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Unlabelled: What's known on the subject? and What does the study add? Epithelial-mesenchymal transition (EMT) is involved in tumor progression where the underlying cellular changes associated with EMT have been identified in in vitro models and confirmed in a limited number of in vivo studies. ZEB1, which targets E-cadherin repression, is a transcriptional regulator that has been implicated in EMT, and is associated with uterine and colorectal cancers. Regulation of ZEB1 expression has been shown to involve different microRNAs (miRNAs), identifying a potential role for miRNA in EMT. In the present study we have identified novel expression of ZEB1 in bladder tumours and shown a role for ZEB1 in enhanced migration and invasion potential in in vitro assays. Confirmation of ZEB1 expression in bladder tumours was shown in tissue microarrays (TMAs)., Objective: To evaluate ZEB1 expression in bladder tumorigenesis and define a possible role for this transcription factor in urothelial carcinomas of the bladder (UCBs)., Materials and Methods: Five hundred and fifty-eight samples were assembled in 10 tissue microarrays (TMAs; 263 non-muscle-invasive Ta/T1/Tis, 295 muscle-invasive T2-T4). All tumours were transitional cell carcinomas (TCCs) and processed for immunohistochemistry to assess nuclear ZEB1 expression. Expression levels of ZEB1 were modulated in bladder carcinoma cell lines CUBIII or UM-UC-3 after forced expression or shRNA knockdown, respectively. Protein expression levels were determined using western blot analysis and transfectants were assessed for migration and invasion potential in standard in vitro assays., Results: Nuclear ZEB1 expression was recorded in 22.8% of non-muscle-invasive UCBs and 21.7% of muscle-invasive UCBs, including 24.1% grade I/II and 21.1% grade III tumours, and absent in normal bladder mucosa. No significant correlation was observed for tumour stage and grade, nodal involvement, vascular invasion, metastasis and overall or cancer-specific survival. The introduction or knockdown of ZEB1 expression in bladder carcinoma cell lines showed enhanced or reduced migration and invasive potential, respectively. Changes in ZEB1 expression were accompanied by altered microRNA (miRNA) expression underlying events linked to epithelial-mesenchymal transition (EMT)., Conclusion: The results in the present study showed novel expression of ZEB1 in bladder cancer in the absence of a link to clinical variables of change, including metastasis and survival. However, in vitro assays showed enhanced or reduced migration and invasion after the introduction or reduction of ZEB1, respectively, in transfected bladder cell lines. Modulation in expression of ZEB1 was closely linked to changes in the miR-200 family along with alternative known prognostic indicators of bladder tumour progression., (© 2010 THE AUTHORS. JOURNAL COMPILATION © 2010 BJU INTERNATIONAL.)

Peptide immunohistochemistry (IHC) controls are a new quality control format for verifying proper IHC assay performance, offering advantages in high throughput automated manufacture and standardization. We previously demonstrated that formalin-fixed peptide epitopes, covalently attached to glass microscope slides, behaved (immunochemically) in a similar fashion to the native protein in tissue sections. To convert this promising idea into a practical clinical laboratory quality control tool, we tested the hypothesis that the quality assurance information provided by peptide IHC controls accurately reflects IHC staining performance among a diverse group of clinical laboratories. To test the hypothesis, we first designed and built an instrument for reproducibly printing the controls on microscope slides and a simple software program to measure the color intensity of stained controls. Automated printing of peptide spots was reproducible, with coefficients of variation of 4% to 8%. Moreover, the peptide controls were stable at

