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Biomedical subjects

R E Anderson

Publications and source records attributed to R E Anderson.

At least 37 records · Page 2Linked to original sources
PubMed2001

Protection of photoreceptor cells in adult rats from light-induced degeneration by adaptation to bright cyclic light.

Light history has been shown to affect the susceptibility of the albino rat retina to the damaging effects of constant light exposure. Retinas of animals raised in relatively bright cyclic light are protected against light-induced degeneration compared with dim-reared animals. These effects were observed in animals raised from birth in bright cyclic light and are part of an adaptive response that protects photoreceptors from stress-induced degeneration. To determine if retinas of adult animals are capable of such adaptive changes or flexibility by switching between different light environments which do not pathologically damage photoreceptor cells, albino rats were maintained in less than 250 lux cyclic light for more than 3 weeks. At 12-13 weeks of age, they were placed into 800 lux cyclic light for 1 week, after which they were exposed to constant illumination of 1500-lux for 1, 3 or 7 days. Retinal function was evaluated by electroretinography and photoreceptor cell death was quantified by measuring outer nuclear layer thickness. After 1 week in bright cyclic light, the retinas were completely protected against 1 day constant light exposure that significantly damaged retinas of animals without 800 lux cyclic light adaptation. Significant protection was also observed in 3 day constant light exposed animals; limited protection occurred after 7 days exposure. These results indicate that the retinas of adult rats adapted to bright cyclic light within certain ranges that did not significantly damage photoreceptor cells are protected from constant light challenge. This phenomenon is a post-developmental response that demonstrates a remarkable plasticity of the retina. The mechanism(s) underlying the ability of this adaptation/flexibility in protecting photoreceptors could involve endogenous molecules that encompass many aspects of retinal cell and molecular biology and physiology. Identification of these molecules may provide insight into the development of therapeutic approaches to treat retinal degeneration.

Adaptation, Ocular↗
PubMed2001

Increase in serum S100A1-B and S100BB during cardiac surgery arises from extracerebral sources.

BACKGROUND: Elevated levels of serum S100B after coronary artery bypass grafting may arise from extracerebral contamination. Serum S100B content was analyzed in several tissues, and the two dimers S100A1-B and S100BB were analyzed separately in blood. METHODS: Serum, shed blood, marrow, fat, and muscle were studied in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass using suction either to the cardiotomy reservoir (group 1, n = 10) or to a cell-saving device (group 2, n = 10), or operated on off-pump (group 3, n = 10). RESULTS: Serum S100B was sixfold higher in group 1 than in groups 2 and 3, which were identical. The same ratio between S100A1-B and S100BB was found in all groups. When compared with serum, S100B was 10(2) to 10(4) times higher in marrow, fat, muscle tissue, and shed blood. CONCLUSIONS: Separate analysis of S100A1-B and S100BB did not distinguish between S100B of cerebral and extracerebral origin. The concept that S100B only originates in astroglial and Schwann cells is wrong. Fat, muscle, and marrow in mediastinal blood contain high levels of S100B. Cardiopulmonary bypass caused no increase in S100B.

Adult↗
PubMed2001

Being awake intermittently during propofol-induced hypnosis: a study of BIS, explicit and implicit memory.

BACKGROUND: Being awake during anaesthesia is a serious complication. An anaesthetic depth monitor must discriminate in real time between wakefulness and unconsciousness. The present study created a period of wakefulness during propofol-induced hypnosis. Bispectral index (BIS), explicit and implicit memories of the awake period were investigated. METHODS: Ten volunteers were studied. The calculated brain concentration of a target controlled infusion of propofol was increased until loss of response (LOR) to verbal command and then propofol was stopped. When fully awake, volunteers were presented with a picture, sound and smell. Propofol infusion was restarted until LOR and then ceased. BIS and the calculated brain concentration of propofol were recorded every minute. A structured interview was conducted for explicit memories after awakening and for explicit as well as implicit memories the day after. RESULTS: Median BIS-index for the transition between awake and asleep and vice versa differed significantly. It was not possible, however, to establish any threshold value or zone for discriminating between wakefulness and LOR due to the large inter-individual variations in BIS-index. No volunteer could explicitly recall any of the stimuli presented during the period of wakefulness. CONCLUSION: The BIS-index decreases with increasing sedation but because of the large individual variations, the real-time BIS-index for the individual subject cannot reliably discriminate wakefulness from unconsciousness during propofol infusion. Propofol causes such profound amnesia that lack of postoperative recall does not assure that episodes of awareness have not occurred during propofol-induced hypnosis.

