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PubMed1999

Nuclear localisation of NOVH protein: a potential role for NOV in the regulation of gene expression.

AIMS: To identify the NOV protein detected by immunofluorescence in the nucleus of human cancer cell lines to establish whether targeting to the nucleus reflects dual paracrine and intracrine biological functions of NOV, as has been reported previously for several signalling peptides and proteins. METHODS: Nuclear and cytoplasmic fractions were prepared from 143 and HeLa cells in which nuclear NOV protein was detected. Western blotting analysis of NOV proteins in both types of fractions was performed using two NOV specific antibodies. Confocal microscopy was used to visualise the nuclear NOV protein in HeLa and 143 cells. A yeast two hybrid screening system was used to isolate cDNAs encoding proteins able to interact with the human NOV protein. RESULTS: A 31/32 kDa doublet of NOV protein was identified in the nuclear fraction of 143 and HeLa cells. Because the antibodies were directed against the C-terminus of NOV, the 31/32 kDa NOV isoform is probably truncated at the N-terminus and might correspond to the secreted 32 kDa NOV isoform detected in cell culture medium. Confocal microscopy indicated that in addition to the cytoplasmic NOV protein already identified, a nuclear NOV protein was present in both the nucleoplasm and nucleoli of Hela and 143 cells. Screening of cDNA libraries prepared from HeLa cells, Epstein-Barr virus transformed lymphocytes, and normal human brain showed that the NOV protein interacts with the rpb7 subunit of RNA polymerase in a yeast two hybrid system. CONCLUSIONS: The NOV protein detected in the nucleus of 143 and HeLa cells is probably an N-terminus truncated isoform of the secreted 48 kDa NOV protein. A growing body of evidence suggests that novH expression is closely associated with differentiation in normal human tissues and that the nov gene encodes a signalling protein that belongs to an emerging family of cell growth regulators. The nuclear localisation of a NOV isoform potentially provides an additional degree of signalling specificity. The interaction of the NOV protein and the rpb7 subunit of RNA polymerase II in the two hybrid system suggests that NOV might be involved in regulating gene expression at the transcriptional level. As has already been suggested for several other nuclearly located cytokines, the NOV protein does not contain a typical nuclear localisation signal. Therefore, it is possible that it combines with either a receptor or a chaperone during its translocation. Disruption of the balance between the secreted and nuclear NOV isoforms might affect the putative autocrine and paracrine functions of NOV and might be of considerable importance in the development of cancers in which the expression of novH has been shown to be impaired.

Base Sequence↗

PubMed1993

Functional interleukin-2 receptor on a Tac negative human leukaemia T-cell line.

Cell line PER-423 was derived from the cells of a patient with an immature acute T-lymphoblastic leukaemia and the growth of this human cell line is strictly dependent on interleukin-2 (IL-2). PER-423 cells express the p75 (beta) subunit of the IL-2 receptor (IL-2R beta), while the p55 chain (IL-2R alpha) is not detectable by immunofluorescence. The analysis of the IL-2R revealed that it is of intermediate affinity and the median effective IL-2 concentration for PER-423 cells (EC50 value) was determined to be 1.44 +/- 0.29 nM. Chemical crosslinking studies showed that the receptor consists of one polypeptide of approximately 95 kDa as well as a doublet of 70 kDa and 60 kDa and does not include the IL-2R alpha-chain. The steady-state mRNA level for the p75 subunit was similar to that present in a cell line expressing an IL-2R alpha+ beta+, while only traces for the alpha-chain were detectable. PER-423 cells can be induced to express the alpha-chain of the IL-2R on the cell surface, concomitant with a much reduced EC50 level. Since cell line PER-423 is functionally dependent on IL-2, it provides an ideal model for IL-2 signal transduction studies and for investigations focusing on the requirements for ligand binding vs activation.

Child, Preschool↗

PubMed2002

Horizontally oriented clusters of multiple chondrons in the superficial zone of ankle, but not knee articular cartilage.

