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Biomedical subjects

K Moore

Publications and source records attributed to K Moore.

At least 217 records · Page 12Linked to original sources
PubMed1987

A comparison of spleen and lymph node cells as fusion partners for the raising of monoclonal antibodies after different routes of immunisation.

Administration of antigen (human insulin) via the rear footpads of BALB/c mice with subsequent fusion of popliteal and inguinal lymph node lymphocytes induced a higher frequency of hybridomas (100%) secreting specific antibody than either intradermal immunisation and lymph node cell fusion (53%) or conventional subcutaneous immunisation and intraperitoneal boost followed by splenic lymphocyte fusion (8%). The rank order of serum antibody titres was found to correlate with the order of fusion efficiencies. Lymph node cell fusions also produced a greater spectrum of antibody specificities. Such differences in fusion efficiencies were also observed using bovine intestinal alkaline phosphatase.

Alkaline Phosphatase↗
PubMed1987

Histone-H1 inhibits translation by reticulocyte lysates with relative mRNA selectivity.

Histone-H1 purified from rat skeletal muscle is a relatively potent inhibitor of peptide chain initiation in a cell free system, the rabbit reticulocyte lysate (50% inhibition at approximately 0.4 microM). H1 does not inhibit formation of the ternary complex nor its attachment to 40S ribosomes; the data are compatible with H1 binding to mRNA. The inhibition shows mRNA selectivity: translation of beta-globin mRNA is more affected than that of alpha-globin mRNA and hepatic albumin mRNA more than total hepatic mRNA. Whether or not histone-H1 plays a role in translational regulation in intact cells is conjectural, it may serve as a useful model for protein-mRNA interactions.

Animals↗
PubMed1987

Glycerol metabolism in higher plants: glycerol kinase.

Glycerol kinase activity was identified in extracts of higher plant seeds and seedlings, and was partially purified and characterized from cucumber radicle tissue. The enzyme was localized in the post-mitochondrial supernatant of the cell, and catalyzed the formation of glycerol-3-phosphate. The pH optiumum was 9.0. ATP, CTP, GTP or UTP could be used as the phosphoryl group donor. The Km for glycerol was 55 microM and Km values for the nucleoside triphosphates were 145-620 microM. The Vmax for the reaction was 40-78 pmol product per min. Kinetic data indicate that the enzyme has a sequential mechanism.

Glycerol↗
PubMed1987

Reconstitution of rabbit thrombomodulin into phospholipid vesicles.

The influence of phospholipid on thrombin-thrombomodulin-catalyzed activation of protein C has been studied by incorporating thrombomodulin into vesicles by dialysis from octyl glucoside-phospholipid mixtures. Thrombomodulin was incorporated into vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (30% phosphatidylserine and 70% phosphatidylcholine). Thrombomodulin is randomly oriented in vesicles of different phospholipid composition. Incorporation of thrombomodulin into phosphatidylcholine, with or without phosphatidylserine, alters the Ca2+ concentration dependence of protein C activation. Soluble thrombomodulin showed a half-maximal rate of activation at 580 microM Ca2+, whereas half-maximal rates of activation of liposome-reconstituted thrombomodulin were obtained between 500 microM Ca2+ and 2 mM Ca2+, depending on the composition (protein:phospholipid) of the liposomes. The Ca2+ dependence of protein C activation fits a simple hyperbola for the soluble activator, while the Ca2+ dependence of the membrane-associated complex is distinctly sigmoidal with a Hill coefficient greater than 2.4. In contrast, the Ca2+ dependence of gamma-carboxyglutamic acid (Gla) domainless protein C activation is unchanged by membrane reconstitution (1/2 max = 53 +/- 10 microM) and fits a simple rectangular hyperbola. Incorporation of thrombomodulin into pure phosphatidylcholine vesicles reduces the Km for protein C from 7.6 +/- 2 to 0.7 +/- 0.2 microM. Increasing phosphatidylserine to 20% decreased the Km for protein C further to 0.1 +/- 0.02 microM. Membrane incorporation has no influence on the activation of protein C from which the Gla residues are removed proteolytically (Km = 6.4 +/- 0.5 microM). The Km for protein C observed on endothelial cells is more similar to the Km observed when thrombomodulin (TM) is incorporated into pure phosphatidylcholine vesicles than into negatively charged vesicles, suggesting that the protein C-binding site on endothelial cells does not involve negatively charged phospholipids. In support of this concept, we observed that prothrombin and fragment 1, which bind to negatively charged phospholipids, do not inhibit protein C activation on endothelial cells or TM incorporated into phosphatidylcholine vesicles, but do inhibit when TM is incorporated into phosphatidylcholine:phosphatidylserine vesicles. These studies suggest that neutral phospholipids lead to exposure of a site, probably on thrombomodulin, capable of recognizing the Gla domain of protein C.

