three cases of this rare tumor by examining expression of selected oncoproteins by immunohistochemistry. Three consecutive cases of primary peritoneal MMMT were examined by paraffin immunohistochemistry for expression of p53, p16, BCL2, CerbB2, and classical cadherins E-cadherin, P-cadherin, and N-cadherin. All three cases expressed p16, but showed less consistent expression of other markers, with one case expressing p53 and one expressing BCL2. All cases were negative for membrane expression of Cerb-B2. The three classical cadherins were expressed in two cases with one case showing only weak N- and P-cadherin expression. No difference in antigen expression was seen in the epithelial compared to sarcomatous components. We conclude that p16 may be a common tumor suppressor gene expressed in peritoneal MMMT. P53 overexpression may be of lesser frequency in peritoneal MMMT compared to MMMT from the ovary and the uterus. We did not observe any difference in antigen expression between areas of epithelial or sarcomatous differentiation, which would support a single pluripotential malignant clone in the histogenesis of these tumors.

Expression of cadherins, p53, and BCL2 in small cell carcinomas of the cervix: potential tumor suppressor role for N-cadherin.

Cadherins are tissue-specific cell adhesion molecules that function as tumor suppressors. Analysis of cadherin expression is useful for differentiation of tumor histogenesis, and because they serve as markers of tumor behavior and prognosis. Since the pattern of cadherin expression is not well characterized for small cell carcinoma of the cervix, we examined cases of these tumors for expression of cadherins, and two other oncoproteins p53 and BCL2. Four cases of small cell neuroendocrine carcinomas were identified from the Gynecologic Oncology Service with diagnoses confirmed by immunohistochemistry for neuroendocrine markers. Archival paraffin blocks were studied by heat-enhanced immunohistochemistry using commercially available antibodies specific for E-cadherin, P-cadherin, and N-cadherin, p53, and BCL2. Sections were examined for specific membrane staining of cadherins, nuclear staining of p53, and cytoplasmic staining of BCL2. E-cadherin was expressed in three of four cases, P-cadherin in one of four, and N-cadherin in none of four cases. P53 was expressed in one of four cases and BCL2 in one of four cases. The four cases showed three different patterns of immunohistochemical staining for the five oncoproteins. Specifically, two cases expressed E-cadherin only; one case lacked all three cadherins, was negative for BCL2, and was only positive for p53; and one case expressed E- and P-cadherin and BCL2. Prior studies of other neuroendocrine and small cell tumors of other organs showed E-cadherin expressed in 98% (42 /43), N-cadherin in 65% (28/43), and P-cadherin in 40% (17/43) of cases. Additionally, one case of vaginal small cell carcinoma showed expression of all three cadherins. The only significant difference between cervical primaries and other primary sites is that N-cadherin was not detected in our four cases vs. 65% expression in other sites (P < 0.001). We conclude that cadherin and oncoprotein profiles in small cell carcinoma of the cervix are different in the four cases analyzed. Additional cases need to be studied to determine the specificity and frequency of these oncoprotein profiles for small cell carcinoma of the cervix. These may possibly represent different oncogenic pathways in development of small cell cancer of the cervix. Also, our results suggest that N-cadherin may be a tumor suppressor gene in these tumors.

Uterine papillary serous carcinoma presenting as distant lymph node metastasis.

BACKGROUND: Uterine papillary serous carcinomas are highly aggressive malignancies that often present with high-stage disease. We report two cases that presented initially as distant metastatic disease. One case was found incidentally at the time of axillary dissection for breast cancer and the second case in the workup of a neck mass. CASES: Clinicopathologic review of the patient material including review of routine H&E pathology and immunohistochemical studies of the patients tumors was performed. Both cases showed high-grade papillary carcinomas with psammoma bodies metastatic to lymph nodes in the axilla or neck. Sampling of the endometrium in these patients confirmed primary uterine papillary serous carcinoma. Patients were treated with adjuvant chemotherapy. CONCLUSIONS: Metastatic uterine papillary serous carcinoma presenting initially in distant sites is an unusual manisfestation of this highly aggressive tumor. This tumor should be considered in the differential diagnosis when patients present with metastatic high-grade papillary serous carcinomas and the primary site is unknown.

Cadherin expression in glandular tumors of the cervix.

