Spondylodiscitis severity code: scoring system for the classification and treatment of non-specific spondylodiscitis.

INTRODUCTION: Established treatment options of spondylodiscitis, a rare but serious infection of the spine, are immobilization and systemic antibiosis. However, the available data for specific treatment recommendations are very heterogeneous. Our intention was to develop a classification of the severity of spondylodiscitis with appropriate treatment recommendations. MATERIALS AND METHODS: From 10/1/1998 until 12/31/2004, 37 cases of spondylodiscitis were examined regarding medical history, gender status, location and extent of spondylodiscitis, type and number of operations. Subsequently, a classification of six grades according to severity has been developed with specific treatment recommendations. The further evaluation of our classification and corresponding treatment modalities from 1/1/2005 to 12/31/2009 including further 132 cases, resulted in a classification of only three grades of severity (the SSC--spondylodiscitis severity code), with a follow-up until 12/31/2011. Between 01/01/2012 and 12/31/2013, a prospective study of 42 cases was carried out. Overall, 296 cases were included in the study. 26 conservatively treated cases were excluded. RESULTS AND CONCLUSION: The main localization of spondylodiscitis was the lumbar spine (55%) followed by the thoracic spine (34%). The classification of patients into 3 grades of severity depends on clinical and laboratory parameters, the morphological vertebral destruction seen in radiological examinations and the current neurological status. Therapies are adapted according to severity and they include a specific surgical management, systemic antibiotic therapy according to culture and sensitivity tests, physiotherapy and initiation of post-hospital follow-up. 40.6% of patients are associated with neurological deficits, classified as severity grade 3 and treated surgically with spinal stabilization and decompression. 46.9% of patients corresponded to severity grade 2, with concomitant vertebral destruction were dorsoventrally stabilized. The 31 patients of severity Grade 1 were treated surgically with dorsal stabilization. From 1998 to 2013, the time from the onset of symptoms to the first surgical treatment was about 69.4 days and has not changed significantly. However, the time from admission to surgical treatment had been reduced to less than 2 days. Also the time of hospitalization was reduced and we see positive effects regarding the sensation of pain. 270 patients underwent surgery. We treated 89% dorsally and 21% dorsoventrally. With the spondylodiscitis severity code, a classification of the severity of spondylodiscitis could be established and used for a severity-based treatment. In addition, specific parameters for the treatment of individual grades of severity can be determined in a clinical pathway.

Loop electrosurgical excision procedure and risk of vaginal infections during pregnancy: an observational study.

OBJECTIVE: To investigate the risk for preterm birth associated with vaginal infections in pregnancies after a loop electrosurgical excision procedure (LEEP), compared with women with no prior LEEP. DESIGN: Multicentre retrospective cohort study. SETTING: USA. POPULATION: Women with LEEP between 1996 and 2006 were compared with two unexposed groups who had cervical biopsy or Pap test, without any other cervical procedure, in the same calendar year. METHODS: The first pregnancy progressing beyond 20 weeks of gestation in women with prior LEEP was compared with pregnancy in women without LEEP. Stratified analysis according to the presence or the absence of vaginal infection during pregnancy was used to investigate whether the risk for preterm birth differed according to the presence or the absence of infection. The interaction between LEEP and vaginal infection was investigated using multivariable logistic regression with interaction terms, as well as the Mantel-Haenszel test for homogeneity. MAIN OUTCOME MEASURES: Spontaneous preterm birth (<37 and <34 weeks of gestation). RESULTS: Of 1727 patients who met the inclusion criteria, 34.4% (n = 598) underwent LEEP prior to an index pregnancy. There was no increased risk for vaginal infections among women with LEEP compared with women without LEEP. Chlamydia infection and LEEP demonstrated significant interaction, suggesting that the presence of chlamydia infection in women with a history of LEEP augments the risk for preterm birth, compared with women with no history of LEEP. CONCLUSIONS: Vaginal infections during pregnancy in women with a history of LEEP may be associated with an increased risk for preterm birth, compared with women with no history of LEEP.

First-trimester growth discordance and adverse pregnancy outcome in dichorionic twins.

