Somatostatin-related therapeutics in ophthalmology: a review.

Somatostatin and its derivatives have been predominantly studied and succesfully used in endocrinological diseases. This article reviews the rationale of the use of somatostatin and its derivatives in ophthalmology based on current understanding of its action in the eye and summarizes previously published controlled studies and case series. The article points out future possible applications. Larger randomised controlled studies are necessary to confirm its current and future use. New ways of application could facilitate its broader use in ophthalmology.

Mutation in the AChR ion channel gate underlies a fast channel congenital myasthenic syndrome.

BACKGROUND: Most congenital myasthenic syndromes (CMS) have postsynaptic defects from mutations within the muscle acetylcholine receptor (AChR). Mutations underlying the slow channel syndrome cause a "gain of function" and usually show dominant inheritance, whereas mutations underlying AChR deficiency or the fast channel syndrome cause a "loss of function" and show recessive inheritance. OBJECTIVE: To characterize the disease mechanism underlying an apparently dominantly inherited CMS that responds to IV edrophonium. METHODS: DNA from CMS patients was analyzed for mutations by single-strand conformation polymorphism analysis, DNA sequence analysis, and restriction endonuclease digestion. Functional analysis of mutations was by alpha-bungarotoxin binding studies and by patch clamp analysis of mutant AChR expressed in human embryonic kidney cells. RESULTS: Analysis of muscle biopsies from father and son in an affected kinship showed normal endplate morphology and AChR number but severely reduced miniature endplate potentials. DNA analysis revealed that each harbors a single missense mutation in the AChR alpha-subunit gene, alphaF256L. Expression studies demonstrate this mutation underlies a fast channel phenotype with fewer and shorter ion channel activations. The major effect of alphaF256L, located within the M2 transmembrane domain, is on channel gating, both reducing the opening and increasing the closure rate. CONCLUSIONS: Mutation alphaF256L results in fast channel kinetics. Expression studies suggest a dominant-negative effect within the AChR pentamer, severely compromising receptor function.

Somatostatin in diabetic retinopathy.

Diabetic retinopathy is a leading cause of legal blindness in the adult population (30-70 year olds). The anatomical changes that occur in the retina during the course of disease are well defined in the literature but the causes are not yet fully understood. Laser photocoagulation of the retina and vitrectomy are currently used to treat diabetic retinopathy but the procedures are invasive and provide only temporary protection. The use of long-acting analogues of the naturally occurring peptide, somatostatin, has been considered by some a promising therapeutic option for retinopathy over the last decade. Experimental evidence supports its use in diabetic retinopathy but further clinical evidence, from larger treatment groups of longer trial duration, is required. Improved analogues with increased selectivity and modified bi-specific analogues are currently emerging and may help to make the use of somatostatin analogues a more realistic option in the treatment of diabetic retinopathy.

Recessive inheritance and variable penetrance of slow-channel congenital myasthenic syndromes.

BACKGROUND: Slow-channel congenital myasthenic syndromes (SCCMS) typically show dominant inheritance. They are caused by missense mutations within the subunits of muscle nicotinic acetylcholine receptors (AChR) that result in prolonged ion channel activations. SCCMS mutations within the AChR subunit are located in various functional domains, whereas fully described mutations in AChR non- subunits have, thus far, been located only in the M2 channel-lining domain. The authors identified and characterized two -subunit mutations, located outside M2, that underlie SCCMS in three kinships. In two of the three kinships, the syndrome showed an atypical inheritance pattern. METHODS: These methods included clinical diagnosis, mutation detection, haplotype analysis, and functional expression studies using single-channel recordings of mutant AChR transiently transfected into HEK293 cells. RESULTS: The authors identified two SCCMS mutations in the AChR subunit, L78P and L221F. Both mutations prolonged ACh-induced ion channel activations. L78P is present in a consanguineous family and appears to be pathogenic only when present on both alleles, and L221F shows variable penetrance in one of the two families that were identified harboring this mutation. CONCLUSION: SCCMS mutations may show a recessive inheritance pattern and variable penetrance. A diagnosis of SCCMS should not be ruled out in cases of CMS with an apparent recessive inheritance pattern.

Acetylcholine receptor delta subunit mutations underlie a fast-channel myasthenic syndrome and arthrogryposis multiplex congenita.

