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Biomedical subjects

S A Moore

Publications and source records attributed to S A Moore.

At least 109 records · Page 6Linked to original sources
PubMed1991

Astrocytes, not neurons, produce docosahexaenoic acid (22:6 omega-3) and arachidonic acid (20:4 omega-6).

Elongated, highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis are not fully characterized. To investigate whether neurons themselves are capable of essential fatty acid elongation and desaturation or are dependent upon the support of other brain cells, primary cultures of rat neurons and astrocytes were incubated with [1-14C] 18:2 omega-6, [1-14C]20:4 omega-6, [1-14C]18:3 omega-3, or [1-14C]20:5 omega-3 and their elongation/desaturation products determined. Neuronal cultures were routinely incapable of producing significant amounts of delta 4-desaturase products. They desaturated fatty acids very poorly at every step of the pathway, producing primarily elongation products of the 18- and 20-carbon precursors. In contrast, astrocytes actively elongated and desaturated the 18- and 20-carbon precursors. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary products from 18:3 omega-3 were 20:5 omega-3, 22:5 omega-3, and 22:6 omega-3. The majority of the long-chain fatty acids formed by astrocyte cultures, particularly 20:4 omega-6 and 22:6 omega-3, was released into the extracellular fluid. Although incapable of producing 20:4 omega-6 and 22:6 omega-3 from precursor fatty acids, neuronal cultures readily took up these fatty acids from the medium. These findings suggest that astrocytes play an important supportive role in the brain by elongating and desaturating omega-6 and omega-3 essential fatty acid precursors to 20:4 omega-6 and 22:6 omega-3, then releasing the long-chain polyunsaturated fatty acids for uptake by neurons.

Animals↗
PubMed1991

Brain microvessel 12-hydroxyeicosatetraenoic acid is the (S) enantiomer and is lipoxygenase derived.

12-Hydroxyeicosatetraenoic acid (12-HETE) production from arachidonic acid by cerebral microvessels isolated from perfused adult murine brain was reduced by the lipoxygenase inhibitors baicalein, esculetin, gossypol, nordihydroguaiaretic acid, and quercetin. Except for quercetin and gossypol, the IC50 did not exceed 10 microM. Each inhibitor, except baicalein, also decreased microvessel prostaglandin production when present in concentrations above their IC50 value for 12-HETE. In contrast, inhibitors of the cytochrome P450 monooxygenase system, clotrimazole, metyrapone, and proadifen (SKF-525A), had little effect on microvessel 12-HETE production. Chiral phase HPLC analysis revealed that only the (S) enantiomer of 12-HETE was formed. The major microvessel metabolite of eicosapentaenoic acid co-eluted with 12-hydroxyeicosapentaenoic acid (12-HEPE) on reverse-phase HPLC and the (S) enantiomer of 12-HEPE on chiral phase HPLC. Furthermore, like 12-HETE, 12-HEPE production was blocked by lipoxygenase inhibitors. These studies demonstrate that brain microvessels produce only the (S) enantiomeric 12-hydroxy derivatives of both arachidonic acid and eicosapentaenoic acid by the action of a lipoxygenase that can be selectively inhibited by baicalein. Since arachidonic acid and eicosapentaenoic acid are available to cerebral blood vessels in certain pathological settings, these 12-hydroxy acid lipoxygenase products may mediate some of the cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1991

Identification and structural characterization of Fc gamma-receptors on pulmonary alveolar macrophages.

Little is known about the structure of the cell surface receptors for the Fc portion of immunoglobulin G (Fc gamma R) on tissue macrophages. Studies on leukocytes indicate the existence of three classes of Fc gamma R, denoted I, II, and III. The purpose of this study was to structurally characterize Fc gamma R on alveolar macrophages obtained by bronchoalveolar lavage, comparing them with Fc gamma R of monocytes, neutrophils, and U937 cells. Flow-cytometric evaluation, utilizing anti-Fc gamma R class-specific monoclonal antibodies, showed that alveolar macrophages expressed three Fc gamma R classes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunopurified Fc gamma R molecules revealed the following apparent molecular mass for each Fc gamma R class: Fc gamma RI, 70 kDa; Fc gamma RII, 44 kDa; and Fc gamma RIII, 57-65 kDa. RNA blot analysis demonstrated a 1.7-kb transcript for Fc gamma RI, 2.5 and 1.6 kb transcripts for Fc gamma RII, and a 2.2 kb mRNA for Fc gamma RIII. Fc gamma RII displayed the high-responder/low-responder polymorphism. Fc gamma RIII did not express the neutrophil antigen-type specific structural polymorphism of the deglycosylated Fc gamma R molecule and appeared to be a transmembrane molecule.

