Recoverin regulates light-dependent phosphodiesterase activity in retinal rods.

The Ca2+-binding protein recoverin may regulate visual transduction in retinal rods and cones, but its functional role and mechanism of action remain controversial. We compared the photoresponses of rods from control mice and from mice in which the recoverin gene was knocked out. Our analysis indicates that Ca2+-recoverin prolongs the dark-adapted flash response and increases the rod's sensitivity to dim steady light. Knockout rods had faster Ca2+ dynamics, indicating that recoverin is a significant Ca2+ buffer in the outer segment, but incorporation of exogenous buffer did not restore wild-type behavior. We infer that Ca2+-recoverin potentiates light-triggered phosphodiesterase activity, probably by effectively prolonging the catalytic activity of photoexcited rhodopsin.

The influence of cross-sectional area on the tensile properties of flexor tendons.

Clinicians have long noted substantial variation in the cross-sectional size of flexor tendons in the hand; however, data indicating that surgical repair techniques of lacerated flexor tendons should be altered according to size are unavailable. Our objectives were to evaluate the cross-sectional size differences among tendons within the same hand and to correlate tendon size with tensile mechanical properties after suture repair. Fifty human cadaver flexor digitorum profundus tendons were measured with digital calipers to determine radioulnar and volardorsal diameters. Twenty tendons were used to measure resistance to suture pull-through; tendons were transected at the A2 pulley, and a transverse double-stranded 4-0 Supramid suture (S. Jackson, Inc, Alexandria, VA) was passed through the radioulnar plane of the tendon 1 cm from the transection site. The remaining tendons were transected and repaired by using a modified Kessler repair with double-stranded 4-0 Supramid suture. Both tendon repairs and tendon-suture pull-through specimens were tested to failure in tension by using a material testing machine. Dorsovolar tendon height and tendon cross-sectional area varied significantly between digits, with an average difference of approximately 40% between the values of the smallest (fifth) and largest (third) fingers. Yield and ultimate force determined by pull-through tests of the simple transverse suture correlated positively with tendon radioulnar width. Tensile properties of tendons repaired with a double-stranded modified Kessler repair, however, did not depend significantly on tendon size. These results indicate that the strength of the commonly used Kessler suture technique is not dependent on tendon cross-sectional size within the clinically relevant range of tendons evaluated.

Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors.

The retina's photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca(2+) regulation of guanylate cyclase (GC) by guanylate cyclase-activating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs-/- retinas showed no Ca(2+) dependence, indicating that Ca(2+) regulation of GCs had indeed been abolished. Flash responses from dark-adapted GCAPs-/- rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs-/- rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs-/- rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rod's operating range.

Under-use of tobacco dependence treatment among Wisconsin's fee-for-service Medicaid recipients.

BACKGROUND: Wisconsin Medicaid enrollees are eligible for treatment for tobacco dependence at minimal charge to the enrollee. This paper describes an evaluation of the use of this treatment within the Wisconsin fee-for-service Medicaid program. METHODS: Pharmaceutical claims data for Medicaid fee-for-service patients were analyzed for the year 1999 to determine rates of treatment use. RESULTS: Of 261,435 adults enrolled in fee-for-service Medicaid for 1 or more months, only 1131 adults received pharmacotherapy for tobacco dependence in 1999 at a modest cost of approximately $135 per treatment user. This represents less than 2% of the adult Medicaid fee-for-service patients who smoke. DISCUSSION: Few Medicaid fee-for-service enrollees are receiving evidence-based treatment for tobacco dependence, the leading preventable cause of illness and death in Wisconsin. The 16,000 Wisconsin physicians caring for these patients are urged to intervene with every Medicaid patient who smokes. To assist in this effort, the Wisconsin Medicaid program's coverage for tobacco dependence treatment is explained and a series of myths are corrected.

Activation, deactivation, and adaptation in vertebrate photoreceptor cells.

