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Biomedical subjects

R K Crouch

Publications and source records attributed to R K Crouch.

107 records · Page 6Linked to original sources
PubMed1984

Computer modeling of the recombination reaction of rhodopsin.

Various mechanistic schemes for the recombination reaction of rhodopsin were designed and tested using computer modeling and simulation with data from kinetics experiments. The reaction schemes were mathematically modeled by systems of nonlinear first-order ordinary differential equations (ODEs) with unknown rate constants. Each model was fitted to the experimental data by using a modified simplex algorithm for parameter (rate constant) estimation and Gear's method for solving stiff systems of ODEs. The recombination reaction of rhodopsin was best modeled by branched, multistep reaction schemes which included formation of noncovalent complexes, acid-base equilibria, and acid and base-catalyzed dehydration of a Schiff base intermediate. The biochemical bases for these models are discussed.

Animals↗
PubMed1984

Localization of copper-zinc superoxide dismutase in the endocrine pancreas.

Immunoelectron microscopy and cellular fractionation on sucrose density gradients have been used to examine the intracellular distribution of copper-zinc superoxide dismutase (SOD) in the canine and rat endocrine pancreas. Using rabbit anti-canine copper-zinc SOD as the primary antiserum, immunostaining in canine beta and non-beta islet cells was significantly greater than that in serial sections of the same islet incubated with preimmune serum from the same rabbit. Within the cells, immunostaining was associated with spherical and crystalloid granules as well as with the cytoplasm. Radioimmune assays of cellular fractions or rat islet-cell preparations showed that the granule-rich fraction containing large amounts of insulin was also rich in SOD. This interesting observation of association of SOD with hormone-containing granules suggests that SOD may play a role in protecting these oxidation-sensitive proteins.

Animals↗
PubMed1984

Localization of corneal superoxide dismutase by biochemical and histocytochemical techniques.

Superoxide dismutase activity is found in the cornea of rats, dogs, rabbits and humans. A superoxide dismutase identical to the Cu-Zn enzyme is identified by biochemical and immunochemical methods in the corneal epithelium and endothelium in relative high abundance (90-100 U mg-1 protein). The enzyme quantities detected by radioimmune assay and bioactivity assay are similar, giving evidence that the enzyme present is biologically functional. Immunohistochemical techniques demonstrate the association of the enzyme with the cytoplasm of these layers. Soluble protein extracts of corneal stroma exhibit weak apparent superoxide dismutase activity which is found to be mainly due to copper associated with serum albumin.

Animals↗
PubMed1984

The effect of age on corneal and lens superoxide dismutase.

The enzymatic activity of copper-zinc superoxide dismutase within the cornea and lens of the juvenile (1 week) and adult (26 week) rat has been measured relative to both total soluble protein and immunoreactive protein. Enzymatic activity of corneal superoxide dismutase, localized in the epithelium and endothelium, remains constant with age relative to total soluble protein and the ratio of catalytically active enzyme to immunoreactive enzyme is near unity indicating that the superoxide dismutase present is enzymatically active. Likewise, in the juvenile rat, the majority of lens superoxide dismutase, which is located in the capsule epithelium, is enzymatically active. However, in the adult rat a large portion of the enzyme present is catalytically inactive. The enzymatic activity also decreases relative to total soluble protein with age in both the rat and the rabbit lens. This decrease in protection against toxic radicals and/or the accumulation of enzymatically defective enzyme may be contributing factors to age related lens disorders.

Age Factors↗
PubMed1984

Regeneration of rhodopsin and isorhodopsin in rod outer segment preparations: absence of effect of solvent parameters.

Regeneration of rhodopsin and isorhodopsin from bleached bovine rod outer segment suspensions and 11- or 9-cis retinal occurs at similar rates (second-order rate constants of 1000 M-1s-1 and 900 M-1s-1, respectively) and to a similar extent (maximal regeneration at pH 5.5-7.0). Formation of both pigments follows pseudo-first order kinetics in an excess of retinal at pH 7.0. These results suggest that the mechanisms of regeneration are similar for the 11-cis and 9-cis retinal isomers. Solvent ionic strength, buffer concentration, viscosity, and polarity have no significant effect on the rate of pigment formation, providing further evidence that the retinal binding site in bleached bovine rod outer segments is well protected from the hydrophilic environment.

