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L R Drewes

Publications and source records attributed to L R Drewes.

At least 55 records · Page 3Linked to original sources
PubMed1987

Characterization of the blood-brain barrier: glycoconjugate receptors of 14 lectins in canine brain, cultured endothelial cells, and blotted membrane proteins.

The avidin-biotinylated peroxidase complex (ABC) method was used to detect binding of 14 lectins in tissue, cultured cells, and nitrocellulose blots. When applied to frozen sections of canine cerebral cortex and pituitary and evaluated by light microscopy, these lectins produced distinct staining patterns as determined by their individual carbohydrate specificities. Major saccharide residues detected in the endothelium of these cerebral tissues include alpha- and beta-galactose, alpha-mannose and/or alpha-glucose, and N-acetylglucosamine. Application to cells cultured from the canine cerebral endothelium gave staining results similar to those of microvessels in tissue. Thus, these characteristics of intact capillaries are retained in cultured cells and define fundamental properties of the blood-brain interface. Visual comparison of these staining patterns to those obtained for electrophoretic blots of solubilized membrane proteins identified multiple glycoprotein receptors and illustrated the vast quantity and variety of surface carbohydrate residues and the complexity of the cerebral endothelial cell glycocalyx. This carbohydrate-rich layer, which extends into the capillary lumen, may be of significant importance to the unique function of the blood-brain barrier.

Animals↗
PubMed1987

Blood-brain transport and regional distribution of bromo-benzodiazepine.

The blood-brain transport and regional distribution of a tritium-labeled, brominated benzodiazepine (BFB) was determined for the rat brain in vivo. The unidirectional transport constant from blood to brain was measured by a graphical, integral method and was found to be 0.83 ml/g/min, a value which indicates that transport is essentially flow-dependent. The apparent volume of distribution increased linearly during the measurement period, suggesting that back transport from brain to blood was zero and that BFB was trapped in the tissue, possibly by specific receptors or acceptors. Under the conditions of these experiments, autoradiography of brain tissue sections indicated a regional distribution of [3H]BFB similar to that expected for regional cerebral blood flow. These results indicate that BFB is a useful blood flow tracer in brain and suggest that BFB radiobrominated with bromine-75 may, under appropriate conditions, be a suitable tracer for in vivo regional blood flow measurement or benzodiazepine receptor mapping by positron-emission tomography.

Animals↗
PubMed1987

Butyrylcholinesterase in pericytes associated with canine brain capillaries.

The distribution of the enzyme butyrylcholinesterase (BChE) in dog brain cortex and cerebellum was studied by light and electron microscopy using a Hatchett's brown technique. Staining was found in neuron cell bodies and processes, the white matter of the cerebellum, and in capillaries and arterioles. Electron microscopy indicated that the enzyme activity associated with vessels was present in pericytes. Reaction product was found at the cell membrane, the intermembranous space of the nuclear envelope, and the Golgi complex of these cells. The finding of BChE in canine brain pericytes and its absence in endothelium does not support the idea that this enzyme is important in blood-brain barrier function. The pericyte in dog brain may be a site of synthesis of this enzyme and is, in this respect, similar to the endothelial cell of rat brain.

Animals↗
PubMed1987

Neurotoxic effects of low-level chronic acephate exposure in rats.

Rats were administered the organophosphorus insecticide acephate at 1.0 or 10.0 mg/kg.day for 15 weeks. Blood and brain samples were collected at the end of the treatment and analyzed for cholinesterase, acetylcholinesterase, and glutamic acid decarboxylase activities and catecholamine and amino acid levels. No significant inhibition in the activity of brain AChE was noted at doses of 1.0 or 10.0 mg/kg.day. Low levels of acephate exposure (1.0 mg/kg.day), which did not alter plasma cholinesterase or RBC acetylcholinesterase activity levels, resulted in a significant elevation of plasma epinephrine and norepinephrine levels. Decreased GABA, dopamine, and tyrosine levels and glutamic acid decarboxylase activity were observed in brains of these rats. Similar changes occurred in rats exposed to 10 mg of acephate/kg.day; however, plasma cholinesterase and RBC acetylcholinesterase activities were inhibited. These observations suggest that chronic exposure to acephate altered the activity of the noncholinergic system without altering the cholinergic activity, and that low-level chronic exposure to organophosphorous compounds cannot be predicted by measuring the ChE or AChE enzyme activities.

