Predicted and measured disorder in peripherin/rds, a retinal tetraspanin.

Vertebrate photoreceptor outer segment (OS) morphogenesis requires peripherin/rds (P/rds). We have characterized this protein's C-terminus and present evidence that suggests it is intrinsically disordered. We propose that structural flexibility may underlie the multifunctionality proposed for this domain previously. The extremely short C-termini present in other tetraspanin family members suggest that intrinsic disorder may also play a role for those proteins.

Folding and subunit assembly of photoreceptor peripherin/rds is mediated by determinants within the extracellular/intradiskal EC2 domain: implications for heterogeneous molecular pathologies.

Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.

Expression and characterization of peripherin/rds-rom-1 complexes and mutants implicated in retinal degenerative diseases.

Nearly 40 disease-linked mutations have been reported for peripherin/rds to date; heterologous expression in tissue culture cells offers a valuable means of efficiently characterizing the biochemical properties of the various mutants. Peripherin/rds is proposed to act as an essential structural element in outer segment disk morphogenesis, and a present transgenic mice offer the sole tractable system in which recombinant peripherin/rds may be examined functionally in situ. Because the generation and characterization of transgenic animals are both expensive and time consuming, heterologous expression in cultured cells offers an important and complementary means of addressing protein structure and function. The immunopurification and detection of the peripherin/rds-rom-1 complex are performed using specific immunochemical reagents, monoclonal and polyclonal antibodies, that are not commonly available. Several laboratories have developed antibodies to peripherin/rds and rom-1 in rabbits and mice, using a variety of immunogens: purified ROS membranes, purified E. coli fusion proteins, and synthetic peptides coupled to proteins. The C-terminal regions appear to be most highly antigenic, although antibodies have been generated to other regions as well. Regardless of their source, antibodies must be thoroughly characterized; specificity is often a function of solution conditions and must be determined empirically. The approach as described here has provided explanations for several instances of peripherin/rds-associated disease, including digenic RP linked to as L185P mutation, and adRP associated with C118/119del and C214S mutations. In addition, the R172W mutation, linked to macular dystrophy and preferential loss in cone function, is shown to behave normally with respect to biosynthesis and subunit assembly; it likely involves a more subtle functional defect that remains to be described. Finally, the methodology reported here has suggested the existence of a novel (homotetrameric) form of peripherin/rds in individuals lacking rom-1; this hypothesis has been confirmed in rom-1 knockout mice. The information obtained thus far demonstrates the utility of using heterologously expressed peripherin/rds and rom-1 to investigate the consequences of disease-linked mutations in these polypeptides. Heterologous cell expression coupled with transgenic mouse methodologies should continue to provide a more detailed understanding of molecular mechanisms underlying inherited retinal degenerative diseases.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

Shifts in tooth maturation patterns in non-French Canadian boys.

AIMS: This study was designed to evaluate the predictability of dental maturation patterns in non-French Canadian boys using the maturation curves developed by Demirjian & Goldstein [1]. SAMPLE: Archival cephalometric radiographs taken during a longitudinal study (1930-1960) of Caucasian Americans were re-evaluated. METHODS: Maturation of the permanent teeth on the left side of the mandible was determined according to the methods described by Demirjian & Goldstein. RESULTS: Of the 79 boys studied, 12 (15.2%) started below Demirjian's median and continued as such during the maturation process. Seven (8.9%) started within the second standard deviation above Demirjian's median and continued as such during the maturation process. The remaining 60 boys (75.9%) started below the median of Demirjian's curves at an early age. They matured at a rate that placed them above the median value at the end of the study period. The shift took place before the age of 6 years in 46 (76.6%) cases and between the ages of 6 and 8 years in another nine (15%) cases. In five of the cases which started below Demirjian's median (8.3%) the shift only took place after the age of 9 years. CONCLUSION: The shift from below median to above median value was considered an important factor in treatment planning. The data indicate that there is considerable risk for treatment planning prior to the age of 8 years. The risk is highest when the children are less than 6 years of age due to growth prediction uncertainties.

