Biomedical subjects
Publications and source records attributed to J M Robinson.
One of the more routine steps associated with cryosectioning of ultra-thin and semi-thin sections for immunocytochemistry is the transfer of sections to EM grids or coverslips. In a study of the granules of human neutrophils, we have found that the composition of the transfer medium was crucial to the success of these experiments.
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Selected regions of the Borrelia burgdorferi flagellin gene (fla) that exhibit high or low homology with related genes from other bacterial species were amplified by the polymerase chain reaction and expressed as fusion proteins in Escherichia coli. Purified fusion proteins were assayed for antibody reactivity in a microtiter plate enzyme-linked immunosorbent assay with sera from Lyme disease patients as well as syphilitic and normal sera. Immunoglobulin G antibody from Lyme disease patient sera reacted predominantly with the central portion of the protein. The region of the flagellin protein encompassing amino acids 64 to 311 detected nearly all of the immunoglobulin G-positive Lyme sera and only reacted with 1 of 26 syphilis patient serum samples. In contrast, 12 of 26 syphilis patient serum samples and 2 of 47 normal serum samples reacted with the amino terminus of the flagellin protein, whereas 4 of 26 syphilis patient serum samples and 7 of 47 normal serum samples reacted with the carboxyl terminus. The central region containing amino acids 64 to 311 may be employed diagnostically to differentiate antibodies to B. burgdorferi from antibodies to Treponema pallidum. In addition, this region also was recognized by immunoglobulin M in the Lyme patient sera, indicating its potential usefulness as a marker for early Lyme disease.
We combined immunocytochemistry and enzyme cytochemistry to localize two different proteins on the same ultrathin cryosection. In this method the immunocytochemical localization is visualized with colloidal gold probes and the enzyme cytochemical detection is achieved with cerium as the capture agent. The immunocytochemistry is conducted first so that any potential adverse effects of the enzyme cytochemical procedure will not alter the antibody binding properties of the cryosections.
Alkaline phosphatase (APase) belongs to a growing family of membrane-associated proteins tethered to the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) anchor. Human neutrophils contain an intracellular pool of APase associated with a novel membrane-bound compartment. Stimulation of neutrophils with the chemotactic peptide formyl-Met-Leu-Phe (fMLP) leads to rapid up-regulation of essentially all of the APase to sites in continuity with the extracellular medium. Pre-treatment of neutrophils with cytochalasin B (cyto B) followed by fMLP likewise leads to expression of the enzyme on the cell surface and a dramatic alteration in cell morphology, but subsequent internalization of the plasmalemma is minimized. Pre-treatment with cyto B and fMLP has been used for isolation and purification of neutrophil APase. Specifically, neutrophils were treated with phosphatidylinositol-specific phospholipase C to release GPI-anchored proteins from the cell surface. APase was purified from supernatants of these preparations by electrophoresis in a non-denaturing gel system and subsequent electroelution. With this approach we rapidly purified neutrophil APase to homogeneity; this protein was then used for immunization. Immunoblotting, ELISA, and immunocytochemical localization were used to characterize the resulting antibodies.
Patients injured during sport or fitness activities can make up a significant portion of a family physician's practice. Asking certain questions while taking the history might help diagnose the injury. Components of the physical examination can also be specific to injured athletes. Specific knowledge about the injury allows more effective management.
Controversy still exists concerning the ultimate fate of transposed or transplanted ossicles in tympanoplasty. Histological studies on autologous and homologous ossicles removed at revision operations, or from animal studies, vary in their conclusions. The viability, of such materials used in the long-term is still uncertain. Whether this has any functional significance is also uncertain. This report contains a histological review of 10 ossicle remnants stored in the 'closed' mastoid cavity for 12 months, following closed cavity mastoidectomy (combined approach tympanoplasty).
Pre-operative Computerized Tomography was performed prior to second look operations in 10 cases to check for cholesteatoma after combined approach tympanoplasty. Three independent opinions were obtained on the CT scans and compared with the operative findings. This small series failed to demonstrate reliable pre-operative radiological detection of cholesteatoma. There was disappointing inter-observer agreement in interpretation of the CT scans.
