Biomedical subjects

D J Moss

Publications and source records attributed to D J Moss.

At least 127 records · Page 7Linked to original sources

PubMed1986

Characterisation of a soluble trypsin fragment of GP130: a neuronal glycoprotein associated with the cytoskeleton.

A neuronal glycoprotein (GP130) that is associated with the cytoskeleton [Ranscht et al: J Cell Biol 99:1803-1813, 1984] remains insoluble in 0.1 M NaOH, a property typical of integral membrane proteins. At present it is possible to solubilise and hence isolate GP130 only under denaturing conditions. However, a large fragment of apparent molecular weight 120K is released into solution by trypsin. The fragment corresponds to the extracellular region of the glycoprotein as shown by the fact that it is released from live cultures of chicken sympathetic neurons and by its retention of concanavalin A-binding activity. The soluble extracellular fragment has been purified using mild biochemical techniques, which are expected to retain its biological activity. Measurement of the sedimentation coefficient, Stokes radius, and frictional ratio in addition to metal shadowing of the fragment show that it has a molecular weight of about 120K and is asymmetric, probably rod-shaped with a long axis of more than 20 nm.

Animals↗

PubMed1986

Induction of Epstein-Barr virus nuclear antigens.

Lymphocytes were infected with the QIMR-WIL strain of Epstein-Barr virus, and the induction of Epstein-Barr virus-associated nuclear antigens was determined by using the protein immunoblot. There was a temporal increase in six antigens, with Epstein-Barr nuclear antigen 2 being detected 1 day after infection. The appearance of these antigens was shown to be independent of cellular DNA synthesis.

Antigens, Viral↗

PubMed1986

T lymphocyte lines from arthritic synovial fluid: establishment and function.

T lymphocyte lines have been established, with the addition of interleukin-2 (IL-2), from the synovial fluid (SF) of patients with arthritis. Characterisation of seven of these SF-derived T cell lines showed them to be T3+, T11+, and to contain a significant proportion of T8+ cells (mean 41%). The proportion of T4+ cells varied among the lines, with a mean T4+/T8+ ratio of 0.6. A significant autologous mixed lymphocyte reaction (AMLR) was observed only in one of four T cell lines assayed. Two of four lines assayed showed natural killer (NK) cell-like activity, while one line displayed a significant suppressor activity, suggesting that the T cell population in SF contained NK-like cells or suppressor cells, or both, either of which may be selected in the establishment of SF-derived T cell lines.

Arthritis↗

PubMed1985

Decreased calcium dependence of lymphoblastoid cell lines compared with Burkitt lymphoma cell lines.

The extracellular calcium level required for proliferation was compared in B lymphoid cell lines from various sources by determining the calcium concentration at which long-term proliferation was inhibited by 50% (CaPD50). Fourteen Burkitt lymphoma (BL) lines had a mean CaPD50 of 44 +/- 28 microM whereas 45 lymphoblastoid cell lines (LCLs) obtained by in vitro transformation of B lymphocytes with Epstein-Barr virus (EBV) had a mean CaPD50 of 3.6 +/- 1.8 microM. This difference applied also to autologous BL lines and LCLs established from the same patient. The decreased calcium requirement of virally-transformed compared with tumour-derived cell lines therefore appears to be a universal phenomenon in mammalian cells. Within the BL group, no correlation was found between the calcium requirement for proliferation and presence or absence of the EBV genome. Arrest of BL lines and LCLs occurred in the G1 phase of the cell cycle and was readily reversed by addition of calcium to the medium. One anomalous LCL was found which showed a high CaPD50 (43 +/- 6 microM) and accumulated in both G1 and G2. These results, in combination with a previous study of EBV transformation in vitro, indicate that the calcium dependence of B lymphocytes generally decreases in the following order: normal cells greater than BL cells = early stage transformation greater than LCL. The 2 transformed phenotypes thus distinguished in human lymphoid cells may offer unique opportunities for defining the status and expression of EBV in vitro and in vivo.

Burkitt Lymphoma↗

PubMed1985

Distinctions between endemic and sporadic forms of Epstein-Barr virus-positive Burkitt's lymphoma.