BACKGROUND: The cadherins are homotypic adhesion proteins that are important in cell sorting during organogenesis. Classic cadherins include several different types that show tissue specific expression. Specific tissue expression of cadherins often is preserved in neoplastic transformation, and cadherin phenotype can be used to differentiate morphologically similar but histogenetically distinct tumors. METHODS: The authors examined by using immunohistochemistry in paraffin sections the expression of E- (epithelial) and P- (placental) cadherin in 39 patients with glandular tumors of the cervix, including invasive adenocarcinoma, villoglandular adenocarcinoma, adenocarcinoma in situ (AIS), and adenoma malignum. RESULTS: In all cases, E-cadherin was expressed in both normal and malignant glands without appreciable differences. P-cadherin, normally confined to basal epithelial cells and not observed in benign glands, was aberrantly expressed in neoplastic glands in 27 cases, including 96%(23 of 24 cases) of invasive cancers, 40% (2 of 5) of villoglandular carcinomas, 25% (2 of 8) of AIS, and 0% (0 of 2) of adenoma malignum. CONCLUSIONS: The authors' results show that E-cadherin is uniformly expressed in glandular tumors of the cervix with no evidence of decreased expression in these tumors. In addition, P-cadherin is aberrantly expressed in most adenocarcinomas and appears to be preferentially expressed in invasive rather than in situ lesions. Thus, aberrant expression of P-cadherin may be a useful marker of invasive or aggressive clinical behavior in glandular lesions of the cervix.

Primary, localized vulvar B-cell lymphoma expressing CD44 variant 6 but not cadherins. A case report.

BACKGROUND: Primary extranodal lymphoma of the vulva is rare, with only 17 prior cases reported. Its immunohistochemical profile has not been characterized beyond B- or T-cell phenotype. CASE: A 64-year-old, white woman presented with a nontender enlargement of the right labium minus and labium majus. Bilateral vulvar punch biopsies revealed an infiltrate of neoplastic lymphocytes that filled the reticular dermis and extended down to the subcutaneous fat. Lymphoma cells were positive for CD20 and expressed CD43 in an aberrant manner. The tumor was examined for adhesion protein expression. There was expression of CD44 standard and variant 6 but not of E-, N- or P-cadherin. No systemic spread of this rare lymphoma was evident after one year. CONCLUSION: Adhesion protein expression in primary vulvar lymphoma may have prognostic implications.

Nuclear localization of E-cadherin expression in Merkel cell carcinoma.

CONTEXT: Cadherins are cell-cell adhesion proteins that act as tumor suppressor genes and have a critical role in cell sorting and tissue formation during organogenesis. The pattern of cadherin expression constitutes a useful diagnostic and prognostic tool in the evaluation of tumors and for determining the histogenesis of tumor cells. We have previously characterized the cell types of several tumors based on the expression of individual cadherins. OBJECTIVE: To investigate the expression of cadherins in Merkel cell carcinomas. DESIGN: Paraffin immunohistochemical analysis of the 3 best-studied cadherins was performed on 35 cases of Merkel cell carcinoma. RESULTS: E-cadherin was expressed in 34 (97%) of 35 Merkel cell carcinomas examined, N-cadherin was expressed in 22 (63%) of 35 cases, and P-cadherin was expressed in 15 (43%) of 35 cases. This frequency of cadherin expression was similar to a group of small cell and neuroendocrine tumors from other primary sites. Interestingly, the localization of E-cadherin expression was unique in Merkel cell carcinomas compared with other primary neuroendocrine tumors. Merkel cell carcinomas showed marked preference for nuclear versus membrane localization, whereas small cell tumors from other sites showed fewer cases of nuclear E-cadherin expression. The nuclear localization of E-cadherin did not correlate with cadherin-associated protein beta-catenin nuclear expression. CONCLUSIONS: Our findings show that E-cadherin is the most frequently expressed cadherin in Merkel cell carcinoma, followed in frequency by N-cadherin then P-cadherin. The pattern of nuclear E-cadherin expression is more frequent for Merkel cell carcinoma than small cell tumors of other primary sites. These observations suggest that E-cadherin expression and function are altered in Merkel cell carcinoma, and this finding has potential use in the differential diagnosis of these tumors.

Coexpression of cytokeratins 7 and 20 confirms urothelial carcinoma presenting as an intrarenal tumor.