Animals↗
PubMed1987

Use of the monoclonal antibody WR17, identifying the CD37 gp40-45 Kd antigen complex, in the diagnosis of B-lymphoid malignancy.

The distribution of the gp40-45 Kd antigen bound by the WR17 monoclonal antibody of IgG2 subclass in normal lymphoid tissue was characterized by immunohistochemistry and immunofluorescence staining with flow cytometric analysis. The predominant staining pattern observed was characteristic of an anti-pan-B-lymphocyte reagent. Weak reactions were observed by immunofluorescence staining of viable cell suspensions with all neutrophils and T-lymphocytes in some normal donors. In tissue sections, B-lymphocytes were stained and no cross reactions were observed with T-lymphocytes, although macrophages stained in some sections. A range of T- and B-cell malignancies were stained with WR17 and the reactivity compared to that observed with other monoclonal antibodies in the CD19, CD21 and CD22 clusters. All B-non-Hodgkin's lymphomas, B-chronic lymphocytic, prolymphocytic and hairy cell leukaemia cells examined were stained by WR17 in indirect immunofluorescence assays, whilst the T-cell tumours were negative. The same pattern was observed in cryostat sections of malignant tissue and in addition some tissue macrophages expressed the CD37 antigen cytoplasmically. Intra-tumour heterogeneity of staining was observed with all the monoclonal antibodies tested, although overall WR17 consistently stained B-cell tumours even when expression of the CD19 pan-B-lymphocyte antigen could not be detected with some monoclonals. Monoclonal antibodies, such as WR17, within the CD37 cluster and binding to the gp 40-45 Kd molecule, bind to mature B-lymphocytes and identify the majority of B-cell malignancies.

Antibodies, Monoclonal↗
PubMed1987

Abnormal subcellular distribution of beta-glucuronidase in mice with a genetic alteration in enzyme structure.

Liver beta-glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of beta-glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 X PAC-Gus(n). Liver beta-glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of beta-glucuronidase in Gus(n)/Gus(n) mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. beta-Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal beta-glucuronidase. The loss of liver lysosomal beta-glucuronidase activity was shown by immunotitration to be due to a decrease in the number of beta-glucuronidase molecules in lysosomes of the congenic strain. The Gus(n) structural alteration likely causes the lowered lysosomal beta-glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus(n)/Gus(b) animals had intermediate levels of liver beta-glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus(n)/Gus(n) mice. Gus(n) is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.

Animals↗
PubMed1987

Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Mouse liver beta-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the beta-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, beta-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway.

Animals↗
PubMed1987

An investigation of the action of disease modifying antirheumatic drugs on the rheumatoid synovial membrane: reduction in T lymphocyte subpopulations and HLA-DP and DQ antigen expression after gold or penicillamine therapy.

Synovial needle biopsy specimens from the knee joints of seven patients with rheumatoid arthritis (RA) were examined immunohistochemically before and after six months' treatment with either gold or penicillamine (disease modifying drugs, DMDs). There were significant reductions in the numbers of infiltrating T lymphocytes and a disproportionate fall in the numbers of lymphocytes of the helper/inducer subset when compared with those of the suppressor/cytotoxic subset. This resulted in a fall in the ratio of helper/inducer to suppressor/cytotoxic cells. The immunohistological changes correlated with improvements in erythrocyte sedimentation rate (ESR), serum immunoglobulins, visual analogue pain assessment, grip strength, and Ritchie articular index. A second group of nine patients with RA, already well established on DMD therapy, did not show similar changes after the six month period. The HLA class II antigens DR, DQ, and DP were widely expressed on lymphocytes, macrophages, and synovial lining cells of a group of patients with RA who had never received disease modifying drug therapy. After treatment there was a significant reduction in the expression of HLA-DP and DQ antigens.