Acoustic Stimulation↗
PubMed2001

A study of bispectral analysis and auditory evoked potential indices during propofol-induced hypnosis in volunteers: the effect of an episode of wakefulness on explicit and implicit memory.

The bispectral index (BIS) and the auditory evoked potential (AEP) index as calculated by the new A-line monitor were measured during hypnosis with propofol, which included an episode of wakefulness. Both indices followed a similar pattern during sedation, with values decreasing with sedation and increasing when awake. Baseline AEP values varied between 60 and 98, and BIS values were between 96 and 98. The AEP-index value was at all times 10-20 points lower than the BIS-index. The transition to loss of response occurred at a mean AEP value of 46 and BIS value of 58. The transition to just responding following a period of unconsciousness occurred at a mean AEP value of 46 and BIS 65. Both monitoring techniques, however, displayed large interindividual variations making it impossible to discriminate in real time between subtle changes of clinical state. The new neurophysiological monitors A-line AEP and BIS are interesting tools for creating a better understanding of the anaesthetic effects of drugs; however, further refinements are required before their relative roles can be fully established in the clinical setting.

Adult↗
PubMed2001

High serum S100B levels for trauma patients without head injuries.

OBJECTIVE: Studies of patients with head trauma have demonstrated a correlation between a serum marker of brain tissue damage, namely S100B, and neuroradiological findings. It was recently demonstrated that the increases in serum S100B levels after heart surgery have extracerebral origins, probably surgically traumatized fat, muscle, and bone marrow. The current study examined multitrauma patients without head trauma, to determine whether soft-tissue and bone damage might confound the interpretation of elevated serum S100B concentrations for patients after head trauma. METHODS: A commercial assay was used to determine serum S100B concentrations for a normal population (n = 459) and multitrauma patients without head injury (n = 17). Concentrations of the two subtypes of S100B (S100A1B and S100BB) were determined using separate noncommercial assays. RESULTS: The mean serum S100B concentration for a normal healthy population was 0.032 microg/L (median, 0.010 microg/L; standard deviation, 0.040 microg/L). The upper 97.5% and 95% reference limits were 0.13 and 0.10 microg/L, respectively. No major age or sex differences were observed. Among trauma patients, serum S100B levels were highest after bone fractures (range, 2-10 microg/L) and thoracic contusions without fractures (range, 0.5-4 microg/L). Burns (range, 0.8-5 microg/L) and minor bruises also produced increased S100B levels. S100A1B and S100BB were detected in all samples. CONCLUSION: Trauma, even in the absence of head trauma, results in high serum concentrations of S100B. Interpretation of elevated S100B concentrations immediately after multitrauma may be difficult because of extracerebral contributions. S100B may have a negative predictive value to exclude brain tissue damage after trauma. Similarly, nonacute S100B measurements may be of greater prognostic value than acute measurements.

Adolescent↗
PubMed2001

Systemic administration of phenyl-N-tert-butylnitrone protects the retina from light damage.

PURPOSE: This study was conducted to test the hypothesis that phenyl-N-tert-butylnitrone (PBN), a spin-trapping agent known to cross the blood-brain barrier and protect the brain from ischemia-reperfusion injury, is incorporated into the retina after intraperitoneal injection and protects photoreceptor cells from the damaging effects of constant visible light. METHODS: Albino rats were injected intraperitoneally with PBN (aqueous solution) or water, or were not injected, and then were placed in constant light (2700 lux) for 24 hours. The incorporation of PBN into the retina was determined by high-performance liquid chromatography. Electroretinograms (ERGs) were recorded before light treatment and 1 and 15 days after the cessation of exposure to constant light. Eyes were taken for histology at each time point and outer nuclear layer (ONL) thickness determined. RESULTS: PBN was incorporated into the retina after intraperitoneal injection. Both control (water-injected and uninjected) groups exposed to constant light maintained only 28% of ONL thickness and 20% of retinal function, compared with the unexposed control group. In contrast, the PBN-treated animals maintained 80% of ONL thickness and exhibited 87% of retinal function. CONCLUSIONS: PBN protects the albino rat retina from the damaging effects of constant light stress. That light-induced and hereditary retinal degenerations share certain morphologic hallmarks and follow a similar apoptotic mechanism of degeneration raises the possibility of pharmacologic therapy for hereditary and environmentally induced neurodegenerative disorders.

