Magnetospheric wind.

An experiment designed to detect the bulk flow of cool plasma within the magnetosphere has been flown on the ATS-1 (Applications Technology Satellite) synchronous satellite. This experiment has yielded evidence for gusts of streaming positive ions in the magnetospheric equatorial plane. This directed ion flow is interpreted as the result of large-scale electric fields of the order of several millivolts per meter.

Immunological heterogeneity of carcinoembryonic antigen: purification from meconium of an antigen related to carcinoembryonic antigen.

Two antigens cross-reactive with carcinoembryonic antigen (CEA) and distinct from the nonspecific cross-reacting antigen were identified in meconium by double immunodiffusion with a conventional goat anti-CEA antiserum. These two antigens together competitively inhibited cross-reacting antibodies against them in CEA radioimmunoassay and contributed to the measurement of meconium CEA levels which averaged 6 times higher than that determined with anti-CEA specific antibody. A purification method for one of these antigens, tentatively designated meconium antigen, is described and uses a combination of ethanol fractionation, ion-exchange and molecular sieve chromatography, and adsorption to an immunoadsorbent containing a cross-reactive murine monoclonal antibody to CEA. Preliminary characterization of the purified meconium antigen showed it to be a glycoprotein, migrating as an alpha-globulin and having a molecular size similar to that of CEA (Mr 185,000 versus 200,000). Antigenically, it lacks at least one determinant present on CEA and differs further from CEA by being weakly reactive with concanavalin A and resistant to proteolytic digestion with Pronase E. Although these properties of meconium antigen suggest that it may be nonspecific cross-reacting antigen 2, additional chemical and antigenic studies are required to establish its relationship to CEA and other CEA-related antigens in meconium.

Antisense-mediated specific inhibition of P120 protein expression prevents G1- to S-phase transition.

A pentadecadeoxyribonucleotide (5'-AAAGCCCCCCACCAC), complementary to a splice junction site of mRNA for human proliferation-associated nucleolar protein P120, inhibited expression of the P120 gene and the mitogen-induced proliferation of human lymphocytes. The inhibition of P120 gene expression and proliferation was concentration dependent and reached 90% at 200 microM, as measured by [3H]thymidine uptake and by densitometric scanning of Northern (mRNA) and Western (protein) blots of P120. Inhibition was not observed in cells treated with the correspondent nonsense oligomer. P120 antisense oligomer treatment prevented S-phase entry of mitogen-stimulated lymphocytes, as determined by flow cytometric analysis, but did not block G0-G1 transition assessed by morphological blast transformation and induction of [3H]uridine incorporation. Results of this study suggest that P120 expression may be required for the upregulation of nucleolar function necessary for cell proliferation.

Identification and partial characterization of a Mr 40,000 nucleolar antigen associated with cell proliferation.

The present study reports the identification and partial characterization of a novel Mr 40,000 nucleolar antigen (P40) by monoclonal antibodies. Monoclonal antibodies to this protein were obtained when a nucleolar protein extract separated from the immunodominant protein C23 was used to immunize BALB/c mice; 12 hybridoma clones produced antibodies to this protein. P40 was not detected in normal human kidney, liver, and leukocytes but was readily demonstrable in a variety of human malignant tissues. This newly identified P40 antigen differs in its specific nucleolar localization from cyclin (proliferating cell nuclear antigen), a Mr 35,000 antigen which is largely in the nucleoplasm. In addition, cyclin appears in the nucleolus in S-phase; P40 appears in the nucleolus 6 h after refeeding serum-starved HeLa cells.

Identification and partial characterization of a Mr 105,000 nucleolar antigen associated with cell proliferation.

This study reports the identification and partial characterization of a novel Mr 105,000 nucleolar antigen (P105) identified by a monoclonal antibody. This monoclonal antibody was obtained when a nucleolar protein extract separated from the immunodominant protein C23 was used as the immunogen. Nucleolar antigen P105 was not detected in normal (resting) human liver, kidney, or peripheral blood lymphocytes but was present in a variety of human malignant cells and tissues. Lymphocyte nucleoli also exhibited specific P105 staining after 72 h of phytohemagglutinin stimulation. Nucleolar antigen P105 was detected in growing and dividing HL 60 cells but was not detected in retinoic acid-induced differentiated HL 60 cells. When HeLa cells were made quiescent by 48 h of serum starvation, the P105 antigen was not detected, but after refeeding with serum-containing medium, the antigen P105 was detected in the HeLa nucleoli within 2 h. These results indicate that nucleolar antigen P105 is a proliferating cell nuclear and nucleolar antigen-like molecule which appears early in the G1-S phase of the cell cycle.

