New p53 target, phosphatase of regenerating liver 1 (PRL-1) downregulates p53.

Most of the p53 target genes, all except MDM2, COP1 and PIRH2, perform functions in apoptosis, differentiation and cell cycle arrest. The aforementioned oncogenes downregulate p53 through a negative feedback mechanism, and thus contribute to tumor development. In this study, we report a new p53 target, PRL-1, which is believed to be a significant regulator in the development and metastasis of a variety of cancer types. Phosphatase of regenerating liver 1 (PRL-1) overexpression reduced the levels of endogenous and exogenous p53 proteins, and inhibited p53-mediated apoptosis. On the other hand, the ablation of PRL-1 by small interfering RNA (siRNA) increased p53 protein levels. The p53 downregulation was mediated by p53 ubiquitination and subsequent proteasomal degradation. Furthermore, p53 ubiquitination by PRL-1 was achieved through two independent pathways, by inducing PIRH2 transcription and by inducing MDM2 phosphorylation through Akt signaling. In addition, we showed that the PRL-1 gene harbors a p53 response element in the first intron, and its transcription is regulated by the p53 protein. These findings imply that the new oncogenic p53 target, PRL-1, may contribute to tumor development by the downregulation of p53 by a negative feedback mechanism.

Trigger digits in children.

Seven thousand, seven hundred newborn children were examined prospectively to determine the congenital incidence of trigger thumb and finger. No cases were found. The case histories of 43 trigger digit cases (35 trigger thumbs and eight trigger fingers) noted in 40 children diagnosed at our center between 1995 and 1998 were reviewed with special reference to the spontaneous recovery rate, treatment outcome, and age at presentation. Of the 35 thumb cases, 23 underwent surgical release and all responded satisfactorily to surgical treatment. Spontaneous recovery was noted in 12 trigger thumb cases and in all eight trigger finger cases. Trigger finger developed earlier in life than trigger thumb and the spontaneous recovery rate was higher in trigger finger than trigger thumb.

Reoperation of Recurrent Gastric Cancer.

PURPOSE: The aim of this study was to evaluate the outcome of reoperation in recurrent gastric cancers. MATERIALS AND METHODS: We conducted a retrospective analysis of 86 patients who underwent reoperation for recurrent gastric cancer. We reviewed the time interval between first operation and reoperation, as well as the recurrence pattern, type of reoperation, and survival following reoperation. RESULTS: the average time to reoperation following curative resection was 27.8+/-25.9 months (median 18.4 months). Fifty-three cases (61.6%) of reoperation were performed within 2 years follwoing the first operation. The most common reason for reoperation was intestinal obstruction followed by gastric remnant recurrence and intra-abdominal mass. Complete resection was possible in 14 cases (16.3%) and a palliative procedure such as partial resection or bypass procedures was performed in 54 cases. In 18 cases (20.9%), simple lapalotomy was done without any aid. The most common site of recurrence was the peritoneum followed by the gastric remnant, distant lymph node and hematogenous liver metastasis. Operative mortality was 10.5%. Excluding the 9 cases of operative mortality, the mean survival time after reoperation was 15.4+/-2.5 months (mean 8.6 months). Survival following complete resection was much longer than palliative procedure and exploration only (37.9+/-8.7 vs 10.9+/-1.5 vs 4.7+/-0.8 months, p=0.000). CONCLUSION: The complete resection of recurrent gastric cancer can prolong survival. Early detection of localized recurrence is important in order to increase the chance of complete resection.

A humanized anti--4-1BB monoclonal antibody suppresses antigen-induced humoral immune response in nonhuman primates.

The interaction of 4-1BB and its ligand plays an important role in the regulation of T-cell-mediated immune responses. In this study, the authors examined the effect of a humanized anti--4-1BB monoclonal antibody (H4B4) on ovalbumin-induced immune responses in baboons. Previously, a mouse monoclonal antibody, 4B4 against the human 4-1BB molecule, was generated and characterized. Based on this antibody, a humanized version of 4B4 monoclonal antibody was constructed and the resultant antibody, H4B4, showed full recovery of the binding activity of the original antibody 4B4: a 1.5-fold increase in affinity for 4-1BB. In addition, H4B4 mediated antibody-dependent cellular cytotoxicity of activated human peripheral blood T cells and CEM cells in a dose-dependent manner. Weekly administration of H4B4 at doses of 1 or 4 mg/kg could suppress immunoglobulin G production against ovalbumin. This was not a result of the overall immune suppression, because the numbers of B and T cells and the total immunoglobulin G production were not altered during treatment with H4B4. These findings suggest that treatment with H4B4 may be a valid therapeutic approach to control unwanted immune responses in persons with autoimmune diseases.

Synergistic inhibitory effect of angiotensin-converting enzyme inhibitor and heparin on intimal hyperplasia after rat aorta injury.