OBJECTIVE: To evaluate the association between first-trimester growth discordance and adverse pregnancy outcome in dichorionic twin pregnancies. METHODS: This was a retrospective cohort study of consecutive women with dichorionic twin pregnancies undergoing an ultrasound scan at our institution between 7 and 14 weeks' gestation. Study groups were defined by the presence or absence of ≥ 11% crown-rump length (CRL) discordance. Pregnancies were excluded if one twin was dead on initial ultrasound or if a termination was performed. The primary outcome was loss of one or both fetuses before 20 weeks. Secondary outcomes included fetal anomaly, fetal demise after 20 weeks (stillbirth), small-for-gestational-age (SGA) at birth, admission to the neonatal intensive care unit (NICU) and preterm delivery before 34 weeks. RESULTS: Of 805 dichorionic twin pregnancies undergoing first-trimester ultrasound, 610 met the inclusion criteria. Eighty-six had ≥ 11% CRL discordance and, of these, nine (10.5%) had a fetal loss at < 20 weeks (risk ratio (RR) 7.8 (95% CI, 3.0-20.5)). In the surviving pregnancies, an increased risk of fetal anomalies was seen (27.3 vs 17.4%, RR 1.6 (95% CI, 1.1-2.4)). In surviving pregnancies unaffected by anomalies, no increased risk of stillbirth, SGA, NICU admission or delivery before 34 weeks was noted in the discordant group. A post-hoc power analysis demonstrated 80% power to detect a five-fold increase in the risk of stillbirth and 90% power to detect a two-fold increase in other outcomes. CONCLUSION: Dichorionic pregnancies in which a CRL discordance of at least 11% is noted are at increased risk for fetal anomalies and fetal loss prior to 20 weeks' gestation. However, patients can be reassured that, in the absence of structural anomalies, CRL discordance does not appear to be associated with other adverse outcomes in continuing pregnancies, although the power to detect small increases in the risk of stillbirth may have been limited by the sample size.

Screening for fetal growth disorders by clinical exam in the era of obesity.

OBJECTIVE: To evaluate the performance of clinical estimation of fetal weight as a screening test for fetal growth disorders and then to estimate the effect of maternal body mass index (BMI) on its screening efficiency. STUDY DESIGN: This was a retrospective cohort study of patients referred for third trimester ultrasound for the indication of 'size unequal to dates'. Patients with medical co-morbidities that may alter their a priori risk for fetal growth disorders were excluded. The incidence of fetal growth disorders as well as amniotic fluid disturbances was determined for each group and then compared across maternal BMI categories of <25 kg m(-2), 25-30 kg m(-2), ≥ 30 kg m(-2) and ≥ 40 kg m(-2). To evaluate the accuracy of clinical estimation of fetal weight in predicting fetal growth disorders, the sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratios, as well as number needed to scan (NNS) was calculated and compared across BMI categories. RESULT: Of 51366 patients, 1623 were referred for the indication of size>dates and 1543 for the indication of size90th percentile and 13.5 and 96.7% for predicting BW<10th percentile. The NNS to detect one neonate with a BW<10th percentile ranged from 5 to 19, whereas the NNS to detect one neonate with a BW>90th percentile ranged from 6 to 13 across BMI categories. CONCLUSION: Overall, clinical estimation of fetal weight yields a low detection rate of fetal growth abnormalities; however, its screening efficiency is not adversely impacted by maternal BMI.

Prediction of intrauterine fetal death in small-for-gestational-age fetuses: impact of including ultrasound biometry in customized models.

