The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems.

Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using µ-calpain knockout (KO) mice in comparison with control wild-type (WT) mice, and evaluate the subsequent effects of silencing this gene on other proteolytic systems. No differences in muscle development between genotypes were observed during the early stages of growth due to the up regulation of other proteolytic systems. The KO mice showed significantly greater m-calpain protein abundance (P < 0.01) and activity (P < 0.001), and greater caspase 3/7 activity (P < 0.05). At 30 wk of age, KO mice showed increased protein:DNA (P < 0.05) and RNA:DNA ratios (P < 0.01), greater protein content (P < 0.01) at the expense of lipid deposition (P < 0.05), and an increase in size and number of fast-twitch glycolytic muscle fibers (P < 0.05), suggesting that KO mice exhibit an increased capacity to accumulate and maintain protein in their skeletal muscle. Also, expression of proteins associated with muscle regeneration (neural cell adhesion molecule and myoD) were both reduced in the mature KO mice (P < 0.05 and P < 0.01, respectively), indicating less muscle regeneration and, therefore, less muscle damage. These findings indicate the concerted action of proteolytic systems to ensure muscle protein homeostasis in vivo. Furthermore, these data contribute to the existing evidence of the importance of the calpain system's involvement in muscle growth, development, and atrophy. Collectively, these data suggest that there are opportunities to target the calpain system to promote the growth and/or restoration of skeletal muscle mass.

Associations of genetic markers in cattle receiving differing implant protocols.

The potential interaction of growth-promoting implants and genetic markers previously reported to be associated with growth, carcass traits, and tenderness was evaluated. Two implant protocols were applied to subsets of steers (n = 383) and heifers (n = 65) that were also genotyped for 47 SNP reported to be associated with variation in growth, fat thickness, LM area, marbling, or tenderness. The "mild" protocol consisted of a single terminal implant [16 mg estradiol benzoate (EB), 80 mg trenbalone acetate (TBA) or 8 mg EB, 80 mg TBA given to steers and heifers, respectively]. The "aggressive" protocol consisted of both a growing implant (8 mg EB, 40 mg TBA) for the lightest half of the animals on the aggressive protocol and 2 successive implants (28 mg EB, 200 mg TBA) given to all animals assigned to the aggressive treatment. Implant protocol had measurable impact on BW and ADG (P < 0.05), with the aggressive protocol increasing these traits before the terminal implant (relative to the mild protocol), whereas the mild protocol increased ADG after the terminal implant so that the final BW and ADG over the experimental period were similar between protocols. Animals on the aggressive protocol had significantly increased (P < 0.05) LM area (1.9 cm(2)), slice shear force (1.4 kg), and intact desmin (0.05 units), but decreased (P < 0.05) marbling score (49 units) and adjusted fat thickness (0.1 cm), and yield grade (0.15 units). Among both treatments, 8 of 9 growth-related SNP were associated with BW or ADG, and 6 of 17 tenderness-related SNP were associated with slice shear force or intact desmin. Favorable growth alleles generally were associated with increased carcass yield traits but decreased tenderness. Similarly, favorable tenderness genotypes for some markers were associated with decreased BW and ADG. Some interactions of implant protocol and genotype were noted, with some growth SNP alleles increasing the effect of the aggressive protocol. In contrast, putative beneficial effects of favorable tenderness SNP alleles were mitigated by the effects of aggressive implant. These type of antagonisms of management variables and genotypes must be accounted for in marker assisted selection (MAS) programs, and our results suggest that MAS could be used to manage, but likely will not eliminate negative impact of implants on quality.

Effects of manipulation of the caspase system on myofibrillar protein degradation in vitro.

