Assembly associated with the cytomatrix.

Assembly in vivo has been studied both for endogenous cytoskeletal proteins and for several classes of viruses. Autoradiography of cytoskeletal proteins has shown that many associate with the cytoskeletal framework close to the time and place of synthesis. The cytoskeletal proteins rearrange after association with the cytoskeletal framework. Rearrangement in symmetrical giant cells occurs in a centrifugal and coherent pattern. Many of the cytoskeletal proteins associate cotranslationally, as shown by their puromycin resistance in a cell-free translation system. The assembly of several groups of viruses has been shown to be associated with various components of the cytoskeleton; whether such assembly is cotranslational has not yet been addressed directly.

Developmental reorganization of the skeletal framework and its surface lamina in fusing muscle cells.

The skeletal framework of cells, composed of internal structural fibers, microtrabeculae, and the surface lamina, is revealed with great clarity after extraction with detergent. When muscle cells fuse to form a multinucleated myotube, their skeletal framework reorganizes extensively. When myoblasts prepare to fuse, the previously continuous surface lamina develops numerous lacunae unique to this stage. The retention of iodinated surface proteins suggests that the lacunae are not formed by the extraction of lamina proteins. The lacunae appear to correspond to extensive patches that do not bind concanavalin A and are probably regions of lipid bilayer devoid of glycoproteins. The lacunae appear to be related to fusion and disappear rapidly after the multinucleated myotube is formed. When muscle cells fuse, their internal structural networks must interconnect to form the framework of the myotube. Transmission electron microscopy of skeletal framework whole mounts shows that proliferating myoblasts have well developed and highly interconnected internal networks. Immediately before fusion, these networks are extensively reorganized and destabilized. After fusion, a stable, extensively cross-linked internal structure is reformed, but with a morphology characteristic of the myotube. Muscle cells therefore undergo extensive reorganization both on the surface and internally at the time of fusion.

Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture.

To monitor the interaction of cell surface acetylcholine (AcCho) receptors with the cytoskeleton, cultured muscle cells were labeled with radioactive or fluorescent alpha-bungarotoxin and extracted with Triton X-100, using conditions that preserve internal structure. A significant population of the AcCho receptors is retained on the skeletal framework remaining after detergent extraction. The skeleton organization responsible for restricting AcCho receptors to a patched region may also result in their retention after detergent extraction.

Biosynthesis of titin in cultured skeletal muscle cells.

Although significant progress has been made regarding the structure and function of titin, little data exist on the biosynthesis of this large protein in developing muscle. Using pulse-labeling with [35S]methionine and immunoprecipitation with an anti-titin mAb, we have examined the biosynthesis of titin in synchronized cultures of skeletal muscle cells derived from day 12 chicken embryos. We find that: (a) titin synthesis increases greater than 4-fold during the first week in culture and during this same time period, synthesis of muscle-specific myosin heavy chain increases greater than 12-fold; (b) newly synthesized titin has a t1/2 of approximately 70 h; (c) titin is resistant to extraction with Triton X-100 both during and immediately after its synthesis. These observations suggest that newly synthesized titin molecules are stable proteins that rapidly associate with the cytoskeleton of developing myotubes.

ISCEV Standard for full-field clinical electroretinography (2008 update).

This document, from the International Society for Clinical Electrophysiology of Vision (ISCEV), presents an updated and revised ISCEV Standard for clinical electroretinography (ERG). The parameters for flash stimulation and background adaptation have been tightened, and responses renamed to indicate the flash strength (in cd x s x m(-2)). The ISCEV Standard specifies five responses: (1) Dark-adapted 0.01 ERG (rod response); (2) Dark-adapted 3.0 ERG (combined rod-cone response); (3) Dark-adapted 3.0 oscillatory potentials; (4) Light-adapted 3.0 ERG (cone response); (5) Light-adapted 3.0 flicker (30 Hz flicker). An additional Dark-adapted 10.0 ERG or Dark-adapted 30.0 ERG response is recommended.

