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D H Anderson

Publications and source records attributed to D H Anderson.

At least 19 recordsLinked to original sources
PubMed1992

Basic fibroblast growth factor: a potential regulator of proliferation and intermediate filament expression in the retina.

Proliferation of astrocytes, and a concomitant increase of intermediate filaments in astrocytes are two fundamental responses of the CNS to injury. We have previously identified these two events in the retina's response to detachment of the neural retina from the adjoining monolayer of retinal pigmented epithelium. In order to analyze the potential role of basic fibroblast growth factor (bFGF) in these responses, we studied cellular proliferation and intermediate filament protein expression in the retinas of cats and rabbits 4 d and 4 weeks after a single intravitreal injection of 1 microgram of bFGF. Our results show that bFGF stimulates both of these processes in an otherwise normal eye. The eyes that received bFGF had significantly elevated numbers of 3H-thymidine-labeled Müller cells, astrocytes, vascular cells, retinal pigmented epithelial cells, microglia, and macrophages by comparison to control eyes. This proliferation was apparent at 4 d after the injection of bFGF but not after 4 weeks. In control eyes, antibodies to glial fibrillary acidic protein and vimentin labeled intermediate filaments only in the inner (vitread) portion of the Müller cells, the specialized radial astrocytes that span the width of the retina. In eyes that had been injected with bFGF, almost the entire Müller cell cytoplasm was labeled at 4 d after injection; after 4 weeks, the cytoplasmic labeling intensity had increased significantly. Release or activation of endogenous stores of bFGF after injury or disease may be involved in the control of cellular proliferation and intermediate filament expression in the retina and elsewhere in the CNS.

Animals↗
PubMed1991

The role of cysteine 206 in allosteric inhibition of Escherichia coli citrate synthase. Studies by chemical modification, site-directed mutagenesis, and 19F NMR.

Escherichia coli citrate synthase is strongly and specifically inhibited by NADH, but this inhibition can be prevented by reacting the enzyme with Ellman's reagent. We have now labeled the single reactive cysteine covalently with monobromobimane and isolated and sequenced the bimane-containing cyanogen bromide peptide and identified the cysteine as Cys-206. Modeling studies suggest that this residue is on the subunit surface, 25-30 A from the active site. Mutation of Cys-206 to serine (C206S), or of Gly-207 to alanine (E207A), weakened NADH binding and inhibition; when these mutations were present together, NADH binding was weaker by 18-fold and inhibition by 250-fold. The mutations also had small effects on substrate binding at the active site. Cys-206 of wild type enzyme and of the mutant E207A was alkylated with 1,1,1-trifluorobromoacetone and the environment of the fluorine nuclei studied by 19F NMR. With wild type enzyme, the NMR spectrum consisted of two peaks of about equal intensity but different line widths, at -8.65 ppm (line width 11.2 +/- 0.5 Hz) and -7.6 ppm (line width 57 +/- 4 Hz). As the labeled wild type citrate synthase was titrated with KCl, the narrow peak converted to the broad one. The same range of KCl concentrations was needed for this conversion as for the allosteric activation of E. coli citrate synthase. The E207A mutant gave the broader NMR peak almost exclusively. We propose that the fluorine label in wild type citrate synthase exists in two conformational states with different mobilities, exchanging slowly on the NMR time scale, and that treatment with KCl, or truncation of the Glu-207 side chain by mutagenesis, stabilizes one of these states. Consistent with this explanation is the finding that Cys-206 reacts more quickly with Ellman's reagent in the presence of KCl, and that this rate is faster yet in the E207A mutant.

Alkylation↗
PubMed1991

Sequestration of basic fibroblast growth factor in the primate retinal interphotoreceptor matrix.

The interphotoreceptor matrix (IPM) occupies the extracellular space between the photoreceptors of the retina and the apical surface of the retinal pigmented epithelium. A large proportion of the IPM is composed of aqueous-insoluble glycoconjugates, including chondroitin sulfate-containing proteoglycans, the distribution of which exhibits both apical-basal and photoreceptor cell type-specific heterogeneities. The precise function of most insoluble IPM constituents is unknown, although the available evidence suggests some may contribute to retinal adhesion or photoreceptor survival. We have now identified basic fibroblast growth factor (bFGF), or an immunologically related protein from the FGF family, within the IPM. The IPM is labeled on sections of primate retinas by a battery of polyclonal antibodies (Abs) directed against various peptide sequences of bFGF and by an Ab to bovine brain bFGF. bFGF Abs also bind to purified preparations of aqueous-insoluble IPM. All bFGF Abs utilized cross-react with equivalent low molecular mass components of 16.5-17.5 kDa on Western blots of insoluble IPM proteins, purified bFGF, and recombinant bFGF. The Abs do not bind any aqueous-soluble IPM components, suggesting that the bFGF is normally bound to an insoluble IPM constituent(s) in situ. The fact that bFGF is sequestered in the IPM and is located in such close proximity to the photoreceptors, the retinal pigmented epithelium, and Mueller's glia raises the strong possibility that it is synthesized by and regulates the activities of one or more of these three cell types in vivo.