Apoptosis via the intrinsic caspase 9 pathway can be induced by oxidative stressors hydrogen peroxide (H2O2) and N-(4 hydroxyphenol) rentinamide (fenretinide), a synthetic retinoid. Accelerated muscle atrophy and proteolysis in muscle-wasting conditions have been linked to oxidative stress and activated protease systems. Therefore, the hypothesis of this study was that proteolysis of myofibrillar proteins could be manipulated through the induction or inhibition of the caspase system. After slaughter, LM and supraspinatus muscles from callipyge (n = 5) and normal (n = 3) lambs were excised, finely diced, and incubated with treatment buffers containing oxidative stressors fenretinide or H2O2, recombinant caspase 3, caspase-specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (DEVD), or control solution. Muscle samples were incubated for 1, 2, 7, and 21 d at 4°C. Activation of the initiator caspase, caspase 9, and myofibrillar protein degradation was determined by SDS-PAGE and Western blotting. Results showed that fenretinide, H2O2, and recombinant caspase 3 increased (P < 0.05) proteolysis of myofibril proteins, whereas DEVD inhibited degradation (P < 0.05). Proteolysis of myofibrillar proteins increased with incubation time (P < 0.0001), and incubation time × treatment interactions (P < 0.05) indicated that the treatment effects did not all occur at the same rate. This study has shown that manipulation of the caspase system through induction or inhibition of activity can affect degradation of myofibrillar proteins, providing further evidence that the caspase system could be involved in postmortem proteolysis and tenderization. However, these stimulated changes were not sufficient to overcome the lack of proteolysis that is characteristic of muscle from callipyge lambs.

Contribution of genetic influences to animal-to-animal variation in myoglobin content and beef lean color stability.

Longissimus thoracis steaks from steers (n = 464) with 0 to 50% inheritance of Angus, Charolais, Gelbvieh, Hereford, Limousin, Red Angus, and Simmental were evaluated during 6 d of display to assess genetic contributions to color stability. Color space values [CIE L* (lightness), a* (redness), b* (yellowness)], chroma, color change (DeltaE), and surface metmyoglobin (K/S 572/525) were determined on d 0 and 6 of display. Myoglobin concentration was highly heritable (0.85), but ultimate pH was weakly heritable (0.06). Day 0 L* values were moderately heritable (0.24). Variation in metmyoglobin, L*, and DeltaE on d 6 was moderately explained by genetic factors (41, 40, and 29%, respectively). Change during display was moderately heritable for a* (0.31), b* (0.23), chroma (0.35), and surface metmyoglobin (0.29). At the start of display, Angus steaks had greater (P < 0.05) L* values than those from all breeds except Charolais. On d 6, Angus steaks had greater (P < 0.05) L* (50.0) values than Gelbvieh, Hereford, and Simmental steaks (46.1, 44.0, and 44.5, respectively). Day 0 values for a*, b*, chroma, and DeltaE were not affected by breed (P > 0.05). On d 6, a* values were greater (P < 0.05) for Charolais and Limousin steaks (31.1 and 30.5) than Angus, Hereford, and Red Angus steaks (27.4, 27.7, and 26.3, respectively). Thus, a* changed less (P < 0.05) in Charolais and Limousin steaks (1.8 and 2.6, respectively) vs. steaks from other breeds. Day 6 b* values were greater (P < 0.05) in Charolais (24.5) and Limousin steaks (24.0) vs. Gelbvieh (22.2), Hereford (21.9), and Red Angus steaks (21.4). Thus, b* values changed less (P < 0.05) in Charolais and Limousin steaks (1.5 and 1.7, respectively) than in Angus, Gelbvieh, Hereford, and Red Angus steaks (4.3, 3.8, 4.4, and 5.1, respectively). After 6 d of display, Charolais and Limousin steaks had greater chroma (P < 0.05; 39.5 and 38.8, respectively) compared with Angus, Hereford, and Red Angus steaks (35.4, 35.3, and 33.9, respectively). Less (P < 0.05) change in chroma occurred for Charolais and Limousin (2.1 and 2.8, respectively) than in Angus, Gelbvieh, Hereford, and Red Angus steaks (7.1, 6.6, 7.4, and 9.0, respectively). Myoglobin concentration was less for Charolais and Limousin (P < 0.05; 2.77 and 2.72, respectively) compared with Gelbvieh, Red Angus, and Simmental steaks (3.62, 3.43, and 3.71, respectively). Breeds did not differ in pH (P > 0.05). These data suggest Charolais- and Limousin-carcasses produced steaks with greater lean color stability than Angus, Hereford, and Red Angus carcasses. Furthermore, these findings suggest that genetics contribute substantially to animal-to-animal variation in lean color, particularly in maintaining color.

