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Biomedical subjects

R L Roberts

Publications and source records attributed to R L Roberts.

At least 91 records · Page 5Linked to original sources
PubMed1988

Lipid tolerance in children receiving long-term parenteral nutrition: a biochemical and immunologic study.

The effect of intravenously administered lipids (intralipid) on immunologic function, complement, and coagulation was prospectively studied over 1 year in 15 children. The mean age of the children was 52.4 +/- 37.9 months; they had received total parenteral nutrition for an average of 3 years. Immunoglobulins (IgA, IgM, IgG), coagulation studies (platelets, prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen, fibrinogen degradation products, factor V) and components of complement (C3, C4, and CH100) were analyzed. Activation of monocytes by opsonized zymosan was measured by chemiluminescence and compared with that of normal control subjects. The clinically stable children had normal monocyte activation and normal complement levels. The PT and PTT values were significantly increased but improved with increased intralipid dose; other coagulation factors were normal. Acutely sick children, however, had decreased fat tolerance with significantly increased serum triglyceride levels and PT and PTT values; their monocyte activation and complement factors remained normal. These data indicate that the dose of intralipid should be lowered during acute illnesses; we suggest close monitoring of PT and PTT values and of serum triglyceride and cholesterol levels to avoid the fat overload syndrome.

Blood Chemical Analysis↗
PubMed1988

Chronic granulomatous disease due to a defect in the cytosolic factor required for nicotinamide adenine dinucleotide phosphate oxidase activation.

The superoxide-generating enzyme of human neutrophils, NADPH oxidase, is present in a dormant state in unstimulated neutrophils. It can be converted to an active form in a cell-free system if both the plasma membrane and cytosol fractions are incubated together in the presence of arachidonic acid. This system was used to determine the nature of the biochemical defect in seven patients with the autosomal recessive, cytochrome b-positive form of chronic granulomatous disease (CGD). A severe deficiency in the cytosol factor was identified in each patient. The defective activity was not caused by the presence of an inhibitor, nor could it be restored to normal by combining cytosol fractions from different patients. In contrast, the membrane fractions from all seven patients contained normal levels of NADPH oxidase when activated in the presence of control cytosol. Of family members tested (obligate heterozygotes for this disorder), seven of eight had intermediate levels of cytosol factor activity. The respiratory burst defect in this form of CGD is caused by an abnormality in the cytosolic factor required for NADPH oxidase activation.

Cytochrome b Group↗
PubMed1988

Clinical features and current management of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a diverse disorder characterized by several clinical presentations and a multitude of metabolic defects. The authors summarize the clinical features and current management of CGD, offer a definition of the disease based on molecular defects of the respiratory burst, and present their experience with trimethoprim/sulfamethoxazole prophylaxis.

Adolescent↗
PubMed1988

Intercellular junctions in the follicular envelope of the teleost, Brachydanio rerio.

The nature and distribution of intercellular junctions in the outer ovarian epithelium (serosa, mesothelium), endothelium, and follicle cells of the teleost oocyte-follicle complex were investigated by freeze-fracture electron microscopy. Tight-junctions were common between outer squamous epithelial cells, sometimes closely associated with intercalated foci of communicating junctions. The tight junctions consisted of one to several sealing strands which possessed focal discontinuities. In addition, the strands existed as loops or as short, free-ending elements; a condition that could indicate lability in their assembly or disassembly. The presence of free-ending strands could also mean that the structure serves for attachment as well as involved in the formation of occluding zonules. The free ends of some bars comprising the tight junctional strands are enlarged slightly. In outer ovarian epithelial or serosal cells, as is the case for mammalian mesothelium described by others, clusters of particles comprising communicating (gap) junctions are often intercalated within or are located in close proximity to tight junctional strands. In freeze-cleaved replicas, the outer squamous epithelial (serosal) cells contained a multitude of micropinocytotic pits (caveolae) and vesicles. Capillary endothelium also contains tight junctional components which are often closely associated with communicating junctions. Tight junctions also exist between follicle cells, but their structure changes during oocyte growth. Communicating junctions between follicle cells tend to be focal in distribution and not closely associated with tight junctions.

Animals↗
PubMed1988

Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease.

