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Biomedical subjects

G D Johnson

Publications and source records attributed to G D Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1994

Expression of a nuclear envelope protein recognized by the monoclonal antibody BU31 in lung tumours: relationship to Ki-67 antigen expression.

The production of the murine monoclonal antibody BU31 is described. This antibody identifies a nuclear envelope protein which is expressed in some but not all cells, and which resembles statin, a protein reported to be expressed by non-proliferating cells. BU31 was applied onto frozen sections of a series of 78 lung tumours and the staining patterns were compared with those obtained with Ki-67. There was an inverse correlation between the proportion of tumour nuclei labelled with the two reagents (r = -0.61, 95 per cent confidence intervals -0.73 to -0.45). However, the four neuroendocrine neoplasms were BU31-negative. Squamous cell carcinomas often showed a peripheral distribution of the cells stained positively with Ki-67, whereas BU31 tended to label centrally situated cells. These observations are consistent with the concept that the antigen recognized by BU31 is expressed by non-proliferating cells in these tumours.

Adult↗
PubMed1994

Immunofluorescent staining of metastatic carcinoma cells in serious fluid with carcinoembryonic antibody, epithelial membrane antibody, AUA-1 and Ber-EP4.

Using an indirect immunofluorescence technique, we assessed the accuracy and clinical usefulness of a panel of monoclonal and polyclonal antibodies. The panel consisted of carcinoembryonic antibody (CEA) and epithelial membrane antibody (EMA), AUA-1, and Ber-EP4 conjugated with fluorescein isothiocyanate. Twenty-six specimens from pleural, peritoneal or pericardial effusions known to contain carcinoma cells (adenocarcinoma or large cell anaplastic carcinoma) and 16 specimens without carcinoma were first examined. The sensitivity and specificity for each of the antibodies were as follows: CEA, 71% and 75%; EMA, 96% and 81%; AUA-1, 80% and 100%; and Ber-EP4, 85% and 100%, respectively. The panel of antibodies was then applied to a group of 14 'problematic' fluids. These had been identified as causing dilemmas in interpretation, either because the cells in the fluids were of equivocal appearance on light microscopy, or the cytological diagnosis was different from that expected in the light of the clinical condition of the patient. Insufficient cellular material was present in one specimen. In five (39%) of the cases the immunochemical staining supported the light microscopic diagnosis. In four (30%) cases, however, the results indicated that the original light microscopic report was incorrect. Two of these were examples of large cell carcinoma of the lung, in which false negative reports had been issued on pleural fluids. The other two were cases of benign ovarian tumours in which a false positive report had been issued. The immunostaining also clarified the final diagnosis in the three patients (23%) on whom 'suspicious' cytological reports had previously been issued. The remaining case, fluid from a patient with a high grade mixed Mullerian tumour of the ovary, was unresolved. We conclude that immunofluorescent staining by AUA-1, EMA and Ber-EP4 is an aid in the cytological interpretation of serous fluids. CEA is much less helpful.

Antibodies, Monoclonal↗
PubMed1994

Classification and localisation of carcinoembryonic antigen (CEA) related antigen expression in normal oesophageal squamous mucosa and squamous carcinoma.

Using a panel of carcinoembryonic antigen (CEA) related antibodies in normal oesophageal squamous mucosa CEA expression is present on suprabasal squames localised to the cell membrane. Immunoblotting shows that this positivity is predominantly due to a glycoprotein of around 180 kDa representing CEA itself. Positivity in squamous carcinomas is confined to cells in foci of squamous differentiation. A shift from membranous localisation to predominant cytoplasmic overexpression is shown between normal and malignant squames using confocal microscopy. The recognition of an adhesive role for CEA and a role in enhancing distant metastases in those tumours expressing CEA highlights the importance of recording CEA expression and changes in subcellular distribution between normal and malignant tissues; CEA expression in oesophageal squamous mucosa has not been well recognised previously and changes in expression may prove of great significance in the spread and dissemination of squamous carcinoma.

Carcinoembryonic Antigen↗
PubMed1994

Distribution of pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PDC-E2) and another mitochondrial marker in salivary gland and biliary epithelium from patients with primary biliary cirrhosis.