BACKGROUND: The differentiation of epithelial tumors arising in the kidney (urothelial vs. renal cell carcinoma) sometimes can be difficult by clinical and radiologic studies. Because urothelial and renal epithelium express unique cytokeratin (CK) 7 and 20 profiles, the authors studied the utility of these markers to confirm the diagnosis of urothelial carcinomas that present clinically as kidney masses. METHODS: Using commercially available monoclonal antibodies, paraffin section immunohistochemistry was used to examine two recent cases of urothelial carcinomas presenting as renal tumors. Tissues were stained for CK7 and CK20 and the expression compared between the tumor and benign tissue. RESULTS: Both cases showed solid renal masses that clinically and radiographically could have been of renal cell origin, but subsequently were confirmed histologically to be extensive renal involvement by urothelial carcinoma. The tumors coexpressed both CK7 and CK20, which is the expected profile for carcinomas of urothelial but not renal origin. CONCLUSIONS: The results of the current study show that coexpression of CK7 and CK20 is a useful diagnostic aid in the differential diagnosis of epithelial kidney tumors of urothelial cell versus renal cell origin.

P-cadherin expression in breast carcinoma indicates poor survival.

BACKGROUND: The cadherin family of cell-cell adhesion molecules and their associated proteins, the catenins, are essential to embryonic development and the maintenance of adult tissues. During development, the homotypic interaction of a particular cadherin with an identical cadherin expressed on a neighboring cell results in the sorting of cells to form distinctive tissues. Cadherins are believed to be tumor suppressors, and their altered expression and function have been associated with tumor development. METHODS: The authors examined the expression of P-cadherin, E-cadherin, and N-cadherin, and alpha-catenin and beta-catenin in 183 cases of invasive breast carcinoma by immunohistochemistry on paraffin sections using specific antibodies and a steam-based antigen retrieval method. RESULTS: P-cadherin was positive in 95 cases and negative in 88 cases of breast carcinoma. Positive P-cadherin expression in breast carcinoma showed a strong correlation with poor patient prognosis. Five years after surgery, 90% of the patients with P-cadherin negative tumors were alive in contrast to only 59% of patients with P-cadherin positive tumors. The difference in survival reached statistical significance (P = 0.0001) as early as 2 years after surgical treatment. Expression of N-cadherin, alpha-catenin, and beta-catenin did not correlate with patient survival. Multivariable statistical analyses of the data showed that expression of P-cadherin was independent of tumor size and lymph node metastases, but correlated inversely with estrogen/progesterone receptor status. In ductal carcinomas, positive P-cadherin expression correlated with a higher histologic grade. In contrast, expression of E-cadherin was low in high grade ductal carcinomas but negative tumors were uncommon. Negative or low E-cadherin expression did not correlate with poor survival. In lobular carcinomas, E-cadherin expression frequently was negative or low, and P-cadherin always was negative. CONCLUSIONS: Expression of P-cadherin in breast carcinoma is associated strongly with poor survival and constitutes an independent prognostic predictor. P-cadherin expression is a better indicator of clinical outcome than alterations in the expression of E-cadherin, N-cadherin, alpha-catenin, or beta-catenin.

Distinct cadherin profiles in special variant carcinomas and other tumors of the breast.

The cadherins are homotypic adhesion proteins that are important in cell sorting during organogenesis. Classical cadherins include several different types that show tissue-specific expression. Cell lineage-specific expression of different cadherin subtypes can differentiate morphologically similar but histogenetically distinct tumors. We examined by immunohistochemistry in paraffin sections, the expression of E (epithelial), N- (neural), and P- (placental) cadherin in 36 unusual tumors of the breast (22 medullary carcinomas, 5 metaplastic carcinomas, 2 carcinosarcomas, 4 phyllodes tumors, and 3 periductal stromal tumors). All carcinomas stain with E-cadherin (22 of 22 medullary and 5 of 5 metaplastic). E-cadherin also stained the epithelial component but not the sarcomatous areas of 2 of 2 cases of carcinosarcomas. E-cadherin was not detected in the stromal tumors (phyllodes, periductal stromal tumor). N-cadherin was most frequently expressed in sarcomatoid metaplastic carcinomas (5 of 5), and variably in other tumors, including the sarcomatous area of carcinosarcoma (1 of 2), and 6 of 22 medullary carcinomas. P-cadherin was frequently identified in medullary carcinomas (20 of 22), in 5 of 5 metaplastic carcinomas, and in the proliferating stroma and benign epithelium of 3 of 3 periductal stromal, but not in phyllodes tumors (0 of 4). All sarcomatoid metaplastic carcinomas co-expressed all 3 classical cadherins. Our results show that these breast tumors have unique patterns of cadherin expression suggesting different histogenetic origin or lines of differentiation. The cadherin profiles in these tumors may be useful for classification and diagnosis.