Activation of 32P-loaded neutrophils with phorbol myristate acetate causes the labeling of a family of three 48K proteins that focus near neutral pH. The relationship between these phosphoproteins and the activation of the respiratory burst has been supported by the previous finding that phosphorylation was defective in the two most common types of chronic granulomatous disease (CGD): X-linked cytochrome-negative (X/-) and autosomal cytochrome-positive (A/+). In this report, these studies have now been extended to the rare A/- and X/+ forms of the disease. In all three patients with A/- CGD examined, the two most acidic 48K proteins failed to undergo enhanced phosphorylation in response to phorbol stimulation, a finding similar to that seen in X/- patients. In contrast, neutrophils from two patients with X/+ CGD appeared to phosphorylate the neutral 48K proteins in a normal fashion. It thus appears that the different phosphorylation patterns seen in chronic granulomatous disease are a reflection of the genetic heterogeneity of this disorder. These findings lend further support to the conclusion that the 48K phosphoprotein family is related to the respiratory burst, although not necessarily in a straightforward manner.

Adult↗
PubMed1987

Purification and properties of porcine polymorphonuclear cells.

A method for the rapid separation of highly purified populations of porcine polymorphonuclear cells from whole blood is described. Porcine blood, anti-coagulated with EDTA, was layered over a discontinuous Percoll gradient (62.5% and 75%) and then centrifuged at 400 X g for 25 min. This results in the formation of a band of cells at the interface of the two Percoll layers which is greater than 99% granulocytes (93.8 +/- 1.8% neutrophils and 5.3 +/- 1.8% eosinophils) with a 77% recovery. The mononuclear cells remain above the 62.5% Percoll layer, and most erythrocytes pellet to the bottom of the tube. The isolated porcine granulocytes were found to respond to opsonized zymosan, phorbol myristate acetate (20 ng/ml), and the calcium ionophore A23187 (10(-5) M) in chemiluminescence assays with kinetics similar to those of human granulocytes. The porcine cells did not respond to the chemotactic peptide N-formyl-methionyl-leucyl-phenalanine (FMLP; 10(-6) M) unlike the human granulocytes which display a very rapid response to FMLP. Both porcine and human granulocytes readily changed shape by elongating and developing pseudopods when exposed to zymosan-activated serum, but only human granulocytes changed on exposure to FMLP. Thus, porcine granulocytes may be rapidly isolated on discontinuous Percoll gradients with little mononuclear cell contamination. Porcine and human PMN have similar oxidative and chemotactic responses, but porcine PMN differ from human granulocytes in the inability of porcine granulocytes to respond to FMLP.

Animals↗
PubMed1987

Transendothelial transfer of macromolecules in vitro.

The transendothelial transfer of macromolecules has been difficult to study because of the complexities of the in vivo models. We have developed a model of an endothelium cultured on a permeable support and used it to characterize the transendothelial transfer of albumin. Porcine pulmonary artery endothelial cells form a single layer of cells lining the gelatin-impregnated polycarbonate micropore filters, and the cells develop junctional structures similar to endothelial tight junctions observed in vivo. The monolayer resists the flow of electrical current, and the resistance is sensitive to extracellular calcium concentrations. Albumin transfer across the cultured monolayers was found to be asymmetric, and the rate of transfer from interstitium to lumen was greater than that from lumen to interstitium. The asymmetric transfer occurred against a concentration gradient and was abolished by treating the monolayer with NaCN. Increasing albumin concentrations increased the rate of interstitial to luminal transfer, and the process demonstrated saturation at an interstitial albumin concentration of 725 microM. These data point out the usefulness of the in vitro preparation to identify potentially important aspects of transendothelial transport that would be difficult to detect in vivo.

Albumins↗
PubMed1986

Glial cells influence membrane-associated enzyme activity at the blood-brain barrier.

Glial cells have been shown to influence several cerebral endothelial cell properties in vitro. A situation similar to the endothelial-astrocyte relationship existing at the blood-brain barrier (BBB) can be produced by growing cultured cerebral endothelium on one side of a filter and glial cells on the other in an enclosed double chamber. In this setting membrane-associated reaction product on the cerebral endothelial cell for both Na+,K+-ATPase and non-specific alkaline phosphatase was markedly increased when the endothelial cells were co-cultured with glial cells. In addition, the distribution of reaction product on the cerebral endothelial cell membrane was similar to that reported in vivo. These observations support a glial influence on enzyme activity at the BBB.

Alkaline Phosphatase↗
PubMed1986

Substrate dependence and oxygen sensitivity of tone and of spontaneous and evoked contractions of the distal colonic muscularis mucosae of opossum.