Antibodies, Monoclonal↗
PubMed1991

Oncogene abnormalities in astrocytomas: EGF-R gene alone appears to be more frequently amplified and rearranged compared with other protooncogenes.

Ten astrocytomas were tested for gene amplification or rearrangement utilizing distinct probes to nine different oncogenes by Southern hybridization. The probes spanned the four major protein-coding classes of oncogenes; growth factor proteins (csis); growth factor receptor/tyrosine kinase-related proteins [erbB1 (epidermal growth factor receptor, EGF-R), neu (HER2/neu, erbB2), mos, yes]; nuclear binding proteins (c-myc, c-fos); and guanosine 5'-triphosphate binding proteins (N-ras, H-ras). Three astrocytomas, all glioblastomas, showed amplification of EGF-R-related sequences, and two of these amplifications were rearranged. Both rearrangements appeared similar by two different restriction endonucleases. Our findings suggest that it is primarily the EGF-R protooncogene (erbB1) that is amplified or rearranged in astrocytic neoplasms. No other oncogenes were amplified or rearranged, although EGF-R and neu cross-hybridization produced a "pseudo-rearranged" pattern for neu in EGF-R-amplified cases. The similar EGF-R restriction endonuclease abnormalities seen in two patients warrant further study.

Adolescent↗
PubMed1990

Kinetic characterization of a prestart cell division control step in yeast. Implications for the mechanism of alpha-factor-induced division arrest.

In Saccharomyces cerevisiae yeast, a cycloheximide-sensitive "prestart" step (Ki = 0.12 +/- 0.05 microM) is passaged greater than or equal to 4 to less than or equal to 13 minutes prior to the alpha-factor-sensitive "Start" step in the cell cycle of normal proliferating cells and in the first and second bud emergence cycles of abnormally large cells that had been arrested for cell division with alpha-factor and allowed to recover. This identifies the chronologically last protein synthetic step of the cell cycle that occurs prior to the completion of Start. This step is named the last synthetic prestart or LSP step. Cells require the completion of the LSP step before they can perform Start during recovery from arrest by alpha-factor. Yet alpha-factor is known to prevent cell division by acting at Start. The combined data suggest that alpha-factor prevents the Start step of cell division by inactivating a protein that is 1) required for the performance of Start, and 2) synthesized shortly prior to Start in the last synthetic prestart step.

Cell Division↗
PubMed1990

Cell size specific binding of the fluorescent dye calcofluor to budding yeast.

The yeast Saccharomyces cerevisiae cell surface outside of the bud scars displayed an increasing fluorescence intensity with increasing cell size (volume), where fluorescence was due to irreversible binding of the fluorescent dye calcofluor. The increase in fluorescence intensity appeared to be due to an increase in the density of fluorescence per unit surface area of the cell. Exposure time measurements from a photomicroscope were used to quantitate fluorescence intensity on individual cells. The cell size dependent increase in fluorescence intensity was displayed by unbudded cells from stationary phase populations, and unbudded and parent cells from exponentially growing populations. Abnormally large cells generated during the arrest of cell division with alpha-factor or restrictive temperature for cdc3, 8, 13, 24, and 28 cell division cycle mutants, displayed significantly greater fluorescence intensity compared to the smaller cells generated during the arrest of division for cdc25, 33, and 35 mutant strains. Fluorescence intensity on newly emerging buds was broadly dependent on both the size of the bud, and the size of the parent cells on which the buds were growing.

