A Polymorphism Cluster at the 2q12 locus May Predict Response to Piromelatine in Patients with Mild Alzheimer's Disease.

BACKGROUND: Piromelatine is a novel melatonin MT1/2/3 and serotonin 5-HT-1A/1D receptors agonist developed for mild Alzheimer's disease (AD). In a randomized, placebo-controlled, dose-ranging study (ReCognition) of piromelatine (5, 20, and 50 mg daily for 6 months) in participants with mild dementia due to AD (n=371, age 60-85 years), no statistically significant differences were found between the piromelatine and placebo-treated groups on the primary (i.e., computerized neuropsychological test battery (cNTB)) and secondary outcomes (ADCS-CGIC, ADCS-MCI-ADL, ADAS-cog14, NPI, and Pittsburgh Sleep Quality Index (PSQI)) nor were there safety concerns (https://clinicaltrials.gov/ct2/show/NCT02615002). OBJECTIVES: This study was aimed at identifying genetic markers predicting piromelatine treatment response using a genome-wide association approach (GWAS). DESIGN: Variant genotyping of a combined whole genome and whole exome sequencing was performed using DNA extracted from lymphocytes from consenting participants. The general case-control allelic test was performed on piromelatine-treated participants, taking "responders" (i.e., >0.125 change from baseline in the cNTB) as cases and "non responders" as controls, using a Cochran-Armitage trend test. SETTING: 58 outpatient clinics in the US. PARTICIPANTS: 371 participants were randomized in the trial; 107 provided informed consent for genotyping. RESULTS: The GWAS sample did not differ from the full study cohort in demographics, baseline characteristics, or response to piromelatine. Six single-nucleotide polymorphisms (SNPs) in chromosome 2q12 (2:107,510,000-107,540,000) were associated with response (p-value < 1x10 -4 each). Post hoc analyses suggested that the carriers of the 2q12 polymorphism cluster (27% of the GWAS sample) improved significantly on the cNTB on piromelatine as compared to placebo but significantly worsened on the ADAS-Cog14 and PSQI. By contrast, "non-carriers" improved significantly with piromelatine compared to placebo on the ADAS-Cog14 ( 2.91 (N=23) with piromelatine 20 mg vs 1.65 (N=19) with placebo (p=0.03)) and PSQI. CONCLUSIONS: The 2q12 (2:107,510,000-107,540,000) 5-6 SNPs cluster may predict efficacy of piromelatine for mild AD. These findings warrant further investigation in a larger, prospective early-stage AD clinical trial for patients who are non-carriers of the 2q12 polymorphism cluster.

Preliminary experience with anterior cervical microdiscectomy and interbody titanium cage fusion (Novus CT-Ti) in patients with cervical disc disease.

BACKGROUND: Although the use of intervertebral fusion after anterior microdiscectomy in cervical disc disease remains controversial, a new surgical device is proposed for use in intervertebral fusion instead of bone graft. METHODS: This retrospective study at the Department of Neurosurgery, Cardarelli Hospital, Naples, from January 1993 to December 1998, compares the results of surgery on 58 patients with anterior microdiscectomy and intervertebral bone graft fusion (Group A) (ADIBG) with a group of 52 patients who underwent anterior microdiscectomy and intervertebral titanium cage fusion (Group B) (ADITC) in cervical radiculopathy and spondylotic myelopathy. In both groups a "radical discectomy" was performed under the operating microscope. In group A, interbody fusion was performed with autologous tricortical bone graft. In group B, a new type of titanium device (Novus CT-Ti) was used (Sofamor Danek Group). RESULTS: There was no collapse or extrusion of the device and no complications at the donor site (the bone fragments used to fill the cage were taken from osteophytes or vertebral body fragments). The use of this device provides immediate stabilization, reduces or eliminates pain, promotes bone fusion between the vertebrae adjacent to the cage by allowing bone growth through the cage, reestablishes and maintains the intervertebral space, reduces the average hospitalization time, and allows a quicker return to work. CONCLUSIONS: Patients who underwent ADITC did well and benefited from the surgery. Those who underwent ADITC did better than those who underwent ADIBG in regard to function, relief from pain, and complications. Early and good stability of the cervical spine seems to be the main advantage of using titanium cages.

