Biochemical characterisation of the D60A mutant of the elongation factor 1α from the archaeon Sulfolobus solfataricus

The D60A mutant of the elongation factor (EF) 1α from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1α; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.     

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.     

P2X receptors: New players in cancer pain

Pain is unfortunately a quite common symptom for cancer patients. Normally pain starts as an episodic experience at early cancer phases to become chronic in later stages. In order to improve the quality of life of oncological patients, anti-cancer treatments are often accompanied by analgesic therapies. The P2X receptor are adenosine triphosphate (ATP) gated ion channels expressed by several cells including neurons, cancer and immune cells. Purinergic signaling through P2X receptors recently emerged as possible common pathway for cancer onset/growth and pain sensitivity. Indeed, tumor microenvironment is rich in extracellular ATP, which has a role in both tumor development and pain sensation. The study of the different mechanisms by which P2X receptors favor cancer progression and relative pain, represents an interesting challenge to design integrated therapeutic strategies for oncological patients. This review summarizes recent findings linking P2X receptors and ATP to cancer growth, progression and related pain. Special attention has been paid to the role of P2X2, P2X3, P2X4 and P2X7 in the genesis of cancer pain and to the function of P2X7 in tumor growth and metastasis. Therapeutic implications of the administration of different P2X receptor blockers to alleviate cancer-associated pain sensations contemporarily reducing tumor progression are also discussed.     

Localization of growth arrest-specific genes on mouse Chromosomes 1, 7, 8, 11, 13, and 16

Growth arrest in NIH3T3 cells is associated with increased expression of a variety of mRNAs, several of which have been isolated as cDNA clones. Six of these growth arrest-specific (Gas) genes were mapped by following the inheritance of DNA restriction fragment length variants (RFLVs) associated with them in panels of recombinant inbred (RI) strains of mice and in the progeny of backcrosses both between laboratory mouse strains and between a laboratory strain and Mus spretus. The six genes are unlinked. Gas-1 maps to Chromosome (Chr) 13, Gas-2 to Chr 7, Gas-3 to Chr 11, Gas-4 to Chr 16, Gas-6 to Chr 8, and Gas-10 to Chr 1.     

The Interactomes of Influenza Virus NS1 and NS2 Proteins Identify New Host Factors and Provide Insights for ADAR1 Playing a Supportive Role in Virus Replication

Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution.     

A novel heterozygous mutation of the AIRE gene in a patient with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED)

Background

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED) is an autosomal recessive disease due to mutations of the autoimmune regulator (AIRE) gene. Typical manifestations include candidiasis, Addison's disease, and hypoparathyroidism. Type 1 diabetes, alopecia, vitiligo, ectodermal dystrophy, celiac disease and other intestinal dysfunctions, chronic atrophic gastritis, chronic active hepatitis, autoimmune thyroid disorders, pernicious anemia and premature ovarian failure are other rare associated diseases although other conditions have been associated with APECED.

Case presentation

What follows is the clinical, endocrinological and molecular data of a female APECED patient coming from Lithuania. The patient was affected by chronic mucocutaneous candidiasis, hypoparathyroidism and pre-clinical Addison's disease. Using direct sequencing of all the 14 exons of the AIRE gene in the patient's DNA, we identified in exon 6 the known mutation c.769 C>T (p.Arg257X) in compound heterozygosity with the newly discovered mutation c.1214delC (p.Pro405fs) in exon 10. The novel mutation results in a frameshift that is predicted to alter the sequence of the protein starting from amino acid 405 as well as to cause its premature truncation, therefore a non-functional Aire protein.

Conclusions

A novel mutation has been described in a patient with APECED with classical clinical components, found in compound heterozygosity with the c.769 C>T variation. Expanded epidemiological investigations based on AIRE gene sequencing are necessary to verify the relevancy of the novel mutation to APECED etiopathogenesis in the Lithuanian population and to prove its diagnostic efficacy in association with clinical and immunological findings.     

Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

Diethylenetriamine pentaacetic acid (DTPA) is a popular chelator agent for enabling the labeling of peptides for their use in structure-activity relationship study and biodistribution analysis. Solid phase peptide synthesis was employed to couple this commercially available chelator at the N-terminus of either the A-chain or B-chain of H2 relaxin. The coupling of the DTPA chelator at the N-terminus of the B-chain and subsequent loading of a lanthanide (europium) ion into the chelator led to a labeled peptide (Eu-DTPA-(B)-H2) in low yield and having very poor water solubility. On the other hand, coupling of the DTPA and loading of Eu at the N-terminus of the A-chain led to a water-soluble peptide (Eu-DTPA-(A)-H2) with a significantly improved final yield. The conjugation of the DTPA chelator at the N-terminus of the A-chain did not have any impact on the secondary structure of the peptide determined by circular dichroism spectroscopy (CD). On the other hand, it was not possible to determine the secondary structure of Eu-DTPA-(B)-H2 because of its insolubility in phosphate buffer. The B-chain labeled peptide Eu-DTPA-(B)-H2 required solubilization in DMSO prior to carrying out binding assays, and showed lower affinity for binding to H2 relaxin receptor, RXFP1, compared to the water-soluble A-chain labeled peptide Eu-DTPA-(A)-H2. The mono-Eu-DTPA labeled A-chain peptide, Eu-DTPA-(A)-H2, thus can be used as a valuable probe to study ligand-receptor interactions of therapeutically important H2 relaxin analogs. Our results show that it is critical to choose an approriate site for incorporating chelators such as DTPA. Otherwise, the bulky size of the chelator, depending on the site of incorporation, can affect yield, solubility, structure and pharmacological profile of the peptide.