Animals↗
PubMed1991

Opsin distribution and protein incorporation in photoreceptors after experimental retinal detachment.

The distribution of opsin was examined immunocytochemically after experimental retinal detachment in adult cats. Retinal detachments were produced by injecting fluid between the retinal pigment epithelium and neural retina. One to 60 days later the animals were killed. Tissue areas from detached and attached retinal regions from the eye with the detached retina, as well as normal (control) retinas, were processed for post-embedding light and electron microscopic immunocytochemistry. In normal and attached retinal regions, anti-opsin labeled the outer segments and Golgi apparatus most heavily, although the entire photoreceptor plasma membrane was labeled at a low level. Beginning at 2 days after retinal detachment, immunolabeling increased in the photoreceptor inner segment, cell body and synaptic terminal plasma membranes. This pattern of anti-opsin labeling continued at all intervals up through the 60-day detachment time-point. Injection of radiolabeled amino acid in detachments from 1 to 30 days show that radiolabeled protein is still transported to the truncated outer segments of the photoreceptor cells. In addition, these outer segment disks label with anti-opsin. These data imply that opsin continues to be transported and incorporated into the outer segments of photoreceptors showing severe degeneration as a result of long-term detachment from the RPE.

Animals↗
PubMed1991

Equilibrium points for nonlinear compartmental models.

Equilibrium points for nonlinear autonomous compartmental models with constant input are discussed. Upper and lower bounds for the steady states are derived. Theorems guaranteeing existence and uniqueness of equilibrium points for a large collection of system are proved. New information relating to mean residence times is developed. Asymptotic results and a section on stability are included. A recursive process is discussed that generates iterates that converge to steady states for certain types of models. An interesting range of models are included as examples. An attempt is made to provide general qualitative theory for such nonlinear compartmental systems.

Acquired Immunodeficiency Syndrome↗
PubMed1991

A mutant of Escherichia coli citrate synthase that affects the allosteric equilibrium.

We describe a mutant of Escherichia coli citrate synthase, CS R319L, in which the arginine residue at position 319 of the sequence has been replaced by leucine. In this mutant, saturation by the substrate acetyl-CoA is changed from sigmoid (Hill parameter = 1.75 +/- 0.2) to hyperbolic (Hill parameter = 1.0 +/- 0.1) and dependence on the activator KCl is greatly reduced. Further mutations at this site and at position 343 (which model building predicts is close enough to allow a side-chain interaction with position 319) are also described. In the wild-type enzyme, the model suggests the possibility of a salt-bridge interaction between Arg-319 (located on the P helix in the small domain) and Glu-343 (in the Q helix in the same domain), but mutation of Glu-343 to Ala (CS E343A) produced a much smaller difference in the kinetic properties than the ARg-319 to Leu mutation did. Small changes in kinetic properties were also obtained with an Arg-319----Glu (CS R319E) mutation. In CS R319L, oxaloacetate, the first substrate to bind, induces an ultraviolet difference spectrum which is obtained with wild-type enzyme only in the presence of KCl. To account for these observations we postulate that wild-type E. coli citrate synthase exists in two conformational states, T and R, which are equilibrium; T state binds NADH, the allosteric inhibitor, while R state binds substrates and can be converted to another substrate-binding state, R', by KCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1991

Intraretinal proliferation induced by retinal detachment.