Acetylcholinesterase↗
PubMed1986

Protection from quinidine or physostigmine against in vitro inhibition by sarin of acetylcholinesterase activity.

We have studied the relative effectiveness of quinidine and physostigmine in protecting against the inhibition of acetylcholinesterase (AChE) by sarin, an organophosphate (OP) compound. The protective effects of these compounds were studied in vitro in both synaptosomal and soluble samples obtained from various regions of sarin-administered or control isolated, perfused canine brain. Although AChE activities in the sarin-administered brain were substantially lower than in the control brain, we observed regional differences in the AChE activity in both. The AChE in the control brain and the AChE remaining in sarin-administered brain had different susceptibilities to inhibition from OP compounds in vitro and, therefore, have different properties. Quinidine partially protected AChE from the inhibitory effects of sarin in vitro possibly by altering the sarin binding sites. Addition of sarin to physostigmine-treated control brain samples allowed partial recovery of the AChE activity. The protective effects of quinidine or physostigmine were lost when samples from sarin-administered brain were treated in vitro with these compounds and then again exposed to sarin. Therefore, both quinidine and physostigmine provided partial protection against the inhibitory effects of sarin in vitro if they were added prior to sarin.

Animals↗
PubMed1986

Light and electron microscopic localization of D-galactosyl residues in capillary endothelial cells of the canine cerebral cortex.

Ricinus communis agglutinin I (RCA-I), a lectin that binds to D-galactosyl residues, intensely stained capillaries in cryostat sections of canine cerebral cortex when evaluated by the avidin-biotin-peroxidase complex method. Of seven lectins tested, only RCA-I gave strong staining of vessels and capillaries with little staining of other cortical cells. Ultrastructural studies using ferritin-, biotin-, and peroxidase-labeled RCA-I indicated that this lectin was bound to the luminal membrane of the cerebral capillary endothelial cell and that lectin receptors were distributed continuously along this membrane. Plasmalemma invaginations that bound RCA-I were also present in endothelial cells. Primary cultures of cerebral capillary endothelial cells grown on plastic or gelatin-coated glass substrates demonstrated staining of the cell membrane and perinuclear structures which appeared to be the Golgi complex and secondary lysosomes. These staining characteristics were retained when the cells were subcultured and were confirmed by ultrastructural studies. In contrast, light microscopy showed that fibronectin was more widely distributed in the cytoplasm, a finding consistent with its occurrence in the endoplasmic reticulum. This work provides support for the concept that lectins may be useful endothelial cell markers in both intact tissue and cell culture.

Animals↗
PubMed1985

Neuron-specific mitochondrial degeneration induced by hyperammonemia and octanoic acidemia.

The neuropathological consequences of acute exposure to the neurotoxicants ammonia and octanoic acid were investigated with the isolated, perfused canine brain preparation. After 1 h of combined hyperammonemia and octanoic acidemia, ultrastructural changes were apparent in all brain regions examined. The cell bodies of neurons were the primary sites of these alterations. Neuronal mitochondria were distended, and the lamellae of the mitochondrial cristae were separated. In some cases the lamellae had completely dispersed, leaving only matrix remnants. Mitochondria of adjacent astrocytes appeared normal. Thus, a characteristic population of brain mitochondria is selectively vulnerable to a combination of hyperammonemia and octanoic acidemia and may be related to the biochemical mechanisms underlying encephalopathies of hepatic origin.

Ammonia↗
PubMed1985

Analysis of soman and sarin in blood utilizing a sensitive gas chromatography-mass spectrometry method.

