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Biomedical subjects

D B Archer

Publications and source records attributed to D B Archer.

At least 91 records · Page 5Linked to original sources
PubMed1995

Characterization of endothelin A (ETA) and endothelin B (ETB) receptors in cultured bovine retinal pericytes.

PURPOSE: The endothelins are a family of structurally similar vasoactive peptides. It has been shown recently that cultured retinal microvascular endothelial cells secrete endothelin-1 (ET-1) and that corresponding pericytes bear receptors and are responsive to this peptide. These findings suggest a role for ET-1 in the autoregulation of retinal blood flow. There are at least two known subtypes of ET receptors, ETA and ETB. The purpose of this study was to characterize endothelin receptor subtypes on cultured bovine retinal pericytes (BRP). METHODS: To characterize the specific binding sites for ET-1 and ET-3 on monolayers of BRP, a radioligand binding assay was performed using [125I] ET-1 and [125I] ET-3. Competition binding studies with ET-1 and ET-3 were used to assess the heterogeneity of the ET-receptor population on BRP. Also, [125I] ET-1 and ET-3 were covalently linked to their corresponding receptors and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. RESULTS: [125I] ET-1 and [125I] ET-3 showed specific binding to BRP and subsequent Scatchard analysis for both labels showed upward concavity, implying two-site ligand binding. Unlabeled ET-1 was found to displace [125I] ET-1 with greater efficiency than ET-3, indicating the presence of the ETA receptor subtype. Conversely, [125I] ET-3 was displaced by ET-1 and ET-3 with equal potency, indicating a component of ETB in the receptor population. Preincubation with BQ123, an ETA selective antagonist, decreased the binding of [125I] ET-1 but had no effect on [125I] ET-3 binding curves. Affinity cross-linking of the receptors showed two distinct protein bands on SDS-PAGE of 66 and 45 kd, corresponding to ETA and ETB. CONCLUSIONS: These results show that BRP possess ETA and ETB receptor subtypes. The function of ETB on BRP may be to modulate the vasoconstrictive effect of ET-1 caused through ETA.

Animals↗
PubMed1994

Crystal structure of the mutant D52S hen egg white lysozyme with an oligosaccharide product.

The crystal structure of a mutant hen egg white lysozyme, in which the key catalytic residue aspartic acid 52 has been changed to a serine residue (D52S HEWL), has been determined and refined to a crystallographic R value of 0.173 for all data F > 0 between 8 and 1.9 A resolution. The D52S HEWL structure is very similar to the native HEWL structure (r.m.s. deviation of main-chain atoms 0.20 A). Small shifts that result from the change in hydrogen bonding pattern on substitution of Asp by Ser were observed in the loop between beta-strands in the region of residues 46 to 49. D52S HEWL exhibits less than 1% activity against the bacterial cell wall substrate. Cocrystallisation experiments with the hexasaccharide substrate beta(1-4) polymer of N-acetyl-D-glucosamine (GlcNAc6) resulted in crystals between 5 days and 14 days after the initial mixing of enzyme and substrate. Analysis by laser absorption mass spectrometry of the oligosaccharides present after incubation with native and D52S HEWL under conditions similar to those used for crystal growth showed that after 14 days with native HEWL complete catalysis to GlcNAc3. GlcNAc2 and GlcNac had occurred but with D52S HEWL only partial catalysis to the major products GlcNAc4 and GlcNAc2 had occurred and at least 50% of the GlcNAc6 remained intact. X-ray analysis of the D52S-oligosaccharide complex crystals showed that they contained the product GlcNAc4. The structure of the D52S HEWL-GlcNAc4 complex has been determined and refined to an R value of 0.160 for data between 8 and 2 A resolution. GlcNAc4 occupies sites A to D in the active site cleft. Careful refinement and examination of 2Fo-Fc electron density maps showed that the sugar in site D has the sofa conformation, a conformation previously observed with the HEWL complex with tetra-N-acetylglucosamine lactone transition state analogue, the HEWL complex with the cell wall trisaccharide and the phage T4 lysozyme complex with a cell wall product. The semi-axial C(5)-C(6) geometry of the sofa is stabilised by hydrogen bonds from the O-6 hydroxyl group to the main-chain N of Val109 and main-chain O of Ala107. The sugar in site D adopts the alpha configuration, seemingly in conflict with the observation that the hydrolysis of beta (1-4) glycosidie linkage by HEWL proceeds with 99.9% retention of beta-configuration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