Microvascular clamps have several potential shortcomings, including the risk of vessel injury. LeGoo, a novel reverse thermosensitive polymer (Pluromed Inc., Woburn, MA), is investigated as a substitute to vascular clamping in a microsurgical model and the technical details are described. Femoral vessels of Sprague Dawley rats were used to evaluate the usefulness of this polymer for performing end-to-end arterioarterial (AA), venovenous (VV), and end-to-side arteriovenous (AV) microvascular anastomoses. The ability to obtain and maintain hemostasis was assessed. Secondary endpoints, including polymer volume, concentration, temperature, infusion technique, ability to reinfuse, blood vessel stenting effect, polymer dissolution characteristics, and reestablishment of flow, were also noted. Initial hemostasis occurred in every case. Mean duration of efficacy (hemostasis) after initial injection was 17.8 minutes (4 minutes to 44.5 minutes) for AA anastomoses and 31.8 minutes (13 minutes to 46 minutes) for VV anastomoses. Mean volume of polymer initially injected was 0.11 mL (0.01 mL to 0.20 mL) and 0.07 mL (0.06 mL to 0.10 mL) for AA and AV arteries, respectively, and 0.14 mL (0.10 mL to 0.20 mL) and 0.20 mL (0.15 mL to 0.27 mL) for VV and AV veins. Use of LeGoo in veins was clearly superior to arterial use with regard to the technical aspects of injecting LeGoo and reestablishing hemostasis, as well as greater vessel stenting effect and less vessel retraction. This novel polymer showed promise for its ability to allow for hemostasis while performing microvascular anastomoses. Improvements were made with regard to injection techniques, appropriate volumes, ability to reliably determine gel plug dissolution, and final vessel patency. Preliminary results demonstrate that this polymer may be a viable substitute for microvascular clamps.

Objective: To identify changes associated with P-cadherin expression in bladder cancer and evaluate the potential role of such events in determining the clinical outcome and cell behaviour, as the function of P-cadherin in normal epithelium is unknown, as is its potential role in neoplastic progression in different cancers., Materials and Methods: In all, 536 bladder tumour specimens from 408 patients were assembled in seven tissue microarrays. Paraffin sections from each array were processed for immunohistochemistry to assess the expression of P-cadherin. The expression of P-cadherin was forced using lipofectin, followed by an assessment of migration and invasion potential using standard in vitro assays., Results: The absence of P-cadherin staining was associated with muscle-invasive disease, grade 3 (P < 0.001) and nodal disease (P = 0.009). Similar results were obtained when considering cytoplasmic and unrestricted localization of P-cadherin (P < 0.001), except for nodal involvement. The group with cytoplasmic location of P-cadherin showed a shorter cancer-specific survival than the group with membrane location of P-cadherin (P = 0.03). Forced expression of P-cadherin in EJ and UM-UC-3 cells, that constitutively lack P-cadherin expression, resulted in modulation of catenin expression and enhanced migration of EJ and UM-UC-3/P-cadherin transfectants (>200%)., Conclusions: These results showed that loss of expression, cytoplasmic relocation or unrestricted tissue location of P-cadherin was associated with a poor clinical outcome and prognosis in bladder cancer. From the in vitro work it is evident that P-cadherin plays a role in regulating the migration potential of bladder carcinoma cells.

Objective: To identify the frequency of change in the expression and localization of p120(ctn) in bladder tumours and its association with clinical outcomes, and to investigate the potential role of p120(ctn) in the migratory and invasive behaviour of bladder carcinoma cells., Materials and Methods: In all, 425 superficial tumour specimens (Ta, Tis and T1) and 305 invasive (T2-T4) tumour specimens from 534 patients were assembled in 10 tissue microarrays. P120(ctn) immunostaining was scored for intensity and cellular localization and correlated with clinical variables and survival analysis. Knockdown of p120(ctn) was achieved using small-interference RNA (siRNA) followed by the assessment of migration and invasion behaviour in standard in vitro assays., Results: The expression levels of p120 catenin inversely correlated with pathological tumour stage (P < 0.001), histological grade (P < 0.001), presence of lymphovascular invasion (P = 0.02) but not lymph node (LN) involvement (P = 0.17). Non-membranous localization of p120(ctn) correlated with stage (P < 0.001), grade (P < 0.001), lymphovascular invasion (P = 0.04) and LN-positive disease (P = 0.02). A low expression level of p120(ctn) was linked to a poor outcome in cancer-specific survival analysis. Knockdown of p120(ctn) using siRNA resulted in a significant reduction in the migration and invasive potential of bladder carcinoma cells., Conclusions: Our findings suggest that p120(ctn) acts as a prognostic factor in bladder tumours and has a primary role to play in the migratory and invasive behaviour of bladder carcinoma cells.