Tumour cell lines were established in vitro from 16 cases of Epstein-Barr (EB) virus genome-positive Burkitt's lymphoma (BL), 7 of "endemic" origin (i.e. from holoendemic malarial areas of Africa and of New Guinea) and 9 of "sporadic" origin (i.e. from outside such high-incidence areas). All the BL cell lines thus established were monoclonal by immunoglobulin isotype expression and displayed a characteristic chromosomal translocation, t(8:14) or t(8:22), confirming their malignant origin. Clear differences observed between the individual BL cell lines appeared to be related to their endemic or sporadic status. All 7 endemic cell lines began growth as a carpet of single cells, often with small, loose clumps appearing in later passage. Whilst 3 lines of sporadic origin displayed a similar pattern to the above, the majority of sporadic lines grew as large, tight clumps of cells from the first passage onwards. These differences in growth pattern were reflected by differences in cell surface phenotype, as defined in indirect immunofluorescence tests using a panel of monoclonal antibodies (MAbs) specific for B-lineage-associated antigens. BL cell lines could be classified into 3 separate groups on the basis of their reactivity with 6 particular antibodies (MHM6, AC2, Ki-1, Ki-24, J5 and 38.13). All 7 endemic BL cell lines and 2 of the 3 sporadic BL cell lines which began growth as single cells showed a group-I cell-surface phenotype (MHM6, AC2, Ki-1, Ki-24 negative; J5, 38.13 positive) in early passage. In contrast, all 6 sporadic BL cell lines which began growth in large clumps displayed a distinct group-II phenotype (MHM6, AC2, Ki-1 positive/negative; Ki-24, J5, 38.13 positive); in later passage most of these sporadic lines progressed to a group-III phenotype (MHM6, AC2, Ki-1, Ki-24 positive; J5, 38.13 negative) without loss of those immunoglobulin and chromosomal markers identifying the cells' malignant origin. These clear differences between endemic BL cell lines on the one hand and the majority of sporadic BL cell lines on the other suggest that endemic BL arises from a more restricted range of progenitor B cells than does the sporadic form of the disease.

Adult↗

PubMed1985

Cell-mediated immunosurveillance mechanisms and the pathogenesis of Burkitt's lymphoma.

Paired Epstein-Barr virus (EBV)-carrying cell lines have been established from Burkitt's lymphoma (BL) patients, one of each pair being the BL cell line derived from the malignant cells of the tumour, the other, the lymphoblastoid cell line (LCL) derived from the patient's normal B cells by experimental infection with the virus. Comparative studies have shown the following: (1) All the lines were to some extent sensitive to in-vitro activated natural-killer cells, individual pairs differing as to whether BL or LCL cells were more susceptible. (2) For six of the seven pairs tested, the BL cell line was clearly sensitive to allo-specific (anti-class 1 HLA) effector T cells, although levels of lysis were slightly below those observed for the corresponding LCL; only one BL cell line showed evidence of a dramatic reduction of HLA antigen expression, and this line was insensitive to allo-specific cytolysis. (3) For two of the three pairs tested to date, EBV-specific cytotoxic T-cell preparations from HLA antigen-matched donors lysed the LCL but not the BL cell line, despite the latter's apparent expression of the relevant restricting antigens. In both of these cases, it was known that the tumour arose in vivo in the face of prevailing EBV-specific T-cell surveillance. An escape of the malignant cells from such surveillance may therefore be important in the overall pathogenesis of EBV genome-positive BL.

Burkitt Lymphoma↗

PubMed1985

T lymphocytes in infectious mononucleosis. I. T cell death in vitro.

A large proportion of T lymphocytes isolated from the peripheral blood of acute infectious mononucleosis (IM) patients rapidly die when cultured in vitro, with greater than 50% dying within 12-15 h of seeding and up to 80% dying within 24 h. The cells die by apoptosis, a morphologically distinct mode of cell death that occurs in circumstances where death is a regulated event such as in embryonic development and hormone-dependent atrophy. In contrast, the level of cell death remained low in cultures of lymphocytes from controls and in the T cell depleted subpopulation from acute IM patients, with less than 2% and 10% of the lymphocytes dying by apoptosis after 36 h in culture, respectively. The rapid death of acute IM T cells in vitro does not involve soluble factors (including the serum fraction) or T cell to T cell contact. It is suggested that this observation may necessitate a re-evaluation of IM T cell function in vitro.