OBJECTIVE: Customized growth charts derived from maternal demographic characteristics alone have been shown to improve the prediction of pregnancy complications compared to population growth curves. We sought to estimate the impact of adding ultrasound biometric parameters to the customized chart for the prediction of intrauterine fetal death (IUFD). METHODS: A retrospective cohort study was undertaken using an ultrasound database including singleton pregnancies followed between 16 and 20 weeks' gestation. After exclusion of preterm births, congenital anomalies, multifetal pregnancies and stillbirths (excluded only from derivation samples), we identified 59 016 births, divided into derivation (34 832) and validation (24 184) samples. Coefficients for significant physiological and pathological variables affecting fetal growth were derived using backward stepwise multiple regression (Cust-chart). The same process was repeated including second-trimester biometric parameters: biparietal diameter, head circumference, femur length and abdominal circumference in the regression models (Cust-plus-USS-chart). The association between small-for-gestational age < 10(th) centile (SGA) pregnancies, defined using the two customized charts or our population-based growth chart (Pop-chart) and IUFD, were compared. Statistical analyses including OR, 95% CI and screening accuracy using each chart were performed. RESULTS: The derived coefficients for fetal growth are comparable to those of previously published series. Of 24 184 pregnancies in the validation sample, IUFD was seen in 169 (0.7%). The pregnancies identified as SGA were: 2482 (10.26%), 2499 (10.33%) and 2634 (10.89%) using the Cust-chart, Cust-plus-USS-chart and Pop-chart, respectively. The OR (95% CI) for the association between SGA defined by the three charts and IUFD was: 7.0 (4.5-11), 6.5 (4.2-10.2) and 2.4 (1.6-3.6) according to the Cust-chart, Cust-plus-USS-chart and Pop-chart, respectively. Screening efficiency for IUFD using both customized charts was similar, with both demonstrating a higher sensitivity compared with the Pop-chart. CONCLUSIONS: Customized charts are more efficient in identifying pregnancies at risk for IUFD compared with population-based charts. However, adding second-trimester ultrasound biometric parameters to the customized model does not improve the prediction of IUFD compared with using maternal characteristics only.

Redefining gigantomastia.

Gigantomastia is a rare but disabling condition characterised by excessive breast growth. Most definitions of gigantomastia refer to a particular weight of excess breast tissue. We speculate that in gigantomastia the weight of the breasts contributes significantly to the BMI, which has implications for healthcare rationing. This study aims to establish the contribution breast tissue makes to BMI in gigantomastia. In so doing, we propose a new definition of gigantomastia. Retrospective data was collected from the case notes of 68 females who underwent breast reduction or therapeutic mastectomy for gigantomastia. For the purposes of patient inclusion, gigantomastia is arbitrarily defined as excessive breast growth of over 1.5kg per breast. The difference between pre- and post-operative BMI is statistically significant (P<0.001). Mean pre-operative BMI is 38.7 with a mean specimen weight of 4506g. Mean contribution of specimen to body weight is 4.29%. There is no correlation between pre-operative body weight and the percentage contribution the breast resection specimen makes to body weight. Based on our data, we define gigantomastia as excess breast tissue that contributes 3% or more to the patient's total body weight, approximately one standard deviation below the mean. We suggest that the estimated excess breast tissue weight is taken into account when calculating pre-operative BMI in the gigantomastia population. The challenge of estimating excess breast weight pre-operatively may be met by 3D photography coupled with computer-assisted volumetry.

New, non-invasive thermographic examination of body surface temperature on tetraplegic and paraplegic patients, as a supplement to existing diagnostic measures.

STUDY DESIGN: Prospective clinical trial of consecutive tetraplegic and paraplegic cases. OBJECTIVES: The detection of the neurological level of paralysis by thermographic imaging. SETTING: Spinal Cord Injury Center in Germany Halle (Saale) and Fraunhofer Institute for Mechanics of Materials in Halle (Saale). METHODS: Twelve tetraplegic and 4 paraplegic patients (ASIA A-C) were examined by thermal imaging with a diagnosis of a temperature difference on the skin surface. RESULTS: A new application of thermography for diagnostic purposes could be demonstrated, especially by the new methodical approaches to evaluate thermographic images. CONCLUSION: Thermography could prospectively be applied in the emergency diagnosis and therapy for accident victims as a supplement to existing diagnostic measures for spinal cord injury.

Sorption of nonpolar aromatic contaminants by chlorosilane surface modified natural minerals.

