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Biological and biochemical properties of Nonidet P40-solubilized and partially purified tumor-specific antigens of the transplantation type from plasma membranes of a methylcholanthrene-induced sarcoma.

Tumor-specific transplantation antigen (TSTA) was solubilized from cell membranes of sarcoma Meth-A with non-ionic detergent Nonidet P40. Soluble TSTA was partially characterized by chromatographic separation and electrophoresis. The antigen responsible for tumor rejection activity had a molecular weight of approximately 70,000 daltons in the presence of detergent and an electrophoretic mobility of alpha-globulin. TSTA was well separated from mouse histocompatibility antigen H-2 by a sequence of procedures, including gel filtration, lectin affinity chromatography, column electrophoresis, and rechromatography on agarose, showed only three major bands on polyacrylamide gel electrophoresis. TSTA was specific for sarcoma Meth-A.

Animals

Isolation of a nucleocapsid polypeptide of herpes simplex virus types 1 and 2 possessing immunologically type-specific and cross-reactive determinants.

A polypeptide (p40) of approximately 40,000 molecular weight was isolated from herpes simplex virus type 1 and 2 nucleocapsids by gel filtration and ion exchange chromatography. This protein appears to be the same as protein 22a described previously (Gibson and Roizman, J. Virol. 10:1044--1052, 1972). Competition immunoassays were developed by using purified p40 and antisera prepared in guinea pigs. The assays indicated that the p40's from herpes simplex virus types 1 and 2 possess both type-specific and cross-reactive antigenic determinants. Antibodies to the p40 cross-reactive determinant reacted with antigens in simian herpes virus SA8-infected cells, but not with antigens induced by pseudorabies virus. Preliminary results indicated that a radioimmunoprecipitation test can be used to detect type-specific herpes simplex virus p40 antibodies in human sera.

Antibodies, Viral

Production of monoclonal antibodies against nucleocapsid proteins of herpes simplex virus types 1 and 2.

We prepared mouse hybrid cell lines which produced antibodies against herpes simplex virus type 1 and 2 nucleocapsids. Cell lines 1D4 and 3E1, respectively, secreted immunoglobulin G1 herpes simplex virus type 1 and immunoglobulin G1 herpes simplex virus type 2 antibodies which immunoprecipitated proteins designated p40 and p45 from homologous nucleocapsid preparations but precipitated no proteins from heterologous preparations. In contrast, guinea pig antisera prepared against either herpes simplex virus type 1 or 2 p40 precipitated p40 and p45 from both homologous and heterologous preparations. These findings suggest that p40 and p45 possess similar antigenic determinants and that the monoclonal antibodies that were tested reacted preferentially with the homologous determinants.

Antibodies, Viral

Rehabilitation following early malnutrition in the rat: body weight, brain size, and cerebral cortex development.

Sprague-Dawley rats were malnourished by giving their mothers an 8% casein diet starting at day 10 of gestation, while controls were fed a 24% casein diet. Starting at postnatal day 20 (P20), rehabilitation of the malnourished animals was attempted by: (1) feeding both mother and young a 24% casein diet, (2) leaving the pups with their mothers until they were 40 days old, and (3) reducing the litter size from 8 to 4 pups. Observations were made on aldehyde-perfused tissue from animals 20, 40 and 70 days old. The somatosensory cortex from one hemisphere was embedded in Araldite, and that from the other side was processed fro Golgi staining. At 20 days of age the body weight of the malnourished animals was 21% that of the controls, but at 70 days it was no longer different. The anterior-posterior length, the width, and the height of the cerebral hemispheres were also significantly reduced at P20, but the differences had disappeared by P70. The thickness of area 3 of the cerebral cortex was measured in 1 micron sections. It was significantly reduced in the malnourished animals at P20, but at P40, following rehabilitation, the difference was no longer statistically significant. In tangential 1 micron sections the fraction of the volume of tissue occupied by neuropil was measured in layers II through IV. At P20 it was significantly reduced only in the upper half of layers II/III of the malnourished animals; at P40 this difference was no longer present. The mean volume of upper layer II/III cell bodies was estimated and found to be significantly reduced in the experimental animals at P20 but not at P40. In the Golgi preparations, pyramidal cells in upper layer II/III were studied. Their estimated volume, as well as the thickness of their basal dendrites, was significantly reduced in the 20 day malnourished animals, but not in the rehabilitated animals. These results show that animals severely malnourished until 20 days of age can reach normal body weight and attain cerebral hemispheres of normal size when proper nutrition is provided. The effects of malnutrition on the cerebral cortex of these animals are most apparent in upper layer II/III which, during the time of nutritional restriction, is the least developed of the cortical layers. However, when proper nutrition is provided, the cerebral cortex may attain normal morphology.