N-cadherin distinguishes pleural mesotheliomas from lung adenocarcinomas: a ThinPrep immunocytochemical study.

BACKGROUND: Cadherins are a family of cell-cell adhesion proteins. The homotypic binding of cadherins is critical for cell sorting and tissue formation during organogenesis. Different cadherin subtypes show lineage specific tissue expression, which has been exploited to differentiate histologically similar tumors of varying ontogeny. By applying immunohistochemistry to tissue sections, the authors have previously documented the utility of N-cadherin in distinguishing between pleural mesotheliomas and lung adenocarcinomas, based on the observation that N-cadherin is expressed in the former disease but not in the latter. Because the diagnosis of these diseases is frequently rendered on cytologic material rather than tissue biopsies, the authors wanted to assess the utility of N-cadherin immunocytochemistry in evaluating material prepared with ThinPrep. METHODS: The authors examined the cytologic material from 12 patients for the expression of N-cadherin using immunocytochemistry. Four patients had mesotheliomas and eight had adenocarcinomas. ThinPrep slides of the patients' pleural fluid were prepared and stained with the monoclonal antibody 13A9, which is specific for N-cadherin. RESULTS: Of the 12 cases studied, all 4 cases of pleural mesothelioma expressed N-cadherin in a specific cell-cell membrane location, and all 8 cases of lung adenocarcinoma were negative for N-cadherin. CONCLUSIONS: These results show that the N-cadherin specific antibody 13A9 is a suitable marker in material prepared with ThinPrep for the differentiation of pleural mesotheliomas from lung adenocarcinomas. This antibody should be included in the diagnostic immunocytochemical panel for evaluation of these malignancies.

Adenocarcinoma arising in extragonadal endometriosis: an immunohistochemical study.

BACKGROUND: Malignant transformation is an infrequent but reported complication of endometriosis. Previous reports of these cases have been limited to clinicopathologic studies based on routine histologic examination of these tumors, whereas, to the authors' knowledge, characterization of these lesions based on immunophenotype and hormone receptor and oncoprotein expression has not been described. METHODS: Using commercially available monoclonal antibodies, the authors studied three recent cases of adenocarcinoma arising in extragonadal endometriosis using paraffin immunohistochemistry. Proteins examined included different cytokeratin (CK) subtypes, as well as hormone receptor status, proliferation rate, and oncoprotein expression. RESULTS: All three cases presented clinically and macroscopically as colonic masses, and the tumors expressed an endometrial CK phenotype (CK7+, CK20-). In contrast, the adjacent benign colonic epithelium expressed the expected opposite phenotype (CK7-, CK20+). Estrogen receptor (ER) and progesterone receptor (PR) were expressed in one of the three tumors. Interestingly, in the ER/PR negative tumors, receptor expression was present in areas of benign endometriosis adjacent to malignancy, suggesting a loss of receptor expression with malignant transformation. The tumors also were examined for proliferation by Ki-67, and the expression of oncoproteins c-erb B-2, p53, cyclin D1, and bcl-2. All cases of malignancy had a high proliferation rate as measured by Ki-67, which was in contrast to areas of benign endometriosis which had a low proliferation rate. Of the other oncoproteins only p53 protein was detected at a significant level in all three cases. Cyclin D1 was overexpressed in two of the three cases. c-erb B2 and bcl-2 overexpression was not detected. CONCLUSIONS: The results of the current study 1) show the utility of CK subtypes in confirming endometrioid phenotype in tumors arising in extragonadal endometriosis with colonic involvement and 2) suggest that loss of hormone receptor expression and p53 oncoprotein abnormalities may be involved as mechanisms in malignant transformation in extragonadal endometriosis.