Gas chromatography with electron impact mass spectrometry and selected ion monitoring provided a simple and sensitive method for measuring organophosphorus compounds sarin and the two isomers of soman (isomer I and isomer II) in blood. These compounds were extracted from blood or isotonic saline using a modification of the method developed by Sass et al. Blood was deproteinized with perchloric acid before extraction. The acid-induced degradation of the organophosphorus compounds could be minimized by neutralizing the acid immediately after deproteinizing. In saline and blood, 81% of the extractable soman and 74% of the extractable sarin was recovered with a single extraction. The overall recovery of added organophosphorus was less in blood than in saline because of the binding of organophosphorus to blood constituents, probably various enzymes and proteins. A time-dependent decrease in extractable organophosphorus was found in whole blood but not in saline. Although soman isomer II was degraded in blood faster than soman isomer I, no significant difference in the affinities of these two isomers to acetylcholinesterase was observed.

Acetylcholinesterase↗
PubMed1984

Cerebral blood flow and regional potassium distribution during focal ischemia of gerbil brain.

In 8 gerbils (Meriones unguiculatus) focal cerebral ischemia was produced by occlusion of the left common carotid artery and the opposite external carotid artery. After two hours blood flow was measured with iodoantipyrine labeled with carbon 14, and evaluated by means of quantitative autoradiography. Thereafter the same brain sections were stained for regional potassium by means of a histochemical technique. Changes in tissue potassium content were assessed by measuring the differences in optical densities in homotopic brain regions of the stained sections. The correlation between blood flow and tissue potassium level revealed that below a flow threshold of about 0.23 ml/gm/min, a definite potassium loss from the tissue was observed. The combination of autoradiographic methods with a technique for measuring the regional distribution of potassium may be useful in providing additional information on the occurrence of disturbed electrolyte homeostasis after the onset of focal ischemia.

Animals↗
PubMed1983

Characterization of the blood-brain barrier: protein composition of the capillary endothelial cell membrane.

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.

Animals↗
PubMed1983

An alpha1-adrenergic receptor-mediated phosphatidylinositol effect in canine cerebral microvessels.

In microvessels isolated from canine cerebral cortex, 32Pi is incorporated into phospholipids when incubated in physiological buffer containing [32Pi]orthophosphate. Norepinephrine (NE) selectively increases 32Pi incorporation into phosphatidylinositol (PI) and phosphatidic acid (PA) 60-200% over control levels. Half-maximal stimulation of PI labeling is observed with 1 microM NE, whereas maximal stimulation occurs at approximately 100 microM. Alpha 1-adrenergic agonists, phenylephrine and methoxamine, mimic the effects of NE, whereas isoproterenol, a beta-adrenergic receptor agonist, is ineffective. A wide variety of other agents tested had no specific effect on 32Pi incorporation into PI or PA. Prazosin, a selective alpha 1-receptor antagonist, at a concentration of 0.05 microM inhibits 50% of the stimulation due to NE (100 microM), whereas 1 microM yohimbine, an alpha 2-selective antagonist, is required to achieve the same effect. These results demonstrate the existence of an alpha 1-adrenergic receptor-mediated PI effect in isolated canine cerebral microvessels.

Adrenergic alpha-Agonists↗
PubMed1980

An improved apparatus for blood perfusion of the canine cerebral vasculature.

An improved perfusion apparatus is described which consists of a membrane oxygenator, roller pump, reservoir, heat exchanger, blood filter, and inert tubing. Heparinized blood may be used and is delivered at flow rates from 10 to 250 ml/min. Dogs are anesthetized with halothane and their cerebral arterial blood supply isolated by the method of Gilboe et al. (8). When the canine brain is perfused for 5 hr using the described apparatus, the rates of cerebral oxygen and glucose consumption are 5.19 +/- 0.12 mg/100 g/min and 39.9 +/- 6.5 mumol/100 g/min, respectively. Of the total glucose consumed by the brain, about 1/4 is contributed by the erythrocytes. An equivalent of about 9% of the consumed glucose is returned to the blood as lactate. Electron microscopic examination of cerebral cortex samples reveals no differences between 5-hr perfused brain and appropriate nonperfused controls. It is concluded that the apparatus is a useful system for organ perfusion and that the canine brain perfused by this method remains physiologically and metabolically active for at least 5 hr.