Animals

Characterization of the minor polypeptides in the foot-and-mouth disease particle.

In addition to the four major polypeptides VP1 and VP4, foot-and-mouth disease virus particles contain two minor polypeptides, mol. wt. 40 X 10(3) (P40) and 52 X 10(3) (P52). Extensive purification procedures failed to remove these minor polypeptides from the virus particles. Polypeptide P40 co-electrophoresed in SDS-polyacrylamide gels with VP0, the probable precursor of VP2 and VP4 and was inaccessible to iodination in situ. The second minor polypeptide, P52, co-electrophoresed with the virus infection associated (VIA) antigen found in large amounts in harvests of the virus grown in BHK 21 cells. Polypeptide P52 was shown to be located near the surface of the virus particle by iodination experiments and by its removal on incubating the particles with trypsin or chymotrypsin. Pactamycin mapping showed that this polypeptide was not a precursor of the structural polypeptides. About one copy of P52 and 4 copies of P40 were found in the virus particles sedimenting at 146S. However a larger number of copies was found in those virus particles sedimenting faster than the 146S peak.

Animals

Ovarian Carcinoma Presenting Mucoepidermoid Carcinoma-Like Features in Association With Seromucinous Borderline Tumor: A Real Seromucinous Carcinoma?

Ovarian seromucinous carcinoma is generally classified within the spectrum of endometrioid carcinoma. We report a rare ovarian carcinoma with mucoepidermoid carcinoma-like features arising in direct association with a seromucinous borderline tumor. A 56-year-old postmenopausal female presented with a 10-cm pelvic multilocular cystic tumor and markedly elevated carbohydrate antigen 19-9. Histopathological examination showed a seromucinous borderline tumor with transition to invasive carcinoma composed of mucinous epithelial cells and p40-positive intermediate-like cells. The tumor was diffusely positive for PAX8 and negative for cytokeratin 20, supporting Mullerian differentiation. The p40-positive cell population persisted in the borderline, invasive, and para-aortic lymph node metastatic components. Comprehensive genomic profiling identified KRAS p.G12D without CTNNB1, PTEN, or ARID1A mutations. The patient developed platinum-resistant recurrence and died 11 months after diagnosis. This case is compatible with a potential endometriosis-independent pathway from seromucinous borderline tumor to an aggressive mucoepidermoid-like carcinoma.

mucoepidermoid carcinoma

Exploring Causal Links Between 91 Circulating Inflammatory Proteins and Hashimoto's Thyroiditis: A Bidirectional Mendelian Randomization Study.

BACKGROUND: Increasing evidence has linked inflammation to Hashimoto's thyroiditis (HT) etiology. However, the causal role of circulating inflammatory proteins in HT remains uncertain. To investigate this, we conducted a bidirectional Mendelian randomization (MR) study. METHODS: Genetic data for 91 inflammatory proteins and HT were sourced from publicly available GWAS databases. The PhenoScanner database was then searched for pleiotropic SNPs associated with potential confounders. Inverse variance weighted (IVW) analysis was used as the primary analysis, simultaneously supplemented by five sensitivity analyses to strengthen the results. RESULTS: The results revealed that, after false discovery rate (FDR) correction, interleukin (IL)-12p40 was causally associated with increased risk of HT (OR [95% CI] = 1.295 [1.172, 1.431], p = 3.66 × 10-7, PFDR = 3.33×10-5). Conversely, none of the inflammatory proteins was a consequence of HT. CONCLUSION: This study suggests that IL-12p40 is probably one of the factors correlated with HT etiology, contributing to a better understanding of the pathogenesis of HT and underscoring the potential for therapeutic interventions targeting inflammatory proteins.