Novel frameshift mutations in CRX associated with Leber congenital amaurosis.

Mutations in CRX, a photoreceptor-specific transcription factor, can cause Leber congenital amaurosis (LCA), cone-rod dystrophy (CORD), and retinitis pigmentosa (RP), all of which feature severe visual impairment. Upon screening 55 patients with Leber congenital amaurosis, 75 patients with cone-rod dystrophy, 13 with cone dystrophy, and 36 with recessive or isolate RP for changes in the CRX sequence, we found two patients with Leber congenital amaurosis who carried heterozygously one of two novel frameshift mutations. The first mutation, Tyr191(1-bp del), was a de novo change and the second change, Pro263(1-bp del) was inherited from the proband's affected father. Both mutations are predicted to encode mutant versions of CRX with altered carboxy termini. We also found a previously reported missense mutation, Arg41Gln, heterozygously in a 47-year-old patient with a form of RP. The missense change Val242Met was found in an isolate case of CORD and no controls; however, its pathogenicity remains uncertain because only limited segregation analysis was possible. A nonpathogenic missense change, Ala158Thr, was found to be a variant present at relatively high frequency among African-Americans.

Specific and quantitative immunoprecipitation of tropomyosin and other cytoskeletal proteins by magnetic separation.

Immunoprecipitation is a powerful technique for purifying many proteins for which specific antibodies exist. Magnetic separation has recently been demonstrated to be effective in the immunoprecipitation of cell-surface proteins. We have used magnetic separation with anti-immunoglobulin or protein A bound to magnetic particles to immunoprecipitate labeled muscle tropomyosin and several other cytoskeletal proteins for which specific antibodies exist. We have not found it necessary to bind antigen-specific antibody to the magnetic particles, increasing the versatility of the technique. The quantitative recovery of tropomyosin from muscle cultures using magnetic separation is superior to Staph A (protein A-positive Staphylococcus aureus cells). The specificity of magnetic separation also compares favorably with Staph A for immunoprecipitation of muscle tropomyosin. Fibroblast tropomyosin, vimentin (from muscle and osteoblast) and myosin heavy chain are other cytoskeletal proteins that are easily recovered with magnetic separation. Magnetic separation, therefore, appears to be a valuable technique for the immunoprecipitation of cytoskeletal proteins from various cell types.

A simple trypsin resistance assay for muscle and other cell fusion.

Muscle cells fusing in vitro have long provided biologists with a tool to study development and gene expression. However, many such studies used morphological assays of cell fusion. We present here a method for assaying fusion at a specific, operationally defined step. Muscle cells grown in monolayer are exposed to trypsin-EDTA solution at 37 degrees C; the trypsin is inactivated, the cells fixed in Lugol's iodine, and 200 to 300 nuclei are counted as being single or multiple. The presence of EDTA is important under standard conditions for muscle culture; however, little difference is seen in divalent cation-depleted cultures. Therefore, for consistency EDTA can be included in all assays. Samples are stable for over 24 hr, with no cell loss from trypsinization or fixation. This assay exploits a specific stage of muscle fusion, trypsin-resistant contact, to provide a rapid, simple, and observer-independent assay for an early state of muscle fusion. The assay can be used to measure fusion between any nucleated cells.

Background adaptation in RCS rats.

The stimulus/response (S/R) relations of scotopic b-waves from dark-adapted RCS-p+ rats and those adapted to steady background lights were investigated in animals ages 14 to 50 days. The shapes of the S/R curves were normal at all ages and in all adaptation conditions. However, dark-adapted sensitivity, amplitude, and latency were abnormal in the second postnatal month. As the disease progressed, background lights varied amplitude and decreased sensitivity less than normal. Thus, the effects of the RCS degeneration on b-wave adaptation were different than previously found in the PCD mouse retinal degeneration studied in a similar fashion. A suggestion is made that investigations of b-wave background adaptation may contribute to elucidation of underlying retinal dysfunction in the human retinal degenerations.