Benzenesulfonates↗
PubMed1990

Activation of CD4+ lymphocytes by syngeneic brain microvascular smooth muscle cells.

Splenocyte proliferation as measured by [3H]thymidine incorporation was detected when brain microvessel smooth muscle cells (SM) were cocultured with syngeneic spleen cells. This report focuses on the role of different lymphocyte populations in this activation. The central role of CD4+ T cells in the proliferation response has been established by different sets of experiments. The phenotypic characterization of splenic lymphocytes before and after the co-culture showed that the only cell type present in higher number after the co-culture than before is the CD4+ T cell. When CD4+ cells were purified by flow microfluorimetry and co-cultured with SM a strong proliferative response was detected. In contrast, purified CD8+ cells in co-culture with SM cells did not proliferate. The activation of CD4+ cells by SM required direct cell-to-cell contact and could be detected on the fourth day, reaching maximal levels at the 6th and 7th days of the co-culture. The activation is more pronounced in the syngeneic system than under allogeneic conditions and is inhibited by anti-MHC II mAb, but not by anti-MHC II mAb. The finding that vascular smooth muscle cells can activate syngeneic T cells may have important implications concerning the mechanism of induction of vasculitis.

Animals↗
PubMed1990

Antigen presentation by brain microvessel smooth muscle and endothelium.

It has been previously reported that cultured brain microvessel smooth muscle cells (SM) express major histocompatibility complex (MHC) class II antigen. Here we report that SM is able to present ovalbumin (OVA) antigen to an OVA-specific T cell hybridoma (A2.2E10) and also presents keyhole limpet hemocyanin (KLH) to a KLH-specific T cell clone (HDK-1). Both the class II expression and the antigen-presenting capacity of SM cells is increased by interferon-gamma stimulation. Antigen presentation by SM is also MHC restricted as it is blocked by anti-Ia monoclonal antibodies. In contrast to SM, brain endothelium (En) presents whole OVA, digested OVA and KLH poorly, to a much lesser degree than SM, to the same antigen-specific T cells.

Animals↗
PubMed1990

Role of the blood-brain barrier in the formation of long-chain omega-3 and omega-6 fatty acids from essential fatty acid precursors.

Elongated, more highly polyunsaturated derivatives of linoleic acid (18:2 omega-6) and linolenic acid (18:3 omega-3) accumulate in brain, but their sites of synthesis and mechanism of entry are not well characterized. To investigate the role of the blood-brain barrier in this process, cultured murine cerebromicrovascular endothelia were incubated with [1-14C]18:2 omega-6 or [1-14C]18:3 omega-3 and their elongation/desaturation products determined. The major metabolite of 18:2 omega-6 was 20:4 omega-6, whereas the primary product from 18:3 omega-3 was 20:5 omega-3. Although these products were found primarily in cell lipids, they were also released from the cells and gradually accumulated in the extracellular fluid. Eicosanoid production was observed from the 20:4 omega-6 and 20:5 omega-3 that were formed. No 22:5 omega-6 or 22:6 omega-3 fatty acids were detected, suggesting that these endothelial cells are not the site of the final desaturation step. Although the uptake of 18:3 omega-3 and 18:2 omega-6 was nearly identical, 18:3 omega-3 was more extensively elongated and desaturated. Competition experiments demonstrated a preference for 18:3 omega-3 by the elongation/desaturation pathway. These findings suggest that the blood-brain barrier can play an important role in the elongation and desaturation of omega-3 and omega-6 essential fatty acids during their transfer from the circulation into the brain.

Acetyltransferases↗
PubMed1990

Permeability of neutral vs. anionic dextrans in cultured brain microvascular endothelium.