A pyruvate decarboxylase gene from Aspergillus parasiticus.

A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase.

Amino Acid Sequence↗
PubMed1994

Altered endothelin-1 induced contraction and second messenger generation in bovine retinal microvascular pericytes cultured in high glucose medium.

The effect of simulated hyperglycaemia on bovine retinal pericytes was studied following culture of these cells for 10 days under normal (5 mmol/l) and elevated (25 mmol/l) glucose conditions in the absence of endothelial cells. Pericytes cultured under high ambient glucose exhibited both a delayed and reduced contractile response following stimulation with endothelin-1. Stimulation with 10(-7) mol/l endothelin-1 for 30 s caused significant contraction in cells grown in both 5 mmol/l and 25 mmol/l glucose. The former also contracted significantly with 10(-8) mol/l endothelin-1. Further, at all concentrations tested, statistical comparison of the time course of contraction showed a significant difference (p < 0.02) in the reduction of planimetric surface area between the two cell groups. Since neither binding of endothelin-1 nor the number of receptors for this peptide were significantly different (p > 0.1) between bovine retinal pericytes grown for 10 days under normo- or hyperglycaemic conditions, it became apparent that the altered contractility in bovine retinal pericytes following culture in high glucose must be due to post-binding intracellular disturbance(s). Indeed, both basal and 15 s post-stimulation with 10(-8) mol/l endothelin-1, levels of inositol trisphosphate were significantly reduced (p < 0.05 and p < 0.02, respectively) in pericytes cultured for 10 days in 25 mmol/l glucose. These results show that endothelial-independent alterations in contractility of pericytes occur when they are grown in conditions which simulate hyperglycaemia. The results also suggest that the observed attenuation in response to endothelin-1 stimulation evident in pericytes grown under simulated hyperglycaemic conditions is not due to alterations in peptide binding.

Animals↗
PubMed1994

Strategies for improving heterologous protein production from filamentous fungi.

Despite the naturally high capacity for protein secretion by many species of filamentous fungi, secreted yields of many heterologous proteins have been comparatively low. The strategies for yield improvement have included the use of strong homologous promoters, increased gene copy number, gene fusions with a gene encoding a naturally well-secreted protein, protease-deficient host strains and screening for high yields following random mutagenesis. Such approaches have been effective with some target heterologous proteins but not others. Approaches used in heterologous protein production from filamentous fungi are discussed and a perspective on emerging strategies is presented.

Animals↗
PubMed1994

Transcriptional and post-transcriptional events affect the production of secreted hen egg white lysozyme by Aspergillus niger.

The relationship between heterologous gene copy number, mRNA and secreted protein yields has been studied in Aspergillus niger transformants containing either the hen egg-white lysozyme (HEWL) cDNA gene or a glucoamylase-HEWL gene fusion (incorporating the A. niger glaA gene). The results support a direct relationship between HEWL gene copy number, mRNA and secreted HEWL protein levels at low (< 25) copy numbers. High protein yields are associated with multiple copies of the recombinant gene at a single site. Fusion of the HEWL gene to the glucoamylase gene resulted in higher steady-state levels of heterologous mRNA. Transformants with the HEWL cDNA alone exhibited a ten-fold higher mRNA:protein ratio than transformants with the gene fusion indicating that post-transcriptional events significantly affect final secreted protein yields.

Animals↗
PubMed1994

Physiological optimization of secreted protein production by Aspergillus niger.

