Serum C-reactive protein in elderly men and women: association with mortality, morbidity and various biochemical values.

OBJECTIVE: The primary aim of this study was to define the distribution and the prognostic value of serum C-reactive protein (s-CRP) measured by a high-sensitivity method in elderly subjects of both genders with special reference to the distribution below 10 mg/l. As a secondary aim, a possible gender difference of s-CRP was examined. MATERIAL AND METHODS: Baseline s-CRP was described in a population-based sample of opposite-sex, twin-pairs (197 F, 189 M available for blood-sampling) aged 71-80 years (mean age 74.5 years), considering mortality through the next 4 years, morbidity (myocardial infarction, angina pectoris, congestive heart failure, arterial hypertension, venous thromboembolism, stroke, diabetes, gout, psoriasis, rheumatoid arthritis) before and after blood sampling, biochemical values (serum levels of urate, urea, ApoA1, ApoB, folate, FSH, LH, oestradiol, progesterone, testosterone, cortisol) and anthropometric measurements (body mass index (BMI), circumference of waist, buttocks and hips). RESULTS: The level of s-CRP did not deviate substantially from what has been reported for younger subjects. Higher values indicated an increased risk of cardiovascular morbidity and diabetes in women but not in men. The s-CRP level was associated with serum levels of urate, progesterone, folate, ApoA1, ApoB and the quotient ApoB/ApoA1 as well as with BMI and waist circumference. CONCLUSIONS: For the 71-80 years age group, s-CRP below the 80th percentile (4.3 mg/l) seems to have prognostic capacity mainly in women. The highest association with mortality as well as with cardiovascular diseases, diabetes and rheumatoid arthritis is found for s-CRP above 10 mg/l, which is the arbitrary lower level for the earlier routine low-sensitivity s-CRP methods. The association of s-CRP with serum urate, folate and the ApoB/ApoA1 quotient should be considered.

Biochemical values in persons older than 82 years of age: report from a population-based study of twins.

According to international standards, reference values for biochemical tests should be obtained in disease-free subjects and for therapies that may influence measurement values. For elderly persons, especially the very old, such a requirement is difficult to meet, since few of these individuals are free of ongoing or former diseases. The present study of a population-based Swedish twin sample comprises 535 individuals (186 males, 349 females) who were at least 82 years of age at blood sampling. Survival over a 6-year period is used as a reference of overall health. Baseline test values for subjects surviving 6 years are compared with results for persons who died in the follow-up period. Results, including mean and median values, standard deviations and ranges, were given for both genders in the total sample. Cases outside the 5th and 95th percentiles were then excluded, as were those who died during the follow-up period. In a third step, cases with disorders and therapies known to influence measurement values were also excluded. The risk of dying during the observation period was calculated across measurements from the lowest and highest 10th and 20th percentiles. The specific impact of proximity to death on the biochemical values, in addition to age effects, was analysed by comparing co-twins discordant for survival over 7 years. Increased mortality was indicated for subjects of both genders with high serum levels of urea, urate, gamma-GT, free thyroxin and plasma homocysteine. In females, low serum values for albumin and total cholesterol were associated with increased mortality. A lower cholesterol level was found 4 years before death, while a lower HDL-cholesterol level was associated with an increased risk of death across the entire 7-year period.

Association of biochemical values with morbidity in the elderly: a population-based Swedish study of persons aged 82 or more years.

BACKGROUND: Various inter-dependent factors influence serum biochemical values. In the elderly, the impact of these factors may differ compared with younger age groups and therefore population-based studies among older people are needed. The specific morbidity in old age, including also various types of drug therapy, should be observed. METHODS: Various biochemical tests in 349 females and 186 males over 81 years of age were carried out and the associations of biochemical values with morbidity, drug therapy, anthropometry and gender were estimated. RESULTS: Biochemical serum values deviate in various diseases, characterized by increased frequency in the elderly, i.e. congestive heart failure, osteoporosis, hip fractures, depression and dementia. All of these diseases present a tendency to increased homocysteine, usually combined with low folate. Cases with intact cognitive function throughout the six years after sampling are characterized by low homocysteine, which is the opposite of what is found in dementia. Furthermore, congestive heart failure is associated with impaired creatinine clearance and increased urea and urate, and osteoporosis and hip fractures are characterized by low albumin and cholesterol. Increased values for urate and impaired creatinine clearance are found in coronary diseases. In gout, multiple biochemical changes take place. For cases with a history of diabetes, arterial hypertension, peptic ulcer and malignancy, few changes are found compared with the values of the total sample. Furosemide therapy is associated with the same pattern as congestive heart failure, and laxative treatment is characterized by low folate and high homocysteine values.