Animals↗
PubMed1983

Attenuation of streptozotocin diabetes with superoxide dismutase-like copper(II)(3,5-diisopropylsalicylate)2 in the rat.

Experimental diabetes can be produced by agents with specific toxicity for pancreatic islet B cells. This effect has been reported to be modified both in vitro and in vivo by various radical scavengers including the enzyme superoxide dismutase. Copper(II)(3,5-diisopropylsalicylate)2 is lipophilic and possesses superoxide dismutase bioactivity. Prior administration of this compound to male rats appeared to attenuate the severity of streptozotocin-induced diabetes as assessed by glycosuria and glucose tolerance. Diisopropylsalicylate, which has no superoxide dismutase activity, did not alter the severity of streptozotocin-induced diabetes. Rats treated with the copper complex, with streptozotocin or with a combination of the two agents gained 50% less weight than untreated controls, or rats treated with diisopropylsalicylate. The attenuation of diabetes by the copper-complex may represent partial protection of the B cells against streptozotocin damage, although an extrapancreatic, toxic effect cannot be ruled out.

Animals↗
PubMed1983

The immunolocalization of copper-zinc superoxide dismutase in canine tissues.

Copper-zinc superoxide dismutase (CuZn SOD) has been localized in paraffin-embedded sections of formalin-fixed, canine tissues. Staining with the immunoenzyme bridge sequence and the technique utilizing avidin-biotin-peroxidase complex revealed high levels of CuZn SOD in a number of sites. These included hepatocytes, colonic absorptive epithelial cells, ciliated bronchiolar epithelial cells, cells lining the thick ascending limb of Henle's loop in the nephron, endocrine cells producing hormones in pancreatic islets, and neurons in several regions of the central nervous system. The above results are compared with the reported distribution of CuZn SOD as determined by biochemical methods in tissues of various animal species.

Animals↗
PubMed1982

Inhibition of rhodopsin regeneration by cyclohexyl derivatives.

Twenty two compounds, bearing some structural similarity either to the ring-end or aldehyde-end of 11-cis-retinal, have been screened for their effectiveness in decreasing the rate of regeneration of rhodopsin in vitro from 11-cis retinal and bovine opsin. The results of these experiments indicate that only those compounds containing a ring and having at least two methyl groups at the 1,5-ring positions are capable of decreasing the rate of regeneration of rhodopsin. Short-chain aldehydes resembling the aldehyde-end of retinal did not affect the rate of pigment formation. There was no additive effect of combining a dimethylcyclohexyl inhibitor and a short chain aldehyde. The methyl groups at the 1,5-positions may play a special role in anchoring the retinal into the protein binding site. This hypothesis is supported by the failure of the ring demethylated analogue, 11-cis, 1,1,5-desmethyl-5,6-dihydroretinal, to form pigment under the same conditions with which good pigment yields are obtained with 11-cis 5,6-dihydroretinal.

Binding Sites↗
PubMed1982

Absence of abnormal erythrocyte superoxide dismutase, copper, or zinc levels in patients with retinitis pigmentosa.

Blood samples obtained from 22 patients with retinitis pigmentosa, 6 unaffected family members, and 8 unrelated controls showed serum copper and zinc to be in the normal range, contrary to the results in earlier reports. Likewise no significant variation of erythrocyte superoxide dismutase (SOD) and catalase levels was found between the 3 groups or when the patients were grouped by sex, age, or genetic distribution. The SOD proteins from controls and retinitis pigmentosa patients had identical mobilities on gel electrophoresis, isoelectric points, and heat stabilities. Our studies do not support the use of copper, zinc, or SOD in the diagnosis or treatment of retinitis pigmentosa.

Adolescent↗
PubMed1982

Protective role of superoxide dismutase against diabetogenic drugs.