A chimeric elongation factor containing the putative guanine nucleotide binding domain of archaeal EF-1 alpha and the M and C domains of eubacterial EF-Tu.

A recombinant chimeric elongation factor containing the region of EF-1 alpha from Sulfolobus solfataricus harboring the site for GDP and GTP binding and GTP hydrolysis (SsG) and domains M and C of Escherichia coli EF-Tu (EcMC) was studied. SsG-EcMC did not sustain poly(Phe) synthesis in either S. solfataricus or E. coli assay system. This was probably due to the inability of the chimera to interact with aa-tRNA. The three-dimensional modeling of SsG-EcMC indicated only small structural differences compared to the Thermus aquaticus EF-Tu in the ternary complex with aa-tRNA and GppNHp, which did not account for the observed inability to interact with aa-tRNA. The addition of the nucleotide exchange factor SsEF-1 beta was not required for poly(Phe) synthesis since the chimera was already able to exchange [(3)H]GDP for GTP at very high rate even at 0 degrees C. Compared to that of SsEF-1 alpha, the affinity of the chimera for guanine nucleotides was increased and the k(cat) of the intrinsic GTPase was 2-fold higher. The heat stability of SsG-EcMC was 3 and 13 degrees C lower than that displayed by SsG and SsEF-1alpha, respectively, but 30 degrees C higher than that of EcEF-Tu. This pattern remained almost the same if the melting curves of the proteins being investigated were considered instead. The chimeric elongation factor was more thermophilic than SsG and SsEF-1 alpha up to 70 degrees C; at higher temperatures, inactivation occurred.

Expression in Escherichia coli of the elongation factor 1beta gene and its nucleotide T160C mutant from the archaeon Sulfolobus solfataricus.

The guanine nucleotide exchange factor EF-1beta gene from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1beta) was amplified by PCR and cloned into the pT7-7 expression vector. One of four selected clones harbored the T160C nucleotide substitution leading to the Y54H amino acid change in a hydrophobic region of SsEF-1beta, caused by a nucleotide misincorporation of the Taq DNA polymerase during PCR. The resulting plasmids were used to transform the Escherichia coli BL21(DE3)pLysE strain. Upon induction with isopropyl beta-d-thiogalactopyranoside about 1.4 mg of the recombinant SsEF-1beta (recSsEF-1beta) and Y54HSsEF-1beta were obtained from 1 liter of cell culture. recSsEF-1beta and Y54HSsEF-1beta were both able to catalyze the GDP/GTP exchange on SsEF-1alpha as observed with the wild-type SsEF-1beta. In addition, the heat inactivation profiles of recSsEF-1beta and Y54HSsEF-1beta were identical, being both half inactivated after 30 min treatment at 105 degrees C. These results suggest that Tyr 54 is not essential for the nucleotide exchange activity and is not involved in the thermostability of SsEF-1beta.

Protein engineering on enzymes of the peptide elongation cycle in Sulfolobus solfataricus.

The present article is a review of the work done on the elongation factors EF-1 alpha, EF-2 and EF-1 beta isolated from the hyperthermophilic archaeon Sulfolobus solfataricus. The molecular, physical and biochemical properties of the intact, truncated, mutant or chimeric forms are described and compared.

Properties of truncated forms of the elongation factor 1alpha from the archaeon Sulfolobus solfataricus.