The caspase proteolytic system in callipyge and normal lambs in longissimus, semimembranosus, and infraspinatus muscles during postmortem storage.

The objective of this experiment was to determine whether the caspase proteolytic system has a role in postmortem tenderization. Six ewes and 6 wethers that were noncarriers and 6 ewes and 6 wethers that were expressing the callipyge gene were used for this study. Caspase activities were determined in LM at 7 different time points during the postmortem storage period: 0 h, 4 h, 8 h, 24 h, 2 d, 7 d, and 21 d and in semimembranosus (SM) and infraspinatus (IS) muscles at 0 h, 8 h, 24 h, and 7 d from callipyge and noncallipyge (normal) lambs. Calpastatin activity was determined at 0 h, 2 d, 7 d, and 21 d and slice shear force measured at 2, 7, and 21 d in the LM. Calpastatin activity and slice shear force were greater in LM from callipyge lambs than normal lambs at each time point (P < 0.001 and P < 0.0001, respectively). Caspases 3 and 7 are executioner caspases, and their combined activity was found to decrease during the postmortem storage period in LM, SM, and IS muscles from callipyge and normal lambs. Similarly, activity of the initiator caspase (caspase 9) decreased (P < 0.05) in all 3 muscles across the postmortem storage period in callipyge and normal lambs, and its decrease in activity preceded that of the executioner caspases 3/7. A positive relationship also was detected between caspase 9 and caspase 3/7 in LM, SM, and IS muscles (P < 0.0001, r = 0.85, r = 0.86, r = 0.84, respectively), which is consistent with caspase 9 being responsible for the cleavage and activation of the executioner caspases (caspase 3/7) downstream. Caspase 3/7 and caspase 9 activities at 8 h in SM were greater in normal lamb than callipyge lamb (P < 0.05), with a trend for caspase 3/7 activity to be greater at 24 h postmortem (P = 0.0841). There also was a trend for caspase 3/7 activity to be greater in LM at 21 d in normal lamb than in callipyge lamb (P = 0.053), although there were no differences detected in caspase activities between genotypes in the IS muscle, which is not affected by the callipyge gene. A negative relationship also was detected between peak caspase 3/7 activity at 8 h in LM from normal lambs and calpastatin activity at 0 and 2 d (r = -0.65, r = -0.68, respectively, P < 0.05). This relationship was not observed in LM from callipyge lambs, suggesting that caspase 3/7 may be cleaving calpastatin in normal lambs but the level of calpastatin in callipyge lambs is such that caspase 3/7 cannot degrade it sufficiently to overcome the increased content of calpastatin, and thus, calpastatin activity is the overriding factor in postmortem proteolysis in these animals. There was no direct evidence from this study that caspases have a significant role in postmortem tenderization, but they may have some role through calpastatin degradation.

The effect of recombinant caspase 3 on myofibrillar proteins in porcine skeletal muscle.

The objective of this study was to investigate the potential role of the caspase protease family in meat tenderisation by examining if caspase 3 was capable of causing myofibril protein degradation. Full-length human recombinant caspase 3 (rC3) was expressed in Escherichia coli and purified. The rC3 was active in the presence of myofibrils isolated from porcine longissimus dorsi muscle (LD) and retained activity in a buffer system closely mimicking post mortem conditions. The effect of increasing concentrations of rC3, incubation temperature, as well as incubation time on the degradation of isolated myofibril proteins were all investigated in this study. Myofibril protein degradation was determined by SDS-PAGE and Western blotting. There was a visible increase in myofibril degradation with a decrease in proteins identified as desmin and troponin I and the detection of protein degradation products at approximately 32, 28 and 18 kDa with increasing concentrations of rC3. These degradation products were analysed using MALDI-TOF mass spectrometry and identified to occur from the proteolysis of actin, troponin T and myosin light chain, respectively. The production of these degradation products was not inhibited by 5 mM EDTA or semi-purified calpastatin but was inhibited by the caspase-specific inhibitor Ac-DEVD-CHO. The temperature at which isolated myofibrils were incubated with rC3 was also found to affect degradation, with increasing incubation temperatures causing increased desmin degradation and cleavage of pro-caspase 3 into its active isoform. Incubation of isolated myofibrils at 4°C for 5 days with rC3 resulted in the visible degradation of a number of myofibril proteins including desmin and troponin I. This study has shown that rC3 is capable of causing myofibril degradation, hydrolysing myofibril proteins under conditions that are similar to those found in muscle in the post mortem conditioning period.