Cysteine residues of photoreceptor peripherin/rds: role in subunit assembly and autosomal dominant retinitis pigmentosa.

Peripherin/rds is a tetraspanning membrane glycoprotein that is essential for the morphogenesis and stabilization of outer segments of vertebrate rod and cone photoreceptor cells. Mutations in the gene for peripherin/rds are responsible for retinal degeneration in the rds mouse and a variety of progressive human retinal degenerative diseases including autosomal dominant retinitis pigmentosa and macular dystrophy. Peripherin/rds associates with rom-1, a homologous subunit, to form a heterotetrameric complex. This study examines the importance of cysteine residues for the structure of peripherin/rds and its assembly with rom-1. Each of the 13 cysteine residues in bovine peripherin/rds was individually replaced with a serine residue by site-directed mutagenesis, and the resulting mutants were expressed individually or together with rom-1 in COS-1 cells. SDS-polyacrylamide gel electrophoresis, immunoprecipitation, and velocity sedimentation were carried out to evaluate the ability of these mutants to form disulfide-linked homodimers, associate with rom-1, and assemble into tetramers characteristic of wild-type peripherin/rds. Substitution of each of the six nonconserved cysteines had no apparent effect on dimer formation, folding, or subunit assembly. In contrast, replacement of any of the seven conserved cysteine residues predicted to lie within a 150 amino acid intradiscal loop significantly altered these properties. Six of these mutants, including a C214S mutant linked to autosomal dominant retinitis pigmentosa, were unable to fold normally, interact with rom-1, or self-assemble into tetramers but instead formed a mixture of large aggregates and a smaller component, most likely a dimer. The C150S mutant, on the other hand, was incapable of forming intermolecular disulfide bonds but did associate with rom-1 into a heterotetramer. These results suggest that (1) the conserved C150 residue is required for intermolecular disulfide bonding but not subunit assembly; (2) the six other conserved cysteine residues are crucial for proper folding and subunit assembly, possibly through formation of intramolecular disulfide bonds; and (3) the misfolding and defective subunit assembly of the C214S mutant is responsible for a form of monogenic autosomal dominant retinitis pigmentosa.

Defective subunit assembly underlies a digenic form of retinitis pigmentosa linked to mutations in peripherin/rds and rom-1.

Retinitis pigmentosa (RP) is a group of progressive retinal dystrophies that include the most common hereditary degenerative disease affecting the retina. Although most disease phenotypes appear to result from defects at single genetic loci (monogenic), at least one instance of RP appears to require a coinheritance of defects in the unlinked peripherin/rds and rom-1 alleles (digenic), which encode the polypeptide subunits of an oligomeric transmembrane protein complex present at photoreceptor outer segment disc rims. Sedimentation velocity analysis was performed upon the affected gene products expressed heterologously in COS-1 cells to examine the assembly of the subunit polypeptides. The results indicate that the missense peripherin/rds mutant, L185P, which segregates with instance of digenically inherited RP, is conditionally defective with respect to its subunit assembly. Unlike wild-type peripherin/rds, the L185P mutant does not form native-like homotetramers on its own; however, the L185P mutant can assemble with wild-type rom-1 to form a structurally normal heterotetrameric complex. These findings provide a novel molecular-based rationale for the unusual digenic disease inheritance pattern and offer insight into regions of peripherin/rds and rom-1, which contribute to subunit-subunit interactions.

Subunit composition of the peripherin/rds-rom-1 disk rim complex from rod photoreceptors: hydrodynamic evidence for a tetrameric quaternary structure.