Two truncated forms of the Sulfolobus solfataricus elongation factor 1alpha (SsEF-1alpha), corresponding to the putative domains G+M, Ss(GM)EF-1alpha, and G, Ss(G)EF-1alpha, have been constructed by gene engineering, produced in Escherichia coli and purified. Neither truncated form was able to sustain poly(Phe) synthesis but they were able to bind guanine nucleotides with an affinity much higher with respect to that of the intact factor. However, the difference in the affinity for GDP and GTP became progressively reduced with the extent of the truncation. The values of kcat and Km for GTP of the intrinsic GTPase of SsEF-1alpha triggered by 3.6 M NaCl were not affected by the deletions. In contrast, both Ss(GM)EF-1alpha and Ss(G)EF-1alpha were less thermostable than the intact factor; the region of the factor most responsible for the loss of resistance against heat inactivation was the C-terminal domain. On the other hand the domain M was the regulator of the thermophilicity of SsEF-1alpha since only Ss(G)EF-1alpha showed a reduced thermophilicity. Remarkably, both Ss(GM)EF-1alpha and Ss(G)EF-1alpha were able to exchange [3H]GDP for GTP at a very high rate so that they were no more sensitive to the stimulatory effect of SsEF-1beta, which is the nucleotide exchange factor of SsEF-1alpha.

Archaeal elongation factor 1 beta is a dimer. Primary structure, molecular and biochemical properties.

The elongation factor 1 beta (EF-1 beta), that in eukarya and archaea promotes the replacement of GDP by GTP on the elongation factor 1 alpha x GDP complex, was purified to homogeneity from the thermoacidophilic archaeon Sulfolobus solfataricus (SsEF-1 beta). Its primary structure was established by sequenced Edman degradation of the entire protein or its proteolytic peptides. The molecular weight of SsEF-1 beta was estimated as about 10000 or 20000 under denaturing or native conditions respectively; this finding suggests that the native protein exists as a dimer. The peptide chain of SsEF-1 beta is much shorter than that of its eukaryotic analogues and homology is found only at their C-terminal region; no homology exists between SsEF-1 beta and eubacterial EF-Ts. At 50 degrees C, at a concentration of SsEF-1 beta 5-fold higher than that of SsEF-1 alpha x [3H]GDP the rate of the exchange of [3H]GDP for GTP becomes about 160-fold faster. An analysis of the values of the energetic parameters indicates that in the presence of SsEF-1 beta the GDP/GTP exchange is entropically favoured. At 100 degrees C the half-life of SsEF-1 beta is about 4 h.

Expression in Escherichia coli of thermostable elongation factor 1 alpha from the archaeon Sulfolobus solfataricus.

The elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was expressed in Escherichia coli and purified. The SsEF-1 alpha gene was amplified by PCR and cloned in the Ndel site of the pT7-7 expression vector, under the control of the promoter of T7 RNA polymerase. Upon induction with isopropyl beta-D-thiogalactopyranoside, the recombinant SsEF-1 alpha (recSsEF-1 alpha) was purified from the E. coli S-100 extract by a two-step procedure. From 1 litre of cell culture, about 2 mg of purified recSsEF-1 alpha was obtained. The N-terminal sequence of the first 30 amino acid residues of recSsEF-1 alpha was identical with that translated from the nucleotide sequence of the corresponding gene, except for the initial residue, which in recSsEF-1 alpha was Ser instead of Met. The M(r) of recSsEF-1 alpha (determined by electrospray MS) was almost coincident with that of the naturally occurring SsEF-1 alpha (SsEF-1 alpha). The thermal-inactivation and thermophilicity profiles of SsEF-1 alpha and recSsEF-1 alpha were identical. Concerning the functional properties, recSsEF-1 alpha was able to support poly(Phe) synthesis in vitro, to bind GDP and GTP and to elicit an NaCl-dependent GTPase activity [Masullo, De Vendittis and Bocchini (1994) J. Biol. Chem. 269, 20376-20379] with the same efficiency as that displayed by SsEF-1 alpha.

The first nucleotide sequence of an archaeal elongation factor 1 beta gene.

