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D H Anderson

Publications and source records attributed to D H Anderson.

At least 37 records · Page 2Linked to original sources
PubMed1997

Charges, hydrogen bonds, and correlated motions in the 1 A resolution refined structure of the mating pheromone Er-1 from Euplotes raikovi.

A detailed description is given of the structure of the small protein mating pheromone Er-1 at atomic resolution. Emphasis is placed on the locations of charges and hydrogen bonds. The model includes all the protein atoms, anisotropic displacement parameters, four disordered side chains, 22 water molecules, a disordered ethanol, and "riding" hydrogen atoms. Analysis of the model revealed that this dense crystal is perfused by hydrogen-bonding networks of solvent and protein atoms. The termini of helices are capped by hydrogen bonding to solvent and protein atoms, and to symmetry-related molecules. An examination of the valencies and charges of the hydrogen-bonding groups suggests that three of the "water" molecules capping the C termini of two helices, and one other, may instead be NH4 ions. Water molecules mediate all but one of the interhelical hydrogen bonds, and many of the lattice interactions. Regions of the molecule where the atomic vibrations deviate from isotropy are identified. There is almost no overall libration of the molecule allowed by the packing, but the side-chains vibrate relative to the backbone. Four side-chains display alternate conformations. Indirect evidence is presented that the switches between their conformations are correlated and driven by protonation of acidic side-chains. These structural features are discussed in the context of function and stability. Equipped with the analysis of the model, we review the course and results of the refinement of the model against 1 A X-ray diffraction data to a crystallographic R-factor of 12.92%.

Amino Acid Sequence↗
PubMed1997

Characterization of drusen-associated glycoconjugates.

BACKGROUND: Drusen are extracellular deposits that accumulate between the basal lamina of the retinal pigment epithelium and the elastic lamina of Bruch membrane in aging human eyes. Although specific types of drusen are recognized as significant risk factors for the development of both the atrophic and exudative forms of age-related macular degeneration, few studies have focused on defining their molecular composition. As an initial step toward identifying the molecular composition of drusen, assessing the biochemical relation between hard and soft drusen, and identifying potential target molecules for detecting drusen clinically, the authors have analyzed their carbohydrate composition using lectin histochemistry. METHODS: Sections of eyes from human donors containing a spectrum of hard and soft drusen were screened with a battery of 22 fluorescein-conjugated lectins. RESULTS: A specific subset of six lectins bind drusen intensely. No significant differences in lectin binding are observed between any subclass of hard and soft drusen. Some drusen exhibit homogeneous, uniform labeling, whereas others show asymmetrical, heterogeneous distribution of glycoconjugates. CONCLUSION: This study shows that glycoconjugates containing specific carbohydrate residues are present in all classes of hard and soft drusen examined. The observation that hard and soft drusen are bound by the same lectins suggests that they may be related compositionally. Identification of the drusen-associated glycoconjugates shown in this investigation will facilitate studies of drusen genesis and their involvement in the pathogenesis of age-related maculopathy. They may also provide a basis for developing avenues of therapeutic intervention.

Aged↗
PubMed1996

The pathophysiology and development of immunity during long-term subclinical infection with Trichostrongylus colubriformis of sheep receiving different nutritional treatments.

The objective of this study was to investigate the relationship between protein nutrition, the pathophysiology, and acquisition and expression of immunity in long-term subclinical intestinal parasitism in sheep. Growing sheep were either uninfected controls or parasitised for 27 weeks with a daily dose of 2500 larvae of Trichostrongylus colubriformis, whilst they were given access to: (1) a low protein food, (2) a high protein food, or (3) a choice between the two foods, where they were allowed to select their diet. Blood samples were taken weekly for determination of serum albumin, total protein, Ca, P, urea and fructosamine concentrations. At the end of the study all sheep received a single (secondary) challenge infection (30,000 T. colubriformis L3) after treatment with anthelmintic to assess their immune status. The concentrations of sheep-mast cell proteinases (SMCP) in intestinal tissue, the number of circulating eosinophils and the total worm numbers recovered from the intestinal tract were used to investigate the effects of previous nutrition on the acquisition and expression of immunity. From the biochemical variables measured over 27 weeks, only serum fructosamine was affected by the interaction between feeding treatment and parasitism: fructosamine concentrations declined only in the parasitised animals on the low protein food during Weeks 6-15 of infection. This casts doubt on the usefulness of plasma fructosamine levels as an indicator of gastrointestinal parasitism, due to its being influenced by the nutritional environment. Total protein, albumin, calcium and phosphorus concentrations in the serum were affected by parasitism, but independently of feeding treatment. During the period of secondary challenge eosinophil numbers and SMCP concentrations were higher in the parasitised animals, reflecting the animals immune responsiveness. The numbers of worms recovered from the intestine of previously parasitised sheep were low; all three indicators of the development of acquired immunity were unaffected by previous nutritional treatment of the sheep. The results do not support the view that the pathophysiology of long term subclinical intestinal parasitism and the expression of acquired immunity induced by a trickle infection could be affected by the feeding treatment of the sheep (protein nutrition).

