Rho kinase and protein kinase C involvement in vascular smooth muscle myofilament calcium sensitization in arteries from diabetic rats.

BACKGROUND AND PURPOSE: Diabetes mellitus (DM) causes multiple dysfunctions including circulatory disorders such as cardiomyopathy, angiopathy, atherosclerosis and arterial hypertension. Rho kinase (ROCK) and protein kinase C (PKC) regulate vascular smooth muscle (VSM) Ca(2+) sensitivity, thus enhancing VSM contraction, and up-regulation of both enzymes in DM is well known. We postulated that in DM, Ca(2+) sensitization occurs in diabetic arteries due to increased ROCK and/or PKC activity. EXPERIMENTAL APPROACH: Rats were rendered hyperglycaemic by i.p. injection of streptozotocin. Age-matched control tissues were used for comparison. Contractile responses to phenylephrine (Phe) and different Ca(2+) concentrations were recorded, respectively, from intact and chemically permeabilized vascular rings from aorta, tail and mesenteric arteries. KEY RESULTS: Diabetic tail and mesenteric arteries demonstrated markedly enhanced sensitivity to Phe while these changes were not observed in aorta. The ROCK inhibitor HA1077, but not the PKC inhibitor chelerythrine, caused significant reduction in sensitivity to agonist in diabetic vessels. Similar changes were observed for myofilament Ca(2+) sensitivity, which was again enhanced in DM in tail and mesenteric arteries, but not in aorta, and could be reduced by both the ROCK and PKC blockers. CONCLUSIONS AND IMPLICATIONS: We conclude that in DM enhanced myofilament Ca(2+) sensitivity is mainly manifested in muscular-type blood vessels and thus likely to contribute to the development of hypertension. Both PKC and, in particular, ROCK are involved in this phenomenon. This highlights their potential usefulness as drug targets in the pharmacological management of DM-associated vascular dysfunction.

Schistosome I/Lamides - A new family of bioactive helminth neuropeptides

Here we report the identification of a new family of helminth neuropeptides with members in both nematodes and flatworms, and include preliminary cell biological and functional characterisation of one of the peptides from the trematode parasite of humans, Schistosoma mansoni. Bioinformatics and Rapid Amplification of cDNA Ends (RACE)-PCR were used to identify the completes. mansoni neuropeptide precursor gene Sm-npp-1, which encodes three pentapeptides bearing the motif (A/G)FVR(I/L).NH2. Similar peptides were identified in three other flatworm species and in 15 nematode species. Quantitative PCR (qPCR) and immunocytochemical (ICC) analyses showed that Sm-npp-1 is constitutively expressed in larval and adult worms. ICC and confocal microscopy were employed to localise one of the schistosome NPP-1 peptides (GFVRIamide) in adult worms and schistosomules; antibodies labelled a pair of neurones in the cerebral ganglia that extend posteriorly along the main nerve cords. GFVRIamide displayed no detectable co-localisation with FMRFamide-like peptides (FLPs), nor was it detectable in muscle innervation. Exogenously applied peptide had a significant inhibitory effect on the mobility of whole adult worm pairs at 10(-5) M (n = 9). Finally, we explored Sm-npp-1 function in schistosomules using RNA interference (RNAi); we successfully achieved specific knockdown of the Sm-npp-1 transcript (54.46 +/- 10.41% knockdown, n = 3), but did not detect any clear, aberrant mobility or morphological phenotypes. NPP-1-like peptides are a new family of helminth peptides with a cell-specific expression pattern distinct from FLPs and a modulatory effect on schistosome muscular activity. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy. © FASEB.