The treatment of tympanic membrane retraction pockets by simple excision is described in 66 ears in 50 patients. After one operation 65 per cent of the retraction pockets were successfully treated (mean follow up 14.1 months). Persisting perforations occurred in five patients after one excision. Retrotympanic cholesteatoma was found in one patient, after two retraction pocket excision operations. The number of myringotomies with grommet insertion and the presence of bilateral disease does not predict the outcome of the procedure. Simple excision should be considered in the first instance, rather than reinforcement tympanoplasty using temporalis fascia or cartilage graft, in the treatment of tympanic membrane retraction pockets.
Soybean plants (Glycine max [L.] Merr. cv Williams), which were symbiotic with Bradyrhizobium japonicum, and which grew well upon reduced nitrogen supplied solely through N(2) fixation processes, often exhibited excess accumulation of starch and sucrose and diminished soluble protein in their source leaves. Nitrate and ammonia, when supplied to the nodulated roots of N(2)-fixing plants, mediated a reduction of foliar starch accumulation and a corresponding increase in soluble protein in the source leaves. This provided an opportunity to examine the potential metabolic adjustments by which NO(3) (-) and NH(4) (+) (N) sufficiency or deficiency exerted an influence upon soybean leaf starch synthesis. When compared with soybean plants supplied with N, elevated starch accumulation was focused in leaf palisade parenchyma tissue of N(2)-fixing plants. Foliar activities of starch synthesis pathway enzymes including fructose-1,6-bisphosphate phosphatase, phosphohexoisomerase, phosphoglucomutase (PGM), as well as adenosine diphosphate glucose pyrophosphorylase (in some leaves) exhibited highest activities in leaf extracts of N(2)-fixing plants when expressed on a leaf protein basis. This was interpreted to mean that there was an adaptation of these enzyme activities in the leaves of N(2)-fixing plants, and this contributed to an increase in starch accumulation. Another major causal factor associated with increased starch accumulation was the elevation in foliar levels of fructose-6-phosphate, glucose-6-phosphate, and glucose-1-phosphate (G1P), which had risen to chloroplast concentrations considerably in excess of the K(m) values for their respective target enzymes associated with starch synthesis, e.g. elevated G1P with respect to adenosine diphosphate glucose pyrophosphorylase (ADPG-PPiase) binding sites. The cofactor glucose-1,6-bisphosphate (G1,6BP) was found to be obligate for maximal PGM activity in soybean leaf extracts of N(2)-fixing as well as N-supplemented plants, and G1,6BP levels in N(2)-fixing plant leaves was twice that of levels in N-supplied treatments. However the concentration of chloroplastic G1,6BP in illuminated leaves was computed to be saturating with respect to PGM in both N(2)-fixing and N-supplemented plants. This suggested that the higher level of this cofactor in N(2)-fixing plant leaves did not confer any higher PGM activation and was not a factor in higher starch synthesis rates. Relative to plants supplied with NO(3) (-) and NH(4) (+), the source leaf glycerate-3-phosphate (3-PGA) and orthophosphate (Pi) concentrations in leaves of N(2)-fixing plants were two to four times higher. Although Pi is a physiological competitive inhibitor of leaf chloroplast ADPG-PPiase, and hence, starch synthesis, elevated chloroplast 3-PGA levels in N(2)-fixing plant leaves apparently prevented interference of Pi with ADPG-PPiase catalysis and starch synthesis.
A randomised cross-over study of 3 bladder washout treatments--saline, Suby G and Solution R--was conducted on 25 elderly females with long-term catheters in order to examine crystal formation and catheter encrustation. With 2 exceptions, all patients produced crystals; only 14 completed the study. While a significant reduction in struvite crystals was found in the returned acidic washout fluid, there was no significant reduction of crystals in the neutral (saline) washout following any of the 3-week periods of treatment. Uric acid crystals appeared with Suby G and Solution R and these solutions were associated with higher red cell deposits in the urine. There was no significant difference in catheter encrustation between the various washouts.
