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R J Harris

Publications and source records attributed to R J Harris.

At least 91 records · Page 5Linked to original sources
PubMed1993

Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody.

A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.

Amino Acid Sequence↗
PubMed1993

Experience with newer techniques for the laboratory detection of Mycoplasma pneumoniae infection: Adelaide, 1978-1992.

Efforts to improve laboratory diagnostic methods for infection due to Mycoplasma pneumoniae have involved the use of a cell-sheet culture method and a modified indirect hemagglutination method for IgM antibody, while direct detection of mycoplasma has employed antigen capture-enzyme immunoassay (Ag-EIA) and polymerase chain reaction (PCR) amplification of sequences within the P1 and 16S ribosomal RNA genes and quantification of the amplified DNA by dot blot hybridization (DBH). Cell-sheet culture was slightly more sensitive and more rapid than culture with cell-free diphasic medium. Indirect hemagglutination detection of IgM antibody to M. pneumoniae was more sensitive than CF and EIA for detection of IgM antibody to mycoplasma. Ag-EIA gave a rapid and reasonably sensitive indication of infection and correlated well with a serological response of patients indicating a current infection. PCR-DBH was a highly sensitive substitute for culture of mycoplasma. Both Ag-EIA and PCR-DBH require confirmation by assessment of serological response to verify that the infection is current and that positive results of PCR-DBH, in particular, are not the result of continuing carriage of the organism from a previous infection, unrelated to the current episode under investigation.

Antigens, Bacterial↗
PubMed1993

O-linked fucose and other post-translational modifications unique to EGF modules.

Three types of unusual post-translational modification have been found within conserved amino acid sequences in epidermal growth factor homology regions (EGF modules) of some multidomain proteins. beta-Hydroxyaspartate and beta-hydroxyasparagine are found within -Cys-Xxx-Asp/Asn-Xxx-Xxx-Xxx-Xxx-Tyr/Phe-Xxx-Cys-Xxx-Cys- sequences. (Xyl alpha 1-->3)Xyl alpha 1-->3Glc beta 1-->O-Ser glycans at conserved sites within -Cys-Xxx-Ser-Xxx-Pro-Cys- sequences have been reported in several proteins. Fuc alpha 1-->O-Thr/Ser modifications have been found at conserved sites within -Cys-Xxx-Xxx-Gly-Gly-Thr/Ser-Cys- sequences. More recently, it has been discovered that the Ser residue corresponding to the potential O-fucosylation site in human factor IX carries the novel tetrasaccharide NeuAc alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Fuc alpha 1-->O-Ser; this tetrasaccharide can be considered to be an extension of the Fuc alpha 1-->O moiety. The consensus sequences for these post-translational modifications are in close proximity to each other; e.g. human factor IX has all three unusual modifications within a 12 amino acid linear sequence. In proteins with multiple EGF modules, the O-glycosidic modifications have been found only within the N-terminal EGF module; beta-hydroxyaspartate/asparagine residues are not restricted in the same fashion. Little is known yet about the functions of, or possible relationships between, any of these modifications.

Amino Acid Sequence↗
PubMed1993

IL-1 beta inhibits adipocyte formation in human long-term bone marrow culture.

Addition of recombinant interleukin-1 beta (IL-1 beta) to human long-term bone marrow culture (LTBMC) was found to have a profound effect on adipocyte formation. Control cultures developed 133 +/- 58 adipocytes/flask compared with 3 +/- 2 adipocytes/flask in IL-1 beta-treated flasks (mean +/- SEM, n = 6 for both groups). In terms of cell number, morphology and colony forming cell, IL-1 beta did not have significant effects on LTBMC, although IL-1 beta slightly enhanced colony-stimulating activity of LTBMC-conditioned medium. To further analyze the effect of IL-1 beta on adipocytes, stromal cells were passaged to reduce hemopoietic cell content, and marrow fibroblasts were converted to adipocytes following addition of hydrocortisone. In both types of assay, IL-1 beta again reduced adipocyte formation: control 1095 +/- 76 adipocytes/flask, IL-1 beta-treated 7 +/- 2 adipocytes/flask (n = 3 for both groups). When preformed adipocytes were treated with IL-1 beta for 1 week, the number of lipid-laden cells decreased to 41 +/- 13% of its value before treatment compared to 87 +/- 9% for untreated controls. The addition of neutralizing concentrations of anti-TNF did not inhibit these effects of IL-1 beta. These results suggest that 1) adipocyte formation is not essential for healthy LTBMC growth, or at least IL-1 beta is an effective substitute for adipocytes; 2) IL-1 beta by itself does not significantly enhance hemopoiesis in LTBMC, and this has implications for clinical trials with this cytokine; 3) IL-1 beta produces major changes in stromal cell development, and 4) overgrowth of fat in marrow may reflect defective cytokine regulation in vivo.