Cell Communication↗

PubMed1985

T lymphocytes in infectious mononucleosis. II. Response in vitro to interleukin-2 and establishment of T cell lines.

The addition of 20% interleukin-2 (IL-2) significantly reduced the percentage of T lymphocytes dying in vitro after being isolated from the peripheral blood of acute infectious mononucleosis (IM) patients. Moreover, the immediate addition of 20% IL-2 to freshly isolated blood allowed IM T cell lines to be readily established from the peripheral blood of acute IM patients. Characterization of seven of these IM T cell lines showed them to be T3+, T11+, T4-, T9- and generally T10-. Over half of the lines characterized were T8+. It will now be possible to re-evaluate IM T cell effector functions as previous assays of IM T cell functions may have been influenced by the presence of rapid and extensive T cell death in vitro.

Cell Line↗

PubMed1984

Paired Epstein-Barr virus-carrying lymphoma and lymphoblastoid cell lines from Burkitt's lymphoma patients: comparative sensitivity to non-specific and to allo-specific cytotoxic responses in vitro.

Paired Epstein-Barr (EB) virus-carrying cell lines have been established from Burkitt's lymphoma (BL) patients; one the BL-cell line derived from the malignant cells of the tumour and bearing the relevant chromosomal translocation, the other the diploid lymphoblastoid cell line (LCL) derived from the patient's normal B cells by experimental infection with the virus. Comparative studies have shown that both BL and LCL cells are relatively resistant to fresh NK cells but are more susceptible to in vitro-activated NK cells; individual pairs differ as to whether the BL or LCL cells are more sensitive to such effectors. With most cell pairs studied, the two cell types were equally efficient at inducing NK-cell activation in vitro. When the in vitro stimulation protocol was changed to favour the induction of allo-specific responses, most BL cells were noticeably poorer stimulators than the corresponding LCL although both cell types induced a similar pattern of cytotoxicity; this appeared to be directed against HLA class I allo-antigens and could be inhibited by class I antigen-specific monoclonal antibodies. Most BL target lines, though susceptible to this allo-specific lysis, were significantly less so than the corresponding LCL However, only 1/7 BL-cell lines tested vis-à-vis the LCL gave evidence of a gross reduction in HLA antigen expression and this was immediately apparent from both serological typing and cellular analysis.

Burkitt Lymphoma↗

PubMed1984

Calcium concentration defines two stages in transformation of lymphocytes by Epstein-Barr virus.

Lymphocytes from Epstein-Barr virus (EBV) seronegative donors were either stimulated with Protein A or infected with EBV and cultured in growth medium containing a range of calcium levels (700 microM to less than 2 microM available calcium). Three calcium end-points for the proliferation of B lymphocytes were defined: 18 microM for mitogenically-stimulated B cells; 8 microM for a pre-12 h event in EBV transformation and less than 2 microM for a post-12 h event in EBV transformation resembling the end-point for the emergent lymphoblastoid cell line. EBV-infected lymphocytes cultured in calcium-depleted medium expressed the EB virus nuclear antigen and proliferated after addition of calcium chloride. The present study should be useful both in defining the sequence of events in EBV transformation and in studying properties specific to the transformed phenotype.

Antigens, Viral↗

PubMed1984

A neuronal surface glycoprotein associated with the cytoskeleton.

A cytoskeleton-associated glycoprotein of 130-kilodalton molecular mass (GP 130) was purified from a nonionic detergent-insoluble fraction of 10-16-d-old chicken embryo brains. GP 130 is tightly associated with other proteins in actin-containing complexes (Moss, D.J., 1983, Eur. J. Biochem., 135:291-297); thus, pure protein preparations were obtained only after the partial dissociation of the complexes with the zwitterionic detergent, dimethyl dodecyl glycine (EMPIGEN BB), followed by ion-exchange chromatography and electrophoresis on preparative SDS polyacrylamide gels. Specific monoclonal and polyclonal antibodies were raised to GP 130 and used to examine its distribution in the developing nervous system. Experiments with these antibodies revealed that GP 130 is confined to nervous tissue and is restricted to the surface of neurons in cultures derived from both the central and peripheral nervous systems. This novel glycoprotein is immunologically unrelated to the neuronal cell adhesion molecule (N-CAM), or to vinculin, a protein of similar molecular mass which has been suggested to link actin filaments to the plasma membrane. In the developing chicken embryo brain, GP 130 is first detectable around day 8 after fertilization and increases to approximately 50% of its adult level by embryonal day 13. In contrast, no increase is observed over a similar developmental period in sciatic nerve. In the adult chicken, GP 130 is most abundant in brain and has a particularly high content in areas rich in dendrites and synapses.

