Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay.

The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation. A maltose-binding protein fusion with the 46 residues of TrpRS (TrpRS46mer) exhibited high activation capacity for several amino acids in the presence of ATP and amino acids, but the activity of an alanine substitution mutant of the first histidine in the HIGH motif (TrpRS46merH15A) was largely reduced. In contrast, pyrophosphate release by HisRS46mer in the histidine activation step was lower than that in the case of TrpRS46mer. Both HisRS46mer and the alanine mutant at the 113th arginine (HisRS46merR113A) showed slightly higher levels of pyrophosphate release than the maltose-binding protein alone. These results do not rule out the Rodin-Ohno hypothesis, but may suggest the necessity of establishing unique evolutionary models from different perspectives.

Glycyl-tRNA synthetase from Nanoarchaeum equitans: The first crystal structure of archaeal GlyRS and analysis of its tRNA glycylation.

This study reports the X-ray crystallographic structure of the glycyl-tRNA synthetase (GlyRS) of Nanoarchaeum equitans - a hyperthermophilic archaeal species. This is the first archaeal GlyRS crystal structure elucidated. The GlyRS comprises an N-terminal catalytic domain and a C-terminal anticodon-binding domain with a long ß-sheet inserted between these domains. An unmodified transcript of the wild-type N. equitans tRNAGly was successfully glycylated using GlyRS. Substitution of the discriminator base A73 of tRNAGly with any other nucleotide caused a significant decrease in glycylation activity. Mutational analysis of the second base-pair C2G71 of the acceptor stem of tRNAGly elucidated the importance of the base-pair, especially G71, as an identity element for recognition by GlyRS. Glycylation assays using tRNAGly G71 substitution mutants and a GlyRS mutant where Arg223 is mutated to alanine strengthen the possibility that the carbonyl oxygen at position 6 of G71 would hydrogen-bond with the guanidine nitrogen of Arg223 in N. equitans GlyRS.

Spinopelvic Alignment and Low Back Pain after Total Hip Replacement Arthroplasty in Patients with Severe Hip Osteoarthritis.

STUDY DESIGN: Retrospective observational study. PURPOSE: We examined change in lumbrosacral spine alignment and low back pain (LBP) following total hip arthroplasty (THA) in patients with severe hip osteoarthritis (OA). OVERVIEW OF LITERATURE: Severe hip osteoarthritis has been reported to cause spine alignment abnormalities and low back pain, and it has been reported that low back pain is improved following THA. METHODS: Our target population included 30 patients (29 female, mean age 63.5 years) with hip OA who underwent direct anterior approach THA. There were 12 cases with bilateral hip disease and 18 cases with unilateral osteoarthritis. Visual analogue scale (VAS) scores for LBP and coxalgia, the Roland-Morris Disability Questionnaire (RDQ), and the Japanese Orthopaedic Association Back Pain Evaluation Questionnaire (JOABPEQ) were assessed before and after surgery. Spinal alignment metrics were measured before and after surgery. RESULTS: VAS for LBP change from preoperative to final postoperative observation was significantly improved (p <0.05), as was VAS for hip pain (p<0.001). RDQ improved significantly (p<0.01). All five domains of JOABPEQ were significantly improved (p<0.05). In terms of coronal alignment, lumbar scoliosis change from preoperative to last observation was significantly reduced (p<0.05). There were no significant changes in the sagittal alignment metrics. In addition, there was a correlation between before and after RDQ difference and before and after lumbar scoliosis difference (p<0.05). VAS for LBP (p<0.05) as well as RDQ (p<0.05) were significantly improved only in unilateral OA. Lumbar scoliosis was significantly improved in cases of unilateral OA (p<0.05), but alignment did not improve in cases of bilateral OA (p=0.29). CONCLUSIONS: The present study demonstrates improvements in VAS for LBP, RDQ, and all domains of JOABPEQ. There were also significant reductions in lumbar scoliosis and an observed correlation of RDQ improvement with lumbar scoliosis improvement. We were able to observe improvements in lumbar scoliosis and low back pain only in cases of unilateral OA. It has been suggested that the mechanism of low back pain improvement following THA is related to compensatory lumbar scoliosis improvement.

Iron-chelating agents never suppress Fenton reaction but participate in quenching spin-trapped radicals.

Hydroxyl radical formation by Fenton reaction in the presence of an iron-chelating agent such as EDTA was traced by two different assay methods; an electron spin resonance (ESR) spin-trapping method with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and high Performance liquid chromatography (HPLC)-fluorescence detection with terephthalic acid (TPA), a fluorescent probe for hydroxyl radicals. From the ESR spin-trapping measurement, it was observed that EDTA seemed to suppress hydroxyl radical formation with the increase of its concentration. On the other hand, hydroxyl radical formation by Fenton reaction was not affected by EDTA monitored by HPLC assay. Similar inconsistent effects of other iron-chelating agents such as nitrylotriacetic acid (NTA), diethylenetriamine penta acetic acid (DTPA), oxalate and citrate were also observed. On the addition of EDTA solution to the reaction mixture 10 min after the Fenton reaction started, when hydroxyl radical formation should have almost ceased but the ESR signal of DMPO-OH radicals could be detected, it was observed that the DMPO-OH* signal disappeared rapidly. With the simultaneous addition of Fe(II) solution and EDTA after the Fenton reaction ceased, the DMPO-OH* signal disappeared more rapidly. The results indicated that these chelating agents should enhance the quenching of [DMPO-OH]* radicals by Fe(II), but they did not suppress Fenton reaction by forming chelates with iron ions.

DMPO-OH radical formation from 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in hot water.

When an aqueous solution of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was heated at 70 degrees C for 30 min, formation of DMPO-OH was observed by ESR. This DMPO-OH radical formation was suppressed under an argon atmosphere. When water was replaced with ultra-pure water for ICP-MS experiments, DMPO-OH radical formation was also diminished. Under an argon atmosphere in ultra-pure water, the intensity of the DMPO-OH signal decreased to about 1/20 of that observed under aerobic conditions with regular purified water. The addition of hydroxyl radical scavengers such as mannitol did not affect the formation of DMPO-OH, but the signal turned faint in the presence of EDTA. We suggest that DMPO reacted with dissolved oxygen to form DMPO-OH.