Animal Nutritional Physiological Phenomena↗
PubMed1996

A challenging case for protein crystal structure determination: the mating pheromone Er-1 from Euplotes raikovi.

Four different phasing methods have been applied to the determination of the crystal structure of the 40 amino-acid mating pheromone of the unicellular ciliated protozoan Euplotes raikovi. The difficulties, failures and successes in attempts to solve the structure by: (1) molecular replacement, (2) direct phasing using the 'Shake and Bake' algorithm, (3) isomorphous replacement, and (4) multiple-wavelength anomalous dispersion are described. The structure was first solved by molecular replacement, and then was the first successful structure determination by 'Shake and Bake' without the direct involvement of its authors. A description of the current status of the high-resolution refinement of the structure is also given. The model is refined against 1 A resolution data to an R factor of 12.9%, and includes H atoms and discretely disordered side chains.

Journal Article↗
PubMed1996

Fate of biotinylated basic fibroblast growth factor in the retina following intravitreal injection.

Exogenous basic fibroblast growth factor (bFGF) stimulates proliferation of non-neuronal retinal cells in vivo. To help understand how this proliferative effect is mediated, we followed the fate of biotinylated bFGF after injection into the vitreous of normal rabbit eyes. The retinal distributions, binding, and processing of biotinylated bFGF (bFGF-biotin) was examined from 2 hr to 7 days after intravitreal injection using laser scanning confocal microscopy, electron microscopy and Western blot analysis. At 2 hr, bFGF-biotin was detected throughout the extracellular space and on retinal basement membranes. At 6 hr, discrete punctate material first appeared within the cytoplasm of Müller cells, astrocytes, endothelial cells, retinal pigment epithelial (RPE) cells, and ganglion cells. Labeling was also present in the invaginations of the photoreceptor synaptic terminals at this time. This general pattern persisted up to 4 days after injection but was greatly attenuated by post-injection day 7. Labeling in the inner retina decreased progressively over the seven days; whereas labeling in the outer retina, primarily within the RPE, increased at 4 days post-injection and then gradually decreased to nearly undetectable levels by 7 days. Western analysis of retinal protein homogenates following injection showed that an 18 kDa component representing intact bFGF, can be identified up to 1 week following injection. This component, as well as a 15 and 9 kDa biotinylated fragment, showed a progressive reduction during the one week post-injection period. Cross-linking experiments demonstrated that bFGF-biotin binds to three putative receptors with approximate molecular weights of 54, 62, and 110 kDa. These data are consistent with binding of exogenous bFGF to: (a) low affinity bFGF receptors associated with retinal basement membranes; (b) invaginations at the base of photoreceptor synapses; and (c) putative high affinity bFGF receptors on the plasma membranes of glial cells, endothelial cells, RPE cells and ganglion cells. bFGF-biotin apparently binds to, and is then internalized by, the same non-neuronal cell types that are stimulated to proliferate following retinal injuries such as detachment.

Animals↗
PubMed1996

Long-term subclinical infection with Trichostrongylus colubriformis: effects on food intake, diet selection and performance of growing lambs.