Purpose: Epithelial to mesenchymal transition (EMT) is reportedly an important transition in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. In this study, we examined the expression pattern of EMT markers in vivo and determined the occurrence and clinical significance of these events in a series of bladder carcinomas., Experimental Design: Eight hundred and twenty-five tumor samples from 572 bladder cancer patients were assembled in 10 tissue microarrays. Paraffin sections from each tissue microarray were subjected to antigen retrieval and processed by immunohistochemistry for the expression of E-cadherin, plakoglobin, beta-catenin, N-cadherin, and vimentin., Results: Pathologic expression of E-cadherin, beta-catenin, plakoglobin, and vimentin were associated with the clinicopathologic variables of grade and stage with only the cytoplasmic localization of plakoglobin found associated with lymph node status. Associations between the aforementioned markers were found significant as determined by the Spearman correlation coefficient with N-cadherin showing no associations in this analysis. In univariate survival analysis involving patients who underwent cystectomy, the reduction or loss of plakoglobin significantly influenced overall survival (P = 0.02) in which the median time to death was 2 years compared with 4 years when a normal level of plakoglobin was recorded. When the analysis was done for cancer-specific survival, low levels of both plakoglobin (P = 0.02) and beta-catenin (P = 0.02) significantly influenced survival., Conclusion: The putative markers of EMT defined within a panel of bladder carcinoma cell lines were recorded in vivo, frequently associated with tumors of high grade and stage. Although multivariate analysis showed no significant influence of the EMT biomarkers on survival, alterations associated with plakoglobin were identified as significant prognostic features in these tumors.

Abnormalities of the p53 tumor-suppressor gene are found in a significant proportion of astrocytic brain tumours. We studied tumour specimens from 74 patients evaluated over 20 years at the Massachusetts General Hospital, where clinical outcome could be determined and sufficient pathologic material was available for immunostaining. p53 expression studies employed an affinity-purified p53 monoclonal antibody, whose specificity was verified in absorption studies and, in a minority of cases, a second antibody recognising a different epitope of p53. Significant overexpression of p53 protein was found in 48% of the 74 tumours included in this series and high levels of expression were associated with higher mortality from astrocytic tumours (P<0.001, log rank). Multivariate analyses revealed that immunohistochemically detected p53 was an independent marker of shortened progression-free and overall actuarial survival in patients with astrocytic tumours, suggesting that increased expression of p53 plays an important role in the pathobiology of these tumours. In a subset of 36 cases, coding regions of the p53 gene were completely sequenced via SSCP and direct DNA sequencing, revealing that overexpression of p53 protein is not always associated with point mutations in conserved exons of the p53 gene. Finally, we confirmed p53 protein expression in early-passage human glioma cell lines of known p53 mutational status and immunostaining scores. Although grade continues to be the strongest prognostic variable, the use of p53 staining as a prognostic indicator, in contrast to mutational DNA analyses, may be a useful adjunct in identifying patients at higher risk of treatment failure.

Human thioredoxin is a putative oncogene that may confer both a growth and survival advantage to tumor cells. Overexpressed thioredoxin mRNA has been found in both primary human lung and colorectal cancers. To determine the intratumor distribution and amount of thioredoxin protein in human primary carcinomas, we developed an immunohistochemical assay for thioredoxin in paraffin-embedded tissue. We then studied 10 patients with primary high-risk gastric carcinoma. To further relate thioredoxin protein overexpression to cell death and survival, we used a paraffin-based in situ end-labeling (ISEL) assay. To delineate proliferation, we used the nuclear proliferation antigen detected by Ki-67. In this survey, we found that thioredoxin was localized to tumor cells and overexpressed compared with normal gastric mucosa in 8 of 10 gastric carcinomas. The thioredoxin was found at high levels in 5 of the 8 overexpressing carcinomas. The overexpression of thioredoxin was typically found in both a nuclear and cytoplasmic location in the neoplastic cells. There was a significant positive correlation (P = .0061) with cancer cell proliferation measured by Ki-67. There was a significant negative correlation (P = .0001) with DNA damage measured by the ISEL assay, suggesting decreased apoptosis and increased carcinoma cell survival. Thus, human primary gastric tumors that are highly expressive of thioredoxin have both a higher proliferative rate and a higher survival rate than tumors that do not express thioredoxin. With these newly developed assays in hand, it is now feasible to question whether this thioredoxin-related combined growth and survival advantage translates into poor clinical outcome.