Expression of P-cadherin identifies prostate-specific-antigen-negative cells in epithelial tissues of male sexual accessory organs and in prostatic carcinomas. Implications for prostate cancer biology.

Cadherins constitute a family of calcium-dependent cell-cell adhesion molecules the individual members of which are essential for the sorting of cells into tissues during development. In this study, we examined the expression of E-cadherin, N-cadherin, and P-cadherin in tissues obtained from radical prostatectomies. Epithelial cells of prostatic glands, ejaculatory ducts, and seminal vesicles expressed E-cadherin but not N-cadherin. P-cadherin was expressed in epithelial cells of the seminal vesicles and ejaculatory ducts. In the prostate it was limited to the basal cells of prostatic acini, glands with basal cell hyperplasia, and atrophic glands denuded of the luminal cells. All P-cadherin-positive cells were negative for prostatic-specific antigen. Prostatic cancers were mostly P-cadherin negative, but some tumors had P-cadherin-positive areas frequently located close to ejaculatory ducts and negative for prostatic-specific antigen. The mutually exclusive expression of P-cadherin and prostatic-specific antigen suggests that these proteins are involved in differential mechanisms of cell regulation in prostate cancer. P-cadherin may become a useful marker in the diagnosis and management of patients with prostate cancer and low levels of prostatic-specific antigen.

Differential expression of N-cadherin in pleural mesotheliomas and E-cadherin in lung adenocarcinomas in formalin-fixed, paraffin-embedded tissues.

The differential diagnosis of pleural mesotheliomas and lung adenocarcinomas presents a continued challenge in the practice of surgical pathology. Paraffin immunohistochemistry (IHC) using different panels of antibodies can be helpful in some cases, but, as yet, no antigen is expressed specifically in mesotheliomas nor in adenocarcinomas. Using well characterized monoclonal antibodies (MAb) that recognized distinct mesenchymal and epithelial adhesion proteins, N-cadherin (13A9 MAb) and E-cadherin (E9 MAb), respectively, we found previously that in frozen-section IHC mesotheliomas and adenocarcinomas had distinct cadherin phenotypes: mesotheliomas were positive for N-cadherin, and lung adenocarcinomas were positive for E-cadherin. Using antigen-retrieval methods, we successfully extended our study to formalin-fixed, paraffin-embedded tissue sections. Tumors from 28 patients (14 originally diagnosed as mesotheliomas, and 14 diagnosed as adenocarcinomas) were stained with 13A9 MAb and E9 MAb. Review of hematoxylin-eosin sections excluded from analysis one case previously diagnosed as mesothelioma, which represented a hemangiopericytoma. Of the remaining 27 cases, 12 of 13 mesotheliomas were positive for N-cadherin and negative for E-cadherin. The exception was a multifocal microscopic papillary tumor of apparent mesothelial origin, which was negative for both N-cadherin and E-cadherin. Conversely, 13 of 14 adenocarcinomas were E-cadherin positive and N-cadherin negative except for one adenocarcinoma with focal N-cadherin expression. One case of a poorly differentiated adenocarcinoma invading skeletal muscle was negative for both 13A9 and E9. These studies confirmed the utility of the cadherin antibodies in distinguishing pleural mesotheliomas from lung adenocarcinomas. The reactivity of the cadherin-specific antibodies with antigens in paraffin sections make them powerful and reliable markers in the practice of diagnostic surgical pathology.

Expression of E-cadherin and N-cadherin in surface epithelial-stromal tumors of the ovary distinguishes mucinous from serous and endometrioid tumors.

This study examines the expression of E-cadherin and N-cadherin in the most common epithelial tumors of the ovary. The homotypic interactions of distinctive members of the cadherin family of cell-cell adhesion molecules segregate cells into tissues during embryonic development, and their expression in tumors can be used to trace the histogenesis of tumor cells. Because the surface epithelium of the ovary is a modified mesothelium, we speculated that the expression of E (epithelial)-cadherin and N (neural, mesodermal)-cadherin may provide clues about the controversial origin of common epithelial ovarian tumors. Immunohistochemistry was performed in paraffin sections using well-characterized monoclonal antibodies to E- and N-cadherin and heat-induced antigen-retrieval methods. We found that serous and endometrioid tumors express both E- and N-cadherin. In contrast, mucinous tumors strongly express E-cadherin, but no N-cadherin. The presence of N-cadherin in serous and endometrioid tumors traces their origin to the mesoderm-derived ovarian surface epithelium. The absence of N-cadherin in mucinous tumors clearly distinguishes them from the former, suggesting histogenesis from a cell lineage other than the ovarian surface epithelium or aberrant differentiation mechanisms associated with neoplastic transformation.