Aging↗

PubMed1984

Identification of multiple Epstein-Barr virus-induced nuclear antigens with sera from patients with rheumatoid arthritis.

By means of the protein immunoblot technique, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) could be identified in a variety of EBV-transformed cell lines with anti-EBNA-positive sera from normal donors. The molecular weight of EBNA expressed in each of the cell lines varied between 70,000 and 75,000 and was dependent upon the strain of infecting virus. In contrast, 15 of 21 sera from patients with rheumatoid arthritis identified antigens in addition to EBNA. The most prominent of these antigens had molecular weights of 110,000 to 115,000 and 92,000. All of the EBV genome-positive cell lines except for QIMR-GOR and cell lines containing the P3HR-1 virus expressed these antigens. The antigens were not present in the EBV genome-negative Ramos and BJAB cell lines, nor were they identified with EBV seronegative sera, indicating that they were EBV related. There was no direct correlation between the presence of antibodies in sera to EBNA, viral capsid antigen or early antigen, and reaction with the 92,000-molecular-weight antigen in immunoblots, indicating that this antigen was distinct from previously described EBV-related antigens.

Animals↗

PubMed1983

Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle.

Förster energy-transfer techniques have been applied to labeled myosin subfragment 1 from rabbit skeletal muscle to determine an intramolecular distance and whether this distance changes during magnesium-dependent ATPase activity. The alkali one light chain was labeled at Cys-177 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and then exchanged into subfragment 1. High specificity of labeling was indicated by high-performance liquid chromatography analysis of a tryptic digest of the labeled light chain. 2'(3')-O-(2,4,6-Trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was bound to the labeled protein at the ATPase active site. The efficiency of energy transfer between the probes was 0.09 when measured by both steady-state and time-resolved fluorescence. Anisotropy measurements of the bound AEDANS indicated considerable freedom of motion of the probe. The probable distance between the probes was 57 A. This distance was unchanged during triphosphatase activity. Two further sites of TNP-ADP interaction with subfragment 1 were found. The effect of these interactions on the energy-transfer measurements was reduced to a minimum by careful choice of reaction conditions.

Adenosine Triphosphatases↗

PubMed1983

Epstein-Barr virus specific T-cell response in nasopharyngeal carcinoma patients.

There is a substantial body of evidence suggesting an association between Epstein-Barr virus (EBV) and undifferentiated nasopharyngeal carcinoma (NPC). The present study has compared a group of NPC patients (newly diagnosed and long-term survivors) and controls for EBV-specific T-cell immunity using the regression of transformation assay. Newly diagnosed patients (17 tested) when compared with either long-term survivors (20 tested) or controls (30 tested) showed a significant impairment in virus-specific T-cell immunity (p = 0.036, p = 0.043 respectively). Furthermore, donors with IgA antibody to EBV showed a significant depression in virus-specific T-cell immunity compared with donors without IgA antibody (19 IgA-positive, 48 IgA-negative; p = 0.0025). These results may be important in explaining the postulated role of EBV in the aetiology of NPC.

Antibodies, Neoplasm↗

PubMed1983

Cytoskeleton-associated glycoproteins from chicken sympathetic neurons and chicken embryo brain.