Although in situ hybridization has been in use for almost 30 years, its technically demanding nature, the requirements for optimal tissue fixation and preservation, and the turnaround time for the experiments have prevented this technique from becoming widely used in the surgical pathology setting. The use of nonisotopic reporter molecules, the possibility of performing hybridization on archival material, and very recently, automation of the procedure have brought in situ hybridization to the forefront of diagnostic and experimental pathology. We describe our experience with nonradioactive, automated in situ hybridization, compare the technique with traditional manual procedures, and briefly outline its potential applications in diagnostic pathology and in the research setting.

The contributing role of hepatocytes and IL-6 in the acute phase-like elevation of plasma type 1 plasminogen activator inhibitor (PAI-1) in vivo is not known. We addressed this question by comparing the effects of two inflammatory stimuli known to increase plasma concentrations of IL-6 on the plasma concentrations and site of synthesis of PAI-1 and cysteine proteinase inhibitor (CPI) in rats. Subcutaneous injection of turpentine results in a sustained increase in plasma IL-6 and CPI Ag levels over a 24-h period. In contrast, plasma PAI-1 activity was not altered by turpentine treatment. Northern blot analysis of poly(A)+ mRNA extracted from freshly isolated hepatocytes of saline- or turpentine-treated animals demonstrated induction of CPI mRNA expression but failed to detect basal or induced PAI-1 or IL-6 mRNA expression. However, PAI-1 mRNA was detected in rat hepatocytes in primary culture for 24 h and was induced by dexamethasone. Intravenous infusion of bacterial LPS (4 mg/kg) induced a sustained increase in plasma CPI Ag and transient increases in plasma IL-6 and PAI-1. Northern blot analysis of freshly isolated, fractionated liver cells indicated that LPS treatment (3 h) induced PAI-1 mRNA expression most significantly in the endothelial and Kupffer cell fractions. IL-6 mRNA expression was induced in Kupffer cells and CPI mRNA was induced in hepatocytes. Immunocytochemical analysis revealed LPS-induced accumulation of PAI-1 Ag associated with the vascular endothelium, subendothelial matrix, and sinusoidal lining cells. Our results indicate that PAI-1 mRNA is not significantly expressed by rat hepatocytes in vivo and that plasma PAI-1 levels are not influenced by increased IL-6 expression in Kupffer cells or in plasma.

Glucocorticoids exert potent inhibitory effects on bone formation. We have previously shown that glucocorticoids suppress plasminogen activator (PA) activity in normal and malignant rat osteoblasts. To clarify the mechanism of this suppression, we investigated the effects of dexamethasone on PA inhibitor-1 (PAI-1), tissue-type PA (tPA), and urokinase-type PA (uPA) expression and also on PAI-1 protein and PA activity in both normal rat calvarial osteoblasts and a clonal osteogenic sarcoma cell line, UMR 106-01. Dexamethasone increased PAI-1 mRNA and protein in both cell types. The increase in PAI-1 protein and the decrease in PA activity were obtained over the same concentration range, with a half-maximally effective concentration of dexamethasone of about 10(-9) M. The increase in PAI-1 mRNA caused by dexamethasone was retained with cycloheximide treatment, but abolished with actinomycin-D. Dexamethasone had no effect on tPA or uPA mRNA in either cell type. The glucocorticoid antagonist RU 486 prevented the effects of dexamethasone on PA activity and PAI-1 protein. Dihydrotestosterone, progesterone, and 17 beta-estradiol did not influence PA activity or PAI-1 formation. Although tPA and uPA protein could not be measured, these results suggest that glucocorticoids suppress PA activity predominantly by increasing PAI-1 synthesis in rat osteoblasts. Suppression of PA activity through actions on PAI-1 formation by glucocorticoids could contribute to the mechanisms by which glucocorticoids inhibit bone formation.