Animals↗
PubMed2001

Low docosahexaenoic acid levels in rod outer segment membranes of mice with rds/peripherin and P216L peripherin mutations.

PURPOSE: Humans with retinitis pigmentosa and dogs with progressive rod-cone degeneration (prcd) have lower than normal blood levels of long-chain polyunsaturated fatty acids, including docosahexaenoic acid (DHA), the major fatty acid found in retinal rod outer segments (ROS). In addition, prcd-affected dogs have lower levels of DHA in their ROS than control animals. The present study was designed to determine whether mice that are heterozygous for the rds mutation and transgenic mice heterozygous for a specific rds/peripherin mutation (P216L) have lower DHA levels in their ROS and other tissues than do control mice. METHODS: Wild-type (rds(+/+)) mice, mice with the rds(-/-) (null) and rds(+/-) mutations, and mice with the P216L rds/peripherin mutation on the rds(+/-) background were maintained in the vivarium under identical husbandry conditions, and tissues were removed from each group for analysis at approximately 2 months of age. Fatty acid compositions of total lipids from plasma, red blood cells, liver, and ROS were determined by gas-liquid chromatography. ROS purity from each group was determined by SDS-PAGE with silver staining. The morphologic status of retinas representing each genotype was analyzed by light and electron microscopy. RESULTS: There was no difference between rds(+/-), P216L on rds(+/-), and rds(+/+) (control) animals in the fatty acid composition of plasma, expressed as relative mole percent or as nanomoles fatty acid per milliliter of plasma. Small but statistically significant differences were found in 18:0 and C-22 polyunsaturated fatty acids of red blood cells. In the liver, the control animals had higher levels of 20:4n-6. In contrast, the ROS of control animals had levels of DHA that were 1.4 times that of ROS from either rds(+/-) or P216L on rds(+/-) mice of the same age. The reduction in DHA was not accompanied by an increase in 22:5n-6, which always occurs in neural tissues of animals deprived of n-3 fatty acids. SDS-PAGE of the three ROS membrane preparations showed that they were of identical purity. CONCLUSIONS: Mice heterozygous for the spontaneous rds/peripherin mutation or mice carrying the P216L mutation on this heterozygous background have a statistically significant reduction of DHA in their ROS membranes. The authors propose that reduction in DHA is an adaptive response to metabolic stress caused by the mutation.

Animals↗
PubMed2001

Impaired synthesis of DHA in patients with X-linked retinitis pigmentosa.

Many patients with X-linked retinitis pigmentosa (XLRP) have lower than normal blood levels of the long-chain polyunsaturated omega3 fatty acid docosahexaenoic acid (DHA; 22:6omega3). This clinical trial was designed to test whether down-regulation of DHA biosynthesis might be responsible for these reduced DHA levels. DHA biosynthesis was assessed in five severely affected patients with XLRP and in five age-matched controls by quantifying conversion of [U-(13)C]alpha-linolenic acid (alpha-LNA) to [(13)C]DHA. Following oral administration of [U-(13)C]alpha-LNA, blood samples were collected at designated intervals for 21 days and isotopic enrichment of all omega3 fatty acids was determined by gas chromatography/mass spectroscopy. Activity of each metabolic step in the conversion of alpha-LNA to DHA was determined by comparison of the ratios of the integrated concentration of (13)C-product to (13)C-precursor in plasma total lipid fractions. The ratio of [(13)C]DHA to [(13)C]18:3omega3 (the entire pathway) and that of [(13)C]20:5omega3 to [(13)C]20:4omega3 (Delta(5)-desaturase) were significantly lower in patients versus controls (P = 0.03 and 0.05, respectively). The estimated biosynthetic rates of [(13)C]20:5omega3, [(13)C]22:5omega3, [(13)C]24:5omega3, [(13)C]24:6omega3, and [(13)C]22:6omega3 were significantly lower in XLRP patients (42%, 43%, 31%, 18%, and 32% of control values, respectively; P < 0.04), supporting down-regulation of Delta(5)-desaturase in XLRP. The disappearance of (13)C-labeled fatty acids from plasma was not greater in XLRP patients compared with controls, suggesting that XLRP was not associated with increased rates of fatty acid oxidation or other routes of catabolism.Thus, despite individual variation among both patients and controls, the data are consistent with a lower rate of Delta(5)-desaturation, suggesting that decreased biosynthesis of DHA may contribute to lower blood levels of DHA in patients with XLRP.