Osteoarthritis is a progressive disease that is initiated at the surface of articular cartilage and proceeds to destroy the entire depth of the cartilage. The prevalence of osteoarthritis varies in different joints; e.g., the ankle joint has a very low prevalence of the disease compared to the knee joint. To better understand any inherent differences between the articular cartilage of the ankle and that of the knee that would account for the difference in occurrence of osteoarthritis, studies were undertaken to examine differences between the superficial zones in these two joint cartilages obtained from human donors. Chondrocytes in the superficial zones of the normal ankle (talocrural) and the normal knee (tibiofemoral) joints were identified with a monoclonal antibody specific for the superficial zone protein (SZP). When the chondrocytes from both joints were compared in serial horizontal sections, the chondrocytes in the superficial zone of the knee cartilage were seen either as isolated single cells or as doublets. However, the chondrocytes within the superficial zone of normal ankle cartilage were arranged in planar clusters containing multiple chondrons composed of 2-13 cells. There were no detectable differences in the chondrocyte clusters in the superficial zone of the ankle with respect to age, gender, or site on the cartilage surface. Adjacent to a lesion in an ankle joint with degenerative changes, the clusters were larger, containing up to 22 chondrocytes. This is the first report documenting the presence of multiple chondrons in the superficial zone of normal human adult articular cartilage.

Adolescent↗

PubMed1995

Antibodies to synovial antigens in recent-onset rheumatoid arthritis.

OBJECTIVE: To identify synovial antigens that bind to serum antibodies from subjects with recent-onset rheumatoid arthritis (RA) (< 6 months of synovitis). METHODS: Soluble and insoluble fractions of Triton X-100 extracts of RA and normal synovial tissue, and normal spleen and placenta, were immunoblotted with sera from 27 patients with recent-onset RA, 13 autoimmune disease control subjects, and 13 blood bank control donors. Bound immunoglobulin was probed with 125I-labeled protein A. RESULTS: Antibodies in the sera of 20 (74%) patients with recent-onset RA recognized at least 1 of 5 antigens in both a disease- and tissue-specific and nonspecific manner. Anti-La antibodies, usually associated with primary Sjögren's syndrome, were detected in 2 sera. Eight sera had increased reactivity to an IgG heavy and light chain dimer. There was strong binding of 8 sera with a 35-kd doublet and of 3 sera with a 55-kd species in RA and normal synovial lysates (insoluble fractions). Two sera uniquely recognized a 45-kd protein only in the RA synovial lysate (soluble fraction). CONCLUSION: IgG antibodies in the sera of patients with recent-onset RA show positive immunoblots for 3 novel synovial antigens of 35-kd, 55-kd, and 45-kd, as well as for 2 previously characterized antigens (La and IgG). Thus, a variety of synovial antigens appear to be recognized by B cells even early in the clinical course of RA.

Adolescent↗

PubMed1999

Insulin-like growth factor binding proteins in cerebrospinal fluid during human development and aging.

We analyzed samples of insulin-like growth factor binding proteins (IGFBPs) in human cerebrospinal fluid (CSF) in neurologically normal patients from one day after birth to age 76 years. CSF samples were separated on SDS-PAGE and then transferred to nitrocellulose membranes where IGFBPs were detected by Western ligand blot using [(125)I]-IGF-II, confirming other reports where we found the presence of IGFBP-2, 3, 4, 5. The 34 kDa IGFBP-2 was present in all samples, and progressively decreased with age. A broad 28- to 30-kDa IGFBP band, having the appearance of IGFBP-5, was triphasic: faint during infancy, barely detectable at 6 months, but intense in adult and aged individuals. The 24-kDa IGFBP-4 band was only seen in neonatal CSF samples, while the IGFBP-3 doublet gradually increased during aging. Thus, these present results show that IGFBP-2, 3, 4 and 5 in CSF are developmentally regulated, suggesting roles for these molecules in the development of the nervous system.

Adolescent↗

PubMedDate not supplied

Histochemical tracing of bismuth in testis from rats exposed intraperitoneally to bismuth subnitrate.

The histochemical silver amplification technique autometallography (AMG), was used to trace bismuth in the testis of Wistar rats injected intraperitoneally with bismuth subnitrate. In the seminiferous tubules, bismuth was located in lysosomes of Sertoli cells closely associated with heads of spermatids in the late stages of the spermatogenesis, i.e. shortly before the release of Step 19 spermatids in Stage XIII. No bismuth-specific AMG silver grains were detected in the spermatogenic cell line. However, tails of free sperm cells located in the tubular lumen showed autometallographic grains in close contact to the nine outer microtubule doublets in the axonema. Leydig cells concentrated huge amounts of AMG-bismuth in their lysosomes. Furthermore, parallel exposure to selenium significantly increased the amount of histochemically traceable bismuth in the rat testis.