Pupillary changes during dark adaptation in human infants.

Pupillary diameter of 10 infants (age 10 weeks) and four adult subjects was measured during 30 min of dark adaptation following exposure to a full-field adapting light. Adult results confirm that, under these conditions, the course of pupillary recovery was reasonably well described by an exponential time course (t0 = 408 sec; SD = 42 sec), as is rhodopsin regeneration. Pupillary recovery of infants also could be described by an exponential course (average t0 = 399 sec; SD = 31 sec). These results, demonstrating similarities between adaptive functions of infants and adults, suggest that pupillographic techniques can be used to investigate dynamic processes accompanying dark adaptation in preverbal children.

The relation of retinal sensitivity and rhodopsin in developing rat retina.

The authors have studied the relations of rhodopsin and retinal sensitivity in developing albino rats, (1) in the dark-adapted state and (2) after exposure to light that bleaches greater than 90% of rhodopsin. Developmental increases in dark-adapted b-wave sensitivity and rhodopsin content were related directly as has been reported previously for rhodopsin and PIII sensitivity of other developing retinas. During dark-adaptation, infant rat log a-wave and log b-wave sensitivities and adults' log b-wave sensitivity were related linearly to the proportion of rhodopsin present. The authors suspect that the sensitivity-rhodopsin relations of infant rat retina are determined mainly by photoreceptor processes. Thus, the infant rat provides a model for investigation of mechanisms underlying both (1) the direct relation of sensitivity and rhodopsin and (2) the log sensitivity-rhodopsin relation.

Behavioral measurement of background adaptation in infants.

Thresholds for detecting blue test flashes in the dark-adapted condition and on steady red background fields were measured in 2- to 18-week-old human infants by a two-alternative forced-choice preferential looking method. The results show that dark-adapted sensitivity increases and background adaptation develops during the early postnatal weeks. Thus, the retinal mechanism that underlie (1) detection of brief flashes and (2) neural processing in background adaptation appear to mature postnatally.

Scotopic optokinetic nystagmus thresholds in 10-week-old infants.

PURPOSE: To compare psychophysical and reflexive optokinetic nystagmus (OKN) estimates of dark-adapted scotopic thresholds mediated by the posterior retina in 10-week-old infants and adults. METHODS: A staircase procedure was used to determine the stimulus intensity needed to produce an OKN response to a moving 19 degrees x 19 degrees grating. In the same subjects, a two-alternative, forced-choice procedure was used to obtain thresholds for detecting 10 degrees diameter, 50 ms duration stimuli. RESULTS: Both OKN and psychophysical thresholds of infants are 0.9 log unit higher than those of adults. CONCLUSION: The infant-adult difference in thresholds mediated by retina at the posterior pole is greater than the infant-adult difference in thresholds for full-field stimuli. It is possible that delayed maturation of the posterior retina is the primary determinant of infants' high OKN and psychophysical thresholds.

The development of scotopic sensitivity.