Expression of nucleolar antigen p145 in bone marrow cells of patients with myeloid leukemias.

The expression of a cell cycle-related nucleolar protein (p145) antigen was examined in the bone marrow aspirates of 45 individuals, three of whom had no malignant disease; 30 had a diagnosis of acute myeloid leukemia (AML), and 12 suffered from chronic myeloid leukemia (CML). While no evidence of p145 expression was found in the three normal bone marrow samples, it was noted to be the highest in patients with active leukemia, be they AML or blastic crisis of CML. There was a direct correlation between the percentage of blasts and the percentage of p145-positive cells in all patients. Double labeling with tritiated thymidine and p145 in AML patients with active leukemia showed that the majority of S-phase cells contained p145. Myeloblasts in both chronic phase and blastic crisis of CML expressed p145. Nine of 12 AML patients studied during remission had less than 5% p145-positive cells, but three showed 11%, 16%, and 33% positive cells. Since functionally/morphologically, these marrows were normal, the appearance of p145 may indicate a proliferative abnormality preceding maturation arrest and development of relapse. Thus we conclude that p145 is more commonly associated with immature cells and may serve as an early indicator of relapse in AML, but requires further study with larger numbers of patients.

Masking of nontumorous antigens for development of human tumor nucleolar antibodies with improved specificity.

Nucleoli purified from HeLa cells were spread by homogenization in low ionic strength buffer and by chelation of divalent cations. The antigenic determinants of these tumor nucleoli that are shared by normal liver nucleoli were masked by addition of rabbit anti-liver nucleolar antisera to form immune complexes. New Zealand White rabbits were immunized with these antibody-nucleolar complexes. The resulting antisera produced nucleolar fluorescence in HeLa cells but not in normal human liver or human kidney cells. One- and two-dimensional immunoblots identified a major Mr 58,000/pl 5.8 antigen in HeLa cells, which was not detected on corresponding immunoblots of normal human liver nucleoli. This method offers an improved approach to selection of tumor-associated antigens.

Intranucleolar localization of human proliferating cell nucleolar antigen p120.

The human proliferation-associated nucleolar antigen p120 was localized to substructures within HeLa cell nucleoli by immunofluorescence and immunoelectron microscopy of cells whose nucleoli were segregated by drug treatment or extracted with nucleases. By indirect immunofluorescence, protein p120 was localized diffusely throughout all interphase nucleoli. However, high resolution immunoelectron microscopy demonstrated that protein p120 staining delineated a network of 20-30-nm diameter beaded fibrils distributed throughout the nucleolus. This distribution was unique compared to that of the nucleolar proteins p145, RNA polymerase I, or B23 which were examined simultaneously. Drug-induced segregation of nucleoli by actinomycin D or dichlorobenzimidazole riboside, followed by immunoelectron microscopy, indicated that protein p120 was concentrated at the periphery of the granular region in segregated nucleoli. In situ nuclease digestion of cells with DNase I and/or RNase A did not release p120 from the nucleolus. Instead, p120 immunoreactivity was retained within phase-dense residual nucleoli. These results provide evidence that protein p120 is associated with, and in fact delineates, a network of fibrils which is retained in the nucleolar residue fraction of proliferating cells.

Optimal loading of scraped HeLa cells with monoclonal antibodies to the proliferation-associated Mr 120,000 nucleolar antigen.

Our laboratory has reported (J. W. Freeman et al., Cancer Res., 48:1244-1251, 1988) a proliferation-associated Mr 120,000 nucleolar antigen (p120), that was found in human tumors but was not detectable in most normal resting tissues and in benign tumors. To study further the function and the localization of this protein, we have investigated various methods of microinjecting p120 monoclonal antibodies into cells. For comparison, we have used a monoclonal antibody to protein C23, a nucleolar protein found in high levels in most cells. To determine optimal conditions for loading of antibodies to nucleolar antigens into mechanically disrupted HeLa cells, we studied the effects of ionic strengths of loading buffers, various antibody concentrations, and optimal time for loading and antibody localization. With ascites fluids in isotonic buffer containing antibodies to nucleolar proteins p120 and C23, a maximum number of cells, 86 and 84%, respectively, were loaded following a 20-min incubation. Hypotonic buffers decreased the percentage of cells loaded (22%); hypertonic buffers reduced cell viability. The optimal concentration of purified antibody yielding a maximum number of loaded cells (81%) was 2.5 mg/ml. Higher concentrations of antibody resulted in residual cytoplasmic staining without increasing the percentage of loaded cells. With antibody concentrations less than 2.5 mg/ml, a linear decrease was noted in the percentage of cells loaded with a decrease in intensity of fluorescence. Following antibody loading, nucleolar fluorescence was observed by 12 h and the intensity increased at 24 h. Localization of the p120 antibody was followed through mitosis where it was perichromosomal and equally divided between the chromosomes at metaphase. A decrease of nucleolar immunofluorescence intensity and percentage of cells labeled were observed in successive cell generations.