Changes in caspase activity during the postmortem conditioning period and its relationship to shear force in porcine longissimus muscle.

The objective of this study was to investigate the protease family caspases in skeletal muscle and their potential contribution to postmortem proteolysis and meat tenderization. Ten Large White gilts were slaughtered, and samples of LM were taken at 0, 2, 4, 8, 16, 32, and 192 h after slaughter and immediately snap frozen in liquid nitrogen. Samples were subsequently analyzed for caspase 3/7 and caspase 9 activity, protein levels of known caspase substrates, alpha II spectrin and poly (ADP-ribose) polymerase (PARP), as well as, at 192 h, shear force. Specific degradation products of alpha II spectrin and PARP, which are known indicators of caspase activity, and apoptosis were detected on immunoblots of muscle samples taken over the postmortem period. The relationships between the changes observed in caspase activities and protein levels of PARP and spectrin across the entire postmortem conditioning period were investigated (n = 70). Protein levels of alpha II spectrin cleavage products across the conditioning period were found to correlate positively to caspase 3/7 activity (r = 0.38, P = 0.003) and caspase 9 activity (r = 0.32, P = 0.012), indicating that caspase-mediated cleavage was occurring in situ. There was a negative relationship between shear force and the 0 to 32 h ratio of caspase 3/7 (r = -0.62, P = 0.053) and caspase 9 activities (r = -0.68, P = 0.044). In addition, there was also a negative relationship between shear force and the level of the caspase-generated alpha II spectrin 120 kDa degradation product (r = -0.75, P = 0.012). The findings of this study indicate that changes in caspase activity and caspase-mediated cleavage take place in muscle during the conditioning period, and this could be associated with the development of tender meat.

Comparison of the relative expression of caspase isoforms in different porcine skeletal muscles.

The present study investigated the relationship between muscle type and components of the caspase protease system in porcine trapezius (TZ), psoas (PS), longissimus dorsi (LD) and semitendinosus (ST) muscles. Muscles were classified according to slow and fast myosin heavy chain (MHC) content determined by western blotting. MHC slow, but not MHC fast protein expression was significantly different between muscles (p<0.001). Protein levels of caspases 3, 8 and 12 and the caspase inhibitor apoptosis repressor with caspase recruitment domain (ARC) were determined. In addition the level of caspase 3 mRNA and activity levels of caspase 3/7 were determined. There was a significant difference in protein levels and activity between muscles (p<0.01), although no difference was observed in mRNA abundance. The data show that multiple components of the caspase system are expressed in porcine skeletal muscle and that their levels are variable, but there is not a distinct association of expression with a particular muscle.

Photoreceptor function in heterozygotes with insertion or deletion mutations in the RDS gene.

PURPOSE: To understand the pathophysiology of human retinal degenerations caused by mutations in the peripherin/RDS gene. METHODS: Three families with autosomal dominant retinal degeneration were found to have mutations in the peripherin/RDS gene. There were two frameshift mutations: a 1-base pair (bp) insertion at codon 32 and a 2-bp deletion at codon 193. For these mutations, the predicted proteins would be truncated by 303 and 131 amino acids, respectively. The third mutation would result in an 8-bp substitution for five nucleotides involving codons 67-69 and would be predicted to disrupt the second transmembrane domain of the protein. Heterozygotes were examined clinically and with rod and cone perimetry, dark adaptometry, and rod- and cone-isolated electroretinograms (ERGs). RESULTS: Rod and cone sensitivity losses were present with perimetric testing in most patients; patients with advanced disease in all three families showed more pericentral than peripheral field dysfunction. The kinetics of dark adaptation were abnormal in all patients. Rod and cone ERG a-waves were normal in maximum amplitude in three younger patients but were reduced in all others; phototransduction was normal in most patients. There was equal loss of rod and cone a-wave amplitudes and equal elevation of rod and cone thresholds. CONCLUSIONS: Heterozygotes with these different peripherin/RDS gene mutations showed variation in clinical presentation but a similar pattern of receptor abnormalities. Results of visual function tests were consistent with a normal amount of rod and cone outer segment membrane in early disease, progressing to reduced outer segments at later stages. There was an equal effect on rod and cone photoreceptor function at all stages of disease. This functional phenotype may represent the human analogue of the rds/+ mouse.