UNLABELLED: PURPOSE. Test the hypothesis that the developmental increases in rod photoreceptor sensitivity and rod-mediated visual sensitivity at 10 degrees, 20 degrees , and 30 degrees eccentric are concurrent. It is known that maturation of the parafoveal (10 degrees eccentric) rod outer segments and visual sensitivity is delayed compared to that at 30 degrees eccentric. METHODS: Rod isolated electroretinographic (ERG) responses to full-field stimuli were obtained from dark-adapted subjects (n = 71), ranging in age from early infancy through middle age. Rod photoreceptor sensitivity was calculated by fitting a model of the activation of phototransduction to the a-wave response. Rod driven b-wave sensitivity was calculated from stimulus-response functions. A logistic growth model was used to summarize the developmental increases in sensitivity of the rod photoreceptors and the b-wave. Previously reported dark-adapted, rod-mediated visual sensitivities at 10 degrees , 20 degrees, and 30 degrees eccentric, obtained using preferential looking procedures, were reanalyzed using the logistic growth model. RESULTS: The logistic growth model accounted for 57% to 85% of the variance of each sensitivity parameter with age in normal subjects. The shape of the growth curve and the age at which sensitivity reaches 50% of the adult value is similar (10.0-13.5 weeks) for the rods, the b-wave, and peripheral visual sensitivity, but is significantly older, 19.5 weeks, for rod-mediated parafoveal visual sensitivity. CONCLUSIONS: Rod photoreceptor sensitivity and peripheral, rod-mediated visual sensitivity develop concurrently. A parsimonious explanation is that rod photoreceptor sensitivity determines dark-adapted, rod-mediated visual sensitivity during development.

The course of maturation of rod-mediated visual thresholds in infants.

PURPOSE: To measure the developmental course of infants' rod-mediated thresholds. METHODS: Thresholds for detecting stimuli (2 degrees diameter, 50 msec duration) presented at 10 degrees (parafoveal site) or 30 degrees (peripheral site) from a central fixation target were estimated using a preferential-looking method. Nine infants were tested at both stimulus positions at ages 10, 18, and 26 weeks. RESULTS: At 10 weeks, infants' thresholds at both sites were significantly higher than those of adults. The infants' average threshold at 10 degrees was 0.5 log unit higher than the infants' average threshold at 30 degrees. Adults' thresholds at the two sites were equal. Thresholds of all infants decreased with age until by age 26 weeks the parafoveal and peripheral thresholds were equal and were the same as those of adults. The rate of change of parafoveal thresholds was significantly faster than the rate of change of peripheral thresholds. CONCLUSIONS: Although postreceptoral factors cannot be ruled out, the results suggest that developmental increases in rod outer segment length and rhodopsin density account for most of the threshold changes during infancy.

Rod-mediated increment threshold functions in infants.

PURPOSE: To obtain and analyze scotopic increment threshold functions to test the hypothesis that rod photoreceptor immaturity accounts for the elevation of infants' over controls' dark-adapted thresholds and elevation of parafoveal over peripheral thresholds in infants. METHODS: Using a preferential looking method, thresholds for detection of 2(o), 50 msec, blue stimuli presented 10(o) (parafoveal) or 30(o) (peripheral) eccentric were measured in the dark and in the presence of steady red backgrounds. Ten 10-week-old infants and four control subjects (8-35 years) were tested. To evaluate pre- and postadaptation site determinants of threshold, a model of the increment threshold function was fit to the data, and the dark-adapted threshold (T(D)) and eigengrau (A(O)) were calculated. The values of T(D) and A(O) were compared between infants and controls and between parafoveal and peripheral eccentricities. RESULTS: At both parafoveal and peripheral eccentricities, infants' values of T(D) and A(O) were significantly higher than those of controls. The locus of the coordinates (A(O), T(D)) differed significantly between parafoveal and peripheral eccentricities. In every infant, the parafoveal value of T(D) was higher (by 0.3-0.6 log unit) and A(O) lower (by 0.2-0.5 log unit) than the peripheral value, whereas controls had no difference in T(D) and A(O) at the two eccentricities. CONCLUSIONS: The results indicate that both receptoral and postreceptoral immaturities have a role in the elevation of infants' over controls' thresholds. In infants, rod photoreceptor immaturity before the site of adaptation accounts for elevation of parafoveal over peripheral thresholds.

Background adaptation in children with a history of mild retinopathy of prematurity.