Previous studies in which quantitative immunofluorescence was used have shown that certain biliary epithelial cells in liver with primary biliary cirrhosis show increased levels of pyruvate dehydrogenase dihydrolipoamide acetyltransferase compared with controls. This study was designed to determine whether the increase in intensity of pyruvate dehydrogenase dihydrolipoamide acetyltransferase in biliary epithelial cells is accounted for by an increase in the number of mitochondria in the same cells. A double-antibody staining technique was used with antibodies specific for pyruvate dehydrogenase dihydrolipoamide acetyltransferase and another mitochondrial inner membrane marker, recognized by the mouse monoclonal antibody MCA151A. Distribution of the antigens was studied in sections of liver and salivary gland, an additional site that is frequently involved in primary biliary cirrhosis. Confocal microscopy was used to quantify the intensity of fluorescence resulting from binding of fluorochrome-labeled antibody. In both liver and salivary glands MCA151A binding was similar in normal and sections with primary biliary cirrhosis and corresponded to the predicted distribution of mitochondria in these tissues. In the liver staining was less intense in biliary epithelial cells than in hepatocytes. In salivary gland binding of both antibodies was predominantly localized to duct cells, with those forming striated ducts, known to be rich in mitochondria, being most intensely stained. There was high coincidence of the two antigens in salivary glands (p < 0.01) and in biliary epithelial cells from normal liver (p = 0.01). However, in liver with primary biliary cirrhosis, despite high coincidence between the antigens on hepatocytes, biliary epithelial cells showed high intensity of pyruvate dehydrogenase dihydrolipoamide acetyltransferase but not MCA151A.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1994

Surgical decompression of Chiari I malformation for isolated progressive sensorineural hearing loss.

Progressive sensorineural hearing loss has been associated with Chiari type I malformation. Retrocochlear features on auditory brainstem response testing have been reported; however, debate exists over the exact location of the defect. Surgical decompression may be beneficial if brainstem or vascular compression have played a significant role in the pathophysiology of the hearing loss. Lack of specific audiologic data before and after surgical decompression has kept surgical decision making theoretic. This report presents the case of a 10-year-old boy with a progressive, asymmetric sensorineural hearing loss without other neurologic abnormalities. He underwent a posterior fossa decompression that resulted in stable hearing over the 2 years he was followed postoperatively. Preoperative and postoperative audiologic and evoked response features are described, and the rationale for operating with symptoms limited to hearing loss is discussed.

Arnold-Chiari Malformation↗
PubMed1993

Quantitative analysis of molecules which distinguish functional compartments within germinal centers.

Five zones in the secondary follicles of human tonsils are described, which are distinguished by the phenotype of their constituent cells. Moving from the apex to the base of the follicle the zones are termed: follicular mantle, outer zone, apical light zone, basal light zone and dark zone. The dark zone contains proliferating, CD77high, centroblasts and thin, widely spaced processes of follicular dendritic cells (FDC). The apical and basal light zones on the other hand contain a dense network of FDC which express CD21 and CD54 strongly. The FDC of the apical light zone differ from those of the basal light zone by their high expression of CD23. Centroblasts of the dark zone give rise to non-proliferating centrocytes which move apically through the light zone (Eur. J. Immunol. 1991. 21:2951). The centrocytes of the basal light zone are more pyroninophilic, more closely-packed and larger than those in the apical light zone. Consequently, by conventional histology the basal light zone appears to be part of the dark zone. The nomenclature adopted, however, adheres to the convention that the dark zone is filled with proliferating centroblasts. Cells undergoing apoptosis were identified both in the dark and light zones, but more than half of these cells were located in the basal light zone. This is consistent with the concept that the progeny of cells which undergo somatic mutation in their immunoglobulin variable region genes in the dark zone migrate to the light zone where they are selected on the basis of their capacity to bind to antigen held on FDC. Cells receiving an antigen-dependent signal survive while those that do not kill themselves by apoptosis. The outer zone does not contain CD23high cells and in this way is distinguished from the adjacent follicular mantle and apical light zone. It contains small lymphoid cells, blasts and plasmacytoid cells. Many cells of the outer zone express CDw75 strongly. The outer zone also extends as a narrow band around the dark zone. Possible roles of FDC and T cells of the light zone and outer zone in inducing centrocytes to differentiate to memory cells or plasmablasts are discussed.

Antigens, CD↗
PubMed1993

A study of protein kinase C isozyme distribution in relation to Bcl-2 expression during apoptosis of epithelial cells in vivo.