An archaeal elongation factor 1 beta gene has been isolated for the first time from a Sulfolobus solfataricus genomic library. The sequenced clone (869 bp) contained two open reading frames, one coding for a protein made of 91 amino acid residues (SsEF-1 beta), the other one encoding a nonidentified product (ORF 115). The amino acid sequences of segments at the N- and C-terminal of the translated SsEF-1 beta were identical to those determined for the native protein. Northern and Southern analyses showed that the SsEF-1 beta gene is represented in S. solfataricus by a unique sequence. Compared to eubacterial or eukaryal corresponding genes the SsEF-1 beta is much shorter.

Glyceraldehyde-3-phosphate dehydrogenase in the hyperthermophilic archaeon Sulfolobus solfataricus: characterization and significance in glucose metabolism.

Glyceraldehyde-3-phosphate dehydrogenase in the archaeon Sulfolobus solfataricus (SsGAPD) has been purified 232 fold with an overall recovery of about 25%. The enzyme is a homomeric tetramer with an M(r) of 41 kDa/subunit. It utilizes either NAD+ or NADP+ as coenzyme but its affinity for the latter is about 50 fold higher. SsGAPD activity is maximum at 87 degrees C. In the range 45-87 degrees C the Arrhenius plot is linear and the activation energy is 55 kJ/mol. The enzyme is thermostable, with a half-life of 45 min at 87 degrees C. The primary structure of SsGAPD shows 35% identity with that of other archaeal GAPDs. Its N-domain shows sequence motifs typical of the dinucleotide binding proteins while the catalytic C-terminal region contains a cysteine residue (C140), required for catalysis, that is conserved in all the archaeal, eukaryal and bacterial GAPDs. These remarks suggest that archaeal GAPDs show a convergent molecular evolution to the eukaryal and eubacterial enzymes in the catalytic region.

Organization of a Sulfolobus solfataricus gene cluster homologous to the Escherichia coli str operon.

The Sulfolobus solfataricus S12, S7 and S10 ribosomal proteins and the elongation factor 1 alpha genes are organized in a sequence analogous to that in the Escherichia coli str operon. Northern analysis showed that the S12 gene belongs to a transcript different from that corresponding to the other three genes. Compared to the Sulfolobus acidocaldarius S12 and to the Methanococcus vannielii S7 proteins, the S. solfataricus S12 and S7 proteins were 33 and 47 amino acids longer respectively. These differences were eliminated if the 5' flanking regions of the S. acidocaldarius S12 and the M. vannielii S7 genes were translated from a different start codon. Despite the structural similarities between the archaeal and the bacterial str operons the S. solfataricus ribosomal proteins S12, S7 and S10 are more similar to the eukaryotic counterparts.

The nucleotide sequence of the gene coding for the elongation factor 1 alpha in Sulfolobus solfataricus. Homology of the product with related proteins.

The cloning and sequencing of the gene coding for the archaebacterial elongation factor 1 alpha (aEF-1 alpha) was performed by screening a Sulfolobus solfataricus genomic library using a probe constructed from the eptapeptide KNMITGA that is conserved in all the EF-1 alpha/EF-Tu known so far. The isolated recombinant phage contained the part of the aEF-1 alpha gene from amino acids 1 to 171. The other part (amino acids 162-435) was obtained through the amplification of the S. solfataricus DNA by PCR. The codon usage by the aEF-1 alpha gene showed a preference for triplets ending in A and/or T. This behavior was almost identical to that of the S. acidocaldarius EF-1 alpha gene but differed greatly from that of EF-1 alpha/EF-Tu genes in other archaebacteria eukaryotes and eubacteria. The translated protein is made of 435 amino acid residues and contains sequence motifs for the binding of GTP, tRNA and ribosome. Alignments of aEF-1 alpha with several EF-1 alpha/EF-Tu revealed that aEF-1 alpha is more similar to its eukaryotic than to its eubacterial counterparts.

Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacterium Sulfolobus solfataricus.

A Sulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (lambda G1-lambda G6) and one of them (lambda G4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of the S. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.