The expression of urokinase-type plasminogen activator (u-PA) and its type-1 inhibitor (PAI-1) was examined in vivo in mouse wounds by in situ hybridization and immunohistochemistry. u-PA mRNA was present in both basal and suprabasal keratinocytes in the regenerative epithelial outgrowths at the edge of the wounds. In the same area, PAI-1 mRNA was only present in the basal keratinocytes. u-PA protein was detected in keratinocytes in several layers of the epithelial outgrowth, whereas PAI-1 protein was confined to the basal keratinocytes and to the area of the basal membrane. The two proteins and their mRNA were not detected in normal epidermis or in normal-looking epidermis adjacent to the wounds. Fibroblast-like cells and fairly large stellate cells (possibly macrophages) in the granulation tissue underneath the wound contained both the two proteins and their mRNA. The large stellate cells, showing a strong hybridization signal for PAI-1 mRNA, were especially abundant at the border between the necrotic wound and the newly formed granulation tissue. The specificity of these results was supported by the use of two different non-overlapping antisense probes, sense mRNA probes, antibody preparations preabsorbed with purified proteins, and Northern analysis of tissue extracts. The localized and regulated expression of u-PA and PAI-1 seen in this study may reflect that plasminogen activation plays a role in the migration of keratinocytes and connective tissue cells during reepithelialization and tissue remodeling in wound healing.

Transforming growth factor beta (TGF beta) treatment of rat osteoblast-rich calvarial cells or of the clonal osteogenic sarcoma cells, UMR 106-01, resulted in dose-dependent inhibition of plasminogen activator (PA) activity, and increased production of 3.2 kb mRNA and protein for PA inhibitor -1 (PAI-1). Although tissue-type PA (tPA) protein was not measured, TGF beta did not influence production of mRNA for tPA. Production of 2.3 kb mRNA for urokinase-type PA (uPA) was also increased by TGF beta in a dose-dependent manner. The effects of TGF beta on synthesis of mRNA for PAI-1 and uPA were maintained when protein synthesis was inhibited, and were abolished by inhibition of RNA synthesis. Although uPA had not been detected previously as a product of rat osteoblasts, treatment of lysates of osteoblast-like cells with plasmin yielded a band of PA activity on reverse fibrin autography, corresponding to a low Mr form of uPA. Untreated conditioned media from normal osteoblasts or UMR 106-01 cells contained no significant TGF beta activity, but activity could be detected in acidified medium. Treatment of conditioned media with plasmin resulted in activation of approximately 50% of the TGF beta detectable in acidified media. The results identify several effects of TGF beta on the PA-PA inhibitor system in osteoblasts. Net regulation of tPA activity through the stimulatory actions of several calciotropic hormones and the promotion of PAI-1 formation by TGF beta could determine the amount of osteoblast-derived TGF beta activated locally in bone. Stimulation of osteoblast production of mRNA for uPA could reflect effects on the synthesis of sc-uPA, a precursor for the active form of the enzyme.

The synthesis and release of plasminogen activators (PAs) in co-cultures of embryonic rat dorsal root ganglion nerve cells (NCs) and Schwann cells (SCs) were examined by metabolic labeling, immunoprecipitation, immunodepletion, SDS-PAGE, zymography, and a two-step esterolytic assay. Metabolic labeling of SC cultures followed by immunoprecipitation of the conditioned medium (CM) demonstrated that cultured SCs synthesized and released tissue type PA (tPA). Failure of amiloride to inhibit PA activity in SCCM indicated that urokinase PA (uPA) was unlikely to contribute significantly to PA activity in SCCM. Experimental manipulation of the NCs and SCs suggested that NCs regulated SC derived PA. Total PA activity increased in SCCM 10-14-fold by 6 days after removal of NCs. Multiple molecular weight forms of PAs were detected by SDS-PAGE followed by zymography. A PA approximately 95 kDa was absent in co-cultures of SCs + NCs but prominent by 4 days postdenervation; PA approximately 50-70 kDa increased through 8 days postdenervation and PA approximately 25 kDa, present in SC + NC cultures, was absent 8 days after removal of NCs. Upon reintroduction of NCs to denervated cultures (SCs), the pattern of PAs detected in culture medium was transitional between innervated and denervated cultures. Immunodepletion experiments using conditioned medium from denervated SC cultures indicated that various molecular weight forms of PA detected in SCCM by zymography were immunologically related to tPA. These studies demonstrate that SCs synthesized and released tPA in a tissue culture model of peripheral nerve and that one mechanism for regulation of PA released by SCs was by association with NCs. This regulation occurred in cultures of both myelinating and nonmyelinating Schwann cells and thus was not dependent on the state of myelination.