Visual transduction captures widespread interest because its G-protein signaling motif recurs throughout nature yet is uniquely accessible for study in the photoreceptor cells. The light-activated currents generated at the photoreceptor outer segment provide an easily observed real-time measure of the output of the signaling cascade, and the ease of obtaining pure samples of outer segments in reasonable quantity facilitates biochemical experiments. A quiet revolution in the study of the mechanism has occurred during the past decade with the advent of gene-targeting techniques. These have made it possible to observe how transduction is perturbed by the deletion, overexpression, or mutation of specific components of the transduction apparatus.

Intrasynovial flexor tendon repair. An experimental study comparing low and high levels of in vivo force during rehabilitation in canines.

BACKGROUND: Rehabilitation methods that generate increased tendon force and motion have been advocated to improve results following intrasynovial flexor tendon repair. However, the effects of rehabilitation force and motion on tendon-healing may be masked by the high stiffness produced by newer suture methods. Our objective was to determine whether the biomechanical properties of tendons repaired by one of two multistrand suture methods were sensitive to an increased level of applied rehabilitation force. METHODS: Two hundred and fourteen flexor digitorum profundus tendons from 107 adult dogs were transected and repaired. Dogs were assigned to one of four groups based on the rehabilitation method (low force [<5 N] or high force [17 N]) and the repair technique (four-strand or eight-strand core suture) and were killed between five and forty-two days after the procedure. Repair-site structural properties were determined by tensile testing, and digital range of motion was assessed with use of a motion-analysis system. RESULTS: Tensile properties did not differ between the low and high-force rehabilitation groups, regardless of the repair technique (p > 0.05). In contrast, tensile properties were strongly affected by the repair technique, with tendons in the eight-strand group having an approximately 35% increase in ultimate force and rigidity compared with those in the four-strand group (p < 0.05). Ultimate force did not change significantly with time during the first twenty-one days (p > 0.05); there was no evidence of softening in either of the repair or rehabilitation groups. Force increased significantly from twenty-one to forty-two days, while rigidity increased throughout the forty-two-day period (p < 0.05). CONCLUSIONS: Increasing the level of force applied during postoperative rehabilitation from 5 to 17 N did not accelerate the time-dependent accrual of stiffness or strength. Suture technique was of primary importance in providing a stiff and strong repair throughout the early healing interval. CLINICAL RELEVANCE: Our findings suggest that there be a reexamination of the concept that increases in force produced by more vigorous mobilization protocols are beneficial to tendon-healing. While more vigorous rehabilitation may help to improve hand function, we found no evidence that it enhances tissue-healing or strength in the context of a modern suture repair.

Rapid and reproducible deactivation of rhodopsin requires multiple phosphorylation sites.

Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein-coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or two (S334/S338) phosphorylation sites generated single-photon responses with greatly prolonged, exponentially distributed durations. Responses from rods expressing mutant rhodopsins bearing more than two phosphorylation sites declined along smooth, reproducible time courses; the rate of recovery increased with increasing numbers of phosphorylation sites. We conclude that multiple phosphorylation of rhodopsin is necessary for rapid and reproducible deactivation.

The resistance of a four- and eight-strand suture technique to gap formation during tensile testing: an experimental study of repaired canine flexor tendons after 10 days of in vivo healing.

There is a high incidence of gap formation at the repair site following tendon repair. Our goal was to determine the resistance of a 4- and an 8-strand suture technique to gap formation during tensile testing. We hypothesized that the 8-strand repair would sustain higher force levels at the onset of 1- and 3-mm gaps than the 4- strand repair. Twenty-two canine flexor tendons were transected, repaired, and tested to failure after 10 days of in vivo healing. Tests were recorded using a 60-Hz video system that allowed frame-by-frame playback for assessment of gap formation. The 8-strand repairs sustained 80% higher force at a gap of 1 mm than the 4-strand repairs (average force, 70 vs 39 N), but the force sustained at a gap of 3 mm did not differ between groups (35 N for both groups). For both repair types, a 1-mm gap typically occurred near the point of ultimate (maximum) force while a 3-mm gap occurred after the ultimate force. We conclude that the 8-strand repair is significantly more resistant to initial gapping during ex vivo tensile testing than the 4-strand repair but that the two repairs are equally susceptible to rupture if a gap of 3 mm or greater forms.

