Cleavage of intron from the standard or non-standard position of the precursor tRNA by the splicing endonuclease of Aeropyrum pernix, a hyper-thermophilic Crenarchaeon, involves a novel RNA recognition site in the Crenarchaea specific loop

In Crenarchaea, several tRNA genes are predicted to express precursor-tRNAs (pre-tRNAs) with canonical or non-canonical introns at various positions. We initially focused on the tRNAThr species of hyperthermophilic crenarchaeon, Aeropyrum pernix (APE) and found that in the living APE cells three tRNAThr species were transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them were cleaved from standard and non-standard positions. Biochemical studies revealed that the APE splicing endonuclease (APE-EndA) removed both types of introns, including the non-canonical introns, without any nucleotide modification. To clarify the underlying reasons for broad substrate specificity of APE-EndA, we determined the crystal structure of wild-type APE-EndA and subsequently compared its structure with that of Archaeaoglobus fulgidus (AFU)-EndA, which has narrow substrate specificity. Remarkably, structural comparison revealed that APE-EndA possesses a Crenarchaea specific loop (CSL). Introduction of CSL into AFU-EndA enhanced its intron-cleaving activity irrespective of the position or motif of the intron. Thus, our biochemical and crystallographic analyses of the chimera-EndA demonstrated that the CSL is responsible for the broad substrate specificity of APE-EndA. Furthermore, mutagenesis studies revealed that Lys44 in CSL functions as the RNA recognition site

A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α4, homodimeric: α2, and heterotetrameric: (αβ)2] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε2) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε2 endonuclease cleaves both canonical and non-canonical bulge–helix–bulge motifs, similar to that of (αβ)2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)2 endonuclease. Thus, the discovery of ε2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes

Discovery of permuted and recently split transfer RNAs in Archaea

Background: As in eukaryotes, precursor transfer RNAs in Archaea often contain introns that are removed in tRNA maturation. Two unrelated archaeal species display unique pre-tRNA processing complexity in the form of split tRNA genes, in which two to three segments of tRNAs are transcribed from different loci, then trans-spliced to form a mature tRNA. Another rare type of pre-tRNA, found only in eukaryotic algae, is permuted, where the 3 ’ half is encoded upstream of the 5 ’ half, and must be processed to be functional. Results: Using an improved version of the gene-finding program tRNAscan-SE, comparative analyses and experimental verifications, we have now identified four novel trans-spliced tRNA genes, each in a different species of the Desulfurococcales branch of the Archaea: tRNA Asp(GUC) in Aeropyrum pernix and Thermosphaera aggregans, and tRNA Lys(CUU) in Staphylothermus hellenicus and Staphylothermus marinus. Each of these includes features surprisingly similar to previously studied split tRNAs, yet comparative genomic context analysis and phylogenetic distribution suggest several independent, relatively recent splitting events. Additionally, we identified the first examples of permuted tRNA genes in Archaea: tRNA iMet(CAU) and tRNA Tyr(GUA) in Thermofilum pendens, which appear to be permuted in the same arrangement seen previously in red alga. Conclusions: Our findings illustrate that split tRNAs are sporadically spread across a major branch of the Archaea

Seismic exploration at Fuji volcano with active sources : The outline of the experiment and the arrival time data

Fuji volcano (altitude 3,776m) is the largest basaltic stratovolcano in Japan. In late August and early September 2003, seismic exploration was conducted around Fuji volcano by the detonation of 500 kg charges of dynamite to investigate the seismic structure of that area. Seismographs with an eigenfrequency of 2 Hz were used for observation, positioned along a WSW-ENE line passing through the summit of the mountain. A total of 469 seismic stations were installed at intervals of 250-500 m. The data were stored in memory on-site using data loggers. The sampling interval was 4 ms. Charges were detonated at 5 points, one at each end of the observation line and 3 along its length. The first arrival times and the later-phase arrival times at each station for each detonation were recorded as data. P-wave velocities in the surface layer were estimated from the travel time curves near the explosion points, with results of 2.5 km/s obtained for the vicinity of Fuji volcano and 4.0 km5/s elsewhere

Rapid improvement of massive bloody pleural effusion after splenectomy for splenic marginal zone lymphoma

Splenic marginal zone lymphoma (SMZL) is a rare malignant lymphoma involving marginal zone B cells, accounting for only 1% of non-Hodgkin lymphomas. No previous reports have documented SMZL accompanied by massive bloody pleural effusion. Herein, the case of a patient with SMZL that was only diagnosed after splenectomy and thoracoscopic pleural biopsy is presented. SMZL in this patient was accompanied by massive bloody pleural effusion. The characteristics of the patient’s pleural effusion changed from bloody to transudative after splenectomy, and rapid improvement was observed. It was considered that the splenectomy was valid for treatment of unidentified pleural effusion with enlarged spleen

Prospective Clinical Evaluation of the Serologic Tuberculous Glycolipid Test in Combination with the Nucleic Acid Amplification Test

We have conducted a prospective controlled multicenter study to evaluate differences in the levels of clinical utility of the tuberculous glycolipid (TBGL) serodiagnostic test and the nucleic acid amplification test in patients with smear-negative active pulmonary tuberculosis (TB). The TBGL test and the PCR test were individually not so useful for the rapid diagnosis of smear-negative active pulmonary TB. However, clinical utility was considerably improved by using the TBGL test and the PCR test in combination, especially in patients with smear-negative and culture-negative active pulmonary TB and in patients with minimally advanced lesions

Clinical and Prognostic Importance of Serotyping Mycobacterium avium-Mycobacterium intracellulare Complex Isolates in Human Immunodeficiency Virus-Negative Patients

We studied whether the serotypes of Mycobacterium avium-Mycobacterium intracellulare complex (MAC) isolates determine the prognosis for pulmonary MAC disease. We prospectively monitored a cohort of 68 patients with pulmonary MAC disease for whom the serotype-specific glycopeptidolipids in isolates were identified using thin-layer chromatography and fast atom bombardment mass-spectrometry in 1990 and 1995. Serovar 4 Mycobacterium avium was detected in 40/68 patients (58.8%). Other serotypes were serotypes 1 (five cases), 6 (three cases), 8 (seven cases), 9 (three cases), 14 (four cases), and 16 (six cases). Patients with serovar 4 were significantly (P < 0.01) younger (63.0 ± 9.8 years) than patients with other serotypes (71.8 ± 10.3). Patients who failed treatment had a significantly poorer prognosis than other patients. There were no cases of MAC-related death in the cured group. Chest radiographic findings progressively worsened in 36 (90%) of patients with serotype 4, and 14/36 died from respiratory failure caused by pulmonary Mycobacterium avium disease. The patients with serotype 4 had a significantly poorer prognosis than patients with other serotypes. These results show that both the outcome of chemotherapy and the serotypes of MAC isolates are important for assessing the prognosis of pulmonary MAC disease