The efficacy of the surface modification of natural diatomite and zeolite material by chlorosilanes is demonstrated. Chlorosilanes used were trimethylchlorosilane (TMSCI), tert-butyldimethylchlorosilane (TBDMSCI), dimethyloctadecylchlorosilane (DMODSCI), and diphenyldichlorosilane (DPDSCI) possessing different headgroups and chemical properties. Silanol groups of the diatomite and zeolite were modified by chemical reaction with the chlorosilanes resulting in a stable covalent attachment of the organosilanes to the mineral surface. The alteration of surface properties of the modified material was proved by measurements of water adsorption capacity, total organic carbon (TOC) content, and thermoanalytical data. The surface modified material showed great stability even when exposed to extremes in ionic strength, pH, and to pure organic solvents. Sorption of toluene, o-xylene, and naphthalene from water was greatly enhanced by the surface modification compared to the untreated materials which showed no measurable sorption of these compounds. The enhanced sorption was dependent on the organic carbon content as well as on chemical characteristics of the chlorosilanes used. Batch sorption experiments showed that the phenyl headgroups of DPDSCI have the best affinity for aromatic compounds. Removal from an aqueous solution of 10 mg/L of naphthalene, o-xylene, and toluene was 71%, 60%, and 30% for surface modified diatomite and 51%, 30%, and 16% for modified clinoptilolite, respectively. Sorption data were well described by the Freundlich isotherm equation, which indicated physical adsorption onto the lipophilic surface rather than partitioning into the surface organic phase. The chlorosilane modified materials have an apparent potential for application in environmental technologies such as permeable reactive barriers (PRB) or wastewater treatment.

Genetic variability at the amyloid-beta precursor protein locus may contribute to the risk of late-onset Alzheimer's disease.

In a series of sibpairs with late onset Alzheimer's disease, we have examined the segregation of the loci involved in the early onset, autosomal dominant form of the disorder by using flanking microsatellite repeat markers: thus we have used APP-PCR3 and D21S210 to examine the segregation of the amyloid-beta precursor protein (APP) gene, the markers DI 4S77 and D14S284 to examine the segregation of the presenilin 1 (PSI) gene and the markers D1S227, D1S249 and D1S419 to examine the segregation of presenilin 2 (PS2). We carried out our analyses on the whole dataset of 291 affected sibpairs, and on subsets comprising those sibpairs in which neither had an apolipoprotein E4 allele (65 affected sibpairs) and those in which both had an apolipoprotein E4 allele (165 affected sibpairs). We used the programs SPLINK to generate allele frequencies and MAPMAKER/SIBS to analyze our results. We examined the segregation of the markers D19S908 and D19S918 that are close to the apolipoprotein E (ApoE) gene as a positive control to assess whether the methods we are employing have the capability to identify known loci. The sibpair approach to the identification of genetic risk loci is relatively insensitive as indicated by the failure of the ApoE locus to reach statistical significance (P = 0.06). Nevertheless, these data suggest that neither the PS1 nor the PS2 gene is a major locus for late-onset AD, but that the APP gene cannot be ruled out as a risk locus in those sibships without an E4 allele (P = 0.014). The possibility that APP is indeed a locus for late onset disease will need confirmation in other series of familial cases.

A full genome scan for late onset Alzheimer's disease.

We have genotyped 292 affected sibling pairs (ASPs) with Alzheimer's disease (AD) according to NINCDS-ADRDA diagnostic criteria and with onset ages of >/=65 years using 237 microsatellite markers separated by an average distance of 16.3 cM. Data were analysed by SPLINK and MAPMAKER/SIBS on the whole sample of 292 ASPs and subsets of 162 ASPs where both members possessed an apolipoprotein E (APOE)straightepsilon4 allele and 63 pairs where neither possessed anstraightepsilon4 allele. Sixteen peaks with a multipoint lod score (MLS) >1 either in the whole sample, the straightepsilon4-positive or -negative subgroups were observed on chromosomes 1 (two peaks), 2, 5, 6, 9 (two peaks), 10 (two peaks), 12, 13, 14, 19, 21 and X (two peaks). Simulation studies revealed that these findings exceeded those expected by chance, although many are likely to be false positives. The highest lod scores on chromosomes 1 (MLS 2.67), 9 (MLS 2.38), 10 (MLS 2.27) and 19 (MLS 1.79) fulfilLander and Kruglyak's definition of 'suggestive' in that they would be expected to occur by chance once or less per genome scan. Several other peaks were only marginally less significant than this, in particular those on chromosomes 14 (MLS 2.16), 5 (MLS 2.00), 12, close to alpha2-macroglobulin (MLS 1.91), and 21, close to amyloid precursor protein (MLS 1.77). This is the largest genome scan to date in AD and shows for the first time that this is a genetically complex disorder involving several, perhaps many, genes in addition to APOE. Moreover, our data will be of interest to those hoping to identify positional candidate genes using information emerging from neurobiological studies of AD.