Adipose Tissue↗
PubMed1992

O-linked fucose is present in the first epidermal growth factor domain of factor XII but not protein C.

Epidermal growth factor (EGF) domains are found in many proteins, particularly those of the coagulation/fibrinolytic system. We and others have demonstrated that tissue plasminogen activator (t-PA) and prourokinase are modified by the attachment of fucose to equivalent threonine residues within their EGF domains. Factor XII and protein C each contain two EGF domains; in both proteins, the EGF domain nearest the N terminus has a threonine residue in a position homologous to that which is fucosylated in t-PA. In protein C, this site is 3 residues from the position of another post-translational modification, beta-hydroxylation of Asp-71. We isolated peptides containing these sites to determine, primarily by mass spectrometric analysis, the presence of O-linked fucose and/or beta-hydroxyaspartate. We found that factor XII is fully fucosylated at Thr-90. Protein C is unmodified at the equivalent site (Thr-68) and is completely beta-hydroxylated at Asp-71. It has been recently reported that the first EGF domain of human factor VII has O-linked fucose at the equivalent position (Ser-60) (Bjoern, S., Foster, D. C., Thim, L., Wiberg, F. C., Christensen, M., Komiyama, Y., Pedersen, A. H., and Kisiel, W. (1991) J. Biol. Chem. 266, 11051-11057), while it is unmodified at Asp-63 despite having the consensus sequence for beta-hydroxylation at the latter site. These observations raise the possibility that O-linked fucosylation and beta-hydroxylation of EGF domains are mutually exclusive post-translational modifications.

Amino Acid Sequence↗
PubMed1992

Laboratory diagnosis of Mycoplasma pneumoniae infection. 4. Antigen capture and PCR-gene amplification for detection of the Mycoplasma: problems of clinical correlation.

Direct detection assays for Mycoplasma pneumoniae were established by PCR amplification of short sequences within the foot protein/adhesin (P1) gene and the 16S ribosomal RNA gene. Specificity and sensitivity was excellent, no hybridization was observed with M. genitalium and other human Mycoplasma species. In nose and throat washings from subjects with respiratory infection a pattern of high counts (c.f.u./ml) of M. pneumoniae (deduced from the amount of amplified PCR product), and a positive antigen capture assay, was found in 83% of subjects with serological evidence of current infection with M. pneumoniae. A small proportion of subjects with serological patterns suggesting infection in the more distant past had positive PCR assays. This was considered to represent either persistence of the organism from a previous infection or perhaps transient carriage during a reinfection, without substantial change in antibody response. PCR-based assay of M. pneumoniae offers a powerful, rapid, and sensitive substitute for culture of the mycoplasma. Antigen capture, while less sensitive than PCR, offers the advantage that it is more often positive with samples from current infection and requires less stringent laboratory organization to contain false positive results. We conclude however that the laboratory diagnosis of a chosen clinical episode should not rest on the PCR or Ag-EIA assays alone, but must also include antibody assays to confirm whether infection is current or represents persistence from past exposure.

Antigens, Bacterial↗
PubMed1992

Rapid detection of respiratory syncytial virus in nasopharyngeal aspirates by reverse transcription and polymerase chain reaction amplification.

A rapid method for detection of respiratory syncytial virus (RSV) in nasopharyngeal aspirates, involving a combination of reverse transcription and polymerase chain reaction amplification (RT-PCR), has been developed. The RT-PCR assay employs oligonucleotide primers specific for the region of the RSV genome which encodes the F1 subunit of the fusion (F) glycoprotein. Other respiratory viruses do not give a positive reaction. The RT-PCR assay was tested on 202 nasopharyngeal aspirates collected from children with clinical signs of respiratory infection, and the results from RT-PCR were compared with those obtained from virus culture and direct detection by enzyme immunoassay (EIA). RT-PCR results were positive in 118 of 125 samples from which RSV was cultured, as well as in 4 of 7 samples which were culture negative but EIA positive. RT-PCR results were negative in 68 of 70 culture-negative, EIA-negative samples, which included 11 samples from which other respiratory viruses were isolated. The speed, sensitivity (94.6%), and specificity (greater than 97%) of the RT-PCR assay suggest that this technique could be useful for rapid detection of RSV in clinical samples.

Base Sequence↗
PubMed1992

Paediatric home care in Tower Hamlets: a working partnership with parents.