Animals↗

PubMed1988

Diagnostic Mr31/32,000 Schistosoma mansoni proteins (Sm31/32): reaction with sera from Sudanese patients infected with S. mansoni or S. haematobium.

Sera of Sudanese patients with active infections of Schistosoma mansoni or S. haematobium were tested in immunoblots for their reactivity with Mr31/32,000 proteins of adult S. mansoni (Sm31/32). All sera from patients with intestinal (n = 123) and all but one from those with urinary schistosomiasis (n = 35) had antibodies against Sm31/32. These and additional data suggest that both specificity and sensitivity of Sm31/32 to detect schistosome infections are close to 100%. Antibodies against these proteins developed also in monkeys after experimental infection with S. haematobium. Sm31/32 antigens reacted in immunoblots as a doublet with most S. haematobium-patient sera and as a broad band with many S. mansoni-sera suggesting that at least two components are present in the molecular weight region of Mr31/32,000. The data demonstrate the potential use of Sm31/32 from adult worms to diagnose patients with intestinal or urinary schistosomiasis in endemic areas.

Adolescent↗

PubMed1981

A study of the magnetic properties of haem a3 in cytochrome c oxidase by using magnetic-circular-dichroism spectroscopy.

M.c.d. (magnetic-circular-dichroism) spectroscopy was used to study the magnetization properties of the haem centres in cytochrome c oxidase with magnetic fields of between 0 and 5.3 T over the temperature range 1.5--200 K. The oxidized, oxidized cyanide and partially reduced cyanide forms of the enzyme were studied. In the oxidized state only cytochrome a3+ is detectable by m.c.d. spectroscopy, and its magnetization characteristics show it to be a low-spin ferric haem. In the partially reduced cyanide form of the enzyme cytochrome a is in the diamagnetic low-spin ferrous form, whereas cytochrome a3--CN is e.p.r.-detectable and gives an m.c.d.-magnetization curve typical of a low-spin ferric haem. In the oxidized cyanide form of the enzyme both cytochrome a and cytochrome a3--CN are detectable by m.c.d. spectroscopy, although only cytochrome a gives an e.p.r. signal. The magnetization characteristics of haem a3--CN show clearly that its ground state is an electronic doublet and that another state, probably a spin singlet, lies greater than 10 cm-1 above this. These features are well accounted for by an electronic state of spin S = 1 with a predominantly axial distortion, which leaves the doublet, Ms = +/- 1, as the ground state and the component Ms = 0 as the excited state. This state would not give an e.p.r. signal. Such an electronic state could arise either from a ferromagnetic coupling between haem a3+(3)-CN and the cupric ion, Cua3, or form a haem in the Fe(IV) state.

Circular Dichroism↗

PubMed1982

Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus.

The p21 transforming protein coded for by the v-ras gene of Harvey murine sarcoma virus (Ha-MuSV) migrates as a doublet band between 21,000 and 23,000 daltons during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lower band of the doublet is designated p21, and the upper band is designated pp21 since it comigrates with the phosphorylated form of p21. By pulse-labeling with [35S] methionine, we detected a p21 precursor, pro-p21, which migrated as if it was approximately 1,000 daltons larger than p21. The precursor-product relationship was established by pulse-chase experiments with [25S] methionine in the presence of 100 micrograms of cycloheximide per ml, which inhibited all de novo protein biosynthesis. Within 4 h, pro-p21 was completely chased into p21, and during the next 24 h pp21 accumulated. Thus, formation of pp21 from p21 did not require de novo protein synthesis. By subcellular fractionation into cytosol amd membrane fractions, we found that pro-p21 was synthesized in a non-membrane-bound state and that shortly after its complete synthesis, the p21 product was associated with the membrane fraction. By selective cleavage of p21 at a unique aspartic acid-proline residue with 70% formic acid or with Staphylococcus aureus V8 protease, we found that the intramolecular site of pro-p21 processing was located in the C-terminal portion of the pro-p21 molecule. The possibilities that the precursor was involved in the assembly of p21 into the plasma membrane and, alternatively, that the processing was a step in the activation of p21 biochemical activities are discussed.