Animals↗
PubMed1977

Cytochalasin B inhibition of brain glucose transport and the influence of blood components on inhibitor concentration.

The effect of cytochalasin B on cerebral glucose transport and metabolism was investigated in 19 isolated perfused dog brain preparations. Cytochalasin B is a potent, non-competitive inhibitor of glucose transport at the blood-brain interface. Both glucose transport into (Ki = 6.6 +/- 1.9 micrometer) and out of the capillary endothelial cell are inhibited. The inhibition is readily reversible by perfusion with blood containing no cytochalasin B. After 2 min of exposure to 30 micrometer cytochalasin B, the cerebral oxygen consumption decreases by 31% probably due to decreased availability of glucose for oxidative metabolism. About one-half of the cytochalasin B that is dissolved in blood is bound to erythrocytes and other blood components while the remainder is free.

Animals↗
PubMed1977

5-O-beta-D-Galactofuranosyl-containing exocellular glycopeptide of Penicillium charlesii. Incorporation of mannose from GPD-D-mannose into glycopeptide.

Three-day-old Penicillium charlesii mycelia were broken with Al2O3 in a buffered system and the membranes were separated on a linear gradient of sucrose concentrations. The most active guanosine 5'-(alpha-D-mannopyranosyl pyrophosphate):glycopeptide mannopyranosyltransferase (GPD-D-mannose mannosyltransferase) was found in two unresolved membrane fractions ( p congruent to 1.1 g/cm3). This preparation incorporated [14C]mannose from GDP-D-[14C]mannose both into endogenous acceptors and added peptidophosphogalactomannan. Mannosyltransferase activity is optimum at pH 7.0 in 0.05 M Tris/maleate buffer, and 17 mM Mn2+. Replacement of Mn2+ with Fe2+, Mg2, Co2+, Ca2+ or Ni2+ greatly reduced the mannosyltransferase activity. [14C]Mannose incorporation from GDP-D-[14C]mannose into acceptors is linearly dependent on enzyme concentration. Mannosyl incorporation into peptidophosphogalactomannan is linear for 3 h and continues for at least an additional 4 h. In contrast, the rate of mannosyl incorporation into endogenous acceptor(s) decreases after 60 min and there is no incorporation after 2 h. A series of possible acceptors related to peptidophosphogalactomannan were tested and it was found that treatment of peptidophosphogalactomannan with 0.01 N HCl at 100 degrees did not appreciably decrease the effectiveness of the acceptor even though this treatment removes the galactofuranosyl residues. In contrast, treatment of peptidophosphogalactomannan with 0.5 N NaOH rendered the products nearly incapable of accepting mannosyl residues from GDP-D-mannose. Derivation of peptidophosphogalactomannan with 2,4-dinitrobenzene also decreased its effectiveness as a mannosyl acceptor. [14C]Mannose from GDP-D-[14C]mannose was incorporated into both the oligosaccharide and phosphogalactomannan regions of peptidophosphogalactomannan. Treatment of the [14C]peptidophosphogalactomannan product, with 0.4 N NaOH released [14C]mannosyl-containing residues which eluted in the mannobiose and polysaccharide fractions from BIo-Gel P2. Approximately 90% of the 14C was in mannobiose. The [14C]mannose was shown to be transferred to the mannosyl-(seryl/threonyl) region of the acceptor. Acetolysis of [14C]peptidophosphogalactomannan resulted in the isolation of [14C]mannose, [14C]mannobiose, and [14C]polysaccharide. Small quantities of 14C were obtained in mannotriose and mannotetraose. The time course of [14C]mannose incorporation into the oligosaccharide region of peptidophosphogalactomannan showed a continual increase over a 4-h interval. In contrast, there was no major increase after 1 h in [14C]mannose incorporated into the polysaccharide region. The enzyme catalyzing incorporation of mannose into the polysaccharide region of peptidophosphogalactomannan was solubilized by treatment of the membranes with Triton X-100.

Cations, Divalent↗