Eyes open to stem cells: safety trial may pave the way for cell therapy to treat retinal disease in patients

A clinical trial using human embryonic stem cell (hESC) therapy for an inherited retinal degenerative disease is about to commence. The Advanced Cell Technology (ACT) trial will treat patients with Stargardt's macular dystrophy using transplanted retinal pigment epithelium derived from hESCs. Currently, no effective treatment is available for Stargardt's disease so a stem cell-based therapy that can slow progression of this blinding condition could represent a significant breakthrough. While there are some hurdles to clear, the ACT trial is a fine example of translational research that could eventually pave the way for a range of stem cell therapies for the retina and other tissues.

Modulation of the motility of the vagina vera of Ascaris suum in vitro by FMRFamide-related peptides

Ascaris suum contains a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8, also called PF3) have been extensively studied and are known to exert actions on somatic muscle strips of the worm. In the present study, the effects of AF1, AF2 and AF8 on the activity of the vagina vera of female A. suum have been examined in vitro. The vagina vera is a muscular tube connecting the uterus and vagina uteri to the gonopore and is probably involved in regulating egg output. The tissue exhibited spontaneous, rhythmic contractions in vitro, which were modulated by each of the FaRPs tested. The effects of each of the peptides were qualitatively and quantitatively different, and in each case were reversible. AF1 (1 mu M) caused a biphasic response in the form of a transient lengthening of the preparation, followed by a shortening; contractions were initially inhibited but resumed 5 min post-addition of the peptide. Lower concentrations (less than or equal to 0.1 mu M) induced a less marked effect, with rhythmic contractions returning 5 min post-addition. AF2 and AF8 reduced contraction frequency at concentrations greater than or equal to 0.1 mu M. Both peptides also caused the tissue to shorten, although the effects of AF8 on baseline tension were inconsistent. The apparent potencies of AF1 and AF8 on contraction frequency of the vagina vera were 10-fold greater than AF2 and, unlike their actions on A. suum somatic body wall muscles, the actions of AF1 and AF2 were qualitatively different. Indeed, the effects of each of these FaRPs on the vagina vera were markedly different from those observed on the somatic muscle.

THE ORGANIZATION OF THE NERVOUS-SYSTEM IN PLATHELMINTHES - THE NEUROPEPTIDE F-IMMUNOREACTIVE PATTERN IN CATENULIDA, MACROSTOMIDA, PROSERIATA

The organization of the nervous system of Archilopsis unipunctata Promonotus schultzei and Paramonotus hamatus (Monocelididae, Proseriata) and Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) was studied by immunocytochemistry, using antibodies to the authentic flatworm neuropeptide F (NPF) (Moniezia expansa). The organization of the nervous system of the Monocelididae was compared to that of the nervous system of Bothriomolus balticus (Otoplanidae), a previously studied species of another family of the Proseriata. The results show that the main nerve cords (MCs), independent of lateral or ventral position in the Monocelididae and the Otoplanidae, correspond to each other. The study also confirms the status of the lateral cords as main cords (MCs) in S. leucops and M. lineare. Common for MCs in the members of the investigated taxa are the following features: MCs consist of many fibres, originate from the brain and are adjoined to 5-HT-positive neurons. In Monocelididae and Otoplanidae, the MCs additionally have the same type of contact to the pharyngeal nervous system. Also common for both proseriate families is the organization of the two lateral nerve cords, with weaker connections to the brain, and the pair of dorsal cords running above the brain. The organization of the minor cords differs. The Monocelididae have a pair of thin ventral cords forming a mirror image of the dorsal pair. Furthermore, an unpaired ventral medial cord connecting medial commissural cells was observed in P. schultzei. Marginal nerve cords, observed in Otoplanidae, are absent in Monocelididae. All minor nerve cords are closely connected to the peripheral nerve plexus. The postulated trends of condensation of plexal fibres to cords and/or the flexibility of the peripheral nerve plexus are discussed. In addition, the immunoreactivity (IR) pattern of NPF was compared to the IR patterns of the neuropeptide RFamide and the indoleamine, 5-HT (serotonin). Significant differences between the distribution of IR to NPF and to 5-HT occur. 5-HT-IR dominates in the submuscular and subepidermal plexuses. In the stomatogastric plexus of M. lineare, only peptidergic IR is observed in the intestinal nerve net. The distribution of NPF-IR in fibres and cells of the intestinal wall in M. lineare indicates a regulatory function for this peptide in the gut, while a relationship with ciliary and muscular locomotion is suggested for the 5-HT-IR occurring in the subepidermal and submuscular nerve plexuses. In M. lineare, the study revealed an NPF- and RFamide-positive cell pair, marking the finished development of new zooids. This finding indicates that constancy of these cells is maintained in this asexually reproducing and regenerating species.