Amino Acid Sequence↗

PubMed1993

Stimulus-associated protein in gastric parietal cell detected using antimelittin antibody.

The bee venom polypeptide melittin binds to and inhibits the gastric hydrogen-potassium-adenosinetriphosphatase (H(+)-K(+)-ATPase). A search for parietal cell proteins with a melittin-like structure was carried out. A 67-kDa (doublet) protein, which reacted with a polyclonal antimelittin antibody, was found in purified rabbit parietal cells. The protein exhibited reversible stimulus-dependent redistribution from cytosol to (total) membranes. It was also found to be associated with H(+)-K(+)-ATPase-containing membranes when isolated from the gastric mucosae of rabbits treated with histamine, but not with cimetidine. The presence of the protein correlated with the ability of the membrane preparations to exhibit ionophore-independent HCl accumulation, a characteristic of gastric membranes from histamine-stimulated animals. The 67-kDa melittin-like protein may play a role in the functional changes in the gastric parietal cell that are involved in stimulation of HCl secretion.

Aminopyrine↗

PubMed1993

Autoantibodies in viral hepatitis-related hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) develop autoantibodies to nuclear and nucleolar antigens (ANAs) which can be readily detected by immunofluorescence on cell substrates. The frequency of ANAs in HCC is 31% (57/184). The identity of three autoantigens was established as: NOR-90, nucleolus organizer region (doublet) polypeptides involved in RNA polymerase I transcription; fibrillarin, a component of nucleolar U3 RNP involved in pre-ribosomal RNA processing, and nucleophosmin/protein B23, a nucleolar protein involved in ribosome maturation and cell proliferation. Changes in ANAs were observed in some patients during transition from chronic liver disease to HCC and were manifested as seroconversion from ANA-negative to ANA-positive status by an increase in titers and changes in ANA specificities. Serum from a patient during this transition period was used to isolate a cDNA clone encoding a novel nuclear protein with structural motifs characteristic of a family of splicing factors. These observations support the notion that ANA responses in HCC might be driven by intracellular events related to transformation from the stage of chronic injury to the stage of malignancy. Changes in ANA profiles which were observed to precede clinically diagnosed HCC in some patients might be early markers of transformation.

Autoantibodies↗

PubMed1990

Identification of B-epitopes in the human papillomavirus 18 E7 open reading frame protein.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.

Amino Acid Sequence↗

PubMed1996

Sex determination of dried blood stains using the polymerase chain reaction (PCR) with homologous X-Y primers of the zinc finger protein gene.

The ability to "sex" unknown dried bloodstains is frequently of evidentiary value in forensic casework. Chelex-extracted DNA from 115 specimens (105 dried blood standards and 10 casework samples) was amplified for specific ZFX and ZFY regions of the X and Y chromosomes and subsequent restriction enzyme digestion. The polymerase chain reaction (PCR) yielded a 209 base pair (bp) product containing a polymorphic position with a Y chromosome portion including an additional Hae III restriction site while the X chromosome portion contains only one. The digested PCR product was separated by polyacrylamide gel electrophoresis (PAGE) and detected by silver staining. Female samples exhibit bands of 172 bp and 37 bp only while male samples (XY) exhibit 2 additional bands appearing as an 88/84 bp doublet. Initially the 105 known bloodstains were typed according to the procedure discussed here and correct gender determination was achieved for all samples therefore establishing the reliability of this method. The 10 casework samples yielded the expected results as well. This assay demonstrates potential in both presumptive and confirmatory capacities.

Base Sequence↗

PubMed1991

CpG islands: features and distribution in the genomes of vertebrates.

We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.

Animals↗

PubMed2004

Rapid screening for S-adenosylmethionine-dependent methylation products by enzyme-transferred isotope patterns analysis.