The luminal surface of vascular endothelium contains glycocalyx residues that establish an overall negative charge. Recent evidence has suggested that local endothelial surface charge properties may account for the permeability properties of various macromolecules. It has also been suggested that altered membrane charge on the luminal side may play a role in thrombogenesis and atherogenesis. The relationship of macromolecule charge to endothelial cell permeability was examined in vitro using mouse brain microvessel endothelial cells grown to confluence on a nitrocellulose filter separating a double-chamber system. Endothelial permeability to 4K and 10K fluorescein-labeled neutral dextrans was compared with the permeability to 4K and 10K fluorescein-labeled anionic dextrans (sulfated). After 1 h, there was significantly greater permeability of neutral fluorescein-labeled dextran than of anionic fluorescein-labeled dextran in each particle size. In addition, there was significantly greater permeability of 4K than 10K fluorescein-labeled dextrans of either charge. The findings indicate that charge in addition to size plays an important role in the movement of macromolecules across cultured microvascular endothelial cells.

Animals↗
PubMed1989

Brain microvessels produce 12-hydroxyeicosatetraenoic acid.

Cerebral microvessels isolated from perfused, adult murine brain produce a compound with the chromatographic properties of a monohydroxyeicosatetraenoic acid when incubated with arachidonic acid or stimulated with calcium ionophore A23187. The formation of this arachidonic acid metabolite is not reduced in the presence of the cyclooxygenase inhibitor ibuprofen, but it is abolished by the lipoxygenase inhibitor nordihydroguaiaretic acid. Analysis by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives of the metabolite, indicate that the compound is 12-hydroxyeicosatetraenoic acid. Fractions of isolated microvessels enriched with capillaries produce 2.1 times more 12-hydroxyeicosatetraenoic acid per microgram of protein than do fractions of microvessels enriched with arterioles. These studies confirm that brain microvessels can produce 12-hydroxyeicosatetraenoic acid and strongly suggest that cerebral endothelia are the primary source of microvessel-derived 12-hydroxyeicosatetraenoic acid. They further suggest that in brain injury, the liberation and accumulation of arachidonic acid in cerebral tissues may lead to the production of 12-hydroxyeicosatetraenoic acid within microvessels. The 12-hydroxyeicosatetraenoic acid formed in this way may mediate some of the blood-brain barrier and cerebrovascular dysfunction that occurs following stroke, brain trauma, or seizures.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation.

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.

Animals↗
PubMed1989

Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled griffonia simplicifolia agglutinin.

Griffonia simplicifolia agglutinin (GSA) is a valuable histochemical tool in the identification of endothelium. In this study GSA labeled with fluorescein isothiocyanate (GSA-FITC) was used to purify cultures of murine cerebral microvascular endothelium. Cultures were stained with GSA-FITC, then sorted using a fluorescence-activated cell sorter (FACS). GSA-positive endothelial cells were collected, re-cultured, and subsequently re-analyzed by FACS using GSA-FITC. Cultures that initially contained 80 +/- 3 to 89 +/- 3% (X +/- SE) GSA-positive cells were purified to 98 +/- 1% positivity. Immunohistochemistry with an anti-muscle-action antibody confirmed that FACS sorting of GSA-FITC-stained cells effectively removed contaminating smooth muscle cells from endothelial cell cultures. Viability, proliferation, and prostaglandin production of the cells was unaltered by lectin staining and FACS sorting. Thus, GSA-FITC can be used in conjunction with flow cytometry to enhance the purity of murine endothelial cell cultures without adversely affecting cell viability, growth, or metabolism.

Actins↗
PubMed1988

Kinetic evidence for a critical rate of protein synthesis in the Saccharomyces cerevisiae yeast cell cycle.

The kinetics of cell cycle initiation were measured at pH 2.7 for cells that had been arrested at the "start" step of cell division with the polypeptide pheromone alpha-factor. Cell cycle initiation was induced by the removal of alpha-factor. The rate at which cells completed start was identical to the rate of subsequent bud emergence. After short times of prearrest with alpha-factor (e.g. 5.2 h), the kinetics of bud emergence were biphasic, indicative of two subpopulations of cells that differed by greater than 10-fold in their rates of cell cycle initiation. The subpopulation that exhibited a slow rate of cell cycle initiation is comprised of cells that resided in G1 prior to start at the time of removal of alpha-factor, whereas the subpopulation that initiated the cell cycle rapidly is comprised of cells that had reached and become blocked at start. A critical concentration of cycloheximide was found to reintroduce slow budding cells into a population of 100% fast budding cells, suggesting that the two subpopulations differ with respect to attainment of a critical rate of protein synthesis that is necessary for the performance of start. Cycloheximide and an increase in the time of prearrest with alpha-factor had opposite effects on both the partitioning of cells between the two subpopulations and the net rate of protein synthesis per cell, consistent with this conclusion. Cell cycle initiation by the subpopulation of fast budding cells required protein synthesis even though the critical rate of protein synthesis had been achieved during arrest. It is concluded that alpha-factor inhibits the synthesis of and/or inactivates specific proteins that are required for the performance of start, but alpha-factor does not prevent attainment of the critical rate of protein synthesis.

