Modulation of mouse rod response decay by rhodopsin kinase and recoverin.

Light isomerizes 11-cis-retinal in a retinal rod and produces an active form of rhodopsin (Rh*) that binds to the G-protein transducin and activates the phototransduction cascade. Rh* is turned off by phosphorylation by rhodopsin kinase [G-protein-coupled receptor kinase 1 (GRK1)] and subsequent binding of arrestin. To evaluate the role of GRK1 in rod light response decay, we have generated the transgenic mouse RKS561L in which GRK1, which is normally present at only 2-3% of rhodopsin, is overexpressed by ∼12-fold. Overexpression of GRK1 increases the rate of Rh* phosphorylation and reduces the exponential decay constant of the response (τ(REC)) and the limiting time constant (τ(D)) both by ∼30%; these decreases are highly significant. Similar decreases are produced in Rv(-/-) rods, in which the GRK1-binding protein recoverin has been genetically deleted. These changes in response decay are produced by acceleration of light-activated phosphodiesterase (PDE*) decay rather than Rh* decay, because light-activated PDE* decay remains rate limiting for response decay in both RKS561L and Rv(-/-) rods. A model incorporating an effect of GRK1 on light-activated PDE* decay rate can satisfactorily account for the changes in response amplitude and waveform. Modulation of response decay in background light is nearly eliminated by deletion of recoverin. Our experiments indicate that rhodopsin kinase and recoverin, in addition to their well-known role in regulating the turning off of Rh*, can also modulate the decay of light-activated PDE*, and the effects of these proteins on light-activated PDE* decay may be responsible for the quickening of response recovery in background light.

Background light produces a recoverin-dependent modulation of activated-rhodopsin lifetime in mouse rods.

The Ca(2+)-binding protein recoverin is thought to regulate rhodopsin kinase and to modulate the lifetime of the photoexcited state of rhodopsin (Rh*), the visual pigment of vertebrate rods. Recoverin has been postulated to inhibit the kinase in darkness, when Ca(2+) is high, and to be released from the disk membrane in light when Ca(2+) is low, accelerating rhodopsin phosphorylation and shortening the lifetime of Rh*. This proposal has remained controversial, in part because the normally rapid turnoff of Rh* has made Rh* modulation difficult to study in an intact rod. To circumvent this problem, we have made mice that underexpress rhodopsin kinase so that Rh* turnoff is rate limiting for the decay of the rod light response. We show that background light speeds the decay of Rh* turnoff, and that this no longer occurs in mice that have had recoverin knocked out. This is the first demonstration in an intact rod that light accelerates Rh* inactivation and that the Ca(2+)-binding protein recoverin may be required for the light-dependent modulation of Rh* lifetime.

Race and Sex Differences in Adult Facial Fracture Risk.

IMPORTANCE: There are well-described racial, sex, and age differences related to osteoporosis and hip and/or extremity fractures. Nonetheless, there has been virtually no inquiry evaluating whether these findings carry over to facial fracture. OBJECTIVE: To characterize the incidence of facial fractures by patient demographics and injury mechanism, focusing on whether differences are noted with race, sex, and advancing age. MAIN OUTCOMES AND MEASURES: Retrospective analysis of the National Electronic Injury Surveillance System (NEISS) was performed in October and November 2015, specifically evaluating adult emergency department (ED) visits from 2012 to 2014 related to facial trauma. Entries were organized by age groups (both individual decades as well as younger adults [18-59 years] vs older adults [60-89 years]), sex, and race (white, black, Asian, other/unspecified). Incidence of facial fractures and mechanism of injury were also evaluated. RESULTS: There were 33 825 NEISS entries correlating to 1 401 196 ED (range, 1 136 048-1 666 344) visits for adult facial injury, with 14.4% involving fracture. A greater proportion of facial injuries among younger men (<60 years) were fractures relative to younger women (15.5% vs 12.5%; difference of the mean [DOM], 3.0%; 95% CI, 2.8%-3.1%; P < .001); however, on comparison by sex in elderly populations (≥ 60 years), women had an increased fracture predilection (15.0% vs 14.0%; DOM, 1.0%; 95% CI, 0.8%-1.2%; P < .001). Also, older women had a significantly greater risk of fracture relative to those younger than 60 years (15.0% vs 12.5%; DOM, 2.5%; 95% CI, 2.4%-2.7%; P < .001), a comparison that was significant among whites and Asians. Black women had a significantly decreased risk of fracture in the older aged population. (8.4% vs 9.1%; DOM, 0.7%; 95% CI, 0.2%- 1.3%; P = .001). Both on individual comparisons of younger and older cohorts, white and Asian individuals of either sex had significantly greater rates of facial fracture injury than blacks. Among younger cohorts in either sex, injuries sustained during participation in recreational activities were a significant factor, replaced largely by injuries due to housing structural elements and falls among older cohorts. CONCLUSIONS AND RELEVANCE: There is an increase in the risk of facial fracture among postmenopausal women sustaining facial injuries, with these results significant among whites and Asians. In contrast, a decreased risk was noted on comparison of younger and older black women. Mechanism of injuries also varied significantly by age, race, and sex. LEVEL OF EVIDENCE: 4.