The tissue-specific distribution of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) and their inhibitor type 1 (PAI-1) was analyzed at mRNA level in five major rat organ tissues. t-PA mRNA was detected in lung, kidney, heart, and liver. u-PA mRNA was detected in kidney and lung. Presence of PA mRNA correlated with the detection of PA activity in extracts of these tissues. PAI-1 mRNA was detected predominantly in heart and lung. Although PAI activity could not be measured directly in tissue extracts, the presence of PAI-1 mRNA correlated with the occurrence of PA.PAI complex in fibrin autography of tissue extracts. Endotoxin injection caused a very large increase in plasma PAI activity. This increase correlated with a marked increase in PAI-1 mRNA in nearly all tissues studied. The increase in PAI-1 mRNA is most pronounced in lung and liver. Endotoxin injection also caused an increased level of t-PA mRNA in heart and kidney, and an increased u-PA mRNA level in kidney. mRNA analysis of freshly isolated and separated subfractionated liver cells showed that the marked increase in PAI-1 mRNA in the liver after endotoxin injection may be due mainly to a strong increase of PAI-1 mRNA in the liver endothelial cells.

Normal rat kidney (NRK) fibroblasts respond to the cell shape-modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52-kDa substrate-associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43-kDa "core" protein (p43), four 50-kDa species (p50-0,1,2,3), and at least seven distinct pI variants of the mature 52-kDa protein. A threshold of 2-4 microM CD was found to be necessary to augment p52 deposition into both the secreted protein- and saponin-resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD-treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type-1 (rPAI-1) and to PAI-1-like proteins of other species by comparative immunoprecipitation, 2-D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI-1 cytoplasmic mRNA abundance, using the rPAI-1 cDNA probe pSS1-3, revealed an induction of rPAI-1 mRNA in NRK/CD cells which paralleled the increased protein production. CD-augmented p52(rPAI-1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape-associated metabolic processes and concomitant RNA synthesis.

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits tissue-type plasminogen activator activity by inducing a specific plasminogen activator-inhibitor (PAI-1). Using immobilized polyclonal antibodies raised against HT-1080 human fibrosarcoma PAI-1, we have purified HTC PAI-1 from serum-free medium conditioned by dexamethasone-treated HTC hepatoma cells and shown it to be antigenically related to human PAI-1. Greater than 100-fold purification with greater than 75% yield was achieved in a single step. The purified PAI-1 migrates on SDS-polyacrylamide gels as a single major band of 49 kDa with a minor band of 46 kDa. Digestion of PAI-1 with endoglycosidase F causes a shift toward faster migrating species which retain inhibitory activity. The purified PAI-1 was stable at pH 2.5, lost 50% of its activity after 15 min at 45 degrees C, and showed marked activation after treatment with SDS or guanidine-HCl. Purified PAI-1 rapidly inhibited and formed complexes with both tissue-type and urokinase-type plasminogen activators. Polyclonal rabbit antirat PAI-1 antibodies were raised which immunoprecipitate both free and complexed PAI-1.

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.

A Mr = 32,000 membrane glycoprotein can be uniquely labeled by galactose oxidase/[3H]sodium borohydride on rat caudal, but not caput, epididymal sperm. It has been suggested that this protein is related to a Mr = 32,000 galactose oxidase-sensitive glycoprotein present in rat caudal epididymal fluid. The tritiated membrane glycoprotein was solubilized and its hydrodynamic properties were determined by conventional gel filtration, high performance gel filtration, sedimentation rate determination in linear sucrose gradients prepared in H2O and D2O, and equilibrium isopycnic centrifugations in CsCl. The Stokes radius and sedimentation coefficient were 4.87 +/- 0.07 nm and 1.73 +/- 0.08 S, respectively. The sedimentation profile in CsCl gradients was asymmetric with a major peak occurring at a density of 1.081 g/cm3 (v = 0.92 cm3/g) and a shoulder at 1.108 g/cm3 (v = 0.90 cm3/g). The glycoprotein did not enter a 5 to 20% linear sucrose gradient prepared in D2O and could be extracted from the intact sperm into acidic chloroform:methanol solutions. These data are consistent with a protein which binds substantial amounts of detergent and/or lipid and has exposed hydrophobic regions. Two-dimensional gel electrophoresis indicated that the membrane protein exhibits charge heterogeneity, with the major components having pI values of 5.4 and 4.9. The fluid glycoprotein was monodisperse on two-dimensional gel electrophoresis having a pI of 3.8. Binding studies failed to demonstrate specific binding of the Mr = 32,000 caudal fluid glycoprotein to caput cells. Moreover, "Western blots" of electrophoretically resolved caput and caudal fluid proteins, followed by immunolabeling with antibodies raised against unfractionated caudal fluid, demonstrated the presence of a Mr = 32,000 protein in caudal fluid which was absent from caput epididymal fluid. Using the same technique, it was shown that antibodies raised against caudal fluid proteins did not cross-react with a Mr = 32,000 caudal membrane glycoprotein. Our data do not support the view that the Mr = 32,000 fluid and membrane proteins are identical.

