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Objective: To analyze the MRI manifestations of Peyronie's disease and investigate the value of high-field MRI in the diagnosis of the disease., Methods: Using a small surface coil, we performed 3.0 Tesla MRI for 14 patients with clinically diagnosed Peyronie's disease. The MRI protocol included routine sequences (T1WI, T2WI, and enhanced T1WI) and susceptibility-weighted imaging (SWI). Each patient had received 2－4 penile ultrasound examinations previously. We compared the MRI findings with the results of ultrasonography., Results: MRI manifested 25 penile plaques in the 14 patients, 3 (7 plaques) with inflammation, 4 (8 plaques) with fibrosis, and the other 7 (10 plaques) with calcification displaying a low signal intensity on SWI. Ultrasonography had revealed the 10 calcified plaques in all the 20 examinations, but exhibited the 7 inflammatory and 8 fibrotic ones in only 3 of the 23 examinations. The combination of MRI SWI sequences was necessitated for the detection of calcified plaques and achieved higher detection rates than ultrasonography for inflammatory and fibrotic plaques (P<0.05)., Conclusions: High-field MRI has high sensitivity and accuracy in the diagnosis of Peyronie's disease, which can effectively display penile plaques of different nature in the early stage through multi-parametric sequences.

Purpose: To investigate the efficacy of diffusion-weighted MRI (DWI) in differentiating recurrent tumor from chronic inflammation and fibrosis after cystectomy or transurethral resection of bladder cancer., Methods: Eleven patients with suspected tumor recurrence underwent pelvic DWI and dynamic contrast-enhanced (DCE) MRI at 3 months to 7 years following bladder cancer resection. The diagnosis was histologically confirmed in all patients by transurethral or cystoscopic resection of 27 lesions within 2 weeks of MR examinations., Results: The accuracies, sensitivities, specificities, and positive predict values of DWI (92.6%, 100%, 81.8%, and 88.9%) were higher than those of DCE MRI (59.3%, 81.3%, 27.3%, and 54.2%) for detecting recurrent tumors. Using receiver operating characteristic analysis, the accuracy of DWI was significantly higher than that of DCE MRI (P < 0.05). There was no significant difference between DWI diagnosis and histopathology (P > 0.05), whereas the difference between diagnosis of DCE MRI and histopathology was significant (P < 0.05). The normalized apparent diffusion coefficients of recurrent tumors (0.697 ± 0.219) were significantly (P < 0.05) lower than those of postoperative inflammation or fibrosis (1.019 ± 0.143)., Conclusions: DWI is superior to DCE MRI for differentiating recurrent bladder tumors from postoperative inflammation or fibrosis. DWI can be included in the follow-up MRI protocol after bladder cancer surgery.