Seven-day extended wear and 30-day continuous wear of high oxygen transmissibility soft silicone hydrogel contact lenses: a randomized 1-year study of 504 patients.

PURPOSE: To compare the performance and complication rates of PureVision (Bausch & Lomb, Inc.) high Dk/t silicone hydrogel lenses in 30-day continuous wear and 7-day extended wear. METHODS: Five-hundred four (504) patients were randomized to 30-day continuous wear (353 patients) or to 7-day extended wear (151 patients) and followed for 12 months. RESULTS: Eighty percent of patients did not report any symptoms/complaints at the follow-up exams. There were no cases of microbial keratitis. The annual incidence of the four most common clinically significant positive slit lamp findings related to lens wear for the 30-day and the 7-day groups, respectively, were as follows: corneal staining, 10.5% and 10.6%; corneal infiltrates, 4.6% and 2.3%; slight epithelial edema, 2.0% and 3.6%; and tarsal conjunctival abnormalities, 1.6% and 3.0%. There was no significant difference in complication rates between the groups. For reasons related to lens wear, 7.6% in the 30-day group and 13.2% in the 7-day group were dropped from the study. The patients' subjective judgment of comfort, visual quality, freedom from dryness, and lens handling varied from 89 to 95 on a scale of 0 to 100. The success rate was 82.9%, slightly better for the 30-day group (84.4%) than for the 7-day group (79.5%). CONCLUSION: The complication rate was low. Sight-threatening events, such as bacterial keratitis did not occur. Inflammatory changes were seen, although at a low rate. Anterior segment changes related to hypoxia were minimal. Limbal and bulbar hyperemiadecreased significantly after switching patients to PureVision lenses. There was no significant difference in complication rates between the 30-day group and the 7-day group.

Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants.

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or alpha-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and alpha-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.

Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content.

Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells. There is an inverse correlation between the melanin content of the eye and the incidence of AMD. Lipofuscin (LF)-accumulation in RPE cells accompanies the process of aging, and may also be related to AMD. This study was designed to evaluate the effect of melanin/melanosomes on the rate of LF formation in cultured rabbit and bovine RPE cells subjected to oxidative stress (40% normobaric O(2)) and daily supplementation with photoreceptor outer segments for 4 weeks. The LF content was measured at 0, 2, and 4 weeks in RPE cells from pigmented and albino rabbits, as well as in pigment-rich and pigment-poor bovine cells. Albino rabbit and pigment-poor bovine cells accumulated significantly higher amounts of LF than pigmented rabbit cells and pigment-rich bovine RPE cells after both 2 and 4 weeks of exposure. Autometallography of melanin-containing cells, without previous exposure to ammonium sulfide, showed a positive outcome, indicating either the occurrence of pre-existing iron-sulphur clusters or an extremely high intrinsic reducing capacity. These results suggest that melanin acts as an efficient antioxidant, perhaps by interacting with transition metals.

Retinal dystrophy of Swedish briard/briard-beagle dogs is due to a 4-bp deletion in RPE65.

The RPE65 gene encodes a 65-kDa microsomal protein expressed exclusively in retinal pigment epithelium (RPE). Mutations in the human RPE65 gene have recently been identified in patients with autosomal recessive, severe, childhood-onset retinal dystrophy. Here we report the characterization of a 2.4-kb canine Rpe65 cDNA. The longest open reading frame predicts a 533-amino-acid protein with a calculated molecular mass of about 61 kDa prior to protein modification. Sequence comparison shows that RPE65 is highly conserved throughout mammalian evolution. We have identified a homozygous 4-bp deletion (485delAAGA) in putative exon 5 of the canine Rpe65 gene in affected animals of a highly inbred kinship of Swedish briard/briard-beagle dogs, in which an autosomal recessive, early-onset, and progressive retinal dystrophy segregates. The deletion results in a frameshift and leads to a premature stop codon after inclusion of 52 canine RPE65-unrelated amino acids from residue 153 onward. More than two-thirds of the wildtype polypeptide chain will be missing, and the mutant protein is most likely nonfunctional (null allele). Clinical features of the canine disease are quite similar to those described in human. Therefore this form of canine retinal dystrophy provides an attractive animal model of the corresponding human disorder with immediate significance for various therapeutic approaches, including RPE transplantation.

Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity.

PURPOSE: Retinal pigment epithelial (RPE) cells slowly accumulate lipofuscin pigment within their acidic vacuolar apparatus as a result of extra- and/or intralysosomal oxidative alterations of phagocytosed photoreceptor outer segments (POS) with consequent imperfect degradation of these structures. In old age, lipofuscin accumulation may become quite substantial. It has been suggested that pronounced accumulation of lipofuscin is related to decreased RPE function and, possibly, to age-related macular degeneration. The aim of the present investigation was to study whether heavy loading with lipofuscin of RPE acidic lysosomes would affect the further phagocytic ability of the cells. METHODS: In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS (artificial lipofuscin) for 4 weeks, resulting in a pronounced lipofuscin accumulation of the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at O and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. The intracellular location of the beads was verified by confocal laser scanning microscopy. RESULTS: Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly (p < 0.001) higher levels in unloaded control cells than in lipofuscin-loaded ones. In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytoplasmic POS emitting reddish fluorescence, while fewer POS were phagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the phagocytic ability of the cells, showed significantly (p < 0.001) higher levels in control cells than in lipofuscin-loaded ones. CONCLUSIONS: Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity. The resulting reduction in ability to cope with the needs of the overlying photoreceptor cells, in order to eliminate the obsolete tips of their POS, may well be of significance in the development of age-related macular degeneration.

Organization of the canine gene encoding the E isoform of retinal guanylate cyclase (cGC-E) and exclusion of its involvement in the inherited retinal dystrophy of the Swedish Briard and Briard-beagle dogs.

Intracellular cyclic GMP concentration is known to change in response to a wide variety of agents, including hormones, neurotransmitters or light. In vertebrate photoreceptors, different membrane-bound guanylate cyclase isoforms are responsible for cGMP synthesis and thus directly involved in termination of light signalling via the phototransduction cascade and recovery of the dark state. We have characterized a 4.7 kb long cDNA for the canine retinal guanylate cyclase isoform E (cGC-E) predicting a polypeptide of 1109 amino acids. The genomic structure and the complete sequence of the canine GC-E gene, which consists of 20 exons and spans about 14.5 kb, has also been determined. Northern blot analysis showed that GC-E was expressed in the canine retina as a 4.7 and 6.1 kb large transcript. RT-PCR analysis also detected low expression in cerebrum (occipital lobe). We performed a sequence analysis of the cGC-E gene in animals of a Swedish Briard and Briard-Beagle dog kinship in which an inherited retinal dystrophy is segregating. Several intragenic DNA polymorphisms were identified and used for segregation analysis which excluded cGC-E as a candidate gene for this type of canine retinal dystrophy.

Prophylactic laser treatment in early age related maculopathy reduced the incidence of exudative complications.