RDS gene mutations causing retinitis pigmentosa or macular degeneration lead to the same abnormality in photoreceptor function.

PURPOSE: To investigate functional abnormalities in mutations in the peripherin (RDS) gene leading to different clinical types of autosomal dominant retinal disease--macular degeneration and retinitis pigmentosa. METHODS: Patients from two families, one with a mutation in codon 167 (Gly167Asp) leading to macular degeneration and another with a mutation in codon 210 (Pro210Ser) leading to retinitis pigmentosa, were studied with clinical examinations and measurements of rod and cone sensitivities and dark adaptation, electroretinography, and rhodopsin levels. RESULTS: Mildly affected patients had sizable rod and cone electroretinograms, reduced levels of rhodopsin, and minor losses of sensitivity. In both mutations, there were delays of rod and cone dark adaptation after bleaching, and the adaptational abnormalities were observed in peripheral and central retinal locations. Analysis of the kinetics of rod adaptation indicates that the underlying abnormalities are similar in both mutations and that the effects of the mutations are similar to those caused by mild systemic vitamin A deficiency. CONCLUSIONS: Patients with the Gly167Asp and Pro210Ser mutations in the peripherin/RDS gene have widely different clinical phenotypes but show the same abnormality, slowed dark adaptation, of rod and cone photoreceptor function. The similarities of the characteristics of the adaptational abnormalities in the two genotypes suggest that, in addition to the structural roles normally assumed for it, peripherin influences or participates in the function of the visual cycle.

Phenotypes of stop codon and splice site rhodopsin mutations causing retinitis pigmentosa.

PURPOSE: To understand the pathophysiology of retinitis pigmentosa caused by mutations in the rhodopsin gene that lead to truncation of the protein. METHODS: Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels. RESULTS: There was a range of severity of disease expression in each family, some heterozygotes having moderate or severe retinal degeneration and others with a mild phenotype. The mildly affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter mutations both had prolonged recovery of sensitivity, but the time course was different in the two genotypes. CONCLUSIONS: There is allele specificity for the pattern of retinal dysfunction in the Q64ter, intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three mutations suggests the mutant opsins interfere with normal rod cell function, and there is subsequent rod and cone cell death.

Retinal morphology and visual pigment levels in 6- and 12-month-old rhesus monkeys fed a taurine-free human infant formula.

Rhesus monkey infants were raised from birth until 6 or 12 months of age on a taurine-free soy protein-based human infant formula or on the same formula supplemented with taurine. An additional group received taurine-free formula until 6 months and then the supplemented diet from 6 until 12 months. The densities of rod and cone visual pigments were measured by fundus reflectometry at 6 and 12 months, and retinal morphology was then examined by light and electron microscopy. The densities of rhodopsin, measured in the near periphery after a white bleach, and of cone pigment, measured in the macula after a red bleach, were significantly reduced in the taurine-deprived monkeys at 6 months but not at 12 months. The retinas of 6-month-old taurine-deprived infants showed degenerative morphological changes in photoreceptors, particularly in cones in the foveal region, which were somewhat less severe than those seen in a previous study at 3 months of age. The prevalence and degree of these abnormalities continued to decrease with age in taurine-deprived animals, but changes persisted in some animals at 12 months. Recovery was more complete in monkeys reversed to the supplemented diet from 6 to 12 months. Thus, monkey infants are dependent on dietary taurine to maintain normal retinal structure until at least 6 months of age; the effects of taurine deprivation regress spontaneously but incompletely by 12 months.

