Establishment, maintenance, and cloning of human dermal fibroblasts.
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Cell Culture Techniques↗
Biomedical subjects
G E Jones
Publications and source records attributed to G E Jones.
Visualization of cell replication using antibody to proliferating cell nuclear antigen.
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Animals↗
Proliferation of murine myoblasts as measured by bromodeoxyuridine incorporation.
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Animals↗
Massage therapy as a workplace intervention for reduction of stress.
This study evaluated the effectiveness of a 15-min. on-site massage while seated in a chair on reducing stress as indicated by blood pressure. 52 employed participants' blood pressures were measured before and after a 15-min. massage at work. Analysis showed a significant reduction in participants' systolic and diastolic blood pressure after receiving the massage although there was no control group.
Adult↗
Diagnosis of ovine enzootic abortion, using a competitive ELISA based on monoclonal antibodies against variable segments 1 and 2 of the major outer membrane protein of Chlamydia psittaci serotype 1.
OBJECTIVE: To develop and evaluate a competitive ELISA (cELISA) specific for detection of antibodies to abortion strains of Chlamydia psittaci and C pecorum that is based on monoclonal antibodies against the 2 segments. PROCEDURA: Monoclonal antibodies were screened for binding to ELISA plates coated with elementary bodies, and were selected on the basis of positive competition with experimentally produced sera against C psittaci and lack of competition with anti-C pecorum sera. The cELISA was evaluated with field sera, and the results were compared with those obtained by complement-fixation testing and by an ELISA containing solubilized outer membrane complexes (A-ELISA). RESULTS: The cELISA detected 9 of 10 C psittaci-infected flocks (57/125 sera, 45.6%), and in 6 of 10 flocks (27.3% of the sera), it specified correctly the infecting chlamydial species. Regarding test sensitivity, the complement-fixation test detected 6 of 10 test-positive (19.2% of the sera) flocks, whereas 7 of 10 test-positive (48.8% of the sera) flocks were detected by use of the A-ELISA. The specificity of the test was satisfactory (100%), compared with the A-ELISA (72.2%). CONCLUSIONS: The new cELISA is a sensitive and specific assay for antibodies against C psittaci abortion strains. It is rapid and easy to perform and does not require serum dilutions. The new cELISA is, therefore, suitable as a routine test for chlamydial diagnosis and seroepidemiologic studies.
Abortion, Veterinary↗
Synthesis, biological activity, and molecular modeling of selective 5-HT(2C/2B) receptor antagonists.
The synthesis and biological activity are reported for a series of analogues of the previously published indole urea 2 (SB-206553), designed to probe the 5-HT(2C) receptor binding site. Small molecule modeling studies have been used to define a region in space which is allowed at the 5-HT(2C) receptor but disallowed at the 5-HT(2A) receptor. In a complementary approach, docking of 2 into our model of the 5-HT(2C) receptor has allowed us to propose a novel primary binding interaction for this series of diaryl ureas, involving a potential double hydrogen-bonding interaction between the urea carbonyl oxygen of the ligand and two serine residues in the receptor. The difference of two valine residues in the 5-HT(2C) receptor for leucine residues in the 5-HT(2A) receptor is believed to account for the observed 5-HT(2C)/5-HT(2A) selectivity with 2.
Animals↗
Identification of a multigene family coding for the 90 kDa proteins of the ovine abortion subtype of Chlamydia psittaci.
While attempting to identify genes and their corresponding antigens that could be used to improve the current methods of diagnosing Chlamydia psittaci infection which causes enzootic abortion in ewes, two candidate clones were isolated from a lambda gt 11 genomic DNA expression library of ovine abortion subtype (strain S26/3) C. psittaci. These clones contained fragments of a gene coding for a group of three chlamydial proteins of approximately 90 kDa which appeared as major immunogens by immunoblotting experiments, indicating their potential as diagnostic or possibly protective antigens. Southern blotting of S26/3 genomic DNA using the two clones as probes identified a family of three or four genes. These represent the first example of protein gene duplication reported in Chlamydia.
Amino Acid Sequence↗
Conversion of dermal fibroblasts to a myogenic lineage is induced by a soluble factor derived from myoblasts.