Humans

Solubilization and molecular characterization of membrane-bound antigens shared by thymocytes and brain.

In order to characterize chemically the serologically well-defined antigens shared by thymocytes and nervous tissue of mice and rats, different solubilization procedures have been tested. Treatment of thymocyte membranes with urea-Nonidet-P40 (NP40) proved to be the most valuable method, since nearly 100% of the antigen was solubilized essentially without loss of the antigenicity. By gel filtration in urea-NP40 the molecular weight was estimated to be 35 000 and the Stokes radius in sodium deoxycholate 31.8 A. In gel filtration studies a homogenous peak was obtained, whereas isoelectric focusing yielded two peaks (isoelectric points pH 4.5-5.0 and pH 5.4). The three determinants of this antigenic system were inseparable in gel filtration and isoelectric focusing, supporting the view put forward on the basis of serological studies that they are part of the same molecule. Extraction of murine thymocyte membranes with organic solvents yielded a considerable loss of activity of the thymocyte-brain antigen. There was some residual activity in the protein fraction, but none in the organic phase. Correspondingly, the highly purified ganglioside G Gtet 2b proved unable to absorb any rabbit anti-CBA brain serum. The reduced antigenic activity of the delipidated protein fraction could be restored by addition of lipids, as well as the nonionic detergent NP40. These observations suggest that interaction of protein and lipid is necessary for the antigenicity of the thymocyte-brain antigen.

Animals

The private specificity H-2.4 and the public specificity H-2.28 of the D region are expressed on two independent polypeptide chains.

The antigenic specificities H-2.4 (private) and H-2.28 (public) in the H-2a haplotype are controlled by the D region of H-2 as defined by the available recombinants. In previous studies we have demonstrated by the antibody-induced redistribution method that the antisera against these specificities contain antibodies against at least two different polypeptide chains. We here report the results of the indirect immunoprecipitation of radiolabeled antigens after solubilization with Nonidet-P40. The antisera against the two specificities precipitated from such extracts two different and independent polypeptide chains, indicating that the products of the D region, as presently defined, comprise at least two different molecules. The molecular weight of both chains is approximately 45 000, which is similar to other molecules bearing private H-2 antigenic specificities. Consequently, the chromosomal segment presently defined by recombination studies as the D region, must contain another locus, controlling the second polypeptide chain which is detectable by anti-H-2.28 antisera, besides the H-2D locus which controls the polypeptide chain bearing the private specificity H-2.4 as well as most of the public specificities.

Animals

Immunization against Marek's disease using Marek's disease virus-specific antigens free from infectious virus.

Immunity against Marek's disease was conferred by the use of non-infectious materials extracted with non-ionic detergents from cells infected with the attenuated strain of Marek's disease virus (MDV). Antibody-free Rhode Island Red chicks were inoculated at 1 week of age with cell extracts emulsified in Freund's complete adjuvant and were given a second inoculation 1 week later without adjuvant. Protection against natural infection was obtained in groups inoculated with both soluble (not sedimented at 100,000 times g/2h) and insoluble antigens present in Nonidet P40 (NP40) extract, but only with the insoluble fraction of deoxycholate extract. The results suggest that the immunizing antigens can be partially solubilized with 0.5% NP40 and that the growth and spread of MDV are reduced in immunized chickens.

Animals

Concentration of lipoproteins containing apolipoprotein B in human peripheral lymph.

The concentration of apolipoprotein B (apoB) in human serum and peripheral lymph was measured by quantitative immunoelectrophoresis with anti-serum to human low-density lipoprotein. In four normal and six hyperlipidaemic subjects, total lymph apob/ml was 5-10% of total serum apoB/ml in the same subject. These ratios were equivalent to lymph apob concentrations of 60-120 microgram/ml. When the assays were carried out under conditions in which unmasking of immunoreactive sites on lymph and serum apoB was assumed to be maximal (delipidation with Nonidet P40), the lymph/serum apoB concentration ratios in three normal subjects were similar to those obtained with untreated lymph and serum.

Adult

Effect of voluntary self-paced movements upon auditory and somatosensory evoked potentials in man.