Abnormal dark adaptation kinetics in autosomal dominant sector retinitis pigmentosa due to rod opsin mutation.

The time course of dark adaptation was measured in 10 subjects from three families with autosomal dominant sector retinitis pigmentosa (RP) due to mutations in the first exon of the rod opsin gene. In each subject cone adaptation and the early part of the recovery of rod sensitivity followed the normal time course, but the later phase of rod adaptation was markedly prolonged. The recovery of rod sensitivity is much slower than that reported in any other outer retinal dystrophy. Using a model based upon primate data of rod outer segment length and turnover, we have calculated that the delayed phase of the recovery of rod sensitivity in the RP patients tested following strong light adaptation could be due in part to formation of new disc membrane with its normal concentration of rhodopsin rather than in situ regeneration of photopigment.

Rhodopsin levels in the central retinas of normal miniature poodles and those with progressive rod-cone degeneration.

Visual pigment in normal miniature poodles and those with progressive rod-cone degeneration (prcd), a late-onset autosomal recessive photoreceptor degeneration, has been studied using imaging fundus reflectometry (IFR). The stage to which the disease had advanced in the animals with prcd was assessed with electroretinography (ERG). Measurements were carried out on seven affected, two heterozygous and three homozygous normal animals. The IFR measurements showed that the in situ difference spectrum of visual pigment measured in the central retina of the normal poodle is typical of vertebrate rhodopsin, with a maximum at about 510 nm. Rhodopsin regeneration following extensive bleaches continues for up to 70 min. In poodles with prcd, rhodopsin is spectrally normal and regenerates at normal rates. In young affected animals under 1 year of age, the final levels of rhodopsin could already be substantially reduced. Serial measurements of visual pigment in these dogs showed differences in the degree and spatial pattern of pigment loss and rate of progression between animals. The extent of visual pigment loss also differed among the older (greater than 4.5 years) affected animals: while in one animal no pigment could be detected, in another a central band of retina was relatively spared, and significant levels of visual pigment were measured within it. Pigment levels measured within the central 25 degrees of the retinas of poodles heterozygous for prcd were lower than those in normal animals, even though their ERGs were within the normal range.

Abnormal dark adaptation and rhodopsin kinetics in Sorsby's fundus dystrophy.

Scotopic visual thresholds and time courses for dark adaptation were determined in eight patients with Sorsby's fundus dystrophy. Rhodopsin regeneration also was recorded in two. All patients had poor night vision and a visible yellow deposit at the level of Bruch's membrane that was confluent in the posterior pole. In retinal regions with the yellow deposit, scotopic thresholds were elevated, the rod-cone break was delayed or indistinct, the time courses for the rod portion of the dark adaptation curve was prolonged, and rhodopsin regeneration was slow in the one patient in whom measurements were made. In regions of ophthalmoscopically normal retina, dark adaptation was affected minimally, and in one patient, rhodopsin was regenerated at a normal rate. It was hypothesized that the abnormal dark adaptation and rhodopsin kinetics might be caused by reduced metabolic exchange across a thickened Bruch's membrane.

Abnormal rod dark adaptation in autosomal dominant retinitis pigmentosa with proline-23-histidine rhodopsin mutation.

We studied rod and cone function in 13 patients from four families with autosomal dominant retinitis pigmentosa and the proline-23-histidine rhodopsin mutation. In patients with early stages of this disease, rod sensitivity was mildly abnormal throughout the retina and cone sensitivity was normal. In more severely affected patients, sensitivity loss varied with retinal region, some regions showing mild rod loss only and other regions having pronounced rod and cone dysfunction. Rhodopsin levels were decreased below normal by amounts that indicated the rod sensitivity loss was determined by the reduced ability to absorb light. The most characteristic abnormality of this genotype was a slowed rod branch of dark adaptation, which was present regardless of the extent or severity of disease. The time required for recovery of rod sensitivity was more than twice the normal time. These findings with dark-adapted perimetry, fundus reflectometry, and dark adaptometry showed intrafamilial and interfamilial consistency.

Retinal function and rhodopsin levels in autosomal dominant retinitis pigmentosa with rhodopsin mutations.