Adult↗
PubMed1987

A translational inhibitor from muscles of diabetic rats: identification as histone H1.

A heat- and acid-stable protein fraction that inhibited peptide chain initiation in rabbit reticulocyte lysates was extracted from frozen, powdered rat skeletal muscles by stepwise trichloroacetic acid precipitation. Streptozotocin-induced diabetes increased the inhibitory activity; this was prevented by insulin therapy. Size-exclusion high-performance liquid chromatography resolved four inhibitory fractions; only one was consistently increased (approximately 2-fold) in muscle extracts from diabetic rats. Polysome profiles of lysates incubated with this fraction indicated peptide chain initiation inhibition. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified inhibitory fraction migrated with apparent Mr 30 and 32 kDa, which on Western blot immunostained with antisera against histone H1/H1(0). Perchloric acid extraction of muscle homogenates yielded approximately twofold more H1 from diabetic than from control rats; yield from diabetics decreased to control values 5 h after subcutaneous insulin injection. Inclusion of detergent during homogenization increased H1 yield more from muscles of control than from diabetic rats and abolished the difference between them. Because H1 affects several biochemical reactions, its facilitated extraction from insulin-deprived tissues can bias interpretation of studies of insulin action.

Animals↗
PubMed1987

Functional heterogeneity of CD4+ T lymphocytes: two subpopulations with counteracting immunoregulatory functions identified with the monoclonal antibodies WR16 and WR19.

Monoclonal antibodies (MoAbs) WR16 and WR19 bound to 47% and 43% of CD4+ tonsil T lymphocytes, respectively. WR16 immunoprecipitated a 220,000 molecular weight (MW) component that was also expressed on CD8+ lymphocytes and on B lymphocytes, whereas binding of WR19 was restricted to CD4+ lymphocytes. Binding of these two MoAbs on freshly prepared tonsil CD4+ T lymphocytes was mutually exclusive, and they were used to prepare reciprocal CD4+ subpopulations by negative depletion using the panning technique. Recombination of subsets isolated in this way with autologous B lymphocytes activated with pokeweed mitogen demonstrated that WR16+/OKT4+ lymphocytes suppressed B lymphocyte Ig secretion, whereas WR19+/OKT4+ lymphocytes helped B lymphocyte Ig secretion. Removal of WR16+ cells from a CD4+ helper cell population enhanced B lymphocyte Ig secretion rate, whilst the re-addition of WR16+ cells to WR19+ helper cells reduced B lymphocyte Ig secretion rates to that seen with non-fractionated CD4+ helper cells. The overall level of helper activity induced by a given CD4+ population was thus a consequence of an interaction between WR19+ helper cells and WR16+ suppressor cells. The WR16+ subset of CD4+ cells exhibited a greater proliferation rate than WR19+/CD4+ cells in response to mitogen. Proliferation of negatively selected WR16+/CD4+ lymphocytes was inhibited by pretreatment with WR16, which also induced a concomitant increase in the level of suppressor activity of these cells. Expression of the antigens identified by these two MoAbs was not constant as phytohaemagglutinin activation of T lymphocytes induced a loss of WR16 binding with a simultaneous increase in the level of WR19 bound/cell and in the proportion of WR19+ cells. T lymphocyte clones selected from a normal population were composed of a disproportionately high number positive for WR19 and negative for WR16. These data indicated that the absence of the WR19 antigen on WR16+ cells may be transient as the antigen was acquired by the majority of CD4+ T lymphocytes after activation. These two MoAbs may therefore be used to predict the functional status of a heterogeneous population of CD4+ lymphocytes and may also prove to be of use in the study of CD4+ T lymphocyte activation.

Antibodies, Monoclonal↗
PubMed1986

Iron deficiency and pseudo-iron deficiency in hospitalized horses.