Adult↗
PubMed2001

Interaction of the insulin receptor beta-subunit with phosphatidylinositol 3-kinase in bovine ROS.

PURPOSE: To identify the tyrosine-phosphorylated protein(s) in bovine rod outer segments (ROS) that are associated with phosphatidylinositol 3-kinase (PI3K). METHODS: Glutathione-S-transferase (GST) fusion proteins containing two SH2 domains of the p85 regulatory subunit of PI3K-GST-p85 (N-SH2), GST-p85 (C-SH2), and respective SH2 mutants (N-SH2, R358A, and C-SH2, R649A)-were prepared and used to pull down tyrosine-phosphorylated proteins in bovine ROS. Protein identity was established by Western blot analysis. PI3K activity was determined in the pull-down mixtures and in immunoprecipitates by incubation with phosphatidylinositol-4,5-bisphosphate (PI-4,5-P(2)) and [gamma(32)P]adenosine triphosphate (ATP). RESULTS: The GST pull-down assays indicated the binding of a 97-kDa protein by GST-p85 (N-SH2) in tyrosine-phosphorylated (PY)-ROS that was not present in nonphosphorylated (N)-ROS. Binding was completely abolished when the Arg 358 in the N-SH2 domain was mutated to Ala. Increased binding of the p110alpha catalytic subunit to GST-p85 (N-SH2) fusion protein was also observed in the presence of the 97-kDa phosphorylated protein. Biochemical evidence indicated that the 97-kDa protein was the beta-subunit of the insulin receptor beta-subunit (IRbeta). Immunoprecipitates of PY-ROS and N-ROS with anti-PY antibodies, probed with anti-IRbeta, indicated the presence of IRbeta only in PY-ROS. Immunoprecipitates of PY-ROS and N-ROS with anti-IRbeta antibodies, probed with anti-p85 and anti-p110alpha antibodies, indicated increased amounts of both p85 and p110alpha in PY-ROS compared to N-ROS. Treatment of ROS with insulin, followed by immunoprecipitation with either anti-IRbeta or anti-PY, resulted in increased PI3K activity. Expression and phosphorylation of the cytoplasmic tail of retina insulin receptor showed direct involvement with the p85 subunit of PI3K in vitro. CONCLUSIONS: Tyrosine phosphorylation of the beta-subunit of the insulin receptor is involved in the regulation of PI3K activity in ROS.

Animals↗
PubMed2000

Techniques of intraoperative cerebral blood flow measurement.

Understanding the physiological mechanisms of cerebral blood flow (CBF) is of great importance to neurosurgeons engaged in the management of patients with cerebrovascular disease. Over the past 50 years, techniques to measure CBF and mathematical methods to calculate CBF have evolved substantially. For the cerebrovascular surgeon, intraoperative CBF monitoring is an important adjunct in certain intra- and extracranial procedures. The authors review current techniques in use for the intraoperative measurement of CBF.

Brain↗
PubMed2000

Tyrosine phosphorylation is involved in phosphatidylinositol 3-kinase activation in bovine rod outer segments.

PURPOSE: We have previously shown that phosphatidylinositol 3-kinase (PI 3-kinase) activity is present in bovine rod outer segments (ROS). The present study was undertaken to investigate the mechanism of PI 3-kinase activation in these membranes. METHODS: Tyrosine-phosphorylated ROS (PY-ROS) were obtained by incubating ROS with ATP, MgCl2, and orthovanadate (Na3VO4), a tyrosine phosphatase inhibitor. Non-phosphorylated ROS (N-ROS) were obtained by incubating ROS under the same conditions, but without ATP and orthovanadate. Both were subjected to immunoprecipitation using antibodies against the regulatory p85 (anti-p85) subunit of PI 3-kinase, the catalytic p110 (anti-p110) subunit of PI 3-kinase, or phosphotyrosine (anti-PY). The immunoprecipitates (IPs) were assayed for PI 3-kinase activity. Enzyme assay products were separated by thin-layer chromatography (TLC), deacylated, and identified by high performance liquid chromatography (HPLC). RESULTS: PI 3-kinase activity in anti-p85 and p110alpha IPs was significantly higher in PY-ROS than in N-ROS. No enzyme activity was recovered in anti-p110beta IPs. PI 3-kinase activity in anti-PY IPs from PY-ROS was six-fold greater than those from N-ROS. Immunoblot analysis showed that the amount of p85 in PY IPs from PY-ROS was significantly higher than those from N-ROS. However, tyrosine phosphorylation of p85 and p110alpha was not observed in anti-p85 and anti-p110alpha IPs that were probed with anti-PY. CONCLUSIONS: This study indicates that the p85/p110alpha complex of PI 3-kinase is present in ROS and tyrosine phosphorylation is involved in the regulation of its activity.

