Inheritable forms of medullary thyroid carcinoma

Medullary thyroid carcinoma (MTC) arises from parafollicular or C cells of the thyroid that produce calcitonin. It accounts for 5-10% of all thyroid cancers. Hereditary MTC represents 20-30% of all MTCs. It can be transmitted with an autosomal dominant pattern, either as a single entity, familial MTC, or it can arise as part of a multiple endocrine neoplasia (MEN) syndrome type 2A or 2B. The identification of hereditary MTC has been facilitated in recent years by the direct analysis of the ret proto-oncogene.     

Determination of hammerhead ribozyme kinetic constants at high molar ratio ribozyme-substrate

 Hammerhead ribozymes provide valuable tools in the field of gene therapy due to their cleavage specificity and the broad range of RNA targets. A major prerequisite for the selection of suitable ribozymes for in vivo application is represented by in vitro determination of ribozyme cleavage kinetic constants. From the experimental cleavage data, kinetic constants are usually calculated under the assumption of rapid conversion of the substrate into the ribozyme-substrate complex. However, this condition is often not satisfied for ribozymes carrying additional RNA stretches, due to cloning strategies or necessary for ribozyme expression in the cell. To overcome this problem, we propose a mathematical model which is able to calculate ribozyme kinetic constants in the case of non-rapid conversion of substrate into ribozyme-substrate complex. In addition, our system gives the opportunity to evaluate the nature of the S conversion into ES through the determination of a model parameter. The validity of the proposed model is restricted to the hypothesis of a ribozyme excess over the substrate at the beginning of the cleavage reaction and to the absence of any mass exchange with the external environment. Received: 1 February 2001 / Revised version: 1 September 2001 / Published online: 23 August 2002     

Mutations altering the cleavage specificity of a homing endonuclease

The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein–DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.     

Congenital fibrosis of the vertically acting extraocular muscles maps to the <Emphasis Type="Italic">FEOM3</Emphasis> locus

The diagnosis of congenital fibrosis of the extraocular muscles (CFEOM) encompasses several different inherited strabismus syndromes characterized by congenital restrictive ophthalmoplegia affecting extraocular muscles innervated by the oculomotor and/or trochlear nerves. The OMIM database (http://www.ncbi.nlm.nih.gov/Omim/) currently contains four familial CFEOM phenotypes: CFEOM1-3, which map to the FEOM1-3 loci (MIM 135600, 602078, 604361), respectively, and congenital fibrosis of the vertically acting extraocular muscles (MIM 600638), reported in a single family without a corresponding genotype. We have had the opportunity to study the reported family with this fourth phenotype and now demonstrate that their phenotype can be reclassified as CFEOM3 and that it maps to FEOM3, flanked by D16S498 to 16qter, with a maximum lod score of 6.0.     

Investigating the association between OPA1 polymorphisms and glaucoma: comparison between normal tension and high tension primary open angle glaucoma

OPA1, the gene responsible for autosomal dominant optic atrophy, represents a good candidate gene for glaucoma, as there are similarities in the clinical phenotype and OPA1 is expressed in the optic nerve. Single nucleotide polymorphisms on intervening sequence (IVS) 8 of the OPA1gene (genotype IVS8+4 C/T;+32T/C) were recently found to be strongly associated with normal tension glaucoma (NTG). In order to investigate whether this association exists in patients with high-tension glaucoma (HTG), 90 well-characterized HTG patients were examined for the presence of these OPA1polymorphisms by PCR amplification followed by bi-directional sequencing. Five out of 90 HTG subjects (5.6%; 95% CI 1.8-12.5) were found to carry the OPA1 genotype IVS 8+4 C/T; +32 T/C, compared with 32/163 (19.6%; 95% CI 13.8-26.6) NTG subjects [chi(2)=9.2, P=0.002, OR 4.1 (95% CI 1.6-11.1)], and 7/186 (3.8%; 95% CI 1.5-7.6) control subjects [chi(2)=0.47, P=0.49, OR 1.5 (95% CI 0.5-4.9)]. These results indicate that unlike NTG, the OPA1 genotype IVS8+4 C/T,+32T/C is not significantly associated with high-tension primary open angle glaucoma, and suggest genetic heterogeneity between the conditions.     

Synaptosome-associated protein of 25 kilodaltons modulates Kv2.1 voltage-dependent K(+) channels in neuroendocrine islet beta-cells through an interaction with the channel N terminus

Insulin secretion is initiated by ionic events involving membrane depolarization and Ca(2+) entry, whereas exocytic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins mediate exocytosis itself. In the present study, we characterize the interaction of the SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) with the beta-cell voltage-dependent K(+) channel Kv2.1. Expression of Kv2.1, SNAP-25, and syntaxin 1A was detected in human islet lysates by Western blot, and coimmunoprecipitation studies showed that heterologously expressed SNAP-25 and syntaxin 1A associate with Kv2.1. SNAP-25 reduced currents from recombinant Kv2.1 channels by approximately 70% without affecting channel localization. This inhibitory effect could be partially alleviated by codialysis of a Kv2.1N-terminal peptide that can bind in vitro SNAP-25, but not the Kv2.1C-terminal peptide. Similarly, SNAP-25 blocked voltage-dependent outward K(+) currents from rat beta-cells by approximately 40%, an effect that was completely reversed by codialysis of the Kv2.1N fragment. Finally, SNAP-25 had no effect on outward K(+) currents in beta-cells where Kv2.1 channels had been functionally knocked out using a dominant-negative approach, indicating that the interaction is specific to Kv2.1 channels as compared with other beta-cell Kv channels. This study demonstrates that SNAP-25 can regulate Kv2.1 through an interaction at the channel N terminus and supports the hypothesis that SNARE proteins modulate secretion through their involvement in regulation of membrane ion channels in addition to exocytic membrane fusion.     

Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition

This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.