Limitation of movement during fetal development may lead to multiple joint contractures in the neonate, termed arthrogryposis multiplex congenita. Neuromuscular disorders are among the many different causes of reduced fetal movement. Many congenital myasthenic syndromes (CMSs) are due to mutations of the adult-specific epsilon subunit of the acetylcholine receptor (AChR), and, thus, functional deficits do not arise until late in gestation. However, an earlier effect on the fetus might be predicted with some defects of other AChR subunits. We studied a child who presented at birth with joint contractures and was subsequently found to have a CMS. Mutational screening revealed heteroallelic mutation within the AChR delta subunit gene, delta 756ins2 and delta E59K. Expression studies demonstrate that delta 756ins2 is a null mutation. By contrast, both fetal and adult AChR containing delta E59K have shorter than normal channel activations that predict fast decay of endplate currents. Thus, delta E59K causes dysfunction of fetal as well as the adult AChR and would explain the presence of joint contractures on the basis of reduced fetal movement. This is the first report of the association of AChR gene mutations with arthrogryposis multiplex congenita. It is probable that mutations that severely disrupt function of fetal AChR will underlie additional cases.

Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria.

The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity.

End-plate gamma- and epsilon-subunit mRNA levels in AChR deficiency syndrome due to epsilon-subunit null mutations.

Acetylcholine receptor (AChR) deficiency is the most common of the congenital myasthenic syndromes (CMS). Typically, the number of AChRs, measured by alpha-bungarotoxin binding, is reduced to 10-30% of normal levels, the miniature end-plate potentials are correspondingly reduced, and there are morphological changes at the motor end-plates. The majority of these syndromes are due to either missense or frameshift mutations within the gene encoding the adult-specific epsilon-subunit. These are often null mutations, but some mutant epsilon-subunits can be incorporated, at low levels, into functional AChRs in transfected cell lines. It is not clear, therefore, whether upregulation of the mutant epsilon-subunit mRNA could generate sufficient AChR to support neuromuscular transmission, albeit at a reduced level. Conversely, it might be that the mutant epsilon-subunit transcripts are subject to mRNA surveillance and 'nonsense-mediated' loss, leading to reduced epsilon-subunit mRNA expression. In either case, it is thought that neuromuscular transmission may be provided partly or entirely by incorporation of the foetal-specific gamma-subunit into end-plate AChR. gamma-Subunit mRNA is expressed at low levels in normal human muscle, but might be upregulated in CMS. The study of mRNA levels for AChR subunits should improve our understanding of genotype-phenotype relationships in CMS. Here we have defined homozygous epsilon-subunit mutations in four unrelated families with AChR deficiency and studied the steady-state levels of mRNA for AChR subunits at the motor end-plates by in situ hybridization. Although we demonstrated that each mutation would lead to almost complete absence of surface adult AChR expression, we detected similar robust expression of alpha- and epsilon-subunit mRNAs at end-plates of patient and control muscles, suggesting that mRNA transcripts for the epsilon-subunit are neither upregulated nor degraded preferentially. Interestingly, we were unable to detect any increase in gamma-subunit mRNA expression at CMS end-plates. Transgenic mice lacking the epsilon-subunit die 2-3 months after birth, suggesting that alpha(2)betadelta(2) pentamers cannot sustain neuromuscular transmission. Therefore, we tentatively conclude that the persistent low level expression of the gamma-subunit, which is present in normal human muscles as well as in AChR deficiency syndromes, is sufficient to enable patients with epsilon-subunit null alleles to survive.

Dominantly inherited familial myasthenia gravis as a separate genetic entity without involvement of defined candidate gene loci.

Myasthenia gravis (MG) is a sporadic autoimmune disorder affecting neuromuscular transmission. Very rarely autoimmune myasthenia gravis may be inherited within a family. We present here the genetic analysis of a Hungarian family where nine members from two generations are affected by myasthenia gravis. Genetic characterisation of this unique Hungarian family using linkage analysis and mutation screening excludes the involvement of defined candidate gene loci. These findings point to familial MG as a separate genetic entity. Identification of the underlying genetic defect in this family may greatly enhance our understanding of the pathogenesis of myasthenia gravis.

Inhibition of glucose transport and direct interactions with type 1 facilitative glucose transporter (GLUT-1) by etomidate, ketamine, and propofol: a comparison with barbiturates.