The effect of voluntary self-paced movements upon auditory (AEPs) and somatosensory (SEPs) evoked potentials has been investigated according to the temporal relationship between movement and delivery of test stimuli. EPs were recorded in 7 subjects and averaged in 10 successive epochs extending from 880 msec before to 2500 msec after movement. AEPs were attenuated in all epochs. The decrease was greatest in the 220 msec epoch just following movement and involved components N85 and P170. SEPs were attenuated similarly to AEPs when movements were performed by the hand contralateral to somatosensory stimulation. Of the 5 SEP components, only P40 failed to reflect the attenuation, while P95 showed the greatest amplitude decrease. When stimulation was ipsilateral, SEP amplitude was attenuated only when close to the movement. N65 and P95 decreased while N130 increased. In all subjects the results were consistent for treatments of AEP and SEP (with contralateral movements), whereas large inter-individual differences were observed for the SEP with ipsilateral movements.

Auditory Cortex

Genomic Characterization of Classic Adamantinoma, Osteofibrous Dysplasia, and Osteofibrous Dysplasia-like Adamantinoma.

Classic adamantinoma, osteofibrous dysplasia (OFD), and OFD-like adamantinoma are rare bone tumors arising primarily in the tibiae. Their distinction can be challenging; data on their molecular pathogenesis remain limited. We searched our pathology files in 2004-2024 for available cases and performed targeted next-generation sequencing along with whole-genome single-nucleotide polymorphism arrays and 3-dimensional genomics/Hi-C sequencing in selected cases. Our cohort included 3 classic adamantinomas (2 females and 1 male; age, 14-56 years), 5 OFDs (3 females and 2 males; age, 9-25 years), and 2 OFD-like adamantinomas (1 female and 1 male; age, 30-41 years). Of the 10 tumors, 9 arose from the tibiae; 1 classic adamantinoma originated from the radius. The 3 classic adamantinomas harbored multiple copy number gains involving chromosome 7, 8, 10, 12, and/or 19. Focal deletion of chromosome 17, intergenic rearrangement involving FGFR1, and NRAS p.G12D were each present in 1 classic adamantinoma. Of the 5 OFDs, KMT2A p.C2441F, KMT2D p.S1040P, PHOX2B p.G213D, and RIF1 deletion were each present in 1 case; no additional copy number/single-nucleotide variants were identified. Of the 2 OFD-like adamantinomas, one case with tumor clusters visible only on cytokeratin immunostain harbored no variants, whereas another case with tumor clusters visible on light microscopy and cytokeratin/p40 immunostains showed gains of chromosome 7, 8, 19, and 20. By Hi-C, 1 classic adamantinoma harbored an approximately 9 Mb tandem duplication on chromosome 12q, 1 OFD harbored a rearrangement with breakpoints near MECOM and HOOK3, and the OFD-like adamantinoma with tumor clusters visible only on cytokeratin immunostain harbored no structural variant. In conclusion, classic adamantinomas and OFD might be genetically distinct. Classic adamantinomas harbored multiple alterations, including chromosome/arm-level copy number gains, the detection of which could aid their distinction from OFDs. Using genomics as the benchmark, OFD-like adamantinomas might be better delineated by light microscopy or p40 than by cytokeratin immunohistochemistry. These data expanded our molecular understanding of these rare bone tumors.

Humans

Nearest-neighbor interactions of the major RNA tumor virus glycoprotein on murine cell surfaces.

Formaldehyde-fixed Staphylococcus aureus and monospecific antiserum to gp70, the major envelope glycoprotein of murine leukemia virus, were used to immunoadsorb gp70 from Nonidet P40 extracts prepared from surface-radioiodinated murine cells. The labeled gp70 molecules in these cells were linked to a protein of approximately 15,000 daltons via native disulfide bonding. Prior treatment of cells with the reversible, bifunctional, crosslinking reagent dimethyl-3,3'-dithiobispropionimidate, followed by immunoadsorption and two-dimensional diagonal electrophoresis, revealed apparent homodimers and homotrimers of the 85,000-dalton complex. Identical treatment of purified type C RNA tumor virus from murine cells also revealed homodimeric and homotrimeric species, demonstrating similar self-associating tendencies of this glycoprotein in both intact virus and the plasma membrane of nonproducing murine cells. One cross-linked product consistently detected on the surfaces of murine cells was not present after crosslinking of a representative strain of murine leukemia virus.