The activity of Cdk1 is the driving force for entry into M-phase during the cell cycle. Activation of Cdk1 requires synthesis and accumulation of cyclin B, binding of cyclin B to Cdk1, and removal of the inhibitory tyr-15-Cdk1 phosphorylation. It was previously shown that oncogenic Ras suppresses Cdk1 activation during the incubation of activated Xenopus egg extracts. However, how oncogenic Ras suppresses Cdk1 remained unclear. Using the histone H1 kinase assay to follow Cdk1 activity and Western blot analysis to assess levels of both cyclin B2 and phosphorylated-tyr-15-Cdk1, how oncogenic Ras suppresses Cdk1 is studied. The results indicate that oncogenic Ras suppresses Cdk1 via induction of persistent phosphorylation of tyr-15-Cdk1. Interestingly, the results reveal that, compared with cyclin B2 in control activated egg extracts, which increased, peaked and then declined during the incubation, oncogenic Ras induced continuous accumulation of cyclin B2. The results also indicate that oncogenic Ras induces continuous accumulation of cyclin B2 primarily through stabilization of cyclin B2, which is mediated by constitutive activation of the Raf-Mek-Erk-p90(rsk) pathway. Taken together, these results indicate that oncogenic Ras suppresses Cdk1 in a complex manner: It induces continuous accumulation of cyclin B2, but also causes persistent inhibitory phosphorylation of tyr-15-Cdk1., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: To evaluate the diagnostic value of 3.0 T diffusion-weighted imaging with background suppression (DWIBS) magnetic resonance (MR) for lymph node metastasis in rectal cancer., Methods: Thirty-five patients with rectal cancer who underwent preoperative routine MRI+DWI examination were enrolled in the study and were treated by rectal cancer resection plus lymph node dissection. Metastatic and non-metastatic lymph nodes were confirmed by postoperative pathology. Apparent diffusion coefficient (ADC) values, long-axis and short-axis diameters of lymph nodes were measured. Receiver operating characteristic (ROC) curve was used to assess the diagnostic efficacy of ADC, long-axis and short-axis diameters for differentiating metastatic lymph nodes from non-metastatic lymph nodes., Results: A total of 151 lymph nodes were confirmed with exact location in 35 patients. Sixty-five metastatic lymph nodes and 86 non-metastatic lymph nodes were identified. The ADC values of metastatic lymph nodes and non-metastatic lymph nodes were(0.86±0.14)×10(-3) and (0.94±0.16)×10(-3) mm(2)/s respectively. The long-axis diameter were(9.78±3.13) and (7.90±1.77) mm, respectively. The short-axis diameter were (7.65±2.00) and (6.45±1.19) mm, respectively. There were statistically significant differences between metastatic and non-metastatic lymph nodes in ADC values, long-axis diameter, and short-axis diameter(all P<0.01). The areas under the ROC curve of ADC value, long-axis diameter, and short-axis diameter were 0.648, 0.706, and 0.692, respectively. Optimal cutoff values for these parameters were 1.05×10(-3) mm(2)/s, 7.95 mm, and 5.90 mm, respectively, and the corresponding sensitivities and specificities were 93.8% and 30.2%, 75.4% and 61.6%, 90.8% and 38.4%., Conclusions: Quantitative measurement of ADC value may reflect the degree of diffusion restriction of metastatic lymph nodes by DWIBS at 3.0 T MR. Accurate diagnosis of metastatic lymph nodes in rectal cancer demands comprehensive evaluation combining ADC value with diameter measurement.

Oncogenic Ras induces cell-cycle arrest in mammalian cells and in fertilized Xenopus eggs. How oncogenic Ras induces cell-cycle arrest remains unclear. We previously showed that oncogenic Ras induces cell-cycle arrest in activated Xenopus egg extracts (cycling extracts) and that the induced cell-cycle arrest correlates with hyperphosphorylation of a 32 kDa protein. However, the identity of the 32 kDa protein was not known. By using a sucrose density-gradient centrifugation, Triton X-100-acetic acid-urea (TAU)-gel electrophoresis, composite agarose-polyacrylamide gel electrophoresis (CAPAGE), SDS-PAGE, and partial tryptic peptide sequence analysis, the 32 kDa protein has now been identified as S6, a 40S subunit ribosomal protein. Hence, our results indicate that the oncogenic Ras-induced cell-cycle arrest is correlated with hyperphosphorylation of S6, suggesting that phosphorylation of S6 plays an important role in the induced cell-cycle arrest. It has been shown that conditional deletion of gene encoding S6 in mammalian cells prevents proliferation, demonstrating the importance of S6 in cell proliferation. The exact role S6 plays in cell proliferation is unclear. However, phosphorylation of S6 has been implicated in the regulation of protein synthesis. Thus, our results are consistent with the concept that oncogenic Ras induces S6 phosphorylation to influence protein synthesis, thereby contributing to the cell-cycle arrest. In addition, our results also demonstrate that composite agarose-polyacrylamide gel electrophoresis is suitable for the separation of large molecular complexes., (Copyright 2004 Wiley-Liss, Inc.)