Cultured resident murine macrophages are incubated in the continuous presence of the fluorescent endocytic marker Lucifer Yellow and a phorbol ester that activates protein kinase C. Under these steady-state labeling conditions the fluorescent tracer was, for the most part, in a tubular/reticular compartment. Enzyme cytochemical localization of acid phosphatase in the same cells showed essentially a one-to-one correlation between the Lucifer Yellow- and acid phosphatase-containing compartments. Procedures for epifluorescence observation and subsequent enzyme cytochemical examination of the same whole mount cells are described. In addition, chemical fixation methods for the preservation of this labile tubular/reticular compartment are presented.
Polymorphonuclear leukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocytic activities. We have developed an in vitro model to evaluate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody complexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% +/- 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% +/- 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% +/- 6.27% for PL+F and 47.2% +/- 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% +/- 8.89% for PL+F and 39.6% +/- 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in vitro (i) occupied a 'pre-defined' apical location; (ii) translocated to a 'newly defined' basal location upon stimulation with immobilized antigen-antibody complex; (iii) and depended on intact MTs for placement of MTOCs in both situations.
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We report the reversible reduction potentials of the purines and pyrimidines of DNA and RNA. These were determined in dimethylsulfoxide using cyclic voltammetry. The absolute electron affinities have been determined from these reduction potentials by calibration with the absolute electron affinities for acridine and anthracene measured in the gas phase. These are the first experimentally determined values of the electron affinities of these purines and pyrimidines and are: Guanine = 1.51 eV, Adenine = 0.95 eV, Uracil = 0.80 eV, Thymine = 0.79 eV and Cytosine = 0.56 eV.
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To investigate NADPH-oxidase activation, we studied the effects of free fatty acid (FFA), their uncharged derivatives, and calcium on membrane lipid structure and superoxide anion (O2-) release from intact neutrophils and in cell-free O2(-)-generating systems. This study determined that in calcium-free phosphate-buffered saline (PBS), cis-unsaturated FFA (cis FFA), trans-unsaturated FFA (trans FFA), and to a limited extent saturated FFA decreased the polarization of the membrane lipid structure probe 1,6-diphenyl-1,3,5-hexatriene (DPH), and these decreases correlated with partitioning of the FFA into the plasmalemma and the release of O2- from intact neutrophils and in cell-free preparations. Although a decrease in DPH polarization was always observed under conditions that resulted in the release of O2-, there was not a direct correlation between the amount of decrease in DPH polarization and the release of O2-. Trans FFA did not induce a dose-dependent decrease or as dramatic a decrease in DPH polarization compared with cis FFA, yet the trans FFA stimulated a greater release of O2- at 2.5, 5, and 10 microM concentrations. In addition, responses of the neutrophil to trans FFA, but not cis FFA, were differentially affected by the presence of calcium. When 0.1 mM calcium was added to the PBS decreases in DPH polarization in response to trans FFA were reduced by greater than 60%, whereas O2- was reduced by only 25-36%. The addition of 0.1 mM calcium 3 min after the trans FFA had partitioned into the membrane also reversed by 50-65% decreases in DPH polarization but did not affect the release of O2-. In the presence of 0.9 mM calcium, only the cis FFA decreased DPH polarization or stimulated the release of O2-. Calcium is known to interact more readily with the ionized carboxyl group of trans FFA, neutralizing the anionic charge through an electrostatic interaction. In support of the requirement for the ionized carboxyl group, structurally similar uncharged cis, trans, and saturated fatty alcohols; methyl esters; and aldehydes decreased DPH polarization in the absence and presence of 0.9 mM calcium; however, none of these compounds stimulated the release of O2-. These results indicate that, in addition to the partitioning of FFA into the plasmalemma, which results in calcium-modulatable decreases in DPH polarization, activation or assembly of the NADPH-oxidase requires the ionized carboxyl group.
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