COMPARATIVE ANALYSES OF THE NEUROPEPTIDE-F (NPF)-RELATED AND FMRFAMIDE-RELATED PEPTIDE (FARP)-IMMUNOREACTIVITIES IN FASCIOLA-HEPATICA AND SCHISTOSOMA SPP

Immunochemical techniques were used to determine the distribution, chemical characteristics and relative abundance of immunoreactivity (IR) to two native platyhelminth neuropeptides, neuropeptide F (NPF) (Moniezia expansa) and the FMRFamide-related peptide (FaRP), GNFFRFamide, in the trematodes, Fasciola hepatica and Schistosoma mansoni; the larger S. margrebowiei was used in the chemical analysis. Extensive immunostaining for the two peptides was demonstrated throughout the nervous systems of both F. hepatica and S. mansoni, with strong IR also in the innervation of muscular structures, including those associated with the egg-forming apparatus. The patterns of immunostaining were similar to those previously described for the vertebrate neuropeptide Y superfamily of peptides and for FMRFamide. Ultrastructurally, gold labelling of NPF- and GNFFRFamide-IRs was localized exclusively to the contents of secretory vesicles in the axons and somatic cytoplasm of neurones. Double-labelling experiments showed an apparent homogeneity of antigenic sites, in all probability due to the demonstrated cross-reactivity of the FaRP antiserum with NPF. Radioimmunoassay of acid-ethanol extracts of the worms detected 8.3 pmol/g and 4.7 pmol/g equivalents of NPF- and FMRFamide-IRs, respectively, for F. hepatica, and corresponding values of 4.9 pmol/g and 4.3 pmol/g equivalents for S. margrebowiei. Gel-permeation chromatography resolved IR to both peptides in discrete peaks and these eluted in similar positions to synthetic NPF (M. expansa) and GNFFRFamide, respectively.

THE GULL-TAPEWORM, DIPHYLLOBOTHRIUM-DENDRITICUM AND NEUROPEPTIDE-F - AN IMMUNOCYTOCHEMICAL STUDY

Neuropeptide F (Moniezia expansa) immunoreactivity (NPF-IR) has been detected in the nervous system of plerocercoid and adult stages of the gull-tapeworm Diphyllobothrium dendriticum, using immunocytochemical methodology. The application of the antiserum for this authentic flatworm neuropeptide to whole-mounts and frozen sections of the worm has resulted in new information about its neuroanatomy. Thus, at regular intervals, transverse nerves extend from the main nerve cords laterally, joining the longitudinal lateral minor cords in the cortical parenchyma. In the adult worm, the transverse nerves are located at the posterior border of each proglottis. The medullary parenchyma lacks NPF-IR. The NPF-immunoreactive cell bodies are bi- to multipolar and preferentially located in the peripheral nervous system, in close association with the holdfast musculature of the scolex and the extensive body musculature. NPF-IR was observed in the innervation to the muscular ducts of the reproductive system. The pattern of NPF-IR was compared with that recorded for RFamide- and 5-HT-IR and double-immunostaining has revealed separate populations of serotoninergic and peptidergic neurones.