Physiological factors affecting hen eggwhite lysozyme and native glucoamylase production by Aspergillus niger have been examined in batch culture. Expression of the genes encoding both proteins was controlled by the glucoamylase promoter. In standard expression medium (ACMS/N/P), secreted lysozyme yields were found to be maximal at 20-25 degrees C (8-10 mg l-1) and markedly reduced at 30-37 degrees C (3-5 mg l-1). Production of lysozyme exhibited similar induction or repression profiles to that of endogenous glucoamylase such that secreted lysozyme yields could be ordered with respect to growth on the following carbon sources: soluble starch > maltose > glucose >> xylose. Significantly higher yields of up to 30-60 mg l-1 were obtained in a richer medium containing soya milk, although in contrast to growth in ACMS/N/P, the highest levels of secreted lysozyme were achieved at 37 degrees C. This improvement is attributed partly to an increase in culture biomass concentration and to a reduction in medium acidification. Growth in this medium produced a markedly different pellet morphology.

Animals↗
PubMed1994

Ionizing radiation and the retina.

This review considers the effects of ionizing radiation on the retina and examines the relationship between the natural course of radiation retinopathy and the radiobiology of the retinal vascular endothelial cell (RVEC). Radiation retinopathy presents clinically as a progressive pattern of degenerative and proliferative vascular changes, chiefly affecting the macula, and ranging from capillary occlusion, dilation, and microaneurysm formation, to telangiectasia, intraretinal microvascular abnormalities, and neovascularization. The total-radiation dose and fractionation schedule are the major determinants for the time of onset, rate of progression, and severity of retinopathy, although other factors such as concomitant chemotherapy and preexisting diabetes may exaggerate the vasculopathy by intensifying the oxygen-derived free-radical assault on the vascular cells. The differential radiosensitivity of RVECs is attributed to their nuclear chromatin conformation, their antioxidant status, and their environment. We propose pathogenetic mechanisms for radiation retinopathy and suggest that the peculiar latency and unique clinical pattern is related to the life cycle of the RVEC. A rationale is also proposed for the use of radiotherapy in the treatment of subneovascularization and age-related macular degeneration.

Chemotherapy, Adjuvant↗
PubMed1994

Selective loss of vascular smooth muscle cells in the retinal microcirculation of diabetic dogs.

This study was undertaken to further characterise the fine structural changes occurring in the retinal circulation in early diabetes. The eyes of eight alloxan/streptozotocin and three spontaneously diabetic dogs were examined by trypsin digest and electron microscopy after durations of diabetes of between 1 and 7 years. Basement membrane (BM) thickening in the retinal capillaries was the only obvious fine structural change identified during the first 3 years of diabetes and was established within 1 year of induction. Widespread pericyte loss was noted after 4 years of diabetes and was paralleled by loss of smooth muscle (SM) cells, in the retinal arterioles. SM cell loss was most obvious in the smaller arterioles of the central retina. No microaneurysms were noted in the experimental diabetic dogs with up to 5 years' duration of diabetes but were widespread in a spontaneously diabetic animal at 7 years. This study has shown that SM cell loss, a hitherto unrecognised feature of diabetic microangiopathy, accompanies pericyte loss in the retinal circulation of diabetic dogs.

Animals↗
PubMed1994

Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments.

Diabetes mellitus was induced in male beagles by a single injection of an alloxan and streptozotocin cocktail and fasting blood sugar levels maintained between 15 and 20 mmol/l. Five years after induction of diabetes, three diabetic animals were sacrificed, together with sex and age-matched controls, and the retinas fixed for either transmission electron microscopy (TEM) or trypsin digestion. In TEM specimens, capillaries in close proximity to the major vessels were designated as either AE (arterial environment) or VE (venous environment) and the thickness of their basement membranes (BMs) measured using an image analyser based two dimensional morphometric analysis system. Results show that the BMs of retinal capillaries from the diabetic dogs were significantly thicker than those from control dogs. Furthermore, within the diabetic group the AE capillaries had thicker BMs than VE capillaries (p < or = 0.05). The controls, however, showed no significant difference in BM thickness between AE and VE capillaries. Although many of the capillaries designated as AE or VE would actually have been derived from the opposite side of the circulation, with respect to BM thickness, they conformed to values of their specific group. The conclusion is that diabetic capillaries are more vulnerable to BM thickening in an arterial environment than in a venous environment.