Peripherin/rds and rom-1 are homologous integral membrane protein subunits found as an oligomeric complex at the rim regions of rod and cone photoreceptor outer segment disks. These proteins are essential for the morphogenesis of normal outer segments and have been linked to a variety of human retinal degenerative diseases. Previous studies have suggested that disulfide-linked homodimers of peripherin/rds and rom-1 can associate noncovalently to form higher order structures. We have characterized the hydrodynamic properties of Triton X-100 solubilized peripherin/rds-rom-1 complexes from bovine ROS membranes by gel exclusion chromatography on Sepharose C1-6B and velocity sedimentation through H2O- and D2O-based sucrose gradients. A single hydrodynamic species is observed which has a Stokes radius of 6.2 nm, a sedimentation coefficient (S20,w) of 5.8 S, and a partial specific volume of 0.83 mL/g. From these data the molecular mass of the detergent-peripherin/rds-rom-1 complex is calculated to be 240 kDa. The protein component of this complex is estimated to be 135 kDa, providing direct evidence that the solubilized peripherin/rds-rom-1 complex is a tetramer. The abundance of this complex as measured by competitive ELISA and immunoaffinity purification is approximately 4% of total bovine ROS membrane protein and indicates that peripherin/rds-rom-1 tetramers are present at a relatively high average surface density (ca. 4100/ microns m2) at the rim surfaces of rod outer segment disks.

Heterologous expression of photoreceptor peripherin/rds and Rom-1 in COS-1 cells: assembly, interactions, and localization of multisubunit complexes.

Peripherin/rds is a 39 kDa integral membrane glycoprotein essential for normal photoreceptor cell development in vertebrates. It has been implicated in several human retinal degenerative diseases including retinitis pigmentosa and macular degeneration and is thought to play a structural role at photoreceptor outer segment disk rims, where it forms a tightly-associated complex with rom-1, a nonglycosylated 37 kDa homologue. Western blot analysis of COS-1 cells transiently transfected with full-length cDNA coding for either peripherin/rds or rom-1 indicates that each protein is expressed primarily as a disulfide-linked homodimer; recombinant peripherin/rds is glycosylated while recombinant rom-1 is not--akin to their counterparts in rod photoreceptor disk membranes. Upon cotransfection of the two cDNAs, the specific assembly of a stable peripherin/rds--rom-1 complex is observed. Immunofluorescence microscopy studies demonstrate that both singly and coexpressed peripherin/rds and rom-1 complexes are localized primarily within internal membranes of transfected cells. Velocity sedimentation data indicate that the recombinant complexes (4.9 S) are assembled with a subunit stoichiometry similar to those extracted from ROS membranes (4.5 S) and are most consistent with a tetrameric arrangement of polypeptides. Sedimentation analyses of individually expressed peripherin/rds (5.1 S) and rom-1 (4.3 S) suggest that each polypeptide can also assemble into a tetrameric form in the absence of its homologue partner. Subunit assembly and interactions are discussed in terms of their potential role in hereditary retinal diseases.

A rabbit lung model for testing reaction to inhaled dental restorative particles.

A study was designed to test the outcome of the deposition of particles of plastic composite dental restorative material in rabbit lungs. Grinding and polishing these restorations in situ produces some particles in the 0.5- to 10-microns size range that easily enter and remain in human lungs and are associated with industrial lung disease. Dental restorative plastic material was ground in the laboratory, suspended in saline solution, and injected transtracheally into four New Zealand white rabbits. Two control rabbits were similarly injected with saline solution transtracheally. Twenty-four hours later, the rabbits were injected with 1 mCi of 67Ga citrate intravenously and subsequently reanesthetized for scanning. Baseline scans were obtained in the six animals prior to the injection of the test particles. Positive gallium scans were obtained 72 h after the administration of particulate material in the four test rabbits. The gallium scans of the control rabbits remained no different from baseline. The study was repeated one month later. The animals were killed seven days after the last gallium scan. Light microscopy and transmission electron microscopy of the lungs of the test animals showed foci of chronic inflammation around particles of the restorative material. Particles were in vacuoles within alveolar macrophages and also free in interstitium. Control animals had normal histologic conditions. Silver amalgam and gold dental restorations have years of clinical use but the new plastic composite restorative materials are rapidly being introduced into human clinical dental practice. Normal use involves polymerization, grinding, and polishing of the material within the mouth. The chronic inflammation in the lungs of rabbits indicates a need to test dental restorative material for lung biocompatibility before further, extensive clinical use.

Solubilization and functional reconstitution of a chloride channel from Torpedo californica electroplax.

