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Biomedical subjects

D Hicks

Publications and source records attributed to D Hicks.

At least 109 records · Page 6Linked to original sources
PubMed1990

The growth and behaviour of rat retinal Müller cells in vitro. 1. An improved method for isolation and culture.

Eyeballs were enucleated from young (postnatal day 8-12) pigmented rats and the retinas were dissected free after soaking the globes overnight in growth medium. The retinas were digested with enzymes, dissociated and maintained in stationary culture in 10% serum supplemented growth medium. Cultures displayed extensive cellular outgrowth after 1-5 days, with abundant fusiform and epithelioid cells. Removal of aggregates and cellular debris after 6-7 days yielded a purified flat cell preparation, which could be maintained either as a primary culture for several weeks or passaged repeatedly as rapidly proliferating epithelioid cells. Staining with monoclonal antibodies RET-G1, G3 and G7, and polyclonal S-100, glutamine synthetase and carbonic acid anhydrase antisera, all markers for Müller cells, showed positive labelling of all cells present in these purified cultures, both primary and passaged cells. This contrasted with the use of RET-G2, anti Factor VIII and anti glial fibrillary acidic protein (GFAP) antibodies. RET-G2, another Müller cell marker, failed to recognize passaged cells. Anti Factor VIII also did not label any cells, and anti GFAP stained very few cells: these remained associated with aggregated material so that vigorous washing to remove loosely adherent tissue from primary cultures resulted in the total absence of GFAP positive cells. In addition, no GFAP positive cells were detected in passaged cells or in cells regrown following freezing and storage. The Müller cell nature of these flat cells in soaked retinal cultures was further supported by the specific uptake of 5-bromo-deoxyuridine by nuclei located within the inner nuclear layer of retinal fragments in vitro. Hence the soaking treatment greatly reduces the number of surviving astrocytes whilst stimulating the rapid growth of cells expressing many properties of mature retinal Müller cells.

Animals↗
PubMed1989

Structural features of CD4 required for binding to HIV.

A soluble form of the human CD4 glycoprotein (sCD4), the cellular receptor for human HIV, was treated with various physical, chemical, and enzymic regimens and tested over a range of concentrations for its capacity to inhibit the binding of HIV to CD4+ T cells. Reduction of disulfide bonds and alkylation in denaturing buffer (8 M urea) destroyed the inhibitory activity of sCD4, whereas reduction and alkylation in PBS had no effect. Derivatization or digestion of carbohydrate groups by periodate oxidation or by glycolytic enzyme digestion did not affect sCD4 inhibitory capacity. Digestion with trypsin or endoproteinase Glu-C destroyed activity. A limited digestion of sCD4 with endoproteinase Glu-C resulted in a mixture of fragments, however, and the mixture had inhibitory activity equivalent to that of intact sCD4. Within this mixture, a fragment of 23 kDa was identified that binds to HIV. Although sCD4 can be digested to yield fully active fragments, the requirement for intrachain disulfide bonding indicates that the minimum sized portion of CD4 that will retain full affinity for HIV will have to be formulated with a proper tertiary structure.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1989

Immunoelectron microscopical examination of the surface distribution of opsin in rat rod photoreceptor cells.

The distribution of the visual pigment protein opsin in postnatal day 8 and adult rat retinal photoreceptor cells was studied using several well characterized monoclonal antibodies against rhodopsin and a sensitive, modified colloidal gold technique. Distribution of opsin was studied in intact tissue, explant cultures and freshly dissociated cell preparation. At PN8 labeling was seen in the plasma membrane overlying the cell body, inner and outer segment. Adult retinas still showed heavy labeling of the cell body and outer segment, but reduced labeling of the inner segment surface. Semi-quantitative estimates of colloidal gold particles bound indicated a ratio of 3-20 times more particles per unit length in the outer vs. the inner segment, depending on tissue age, preparation and antibody. Together with the subcellular localization of other photoreceptor plasma membrane proteins, these data reveal a complex system of membrane domains into which different protein components are segregated.

Animals↗
PubMed1988

Acidic fibroblast growth factor stimulates opsin levels in retinal photoreceptor cells in vitro.