The muscularis mucosae of the opossum distal colon fails to maintain tone and to generate spontaneous contractions in an anaerobic environment in vitro. To investigate this phenomenon further, dose-response curves to agonists under different oxygen concentrations were examined. In other experiments the tissue was actively exhausted (i.e., repeatedly stimulated in a substrate-free environment until no response to K+, acetylcholine, or histamine could be observed) and then equilibrated with an alternative substrate for 3 h, when the ability of the tissue to contract to agonists was reevaluated. Reducing the available oxygen abolished tone and caused a graded loss of responses to histamine; in contrast, at 0% oxygen maximum responses to acetylcholine were reduced by only 75%. Abolition of tone by the addition of cyanide to the bathing medium did not affect the amplitude of responses to histamine and acetylcholine. The tissue was able to use all the chosen alternative substrates to different degrees for acetylcholine-induced contraction; similarly, with the exception of beta-hydroxybutyrate it could use all substrates to some degree to support responses to histamine. The evidence suggests that the source of energy required to maintain tone is different from the source required to sustain K+ or agonist-induced contractions, and that the dependence of isotonic contractions on exogenous substrates varies with the pharmacologic stimulus.

Acetylcholine↗
PubMed1986

Staining of eosinophils with nitroblue tetrazolium in patients with chronic granulomatous disease.

The nitroblue tetrazolium (NBT) test is the primary screening test for chronic granulomatous disease (CGD). Neutrophils and other phagocytic cells readily reduce NBT to blue formazan after oxidative stimulation whereas CGD cells remain colorless. In the present study purified eosinophil populations were obtained from CGD patients and normals by exposing peripheral blood to the peptide N-formyl-methionyl-leucyl-phenylalanine and then centrifuged over a discontinuous Percoll gradient. The eosinophils were then incubated in 0.1% NBT (37 degrees C, 15 min) with either phorbol myristate acetate or buffer alone (HEPES with calcium and magnesium). Blue staining characteristic of NBT reduction occurred in the phorbol myristate acetate-stimulated eosinophils of half (4/8) of the CGD patients tested. The staining was most intense between the nuclear lobes of the eosinophil with little staining near the cell periphery. The staining pattern was present in 75.5 +/- 5.3% of the purified eosinophils in those patients in which the phenomenon occurred and was reproducible in the same patients over a 6-month period. Eosinophils from normal individuals tested with NBT and phorbol myristate acetate showed intense staining over the entire cell cytoplasm as did normal neutrophils. Purified CGD eosinophils that did show the staining pattern were not able to produce superoxide (as measured by cytochrome C reduction) when stimulated by phorbol myristate acetate indicating the staining was probably not related to superoxide production. Patients with CGD have a mild eosinophilia (4.6 +/- 0.7% in 11 patients at the National Institutes of Health) and eosinophil staining may account for the small number of positive NBT cells reported in some patients.

Adolescent↗
PubMed1985

Rapid method for isolation of normal human peripheral blood eosinophils on discontinuous Percoll gradients and comparison with neutrophils.

Previous studies on human eosinophils often have used cells from patients with hypereosinophilia syndrome or parasitosis owing to the difficulty in isolating pure populations of eosinophils from normal individuals. In the present study, human eosinophils were isolated with a purity of 97%, with 70% recovery from normal individuals with blood eosinophil counts of less than 3%. Human eosinophils are denser than neutrophils, but the range of densities of the two cell types overlap, making purification of eosinophils by density-gradient centrifugation difficult. However, if neutrophils were exposed to the chemotactic peptide (f-Met-Leu-Phe), which did not stimulate eosinophils, the neutrophils' density decreased, shifting them away from the density of eosinophils. Whole normal blood anticoagulated with EDTA was incubated at 37 degrees C for 15 minutes with 10(-6) mol/L f-Met-Leu-Phe and then layered over a discontinuous Percoll gradient (65% and 75% in diluted phosphate-buffered saline) and centrifuged at 400 g for 25 minutes at 22 degrees C. The cell layer between the 65% and 75% Percoll was collected and washed, and hypotonic lysis was used to remove erythrocytes. This cell layer contained 97.3 +/- 0.7% eosinophils (N = 8) with a yield of 4.9 X 10(4) eosinophils per milliliter of whole blood, or 70% of the total eosinophil count. The isolated eosinophils were in a quiescent state but responded to Escherichia coli endotoxin-activated serum with shape change and chemotaxis, membrane depolarization, and reduced nitroblue tetrazolium (96.0 +/- 1.0%), when stimulated with phorbol myristate acetate. In phagocytic assays, 89.3 +/- 1.3% of the eosinophils ingested Candida albicans v 96.0% +/- 1.0% of neutrophils. In contrast, the eosinophils did not respond chemotactically, alter membrane potential, or reduce nitroblue tetrazolium when treated with f-Met-Leu-Phe, and studies with f-Met-Leu-[3H]Phe showed that normal eosinophils lacked expression of receptors for f-Met-Leu-Phe. In control studies, normal eosinophils that were not exposed to f-Met-Leu-Phe during purification also failed to respond to f-Met-Leu-Phe, indicating intrinsic differences between normal eosinophils and neutrophils. Thus, exposure of whole blood to f-Met-Leu-Phe, followed by separation on Percoll is a simple method for rapid isolation of normal human eosinophils.