AIM: To investigate the effect of prophylactic laser treatment on drusen area and incidence of exudative lesions in patients with soft drusen maculopathy. METHODS: In a prospective study, patients with early age related maculopathy (ARM) and good visual acuity were randomized to laser treatment or to a control group. Each group consisted of two subgroups: a fellow eye group and a bilateral drusen group. At 3 years, 36 of 38 enrolled patients remained in the study. Photocoagulation was performed with an argon green laser, approximately 100 mild laser burns being placed on and between the drusen in a perifoveal temporal horseshoe-shaped area. Both cases and controls were subjected to fundus colour photographs and fluorescein angiograms at regular intervals, and the drusen area was calculated in both photographs and angiograms. At baseline, there were no significant differences (p > 0.3-0.8) in drusen area between the groups. RESULTS: In the treatment group, mean drusen area decreased significantly in the fundus photographs as well as in the angiograms (p < 0.001). Visual acuity and colour contrast sensitivity (CCS) did not change significantly. All these results are valid also for the subgroups. In the control group, however, mean drusen area increased significantly (p < 0.001). Mean visual acuity decreased significantly (p < 0.01) as did the colour contrast sensitivity along the tritan axis (p = 0.02). For the fellow eye control group (n = 7), the increase in drusen area in fundus photographs and the decrease in CCS along the tritan axis were not statistically significant (p = 0.57 and p = 0.37, respectively). Furthermore, at 3 years, five patients in the control group showed exudative lesions (1/7 in the fellow eye group and 4/12 in the bilateral drusen group), whereas no such complications occurred in the treatment group. One patient developed a small atrophy, however. Thus, there is now a significant difference (p = 0.047), however with a large 95% confidence interval, 0.06-0.46, regarding exudative complications between the treated group and the control group in our small patient material. CONCLUSION: Perifoveal mild laser treatment causes a reduction in drusen area in patients with soft drusen maculopathy and may lower the incidence of exudative lesions.

Location and stability of a newly established eccentric retinal locus suitable for reading, achieved through training of patients with a dense central scotoma.

Six patients, median age 71 years, with a dense central scotoma in one eye and a median visual acuity of 0.06 (20/330) in the same eye, were all (100%) shown by means of fundus photography including a fixation target to preferably use an unfavorable retinal locus for fixation, i.e., within the lesion (scotoma). None of the patients was able to read novel text with the affected eye. A computer and video display system were used to determine the most suitable area above or below the visual field scotoma (below or above the retinal lesion) for reading and the magnification needed at this eccentricity. The same setup was also used for an introductory training in reading single words as well as scrolled text with the aim of establishing a preferred retinal locus (PRL) at a favorable, eccentric position, the trained retinal locus (TRL). Thereafter, the patients were provided with strong positive lenses (median power, 40 D) for reading printed text at a very short reading distance (median, 2.5 cm), first single words, above and below which help lines were printed to facilitate eccentric fixation, and finally, novel text. The total training time was 4 to 5 h. Thereafter, fundus photography showed that five of the patients (83%) used their TRL as their PRL. Reading speed was 71 words per minute (median). Our results seem to indicate that an eccentric PRL favorable for effective reading can be established through training and that a fairly low number of training sessions is required.

The effect of high and low Dk/L soft contact lenses on the glycocalyx layer of the corneal epithelium and on the membrane associated receptors for lectins.

PURPOSE: Bacterial adherence or binding to the target cell is a prerequisite for the initial stage of most infections and seems to be mediated by lectin-like ligands on the bacterial surface and specific receptors on the target cell membrane. The purpose of this study was to establish whether contact lens wear under closed eye conditions changes the glycocalyx layer, whether it exposes more lectin receptors than eye closure without a contact lens, and whether wear of low oxygen transmissibility (Dk/L) contact lenses exposes more receptors than high Dk/L contact lenses. METHODS: The eyes of six rabbits under general anesthesia were fit with either a high Dk/L soft contact lens (40 x 10(-9), boundary corrected) or a low Dk/L soft contact lens (2 x 10[-9]) or were left without a lens as controls. All eyes were kept closed by suturing for 24 hours. After removal of the contact lenses, all corneas were excised, put in glutaraldehydeforfixation, rinsed, incubated with plant-derived lectins (wheat-germ agglutinin [WGA]) conjugated with gold particles, and prepared for electron microscopy. Membrane associated gold particles were counted and the results were processed statistically. RESULTS: After 24 hours of lens wear under closed eye conditions, the glycocalyx layer showed physical changes in the form of thinning or compression and signs of biochemical changes reflected as an increase in number of WGA receptors. The average number of membrane associated gold particles per 750 micro length of corneal epithelium in control corneas was 1,287.5 +/- 92.5. Particles were significantly (P<0.001) more numerous after wear of high Dk/L contact lenses (3,230.0 +/- 294.5) and after wear of low Dk/L contact lenses (4,611.3 +/- 223.3). The figure after wear of low Dk/L contact lenses was significantly (P<0.01) higher than the figure after wear of high Dk/L contact lenses. CONCLUSION: Our results indicate that lens wear under closed eye conditions seems to change the corneal glycocalyx layer physically as well as biochemically. Significantly larger numbers of WGA receptors were exposed after contact lens wear than without a contact lens. Significantly more receptors were exposed after wear of low Dk/L contact lenses than after wear of high Dk/L contact lenses. These changes may be of importance in relation to the risk of bacterial keratitis.