Growing lambs (mean liveweight 22.4 kg) were used to test for the effects of long-term subclinical infection with Trichostrongylus colubriformis (2500 larvae per day) on the voluntary food intake (VFI), diet selection and performance of sheep given a choice between two foods of different protein content (low versus high). Sheep were either uninfected controls or parasitised for 27 weeks (Group 27P). Additional sheep were infected for either 9 or 18 weeks (before and after the development of complete resistance to parasites, as assessed by faecal egg counts) and then treated with anthelmintic to remove the parasites (Groups 9P and 18P). Group 18P remained uninfected after anthelmintic treatment whereas Group 9P was infected again for the last 9 weeks after a 9 week worm-free period. The VFI declined or remained static from Week 5 up to Week 11/12 of continuous infection, for the 27P and 18P groups. For 9P sheep, VFI recovered within a few days after the anthelmintic administration and was unaffected during re-infection over the last 9 weeks of the experiment. Diet selection changed in such a pattern that the proportion of the high protein food (prop H) in the selected diet started to increase from Week 6 of the infection. The prop H selected remained higher over the interval 10-18 weeks of the continuous infection for 27P and 18P groups than the uninfected controls. There were no significant differences in diet selection among any of the groups beyond Week 18 of the experiment. The infection caused a growth check from Week 7 to approximately Week 12/14 of dosing; these was no evidence of compensatory growth following the development of resistance or recovery. The results are consistent with the view that sheep given a choice between two appropriate foods will modify their diet selection in order to moderate the adverse effects of subclinical nematode infection. The performance and diet selection of all sheep beyond Week 18 of infection suggest that no obvious nutritional penalties are incurred with the continuation of the infection once the animals have become resistant to parasites.

Animals↗
PubMed1995

A cooperative model for receptor recognition and cell adhesion: evidence from the molecular packing in the 1.6-A crystal structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The crystal structure of the pheromone Er-1 from the unicellular eukaryotic organism Euplotes raikovi was determined at 1.6 A resolution and refined to a crystallographic R factor of 19.9%. In the tightly packed crystal, two extensive intermolecular helix-helix interactions arrange the Er-1 molecules into layers. Since the putative receptor of the pheromone is a membrane-bound protein, whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromone, the interactions found in the crystal may mimic the pheromone-receptor interactions as they occur on a cell surface. Based on this, we propose a model for the interaction between soluble pheromone molecules and their receptors. In this model, strong pheromone-receptor binding emerges as a consequence of the cooperative utilization of several weak interactions. The model offers an explanation for the results of binding studies and may also explain the adhesion between cells that occurs during mating.

Amino Acid Sequence↗
PubMed1995

Vitronectin receptor expression and distribution at the photoreceptor-retinal pigment epithelial interface.

Laser scanning confocal microscopy was employed to map the distribution of integrin immunoreactivity at the photoreceptor-retinal pigment epithelial (RPE) interface of the primate retina, and to determine its relationship to the actin cytoskeleton. Immunolabeling using a polyclonal antibody to the human vitronectin receptor (VnR), a heterodimer containing the alpha v subunit in combination with either the beta 3 or beta 5 subunits, is detected primarily on the apical surface of the retinal pigment epithelium (RPE) in vivo and in vitro. It is also associated with the photoreceptor inner and outer segment cell surfaces. In contrast, immunolabeling using a polyclonal antibody to the human fibronectin receptor (FnR), a heterodimer containing the alpha 5 and beta 1 subunits, is detected principally on the basolateral surface of the RPE and is virtually absent in photoreceptors. A partial three-dimensional reconstruction of the anti-VnR labeling pattern in cone photoreceptors reveals cell surface labeling that originates at the level of the myoid just distal to the outer limiting membrane. It extends distally toward the ellipsoid and terminates at the level of the cone outer segment. Approximately 20-22 immunoreactive foci are distributed evenly around the perimeter of the cone ellipsoid. These foci correspond in number and location to the calycal processes that protrude from the distal portion of the ellipsoid. A double-labeling procedure, employing VnR antibody and a fluorescently labeled phallotoxin (phalloidin), was used to identify regions of VnR co-distribution with filamentous actin (F-actin). One such region includes the VnR-immunoreactive foci at the margins of the cone inner segments and the actin cables that course through the photoreceptor ellipsoid and terminate within the calycal processes. A second zone of co-distribution coincides with the actin-containing, circumferential bundle at the lateral borders of the RPE cells, and a third zone is associated with the apical microvilli of the RPE that ensheath cone outer segments. In order to help identify the specific subunits underlying VnR (alpha v beta 3/5) immunoreactivity, Northern blots of retinal-RPE RNA were probed with alpha 32P-cDNAs to the human alpha v, beta 3, and beta 5 subunits and additional immunolocalization studies were performed using integrin human alpha or beta subunit-specific antisera. The results from these studies strongly suggest that one or more integrins, containing the alpha v and/or beta 5 subunits, are expressed by the photoreceptors and RPE.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1995

Distribution of transforming growth factor-beta isoforms in the mammalian retina.