An analytical method for the selective retrieval of surface plasma membrane proteins which have been covalently "tagged" with the low-molecular-weight ligand 2-iminobiotin has been developed. Retrieval is based upon the specific interaction between the 2-iminobiotin molecule, avidin, antiavidin antibody, and insoluble protein A from Staphylococcus aureus. Conditions for the reaction include moderately basic pH (8.0-9.0) and moderately high ionic strength (300 mM NaCl). The retrieval reaction is insensitive to 4% (w/v) Triton X-100, NP-40, and Lubrols PX and WX and is inhibited by octyl-beta-D-glucopyranoside and Ammonyx-LO. Large numbers of samples can be processed quickly and simultaneously. Immunoprecipitated proteins can be readily released into solution by incubation in the presence of either low pH, biotin, or sodium dodecyl sulfate.

Full-length cDNA for plasminogen activator inhibitor (PAI-1) was isolated from a human umbilical vein endothelial cell (HUVEC) lambda gt11 cDNA library. Three overlapping clones were identified by immunologic screening of 10(6) recombinant phage using a rabbit anti-human fibrosarcoma PAI-1 antiserum. The fusion proteins encoded by these three clones also react strongly with a monoclonal mouse anti-human fibrosarcoma PAI-1 antibody. By nucleotide sequence analysis, PAI-1 cDNA encodes a protein containing 402 amino acids with a predicted, nonglycosylated molecular mass of 45 kD. Identity of this material as authentic PAI-1 was confirmed by the presence of high level homology with the primary amino acid sequence of an internal peptide prepared from purified rat hepatoma PAI-1. The predicted amino acid sequence also reveals extensive homology with other members of the serine protease inhibitor gene family. Cultured HUVECs contain two PAI-1 mRNA species, both encoded by a single gene, differing by 1 kb in the 3' untranslated region. The PAI-1 gene is located on human chromosome 7.

Three drug-resistant sublines of the murine macrophage-like cell line J774.2 were selected in vitro for their ability to grow in high concentrations of either taxol, vinblastine, or colchicine. Each contains a major plasma membrane glycoprotein (130-150 kDa), which is barely seen in the drug-sensitive parental cell line. Polyclonal antibodies, raised against the glycoproteins present in the colchicine- and vinblastine-resistant cells, were used to probe for relationships among the three glycoproteins. Our observations suggest that the glycoproteins from the different drug-resistant cell lines share many common domains but are not identical.

Incubation of HTC rat hepatoma cells with the synthetic glucocorticoid dexamethasone rapidly inhibits plasminogen activator (PA) activity and reveals the presence of a specific PA inhibitor (PAI-1). To determine whether the hormonal inhibition of PA activity reflects a decrease in the amount of PA or an increased amount of the inhibitor, or both, we have assayed PA and PAI-1 immunologically. HTC PA was determined to be entirely of the tissue type (tPA), and both free and complexed antigen was quantified by a RIA using rabbit antirat tPA, with rat insulinoma tPA as tracer and standard. PAI-1 was quantified by a Western blot assay using rabbit anti-HTC PAI-1 antibody and purified HTC PAI-1 as standard. Under conditions in which dexamethasone inhibited PA activity by 90%, there was no decrease in the cellular content of tPA antigen. Paradoxically, dexamethasone increased tPA antigen approximately 1.5-fold. Under these same conditions, dexamethasone increased PAI-1 antigen 4- to 5-fold. We conclude that the glucocorticoid inhibition of tPA activity in HTC cells is not secondary to a decrease in the amount of tPA but is secondary to the induction of a specific PA inhibitor.