An unexpectedly labile mitochondrially encoded protein is required for Mta expression.

Maternally transmitted antigen (Mta) is a mouse major histocompatibility antigen recognized by cytotoxic T lymphocytes. A role for mitochondria in expression of this class I-like cell surface antigen has been previously established. We now show that a labile product of mitochondrial protein synthesis is required for Mta expression. Reexpression of Mta determinants after enzymatic removal occurred within 24 h, and the regeneration process was sensitive to chloramphenicol (CAP), a selective inhibitor of mitochondrial protein synthesis. Additionally, target cells treated with CAP for as little as 18 h showed diminished expression of Mta. The estimated half-life for Mtf products ranged from 6 to 15 h, less than the half-lives of known mitochondrial translation products. This suggests that the Mtf product is not generated by the normal turnover of stable mitochondrial respiratory proteins. Instead, these results indicate the existence of either labile unknown mitochondrially encoded peptides or a rapid turnover pathway for known mitochondrial products.

Activation of branched-chain alpha-ketoacid dehydrogenase in isolated hepatocytes by branched-chain alpha-ketoacids.

The effects of branched-chain alpha-ketoacids on flux through and activity state of the branched-chain alpha-ketoacid dehydrogenase complex were studied in hepatocytes prepared from chow-fed, starved, and low-protein-diet-fed rats. Very low concentrations of alpha-ketoisocaproate caused a dramatic stimulation (50% activation at 20 microM) of alpha-ketoisovalerate decarboxylation in hepatocytes from low-protein-fed rats. alpha-Keto-beta-methylvalerate was also effective, but less so than alpha-ketoisocaproate. alpha-Ketoisocaproate did not stimulate alpha-ketoisovalerate decarboxylation by hepatocytes from chow-fed or starved rats. To a smaller degree, alpha-keto-beta-methylvalerate and alpha-ketoisovalerate stimulated alpha-ketoisocaproate decarboxylation by hepatocytes from low-protein-fed rats. The implied order of potency of stimulation of flux through branched-chain alpha-ketoacid dehydrogenase was alpha-ketoisocaproate greater than alpha-keto-beta-methylvalerate greater than alpha-ketoisovalerate, i.e., the same order of potency of these compounds as branched-chain alpha-ketoacid dehydrogenase kinase inhibitors. Fluoride, known to inhibit branched-chain alpha-ketoacid dehydrogenase phosphatase, largely prevented alpha-ketoisocaproate and alpha-chloroisocaproate activation of flux through the branched-chain alpha-ketoacid dehydrogenase. Assay of the branched-chain alpha-ketoacid complex in cell-free extracts of hepatocytes isolated from low-protein-diet-fed rats confirmed that alpha-ketoacids affected the activity state of the complex. Branched-chain alpha-ketoacids failed to activate flux in hepatocytes prepared from chow-fed and starved rats because essentially all of the complex was already in the dephosphorylated, active state. These findings indicate that inhibition of branched-chain alpha-ketoacid dehydrogenase kinase activity by branched-chain alpha-ketoacids is important for regulation of the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase.

Unglycosylated Mtaa expresses an Mtab-like determinant.

Antigenic polymorphism of the class I-like maternally transmitted antigen (Mta) is controlled by a maternally transmitted factor (Mtf) thought to reside in mitochondria. However, the mechanisms by which Mtf generates antigenic polymorphism are not known. To address this issue, we investigated a possible role of posttranslational oligosaccharide addition in the formation of Mta determinants. We examined the expression of Mta on cytotoxic T lymphocyte (CTL) target cells cultured in tunicamycin (TM), a known inhibitor of asparagine(N)-linked glycosylation. Of 18 Mtab-specific CTL lines, 8 lysed TM-treated Mtaa targets. Furthermore, a subclone of one of these eight lines, 17D5.G2, lysed TM-treated targets from all Mtaa strains tested, regardless of H-2K/D haplotype. On the other hand, this CTL clone did not lyse TM-treated target cells from the Mta null, but H-2 expressing strain B10.CAS2. Therefore expression of this Mtab-like determinant is concordant with the expression of Mtaa and seems unlikely to represent a cross-reactive H-2K/D epitope. Our data suggest that an Mtab-like determinant is expressed on unglycosylated Mtaa molecules. Thus, N-linked oligosaccharides probably prevent the expression of an Mtab-like determinant on the Mtaa molecule. We discuss how Mtf may contribute to Mta polymorphism through glycosylation.