Genetic studies on chromosome 12 in late-onset Alzheimer disease.

CONTEXT: The only genetic locus universally accepted to be important as a risk factor for late-onset Alzheimer disease (AD) is the apolipoprotein E (APOE) locus on chromosome 19. However, this locus does not account for all the risk in late-onset disease, and a recent report has suggested a second locus on chromosome 12p11-12. OBJECTIVE: To look for evidence of linkage on chromosome 12 and to test for the presence of the new locus in an independent sample of familial late-onset AD cases. DESIGN: Retrospective cohort study. As part of a 20-centimorgan genome screen (approximately equal to 200 markers), we tested a series of 18 genetic markers on chromosome 12 and carried out multipoint, nonparametric lod score and exclusion analyses. SETTING: Clinic populations in the continental United States selected from the National Institute of Mental Health AD Genetics Consortium. PATIENTS: We selected samples for DNA analysis from affected sibling pairs, 497 subjects from 230 families with 2 or more affected individuals with probable or definite AD with onset ages older than 60 years (mean+/-SD, 75+/-6 years). Within the families, we used the 2 probable or definitely affected individuals. In families with more than 2 such cases available, we used all of them; in families with only 2 such cases in which unaffected individuals were available, we also sampled the oldest unaffected individual and used genotype data from this unaffected individual to check for nonpaternity and genotyping errors. MAIN OUTCOME MEASURE: Presence of linkage or locus on chromosome 12. RESULTS: Although linkage analyses confirmed the presence of a genetic susceptibility factor at the APOE locus in these families with late-onset AD, we were unable to confirm the presence of a locus close to the marker D12S1042. A moderate lod score (1.91) was found near D12S98 close to the alpha2-macroglobulin locus in the affected pairs in which both members did not possess an APOE epsilon4 allele. CONCLUSIONS: APOE remains the only locus established to be a risk factor for late-onset AD. We were unable to confirm that a locus on chromosome 12p11-12 has a major effect on risk for late-onset AD, although an effect smaller than that for APOE cannot be excluded.

Stimulation of cell elongation in teleost rod photoreceptors by distinct protein kinase inhibitors.

The rod photoreceptors of teleost retinas elongate in the light. To characterize the role of protein kinases in elongation, pharmacological studies were carried out with rod fragments consisting of the motile inner segment and photosensory outer segment (RIS-ROS). Isolated RIS-ROS were cultured in the presence of membrane-permeant inhibitors that exhibit selective activity toward specific serine/threonine protein kinases. We report that three distinct classes of protein kinase inhibitors stimulated elongation in darkness: (1) cyclic-AMP-dependent protein kinase (PKA)-selective inhibitors (H-89 and KT5720), (2) a protein kinase C (PKC)-selective inhibitor (GF 109203X) that affects most PKC isoforms, and (3) a kinase inhibitor (H-85) that does not affect PKC and PKA in vitro. Other kinase inhibitors tested neither stimulated elongation in darkness nor inhibited light-induced elongation; these include the myosin light chain kinase inhibitors ML-7 and ML-9, the calcium-calmodulin kinase II inhibitor KN-62, and inhibitors or activators of diacylglycerol-dependent PKCs (sphingosine, calphostin C, chelerythrine, and phorbol esters). The myosin light chain kinase inhibitors as well as the PKA and PKC inhibitors H-89 and GF 109203X all enhanced light-induced elongation. These observations suggest that light-induced RIS-ROS elongation is inhibited by both PKA and an unidentified kinase or kinases, possibly a diacylglycerol-independent form of PKC.