The steady-state population of stratified squamous epithelium is maintained by balanced cell proliferation and apoptosis. Protein kinase C (PKC) is intimately involved in the regulation of cell proliferation and cell survival. In order to gain insight into the mechanisms regulating apoptosis, the immunocytochemical localization of six PKC isozymes (PKC-alpha, -beta, -delta, -epsilon, -gamma, and -zeta) were studied in the surface epithelium of the human tonsil by immunofluorescence staining and confocal laser scanning microscopy. All cells expressed cytoplasmic PKC-alpha, -beta, -delta, -epsilon, and -zeta; PKC-delta and -epsilon were most abundant in viable epithelial cells while PKC-alpha and -beta expression was most intense in cells undergoing apoptosis. PKC-beta and -delta were also present in the nucleus of viable epithelial cells coexpressing cytoplasmic Bcl-2, an oncogene product which protects from apoptosis. Nuclear expression of these isozymes did not correlate with epithelial cell mitosis, as defined by proliferating cell nuclear antigen. Thus, differential subcellular localization of PKC isoforms is associated with the regulation of epithelial cell apoptosis in situ.

Apoptosis↗
PubMed1993

Cutaneous and mucosal human papillomavirus E4 proteins form intermediate filament-like structures in epithelial cells.

Human papillomavirus (HPV) type 1 (HPV 1) is associated with benign cutaneous warts and HPV type 16 (HPV 16) with mucosal epithelial lesions that can progress to invasive carcinoma. The primary structure of the HPV E4 proteins is not highly conserved between types and their role in the viral life cycle is still unknown. A large panel of Simian virus 40 (SV40)-transformed human and monkey epithelial and fibroblast cell lines were infected with recombinant SV40/HPV1 E4 or SV40/HPV 16 E4 viruses and the expression of the viral proteins was analyzed by indirect immunofluorescence. Both HPV 1 and HPV 16 E4 proteins formed extensive and organized filamentous cytoplasmic networks that co-localized with the cytokeratin intermediate filaments. However, only HPV 16 E4 induced the collapse of the cytokeratin filaments. Furthermore, when both virus type E4 proteins were expressed within the same cell the collapse of the HPV 16 E4 filaments did not induced the collapse of the HPV 1 E4 network. Similar E4 filamentous structures were also observed in the cytoplasm of cells of the parabasal layer of an HPV 1-induced experimental wart. The HPV 16 E4 protein formed cytoplasmic networks in all SV40-transformed cell lines examined, but HPV 1 E4 only formed filamentous networks in human keratinocytes and in a monkey stomach epithelial cell line. In keratinocyte cells HPV 1 E4 species of 16, 17, 32, and 34 kDa were expressed, while in Cos-1 cells (in which no E4 networks are formed) only the 17 and 34 kDa polypeptides were found. The specific behavior of E4 proteins of cutaneous and mucosal HPVs expressed in cultured cells may suggest that these viral proteins have evolved to perform a similar function at different epithelial sites.

Animals↗
PubMed1993

Structure-activity relationships in corpora allata of the cockroach Diploptera punctata: roles of mating and the ovary.

Morphometric studies were made on corpora allata of the cockroach Diploptera punctata from animals in which increasing gland size is not coupled to hormone synthesis (ovariectomized mated females; last-instar larvae) and in which gland size is coupled to hormone synthesis (normal mated and virgin females; penultimate-instar larvae). Cell number, gland volume, and juvenile hormone synthesis were measured. From electron micrographs, nuclear, cytoplasmic, and extracellular volumes; and cell membrane area were calculated; and fine structure described. Low-activity glands of ovariectomized mated females resembled high-activity glands from mated females in high cell number, large overall and cytoplasmic volume, and low nuclear-cytoplasmic ratio; they differed in having organelles typical of low-activity glands, mitochondria with dense matrices and large whorls of smooth endoplasmic reticulum. Inactive last-instar larval glands resembled mated ovariectomized female glands in increased cell number and organelles characteristic of inactive glands; however, their nuclear-cytoplasmic volume ratio was much higher. Penultimate larval glands with high activity per cell resembled active glands of normal mated females. Ovariectomy did not change morphometric parameters of virgin female glands; thus mating results in increase in size of adult female glands whereas the growing ovary is needed for changes in mitochondria and endoplasmic reticulum associated with high juvenile hormone synthesis.