The limb and axial skeletal muscles of mammals originate from somitic dermomyotome, which during early development separates to form two discrete structures, the dermatome and the myotome. The latter cell mass gives rise to the muscle-forming lineage while cells of the dermatome will form the skin dermal fibroblast population of the dorsal regions of the body. It has been generally accepted for some time that myotome-derived myoblasts were the sole source of muscle fibre nuclei, but evidence has recently been presented from several laboratories that fibroblasts can fuse with myoblasts to contribute active nuclei to the resulting myotubes. We report here an investigation into the myogenic capacity of fibroblasts. Confluent monocultures of mouse dermal fibroblasts, muscle fibroblasts, and C2C12 myoblasts each retain their individual phenotype when maintained for periods up to 7 days in culture. We also grew isolated colonies of fibroblasts and myoblasts in an arrangement which allowed free exchange of tissue culture medium between the 2 cell types. We found evidence of the conversion of dermal fibroblasts to a myogenic lineage as measured by the appearance of MyoD-positive cells expressing the muscle-specific intermediate filament desmin. In addition, dermal fibroblast cultures contained multinucleate syncytia positive for MyoD and containing sarcomeric myosin heavy chain. In contrast, muscle-derived fibroblasts showed no evidence of myogenic conversion when maintained in identical culture conditions. We prepared conditioned medium from confluent cultures of C2C12 myoblasts and added this material to confluent monocultures of either dermal or muscle fibroblasts. While muscle fibroblasts showed no phenotypic alterations, cultures of dermal fibroblasts responded to myoblast conditioned medium by converting to a myogenic lineage as judged by expression of MyoD and desmin. We conclude that a proportion of dermal fibroblasts retain a myogenic capacity into stages well beyond their early association with myoblasts in the dermomyotome.
Animals↗
Calreticulin binding affinity for glycosylated laminin.
Several lines of evidence indicate that calreticulin has lectin-like properties. As a molecular chaperone, calreticulin binds preferentially to nascent glycoproteins via their immature carbohydrates; this property closely resembles that seen for calnexin, a chaperone with extensive molecular identity to calreticulin. A cell surface form of calreticulin also exhibits lectin-like properties, binding specific oligomannosides including those covalently linked to laminin. In the present study we examined the interaction between calreticulin and laminin by means of surface plasmon resonance. The results show that calreticulin specifically binds to glycosylated laminin but fails to specifically bind tunicamycin-derived unglycosylated laminin or bovine serum albumin. Calreticulin binding to glycosylated laminin requires calcium and is abolished in the presence of EDTA. Scatchard analysis of binding yields an apparent association constant, Ka, of 2.1 +/- 0.9 x 10(6) m-1 while kinetic analysis yields an estimate of the association on rate, (Kassoc), as 2 x 10(5) m-1 s-1. The composite results support calreticulin's lectin-like properties as well as its proposed role in laminin recognition, both in the cell interior and on the cell surface.
Animals↗
Utrophin-dystroglycan complex in membranes of adherent cultured cells.
In skeletal muscle, dystrophin binds to an oligomeric, transmembrane complex (DAGc; dystrophin-associated glycoprotein complex) which interacts with laminin in the extracellular matrix. We now present biochemical evidence for an association between utrophin (dystrophin-related protein, DRP) and a major DAGc component, beta-dystroglycan (43DAG) in cultured cell lines which contain little if any dystrophin. We have shown also that utrophin and beta-dystroglycan co-localise at or near the plasma membrane and that they co-sediment in large complexes on sucrose density gradients. On the lower plasma membrane, in contact with the substratum, part of the utrophin and beta-dystroglycan staining co-localised with alpha-actinin in a punctate distribution outside classical vinculin-rich focal adhesions. beta-dystroglycan, utrophin, syntrophin (59DAP), and alpha-actinin were found in all adhesion-competent cell lines studied, but levels of the last three proteins were greatly reduced in myeloma cells, which cannot readily attach to substrata. Possible roles for utrophin in cultured cells are considered in the light of recent evidence for involvement of utrophin-glycoprotein complexes in muscle in signal transduction and recruitment of acetylcholine receptors to neuromuscular junctions.
Actinin↗
Pathogenesis of Chlamydia psittaci infection in sheep: detection of the organism in a serial study of the lymph node.