Isolation of canine retinal arrestin cDNA and exclusion of three candidate genes for Swedish Briard retinal dystrophy.

PURPOSE: Mutations of genes encoding various retina-specific proteins are known to cause a wide spectrum of inherited retinal dystrophies in different species. In the canine, several types of genetic retinal dystrophies have been described affecting primarily the photoreceptors and/or the retinal pigment epithelium. We are performing a systematic analysis of canine candidate genes for such diseases to identify the one mutated in the retinal dystrophy in Swedish Briard dogs. METHODS: We isolated and characterised the full length cDNA of canine retinal arrestin by the method of rapid amplification of cDNA ends (RACE). RESULTS: The full length cDNA isolated by us is 1,575 base pairs (bp) long and contains a 1,218 bp-long open reading frame. CONCLUSIONS: The homology of the canine arrestin protein is highest with the human analogue (88.9%) and lowest with mouse arrestin (85.3%). The most obvious sequence differences among the different arrestins are in the extreme carboxyl terminus. PCR-SSCP (single strand conformation polymorphism) analysis and direct sequencing of retinal cDNA didn't provide any evidence that mutations in the canine arrestin gene are responsible for the retinal dystrophy seen in the Swedish strain of Briard dogs. Similar data were obtained for the genes encoding rhodopsin and the beta-subunit of photoreceptor-specific phosphodiesterase by segregation analysis.

Lipids of plasma, retina, and retinal pigment epithelium in Swedish briard dogs with a slowly progressive retinal dystrophy.

Reduced blood levels of long chain polyunsaturated fatty acids have been reported in humans and animals with inherited retinal degenerations. The lipid and fatty acid compositions of plasma, retina, and retinal pigment epithelium of the Swedish Briard dog, which has a very slowly progressive retinal dystrophy that is inherited in an autosomal recessive manner were analysed. The lipid class composition of the pigment epithelium was not different between affected and normal dogs; however, significant differences were found between the retinas of the two groups. Affected dogs had relatively more phosphatidylethanolamine and phosphatidylinositol and less phosphatidylcholine than normal dogs. There was no difference in the fatty acid compositions of plasma and retinal pigment epithelium between affected and normal dogs. However, the retinas of affected dogs had significantly lower levels of 22:5n-3 and 22:6n-3 and higher levels of 18:2n-6, 20:4n-6, and 22:5n-6. The total n-3 fatty acid content was significantly lower in affected retinas (P < 0.001), whereas the content of n-6 fatty acids was significantly higher in affected retinas (P < 0.001). These studies provide evidence for yet another animal model of inherited retinal degeneration with a defect in retinal polyunsaturated fatty acid metabolism. The fatty acid pattern in affected dogs resembles that seen in the retina in n-3 fatty acid deficiency.

Electrophysiology in some animal and human hereditary diseases involving the retinal pigment epithelium.

The present paper surveys slow electrophysiological responses recorded by a d.c. technique in some hereditary eye diseases involving the retinal pigment epithelium (RPE) in animals (English setter dogs and Polish Owczarec Nizinny (PON) dogs with ceroid lipofuscinosis and Briard dogs with a slowly progressive rod-cone dystrophy associated with RPE inclusions) and in humans (Best's disease). The electroretinogram c-wave was typically either decreased in amplitude, lacking or replaced by a negative wave. These c-wave changes could be seen at fairly early stages of disease, when the a- and b-waves of the electroretinogram were still within normal limits.

Ten years of disposable contact lenses--a review of benefits and risks.