We have shown previously that oncogenic Ras induces cell cycle arrest in activated Xenopus egg extracts [Pan, Chen and Lin (1994) J. Biol. Chem. 269, 5968-5975]. The cell cycle arrest correlates with the stimulation of a protein kinase activity that phosphorylates histone H2b in vitro (designated p96(h2bk)) [Chen and Pan (1994) J. Biol. Chem. 269, 28034-28043]. We report here that p96(h2bk) is likely to be p96(ram), a protein of approx. 96 kDa that immunoreacts with a monoclonal antibody (Mk-1) raised against a synthetic peptide derived from a sequence highly conserved in Erk1/Erk2 (where Erk is extracellular-signal-regulated kinase). This is supported by two lines of evidence. First, activation/inactivation of p96(h2bk) correlates with upward/downward bandshifts of p96(ram) in polyacrylamide gels. Secondly, both p96(h2bk) and p96(ram) can be immunoprecipitated by antibody Mk-1. We also studied the activity of p96(h2bk)/p96(ram) in Xenopus oocytes and eggs. p96(h2bk)/p96(ram) was inactive in stage 6 oocytes, was active in unfertilized eggs, and became inactive again in eggs after fertilization. Since stage 6 oocytes are at G2-phase of the cell cycle, unfertilized eggs arrest at M-phase and eggs exit M-phase arrest after fertilization, the results thus indicate that p96(h2bk)/p96(ram) activity is cell cycle dependent. Moreover, microinjection of oncogenic Ras into fertilized eggs at the one-cell stage arrests the embryos at the two-cell stage, and this induced arrest is correlated with an inappropriate activation of p96(h2bk)/p96(ram). The data are consistent with the concept that inappropriate activation of p96(h2bk)/p96(ram) plays a role in the cell cycle arrest induced by oncogenic Ras.

In the Xenopus egg and oocyte system, oncogenic Ras protein can induce cell cycle arrest. The effect of oncogenic Ras on the cell cycle seems to be mediated by the Raf-Mek-Erk pathway of Ras signal transduction since constitutively active Raf, Mek, or Erk can mimic the effect of oncogenic Ras protein and since specific inhibition of these kinases can block the effect of oncogenic Ras. Using activated Xenopus egg extracts, we previously reported that the cell cycle arrest induced by oncogenic Ras correlates with the activation of a 96 kDa protein that phosphorylates histone h2b in vitro. This result raised the possibility that the 96 kDa kinase (designated as p96h2bk) is a potential target of the Raf-Mek-Erk pathway that links the pathway to the control of the cell cycle. We report here that constitutively active Mek1 could activate p96h2bk in the absence of oncogenic Ras. Moreover, inhibition of endogenous Mek by a specific inhibitor, PD 098059, suppressed the activation of p96h2bk by oncogenic Ras. These results are consistent with the concept that p96h2bk is a component or target of the Raf-Mek-Erk pathway. Furthermore, we have shown that activation of p96h2bk requires serine/threonine phosphorylation of p96h2bk.

Activated Xenopus egg extracts are capable of undergoing cell-free cell cycling. Using these activated extracts, we previously showed that purified, bacterially expressed oncogenic human RasH protein arrests cell cycle progression. Because oncogenic Ras activates many serine/threonine protein kinases in Xenopus oocytes and egg extracts, it is possible that induction of cell cycle arrest involves the action of oncogenic Ras-activated kinases. Thus, the identification of the physiological substrates for oncogenic Ras-activated kinases is important for elucidating the molecular mechanism underlying oncogenic Ras-induced cell cycle arrest. We used 32P-orthophosphate as a label to identify the potential substrates. Our results demonstrated that the 32P-labeling of both a 32 and a 33 kDa protein were greatly enhanced by oncogenic Ras during the incubation of activated Xenopus egg extracts. The enhanced labeling correlated with the induced cell cycle arrest and was contributed by serine phosphorylation. Moreover, the 33 kDa protein was detected only in the presence of oncogenic Ras and was a serine-hyperphosphorylated form of the 32 kDa protein. Furthermore, new protein synthesis was not required for the enhanced labeling, consistent with the concept that the enhanced serine phosphorylation of the 32 kDa protein is by oncogenic Ras-activated protein kinases. In addition to serine phosphorylation, our results also suggested that an as yet unidentified modification of the 32 kDa protein might also be induced by oncogenic Ras. Our results suggest that the 32 kDa protein is a potential physiological substrate for oncogenic Ras-activated protein kinases.