The respective efficacies of angiotensin-converting enzyme (ACE) inhibitor and standard heparin were investigated with respect to their inhibitory effects on intimal hyperplasia after balloon denudation of rat aorta. Local angiotensin II effects in the artery wall may participate in regulation of the vascular response to arterial injury, apparently independent of the plasma renin and angiotensin system. ACE inhibitors have been shown to block intimal hyperplasia after arterial injury in rats. Increasing evidence points toward an inhibitory effect of heparin on intimal hyperplasia independent of anticoagulation. Balloon catheter aortic denudation was performed in 25 rats pharmacologically treated from six days or one day before to fourteen days after surgery and split into four groups: group A (control group), normal feeding; group B (ramipril group), ramipril 10 mg/kg/day orally; group C (heparin group), heparin 1200 IU/kg/day subcutaneously; group D (combined group), both ramipril and heparin. Animals were killed and aortas were perfused and fixed at physiologic pressure fourteen days after denudation. Cross-sectional intima-to-media area ratios (I-M ratio) were calculated by an image analyze system.

CT analysis of intestinal obstruction due to adhesions: early detection of strangulation.

We evaluated the CT of intestinal obstruction due to adhesions in 20 postoperative patients, with emphasis on early detection of strangulation. Ten patients with surgically proven strangulated obstruction (strangulation group) were compared with another ten patients (nonstrangulation group) in whom seven improved with conservative management and three had confirmed simple obstruction on surgical exploration. Beak-like luminal narrowing ("beak") was the most common CT finding at the obstructed site in both groups. The CT findings that suggested strangulated obstruction were serrated beaks, mesenteric edema or vascular engorgement, and moderate to severe bowel wall thickening. In contrast, simple obstruction could be assumed when the beak was smooth, there were no mesenteric changes, and the bowel wall was normal or mildly thickened. Computed tomography is a useful tool for detecting strangulation in patients with postoperative adhesive intestinal obstruction.

Magnetic circular dichroism studies of the active site structure of hemoprotein H-450: comparison to cytochrome P-450 and sensitivity to pH effects.

Ferric, ferrous and ferrous-CO hemoprotein H-450 from rat liver have been examined with magnetic circular dichroism spectroscopy under alkaline (pH 8.0) and acidic (pH 6.0) conditions. The spectral properties of these species require that one of the axial heme iron ligands in the alkaline ferric and ferrous states must be a thiolate sulfur, presumably from cysteine. The data are most consistent with the ligand trans to thiolate being either histidine or methionine. The reversible pH effects on the spectral properties of the ferrous protein, but not of the ferric protein, appear to involve protonation or displacement of the thiolate. As treatment of the ferrous protein with CO does not yield a thiolate-ligated ferrous-CO adduct, CO either displaces the thiolate or its addition is accompanied by protonation of the thiolate.

The production of transforming growth factor-beta activity by rat granulosa cell cultures.

We have examined whether granulosa cells (GC) secrete transforming growth factor-beta (TGF beta)-like activity using cell cultures prepared from diethylstilbestrol-primed female rats. Our results indicate that a significant level of active as well as latent TGF beta activity is found in defined GC culture medium as assessed by 1) potentiation of FSH-induced differentiation of rat GC, 2) neutralization of its activity by anti-TGF beta immunoglobulin, 3) inhibition of DNA synthesis in mink lung epithelial cells (CCl 64), and 4) activation of latent TGF beta activity by either acid or heat treatment. TGF beta production was more pronounced when the cells were seeded on fibronectin-coated plates. There was no difference in the level of TGF beta secretion by GC preparations derived from either diethylstilbestrol-primed immature or normal immature rats or adult rats. Furthermore, rat GC-conditioned medium contained much more TGF beta activity than medium from normal rat kidney cells (NRK 49-F), human prostatic adenocarcinoma cells (PC-3), or porcine GC. Rat thecal/interstitial cell culture medium contained activity comparable to that of GC medium. We conclude that rat GC preparations secrete a high level of TGF beta activity in vitro. Taken together with previous results, this indicates the possibility that TGF beta may be an autocrine regulator as well as a paracrine one within the ovarian follicle. Moreover, because of the high level of TGF beta activity produced, the rat GC culture system appears to be a useful experimental model for further exploring relationships between TGF beta production and its action.

Identification of trophoblast membrane-originated proteins in human placental blood.

The human placenta secretes various fetoplacental proteins, including a trophoblast membrane (TM)-bound alkaline phosphatase. Five TM-originated proteins (TOPs), molecular weights 100, 39, 37, 35, and 26 kilodaltons (kDa), were isolated from placental blood by using an anti-TM Sepharose immunoaffinity column. Apparently, these five TOPs are secreted from TM into the maternal circulation during pregnancy. One of these five TOP bands (26 kDa) was identified by Western blot analysis to be human placental lactogen. The other four bands are newly identified as trophoblast-specific proteins and, therefore, could have potential diagnostic applications as serum markers for monitoring tumor development.