Copper-zinc superoxide dismutase (SOD) is present in relatively high concentrations in the beta-cells of human islets. The activity of the extracted enzyme is partially inhibited upon incubation with the diabetogenic drugs alloxan, streptozotocin, or Vacor. The role of this enzyme in protecting beta-cells against chemically induced diabetes was further investigated. Incubation of intact canine islets with alloxan (0.2 mg/ml) and 4 mM glucose decreased the insulin secretory response by 87% during subsequent exposure to 28 mM glucose. Concomitantly the SOD-specific activity (units of enzyme activity per milligram immunoreactive SOD) decreased 50% in alloxan-exposed islets. When islets were protected from alloxan toxicity by including 28 mM glucose with alloxan, the insulin secretory response and SOD specific activity remained identical to controls. Thus, SOD specific activity correlates with maintenance of beta-cell function. To test the effectiveness of SOD against streptozotocin in vitro, canine islets were incubated 10 min with or without streptozotocin (0.1 mg/ml) with 4 mM glucose; their functional integrity was tested subsequently as the insulin secretory response to 28 mM glucose. Exposure to streptozotocin alone decreased the response by 70%; inclusion of SOD (1.5 mg/ml) before and during exposure to streptozotocin completely prevented this effect. Cyanide-inactivated SOD was not effective. The potential of SOD to prevent streptozotocin-induced diabetes was tested in rats in vivo. SOD injected 10 s or 50 min before streptozotocin prevented or significantly attenuated diabetes. Injection of SOD and streptozotocin simultaneously was much less effective, and cyanide-inactivated SOD was ineffective. No protection was afforded by injection of SOD 12 or 24 h before streptozotocin. Our results support hypotheses that (a) oxygen radicals mediate the beta-cell toxicity of both alloxan and streptozotocin, and (b) beta-cells may be particularly vulnerable to oxygen radical damage.

Alloxan↗
PubMedDate not supplied

Salamander rods and cones contain distinct transducin alpha subunits.

The mammalian retina is known to contain two distinct transducins that interact with their respective rod and cone pigments. However, there are no reports of a nonmammalian species having two distinct transducins. In the present study, we report the cloning and cellular localization of two transducin a subunits (G alpha t) from the tiger salamander. Through degenerate polymerase chain reaction (PCR) and subsequent screening of a salamander retina cDNA library, we have identified two forms of G alpha t. When compared to existing sequences in GenBank, the cloned subunits showed high similarity to rod and cone transducins. The salamander G alpha t-1 has 91.2-93.7% amino acid sequence identity to mammalian rod G alpha t subunits and 79.7-80.9% to mammalian cone Gats. The salamander G alpha t-2 has 86.2-87.9% sequence identity to mammalian cone G alpha ts and 78.9-80.9% to mammalian rod G alpha ts at the amino acid level. The G alpha t-1 cDNA encodes 350 amino acids while the G alpha t-2 cDNA encodes 354 residues, which is typical for rod and cone G alpha ts, respectively, and we thus identified the G alpha t- 1 as rod and G alpha t-2 as cone G alpha t. Sequences identified as effector binding sites and GTPase activity regions are highly conserved between the two subunits. Genomic Southern blot analysis showed that rod and cone G alpha t subunits are both encoded by single-copy genes. Northern blot analysis identified retina-specific transcripts of 3.0 kb for rod G alpha t and 2.6 kb for cone G alpha t. Immunohistochemistry in the flat-mounted salamander retina demonstrated that rod G alpha t is localized to rods, predominantly in the outer segments; similarly, cone G alpha t is localized to cone outer segments. The results confirm that the two sequences encode rod and cone transducins and demonstrate that this lower vertebrate contains two distinct transducins that are localized specifically to rod and cone photoreceptors.

Amino Acid Sequence↗
PubMedDate not supplied

Salamander UV cone pigment: sequence, expression, and spectral properties.

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (lambdamax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a lambdamax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of lambdamax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.

Amino Acid Sequence↗
PubMedDate not supplied

Rhodopsin phosphorylation in rats exposed to intense light.

The damaging effects of intense light on the rat retina are known to vary depending on the time of day of exposure. The purpose of this study was to determine if rhodopsin phosphorylation patterns, a measure of the activity of the pigment, varied in a similar manner. After 10 min in strong light (1400 lux), all six threonine and serine sites in the rat rhodopsin C-terminus were phosphorylated, with mono- to tetraphosphorylation being substantially more prominent than penta- to hexaphosphorylation. The level and multiplicity of rhodopsin phosphorylations were reduced both with the duration of light exposure and the duration of subsequent darkness. Although showing vast differences in susceptibility to light damage, rats exposed at 5 P.M. or 1 A.M. showed similar rhodopsin phosphorylation levels and patterns. These data indicate that a process controlled by circadian rhythm other than rhodopsin phosphorylation is involved either in damaging or mediating the damage evoked by intense light exposure.

Animals↗