Localization of kinesin superfamily proteins to the connecting cilium of fish photoreceptors.

Kinesin superfamily proteins (KIFs) are probable motors in vesicular and non-vesicular transport along microtubular tracks. Since a variety of KIFs have been recently identified in the motile flagella of Chlamydomonas, we sought to ascertain whether KIFs are also associated with the connecting cilia of vertebrate rod photoreceptors. As the only structural link between the rod inner segment and the photosensitive rod outer segment, the connecting cilium is thought to be the channel through which all material passes into and out of the outer segment from the rod cell body. We have performed immunological tests on isolated sunfish rod inner-outer segments (RIS-ROS) using two antibodies that recognize the conserved motor domain of numerous KIFs (anti-LAGSE, a peptide antibody, and anti-Klp1 head, generated against the N terminus of Chlamydomonas Klp1) as well as an antibody specific to a neuronal KIF, KIF3A. On immunoblots of RIS-ROS, LAGSE antibody detected a prominent band at approximately 117 kDa, which is likely to be kinesin heavy chain, and Klp1 head antibody detected a single band at approximately 170 kDa; KIF3A antibody detected a polypeptide at approximately 85 kDa which co-migrated with mammalian KIF3A and displayed ATP-dependent release from rod cytoskeletons. Immunofluorescence localizations with anti-LAGSE and anti-Klp1 head antibodies detected epitopes in the axoneme and ellipsoid, and immunoelectron microscopy with the LAGSE antibody showed that the connecting cilium region was particularly antigenic. Immunofluorescence with anti-KIF3A showed prominent labelling of the connecting cilium and the area surrounding its basal body; the outer segment axoneme and parts of the inner segment coincident with microtubules were also labelled. We propose that these putative kinesin superfamily proteins may be involved in the translocation of material between the rod inner and outer segments.

Phosducin and PP33 are in vivo targets of PKA and type 1 or 2A phosphatases, regulators of cell elongation in teleost rod inner-outer segments.

Teleost rod photoreceptors elongate in the light and shorten in darkness. We are investigating the role of cAMP-dependent protein kinase (PKA), phosphatases and target phosphoproteins in the regulation of photoreceptor cell shape. Preparations of rod fragments, consisting of the motile inner segment with attached photosensory outer segment (RIS-ROS), undergo light-stimulated elongation in culture. The PKA-selective inhibitor, H89, enhanced RIS-ROS elongation in both light and darkness, suggesting that elongation is associated with dephosphorylation of PKA substrates. Okadaic acid and calyculin A, inhibitors of type 1 and 2A phosphatases, blocked light-dependent and light-independent elongation with relative potencies suggesting that elongation requires dephosphorylation by type 1 phosphatase in light and type 2A phosphatase in darkness. To identify targets of PKA and phosphatases, RIS-ROS were isolated from retinas prelabeled with 32P-orthophosphate, and then incubated in the presence of kinase inhibitors or phosphatase inhibitors. Two phosphoproteins, PP33 and PP35, were phosphorylated by PKA and dephosphorylated by type 1 or 2A phosphatases in light- and dark-cultured RIS-ROS. PP35 (but not PP33) was immunoprecipitated by an antibody to phosducin, a PKA-regulated modulator of phototransduction (Lee et al., 1992); PP35 was also phosphorylated in vitro by a Ca2+ calmodulin-activated kinase. PP33 further differed from PP35 in its phosphopeptide maps and phosphorylation by PKC. We conclude that RIS-ROS elongation is correlated with the dephosphorylation of PKA substrates by type 1 or 2A phosphatases. Candidate mediator proteins include PP35, a fish phosducin homolog, and PP33, a newly described photoreceptor phosphoprotein.

Identification of cyclic nucleotide-regulated phosphoproteins, including phosducin, in motile rod inner-outer segments of teleosts.