The performance and benefits as well as the risks and complications of disposable soft lenses (single use disposable or re-used 2-4 weeks) are reviewed in relation to corneal physiology. Modern designs and advanced technology have resulted in disposable lenses providing excellent visual performance. Fewer subjective symptoms (e.g. dryness and grittiness), slit-lamp findings (such as injection and tarsal abnormalities), and inflammatory responses (papillary conjunctivitis; contact lens-induced acute red eye) have been reported with disposable lenses than with conventional soft lenses. The risk of contact lens-induced keratitis (CLIK) has been reported to be higher for daily wear of disposable lenses than for other lens types by UK investigators. Other studies have shown the risk of CLIK with disposable lenses to be equal to that with conventional soft lenses, and in two extensive Swedish studies, the incidence of severe keratitis was found to be significantly lower for daily wear (DW) of disposable lenses than for DW of conventional soft and rigid gas permeable lenses. Both the total complication rate and the number of unscheduled visits are found to be significantly lower for disposable lenses than for conventional soft lenses in several studies. Comfort has been reported to be significantly better and overall satisfaction significantly greater with disposable lenses than with conventional soft lenses according to many studies. These may be important reasons why the success rate for disposable lens wear is reported to be fairly high, generally 70-90%. The importance of appropriate care for 're-usable disposable lenses' must be stressed. One day disposable lenses, if used as directed, will eliminate the risk factors of inadequate cleaning and disinfection as well as contaminated lens cases.

Lipofuscin accumulation in cultured retinal pigment epithelial cells causes enhanced sensitivity to blue light irradiation.

Lipofuscin accumulates with age within secondary lysosomes of retinal pigment epithelial (RPE) cells of humans and many animals. The autofluorescent lipofuscin pigment has an excitation maximum within the range of visible blue light, while it is emitting in the yellow-orange area. This physico-chemical property of the pigment indicates that it may have a photo-oxidative capacity and, consequently, then should destabilize lysosomal membranes of blue-light exposed RPE. To test this hypothesis, being of relevance to the understanding of age-related macular degeneration, cultures of heavily lipofuscin-loaded RPE cells were blue-light-irradiated and compared with respect to lysosomal stability and cell viability to relevant controls. To rapidly convert primary cultures of RPE, obtained from neonatal rabbits, into aged, lipofuscin-loaded cells, they were allowed to phagocytize artificial lipofuscin that was prepared from outer segments of bovine rods and cones. Following blue-light irradiation, lysosomal membrane stability was measured by vital staining with the lysosomotropic weak base, and metachromatic fluorochrome, acridine orange (AO). Quantifying red (high AO concentration within intact lysosomes with preserved proton gradient over their membranes) and green fluorescence (low AO concentration in nuclei, damaged lysosomes with decreased or lost proton gradients, and in the cytosol) allowed an estimation of the lysosomal membrane stability after blue-light irradiation. Cellular viability was estimated with the delayed trypan blue dye exclusion test. Lipofuscin-loaded blue-light-exposed RPE cells showed a considerably enhanced loss of both lysosomal stability and viability when compared to control cells. It is concluded that the accumulation of lipofuscin within secondary lysosomes of RPE sensitizes these cells to blue light by inducing photo-oxidative alterations of their lysosomal membranes resulting in a presumed leakage of lysosomal contents to the cytosol with ensuing cellular degeneration of apoptotic type. The suggested mechanism may have bearings on the development of age-related macular degeneration.

Characterization of canine rod photoreceptor cGMP-gated cation channel alpha-subunit gene and exclusion of its involvement in the hereditary retinal dystrophy of Swedish Briards.

The nucleotide sequence of the canine rod photoreceptor cGMP-gated cation channel alpha-subunit (cCNCG1) cDNA has been determined. The open reading frame consists of 2073 nucleotides, which encode a putative protein of 691 amino acids. In addition, we have established the exon/intron boundaries of the cCNCG1 gene and determined the complete sequence of six introns of a total of eight. The exon/intron organization (location and length of exons and introns) of the cCNCG1 gene is very similar to that of the human rod photoreceptor cGMP-gated cation channel alpha-subunit gene. We used single-strand conformation polymorphism analysis to search for potential pathogenic sequence changes in the cCNCG1 gene in a Swedish Briard and Briard-Beagle dog kindred, in which an autosomal recessive retinal dystrophy is segregating, a disease which shows phenotypic similarities to retinitis pigmentosa, a heterogeneous group of hereditary and progressive retinal degeneration in human. In intron 3, we found several DNA polymorphisms, which do not cosegregate with the affected status of the dogs, thus excluding cCNCG1 as a candidate gene for the retinal dystrophy in this strain of Swedish Briards.