Sixteen sheep were inoculated subcutaneously over the left prefemoral lymph node with an abortifacient strain of Chlamydia psittaci. Groups of four animals were killed after 3, 6, 12 and 18 days. Four of eight sheep which received a control inoculum were killed on day 6 and four on day 18. The left and right prefemoral lymph nodes were removed and weighed and portions taken from each for examination by the polymerase chain reaction (PCR), by culture, and by histopathological and immunohistochemical methods. The left prefemoral lymph nodes enlarged after the injection of C. psittaci, with the group mean weight on day 6 being the greatest and that on day 18 being normal. Examination by "nested" PCR showed samples from these nodes to be positive, except for one animal killed on day 3 and one on day 12. Live organisms, however, were not cultured from any of the samples collected. C. psittaci antigen was detected immunohistochemically in three of four nodes on day 3, in each of four on day 6, and in two of four on both days 12 and 18. Nodes from the contralateral side remained normal, as did those from unchallenged control sheep, and no antigen or DNA was detected in them.
Animals↗
Intestinal Chlamydia in finishing pigs.
Gut and blood samples from 119 finishing pigs derived from 11 farms were collected during routine slaughter at an abattoir. Sections of formalin-fixed, paraffin-embedded tissues were labeled immunohistochemically using genus-specific, mouse monoclonal antibody against chlamydial lipopolysaccharide; goat polyclonal antiserum against the major outer membrane protein of Chlamydia trachomatis; and mouse monoclonal antibody against the ovine abortion subtype of C. psittaci. Gut samples from 33 of 111 (29.7%) individual pigs stained positive with the genus-specific monoclonal antibody, and of these 30 of 32 (93.7%) also reacted with the C. trachomatis-specific antiserum. Labeled inclusions were restricted to mature enterocytes of the large intestine in 33 of 111 cases. Infection of small intestinal enterocytes was noted in only one of 82 ileal samples. The blood samples were tested for antichlamydial antibodies by enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT). With ELISA, 95 of the 115 sera tested (82.6%) yielded positive antichlamydial reactions. With CFT, 34 of the 119 sera tested (28.6%) were unequivocally positive (> or = 1:10, 100% binding), and 10 (7.6%) yielded doubtful positive reactions (1:10, 50-75% binding). Positive ELISA and CFT titers showed poor agreement (kappa = 0.112), whereas the agreement between positive findings by immunohistochemical labeling and CFT was fair (kappa = 0.205).
Animals↗
Direct observation and quantification of macrophage chemoattraction to the growth factor CSF-1.
The cloned mouse macrophage cell line, BAC1.25F, resembles primary macrophages in its dependence on colony stimulating factor-1 (CSF-1) for both viability and proliferation. Re-addition of CSF-1 to cytokine-deprived cells, which are rounded with diffusely organised F-actin, stimulates rapid cell spreading and cell polarisation. Using the Dunn chemotaxis chamber the movement of stimulated macrophages was monitored over a 2 hour period. Cells restimulated with 1.32 nM human recombinant CSF-1 migrated at a mean rate of 7.71 microns per hour, but showed no directional preferences. In a linear concentration gradient of CSF-1, cytokine-deprived cells were again stimulated to migrate and the mean rate of cell motility, at 6.88 microns per hour, was not significantly different from that measured in an isotropic environment of CSF-1. However, there was a strong preference for the cells to orientate so that their long axes aligned with the CSF-1 gradient and they migrated preferentially towards the source of CSF-1. Migrating cells contained abundant F-actin within the leading lamellae as judged by confocal imaging of fluorescent phalloidin, but the actin was not arranged into stress fibre-like structures. These data support the proposition that CSF-1 is both a chemokinetic and chemotactic agent for macrophages. Tumour necrosis factor (TNF-alpha) failed to stimulate cell migration and thus was neither chemokinetic nor a chemotactic agent. However, cells exposed to a dual concentration gradient of both TNF-alpha and CSF-1 did migrate successfully, although the chemotactic response to CSF-1 was abolished.
Actins↗
Identification of genetic markers for initial sensitivity and rapid tolerance to ethanol-induced ataxia using quantitative trait locus analysis in BXD recombinant inbred mice.