Human platelet-derived growth factor preparations contain a separate activity which potentiates follicle-stimulating hormone-mediated induction of luteinizing hormone receptor in cultured rat granulosa cells: evidence for transforming growth factor-beta.

The ability of platelet-derived growth factor (PDGF) preparations to potentiate FSH-mediated LH receptor induction in rat granulosa cell cultures was shown to be due to a component distinct from PDGF. Purification of heat-treated platelet lysate by carboxymethyl-Sephadex C-50 and Cibacron blue-Sepharose chromatography, followed by Bio-Gel P-60 chromatography, resulted in the separation of two activities: 1) a growth-promoting activity, P60-PDGF, defined on the basis of increased DNA synthesis in BALB/c-3T3 cells, and 2) a differentiation-promoting activity which enhanced FSH-dependent LH receptor induction in granulosa cells. On the basis of electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels, inhibition of tritiated thymidine uptake by epithelial cells, and attenuation of LH/hCG receptor expression in the presence of antitransforming growth factor-beta (anti-TGF beta) immunoglobulin G, the differentiation-promoting component of the preparations appears to be TGF beta. The Bio-Gel fractions that contained TGF beta did not stimulate LH receptor induction of cAMP production in the absence of FSH. PDGF prepared free of TGF beta did not potentiate receptor induction. We conclude, therefore, that the differentiative effects of PDGF previously described in this system are due to TGF beta.

Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells. A 92-kDa Glycoprotein.

The structure of the lutropin/choriogonadotropin (LH/CG) receptor has been studied by immunoprecipitating the receptor from biosynthetically labeled cultured Leydig tumor cells (designated MA-10). This was performed by binding human choriogonadotropin (hCG) to the labeled cells, solubilizing the hormone-receptor complex, partially purifying the complex by lectin chromatography, and immunoprecipitating the complex with an antibody that recognizes receptor-bound hCG. The conditions used for the release of the radiolabeled receptor from the immunoprecipitate and the subsequent analysis of this material on sodium dodecyl sulfate gels allowed us to determine directly the structure of the free (not hormone-occupied) LH/CG receptor. From experiments using cells labeled with [35S]methionine and [35S]cysteine, we show that the LH/CG receptor is composed of a single polypeptide chain that migrates as a 92-kDa protein on sodium dodecyl sulfate gels whether analyzed in the absence or presence of reducing agents. Other studies presented demonstrate that the LH/CG receptor is a glycoprotein.

Antigenic analysis of testicular cytochromes c using monoclonal antibodies.

Testicular cytochrome c (cyt ct) was isolated from testes of sexually mature, rat, mouse, rabbit, and bull, among which rat testis is highly rich in cyt ct. By fusion of NS-1 myeloma cells and spleen cells of mice immunized with rat cyt ct, 11 stable mouse hybridoma cell lines were established. Using an enzyme-linked immunosorbent assay, it was determined that 4 of the 11 anti-rat cyt ct monoclonal antibodies (McAb) did not bind to somatic cyt c (cyt cs) of vertebrates nor to cyt ct of mouse, rabbit, and bull. Four other McAb showed no binding to cyt cs but showed different patterns of cross-reactivity with these four cyt ct. Therefore, these McAb appear to be very sensitive and useful probes for the discrimination or identification of extremely similar isocytochromes c. Although the primary amino acid sequences between cyt cs of rat and mouse are identical, the antigenic structure of cyt ct of rat and mouse are clearly distinct with regard to cross-reactivity with some anti-rat cyt ct McAb. Furthermore, these McAb also reveal that the primary amino acid sequences of cyt ct, which reflect differences in the surface conformation of the molecule, are probably species specific.

Properties of nonheme iron in a cell envelope fraction from Escherichia coli.

The reaction with o-phenanthroline of nonheme iron found in cell envelope fractions from Escherichia coli has been investigated. About 20% of the total nonheme iron reacts directly with o-phenanthroline. This iron appears to be in the ferric state but is reducible by protein sulfhydryl groups in the presence of the chelating agent. A further 20% of the nonheme iron will react with o-phenanthroline only in the presence of dithionite. Succinate can replace dithionite but only produces about 35% of the reaction given by dithionite. The reduction of cytochrome b(1) of the respiratory chain by succinate shows similar behavior to the reaction of iron with o-phenanthroline in the presence of succinate. Both of these components react completely only in the presence of 2-heptyl-4-hydroxyquinoline-N-oxide. The remaining 60% of the nonheme iron of the cell envelope will not react with o-phenanthroline even in the presence of dithionite or 6 m urea. Triton X-100 with dithionite will permit a small part (10%) of this iron to react with o-phenanthroline. The iron which does not react with o-phenanthroline is not associated with succinate, reduced nicotinamide adenine dinucleotide, or d-lactate dehydrogenases.