In teleost retinas, rods elongate in the light and shorten in the dark. Rod motility is mediated by the actin cytoskeleton of the inner segment and is regulated by cyclic AMP- or cyclic GMP-stimulated phosphorylation of target proteins. In this study, we have identified the target proteins of cyclic nucleotide-dependent kinases in rods, using preparations of isolated, motile rod inner-outer segments (RIS-ROS). Five proteins found in Percoll-purified RIS-ROS were phosphorylated in the presence of cAMP (> 10 nM), cGMP (> or = 10 microM) and exogenous catalytic subunit of cAMP-dependent protein kinase (PKA). The PKA inhibitor, PKI, blocked stimulation of phosphorylation by both cAMP and cGMP. Three cAMP-stimulated phosphoproteins were detected in cytoskeletal fractions of light- and dark-adapted RIS-ROS. One of these, PP33, appears to be a fish homologue of mammalian phosducin, based on immunolabeling by two different antibodies against mammalian phosducin and on electrophoretic characteristics in 2-D gels. Two additional phosducin immunoreactive bands were detected in Western blots. One, at 35 kDa, comigrated with a second cAMP-stimulated RIS-ROS phosphoprotein, PP35, which was also detected in the cytoskeleton. The other, at 37 kDa, was present in whole teleost retinas but not in purified RIS-ROS. Our results suggest that the effects of both cAMP and cGMP on teleost rod motility are mediated through PKA modulation of target phosphoproteins. These phosphoproteins include a cytoskeleton-associated phosducin homologue.

Retinomotor movements in isolated teleost retinal cone inner-outer segment preparations (CIS-COS): effects of light, dark and dopamine.

Teleost cone inner segments elongate and contract in response to light and circadian signals. Previous studies have shown that teleost cone contraction is triggered by light or dopamine, while cone elongation is triggered by darkness or experimental elevation of cAMP. We have developed procedures for isolating and purifying motile cone fragments consisting of inner and outer segments (CIS-COS) to permit more detailed analysis of light and dopamine regulation of cone retinomotor movements. When retinas are dissected from long-term dark-adapted fish, CIS-COS break off at the base of the ellipsoid and remain attached to the RPE. CIS-COS can be detached from the RPE by brief protease treatment, thereby generating a highly enriched CIS-COS suspension. CIS-COS retain normal morphology and extend new myoids when cultured in darkness or in light plus forskolin, an activator of adenylate cyclase. The microtubule and actin cytoskeletons of the new myoids resemble those of intact cone myoids in vivo. Light inhibits CIS-COS myoid elongation, suggesting that light reception by the outer segment can directly influence cone motility. In dark-cultured CIS-COS, myoid elongation is inhibited half-maximally by nanomolar concentrations of dopamine, suggesting that dopamine effects on motility are mediated by D2-family receptors present on the cone inner and/or outer segment. After dark-induced elongation in culture, CIS-COS myoids can be induced to contract by subsequent culture in the light or with dopamine. Thus isolated cone inner and outer segments possess sufficient cytoskeletal and regulatory machinery to exhibit light- and dopamine-regulation retinomotor movement similar to that observed in intact cones in situ.

Shortening of the calycal process actin cytoskeleton is correlated with myoid elongation in teleost rods.