Cellular proliferation after retinal detachment was studied by 3H-thymidine light microscopic autoradiography in cats that had experimental detachments of 0.5-180 days duration. The animals underwent labeling 2 hr before death with an intraocular injection of 200 microCi of 3H-thymidine. The number of labeled nuclei were counted in 1-micron thick tissue sections in regions of detachment, in regions of the experimental eyes that remained attached, and in control eyes that had no detachments. In the normal eye, in one that had only the lens and vitreous removed, and in the eyes with 0.5- and 1-day detachments, the number of labeled nuclei ranged from 0/mm (0.5-day detachment) to 0.38/mm (lens and vitreous removed only). By 2 days postdetachment, the number of labeled nuclei increased to 2.09/mm. The highest levels of labeling occurred in two animals with detachments of 3 (7.86/mm) and 4 (7.09/mm) days. Thereafter, the numbers declined steadily until near-baseline counts were obtained at 14 days. The number of labeled nuclei was slightly elevated in the attached regions of two animals with 3-day detachments. Labeled cell types included: Müller cells, astrocytes, pericytes, and endothelial cells of the retinal vasculature, and both resident (microglial cells) and invading macrophages. In an earlier study RPE cells were also shown to proliferate in response to detachment. Thus, these data show that proliferation is a rapid response to detachment, reaching a maximum within 4 days, and that virtually every nonneuronal cell type in the retina can participate in this response. The data suggest that events leading to such clinical manifestations as proliferative vitreoretinopathy and subretinal fibrosis may have their beginnings in this very early proliferative response.

Animals↗
PubMed1990

Crystal structure of alpha 1: implications for protein design.

X-ray diffraction shows the structure of a synthetic protein model, formed from noncovalent self-association of a 12-residue peptide and of sulfate ions at low pH. This peptide is a fragment of a 16-residue polypeptide that was designed to form an amphiphilic alpha helix with a ridge of Leu residues along one helical face. By interdigitation of the leucines of four such helices, the design called for self-association into a four-alpha-helical bundle. The crystal structure (2.7 angstrom resolution; R factor = 0.215) reveals a structure more complex than the design, with both a tetramer and a hexamer. The alpha-helical tetramer with leucine interior has more oblique crossing angles than most four-alpha-helical bundles; the hexamer has a globular hydrophobic core of 12 leucine residues and three associated sulfate ions. Computational analysis suggests that the hexameric association is tighter than the tetrameric one. The consistency of the structure with the design is discussed, as well as the divergence.

Macromolecular Substances↗
PubMed1990

Comparison of photoreceptor-specific matrix domains in the cat and monkey retinas.

Two lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), were used to compare domains within the interphotoreceptor matrices (IPM) of the cat and monkey, two species where the morphological relationship between the retinal pigment epithelium (RPE) and photoreceptors is distinctly different. In the monkey, PNA labeling was heaviest over the cone outer segments and a discrete region of the interphotoreceptor matrix bordering the cone inner and outer segments--a region which has been termed the cone matrix sheath. Near the apical border of the RPE, the outer margin of the PNA-labeled matrix is surrounded by a circular array of apical microvilli. In the cat retina, PNA labeling was highest over a region of the IPM lying between the outer margin of the cone sheath processes and surrounding rod matrix. In contrast, intracellular labeling of cone inner and outer segments was sparse. The RPE apical processes forming the cone sheath were not labeled. In the monkey retina, WGA preferentially labeled rod outer segments and the region of the IPM around rod inner and outer segments. The cone matrix sheath was not preferentially labeled using this lectin. Rod inner segments and cone inner and outer segments were labeled moderately. In the cat retina, WGA labeling was dense over both rod outer segments and cone outer segments as well as the cone sheath. Rod and cone inner segments, as well as the IPM around both rods and cones, were moderately labeled. These observations suggest that the specialized processes arising from the apical surface of retinal pigment epithelial cells, together with photoreceptor-specific extracellular matrix domains, contribute to the formation of specific microenvironments around rod and cone photoreceptor cells.

Animals↗
PubMed1990

Identification and localization of a beta-1 receptor from the integrin family in mammalian retinal pigment epithelial cells.