Animals↗
PubMed1993

Diazepam's effect on systemic vascular resistance during cardiopulmonary bypass is not caused by its vehicle (alcohol-propylene glycol).

Control of hemodynamic parameters during cardiopulmonary bypass (CPB) is a desirable goal of the anesthesiologist. Diazepam is known to reduce systemic vascular resistance (SVR) during CPB. This study tested the hypothesis that diazepam reduces SVR during CPB through the action of its vehicle, alcohol-propylene glycol (APG). The study protocol was approved by the Institution's Human Experimentation Review Board and all patients gave informed consent. Premedication, induction, and maintenance of anesthesia were standardized. Thirty consecutive patients undergoing nonemergent cardiac surgical procedures were randomized to receive a standardized amount of diazepam, diazemuls, or alcohol-propylene glycol during stable CPB. SVR in the diazepam group decreased significantly (1,242 to 968 dyne.s.cm-5, P < 0.05) compared to baseline; whereas there was no significant change in SVR in the diazemul group, and a statistically significant increase in SVR (1,217 to 1,537 dyne.s.cm-5, P < 0.05) in the APG group. It is concluded that diazepam reduces SVR during CPB. Diazepam's vehicle does not reduce SVR during CPB, and is in fact associated with an increase in SVR.

Aged↗
PubMed1993

IL-2 expands and maintains IgM plasmablasts from a CD5+ subset contained within the germinal centre cell-enriched (surface IgD-/CD39- buoyant) fraction of human tonsil.

IL-2 was found to promote the rapid growth of a minority population contained within the germinal centre (GC) cell-enriched (CD39- and/or IgD- buoyant) fraction of human tonsillar B lymphocytes. The cells emerging in response to IL-2 had a high mitotic index and morphologically resembled plasmablasts. Cultures could be maintained in the absence of feeder cells for up to 3 weeks in IL-2 and were characterized by large amounts of IgM in their supernatants: approximately 40% of the cells contained readily detectable cytoplasmic IgM by day 10 of culture. Negligible quantities of IgG and IgA were found. The target population for IL-2-driven expansion and IgM secretion was smIg+/CD38+ and was subject to suppression by anti-IgM antibody. While only 8% of cells within the GC cell-enriched fraction were CD5+ (compared with 15% of high density resting B cells), their removal led to an 83% reduction in the amount of IgM produced in response to IL-2, IL2 selectively expanded this minor CD5+ subset such that by day 6 of culture they comprised 57% of all viable cells. Cultures established with IL-2 showed increasing expression of cytoplasmic Bcl-2 and withdrawal of growth factor resulted in cell death via apoptosis. We discuss these results in relation to CD5+ B cells and their potential role in antibody responses to TD antigens.

Adenosine Triphosphatases↗
PubMed1993

Distribution of cAMP in secondary follicles and its expression in B cell apoptosis and CD40-mediated survival.

Apoptosis occurs at a high rate among B cells in germinal centres (GCs). Isolated GC B cells undergo apoptosis spontaneously when cultured in vitro: such self-destruction can be arrested by protein kinase C-activating phorbol esters and by ligating surface CD40. This study sought to explore whether the cAMP-dependent second messenger system played a role in the regulation of GC cell apoptosis and in ligand-promoted survival. First, the distribution of cAMP in GCs was analysed by immunofluorescence staining and confocal laser scanning microscopy in situ: cytoplasmic cAMP was expressed by all cells but was most abundant in the constitutive cells of the light zones, where GC B cell apoptosis and rescue occur. Isolated GC B cells were found to express higher levels of cAMP than quiescent B lymphocytes as assessed both by a competitive binding assay and a newly developed flow cytometric method. Culturing GC B cells alone increased cAMP level, while those cells rescued with the phorbol ester phorbol myristate acetate (PMA) and anti-CD40 exhibited decreased cAMP levels. Resting B lymphocytes showed no change in cAMP level following culture alone, but a significant increase in cAMP levels when co-cultured with PMA and anti-CD40. These data suggest that given identical signals, resting B cells and GC B cells exhibit differential regulation of the cAMP-dependent second messenger system; moreover, this second messenger system appears to be involved in the regulation of apoptosis in GC B cells.

Antigens, CD↗
PubMed1992

Prevention of programmed cell death in Burkitt lymphoma cell lines by bcl-2-dependent and -independent mechanisms.