Reversion of transcriptional repression of Sp1 by 5 aza-2' deoxycytidine restores TGF-beta type II receptor expression in the pancreatic cancer cell line MIA PaCa-2.

The pancreatic cancer cell line, MIA PaCa-2 is not responsive to transforming growth factor beta (TGF-beta) because of a lack of expression of the TGF-beta type II receptor (RII). We show that the lack of RII expression is caused by a deficit of the transcription factor Sp1. Nuclear run-off assays and Western immunoblot showed low levels of transcription and protein levels of Sp1, respectively. Treatment of MIA PaCa-2 cells with the DNA methyl transferase inhibitor, 5-aza-2'-deoxycytidine, resulted in an increase in the rate of Sp1 transcription, in Sp1 protein expression, and in the binding of Sp1 to the RII promoter. Ectopic expression of Sp1 cDNA in MIA PaCa-2 cells led to an increase in RII promoter-chloramphenicol acetyltransferase activity and RII expression. Expression of Sp1 cDNA also caused a reduction in both growth and clonogenicity that was associated with restoration of responsiveness to TGF-beta. Conversely, cells that express RII (BxPC-3 and MIA PaCa-2 Sp1 transfectants) when treated with mithramycin, an inhibitor of Sp1 binding, showed a reduction in RII mRNA expression. The reduction of RII mRNA was attributed to a decrease in RII promoter-chloramphenicol acetyltransferase activity that was associated with a decrease in Sp1 binding to the RII promoter. These data indicate that transcriptional repression of the Sp1 gene in MIA PaCa-2 cells plays a role in the transcriptional inactivation of the RII gene and thus lack of responsiveness to TGF-beta.

Novel cell cycle-related nuclear proteins found in rat and human cells with monoclonal antibodies.

Monoclonal antibodies which react specifically with the nuclei of interphase cells recognized three nuclear antigens with molecular weights of 110,000 (p110), 85,000 (p85), and 18,000 (p18). p110 and p85 were found in eight tumor cell lines but were not found in resting lymphocytes. p18 was found in resting lymphocytes as well as the tumor cell lines. Protein p85 appeared in phytohemagglutinin-stimulated lymphocytes in the G1 phase and protein p110 appeared in the S phase. p110 and p85 were localized to the extranucleolar chromatin while p18 was distributed throughout the nucleus and was determined by microscopic and DNase digestion studies to be DNA associated. The anti-p110 antibody recognized a component of the DNA polymerase alpha 2 complex. Three novel nuclear proteins were identified using monoclonal antibodies. Two of these proteins (p110 and p85) are proliferating cell nuclear and nucleolar antigen-like while the third (p18) is not cell cycle dependent.

Effect of nucleolar P120 expression level on the proliferation capacity of breast cancer cells.

Steady-state level of nucleolar P120 protein and P120 mRNA was compared to the doubling time and S-phase fraction in human breast cancer cell lines growing exponentially and in similar cells treated with a single dose of P120 antisense oligodeoxynucleotides. The study included six breast cancer cell lines and one nontransformed breast cell line with doubling times from 1.1 to 5.5 days and with S-phase fractions from 35 to 9%. P120 expression level was determined by densitometric computerized evaluation of protein and mRNA blots and with a quantitative 32P-reverse transcriptase-polymerase chain reaction method developed for small-scale samples. In the slowest growing normal cell line, P120 expression level was only about 10% of the level found in the most rapidly growing cancer cell line. The amount of P120 mRNA was highly correlated with the amount of P120 protein (P = 0.0001), indicating that P120 accumulation is regulated in these cells primarily at a transcriptional level. There was also a significant positive correlation between the level of P120 protein/mRNA and doubling time of cell lines (P = 0.0008) or percentage of S-phase cells (P = 0.210). P120 antisense oligomer treatment decreased the growth rate of cells in a dose-dependent manner, and the inhibition reached 70% at 100 microM concentration. Both P120 mRNA and P120 protein levels were also decreased by approximately 70% in cells treated with 100 microM P120 antisense oligomer. Slowly growing cells exhibited 50% inhibition by treatment at a proportionally lower concentration of P120 antisense oligomer than fast growing cells. This study shows that the expression of P120, measured either at the protein or the mRNA level, correlates with proliferation rate, identifying P120 as a cell proliferation marker.