Animals↗
PubMed1994

Stereological estimation of Weibel-Palade bodies in the retinal vasculature of normal and diabetic dogs.

The absolute volume of Weibel-Palade (WP) bodies, the storage organelles of von Willebrand factor (vWF), was estimated by a stereological method in a known volume of central retina from normal and 5-year diabetic dogs. The results showed that the volume of WP bodies present in the endothelium of the retinal vasculature varies with blood vessel type and in diabetes. In both diabetic and normal dogs the endothelium of the retinal veins contained a higher volume of WP bodies than that of the retinal arteries. In dogs which had been diabetic for a duration of 5 years the volume of WP bodies present in the endothelium of retinal veins was significantly greater than in the endothelium of veins from the control animals. However, there was no significant difference in the volume of WP bodies present in the endothelium of retinal arteries or capillaries between the two groups of animals.

Animals↗
PubMed1994

The combined effects of diabetes and ionising radiation on the rat retina: an ultrastructural study.

The combined effect of STZ-diabetes and ionising radiation on the rat retina was investigated. Wistar rats, which had been diabetic for 6 months, were irradiated with a single dose of x-rays (1500 cGy) and the ultrastructural effects evaluated at 4-10 mths post-irradiation. At 4 months post-irradiation, the outer nuclear layer of the retina was greatly reduced in thickness and the photoreceptor outer segments were disorganised and reduced in length. In addition, the nerve fibre layer contained many cytoid bodies and there were many redundant basement membrane tubes throughout the inner retina. By 6 months post-irradiation, the photoreceptor cells were virtually absent, bringing the external limiting membrane into close apposition to the RPE. Throughout large areas of the outer retina, RPE cells were hypertrophic and some had proliferated into the inner retina. In many regions, proliferating retinal capillaries were observed within the RPE layer, and at 8 months post-irradiation, some vessels extended into the inner retina accompanied by RPE cells. At 10 months post-irradiation, the RPE was atrophic and degenerative with retinal glial cells coming into contact with Bruch's membrane. In some areas, the glia which had breached Bruch's membrane had invaded the underlying choroid. Where glial cells contacted the choriocapillaries, the vessels assumed the appearance of retinal vessels with plump endothelia and no fenestrations. This study has described a progressive inner retinal ischemia, with cytoid bodies, capillary non-perfusion and general atrophy of the inner retina intensifying markedly with increasing post-irradiation time.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Enzyme production by recombinant Aspergillus.

The use of molds, principally A. niger and T. reesei, in the production of heterologous proteins is becoming more successful. The high levels expected for commercial success have already been attained with a limited number of proteins, and other examples are bound to follow. It must be remembered that, although yields will always need to be maximized, the yield required for a bulk enzyme will be much higher than that for a high-value therapeutic protein. A commercial application is not the only driving force for the use of molds as hosts for heterologous gene expression and protein production. Amenable hosts are necessary for production of sufficient protein for many structural investigations, especially for engineered proteins, and deficiencies are often encountered in alternative hosts. For the expression and production of some proteins, there is no doubt that filamentous fungi will offer the system of choice and be serious contenders for the production of other proteins. Severe problems have been encountered in the development of molds as hosts for heterologous protein production, but I am greatly encouraged in the way these obstacles have been overcome. Problems remain, and it is these that will shape the direction of future research. Optimization of yield still needs to tackled for each protein, and it is true, for example, that fungal proteins tend to be secreted at higher yield than nonfungal proteins. In some cases there is evidence that gene expression is a limiting factor in protein production, but in the main, and especially with A. niger, secretion is the major limitation. Unraveling the secretion process in A. niger and understanding the importance of protein motifs in folding in vivo will be major areas of research activity. Authenticity of the target protein is equally important, with the major limitations being proteolytic modification and glycosylation. Although the degradation of secreted proteins in cultures is a problem, it has already been tackled successfully with the development of proteinase-deficient strains. Proteolytic degradation of proteins during the secretion process and the control of glycosylation during secretion are areas for detailed study. There will undoubtedly be major advances made in the coming years.