Immunohistochemical localization of a Shaker-related voltage-gated potassium channel protein in Schistosoma mansoni (Trematoda: Digenea)

We have recently isolated a cDNA (SKV1.1) encoding a Shakei-related K+ channel from the human parasitic trematode Schistosoma mansoni. In order to better understand the functions of SKv1.1 protein, the distribution of SKv1.1 protein in adult S. mansoni was analyzed by immunohistochemistry using a region-specific antibody. SKV1.1 proteins were widely expressed in the nervous and muscular systems. The strongest immunoreactivity (IR) was observed in the nervous system of both male and female. In the nervous system, IR for SKv1.1 proteins was localized in cell bodies and nerve fibers of the anterior ganglia, the central commissure, and the main nerve cords. IR was also observed in the dorsal and the ventral peripheral nerve nets, fine nerve fibers entering into a variety of structures such as the dorsal tubercles, longitudinal and ventral muscle fibers, and oral and ventral suckers. In the muscular system, SKv1.1 proteins were localized to the longitudinal, circular, and ventral muscle fibers of male as well as in isolated muscle fibers where native A-type K+ currents were measured. Moderate IR was also seen in a large number of cell bodies in the parenchyma. These results indicate that SKv1.1 protein may play an important role in the regulation of the excitability of neurons and muscle cells of S. mansoni. (C) 1995 Academic Press, Inc.

Indications for penetrating keratoplasty and associated procedures, 1989-1995

Purpose. To identify changing trends in indications for penetrating keratoplasty and associated surgical procedures. Methods. Review of charts from all patients who underwent penetrating keratoplasty at Wills Eye Hospital from January 1, 1989 through December 31, 1995. Results. A total of 2,442 corneal transplants were performed in 2,186 patients. The leading indication for penetrating keratoplasty was pseudophakic corneal edema, accounting for 634 cases (26.0%); 54.7% of them were associated with anterior chamber intraocular lenses, 36.4% with posterior chamber intraocular lenses, and 3.1% with iris-fixated intraocular lenses. Regraft (17.8%), Fuchs' dystrophy (15.7%), and keratoconus (13.2%) followed pseudophakic corneal edema in frequency. Cataract extraction, with or without intraocular lens implantation, was combined with penetrating keratoplasty in 439 cases of 1,264 phakic eyes (34.7%). Intraocular lens exchange was performed in 285 of the 634 cases of pseudophakic cornea edema (44.9%). Conclusion. Pseudophakic corneal edema was the leading indication for penetrating keratoplasty, with an increasing number of cases associated with posterior chamber intraocular lenses during the study period (p = 0.001). The number of regrafts steadily increased between 1989 and 1995 (p = 0.001), being the second most common indication for corneal transplantation since 1992.

An analysis of ABCR mutations in British patients with recessive retinal dystrophies

PURPOSE. Several reports have shown that mutations in the ABCR gene can lead to Stargardt disease (STGD)/fundus flavimaculatus (FFM), autosomal recessive retinitis pigmentosa (arRP), and autosomal recessive cone-rod dystrophy (arCRD). To assess the involvement of ABCR in these retinal dystrophies, the gene was screened in a panel of 70 patients of British origin. METHODS. Fifty-six patients exhibiting the STGD/FFM phenotype, 6 with arRP, and 8 with arCRD, were screened for mutations in the 50 exons of the ABCR gene by heteroduplex analysis and direct sequencing. Microsatellite marker haplotyping was used to determine ancestry. RESULTS. In the 70 patients analyzed, 31 sequence changes were identified, of which 20 were considered to be novel mutations, in a variety of phenotypes. An identical haplotype was associated with the same pair of in-cis alterations in 5 seemingly unrelated patients and their affected siblings with STGD/FFM. Four of the aforementioned patients were found to carry three alterations in the coding sequence of the ABCR gene, with two of them being in-cis. CONCLUSIONS. These results suggest that ABCR is a relatively polymorphic gene. Because putative mutations have been identified thus far only in 25 of 70 patients, of whom only 8 are compound heterozygotes, a large number of mutations have yet to be ascertained. The disease haplotype seen in the 5 patients carrying the same 'complex' allele is consistent with the presence of a common ancestor.