Burkitt lymphoma (BL) cell lines which retain the phenotypic characteristics of the freshly-isolated tumour cells (group I cells) readily enter programmed cell death (apoptosis) in response to a variety of triggers. By contrast, isogenic BL cells which are phenotypically altered as a result of activation of their resident EBV genome (group-III cells) are highly protected from apoptosis. Phenotypic changes in group-III cells include the up-regulation of the oncogene, bcl-2. Expression of the 26-kDa bcl-2 protein in group-I BL cells following gene transfer was found to afford protection from apoptosis: the degree of protection was proportional to the amount of bcl-2 protein expressed. When group-I bcl-2 transfectants were compared with their group-III counterparts it was found that, whilst bcl-2 made a significant contribution in protecting from entry into apoptosis, hyper-expression of bcl-2 protein in group-I cells (well beyond that of group-III cells) was necessary to attain the high levels of protection observed in group-III cells. These results suggested that additional, bcl-2-independent, survival mechanisms could operate in BL cells. In support of this notion it was also found that: (1) prolonged culture of group-I lines in vitro resulted in enhanced survival in the absence of bcl-2 up-regulation, and (2) exposure of group-I cells to interferon-alpha triggered a bcl-2-independent protective response. The molecular mechanisms of both the bcl-2-dependent and -independent survival pathways remain to be determined.

Apoptosis↗
PubMed1992

Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors. Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity. In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding. Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A). Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved. The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.

Alanine↗
PubMed1992

Cloning a rat meprin cDNA reveals the enzyme is a heterodimer.

The structure of the kidney microvillar membrane metallopeptidase meprin (EC 3.4.24.18) from rats has been examined. Previously reported to be a homotetramer, we demonstrate that the enzyme is composed of two similar but distinct subunits through tryptic peptide mapping and the sequencing of peptides of the papain solubilized form of the enzyme. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that the native rat meprin tetramer is dissociated by detergent into disulfide-linked heterodimers. A full-length cDNA clone encoding one of the meprin subunits has been isolated and sequenced. The cDNA contains an open reading frame of 668 amino acids, coding for a polypeptide of molecular weight 75,054. The enzyme contains the zinc binding sequence HEFLH and a potential membrane-spanning region near its amino terminus. Comparison of this clone with peptide sequences from mouse meprins A and B shows that the clone is a B type or beta subunit. Northern blot analysis is consistent with the existence of two distinct subunits and further indicates that rat meprin subunits may be differentially expressed in various rat tissues.

Amino Acid Sequence↗
PubMed1992

Membrane dihydrolipoamide acetyltransferase (E2) on human biliary epithelial cells in primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is associated with serum antibodies that react with the dihydrolipoamide acetyltransferase component (E2) of the mitochondrial pyruvate dehydrogenase complex. We have sought the presence of E2 on the surface of human intrahepatic biliary epithelial cells (BEC). Cultured BECs from PBC but not normal liver were found to have E2 on the membrane after three days' culture. Isolated, viable cells examined by laser-scanning confocal microscopy revealed the pattern of E2 staining on the membrane to be similar to that seen with the membrane glycoprotein marker, HEA-125. By contrast, BECs from normal liver showed membrane staining only with HEA-125. When BECs were fixed before incubation with antibody to E2, cytoplasmic staining was observed. Our results suggest that E2 is present on the surface of biliary epithelial cells in PBC, and support the idea of a pathogenetic association between antimitochondrial antibodies and bileduct damage.

Acetyltransferases↗
PubMed1992

Confocal microscopic and other observations on the distal end of the thick limb of the human loop of Henle.

Various antibodies and lectins were used in a histological study of the human renal tubule, particularly of the distal end of the thick limb of the loop of Henle. The thick limb, identified by antibody to Tamm-Horsfall protein, ended abruptly, either at the macula densa or at a variable distance after it. At this point there was an abrupt change in cell size. Confocal microscopy and other techniques showed that this point marked an abrupt beginning of tubular staining by the cytokeratin antibody PKK2 and the lectin UEA 1, with an abrupt end of staining by the lectin DBA. Distal from this point, there were gradual changes in staining of the tubule by various reagents including other antibodies to cytokeratins. These structural findings suggest that there is a fundamental change in the tubule at the end of the thick limb. The abrupt end to the thick limb in man resembles that seen in the rat and the rabbit.

Biomarkers↗