Adenosine Triphosphate↗
PubMed2000

Gender differences and the effects of synthetic exogenous and non-synthetic estrogens in focal cerebral ischemia.

The role of gender difference and estrogen in ischemic cerebrovascular events is controversial. Evidence is lacking as to whether or not there are significant gender differences in the incidence and outcome of stroke in the clinical setting. Recent clinical epidemiological studies have demonstrated that there is no significant association between the use of hormonal replacement therapy and the risk of stroke. However, several animal studies have shown that there are gender differences in stroke outcome and that exogenous administered estrogens are neuroprotective. In this study, the influence of gender differences and the effects of synthetic and non-synthetic estrogens were examined in a model of focal cerebral ischemia using 210 male, intact female, and ovariectomized female rats. All animals underwent 3 h of middle cerebral artery and bilateral common carotid artery occlusion. After 72 h, the rats were sacrificed and stained for histological assessment of infarction. There were no gender differences in infarction volume. Intravenous administration of either low or high dose 17 beta-estradiol or tibolone did not alter infarct volume. Subcutaneous administration of low and high dose 17beta-estradiol using 7-day release pellets did not alter infarct volume. Low dose tibolone using implanted 7-day release pellets did not alter infarct volume. However, high dose tibolone using implanted 7-day release pellets significantly (P<0.05) reduced infarct volume only in ovariectomized female rats. These results demonstrate that estrogen therapy has no effect on infarction volume following severe focal cerebral ischemia.

Animals↗
PubMed2000

Exogenous spermine reduces ischemic damage in a model of focal cerebral ischemia in the rat.

Alterations in polyamine metabolism during and after global or focal cerebral ischemia can produce a multiplicity of effects on brain such as modification in mitochondria calcium buffering capacity, exacerbating glutamate-mediated neurotoxicity, and impairment of the blood-brain barrier. In this study, the endogenous polyamine spermine was administered intravenously 30 min prior to temporary focal cerebral ischemia in rats induced by clipping of the left middle cerebral and bilateral common carotid arteries for 3 h. Three days after removal of the microclips, intracardiac perfusion with 2% 2,3,5-triphenyl tetrazolium chloride was performed. Coronal slices were cut, photographed, and examined for cortical infarct volume. Spermine reduced infarct volume in a dose-dependent fashion. This study demonstrates that the use of polyamines may be considered as a powerful tool in prevention of ischemic tissue damage following focal cerebral ischemia.

Animals↗
PubMed2000

Is intracellular brain pH a dependent factor in NOS inhibition during focal cerebral ischemia?

The interaction between nitric oxide (NO.) and focal cerebral ischemia is multifaceted. Experiments have shown that inhibition of nitric oxide synthase (NOS) either ameliorates or exacerbates focal cerebral ischemia. Recent in vitro experiments have shown that NOS activity is pH-dependent. Previous work from this laboratory has demonstrated that N(G)-nitro-L-arginine-methyl-ester (L-NAME) mitigated cerebral ischemia independent from regional cerebral blood flow (rCBF) changes during moderate focal cerebral ischemia. This study examined the effects of L-NAME inhibition on brain pH(i), rCBF, and NADH redox state during 3 h of severe focal cerebral ischemia. Fifteen fasted rabbits under 1.5% halothane were equally divided into three groups: ischemic controls and two drug groups receiving either 1.0 or 10 mg/kg L-NAME intravenously 30 min prior to ischemia. In the ischemic controls, brain pH(i) declined from 6.95+/-0.04 to 6.60+/-0.05, rCBF declined from 48+/-7 to 10+/-3 ml/100 g/min, and NADH fluorescence increased by 149+/-15% 3 h after onset of ischemia (p<0.01 for all three parameters). L-NAME at either dose did not significantly alter these values. Infarct volume was not significantly different between both the L-NAME treated groups and the ischemic control group. This data suggests that during severe focal cerebral ischemia, NO. mechanisms of injury have a less important punitive role. One possible explanation is that the severity of acidosis secondary to anaerobic metabolism during severe focal cerebral ischemia attenuates NOS activity.