PURPOSE: In children with a history of mild retinopathy of prematurity (ROP), test the hypothesis that elevation of the parafoveal over peripheral dark-adapted threshold is due to photoreceptor rather than postreceptor dysfunction. METHODS: A forced choice procedure was used to measure thresholds, for detection of 2 degrees diameter, 50 msec, blue stimuli presented 10 degrees (parafoveal) or 30 degrees (peripheral) eccentric in the dark and in the presence of steady red backgrounds (-4 to +2 log scot td). Four ROP and four control subjects were tested at both eccentricities. A model of the increment threshold function was fit to the data to calculate the eigengrau and dark-adapted threshold. RESULTS: Both ROP subjects with elevated parafoveal thresholds also have elevated parafoveal eigengraus. On the other hand, parafoveal and peripheral eigengraus are equal in ROP subjects without parafoveal threshold elevation. Nevertheless, the dark-adapted thresholds of all ROP subjects are higher than those of any control subject at both sites. CONCLUSIONS: The parafoveal threshold elevation is due to rod dysfunction. There is also evidence of peripheral rod photoreceptor involvement in the subjects with ROP.

Cycloplegic refractions of infants and young children: the axis of astigmatism.

Review of the cycloplegic refractions of all children who were first examined at Children's Hospital Medical Center in Boston between 1968 and 1978 revealed that 281 children between the ages of 0 and 9.5 years had astigmatism of 1 diopter (D) or greater but no other ophthalmological or neurological problems. In the 85 children under 3.5 years of age, against-the-rule astigmatism was 2.5 times more common than with-the-rule astigmatism, whereas in the 103 children over 5.5 years of age, with-the-rule astigmatism was three times as common as against-the-rule astigmatism. Eleven children who had been astigmatic as infants were recalled; follow-up refraction at ages 5-11 years revealed that all but three had at least a 0.75 D decrease in astigmatism. The remaining three had astigmatism equal in magnitude and axis to the astigmatism they had as infants. Thus, there is a high prevalence of against-the-rule astigmatism in infants and toddlers, which disappears by the time the children reach school age.

A behavioral method for efficient screening of visual acuity in young infants. II. Clinical application.

Visual performance in 130 infants was assessed in a clinical setting with the forced-choice preferential looking (FPL) method described by Dobson et al.5 Over 90% of infants completed testing. Testing usually required less than 10 min. One group of infants tested also underwent complete ophthalmic examination. The second, larger group was screened with handlight examinations and FPL testing; any abnormalities detected were evaluated by full ophthalmic examination. Results so far indicate that the FPL test accurately identifies babies with binocular visual problems and that, when coupled with a handlight examination, it provides efficient screening for ocular problems in infants 0 through 16 weeks of age (postterm).

The effects of acetazolamide on the electroretinographic responses in rats.

PURPOSE: To study the mechanisms and sites of action of the carbonic anhydrase inhibitor, acetazolamide (AZM), on the rod- and cone-mediated electroretinogram (ERG) of the dark-adapted rat. METHODS: After a within-subjects design, ERG responses to brief, full-field flashes were recorded from adult (60 to 90 days old) albino rats, with and without AZM (5 mg/100 g, intraperitoneally). Flickering stimuli (6 and 26 Hz) were used to study rod- and cone-mediated responses. Aspartate-isolated responses of the isolated retina were recorded with and without AZM in the superfusate. The a-wave and PIII responses were fitted with a model of the rod's response by estimating the maximum response (Rmp3), sensitivity (S), and delay td. The b-wave response amplitude and implicit time were examined as a function of stimulus energy. The parameters obtained in the AZM-treated and untreated conditions were compared. RESULTS: Acetazolamide causes a significant decrease in saturated rod response, b-wave amplitude, aspartate-isolated PIII, and the rod- and cone-mediated responses to flickering light. The estimated sensitivity of the rod response (S), b-wave sensitivity, and b-wave implicit time are not altered significantly by AZM. CONCLUSION: Acetazolamide, probably through mechanisms that acidify the retina, attenuates the amplitudes of the retinal responses without significant effect on sensitivity.