Slowed recovery of rod photoresponse in mice lacking the GTPase accelerating protein RGS9-1.

Timely deactivation of the alpha-subunit of the rod G-protein transducin (Galphat) is essential for the temporal resolution of rod vision. Regulators of G-protein signalling (RGS) proteins accelerate hydrolysis of GTP by the alpha-subunits of heterotrimeric G proteins in vitro. Several retinal RGS proteins can act in vitro as GTPase accelerating proteins (GAP) for Galphat. Recent reconstitution experiments indicate that one of these, RGS9-1, may account for much of the Galphat GAP activity in rod outer segments (ROS). Here we report that ROS membranes from mice lacking RGS9-1 hydrolyse GTP more slowly than ROS membranes from control mice. The Gbeta5-L protein that forms a complex with RGS9-1 was absent from RGS9-/- retinas, although Gbeta5-L messenger RNA was still present. The flash responses of RGS9-/- rods rose normally, but recovered much more slowly than normal. We conclude that RGS9-1, probably in a complex with Gbeta5-L, is essential for acceleration of hydrolysis of GTP by Galphat and for normal recovery of the photoresponse.

Proteins involved in synaptic vesicle trafficking.

Neurotransmitter release relies on a series of synaptic vesicle trafficking reactions. We have determined the molecular basis of these reactions by microinjecting, into 'giant' nerve terminals of squid, probes that interfere with presynaptic proteins. These probes affect neurotransmitter release and disrupt nerve terminal structure. From the nature of these lesions, it is possible to deduce the roles of individual proteins in specific vesicle trafficking reactions. This approach has revealed the function of more than a dozen presynaptic proteins and we hypothesize that neurotransmitter release requires the coordinated action of perhaps 50-100 proteins.

Tobacco and alcohol abuse: clinical opportunities for effective intervention.

No behaviors are more costly to the United States from a health or economic perspective than tobacco and alcohol use. One of the primary strategies available to mitigate this exacting toll is to identify and clinically treat the 25% of adults in America who smoke and the 20% of adults who drink alcohol above recommended limits. During the last two decades, researchers have identified a series of brief clinical interventions that can markedly reduce alcohol and tobacco use and significantly decrease the health burdens resulting from such use. This review outlines office-based clinical interventions and the organizational policies that support these interventions that have been shown to decrease tobacco and alcohol use.

Abnormal photoresponses and light-induced apoptosis in rods lacking rhodopsin kinase.

Phosphorylation is thought to be an essential first step in the prompt deactivation of photoexcited rhodopsin. In vitro, the phosphorylation can be catalyzed either by rhodopsin kinase (RK) or by protein kinase C (PKC). To investigate the specific role of RK, we inactivated both alleles of the RK gene in mice. This eliminated the light-dependent phosphorylation of rhodopsin and caused the single-photon response to become larger and longer lasting than normal. These results demonstrate that RK is required for normal rhodopsin deactivation. When the photon responses of RK-/- rods did finally turn off, they did so abruptly and stochastically, revealing a first-order backup mechanism for rhodopsin deactivation. The rod outer segments of RK-/- mice raised in 12-hr cyclic illumination were 50% shorter than those of normal (RK+/+) rods or rods from RK-/- mice raised in constant darkness. One day of constant light caused the rods in the RK-/- mouse retina to undergo apoptotic degeneration. Mice lacking RK provide a valuable model for the study of Oguchi disease, a human RK deficiency that causes congenital stationary night blindness.

