Subclinical TRAPS treated with canakinumab.

Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is a rare autoinflammatory disease characterized by recurrent episodes of fever and systemic inflammation. We describe the case of a 19-year-old patient who was referred to our attention with frequent subclinical TRAPS episodes characterized by mild arthralgias and crampy abdominal pain, without fever. Inflammatory markers, including serum amyloid A which increases the risk of long-term amyloidosis, were persistently high also when the patient was in good general conditions. Therapy with human anti-interleukin 1ß monoclonal antibody, canakinumab, led to disease control and normalization of the inflammatory markers, which are currently still normal. This clinical case supports the need to treat also subclinical TRAPS. In this respect, canakinumab is effective and reduces the risk of developing amyloidosis.

Cardiovascular risk factors in childhood obesity.

Childhood obesity is the "disease of the century". This article reviews the early cardiovascular risk factors and the recommendations to prevent them in the overweight and obese children. A comprehensive search of published literature was carried out to identify all articles published on this topic in English and Italian from 1999 to 2020.

The A26G replacement in the consensus sequence A-X-X-X-X-G-K-[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon Sulfolobus solfataricus.

A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF-2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A-X-X-X-X-G-K-[T,S] of the elongation factors EF-G or EF-2 into the corresponding G-X-X-X-X-G-K-[T,S] motif which is present in all the other GTP-binding proteins. The rate of poly(U)-directed poly(Phe) synthesis and the ribosome-dependent GTPase activity of A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacement partially affected the function of SsEF-2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78, 832-837]. Surprisingly, A26GSsEF-2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF-2. The involvement of this alanine residue in the guanine nucleotide-binding pocket of EF-2 or EF-G is discussed.

Heterologous expression in Escherichia coli of the gene encoding an archaeal thermoacidophilic elongation factor 2. Properties of the recombinant protein.

The gene encoding the elongation factor 2 from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-2) was expressed in Escherichia coli using the pT7-7 expression vector. The synthesis of the heterologous product did not increase upon addition of isopropyl-beta-thiogalactopyranoside. The amount of purified intact recombinant SsEF-2 (SsEF-2rec) was about 3 mg from 60 g of transformed wet cells. Recombinant and naturally occurring SsEF-2 showed identical electrophoretic mobility, immunological properties and the N-terminal amino acid sequence; both were lacking the initial methionine. Differently from SsEF-2, SsEF-2rec did not undergo post-translational modification of His603 into diphthamide, as indicated by its inability to be ADP-ribosylated. SsEF-2rec appeared indistinguishable from SsEF-2 in the fulfillment of its biological functions; in fact, it was fully capable to support poly(Phe) synthesis, to bind GDP and to display either the intrinsic or the ribosome-dependent GTPase. Finally, SsEF-2rec was endowed with the same heat stability as SsEF-2. Altogether these findings proved that SsEF-2rec was functionally active as SsEF-2. The used expression system could allow to produce mutated forms of SsEF-2 obtained by mutagenesis of the corresponding gene.

Cloning and sequencing of the gene encoding thermostable elongation factor 2 in Sulfolobus solfataricus.

The gene (aEF-2) coding for the translation elongation factor 2 (aEF-2) in the thermoacidophilic archaebacterium, Sulfolobus solfataricus, has been cloned and sequenced. The deduced primary structure of aEF-2 is composed of 735 amino acids (aa), excluding the Met start residue. There are no Cys residues and the calculated M(r) is 81,699. In the coding region of aEF-2, the high A + T content greatly influences the codon usage. From the alignment of the primary structure of aEF-2 with that of the analogous factors from the three kingdoms, aa identities were derived. The greatest identity (82%) was found with EF-2 from Sulfolobus acidocaldarius; lower values were observed with other archaebacterial EF-2 (45-47%), eukaryotic EF-2 (38-40%) and with the functional eubacterial analogue EF-G (28-31%). aEF-2 possesses the consensus sequences required for a GTP-binding protein and the four regions which are supposed to be involved in the functional regulation of EF-2/EF-G. These data should have phylogenetic implications.