We report here an isotopic labeling and mass spectrometric method to rapidly identify S-adenosylmethionine (AdoMet)-dependent methylation products. In the presence of CH(3)- and CD(3)-labeled AdoMet, a methyl transfer product appears as a doublet separated by 3 Da in a mass spectrum, while other compounds show their normal isotopic distribution. Based on this unique isotopic pattern, methylation product(s) can be easily detected even from a mixture of cellular components. To validate our method, the product of human thiopurine methyltransferase (TPMT, EC 2.1.1.67) has been successfully identified from both an in vitro assay and a whole-cell assay. This method is generally applicable to AdoMet-dependent transmethylation and other group-transfer reactions, and constitutes the first example of a general strategy of enzyme-transferred isotope patterns (ETIPs) analysis.

Deuterium↗

PubMed1998

Novel non-radioisotope immunoprecipitation studies indicate involvement of pemphigus vulgaris antigen in paraneoplastic pemphigus.

We have developed two different novel immunoprecipitation assays in which radioisotopes are not used, and have examined antigens for four cases of paraneoplastic pemphigus (PNP) including three new patients. The PNP sera showed a clear reactivity with transitional epithelia of rat urinary bladder by immunofluorescence, and reacted with a characteristic doublet of the 210 and 190 kD proteins by immunoblotting of normal human epidermal extract, confirming the diagnosis of PNP. In addition, by immunoprecipitation using silver-stain to detect immunoprecipitated proteins, the PNP sera detected the 250, 210 and 190 kD proteins, while control bullous pemphigoid sera detected only the 230 kD bullous pemphigoid antigen. Furthermore, with another immunoprecipitation using cell surface biotinylation, three of the four PNP sera specifically reacted with the 130 kD pemphigus vulgaris antigen (Dsg3), indicating that pemphigus vulgaris antigen may be involved in PNP. This reactivity was further suggested by the immunoblot analysis using recombinant pemphigus vulgaris antigen. In future, these non-radioisotope immunoprecipitation assays should become a useful tool not only to unravel the complex situation for the PNP antigens, but also to study antigens in other autoimmune bullous skin diseases.

Animals↗

PubMedDate not supplied

The retinoblastoma susceptibility gene encodes a nuclear phosphoprotein associated with DNA binding activity.

The human gene (RB) that determines susceptibility to hereditary retinoblastoma has been identified recently by molecular genetic techniques. Previous results indicate that complete inactivation of the RB gene is required for tumour formation. As a 'cancer suppressor' gene, RB thus functions in a manner opposite to that of most other oncogenes. Sequence analysis of RB complementary DNA clones demonstrated a long open reading frame encoding a hypothetical protein with features suggestive of a DNA-binding function. To further substantiate and identify the RB protein, we have prepared rabbit antisera against a trypE-RB fusion protein. The purified anti-RB IgG immunoprecipitates a protein doublet with apparent relative molecular mass (Mr) of 110,000-114,000. The specific protein(s) are present in all cell lines expressing normal RB mRNA, but are not detected in five retinoblastoma cell lines examined. The RB protein can be metabolically labelled with 32P-phosphoric acid, indicating that it is a phosphoprotein. Biochemical fractionation and immunofluorescence studies demonstrate that the majority of the protein is located within the nucleus. Furthermore, the protein can be retained by and eluted from DNA-cellulose columns, suggesting that it is associated with DNA binding activity. Taken together, these results imply that the RB gene product may function in regulating other genes within the cell.

Base Sequence↗

PubMed1981

Characterization, by immunoprecipitation, of myeloid- and monocyte-specific antigens present on the human promyelocytic cell line (HL-60) in three stages of differentiation.

The human promyelocytic leukemia cell line HL-60 is reactive with an antiserum raised against normal human granulocytes (AGS). Immunoprecipitation with AGS on [35S]methionine-labeled HL-60 cell lysates with subsequent analysis by NaDodSO4/polyacrylamide slab gel electrophoresis shows a major antigenic doublet with molecular weights of 88,000 and 86,000, together with some minor antigens of lower molecular weight. Upon stimulation with dimethyl sulfoxide or 12-O-tetradecanoylphorbol 13-acetate, which induces HL-60 to differentiate to mature granulocytes or monocytes/macrophages, respectively, this antigenic doublet disappears. 12-O-Tetradecanoylphorbol 13-acetate induces the synthesis of an antigen, molecular weight 83,000, reactive with an antimonocyte serum. Neutrophil-specific alloantigens were not detected on HL-60 or its differentiated derivatives.

Animals↗