Shoulder and elbow injuries in the skeletally immature athlete.

The intensity of training and competition among young athletes can place them at increased risk of acute and chronic injuries, which occur in patterns unique to the skeletally immature athlete. Prompt recognition and treatment of these injuries are critical to prevent long-term functional disability and deformity. Children and adolescents participating in recreational and organized sports are particularly susceptible to a broad spectrum of shoulder and elbow injuries involving both osseous and soft-tissue structures. Understanding the relevant functional anatomy, biomechanics of throwing, and pathophysiology of injury can help the clinician manage common acute traumatic injuries, some of which may result in chronic problems. Over-use injuries occur more frequently than do acute, traumatic injuries, and early recognition, coupled with appropriate treatment or prevention, can help restore and maintain normal shoulder and elbow function.

Matrix-matrix interaction of cartilage oligomeric matrix protein and fibronectin.

Recent work indicates that cartilage oligomeric matrix protein (COMP) plays an important role in extracellular matrix assembly and matrix-matrix protein interactions. In order to identify the proteins in extracellular matrix that interact with COMP, we used an ELISA-based solid-phase binding assay, which revealed a specific, high-affinity interaction between COMP and fibronectin. This interaction is concentration-dependent and saturable, and appears to occur under physiologically relevant conditions. Electron microscopy after negative staining and fragment binding analysis using the solid-phase assay revealed a predominant binding site for the COMP C-terminal globular domain to a molecular domain approximately 14 nm from the N-terminal domain of fibronectin, which can be inhibited by the presence of a polyclonal antibody specific for the C-terminal heptadecapeptide of COMP. This interaction is further demonstrated in vivo by colocalization of both COMP and fibronectin in the chondrocyte pericellular matrix by laser confocal microscopy of chondrocytes grown in agarose culture, and by appositional and colocalization of these proteins in the growth plate of primates by immunohistochemistry.

Functional redundancy of R7 RGS proteins in ON-bipolar cell dendrites.

PURPOSE: In the Gbeta5(-/-) mouse, the electroretinogram (ERG) b-wave is absent, and the R7 subfamily of regulators of G protein signaling (RGS), which includes RGS6, -7, -9, and -11, is downregulated. Mutant mouse strains deficient in RGS7 or -11 were characterized, and the SG711 strain which is deficient in both proteins was examined, to learn whether the loss of some of these RGS proteins causes the absence of the ERG b-wave. METHODS: Antibodies to RGS7 and -11 were generated to determine their expression levels and localizations in retinas with various genetic backgrounds by Western blot analysis and immunohistochemistry, respectively. The implicit times and amplitudes of ERG a- and b-waves were analyzed to examine photoreceptor and bipolar cell functions. RESULTS: RGS7 and -11 co-localized to the dendritic tips of the ON-bipolar cells. In the RGS11(-/-) mouse, the level of RGS7 protein increased. However, the level of RGS11 protein remained unchanged in the RGS7 mutant mouse, where a truncated RGS7 protein was expressed due to the deletion of exon 10. In the SG711 mouse retina, the Gbeta5-S protein level was reduced. The ERG b-wave of SG711 mice was markedly delayed. In contrast, RGS11(-/-) mice showed a moderately delayed b-wave, whereas the RGS7 mutant mice showed normal ERG responses. CONCLUSIONS: The data demonstrate the presence of a delayed ERG b-wave in SG711 mice and a functionally redundant role for RGS11 and -7 at the tips of ON-bipolar cell dendrites. These results suggest that RGS11 or -7 works as the major physiological GAP (GTPase acceleration protein) for Galphao1 in ON-bipolar cells.

In vitro assembly of a prohead-like structure of the Rhodobacter capsulatus gene transfer agent.

The gene transfer agent (GTA) is a phage-like particle capable of exchanging double-stranded DNA fragments between cells of the photosynthetic bacterium Rhodobacter capsulatus. Here we show that the major capsid protein of GTA, expressed in E. coli, can be assembled into prohead-like structures in the presence of calcium ions in vitro. Transmission electron microscopy (TEM) of uranyl acetate staining material and thin sections of glutaraldehyde-fixed material demonstrates that these associates have spherical structures with diameters in the range of 27-35 nm. The analysis of scanning TEM images revealed particles of mass approximately 4.3 MDa, representing 101+/-11 copies of the monomeric subunit. The establishment of this simple and rapid method to form prohead-like particles permits the GTA system to be used for genome manipulation within the photosynthetic bacterium, for specific targeted drug delivery, and for the construction of biologically based distributed autonomous sensors for environmental monitoring.