It is demonstrated that newborn rat retinal photoreceptor cells can differentiate in monolayer culture, and synthesize de novo photoreceptor-specific proteins such as opsin. When maintained in serum supplemented medium on a laminin substrate, these cells survive for up to 3 weeks. The addition of acidic fibroblast growth factor stimulates an increase in the levels of opsin of 5-10-fold control values, and prolongs cell survival by up to 6 days.

Animals↗
PubMed1988

Detection of DNA transcripts of measles genes in multiple sclerosis brain tissue by transfection.

Transfection studies on DNA obtained from 16 multiple sclerosis (MS) and 13 non-MS brain tissues suggested that 3/16 (19%) MS and 3/13 (23%) non-MS samples contained contaminating measles virus double-stranded RNA, and that 6/16 (38%) MS and 0/13 non-MS samples contained DNA transcripts of measles genes. It is argued that the DNA transcripts most probably originated from dual retrovirus and measles infections in the subjects.

Adult↗
PubMed1987

Histiotypic organization and cell differentiation in rat retinal reaggregate cultures.

Reaggregate cultures have been formed from cell suspensions of neonatal rat retinas. Histological sections of the reaggregates showed evidence of lamination with central rosettes formed around a lumen, a clear neuropil layer and an outer cellular layer. Each of the major retinal cell types, except ganglion cells, could be positively identified using cell type-specific antibodies to label cryostat sections. Many of these were found to occupy positions within the reaggregates similar to those found in the intact retina. Electron microscopic observations showed abundant immature and mature synaptic endings within the neuropil layer, including a number of ribbon synapses. Examination of the rosettes showed an arrangement of Müller glia and photoreceptors that closely resembled that of the intact retina. Within the lumen of rosettes, photoreceptors were found to contain stacks of disc-like membranes bounded by a plasma membrane, analogous to immature outer segments. The photoreceptors within rosettes also underwent molecular differentiation and expressed an outer segment specific marker. The findings suggest that retinal cells have intrinsic properties that allow them to organize themselves into a correctly laminated structure and that particular cell interactions are necessary for continued differentiation of at least rod photoreceptors and Müller cells.

Animals↗
PubMed1987

Role of circulating complement and polymorphonuclear leukocyte transfusion in treatment and outcome in critically ill neonates with sepsis.

We examined the effects of early administration of polymorphonuclear leukocyte (PMN) transfusions in neonates with sepsis by prospectively randomizing 35 consecutive critically ill infants with sepsis, 21 of whom received PMN transfusions in addition to supportive care, one transfusion every 12 hours for a total of five transfusions. Each transfusion consisted of 15 mL/kg containing 0.5 to 1.0 X 10(9) PMN with less than 10% lymphocytes, and was subjected to 1500 rads. PMNs were obtained by continuous-flow centrifugation leukopheresis. Pretreatment values that did not significantly affect survival included weight, gestational age, sex, prematurity, C-reactive protein, initial hematocrit, platelet count and absolute granulocyte count (AGC less than or equal to 1500/mm3), IgM, IgG, IgA, neutrophil supply pool depletion, hypoxia, acidosis, and hypotension. Postnatal age was significantly lower in the nontransfused group than in the transfused group; 2.3 +/- 0.6 vs 6.1 +/- 2.2, (P less than 0.001). Positive blood cultures were obtained in 80% of both groups. Low circulating levels of total hemolytic complement were associated with a poor outcome and higher mortality: 56 +/- 4.0 IU in survivors vs 31 +/- 4.4 IU in nonsurvivors (P less than 0.01). Survival was significantly greater in the PMN transfused group than in the nontransfused group: 20 (95%) of 21 vs nine (64%) of 14 (P less than or equal to 0.05). No untoward effects were attributable to PMN transfusions, either during the study or on subsequent follow-up visits. These preliminary data suggest that early treatment with PMN transfusions improves survival in neonates with overwhelming sepsis. In addition, depleted or low circulating levels of complement may influence prognosis and thus future treatment strategies for neonatal sepsis.

Acute Disease↗
PubMed1987

Different rhodopsin monoclonal antibodies reveal different binding patterns on developing and adult rat retina.