Mutational spectrum of the ZEB1 gene in corneal dystrophies supports a genotype-phenotype correlation

Mutations in ZEB1 have been reported in posterior polymorphous corneal dystrophy (PPCD3; MIM #609141) and Fuchs' endothelial corneal dystrophy (FECD6; MIM #613270). Although PPCD and keratoconus are clinically and pathologically distinct, PPCD has been associated with keratoconus, suggesting a common genetic basis. The purpose of our study was to perform mutational screening of the ZEB1 gene in patients affected with keratoconus or PPCD.

Next generation sequencing-based molecular diagnosis of retinitis pigmentosa: identification of a novel genotype-phenotype correlation and clinical refinements

Retinitis pigmentosa (RP) is a devastating form of retinal degeneration, with significant social and professional consequences. Molecular genetic information is invaluable for an accurate clinical diagnosis of RP due to its high genetic and clinical heterogeneity. Using a gene capture panel that covers 163 of the currently known retinal disease genes, including 48 RP genes, we performed a comprehensive molecular screening in a collection of 123 RP unsettled probands from a wide variety of ethnic backgrounds, including 113 unrelated simplex and 10 autosomal recessive RP (arRP) cases. As a result, 61 mutations were identified in 45 probands, including 38 novel pathogenic alleles. Interestingly, we observed that phenotype and genotype were not in full agreement in 21 probands. Among them, eight probands were clinically reassessed, resulting in refinement of clinical diagnoses for six of these patients. Finally, recessive mutations in CLN3 were identified in five retinal degeneration patients, including four RP probands and one cone-rod dystrophy patient, suggesting that CLN3 is a novel non-syndromic retinal disease gene. Collectively, our results underscore that, due to the high molecular and clinical heterogeneity of RP, comprehensive screening of all retinal disease genes is effective in identifying novel pathogenic mutations and provides an opportunity to discover new genotype-phenotype correlations. Information gained from this genetic screening will directly aid in patient diagnosis, prognosis, and treatment, as well as allowing appropriate family planning and counseling.

Upper limb deep vein thrombosis due to Langer's axillary arch

Langer's axillary arch is a recognized muscular anomaly characterized by an accessory muscular band crossing the axilla that rarely causes symptoms. We describe a patient who presented with an upper limb deep vein thrombosis caused by this aberrant muscle, which we believe is the first reported case. Axillary surgery with division of the aberrant muscle relieved upper limb venous obstruction in this patient. (J Vase Surg 2012;55:234-6.)

Fruit and vegetable consumption and muscle strength and power during adolescence: a cross-sectional analysis of the Northern Ireland Young Hearts Project 1999-2001

Objectives: To examine the association between fruit and vegetable (FV) consumption and muscle strength and power in an adolescent population. Methods: We conducted a cross-sectional analysis among 1019 boys and 998 girls, aged 12 and 15 years, who participated in The Young Hearts Project. FV consumption (excluding potatoes) was assessed by 7-d diet history. Grip strength and jump power was assessed with a dynamometer and Jump-MD meter, respectively. Associations between FV consumption and strength and power were assessed by regression modelling. Results: Boys and girls with the highest FV intakes (>237.71 g/d and >267.57 g/d, respectively, based on the highest tertile) had significantly higher jump power than those with the lowest intakes (<135.09 g/d and <147.43 g/d, respectively), after adjustment for confounding factors. Although girls with the highest FV intakes had higher grip strength than those with the lowest intakes, no significant independent associations were evident between FV intake and grip strength in boys or girls. Similar findings were observed when FV were analysed separately.Conclusions: Higher FV consumption in this group of adolescents was positively associated with muscle power. There was no independent association between higher FV consumption and muscle strength. Intervention studies are required to determine whether muscle strength and power can be improved through increased FV consumption.