The distribution of transforming growth factor-beta (TGF-beta) was examined in the posterior segment of the monkey, human, and feline eye using antisera to TGF-beta 1, TGF-beta 2, or TGF-beta 3. A number of different antibodies, tissue processing methods, immunolocalization techniques, and microscopic imaging systems were used in an attempt to gain a more comprehensive picture of TGF-beta isoform distribution in the retina and retinal pigmented epithelium (RPE). The results are generally consistent in identifying one or more of the three TGF-beta isoforms in the cytoplasm of a small, overlapping subset of cells. RPE cells, photoreceptors, Mueller cells, ganglion cells, hyalocytes, and cells associated with choroidal and retinal vessels are all represented in this immunoreactive population. No evidence of extracellular labeling was noted. The intracellular distribution of the three isoforms is distinctly different in photoreceptors. Anti-TGF-beta 1 precursor and anti-TGF-beta 2 immunoreactivity is confined primarily to rod outer segments, whereas anti-TGF-beta 3 immunoreactivities are restricted to mitochondria within inner segments. In the RPE, clusters of anti-TGF-beta 2 positive cytoplasmic granules are located near the cells' lateral borders, whereas anti-TGF-beta 3 labeling is concentrated apically. These results provide baseline information from which new hypotheses regarding the function(s) of TGF-beta isoforms in the retina can be formulated.

Animals↗
PubMed1995

Use of the MIB-1 antibody for detecting proliferating cells in the retina.

PURPOSE: To study intraretinal proliferation as a response to experimental retinal detachment using an antibody that recognizes the nuclear specific antigen Ki-67 in proliferating cells. METHODS: Experimental retinal detachments were produced in cats (1, 3, 7, and 28 days) and rabbits (1, 3, and 7 days). The animals were killed and the eyes were fixed and embedded in paraffin. Histologic sections were processed for immunohistochemistry using the MIB-1 antibody to detect the Ki-67 protein. Labeled cells were identified, and the proliferative response was quantified. RESULTS: In normal cat retina, approximately 0.05 cells per millimeter of retina are labeled. In cat retina detached for 1, 3, 7, or 28 days, the number of cells labeled by MIB-1 is 0.06, 5.03, 1.38, and 0.23 cells per millimeter of retina, respectively. MIB-1 labeling yields an approximate fivefold increase over the number of proliferating cells detected in retinal sections using 3H-thymidine autoradiography. Detachment of the rabbit retina elicits a similar response as measured by MIB-1 immunohistochemistry. CONCLUSIONS: In contrast to 3H-thymidine, which labels cells in S-phase only, the MIB-1 antibody labels proliferating cells regardless of their location within the cell cycle. MIB-1 labeling, therefore, is a more accurate means of evaluating cellular proliferation in the retina and elsewhere in the central nervous system, and it is a relatively simple way of evaluating the effects of agents that may affect this response.

Animals↗
PubMed1994

Rapid changes in the expression of glial cell proteins caused by experimental retinal detachment.

We examined the expression of several proteins normally present in Müller's glia after the production of experimental retinal detachment in adult cats. Retinas were detached for one-half to seven days, after which the tissue was processed for correlative immunocytochemistry and biochemistry. Previous studies demonstrated that the intermediate filament proteins glial fibrillary acidic protein and vimentin, increase after long-term retinal detachment (30 to 60 days), whereas glutamine synthetase, carbonic anhydrase C, and cellular retinaldehyde-binding protein all decrease to barely detectable levels. Alterations in Müller cell protein expression are rapid and specific events that can be detected as early as two days after retinal detachment. By seven days, levels of protein expression are similar to those in the long-term retinal detachments. Within the first week after injury the Müller cell processes hypertrophy and begin forming glial scars, which indicates that early intervention may be required to halt or reverse the effects of detachment.

Animals↗
PubMed1994

Transforming growth factor beta 2 is the predominant isoform in the neural retina, retinal pigment epithelium-choroid and vitreous of the monkey eye.

Several techniques were utilized to assess the levels, disposition and cellular sources of isoforms 1 and 2 of transforming growth factor beta (TGF-beta) in the posterior pole of the monkey eye. Freshly dissected tissues, as well as the saline vehicles in which dissections were performed, were analysed by sandwich enzyme-linked immunosorbent assay. In all tissues TGF-beta 2 was the predominant isoform, with beta 2:beta 1 ratios of 6:1 for neural retina (as ng g-1) and 425:1 for vitreous (as pmol l-1). Retinal pigment epithelium (RPE)-Bruch's membrane-choroid complex contained approximately 10 times the amount of both TGF-beta isoforms as neural retina. For first passage cultures of monkey RPE, TGF-beta 2, but not TGF-beta 1, accumulated over time in conditioned media samples. Immunoreactivity for TGF-beta 2 was detected both in tissue sections of posterior pole, specifically in rod outer segments and RPE, and also in the first passage cultures of RPE. Antibodies to specific peptide sequences of both isoforms localized TGF-beta to the outer segments of rod photoreceptors. The apparent sequestration of TGF-beta 2 in photoreceptor outer segments, as well as the in vitro evidence for possible synthesis and release by RPE, suggest that TGF-beta 2 is an important modulator of visual function acting at the retina-RPE interface.