Binding Sites↗
PubMed1984

Purification of human blood basophils by single step isopycnic banding on Percoll.

Human venous blood, anticoagulated with EDTA, was layered onto a discontinuous Percoll gradient, made from solutions of density 1.088, 1.079, and 1.070 gm/ml. After centrifugation at 700g for 15 min at 22 degrees C, the majority of the blood basophils was found in a narrow band at the density 1.070-1.079 interface (Percoll band 2). For 15 normal donors, mean total basophil number recovered from all locations in the gradient was 3.8 +/- 1.2 (SD) X 10(4) basophils per ml of blood applied. Thus, 95% of the values ranged from 1.5 to 6 X 10(4), which compares favorably with the reported range of 1 to 8 X 10(4) basophils per ml for normal subjects. In the basophil-rich Percoll band 2, 2.8 +/- 0.8 X 10(4) basophils were recovered per ml of blood applied. The mean percentage of basophils in Percoll band 2 was 19%, with a range of 5 to 53%. Monocytes and neutrophils were present in very small numbers; the majority of accompanying cells were small lymphocytes.

Basophils↗
PubMed1984

Increasing extracellular potassium causes calcium-dependent shape change and facilitates concanavalin A capping in human neutrophils.

Stimulation of human polymorphonuclear leukocytes (PMN) by chemoattractants is associated with changes in membrane potential, and it has been speculated that these changes are important in PMN function. In the present study, human PMN isolated on Percoll gradients were exposed to extracellular K+ concentrations, which have been shown to depolarize PMN. Incubation of PMN at 37 degrees C for 15 min in 125 mM K+ increased the number that capped fluoresceinated concanavalin A (Con A) to 78% +/- 5.1 with 6.9% +/- 0.8 forming Con A caps in 5 mM K+ (p less than 0.001). In the absence of lectins, high K+ induced polarization of PMN shape with pseudopod and uropod formation (2.4% +/- 0.1 polarized in 5 mM K+ vs 83.4% +/- 1.0 polarized in 125 mM K+ p less than 0.001). Control studies, in which Na+ was decreased by substituting choline chloride or sucrose, indicated that the K+ effects were specific and not due to lowering of Na+. Increasing K+ also stimulated preferential secretion of the specific (secondary) granule constituents, caused significant mobilization of receptors for the peptide N-formyl-methionyl-leucyl-phenylalanine (fmet-leu-phe), and increased the number of PMN migrating in chemotactic assays. Unlike the capping of Con A and shape change, however, the increase in fmet-leu-phe binding, degranulation, and locomotion was not specific for raising extracellular K+ and occurred when choline chloride was substituted for sodium chloride, suggesting that these effects may be due to the lowering of Na+ or the changing of the Na+ to K+ ratio. In related studies designed to examine the mechanism of these K+-induced effects, shape change and Con A cap formation were inhibited by the absence of extracellular calcium or the presence of the calcium channel blockers, methoxyverapamil (100 microM), manganese (2 mM), or cobalt (2 mM), suggesting that the K+-induced changes depend on an influx of calcium. Thus, the increase of extracellular K+ induces calcium-dependent membrane changes in PMN similar to those seen when PMN are stimulated by a chemoattractant.

Adult↗
PubMed1983

Vectorial synthesis of a polysaccharide by isolated plasma membranes.