The distribution of an adhesion receptor from the integrin family was mapped in cultured mammalian retinal pigment epithelial cells (RPE), and in primate RPE cells in vivo, using antibodies to the human fibronectin receptor (FnR) in conjunction with indirect immunofluorescence and immunoelectron microscopy. Protein homogenates from human RPE or MG-63 cells were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. On Western blots of proteins from both cell types, anti-FnR and a monoclonal antibody to the beta 1 subunit of human FnR each recognized a single band with a molecular mass of approximately 115 kDa. After 1-6 weeks in culture human, monkey and feline RPE cells gradually acquired a morphology and cytoskeletal arrangement typical of a mature cuboidal epithelium. The distribution of anti-FnR labeling changed dramatically in accordance with the cells' phenotype. In preconfluent cells, labeling consisted of streaks and flecks of fluorescence at the termini of stress fibers and at putative sites of cell substratum attachment. As the cells became confluent and acquired an epithelioid morphology, the bulk of anti-FnR labeling shifted to the peripheral cytoplasm and appeared as a cross-hatched meshwork. In fully differentiated RPE cells anti-FnR labeling consisted of a dense punctate pattern on or close to the apical cell surface that coincided with the distribution of f-actin as shown by phalloidin staining, and to the distribution of apical microvilli as identified by scanning electron microscopy. In addition, a compacted rim of fluorescence appeared at the cells' lateral margins that was also virtually identical to the phalloidin staining pattern. No basal surface labeling was apparent at this stage. At the ultrastructural level, FnR was localized to the apical surface of nonpermeabilized RPE cells and, in particular, to the plasma membrane of apical microvilli in vitro and in vivo using an indirect, pre-embedding method. The results strongly suggest that: (1) a membrane receptor/s containing the integrin beta 1 subunit is normally present on the plasma membrane of apical microvilli and on the lateral cell surfaces of cultured mammalian RPE cells; and (2) this receptor also is present on the plasmalemma of RPE apical microvilli in vivo.

Animals↗
PubMed1990

Cytoskeletal redifferentiation of feline, monkey, and human RPE cells in culture.

Retinal pigment epithelial (RPE) cells in vivo have a polarized structure with specialized apical and basal faces. Isolated RPE cells lose but eventually regain their epithelial morphology under appropriate culture conditions. We evaluated the ability of isolated feline, primate, and human RPE cells to regain this morphology in culture with scanning electron, transmission electron, phase contrast, and immunofluorescence microscopy. In culture, isolated RPE cells lose their cuboidal shape, their apical microvilli, and their in vivo cytoskeletal organization. Stress fibers from in these cells; microtubules radiate from the cells' center to their periphery; and vimentin filaments radiate from the cells' nucleus to their periphery. As cultures become confluent, RPE cells aggregate into small groups, gradually regaining a cuboidal shape and acquiring microvilli on their apical surface. Filamentous actin redistributes to the apical face where it presumably forms the cytoskeletal core normally present in RPE microvilli. Stress fibers disappear and are replaced by a circumferential microfilament bundle (CMB). Confluent cells surrounding the colonies of differentiated RPE attain a cuboidal shape but do not show complete cytoskeletal redifferentiation. Such cells, while appearing to be differentiated by phase contrast microscopy, fail to develop a compacted CMB. In these cells, f-actin is organized as a loose peripheral band within the cell cytoplasm. Our observations indicate that confluency cannot be equated with the end stage of morphologic differentiation, and that cytoskeletal organization provides a more accurate gauge of RPE maturation in culture.

Actins↗
PubMed1990

Changes in intermediate filament immunolabeling occur in response to retinal detachment and reattachment in primates.

The immunolabeling patterns for vimentin and glial fibrillary acidic protein (GFAP) were studied in five rhesus monkeys that had undergone retinal detachment or detachment and reattachment. Anti-vimentin and anti-GFAP labeling intensity increased in Müller cells after 2 days of detachment. Weak anti-vimentin labeling of the basal RPE cytoplasm, which was absent in control tissue, was detected 2 days after detachment. After detachment for 7 days and reattachment for 7 or 14 days, the pattern and extent of intermediate filament (IF) labeling changed. In Müller cells, the labeling, which in controls was restricted to processes near the vitreal border of the retina, was present in Müller processes spanning the entire retina. In retinal pigment epithelium (RPE) cells, prominent anti-vimentin labeling was identified in the basal and basolateral cytoplasm. The extent of RPE and Müller cell IF labeling in two animals whose retinas had been detached and then reattached for 150 days was different from that found at either the 7- or 14-day reattachment time points. This suggests that the abnormal IF distribution triggered by detachment may be attenuated after a lengthy period of reattachment.

Animals↗
PubMed1990

Immunocytochemical identification of Müller's glia as a component of human epiretinal membranes.