Animals↗
PubMed1993

Crystal structure of phospholipase A2 from Indian cobra reveals a trimeric association.

Phospholipase A2 (PLA2) from Indian cobra venom (Naja naja naja) was crystallized from ethanol in space group P4(3)2(1)2 in the presence of Ca2+. The x-ray crystal structure was determined to 2.3-A resolution by molecular replacement techniques using a theoretical model constructed from homologous segments of the bovine pancreatic, porcine pancreatic, and rattlesnake venom crystal structures. The structure was refined to an R value of 0.174 for 17,542 reflections between 6.0- and 2.3-A resolution (F > 2 sigma), including 148 water molecules. The 119-amino acid enzyme has an overall architecture strikingly similar to the other known PLA2 structures with regions implicated in catalysis showing the greatest structural conservation. Unexpectedly, three monomers were found to occupy the asymmetric unit and are oriented with their catalytic sites facing the pseudo-threefold axis with approximately 15% of the solvent accessible surface of each monomer buried in trimer contacts. The majority of the interactions at the subunit interfaces are made by residues unique to PLA2 sequences from cobra and krait venoms. The possible relevance of this unique trimeric structure is considered.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of a MAP kinase homologue from pea.

The cdc2 kinases are important cell cycle regulators in all eukaryotes. MAP kinases, a closely related family of protein kinases, are involved in cell cycle regulation in yeasts and vertebrates, but previously have not been documented in plants. We used PCR to amplify Brassica napus DNA sequences using primers corresponding to amino sequences that are common to all known protein kinases. One sequence was highly similar to KSS1, a MAP kinase from Saccharomyces cerevisiae. This sequence was used to isolate a full-length MAP kinase-like clone from a pea cDNA library. The pea clone, called D5, shared approximately 50% amino acid identity with MAP kinases from yeasts and vertebrates and about 41% identity with plant cdc2 kinases. An expression protein encoded by D5 was recognized by an antiserum specific to human MAP kinases (ERKs). Messenger RNA corresponding to D5 was present at similar levels in all tissues examined, without regard to whether cell division or elongation were occurring in those tissues.

Amino Acid Sequence↗
PubMed1993

Recovery of photoreceptor outer segment length and analysis of membrane assembly rates in regenerating primate photoreceptor outer segments.

PURPOSE: Photoreceptor outer segments are in a dynamic state of membrane addition and disposal. This study was undertaken to determine how a standardized period of retinal detachment and varying periods of reattachment affect the renewal process. METHODS: To investigate the effects that retinal detachment and reattachment may have on this process, 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. After a standardized detachment period of 7 days, the two tissue layers were reapposed. Animals were labeled with 3H-fucose and killed at times ranging from 3-150 days after reattachment. RESULTS: During the 7 day detachment period, the majority of rod outer segments (ROS) and cone outer segments (COS) degenerated, but inner segments remained intact. During the first week after reattachment, a rapid increase in rod and cone outer segment length occurred in the absence of disc shedding. This was accompanied by re-establishment of a modified morphologic relationship between the apical processes of the RPE and the regenerating outer segments. ROS and COS regained approximately 40% of their control lengths after a 2 wk reattachment period. By 30 days of reattachment, ROS had regained 72% of their normal length and COS had regained approximately 48%. After 150 days of reattachment, photoreceptor outer segment mean length was not statistically different from control areas. Autoradiographic results confirmed that new disc membranes were synthesized after reattachment. The rate of ROS membrane assembly was subnormal at reattachment time points up to 30 days. CONCLUSIONS: Retinal detachment leads to a reduction in photoreceptor outer segment absolute length and membrane assembly rates. Increasing time of retinal reattachment is positively correlated with an increase in outer segment absolute length and a corresponding increase in membrane assembly rates. This recovery pattern in eyes without underlying pathology and after a relatively brief detachment interval may represent the upper limit of the recovery process.

Animals↗
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↗