Light activates the elongation of rods within teleost retinas. Rod cell elongation is mediated by actin-dependent length changes of the myoid portion of the inner segment. The actin cytoskeleton of the inner segment consists of filament bundles, which run parallel to the long axis of the rod, from the calycal processes, through the ellipsoid and into the myoid. In isolated rod inner/outer segments (RIS-ROS), myoid elongation was found to occur in the absence of net polymerization of actin into filaments. Outgrowth of actin filaments within the myoid was counterbalanced by a shortening of actin filaments within the calycal processes. In this study, we have further examined light-activated modifications of the rod cytoskeleton using rhodamine-phalloidin to stain actin filaments within retinal cryosections as well as in isolated RIS-ROS. In RIS-ROS isolated from dark-adapted green sunfish, the phalloidin-stained calycal processes appeared as long, brush-like structures, averaging 4.2 microns in length. In light-cultured RIS-ROS populations, the calycal process actin cytoskeleton shortened from 4.2 microns to 1.7 microns. In control, dark-cultured populations, RIS-ROS that did not elongate maintained long calycal process actin cytoskeletons. However, in cases where dark-cultured RIS-ROS did elongate, despite the absence of a light stimulus, myoid elongation was accompanied by a shortening of the calycal process actin cytoskeleton, suggesting that the two events are correlated with one another. In light-adapted green sunfish and in light-cultured retinas from green sunfish and the Midas cichlid, the calycal process cytoskeleton of intact rods shortened by 40-60%. Within the two-tiered retina of green sunfish, shortening of the calycal process cytoskeleton, from 5.1 microns to 2.1-3.1 microns, was only evident in the shorter, inner tier of rods. The calycal process actin cytoskeleton did not appear to shorten within the longer, outer tier of rods; here, stained processes were short, averaging 2.3 microns in length, within dark-adapted retinas. Using scanning and transmission electron microscopy, we present evidence to suggest that the plasmalemmal surface of the calycal processes shortens along with the cytoskeletal actin core. We conclude that calycal processes of teleost rods are dynamic structures which shorten during light-activated myoid elongation.

Actin-dependent myoid elongation in teleost rod inner/outer segments occurs in the absence of net actin polymerization.

In the retinas of teleost fish, rod photoreceptors elongate in response to light. Light-activated elongation is mediated by the myoid of the rod inner segment and is actin-dependent. Inner segment F-actin filaments form bundles running parallel to the cell's long axis. We examined the mechanism of rod elongation using mechanically-detached rod fragments, consisting of the motile inner segment and sensory outer segment (RIS-ROS). When RIS-ROS are isolated from dark-adapted green sunfish and cultured in the light, they elongate 15 microns at 0.3-0.6 microns/min. Elongation was inhibited 65% by 0.1 microM Cytochalasin D, suggesting a requirement for actin assembly. To determine the extent of assembly during elongation, we used three approaches to measure the F-actin content in RIS-ROS: detection of pelletable actin by SDS-PAGE after detergent-extraction of RIS-ROS; quantification of fluorescein-phalloidin binding by fluorimetry, fluorescence-activated cell sorting and image analysis; estimation of total F-actin filament length by electron microscopy. All three assays indicated that no net assembly of RIS-ROS F-actin accompanied myoid elongation. An increase in F-actin content within the elongated myoid was counterbalanced by a decrease in F-actin content within the 13 microvillus-like calycal processes located at the end of the inner segment opposite to the growing myoid. O'Connor and Burnside (Journal of Cell Biology 89:517-524, 1981) showed that minus-ends of rod F-actin filaments are oriented towards the elongating myoid while plus-ends are oriented towards the shortening calycal processes. Our observations suggest that RIS-ROS elongation entails actin polymerization at the minus-ends of filaments coupled with depolymerization at the filament plus-ends.

Posttranslational modifications of tubulin in teleost photoreceptor cytoskeletons.

1. Posttranslational modifications of tubulin by acetylation and detyrosination have been correlated previously with microtubule stability in numerous cell types. 2. In this study, posttranslational modifications of tubulin and their regional distribution within teleost photoreceptor cones and rods are demonstrated immunohistochemically using antibodies specific for acetylated, detyrosinated, or tyrosinated tubulin. 3. Immunolocalization was carried out on isolated whole cones and mechanically detached rod and cone inner/outer segments. 4. Acetylated tubulin within rods and cones is found only in microtubules of the ciliary axoneme of the outer segment. Detyrosinated tubulin is also enriched in axonemes of both rod and cone outer segments. 5. Distributions of tyrosinated and detyrosinated cytoplasmic microtubules differ within cones and rods. In cones, detyrosinated and tyrosinated tubulins are both abundant throughout the cell body. In rods, the ellipsoid and myoid contain much more tyrosinated tubulin than detyrosinated tubulin. Comparisons between whole cones and cone fragments suggest that detyrosinated microtubules are more stable than tyrosinated microtubules in teleost photoreceptors. 6. Our findings provide further evidence that microtubules of teleost cones differ from rod microtubules in their stabilities and rapidity of turnover within the photoreceptor inner segment.