A non-ionic detergent-insoluble fraction was obtained from pure cultures of chicken sympathetic neurons and further purified at the 10%-30% interface of a discontinuous density gradient. This fraction contains actin as its major component and approximately 20 further polypeptides some of which are glycosylated. Two conspicuous glycoproteins in this fraction, of molecular masses 130 kDa and 90 kDa, have been shown to bind to concanavalin A; in cultured neurons the 130-kDa glycoprotein may also be labelled with [3H]glucosamine and [3H]fucose. Both are restricted to one interface of the stepped sucrose gradient when cells are lysed in low ionic strength buffer and eluted with actin in the void volume of a Sepharose 6B column. Glycoproteins of the same molecular weight have been obtained by the same isolation procedure from 10-day-old chicken embryo brains. One-dimensional peptide maps show that the carbohydrate-containing peptides from brain and sympathetic neurons are closely similar if not identical. The glycoproteins are also present in sciatic nerve but cannot be detected in a detergent-insoluble form in rounded neurons - lacking axons - or fibroblasts. They might, therefore, be involved in the linkage of the axonal cytoskeleton to the plasma membrane.

Animals↗

PubMed1983

A comparison of Epstein-Barr virus-specific T-cell immunity in malaria-endemic and -nonendemic regions of Papua New Guinea.

Epstein-Barr virus genome-positive Burkitt's lymphoma is endemic in Africa and Papua New Guinea and in both countries the tumour is restricted to regions with holoendemic malaria. The present work has compared groups of healthy indigenous individuals living in malarious and non-malarious regions of Papua New Guinea for Epstein-Barr virus-specific T-cell-mediated immunity using the in vitro regression assay. Residents of the malarious region (55 tested), when compared with either residents of the non-malarious area (35 tested) or Caucasian controls (27 tested) showed a significant (p less than 0.0001) impairment of virus-specific T-cell immunity but no obvious disturbance (p greater than 0.05) of anti-viral antibody titres. These results may be important in explaining the postulated role of malarial infection as a co-factor in the pathogenesis of Burkitt's lymphoma.

Adolescent↗

PubMed1983

Actin polymerization and synthesis in cultured neurones.

The protein content of sympathetic neurones explanted from 10-11-day old chick embryos into culture medium containing nerve growth factor (NGF) increases steadily from about 100 to about 400 pg/cell in 7 days. Actin remains at close to 5% of the total protein during this period, but the proportion of unpolymerized actin falls. As measured by the inhibition of DNase I activity, rounded neurones without neurites contain 70 +/- 7% of their total actin in monomeric form, whereas cells in mature, neurite-bearing cultures contain 39 +/- 7%. When allowance is made for the increase in size of the neuronal cell bodies, the actin present in the neurites ('axons') alone is found to be almost entirely in filamentous form. Cultures exposed to radioactive leucine rapidly incorporate radioactivity into both sedimentable and non-sedimentable forms of actin. Actin-specific activities in the two fractions--estimated after isolation of the actin on small DNase I--Sepharose affinity columns--are similar after labelling for less than 1 h. Direct incorporation of newly-synthesized actin into filaments is suggested from these results. Pulse-chase experiments show that non-sedimentable protein in cultured sympathetic neurones turns over more rapidly than sedimentable protein. However, this is not true for actin, which shows a similar specific activity in sedimentable and non-sedimentable forms--even after 6 days of cold chase. This anomalous behaviour is simply explained by an exchange of actin molecules between filamentous and non-filamentous forms. Control experiments indicate that exchange does not occur to this degree during preparation of subcellular fractions. It is consequently attributed to exchange processes in the living cell.

Actins↗

PubMed1983

A comparison of Epstein-Barr virus-specific T-cell immunity in rheumatoid arthritis and osteoarthritis patients.

The level of Epstein-Barr virus (EBV)-specific T-cell-mediated immunity in 20 rheumatoid arthritis (RA) patients was compared with 16 age- and sex-matched osteoarthritis (OA) patients using the regression of EBV-transformation assay. The results show that the level of EBV-specific T-cell immunity in RA patients is significantly depressed compared with OA patients (P less than .001) or healthy laboratory controls (P less than .001). In contrast, lymphocytes from RA and OA patients showed a similar ability to act as a responder population in the mixed leucocyte reaction. It is unlikely that the difference in EBV-specific immunity is due to a general T-cell defect in RA patients since there was no correlation between EBV-specific T-cell immunity and mixed leucocyte reactivity. There was no correlation between EBV-specific T-cell immunity and any of the indicators of disease activity nor was there any difference in the anti-EBV antibody titre between both groups of patients. These results indicate that RA patients are deficient in the EBV-specific cytotoxic T-cell precursor population and may explain some of the reported observations of the involvement of EBV in this disease.

Aged↗