OBJECTIVES: To describe the first two years of a paediatric home care service. DESIGN: Observational cross sectional study, 1989-91. SETTING: One inner London health district. PATIENTS: 611 children referred to the service; 50 children selected from those referred during the first year, whose parents were interviewed and whose general practitioners were invited to complete a questionnaire. MAIN MEASURES: Description and costs of service; views of parents and general practitioners of selected sample of children. RESULTS: In its second year the team received 303 referrals and made 4004 visits at a salary cost of 98000 pounds, an average of 323 pounds/referral and 24 pounds/visit. This represented a referral rate of 3.2% (258/7939) of inpatient episodes from the main referring hospital between 1 December 1989 and 30 November 1990. Of all referrals to the service, 343(56%) came from hospital inpatient wards. The service was used by disadvantaged and ethnic minority families. The children's parents (in 28(61%) families) and the home care team did a wide range of nursing tasks in the home. Parents of 47(94%) children sampled agreed to be interviewed, and those of 43(91%) found the service useful; guidance and support were most commonly appreciated (33, 70%). Parents of 25(53%) children said that hospital stay or attendance had been reduced or avoided. Parents and general practitioners disagreed on clinical responsibility in 10 children, and communication was a problem for some general practitioners. CONCLUSIONS: The service enabled children to receive advanced nursing care at home. Clinical responsibility should be agreed between parents and professionals at referral.

Attitude of Health Personnel↗
PubMed1992

The connective tissue and partial thickness double pedicle graft: a predictable method of obtaining root coverage.

Obtaining predictable root coverage has been a goal of periodontal therapy for sometime. The purpose of this study is to present a technique for obtaining root coverage. This study reports the results of 20 patients (30 defects) treated with a connective tissue and partial thickness double pedicle graft. Root coverage of 100% was obtained in 24 of 30 defects, or 80% of the time. In all the treated defects the root coverage obtained was to within 0.5 mm of the cemento-enamel junction. The mean percent root coverage was 97.4%. The mean amount of exposed root surface initially was 3.6 mm. At the final postoperative appointment the mean exposed root surface was 0.1 mm. This change represents a net root coverage of 3.5 mm or 97.2%. The number of sites with bleeding on probing and plaque present decreased. The esthetics, both color match and tissue contours, were acceptable to all the patients in all cases. With this technique root coverage can be accomplished in a predictable manner.

Adolescent↗
PubMed1991

Tissue plasminogen activator has an O-linked fucose attached to threonine-61 in the epidermal growth factor domain.

An unusual type of glycosylation has been observed for tissue plasminogen activator (t-PA). The monosaccharide fucose is glycosidically linked to threonine-61 in the epidermal growth factor region of t-PA. The presence of O-linked fucose was demonstrated by carbohydrate analysis and mass spectrometry of tryptic and chymotryptic peptides that contain this site. The susceptibility of the fucose residue to alpha-fucosidase indicated that it was in the alpha-anomeric configuration. Fucosylation of threonine-61 was observed in t-PA isolated from the Bowes melanoma cell line and from recombinant expression systems using Chinese hamster ovary or human embryonic kidney cells. Fucosylation of the homologous residue in prourokinase has also been reported recently. Our results indicate that this novel type of glycosylation may be common to the epidermal growth factor domains found in coagulation and fibrinolytic proteins and, therefore, suggest that the modification may have functional significance.

Amino Acid Sequence↗
PubMed1991

Organ donation terminology: are we communicating life or death?

The continued functioning of organ transplantation depends on obtaining the permission of the next of kin. This communication process between medical or transplant professionals and a donor family hinges heavily on the understanding of certain critical terms like brain dead, life support, and transplantation. Communication issues in obtaining organ donation consent were examined, with particular focus on what are literally life-and-death decisions. Using an experimental simulation methodology, data are offered in support of the claim that much miscommunication occurs in such situations. Directions for improving such communication by allaying latent fears and more carefully defining crucial terminology are offered.

Adolescent↗
PubMed1991

Laparoscopic appendectomy for acute appendicitis: indications and current use.

Laparoscopic evaluation was performed in 43 consecutive patients with right lower abdominal pain and preoperative diagnosis of possible appendicitis. Patients with generalized peritonitis and evidence of perforation of the appendix were not considered for laparoscopy. Visualization was sufficient for making a diagnosis in 97.7% of the cases. In 95%, laparoscopic findings were compatible with the pathology report. Thirty-five patients underwent successful laparoscopic appendectomy with neither intraoperative nor postoperative complications. No further surgery was required; slightly elevated temperatures in 6 patients responded to treatment with antibiotics, and there were no wound infections. Laparoscopic appendectomy is minimally invasive and results in less postoperative pain and morbidity and fewer adhesions and other long-term sequelae than conventional laparotomy. It is associated with superior cosmetic results, a shorter hospital stay, and faster return to normal activities. This experience suggests that if there is no evidence that the appendix is perforated or that generalized peritonitis exists and if qualified physicians and adequate facilities are available, patients presenting with right lower quadrant abdominal pain and possible appendicitis are best evaluated and treated with laparoscopic technique.