Identification and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120,000 expressed in early G1 phase.

Tumor nucleoli were treated with polyclonal antisera to normal human tissue nucleoli to block some determinants common to tumor and normal tissue nucleoli. Immunization of mice with these immune complexes resulted in the development of a monoclonal antibody (FB2) to a novel Mr 120,000 nucleolar proliferation-associated antigen. By indirect immunofluorescence, antibody FB2 produced bright nucleolar staining in a variety of malignant tumors, including cancers of the breast, liver, gastrointestinal tract, genitourinary tract, blood, lymph system, lung, and brain. Although specific nucleolar immunofluorescence was not detectable in most normal tissues, it was detectable in some proliferating nonmalignant tissues including spermatogonia of the testes, ductal regions of hypertrophied prostates, and phytohemagglutinin-stimulated lymphocytes. The Mr 120,000 antigen was not detectable in 48-h serum-deprived HeLa cells but was readily detectable (within 30 min) following serum refeeding. The Mr 120,000 antigen was not detected in retinoic acid-treated HL-60 cells following morphological differentiation but was detectable in 48-h phytohemagglutinin-treated lymphocytes. These studies suggest that the Mr 120,000 antigen is a proliferation-associated antigen which plays a role in the early G1 phase of the cell cycle.

Effect of differentiation on the expression of a nucleolar antigen with a molecular weight of 145,000 in HL-60 cells.

Mr 145,000 nucleolar protein antigen (p145) is associated with growing cells (R. L. Ochs et al., J. Cell Biol., 101: 211a, 1985) and has been found in a broad range of human cancers (J. W. Freeman et al., Cancer Res., 46: 3593-3598, 1986). In this study the presence of nucleolar antigen p145 was examined in the human promyelocytic tumor cell line HL-60 which was induced to differentiate by retinoic acid. Differentiation was monitored by morphological changes, [3H]thymidine accumulation, the ability of cells to reduce nitroblue tetrazolium, and cell number. The monoclonal antibody to nucleolar antigen p145 produced bright immunofluorescence in all cycling interphase HL-60 cells; during mitosis only diffuse staining was detected. Nucleolar antigen p145 in HL-60 cells was undetectable after 132 h of treatment with retinoic acid. The absence of nucleolar antigen p145 was associated with an 81% decline in thymidine accumulation and apparent inactivation of ribosomal and nonribosomal DNA transcription as observed by electron microscopy. The loss in expression of the antigen also correlated with increased nitroblue tetrazolium-positive cells, appearance of morphologically distinct myeloid cells, and termination of cell proliferation. These data indicate that the expression of nucleolar antigen p145 occurred in cycling HL-60 cells but not in terminally differentiated noncycling HL-60 cells.

Identification and partial characterization of a nucleolar antigen with a molecular weight of 145,000 found in a broad range of human cancers.

Previous studies in our laboratory have indicated the presence of nucleolar antigens in tumors which were not detected in normal tissues. Some of the polyclonal antisera produced in these studies were shown to identify a Mr, 145,000 nucleolar antigen on immunoblots of tumor nucleoli but not in normal human liver nucleoli. A monoclonal antibody to a Mr 145,000 nucleolar protein (p145) was produced by immunization of mice with a nucleolar extract of HeLa cells which is enriched with this antigen. The monoclonal antibody showed bright nucleolar immunofluorescence localization in a broad range of human tumors including cancers of the gastrointestinal tract, genitourinary tract, lung, liver, muscle, cartilage, and blood. The p145 nucleolar antigen was not detected in most normal human tissues or in benign tumors, with only weak nucleolar staining observed in spermatogonia of the testes and in ductal regions of some hypertrophied prostates. Nucleolar antigen p145 was extracted from HeLa cell nucleoli by homogenization in a 0.01 M Tris buffer containing 0.2% deoxycholate. On sucrose density gradient centrifugation, the antigen remained sedimented with the nucleolar ribonucleoprotein fraction. Nucleolar antigen p145 was released from ribonucleoproteins following treatment with 4 M guanidinium hydrochloride or RNase. Peptide mapping of nucleolar antigen p145 showed that it was distinct from other known nucleolar antigens. Although it remains to be determined if the p145 antigen plays a role in cell transformation, maintenance of the malignant phenotype, or in cell division, it may have value as a tumor marker or as a therapeutic target.