Control of rhodopsin activity in vision.

Although rhodopsin's role in activating the phototransduction cascade is well known, the processes that deactivate rhodopsin, and thus the rest of the cascade, are less well understood. At least three proteins appear to play a role: rhodopsin kinase, arrestin and recoverin. Here we review recent physiological studies of the molecular mechanisms of rhodopsin deactivation. The approach was to monitor the light responses of individual mouse rods in which rhodopsin was altered or arrestin was deleted by transgenic techniques. Removal of rhodopsin's carboxy-terminal residues which contain phosphorylation sites implicated in deactivation, prolonged the flash response 20-fold and caused it to become highly variable. In rods that did not express arrestin the flash response recovered partially, but final recovery was slowed over 100-fold. These results are consistent with the notion that phosphorylation initiates rhodopsin deactivation and that arrestin binding completes the process. The stationary night blindness of Oguchi disease, associated with null mutations in the genes for arrestin or rhodopsin kinase, presumably results from impaired rhodopsin deactivation, like that revealed by the experiments on transgenic animals.

Role for the target enzyme in deactivation of photoreceptor G protein in vivo.

Heterotrimeric guanosine 5'-triphosphate (GTP)-binding proteins (G proteins) are deactivated by hydrolysis of the GTP that they bind when activated by transmembrane receptors. Transducin, the G protein that relays visual excitation from rhodopsin to the cyclic guanosine 3',5'-monophosphate phosphodiesterase (PDE) in retinal photoreceptors, must be deactivated for the light response to recover. A point mutation in the gamma subunit of PDE impaired transducin-PDE interactions and slowed the recovery rate of the flash response in transgenic mouse rods. These results indicate that the normal deactivation of transducin in vivo requires the G protein to interact with its target enzyme.

A neuronal Sec1 homolog regulates neurotransmitter release at the squid giant synapse.

Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the squid giant synapse. Microinjection of s-Sec1 into the presynaptic terminal of the giant synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro, completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites ("active zones"), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin-at a stage blocked by the peptide-is necessary for docked vesicles to fuse.

Rabphilin-3A: a multifunctional regulator of synaptic vesicle traffic.

We have investigated the function of the synaptic vesicle protein Rabphilin-3A in neurotransmitter release at the squid giant synapse. Presynaptic microinjection of recombinant Rabphilin-3A reversibly inhibited the exocytotic release of neurotransmitter. Injection of fragments of Rabphilin-3A indicate that at least two distinct regions of the protein inhibit neurotransmitter release: the NH2-terminal region that binds Rab3A and is phosphorylated by protein kinases and the two C2 domains that interact with calcium, phospholipid, and beta-adducin. Each of the inhibitory fragments and the full-length protein had separate effects on presynaptic morphology, suggesting that individual domains were inhibiting a subset of the reactions in which the full-length protein participates. In addition to inhibiting exocytosis, constructs containing the NH2 terminus of Rabphilin-3A also perturbed the endocytotic pathway, as indicated by changes in the membrane areas of endosomes, coated vesicles, and the plasma membrane. These results indicate that Rabphilin-3A regulates synaptic vesicle traffic and appears to do so at distinct stages of both the exocytotic and endocytotic pathways.

Exocytosis: proteins and perturbations.

Exocytosis is the primary means of cellular secretion. Because exocytosis involves fusion between the plasma membrane and the membrane of secretory vesicles, it is likely that proteins on these two membranes, as well as additional proteins in cellular cytoplasm, mediate exocytosis. Although we know much about the proteins of secretory cells, we still have much to learn about how these proteins participate in exocytosis; in no case has an unambiguous exocytotic function been assigned to any of these proteins. To identify the roles of proteins in exocytosis it is necessary to perturb protein function in living secretory cells. We review a number of perturbation strategies and summarize what this approach has taught us about the functional roles of proteins in exocytosis, concluding with a molecular model of protein dynamics during exocytosis.