Acute Disease↗
PubMed1991

Ethnic and gender differences in Southwestern students' sources of information about health.

Students from several rural, Southwestern schools rated family, television, teachers and doctors as the most important of 11 sources of information about various health topics. However, doctors were only the fifth-ranked source of information about sex and reproduction and the seventh-ranked source of information about drugs, with friends being the fourth- and third-ranked sources of information about these two more personal and sensitive topics. American Indians reported even stronger reliance on doctors than did Hispanic and Anglo students, and rated clinic nurses as much more important sources of health information than did the other two ethnic groups. Females showed greater reliance on social sources and less on electronic media than did male students, and high school students relied more on print media and less on television, nurses and family than did younger children. Students felt more knowledgeable about smoking, alcohol and exercise than about diet, the heart-blood system and cancer; however, American Indians felt less informed about smoking and alcohol than did Anglos or Hispanics. These findings (especially when compared to previous results) suggest that effective health education programs must be tailored to particular populations and based on specific knowledge of their attitudes, behavior and environment.

Adolescent↗
PubMed1990

Structural characterization of a recombinant CD4-IgG hybrid molecule.

CD4-IgG is a homodimer of a hybrid polypeptide consisting of the two amino-terminal domains (residues 1-180) of human CD4 fused to the hinge region and the second and third constant-sequence (CH2 and CH3) Fc domains (residues 216-441) of human immunoglobulin G (IgG-1). This antibody-like molecule, termed an immunoadhesin, was produced in an effort to combine the binding specificity of CD4 with several potentially desirable properties of IgG molecules [Capon et al. (1989) Nature 337, 525-531]. The structural characteristics of the molecule have been evaluated to demonstrate that CD4-IgG has the same features as the N-terminal region of soluble CD4, while retaining those expected for the Fc portion of human IgG. Identification of peptides recovered from the tryptic map confirmed 98.8% of the expected structure of CD4-IgG. The detection of glucosamine in peptides containing Asn257 and the retention time shift of this tryptic peptide after deglycosylation confirmed the presence of Asn-linked oligosaccharides at this position. Four pairs of intrachain and two interchain disulfide bonds were also established.

Amino Acid Sequence↗
PubMed1990

Post-translational modifications of recombinant human tissue factor.

Recombinant human tissue factor (rTF) purified from transfected mammalian cells is a glycoprotein that contains N-linked, but not O-linked oligosaccharides. Two of the three potential N-linked sites in the extracellular portion are fully glycosylated, while one site is approximately 90% utilized. These sites have complex-type oligosaccharides attached. The potential N-linked site in the cytoplasmic domain near the C-terminus is not glycosylated. Characterization of the tryptic map of rTF confirmed most of the proposed amino acid sequence. In addition, the disulfide bonds (between Cys-49 and Cys-57 and between Cys-186 and Cys-209) were demonstrated by FAB-MS analysis of cysteine-containing fragments obtained from the tryptic map.

Amino Acid Sequence↗
PubMed1990

Enzymatic cleavage of a CD4 immunoadhesin generates crystallizable, biologically active Fd-like fragments.

CD4, the cell-surface receptor for the human immunodeficiency virus (HIV), is a member of the immunoglobulin (Ig) gene superfamily. It contains four extracellular sequences homologous to Ig VL domains. The first of these (V1) is sufficient for binding to HIV; however, the structural basis for this binding has yet to be elucidated. While several models for the structure of Ig-like domains in CD4 have been proposed on the basis of crystal structures of Ig VL domains, direct evidence that CD4 and VL domains fold similarly has not been obtained. To produce individual domains of CD4 for structural studies, we used molecular fusions of such domains with Ig heavy chain (CD4 immunoadhesins), which are very efficiently expressed and secreted in mammalian cells and can be easily isolated in single-step purification with protein A. Since these fusion molecules are antibody-like homodimeric proteins, we investigated the possibility that they might be cleaved enzymatically to produce Fd-like and Fc fragments. We found that cleavage with papain releases an Fd-like fragment containing the V1 and V2 CD4 domains; this fragment fully retains the ability to bind to the HIV-1 envelope glycoprotein gp120 and to block HIV infection in vitro. Moreover, folding of the CD4 domains in the Fd-like fragment and in the parent immunoadhesin is indistinguishable, as indicated by circular dichroism. Spectral analysis of the Fd-like fragment suggests that secondary structure content is identical with that predicted from the known structure of Ig VL domains; this directly supports the hypothesis that the V1 and V2 domains of CD4 fold similarly to Ig VL domains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