We previously showed that purified, bacterially expressed oncogenic human rasH protein blocks or delays the progression of the embryonic cell cycle into M-phase in activated Xenopus egg extracts. This block correlates with the suppression of the activation of p34cdc2 kinase (Pan, B.-T., Chen, C.-T., and Lin, S.-M. (1994) J. Biol. Chem. 269, 5968-5975). In an attempt to identify kinases that are involved in mediating the effect of oncogenic Ras on the cell cycle, we assayed aliquots of activated Xenopus egg extracts, which were incubated at 25 degrees C for various times in the absence and presence of oncogenic Ras, for their kinase activities toward a calf thymus histone fraction (Hf1). We find that the suppression of the histone H1 kinase activity of p34cdc2 by oncogenic Ras correlates with a simultaneous stimulation of a histone H2b serine kinase activity. Using a histone H2b in-the-gel kinase assay, we further show that the stimulated histone H2b kinase activity is attributed mainly to a 96-kDa kinase and slightly to p42mapk. Although Xenopus p90rsk is also activated by oncogenic Ras, we demonstrate that activated p90rsk is not responsible for the 96-kDa histone H2b kinase activity. The identity of the 96-kDa kinase remains unclear. Our data suggests that the 96-kDa kinase may be involved in mediating the effect of oncogenic Ras on the embryonic cell cycle of Xenopus.

The effect of purified, bacterially expressed human RasH proteins on embryonic cell cycle progression in activated Xenopus egg extracts was studied. Bacterially expressed human oncogenic RasH protein is able to block the progression of the Xenopus embryonic cell cycle into M-phase. In contrast, the corresponding normal human Ras protein is relatively ineffective when assayed in a like manner. The observed arresting activity can be blocked by the addition of Y13-259 anti-Ras monoclonal antibody but not by nonspecific IgG. Oncogenic Ras also induces unique morphological changes in the reconstituted nuclei; the nuclei appear to be enlarged, and the chromatin partially condenses into fiber-like structures. This induced arrest is associated with suppression of p34cdc2 kinase activity, indicating that the oncogenic Ras protein induces the arrest by inactivating p34cdc2. This inactivation by oncogenic Ras protein does not result from inhibition of the synthesis of cyclin B or of the binding of the newly synthesized cyclin B to the p34cdc2.

Microinjection of Xenopus oocytes with ras protein (p21) was used to investigate the role of phospholipid metabolism in ras-induced meiotic maturation. Induction of meiosis by ras was compared with induction by progesterone, insulin, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Neomycin, which specifically binds to phosphatidylinositides and inhibits their metabolism, blocked meiotic maturation induced by ras or insulin but not by progesterone or TPA. In addition, p21 and TPA, but not insulin or progesterone, stimulated the incorporation of 32Pi into oocyte lipids. ras protein specifically stimulated 32P incorporation into phosphatidylinositides, whereas both ras and TPA stimulated 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. The stimulatory effect of p21 on phosphatidylinositide metabolism correlated with the dose response and kinetics of ras-induced meiotic maturation. In addition, the ras oncogene protein was more potent than the proto-oncogene protein both in inducing meiotic maturation and in stimulating phosphatidylinositide metabolism. These results indicate that phosphatidylinositide turnover is required for ras-induced meiosis and suggest that phosphatidylinositide-derived second messengers mediate the biological activity of ras in Xenopus oocytes.

We review the use of functional assays for the ras protein, p21, that have allowed us to screen for mutant ras genes encoding proteins defective in either interactions with guanine nucleotides or transforming activity. GTP binding and GTP-dependent autokinase activities were assayed directly on lysed bacterial colonies expressing p21. Mutants encoding ras proteins deficient in these activities were isolated after randomly mutagenizing a v-rasH expression vector. Transformation defective mutants were isolated by randomly mutagenizing a v-rasH retroviral shuttle vector. NIH cells were then infected with a stock of nonreplicating mutagenized retroviruses and nontransformed infected colonies were isolated. The mutant ras genes were then rescued from these cells for analysis. Characterization of these mutants defines domains of p21 involved in both biochemical and biological activities and addresses the role of guanine nucleotide binding in p21 function.