Rapid tolerance to rotarod ataxia has previously been demonstrated in mice after sequential ethanol injections. Here we tested DBA/2J and C57BL/6J mice for initial ethanol sensitivity; DBA/2J mice were more sensitive (0.40 +/- 0.17 mg/g brain) than C57BL/6J mice (1.44 +/- 0.12 mg/g). We then monitored the development of tolerance by quantifying blood ethanol concentrations at the recovery from ataxia over five sequential injections; tolerance reached a plateau in about 5 hr. DBA/2J mice became very tolerant (final ethanol threshold 3.47 +/- 0.16 mg/ml, an increase of 3.07 mg/ml, or 8.7-fold above base line); B6 became slightly tolerant (final ethanol threshold 2.62 +/- 12 mg/ml, and increase of 1.18, or 1.8-fold above base line). Therefore, by the end of the treatment regimen, the rank order of sensitivity of the two strains had reversed. We then tested 25 recombinant inbred strains from among strains representing a cross between C57BL/6J and DBA/2J inbred strains, followed by a quantitative trait locus analysis with a database of 1522 markers to identify provisional loci. This procedure identified 19 markers on 11 chromosomes for initial sensitivity, 18 markers on 9 chromosomes for tolerance (delta) and 21 markers on 11 chromosomes for tolerance (fold-increase). Of these, 17 markers were in common, which suggests that initial sensitivity and tolerance share substantial genetic codetermination. Major candidate loci will be confirmed by genotyping B6D2F2 offspring that have been tested for initial sensitivity and tolerance.
Animals↗
Some blood chemical, electrolyte and mineral values from young ostriches.
Haematocrit, plasma osmolality and plasma calcium, inorganic phosphorus and magnesium concentrations were measured in 10 healthy ostrich chicks at 2-weekly intervals between 8 and 26 weeks of age. Plasma sodium, potassium and zinc concentrations were measured in an additional 50 chicks between 2 and 6 months of age. With the exception of calcium, blood constituents did not change significantly with age. Overall, haematocrit was lower and phosphorus and magnesium were slightly higher than previously measured for adults, which is consistent with previous measurements for chicks.
Age Factors↗
N-(1-methyl-5-indolyl)-N'-(3-methyl-5-isothiazolyl)urea: a novel, high-affinity 5-HT2B receptor antagonist.
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Animals↗
Efficacy trials with tissue-culture grown, inactivated vaccines against chlamydial abortion in sheep.
Vaccines containing inactivated, semi-purified antigen of ovine abortifacient Chlamydia psittaci were prepared from tissue-culture grown harvests of two strains. These were evaluated for efficacy against experimentally reproduced enzootic abortion of ewes (EAE), the protection index (PI) of each vaccine being calculated from three variables. Trial 1 tested the adjuvants Marcol 52/Arlacel A, ISCOM matrix, Novasomes and Alhydrogel, each administered with a total antigen dose of 4 micrograms chlamydial protein (cp), against challenge with the two homologous vaccine strains. The first three formulations yielded PIs of 39-42%, while that of Alhydrogel was 23%. Marked granuloma and/or abscess formation occurred in the Marcol 52/Arlacel A group but in none of the others, and only this group seroconverted following vaccination. In Trial 2, ISCOM matrix containing 16 micrograms cp per dose was administered once or twice. Two challenges were used, one involving the two vaccine strains and the other these two strains plus five others, but no significant differences were seen between them. The double dose schedule, which was better than the single dose and also several Novasomes formulations tested concurrently, gave PIs of 64 and 77% against the two challenges (combined value 71.5%). Trial 3 studied duration of immunity over two lambing seasons using three different administration schedules of 16 micrograms cp in ISCOM matrix, namely single dose in year 1, single dose in year 2 (Group 1), double dose then booster (Group 2), and double dose without booster (Group 3). PIs against the homologous bivalent challenge were 79-80% for Groups 1 and 3 and 91% for Group 2.(ABSTRACT TRUNCATED AT 250 WORDS)
Abortion, Veterinary↗
Development and evaluation of an indirect ELISA to detect antibodies to abortion strains of Chlamydia psittaci in sheep sera.
A novel indirect enzyme-linked immunosorbent assay (ELISA) for antibodies against abortion strains of Chlamydia psittaci (C. psittaci) has been developed. The antigen used was chlamydial elementary bodies treated sequentially with N-lauroyl sarcosine and n-octyl-beta-D-glucopyranoside and finally solubilized with N-lauroyl sarcosine and dithiothreitol. Treating the antigen with sodium periodate after coating of the plates increased the specificity for antibodies to abortion strains. The test was evaluated initially with sera from experimentally infected sheep and an uninfected control group. These sheep were monitored for lambing performance and infection status. When used in conjunction with the indirect micro-immunofluorescence test (MIF), the ELISA was able to identify as negative all twenty-five sera from ewes that had no typical placental lesions and identified as positive twenty of twenty-one sera from infected ewes that had either typical placental lesions or had been found positive by isolation of chlamydia in cell culture. The combination of ELISA and MIF was also able to discriminate correctly groups of sera from six flocks with a history of infection from four known uninfected flocks.
Abortion, Veterinary↗