We used a battery of 10 monoclonal antibodies directed against different identified peptide sequences within the carboxyl, transmembrane loop, and amino terminal regions of rhodopsin to label retinas from early postnatal and adult rats. Intensity of label, age of initial appearance of staining, and distribution of label varied depending on the antibody. Most antibodies showed detectable labeling at postnatal day 1, and were eventually observed binding to the cell bodies and the inner and outer segments of the photoreceptors. One amino terminal and two carboxyl terminal antibodies, however, showed no detectable labeling until postnatal day 5 and were only transiently detectable in the cell body region. These patterns cannot be explained by accessibility of binding site, binding affinity, fixation artifact, or crossreactivity. The results indicate that physiological and experimental parameters can alter the apparent immunocytochemical localization of conformationally active molecules such as rhodopsin. The results also suggest that rhodopsin can undergo light-dependent conformational changes in several different compartments within rat retinal photoreceptors before the time of eye opening.

Aging↗
PubMed1987

Peripherin. A rim-specific membrane protein of rod outer segment discs.

Monoclonal antibodies were used with radioimmune assays and immunocytochemical techniques to identify and localize membrane proteins in bovine rod outer segment (ROS) disc membranes. When ROS membrane proteins were separated by SDS-polyacrylamide gel electrophoresis in the presence of the sulfhydryl reducing agent, 2-mercaptoethanol, two monoclonal antibodies designated as 3B6 and 2B6 were found to bind to a polypeptide having an apparent molecular weight (Mr) of 33,000 daltons. In the absence of 2-mercaptoethanol, these monoclonal antibodies bound to a doublet having Mr of 67,000 and 69,000. Immunogold-dextran labeling of ROS sections embedded in Lowicryl resin indicated that this protein is localized around the periphery of the ROS organelle where the discs come in close contact to the ROS plasma membrane. Immunogold labeling of morphologically intact isolated discs prepared by mild trypsinization of ROS fragments confirmed that this disc membrane protein is localized along the rim region of discs. On the basis of these localization studies, the authors have named this protein peripherin. Immunogold-dextran markers were also used with previously characterized antirhodopsin monoclonal antibodies to visualize the distribution of rhodopsin on isolated discs. Dense labeling was observed along the lamellar region of the discs, but little if any labeling was observed on the extreme edges of the discs. These results are consistent with the view that the lamellar region of discs containing rhodopsin is a distinct membrane domain from the rim region which contains peripherin, a high Mr rim protein and possibly other proteins involved in disc-disc and disc-plasma membrane interactions.

Animals↗
PubMed1987

A phosphorylation-sensitive anti-rhodopsin monoclonal antibody reveals light-induced phosphorylation of rhodopsin in the photoreceptor cell body.

Rho-1C5, a monoclonal antibody sensitive to phosphorylation of rhodopsin, bound to the retinal photoreceptor cell body region of dark-adapted but not light-adapted 8 to 13-day-old-rats. There was no cell body labeling visible either before or after this time, although the photoreceptor outer segments were labeled at all times from postnatal day 5 (PN5) onwards, in both light and dark adapted retinas. However, opsin was detectable in the photoreceptor cell body region from birth onwards using another rhodopsin antibody binding to a site unaffected by phosphorylation. Competitive inhibition radioimmunoassays also indicated light-dependent differences in Rho-1C5 binding at PN8 and adult. Biochemical studies showed light-dependent phosphorylation of rhodopsin at PN8, PN13 (just after eye opening) and adult. These data indicate that rhodopsin can be phosphorylated in a light-regulated manner early in development before eye opening and imply that photoactive chromophores can attach to opsin in the cell body as well as the outer segment.

Animals↗
PubMed1986

Lectin and antibody labelling of developing rat photoreceptor cells: an electron microscope immunocytochemical study.