Ketamine, etomidate, propofol, and pentobarbital were compared for effects on and interactions with the type 1 facilitative glucose transporter (GLUT-1). Fluxes of radiolabeled hexoses were used to determine the effects of anesthetics on GLUT-1 function. Hypotonic hemolysis of human erythrocytes was used to assess perturbations of membrane integrity. Quenching of intrinsic protein fluorescence was used to assess the direct interactions of anesthetics with purified GLUT-1. Pentobarbital, ketamine, etomidate, and propofol inhibited glucose transport in murine fibroblasts with IC(50) values of 0.8, 1. 6, 0.1, and 0.4 mM, respectively. Pentobarbital, ketamine, etomidate, and propofol also inhibited sugar transport in human erythrocytes. The IC(50) values for pentobarbital and ketamine exhibited substrate dependence for equilibrium exchange but not unidirectional effluxes. This was not observed for etomidate. Propofol did not inhibit equilibrium exchange but did inhibit unidirectional efflux with little substrate dependence. Pentobarbital protected against hemolysis, whereas etomidate and ketamine promoted hemolysis of erythrocytes. Propofol had no effect on membrane integrity. Pentobarbital, ketamine, and etomidate all interacted directly with GLUT-1, with apparent K(d) values of 2.2, 0.8, and 0.5 mM, respectively. Like barbiturates, ketamine, etomidate, and propofol inhibited GLUT-1 at concentrations near to those used pharmacologically. Inhibition of GLUT-1 by these intravenous general anesthetics was complex, exhibiting differential kinetic effects on equilibrium exchange versus unidirectional fluxes and contrasting substrate dependencies. Like barbiturates, ketamine and etomidate bound to GLUT-1 with affinities that paralleled inhibition of glucose transport. As a class, intravenous general anesthetics, in contrast to inhalation anesthetics, inhibit GLUT-1-mediated glucose transport.

Novel functional epsilon-subunit polypeptide generated by a single nucleotide deletion in acetylcholine receptor deficiency congenital myasthenic syndrome.

Acetylcholine receptor (AChR) deficiency is a recessively inherited congenital myasthenic syndrome in which fatigable muscle weakness results from impaired neuromuscular transmission caused by reduced AChR numbers. In mature muscle, AChRs consist of alpha2 betadelta together with the adult-specific epsilon subunit. We have identified a deletion of the first nucleotide in exon 12 of the AChR epsilon-subunit gene (epsilon1267delG) and demonstrate its recessive inheritance segregates with disease in 6 unrelated cases of AChR deficiency. In addition, we found that both healthy and AChR-deficient muscle contain a population of AChR epsilon-subunit mRNA transcripts that retain intron 11. We investigated the possible consequences of combining this mutation with the alternative mRNA species through AChR expression studies in human embryonic kidney cells and Xenopus oocytes. Epsilon1267delG generates a polypeptide that lacks M4 and is not detected in surface AChR, whereas retention of intron 11 in the RNA transcript restores the reading frame, conserves M4, and generates a polypeptide that is incorporated into functional surface AChR, although at a reduced level, consistent with the disease phenotype. Our results indicate that for some AChR deficiency mutations located between M3 and M4, the retention of intron 11 in the epsilon-subunit mRNA transcripts may rescue adult AChR function.

Mapping of autosomal dominant progressive external ophthalmoplegia to a 7-cM critical region on 10q24.

OBJECTIVE: To map the gene responsible for autosomal dominant progressive external opthalmoplegia. BACKGROUND: The pathogenesis of progressive external ophthalmoplegia (PEO) can be associated with multiple deletions of mitochondrial DNA (mtDNA). PEO may show autosomal dominant (adPEO) or autosomal recessive (arPEO) patterns of inheritance, indicating that the genetic defect has a Mendelian basis and most likely involves a nuclear gene encoding a protein that interacts with the mitochondrial genome. adPEO is heterogeneous genetically, and thus far disease loci have been identified on chromosomes 3 and 10. The locus on chromosome 10q23-q25 was assigned by linkage analysis in a single Finnish family. METHODS: Samples from a large Pakistani family with adPEO, in which clinical symptoms are bilateral ptosis, limitations of eye movements, and varying degrees of proximal muscle weakness, were collected. Muscle biopsy and mtDNA rearrangement analysis was used to confirm the diagnosis. Genomewide linkage analysis was set up using a set of 391 microsatellite markers. RESULTS: The muscle biopsy from an affected member showed ragged red fibers, increased succinic dehydrogenase staining, lack of cytochrome oxidase activity, and multiple deletions of mtDNA. The disease locus was mapped to 10q23.31-q25.1 by linkage analysis, and a maximum lod score of 5.72 was obtained with D10S1267. CONCLUSION: By analysis of meiotic recombinations in affected individuals, the critical region was restricted to the 7-cM interval between D10S198 and D10S1795.

Effects of barbiturates on facilitative glucose transporters are pharmacologically specific and isoform selective.

Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [3H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC50s of 0.2-2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all 10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms.

Mutation of the acetylcholine receptor epsilon-subunit promoter in congenital myasthenic syndrome.

Congenital myasthenic syndrome comprises a heterogeneous group of inherited disorders of neuromuscular transmission. Acetylcholine receptor (AChR) deficiency is the most common form of congenital myasthenic syndrome and in most cases results from mutations within the coding region of the AChR epsilon subunit. However, studies in mice have established that synapse-specific expression of AChR is dependent on a sequence contained within the AChR-subunit promoter regions, termed an N-box. We describe a consanguineous family in which 2 of 7 siblings had clinical and electromyographic features consistent with AChR deficiency. Muscle biopsy demonstrated low AChR numbers, establishing the disorder as postsynaptic. Single-strand conformational polymorphism analysis identified an abnormal conformer in the AChR epsilon-subunit gene promoter of the patients. DNA sequence and restriction endonuclease analysis shows that the disorder cosegregates with recessive inheritance of a single point mutation, a transition (C-->T) in the N-box of the epsilon-subunit promoter. Analysis of an intercostal biopsy from 1 of the patients showed a dramatic reduction in epsilon-subunit mRNA levels compared with disease and normal controls. This is the first evidence in humans that an N-box mutation can lead to disruption of epsilon-subunit transcription, resulting in the loss of adult AChR synthesis and the clinical phenotype of AChR-deficiency congenital myasthenic syndrome.

Mutations in different functional domains of the human muscle acetylcholine receptor alpha subunit in patients with the slow-channel congenital myasthenic syndrome.

Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slow-channel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine receptor (AChR). We have identified three new SCCMS mutations and a further familial case of the alpha G153S mutation. Single channel recordings from wild-type and mutant human AChR expressed in Xenopus oocytes demonstrate that each mutation prolongs channel activation episodes. The novel mutations alpha V156M, alpha T254I and alpha S269I are in different functional domains of the AChR alpha subunit. Whereas alpha T254I is in the pore-lining region, like five of six previously reported SCCMS mutations, alpha S269I and alpha V156M are in extracellular domains. alpha S269I lies within the short extracellular sequence between M2 and M3, and identifies a new region of muscle AChR involved in ACh binding/channel gating. alpha V156M, although located close to alpha G153S which has been shown to increase ACh binding affinity, appears to alter channel function through a different molecular mechanism. Our results demonstrate heterogeneity in the SCCMS, indicate new regions of the AChR involved in ACh binding/channel gating and highlight the potential role of mutations outside the pore-lining regions in altering channel function in other ion channel disorders.

Genes at the junction--candidates for congenital myasthenic syndromes.

The neuromuscular junction is the site of several myasthenic (mys, muscle; aesthenia, weakness) disorders of autoimmune and genetic origin. The acquired autoimmune conditions are mainly adult-onset and caused by antibodies to specific neuronal and muscle ion channels, but can occur neonatally due to placental transfer of maternal antibodies. This review focuses on the rarer genetic conditions, called congenital myasthenic syndromes (CMS), that often present at birth. Mutations have yet to be characterized for familial infantile myasthenia, acetylcholinesterase deficiency and ACh-receptor deficiency; but genes encoding both structural and functional NMJ protiens should be considered. Other syndromes have recently been shown to involve defects in the functioning of the ACh receptor itself. In particular, eight different mutations have been reported in cases of the slow channel syndrome, a dominant condition associated with point mutations that generate single amino acid changes within the ACh receptor and result in prolonged channel activations. These investigations are providing new insights into the structure and function of the ACh receptor. Further studies of CMS should pave the way for analysis and treatment of disorders involving other synapses in the peripheral and central nervous system.

Isolation of an Ustilago maydis gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase and expression of a C-terminal-truncated form in Escherichia coli.

A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was isolated from the maize fungal pathogen Ustilago maydis. This was accomplished by identifying cDNA and genomic clones that hybridized to an internal fragment of the gene, amplified from U. maydis genomic DNA by PCR. The nature of the gene was determined by nucleotide sequence analysis, and by comparing the derived amino acid sequence of the gene with HMG-CoA reductases from yeast, and from other organisms. The hydrophobic nature of the N-terminal region of the deduced protein sequence also supported the view that this gene encoded HMG-CoA reductase. A C-terminal-truncated fragment of the U. maydis HMG-CoA reductase gene was shown to be expressed in Escherichia coli in a catalytically active form. The expressed protein was also shown to be sensitive to an inhibitor of mammalian HMG-CoA reductase activity.