Cells, Cultured

Biologic and biochemical properties of detergent-solubilized tumor-specific transplantation antigen from a simian virus 40-induced neoplasm: brief communication.

The detergent Nonidet P40 was used to solubilize tumor-specific transplantation antigens (TSTA) from crude membranes obtained from dissociated cells of simian virus 40-induced sarcoma of BALB/c mice. A good recovery of specific tumor rejection activity was observed. One fraction, fraction V, was obtained following polyacrylamide-agarose filtration of the solubilized material, and this fraction contained most of the activity. An increased specific activity followed gel filtration. Preliminary data from lectin column chromatography of the active fraction V indicated a separation of TSTA activity from H-2 activity.

Animals

Lung Squamous Cell Carcinoma Harbouring a Novel PAX8::PPARγ Fusion and a FGFR2 Exon 7 Missense Mutation.

Comprehensive molecular profiling is now routinely performed in newly diagnosed non-small cell lung carcinomas (NSCLCs) to identify actionable genomic alterations. Although numerous molecular abnormalities have been described in lung carcinomas, rare and unexpected gene fusions may create significant diagnostic challenges, particularly when they are characteristically associated with tumours of different lineages. To our knowledge, this is the first reported case of a primary lung squamous cell carcinoma harbouring an in-frame PAX8::PPARγ fusion with a concurrent FGFR2 exon 7 missense mutation (p.W290C). An 80-year-old man with a smoking history exceeding 50 years presented with a rapidly enlarging PET-avid right upper lobe pulmonary mass. Bronchial brushing cytology demonstrated a hypercellular malignant neoplasm composed of pleomorphic squamoid cells with hyperchromatic nuclei, dense cytoplasm and extensive necrosis. Cell block material showed squamous morphology and diffuse p40 positivity, supporting squamous differentiation. Reflex next-generation sequencing identified an FGFR2 exon 7 missense mutation (p.W290C; c.870G>C) and targeted RNA fusion analysis demonstrated an in-frame PAX8::PPARγ fusion resulting from a t(2;3)(q13;p25.2) translocation. Because PAX8::PPARγ rearrangements are strongly associated with follicular thyroid neoplasms, extensive clinicoradiologic and immunohistochemical correlation was performed to exclude metastatic thyroid carcinoma. Imaging studies showed no thyroid lesion or residual thyroid tissue, and tumour cells were negative for thyroglobulin, TTF-1 and PAX8. Correlation of the clinical history, radiologic findings, cytomorphology, immunophenotype and molecular profile supported the diagnosis of primary lung squamous cell carcinoma. This case expands the molecular spectrum of lung squamous cell carcinoma and highlights the importance of integrated cytopathologic, immunohistochemical, molecular and radiologic evaluation when unexpected gene fusions are identified in cytology specimens.

FGFR2 exon 7 missense mutation

Partial characterization of cell surface idiotypes on alloantigen-activated T lymphoblasts.

T lymphoblasts of specificity Lewis anti-DA, Lewis anti-BN, BN anti-DA and L.BN anti-DA were purified on Ficoll-Paque from mixed leucocyte cultures on day 5. Blasts were radiolabelled with iodine-125 by the lactoperoxidase method and lysed by the aid of Nonidet P40. Idiotypic molecules were puried from the lysates by the use of anti-idiotypic antiserum of specificity anti-(Lewis anti-DA) and Staphylococcus aureus bearing protein-A. In this way, molecules with a molecular weight of 150,000 and 75,000 daltons could be isolated from Lewis anti-DA and L.BN anti-DA T lymphoblasts but no significant radioactivity was brought down by the same procedure from Lewis Lewis anti-BN or BN anti-DA T lymphoblasts. No additional molecules were detected on the lower molecular weight regions where light chains of Ig type as well as conventional products of the MHC genes would appear. The 150,000 dalton molecules are composed of two single polypeptide chains with a molecular weight of around 75,000 daltons. These data are in complete agreement with earlier results on antigen-binding, idiotypic receptors obtained from normal rat T lymphocytes.

Animals