We have developed a strategy to isolate mutant ras genes encoding proteins defective in GTP binding. Random in vitro mutagenesis of a v-Harvey (Ha)-ras expression vector was followed by an in situ GTP-binding assay on lysed bacterial colonies. Single amino acid substitutions at ras codon 83, 119, or 144 decreased the affinity of p21 for GTP by a factor of 25-100 primarily as a consequence of increased rates of dissociation of GTP from p21. Nevertheless, these mutant genes induced transformation of NIH 3T3 cells with efficiencies comparable to wild-type v-Ha-ras. In transformed cells, mutant p21s were phosphorylated to a degree similar to that of wild-type v-Ha-ras p21, suggesting that a decrease in affinity by a factor of 100 did not prevent the mutant ras protein from binding GTP in vivo. These results are discussed with respect to the role of GTP in the regulation of p21 function.

The transferrin receptor of sheep reticulocytes is excised from the cell during in vitro maturation to erythrocytes. The excised receptor may be recovered from the medium by centrifugation at 100 000 X g. Loss of transferrin-binding activity parallels the loss of binding of anti-receptor antibody, as well as RNA content. The released receptor retains the molecular size of the receptor isolated from the plasma membranes (93 000 monomer, 186 000 dimer), has an identical iodotyrosyl peptide map, and is still capable of binding transferrin, as well as an antibody directed against the receptor. The receptor is released in a vesicular form. The major peptides of the vesicles are the receptor and an unidentified peptide of 70 000 whose iodotyrosyl peptide map is distinct from that of the receptor. Although the transferrin receptor has been shown to undergo posttranslational modification (phosphorylation, acylation, and glycosylation) in cultured cells, it has not been established whether any of the transformations are retained in nongrowing cells. In the present communication, it is shown that isolated reticulocyte plasma membranes are capable of receptor phosphorylation, a process previously shown only with intact, cultured cells. The phosphorylating activity is retained in immunoprecipitates of the receptor, but is absent in the vesicles released during maturation. No evidence has been obtained for an effect of either transferrin or an anti-receptor antibody on receptor phosphorylation in intact cells or isolated membranes.

The transferrin receptor of sheep reticulocytes is released in vesicular form during in vitro incubation of the reticulocytes. A polyclonal antibody against the transferrin receptor slows down the release of the vesicles bearing the receptor, whereas transferrin and calf serum accelerate vesicle release. Vesicle formation and receptor release are inhibited at low temperatures and by the presence of inhibitors of ATP formation. In addition, lysosomotropic agents or transglutaminase inhibitors block receptor externalization. The externalized receptor has the same molecular size and peptide map as the receptor isolated from the membrane, suggesting that an intact receptor is removed and released from the cell. An unidentified peptide of 70 kDa is externalized with the transferrin receptor. Peptide maps show that the 70-kDa species is not a degradation product of the receptor. No function has yet been assigned to the 70-kDa peptide.

Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37 degrees C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5 micron. Inside these large MVEs are round bodies of approximately 50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nm bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the approximately 50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation.

The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and 125I-labeled anti-transferrin-receptor antibodies. Vesicles containing peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with anti-transferrin-receptor antibodies. Vesicle formation does not require the presence of the anti-transferrin-receptor antibodies. Using 125I-surface-labeled reticulocytes, it can be shown that the 125I-labeled material which is released is retained by an immunoaffinity column of the anti-transferrin-receptor antibody. Using reticulocytes tagged with 125I-labeled anti-transferrin-receptor antibodies to follow the formation of vesicles, it can be shown that at 0 degree C or in phosphate-buffered saline the rate of vesicle release is less than that at 37 degrees C in culture medium. There is selective externalization of the antibody-receptor complex since few other membrane proteins are found in the externalized vesicles. The anti-transferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.

Single amino acid substitutions were introduced into a region of the rasH protein (residues 116, 117, and 119) homologous to a variety of diverse GTP-binding proteins. Each of the mutant p21 proteins displayed a significant reduction (10- to 5,000-fold) in GTP binding affinity. Activated rasH proteins deficient in GTP binding were unaltered in their ability to morphologically transform NIH 3T3 cells.

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.