Animals↗
PubMed2000

The effect of cardiotomy suction on the brain injury marker S100beta after cardiopulmonary bypass.

BACKGROUND: An increase of S100beta in serum during cardiopulmonary bypass (CPB) has been interpreted as a sign of brain injury. Cardiotomy suction may cause fat embolization, and its role in the S100beta increase was examined. METHODS: Twenty coronary artery operation patients were randomly assigned to two groups, 10 with suction during CPB to cardiotomy reservoir (CR), 10 to cell saving device (CS). S100beta was measured (immunoassay) in blood from the patients and from cell saving device after processing. In 7 additional patients S100beta was measured in the cell saving device before processing and directly from the wound at sternotomy. RESULTS: Before anesthesia, serum S100beta was 0.03+/-0.06 microg/L. At the end of CPB it was 2.47+/-1.31 microg/L and 0.44+/-0.27 microg/L (CR vs CS; p < 0.001). S100beta was 33+/-12 microg/L in CS reservoir and 42+/-18 microg/L in blood from the wound. CONCLUSIONS: Most serum S100beta after CPB with cardiotomy suction may be of extracerebral origin. S100beta after CPB with cell saving device was the same as after off-pump operation. The interpretation that an increase in S100beta during CPB in patients reflects cerebral injury must be questioned.

Aged↗
PubMed2000

Effects on the bispectral index during medium-high dose fentanyl induction with or without propofol supplement.

BACKGROUND: The search for a drug-independent monitor to determine depth of anaesthesia and hypnosis continues. The bispectral analysis (BIS) of the EEG correlates well with the clinical dose-response of hypnotic drugs during induction, but the effect on BIS of an opiate induction, as for coronary bypass surgery, is not known. METHODS: Fourteen patients scheduled for elective coronary bypass surgery were studied. BIS was recorded during induction in 7 patients receiving 10 microg/kg fentanyl without any hypnotic agent and in 7 patients receiving 0.5 mg/kg propofol before the fentanyl dose. RESULTS: The effect of fentanyl was very variable both regarding BIS and clinical response. Five of the 7 patients that received only fentanyl lost their response to verbal command within 8 min. BIS values at loss of response varied between 45 and 94. One patient remained awake with BIS 43. All 7 patients receiving propofol before the fentanyl dose lost their response to verbal command within 5 min. BIS values at the time for loss of response varied between 78 and 98. CONCLUSION: Loss of response to verbal command when a medium-high dose of fentanyl is used for induction cannot be distinguished from wakefulness with adequate sensitivity by BIS. The current BIS algorithm seems not to accurately reflect the hypnotic effects of fentanyl.

Aged↗
PubMed2000

Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate is regulated by light in retinal rod outer segment (ROS) membranes. We recently reported that the activities of phosphatidylinositol synthetase and phosphatidylinositol 3-kinase are also higher in bleached (light-exposed) ROS (B-ROS). In this study, we investigated the effect of bleaching on diacylglycerol (DAG) kinase (DAG-kinase) activity in bovine and rat ROS membranes prepared from dark-adapted (D-ROS) or bleached (B-ROS) retinas. In bovine ROS, DAG-kinase activity toward endogenous DAG substrate was higher in B-ROS than in D-ROS. Quantification of DAG in both sets of membranes showed that the levels were the same, eliminating the possibility that the greater DAG-kinase activity was due to higher levels of endogenous substrate in B-ROS. DAG-kinase activity was also higher in B-ROS against an exogenous, water-soluable substrate (1, 2-didecanoyl-rac-glycerol), which competed with endogenous DAG substrate and saturated at approximately 2 mM. Immunoblot analysis with an anti-DAG-kinase gamma polyclonal antibody demonstrated that the gamma isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal sections confirmed the presence of DAG-kinase gamma immunoreactivity in ROS, as well as other retinal cells. Quantification of the immunoreactive products on western blots showed that more DAG-kinase gamma was present in B-ROS than in D-ROS. In an in vivo experiment, ROS prepared from rats exposed to 30 min of room light had greater DAG-kinase activity than ROS prepared from dark-adapted animals. Taken together, these data suggest that light exposure leads to the translocation of DAG-kinase from the cytosol to ROS membranes and that the greater DAG-kinase activity in B-ROS is due to the presence of more protein associated with ROS membranes.

Animals↗