Lectin and rhodopsin antibody binding sites were studied in developing and adult rat photoreceptors in order to compare changes in the total carbohydrate pool with the movement of a known glycoprotein rhodopsin. Electron microscope immunocytochemical techniques utilizing modified colloidal gold methods were used. At birth, all three lectins - Concanavalin A (ConA), Ricinus communis agglutinin II (RCA II) and wheat germ agglutinin (WGA) - showed heavy labelling of the photoreceptor surface scleral to the outer limiting membrane. At the same age, a monoclonal antibody against rhodopsin, RET-P1, revealed sparse labelling of only occasional immature photoreceptor surfaces. At postnatal day 4(P4), all three lectins showed variable binding to the inner segment and along the length of the newly forming connecting cilium. There was generally a region of more intense label at the base of the cilium. RET-P1 binding to P4 retina showed a discontinuous distribution, with heavily labelled inner segments being adjacent to unlabelled inner segments. This pattern indicates that the initial expression of rhodopsin is not a coordinate event but occurs in discrete cells, possibly related to the end of mitosis. RET-P1 binding at this age was reduced or absent from the proximal connecting cilium. AT P7, when the outer segments are beginning to develop, all the lectins and RET-P1 showed reduced binding to the inner segment plasma membrane and heavy labelling of the outer segment surface. In favourable sections, heavy labelling of the photoreceptor cell body plasma membrane by ConA and RCA II was also observed, terminating abruptly at the outer limiting membrane. The variation in ligand binding between different cellular compartments which are all formed from a continuous plasma membrane may indicate the presence of special barriers to diffusion of membrane components. This labelling pattern persisted into maturity. RET-P1 and lectin binding did not always correspond in developing retina, indicating that at least part of the observed lectin label must be due to other glycoproteins or glycolipids. Post-embedding thin section labelling of adult rat retina revealed a uniform binding pattern across the outer segment for ConA, WGA and RET-P1. However, RCA II exhibited labelling only along the basal edge of outer segments. Labelling of isolated, opened discs from bovine rod outer segments revealed binding to a single surface for ConA, WGA and RET-P1, but RCA II only labelled a small amount of membrane. Hence RCA II seems to recognize a determinant present only on the outer segment plasma membrane.

Animals↗
PubMed1986

Differential immunogold-dextran labeling of bovine and frog rod and cone cells using monoclonal antibodies against bovine rhodopsin.

Eleven monoclonal antibodies against different segments of bovine rhodopsin were used with immunogold-dextran markers to label Lowicryl thin sections of bovine and frog retinal photoreceptor cells for visualization by transmission electron microscopy. Antibodies against the C-terminus, F1-F2 loop and N-terminus of rhodopsin were all observed to label bovine rod outer segments (ROS) densely, but to label rod inner segments (RIS) only sparsely. Most antibodies bound 200-600 gold particles per micron2 in the ROS, 10-60 gold particles per micron2 in the RIS and 5-20 particles per micron2 on the Lowicryl resin. One antibody against the N-terminus and one antibody against the C-terminus resulted in the binding of over 1000 particles per micron2 in bovine ROS. Cone outer segments (COS) were labeled with only one antibody, rho 3D6, having a specificity for the 1'-4' C-terminus of bovine rhodopsin. Ninety per cent of the COS were observed to be labeled with this antibody. Immunogold-dextran labeling was also used to study the cross-reactivity of these antibodies to rhodopsin in red and green frog ROS and COS. Monoclonal antibodies directed against sites along the F1-F2 loop, and the N-terminus labeled red frog ROS densely, but did not label either green ROS or COS. Three C-terminal specific antibodies against binding sites along the 1'-8' segment labeled both green and red ROS, but a higher extent of labeling was observed on the green ROS. Antibody rho 3D6, which bound to bovine COS, also labeled frog COS. These results indicate that the F1-F2 loop and segments along to the N-terminus and the C-terminus of bovine rhodopsin show a high degree of homology with corresponding regions of frog rhodopsin from red ROS; the C-terminal 1'-8' segment of bovine rhodopsin is closely related to the corresponding segment of frog rhodopsin from green ROS; and the 1'-4' C-terminus of bovine rhodopsin is highly homologous to segments of opsin from most bovine and frog COS. The labeling of frog red ROS in relation to multiple forms of rhodopsin observed by SDS-gel electrophoresis is discussed.

Animals↗
PubMed1986

Etodolac kinetics in the elderly.

The effects of age and chronic dosing on the pharmacokinetics of the anti-inflammatory drug etodolac were evaluated in healthy young subjects, healthy elderly subjects, and elderly patients with osteoarthritis. After either single or chronic (7 days) dosing, both the healthy elderly subjects and the elderly patients with osteoarthritis had values for etodolac peak concentration, time to reach peak concentration, the AUC from 0 to 24 hours, elimination t1/2, and free fraction that did not differ significantly from those in the young (control) subjects. Despite the expected increases in the peak concentration and AUC from 0 to 24 hours for all groups after chronic dosing, there were no changes in etodolac free fraction, time to peak concentration, or t1/2. Because significant accumulation of etodolac was not observed in our elderly participants, adjustment of dosage when elderly subjects receive etodolac therapy is not indicated.

Absorption↗