Values for serum ferritin, serum iron, and serum total-iron binding capacity were determined in 100 hospitalized horses. Six foals had iron deficiency, as indicated by low serum ferritin and iron concentrations. Twenty-eight horses had low serum iron content and normal or high serum ferritin content and were considered to have pseudo-iron deficiency, possibly in response to infection. Ten horses had serum ferritin concentrations that were quite high, probably indicating iron overload.

Age Factors↗
PubMed1986

Identification and isolation of OKT4+ suppressor cells with the monoclonal antibody WR16.

The monoclonal antibody WR16 was secreted by a hybridoma produced by fusing splenocytes from a BALB/c mouse immunized with human T-cell chronic lymphocytic leukaemia cells and the murine myeloma cell line NS-O. WR16 reacts specifically with human lymphocytes and binds to 48% of OKT4+ T lymphocytes and the majority of B lymphocytes. Human OKT4+ tonsil lymphocytes were subfractionated into WR16-/OKT4+ and WR16+/OKT4+ subpopulations by the panning technique. The capacity of these cells to help or suppress pokeweed mitogen-induced immunoglobulin (Ig) secretion by autologous B lymphocytes was monitored after 9 days of coculture. Cells of phenotype WR16-/OKT4+ enhanced Ig secretion in excess of that found with non-fractionated OKT4+ lymphocytes, whilst WR16+/OKT4+ lymphocytes suppressed Ig secretion when added to a mixture of B lymphocytes and non-fractionated OKT4+ cells. The WR16-/OKT4+ subpopulation was further fractionated by use of the MoAb Leu 8. Forty-two percent of WR16-/OKT4+ lymphocytes bound Leu 8, and cells of the phenotype WR16-/OKT4+/Leu 8+ were found to induce B-lymphocyte Ig secretion, whilst WR16-/OKT4+/Leu 8- lymphocytes were less active in this system. These data confirm the heterogeneity of the human T helper/inducer subset and indicate the existence of a population of OKT4+ lymphocytes that can suppress Ig secretion in the absence of OKT8+ lymphocytes.

Antibodies, Monoclonal↗
PubMed1985

Clinicopathologic correlate of a fresh eyelid pigment implantation.

An eyelid with freshly applied black eyeliner pigment was examined histologically. X-ray microanalysis of the pigment suspension from the manufacturer's vial indicated that its composition was 98% iron and 2% titanium. Transmission electron microscopic examination disclosed that particles were in the extracellular matrix; intracellular particles were not seen. By light microscopy, implant material was detected in various levels of the dermis and was found in dermal lymphatics as well as within and surrounding a hair follicle. Our study suggests that systemic exposure to the implant material is possible and offers explanations for permanent eyelash loss, which we have seen following this procedure.

Aged↗
PubMed1985

Properties of rat and mouse beta-glucuronidase mRNA and cDNA, including evidence for sequence polymorphism and genetic regulation of mRNA levels.

cDNA clones containing partial sequences for beta-glucuronidase (beta G) were constructed from rat preputial gland RNA and identified by their ability to selectively hybridize beta G mRNA. One such rat clone was used to isolate several cross-hybridizing clones from a mouse-cDNA library prepared from kidney RNA from androgen-treated animals. Together, the set of mouse clones spans about 2.0 kb of the 2.6-kb beta G mRNA. Using these cDNA clones as probes, a genomic polymorphism for DNA restriction fragment size was found that proved to be genetically linked to the beta G gene complex. A fragment of beta G cDNA was subcloned into a vector carrying an SP6 polymerase promoter to provide a template for the in vitro synthesis of single-stranded RNA complementary to beta G mRNA. This provided an extremely sensitive probe for the assay of beta G mRNA sequences. Using either nick-translated cDNA or transcribed RNA as a hybridization probe, we found that mouse beta G RNA levels are strongly induced by testosterone, and that induction by testosterone is pituitary-dependent. During the lag period preceding induction, during the induction period itself, and during deinduction following removal of testosterone, beta G mRNA levels paralleled rates of beta G synthesis previously measured by in vivo pulse-labelling experiments. Genetic variation in the extent of induction affected either the level of beta G mRNA or its efficiency of translation depending on the strain of mice tested.

Amino Acid Sequence↗