Cell Cycle↗
PubMed1988

Murine cerebral microvascular endothelium incorporate and metabolize 12-hydroxyeicosatetraenoic acid.

Cultured murine cerebromicrovascular endothelial cells were employed to study the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) in an in vitro model of the blood-brain barrier. These endothelial cells convert 12-HETE to at least four, more polar compounds. Analysis of the least polar and predominant metabolite by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives indicate that the compound is 8-hydroxyhexadecatrienoic acid (8-HHDTrE). The uptake of 12-HETE into cell phospholipids peaks at 2 hr, and is not saturable up to the highest concentration tested, 5 microM. Seventy-five to 92% of this 12-HETE is incorporated into phosphatidylcholine, while the remainder is divided between the inositol and ethanolamine phospholipids. Incorporation into neutral lipids is slower, with radioactivity gradually accumulating in triglycerides over 24 hr. Saponification of cell lipids demonstrated that not only 12-HETE, but also its major metabolite, 8-HHDTrE, is incorporated into the cell lipids. Prostacyclin and prostaglandin E2 production by the cerebral endothelial cells is inhibited by up to 56% with 1 microM and 90% with 5 microM 12-HETE. These data demonstrate that 12-HETE is actively metabolized by cerebral endothelium and suggest at least two mechanisms through which 12-HETE may alter cerebromicrovascular function: 1) incorporation into cerebral endothelial membranes and 2) inhibition of cerebral endothelial prostaglandin production. Conversion of 12-HETE to more polar compounds, particularly 8-HHDTrE, may be interpreted as either the inactivation of 12-HETE or the production of additional, biological mediators.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1988

Automated spectrophotometric assay for cell division regulation in yeast.

A spectrophotometric assay is presented for monitoring the regulation of cell division by the polypeptide alpha-factor in cultures of living cells of Saccharomyces cerevisiae yeast. This assay is simple, automated, and may have wider application in the study of other eucaryotic cells that do not require anchorage for cell growth. The kinetics of absorbance change were monitored continuously over time in yeast cell cultures that were mixed and aerated in cuvettes fitted with top-loading propeller stirrers. The absorbance doubling time. TD(Abs), was identical to the cell number doubling time in the absence of cell division arrest by alpha-factor. alpha-Factor lengthened the TD(Abs) during division arrest. At pH 5.8, 10(5) 381G cells/ml, the Khalf-maximal was 250 +/- 50 nM alpha-factor for the TD(Abs) increase during arrest, with a maximum increase of five-fold. After a period of time the TD(Abs) abruptly shortened. This is defined as the spectrophotometric recovery time (RTspec) and was compared to the time of recovery that is due to the reinitiation of cell division monitored by bud emergence (RTBE). RTBE occurred 40 +/- 5 min prior to RTspec when recovery was spontaneous or was artificially induced by the removal of alpha-factor (pH 5.8, 381G). The difference between RTBE and RTspec was independent of alpha-factor concentration between 0.05 and 1 microM and cell concentration between 1 and greater than or equal to 25 x 10(5) cells/ml (pH 5.8, 381G) but was both pH and cell strain dependent. At pH 5.8 and 2.7 the recovery from arrest occurred by inactivation of alpha-factor. The TD(Abs) increase during arrest appears to be due to an alpha-factor-induced inhibition of net cell mass increase, an effect that has not been reported previously. Evidence is presented that this process is also correlated with the formation of cell projections.

Autoanalysis↗