Aspergillus↗
PubMed1994

Estimation of the surface area and volume of the retinal capillary basement membrane using the stereologic method of vertical sections.

The modern stereologic method of vertical sections was applied to the retina as a means of generating unbiased estimates of three-dimensional structure. The method is illustrated with real data on the volume and surface area of the capillary basement membrane from the central retina of the rat. Novel methods of estimating the volume of retina sampled and of creating accurate vertical sections are described. The advantages of using stereologic methods to generate quantitative information on the three-dimensional structure of the retina are discussed and compared to those of previous quantitative methods that provide data on two-dimensional structure only.

Animals↗
PubMed1994

Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

PURPOSE: The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). METHODS: Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. RESULTS: Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. CONCLUSION: These results illustrate the internalization and intracellular trafficking by RVECs of insulin and LDL through highly efficient RME, and they provide evidence for at least two possible fates for the endocytosed ligands. This study outlines a route by which vital macromolecules may cross the inner blood-retinal barrier.

Animals↗
PubMed1994

Immunoreactive endothelin distribution in ocular tissues.

PURPOSE: To study the distribution of the endothelins in the eye using ocular tissues of human, rat, and porcine origin. METHODS: Extracts of ocular tissues were examined for immunoreactivity to endothelin 1 and 3 and pro-endothelin 1 using radioimmunoassay. Characterization of immunoreactivity was verified using high-performance liquid chromatography. RESULTS: Immunoreactivity to endothelin 1 and endothelin 3 was found in all ocular tissues except the cornea, which contained no immunoreactivity to endothelin 3. Highest levels were found in the choroid, although species-related differences were present. Immunoreactivity to endothelin 3 was generally twofold to threefold higher than immunoreactivity to endothelin 1. The majority of endothelin-like-immunoreactivity in the retina was blood vessel-associated. High-pressure liquid chromatography (HPLC) confirmed that immunoreactive endothelin 1 and endothelin 3 in the tissue extracts eluted in identical positions to their respective standard synthetic peptides. CONCLUSIONS: The endothelins are abundantly distributed in the eye. Endothelin 1 is present in its mature, 21 amino acid form, and only minimal amounts of the precursor pro-endothelin is found in ocular tissues. The wide distribution patterns point to complex roles for these peptides in blood vessel physiology and in such other functions as regulation of aqueous outflow, and in neurotransmission or modulation.

Aged↗
PubMed1993

A truncated glucoamylase gene fusion for heterologous protein secretion from Aspergillus niger.

The secreted yield of hen egg-white lysozyme (HEWL) from the filamentous fungus Aspergillus niger was increased 10-20-fold by constructing a novel gene fusion. The cDNA sequence encoding mature HEWL was fused in frame to part of the native A. niger gene encoding glucoamylase (glaA), separated by a proteolytic cleavage site for in vivo processing. Using this construct, peak secreted HEWL yields of 1 g/l were obtained in A. niger shake flask cultures compared to about 50 mg/l when using an expression cassette lacking any glaA coding sequence. The portion of glaA used in the gene fusion encoded the first 498 amino acids of glucoamylase (G498) and comprised its secretion signal, the catalytic domain and most of the O-glycosylated linker region which, in the entire glucoamylase molecule, spatially separates and links the catalytic and starch-binding domains.

Animals↗