To ascertain the directionality of chitin synthesis by yeast plasma membranes, the external surface of Saccharomyces cerevisiae protoplasts was labeled with ferritin--concanavalin A. After protoplast lysis, plasma membranes were isolated and treated with trypsin to activate chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyl-transferase, EC 2.4.1.16). The membranes were then enrobed in agar and allowed to synthesize chitin from UDP-N-acetylglucosamine. After fixation and embedding in Epon, thin sections were stained for chitin with wheat germ agglutinin--colloidal gold complexes. The chitin marker was found near the ferritin-labeled external face of the membrane--i.e., the polysaccharide was located on the outside of the membrane, as it is in the intact cell. Chitin synthase activity was not detected in intact protoplasts before or after treatment with trypsin. The enzyme became available to trypsin activation after lysis of the protoplasts. Together with similar, previously reported experiments on the inactivation of chitin synthase by glutaraldehyde, these results indicate that the enzyme faces the interior of the cell. We conclude that, both in vivo and in vitro, the synthase receives N-acetylglucosamine residues from UDP-N-acetylglucosamine at the cytoplasmic face of the membrane and transfers them vectorially to a growing chain of chitin that is concomitantly extruded to the outside.

Cell Membrane↗
PubMed1983

Chitin synthesis and localization in cell division cycle mutants of Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae cell cycle mutants cdc3, cdc4, cdc7, cdc24, and cdc28 at a nonpermissive temperature (37 degrees C) resulted in increased accumulation of chitin relative to other cell wall components, as compared with that observed at a permissive temperature (25 degrees C). Wild-type cells showed the same chitin/carbohydrate ratio at both temperatures, whereas mutants cdc13 and cdc21 yielded only a small increase in the ratio at 37 degrees C. These results confirm and extend those reported by B. F. Sloat and J. R. Pringle (Science 200:1171-1173, 1978) for mutant cdc24. The distribution of chitin in the cell wall was studied by electron microscopy, by specific staining with wheat germ agglutinin-colloidal gold complexes. At the permissive temperature, chitin was restricted to the septal region in all strains, whereas at 37 degrees C a generalized distribution of chitin in the cell wall was observed in all mutants. These results do not support a unique interdependence between the product of the cdc24 gene and localization of chitin deposition; they suggest that unbalanced conditions created in the cell by arresting the cycle at different stages result in generalized activation of the chitin synthetase zymogen. Thus, blockage of an event in the cell cycle may lead to consequences that are not functionally related to that event under normal conditions.

Cell Cycle↗
PubMed1983

Differences between esophageal body and lower esophageal sphincter in mitochondria of smooth muscle in opossum.

In opossum, contractions of smooth muscle of the circular layer of the esophageal body resist oxygen deprivation, while tone of the lower esophageal sphincter requires oxygen. Thus, morphologic differences in mitochondria could distinguish the two types of muscle. Transverse strips, cut at regular intervals along the smooth-muscle segment of the esophageal body, were physiologically identified as muscle of body or sphincter. Transverse sections of the circular muscle layer were examined by transmission electron microscopy. Muscle cell profiles located between the ends of the nucleus and the tips of the cells were used to evaluate the number of mitochondrial profiles per muscle cell profile, mitochondrial mass (relative total area of mitochondrial profiles in each muscle cell profile), mitochondrial position (distance from the center of each mitochondrial profile to the nearest point on the cell membrane relative to mean radius of that muscle cell profile), and mass of smooth endoplasmic reticulum (relative total area of profiles in each muscle cell profile). In the nonnucleated part of the muscle cells, sphincter muscle had a greater ratio of mitochondrial profile area to cell profile area (p less than 0.05), mitochondrial profiles in sphincter muscle were more central in location than in esophageal body muscle (p less than 0.05), and sphincter muscle contained more endoplasmic reticulum than body muscle (p less than 0.05). Sphincter muscle did not differ from body muscle in mean muscle cell profile area and number of mitochondrial profiles per muscle cell profile in this region of the muscle cell (p greater than 0.05).

Animals↗
PubMed1982

Paranasal sinus obliteration in Wegener granulomatosis.

The authors report 14 cases of Wegener granulomatosis in which one or more paranasal sinuses were obliterated by bone. The maxillary antra were involved in all cases, with the other sinuses being affected less frequently. These changes are thought to result from chronic bacterial sinusitis superimposed on the granulomatous vasculitic process. Computed tomography dramatically demonstrated the bone changes, consisting of a combination of sinus wall thickening and trabeculated new bone formation within the sinuses.

Adolescent↗