We studied rod and cone function in 20 patients from six families with autosomal dominant retinitis pigmentosa, who represented five different point mutations in the gene encoding rhodopsin. In a family with a stop codon mutation at the carboxyl end of the molecule (glutamine-344), young members with the mutation were asymptomatic and clinically unaffected but showed about 1 log unit of rod sensitivity loss across the visual field and decreased rhodopsin levels; at this stage, cone function was essentially normal. In three families with mutations at the border of a transmembrane segment (arginine-135-leucine and arginine-135-tryptophan), there was neither detectable rod function nor measurable rhodopsin; cone function was variably impaired. Two families carrying different mutations (threonine-17-methionine and threonine-58-arginine) had altitudinal visual field defects with less impaired rod and cone function in the inferior than in the superior field. Rod adaptation was abnormal in both families, but the time course of adaptation differed between patients with the two mutations. Differences in the pattern of retinal dysfunction were therefore discernible in patients with different rhodopsin mutations.

The distribution and kinetics of visual pigments in the owl monkey retina.

An imaging fundus reflectometer has been used to study the distribution and regeneration of visual pigments in the retina of the owl monkey, Aotes trivirgatus. Measurements were made over an area of retina from 10 degrees nasal to 30 degrees temporal on the horizontal meridian, and from 5 degrees inferior to 30 degrees superior on the vertical meridian. The measured density differences vary with retinal location, with values in the central retina higher than those in more peripheral regions. The area of high density differences is roughly circular, with the highest values (approximately 0.3 log units) centred on or near the area centralis. Spectral measurements are consistent with a rod visual pigment absorbing maximally at about 518 nm, and indicate that the contribution of cone pigments to the imaging fundus reflectometer (IFR) data is negligible everywhere within the retinal area studied. The distribution of density differences is shown to correlate well with anatomical data for receptor and ganglion cell populations. Bleaching the visual pigment with brief intense lights leads to the extensive formation of the long-lived photoproduct metarhodopsin 3. Complete regeneration of rhodopsin following a full bleaching exposure (whether of brief or extended duration) takes more than 60 min and the time course of its recovery cannot be described accurately by first order kinetics.

Electrophysiologic characteristics of human and rat retinas in vitro.

To promote studies on the human retina, we investigated the survival of function in postmortem specimens. Visual pigment has been regenerated in normal human retinas, 5 to 58 hours postmortem, by exposure to retinal isomers in the dark. Levels from 0.1 to 0.41 nmol/mg protein were reached. Photoresponses were obtained in 9 of 13 retinas: P III maximum amplitudes ranged from 20-398 microV and thresholds, taking the criterion amplitude as 3 microV, ranged from 8.8-1340 quanta/micros2. In three cases, the b-wave was also seen. The P III amplitude vs. log intensity curves gave values of n between 0.6 and 1.0, and sigma (the stimulus intensity for a half maximal response) between 132-3700 quanta/microns2. Recovery of sensitivity did not always correspond to that of maximum response.

SWS (blue) cone hypersensitivity in a newly identified retinal degeneration.

Photoreceptor-mediated mechanisms were studied in patients with a recently identified retinopathy typified by night blindness, cystoid maculopathy, and similar scotopic and photopic electroretinograms (ERGs). Dark-adapted spectral sensitivity functions were only partly explained as composites of rod and cone curves shifted to lower sensitivities; there was unusually high sensitivity from 400-460 nm. A rod mechanism, reduced in sensitivity by at least 3 log units, was detectable with dark adaptometry. No measurable rhodopsin was found with fundus reflectometry. Light-adapted spectral sensitivities were subnormal for wavelengths greater than 500 nm but supernormal from 420-460 nm. On a yellow adapting field, the supernormal spectrum approximated that of the short-wavelength-sensitive (SWS) cone system. With spectral ERGs, two mechanisms were demonstrated. Dark- and light-adapted ERGs to green, orange-yellow, and red stimuli had similar waveforms and coincident intensity-response functions on a photopic intensity axis. ERGs to blue and blue-green stimuli were similar, and intensity-response functions coincided on a SWS cone intensity axis. Patients varied in the degree to which rod and midspectral cone function were decreased and SWS cone function was increased.