Prognostic significance of proliferation associated nucleolar antigen P120 in human breast carcinoma.

Nucleolar antigen P120 is detected in rapidly proliferating cells but not in normal resting cells or in many benign and slowly growing malignant tumors. The objective of the study was to determine whether the expression of P120 in breast cancer correlated with histopathological or biological properties associated with prognosis. In this retrospective study, 120 primary breast tumors were analyzed for P120; 114 of these tumors were also stained for the erbB-2 protein. Immunopositive staining was correlated with patient survival, nodal status, estrogen receptor levels, and number of mitoses. Sixty-nine % (83 of 120) of the tumors were positive for P120; 25% (28 of 114) stained positively for erbB-2. Of the 28 erbB-2 positive tumors 26 were also positive for the P120 protein. Forty-six % (55 of 120) of the specimens were from patients who later died from recurrent breast cancer; P120 was detected in 89% (49 of 55) of these specimens. In 52% of the survivors the P120 protein was also expressed. P120 negative tumors were highly correlative with survival (P = 0.0001); 84% (32 of 37) of patients with P120 negative tumors survived more than 7 years without evidence of recurrent disease. Multivariate analysis showed that the worst prognosis was for patients who had tumor positive nodes and expressed P120 (P = 0.0001); death occurred in 73% (30 of 41) of these patients. For the node negative patients who did not express P120, 5-year survival was 90% (19 of 21 patients); 5-year survival for the node negative patients who expressed P120 was significantly less (67%; 28 of 42 patients). Patients with P120 negative tumors had a good prognosis, irrespective of their nodal status. In this group, survival of node negative patients was 86% (18 of 21) and for those with positive nodes survival was 82% (13 of 16). A poor prognosis was found for patients with intense erbB-2 stained tumors (5 of 7 patients died). Weak staining of erbB-2 tumors (21 specimens) was not correlated with patient survival. Compared to P120 negative tumors, P120 positive tumors had greater numbers of mitoses (9.06 versus 6.65) and an almost 2-fold increase in the occurrence of positive nodes (one of every 4.67 versus one of every 8.81). The number of P120 positive tumors was greater in estrogen receptor positive tumors (75%) than in estrogen negative tumors (54%).(ABSTRACT TRUNCATED AT 250 WORDS)

Aberrant expression and activation of insulin-like growth factor-1 receptor (IGF-1R) are mediated by an induction of IGF-1R promoter activity and stabilization of IGF-1R mRNA and contributes to growth factor independence and increased survival of the pancreatic cancer cell line MIA PaCa-2.

In the present study we investigated the mechanisms responsible for and the biological consequences of the constitutive activation of the insulin-like growth factor-1 receptor (IGF-1R) in the MIA PaCa-2 cells. An aberrant increase in the expression and activation of the IGF-1R was observed during the transition of growth states from exponential to quiescent. The increase in IGF-1R expression is preceded by an increase in IGF-1R mRNA transcript and is associated with an increase in the IGF-1R promoter activity. Inhibition of de novo transcription by actinomycin D increased the stability of IGF-1R mRNA in exponentially growing cells, thereby increasing the expression of IGF-1R to a level similar to that seen in quiescent cells. Increased IGF-1R signaling mediated the growth factor independence of quiescent MIA PaCa-2 cells through the constitutive activation of mitogen-activated protein kinase (MAPK). Exogenous IGF-1 increased cell proliferation and activated MAPK and AKT signaling pathways. The resistance of cells to apoptosis by IGF-1R signaling was mediated through MAPK and phosphatidylinositol 3-kinase (PI3K) pathways and a yet unidentified pathway(s). Thus, aberrant regulation of IGF-1R signaling is required for resistance to apoptosis and growth factor independence of MIA PaCa-2 cells. This likely protects cells from unfavorable conditions and allows cells to rapidly re-enter the cell cycle when conditions are favorable.

Craniopharyngioma simulating bilateral internal ophthalmoplegia.

A 15-year-old girl had rapid onset of an apparent bilateral internal ophthalmoplegia. Subsequent evaluation revealed a large craniopharyngioma. It is uncommon for a mass to cause such eye findings and unique for a craniopharyngioma to manifest in this fashion.

Chronic meningitis caused by Sporotrichum schenckii.

The fungus Sporotrichum schenckii caused chronic meningitis in a 48-year-old man. Only three other firmly diagnosed cases were reported previously.

Prolonged encephalopathy with arsenic poisoning.

A case of prolonged reversible encephalopathy in a woman with arsenic poisoning is described. Previous descriptions of extended encephalopathy due to arsenic are rare.