Regulation of branched-chain alpha-ketoacid dehydrogenase complex by covalent modification.

The branched-chain alpha-ketoacid dehydrogenase complex, like the pyruvate dehydrogenase complex, is an intramitochondrial enzyme subject to regulation by covalent modification. Phosphorylation causes inactivation and dephosphorylation causes activation of both complexes. The branched-chain alpha-ketoacid dehydrogenase kinase, believed distinct from pyruvate dehydrogenase kinase, is an integral component of the branched-chain alpha-ketoacid dehydrogenase complex and is sensitive to inhibition by branched-chain alpha-ketoacids, alpha-chloroisocaproate, phenylpyruvate, clofibric acid, octanoate and dichloroacetate. Phosphorylation of branched-chain alpha-ketoacid dehydrogenase occurs at two closely-linked serine residues (sites 1 and 2) of the alpha-subunit of the decarboxylase. HPLC and sequence data suggest homology of the amino acid sequence adjacent to phosphorylation sites 1 and 2 of complexes isolated from several different tissues. Stoichiometry for phosphorylation of all of the complexes studies was about 1 mol P/mol alpha-subunit for 95% inactivation and 1.5 mol P/mol alpha-subunit for maximally phosphorylated complex. Site 1 and site 2 were phosphorylated at similar rates until total phosphorylation exceeded 1 mol P/mol alpha-subunit. The complexes from rabbit kidney, rabbit heart, and rat heart showed 30-40% additional phosphorylation of the alpha-subunit beyond 95% inactivation. Site specificity studies carried out with the kinase partially inhibited with alpha-chloroisocaproate suggest that phosphorylation of site 1 is primarily responsible for regulation of the complex. The capacity of the branched-chain alpha-ketoacid dehydrogenase to oxidize pyruvate (Km = 0.8 mM, Vmax = 20% of that of alpha-ketoisovalerate) interferes with the estimation of activity state of the hepatic pyruvate dehydrogenase complex. The disparity between the activity states of the two complexes in most physiologic states contributes to this interference. An inhibitory antibody for branched-chain alpha-ketoacid dehydrogenase can be used to prevent interference with the pyruvate dehydrogenase assay. Almost all of the hepatic branched-chain alpha-ketoacid dehydrogenase in chow-fed rats is active (greater than 90% dephosphorylated). In contrast, almost all of the hepatic enzyme of rats fed a low-protein (8%) diet is inactive (greater than 85% phosphorylated). Fasting of chow-fed rats has no effect on the activity state of hepatic branched-chain alpha-ketoacid dehydrogenase, i.e. greater than 90% of the enzyme remains in the active state. However, fasting of rats maintained on low-protein diets greatly activates the hepatic enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Purification of testosterone-oestradiol-binding globulins from mammalian sera by anion-exchange high performance liquid chromatography.

Testosterone-oestradiol-binding globulin (TeBG) has been isolated from serum or plasma of several species using procedures that yielded highly purified protein, but which required multiple and tedious chromatographic steps. In this report we describe a procedure for the isolation of TeBG which involves two chromatographic steps: androgen affinity chromatography followed by anion-exchange high performance liquid chromatography (anion-exchange HPLC). The purity of the final product was confirmed by silver staining following fractionation on sodium dodecyl sulphate-containing polyacrylamide gels. The size heterogeneity and specific binding activity of TeBGs purified from human, rabbit, or bull serum (or plasma) by this technique was indistinguishable from preparations obtained by conventional chromatography. The present technique shortened the entire purification procedure to about 5 working days and yielded milligram quantities of highly purified protein. Bases on our experience with serum or plasma from the human, rabbit, and bull, this approach should be suitable for isolation of TeBG from a wide range of species.