Chloride channels were detergent-extracted from Torpedo electroplax plasma membrane vesicles and reconstituted into liposomes by rapid detergent removal and a freeze-thaw-sonication procedure. Concentrative uptake of 36Cl-, driven by a Cl- gradient was used to determine conductance properties of reconstituted channels. Chloride flux assayed by this method is strongly selective for Cl- over cations, is blocked by SCN-, inactivated by treatment with DIDS, and exhibits an anion selectivity sequence Cl- greater than Br- greater than F- greater than SO4(2-), as does the voltage-gated Cl- channel from Torpedo observed in planar lipid bilayers. The channels are localized to the noninnervated face of the electrocyte, and a novel trapped-volume method is used to estimate a channel density on the order of 500 pmol/mg protein. An initial fractionation of the membrane extract by anion exchange chromatography yields fivefold enrichment of the channel activity.

Hodgkin's disease and AIDS. Twenty-three new cases and a review of the literature.

Despite numerous reports suggesting an association of Hodgkin's disease (HD) with the acquired immunodeficiency syndrome (AIDS), HD in an individual seropositive for the human immunodeficiency virus (HIV) still is not considered a criterion for the diagnosis of AIDS. The authors report 23 new cases of HD in individuals at risk for AIDS and review the literature. As a group, individuals at risk for AIDS who develop HD have a more aggressive form of the illness (82% with stage III or IV), have or develop AIDS-related opportunistic infections (54%), second neoplasms (10%), and /or profound cytopenias (32%), and 85 to 90% are HIV positive when tested. More than two thirds die within 1 year of the diagnosis of HD. The authors conclude that HIV infection alters the clinical course of HD, that advanced or high-grade HD in HIV-infected individuals should be considered indicative of AIDS, and all patients with HD should be tested for HIV.

Effects of human growth hormone in men over 60 years old.

BACKGROUND: The declining activity of the growth hormone--insulin-like growth factor I (IGF-I) axis with advancing age may contribute to the decrease in lean body mass and the increase in mass of adipose tissue that occur with aging. METHODS: To test this hypothesis, we studied 21 healthy men from 61 to 81 years old who had plasma IGF-I concentrations of less than 350 U per liter during a six-month base-line period and a six-month treatment period that followed. During the treatment period, 12 men (group 1) received approximately 0.03 mg of biosynthetic human growth hormone per kilogram of body weight subcutaneously three times a week, and 9 men (group 2) received no treatment. Plasma IGF-I levels were measured monthly. At the end of each period we measured lean body mass, the mass of adipose tissue, skin thickness (epidermis plus dermis), and bone density at nine skeletal sites. RESULTS: In group 1, the mean plasma IGF-I level rose into the youthful range of 500 to 1500 U per liter during treatment, whereas in group 2 it remained below 350 U per liter. The administration of human growth hormone for six months in group 1 was accompanied by an 8.8 percent increase in lean body mass, a 14.4 percent decrease in adipose-tissue mass, and a 1.6 percent increase in average lumbar vertebral bone density (P less than 0.05 in each instance). Skin thickness increased 7.1 percent (P = 0.07). There was no significant change in the bone density of the radius or proximal femur. In group 2 there was no significant change in lean body mass, the mass of adipose tissue, skin thickness, or bone density during treatment. CONCLUSIONS: Diminished secretion of growth hormone is responsible in part for the decrease of lean body mass, the expansion of adipose-tissue mass, and the thinning of the skin that occur in old age.

The relationship of growth hormone to alveolar ridge atrophy in an older male nursing home population.

Alveolar bone development has been shown by other investigators to be sensitive to growth hormone concentration in both humans and rodents. Many elderly people are deficient in growth hormone. The cyclic variation in plasma growth hormone concentration that complicates growth hormone measurement is not seen in the plasma somatomedin C (SmC) concentration. Furthermore, SmC is a dependable indicator of growth hormone secretion. It has been established that an SmC level of 0.24 or less is associated with decreased muscle mass and kidney size. We have investigated the relationship of the alveolar bone/supporting (basal) bone (A/B) ratio to the SmC in an elderly male population. These data suggest that growth hormone secretion is not the sole determinant of alveolar bone retention.