Sections of seven human epiretinal membranes of diverse pathologic origin were labeled with antibodies to cellular retinaldehyde binding protein (CRALBP) and two intermediate filament proteins: glial fibrillary acidic protein (GFAP) and vimentin. All of the membranes studied contained heterogeneous cell populations that exhibited diverse morphologic characteristics. Double labeling with both anti-GFAP and anti-CRALBP positively identified one of the cellular components in these membranes as Müller's glia. In addition, other epiretinal cells exhibited immunolabeling patterns consistent with those found in fibrous astrocytes and retinal pigment epithelial (RPE) cells normally. The results demonstrate that a double-labeling method using CRALBP antibodies, in combination with antibodies to other appropriate antigens, can be used to distinguish between the different epiretinal cell types.

Adult↗
PubMed1989

Changes in the expression of specific Müller cell proteins during long-term retinal detachment.

Retinal detachments were produced in domestic cats by injecting fluid between the retinal pigment epithelium and neural retina. Retinas were allowed to remain detached for 30 or 60 days at which time the animals were killed. Tissue areas from detached and attached retinal regions from the same eye were processed for correlative biochemical and structural analysis, i.e. SDS-PAGE and Western blots of tissue homogenates were correlated with tissue processed for postembedding immunoelectron microscopy. Antibodies to six proteins were used as probes. Glial fibrillary acidic protein in Müller cells has previously been shown to increase after retinal detachment; here we show that vimentin, another intermediate filament protein present in Müller cells, also increases after detachment. In contrast, cellular retinaldehyde binding protein, cellular retinol binding protein, glutamine synthetase, and carbonic anhydrase C--all normally found in Müller cells--decrease after detachment. The results of this study indicate that retinal Müller cells dramatically alter their expression of proteins in response to retinal detachment.

Animals↗
PubMed1989

Nucleotide sequence and expression in Escherichia coli of the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene of Pseudomonas mevalonii.

The mva operon of Pseudomonas mevalonii encodes two enzymes that can convert internalized mevalonate into acetoacetate and acetyl-coenzyme A (CoA). The promoter-proximal gene of this operon is mvaA, the structural gene for 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (EC 1.1.1.88). The cloning, characterization, and expression of mvaA has been reported (M. J. Beach and V. W. Rodwell, J. Bacteriol. 171:2994-3001, 1989). We report here the nucleotide sequence of another gene of this operon, mvaB, its expression in Escherichia coli, and its identification as the structural gene for HMG-CoA lyase (EC 4.1.3.4). P. mevalonii HMG-CoA lyase is a cytosolic protein with 301 amino acid residues and a molecular weight of 31,600. This represents the first reported sequence of an HMG-CoA lyase from any source.

Amino Acid Sequence↗
PubMed1989

Retinal reattachment of the primate macula. Photoreceptor recovery after short-term detachment.

The macula of the neural retina from 12 adult rhesus monkeys (Macaca mulatta) was detached from the overlying retinal pigment epithelium (RPE) by subretinal injection of a balanced salt solution. Seven days later, the two layers were reapposed by draining fluid from the vitreous cavity and replacing it with a 3:1 mixture of sulphur hexafluride gas and air. Animals were sacrificed at 1 hr, 2 days and 7 days after detachment, and at periods ranging from 3 to 14 days after reattachment. At 2-7 days prior to sacrifice, some eyes received an intravitreal injection of 3H-L-fucose. The eyes were then fixed for light and electron microscopy (EM), and tissue sections were processed for autoradiography (ARG) or immunocytochemistry. During the 7-day detachment interval, rod outer segments (ROSs) and cone outer segments (COSs) degenerated, but inner segments remained intact and the rest of the retina appeared normal. The apical RPE surface dedifferentiated during the detachment interval. At 3 days after reattachment, a regrowth of rudimentary ROSs and COSs had occurred, but the disc stacking was clearly abnormal. ROSs and COSs both showed an increase in length and a tendency to return to their normal configurations with increasing time after reattachment. ROSs and COSs regained approximately 40% of their normal lengths after a 2-week reattachment period; however, persistent outer segment abnormalities were frequently found in otherwise well regenerated areas. Autoradiographic results confirmed that new disc members were synthesized subsequent to reattachment. Newly synthesized rod disc membranes were uniformly labeled using antibodies to bovine opsin. Regenerating outer segments interdigitated with newly formed apical RPE processes, and radiolabeled phagosomes were identified within the RPE cytoplasm by 1 week after reattachment. Proliferation of the RPE cell layer was identified at some locations in all animals, and was strongly correlated with a lack of underlying outer segment regeneration. Because of the short detachment interval, and the absence of underlying pathology or trauma, the recovery process described here probably represents an example of optimum recovery after retinal reattachment.

Animals↗