Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein-tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.     

Structure of genomic DNA for rat platelet phospholipase A2

A genomic DNA for rat platelet phospholipase A2 was isolated by screening a rat genomic library with oligonucleotide probes based on its published amino acid sequence. The rat platelet phospholipase A2 gene had a total length of about 2.5 kb and contained five exons and four introns. The intron-exon structure of the rate gene was similar to that of human non-pancreatic phospholipase A2.     

Structural analysis and biosynthesis gene cluster of an antigenic glycopeptidolipid from Mycobacterium intracellulare

Mycobacterium avium-Mycobacterium intracellulare complex (MAC) is the most common isolate of nontuberculous mycobacteria and causes pulmonary and extrapulmonary diseases. MAC species can be grouped into 31 serotypes by the epitopic oligosaccharide structure of the species-specific glycopeptidolipid (GPL) antigen. The GPL consists of a serotype-common fatty acyl peptide core with 3,4-di-O-methyl-rhamnose at the terminal alaninol and a 6-deoxy-talose at the allo-threonine and serotype-specific oligosaccharides extending from the 6-deoxy-talose. Although the complete structures of 15 serotype-specific GPLs have been defined, the serotype 16-specific GPL structure has not yet been elucidated. In this study, the chemical structure of the serotype 16 GPL derived from M. intracellulare was determined by using chromatography, mass spectrometry, and nuclear magnetic resonance analyses. The result indicates that the terminal carbohydrate epitope of the oligosaccharide is a novel N-acyl-dideoxy-hexose. By the combined linkage analysis, the oligosaccharide structure of serotype 16 GPL was determined to be 3-2'-methyl-3'-hydroxy-4'-methoxy-pentanoyl-amido-3,6-dideoxy-beta-hexose-(1-->3)-4-O-methyl-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->3)-alpha-L-rhamnose-(1-->2)-6-deoxy-alpha-L-talose. Next, the 22.9-kb serotype 16-specific gene cluster involved in the glycosylation of oligosaccharide was isolated and sequenced. The cluster contained 17 open reading frames (ORFs). Based on the similarity of the deduced amino acid sequences, it was assumed that the ORF functions include encoding three glycosyltransferases, an acyltransferase, an aminotransferase, and a methyltransferase. An M. avium serotype 1 strain was transformed with cosmid clone no. 253 containing gtfB-drrC of M. intracellulare serotype 16, and the transformant produced serotype 16 GPL. Together, the ORFs of this serotype 16-specific gene cluster are responsible for the biosynthesis of serotype 16 GPL.     

Isolation and characterization of a novel thermostable neopullulanase-like enzyme from a hot spring in Thailand

A gene encoding a thermostable pullulan-hydrolyzing enzyme was isolated from environmental genomic DNA extracted from soil sediments of Bor Khleung hot spring in Thailand. Sequence comparison with related enzymes suggested that the isolated enzyme, designated Env Npu193A, was most likely a neopullulanase-like enzyme. Env Npu193A was expressed in Pichia pastoris as a monomeric recombinant protein. The purified Env Npu193A exhibited pH stability ranging from 3 to 9. More than 60% of enzyme activity was retained after incubation at 60 degrees C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and gamma-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75 degrees C with K(m) and V(max) toward pullulan of 1.22+/-0.3% and 23.24+/-1.7 U/mg respectively. Env Npu193A exhibited distinct biochemical characteristics as compared with the previously isolated enzyme from the same source. Thus, a culture-independent approach with sequence-basing was found to be an effective way to discover novel enzymes displaying unique substrate specificity and high thermostability from natural bioresources.     

Important roles of Tyr43 at the putative heme distal side in the oxygen recognition and stability of the Fe(II)-O2 complex of YddV, a globin-coupled heme-based oxygen sensor diguanylate cyclase

YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O(2) and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was >150 s(-1), which is significantly larger than that of the wild-type protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O(2) and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O(2) recognition and stability of the Fe(II)-O(2) complex, because the hydroxyl group of the residue appears to interact electrostatically with the O(2) molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.     

Cytological study of early cervical adenocarcinoma: special reference to the depth of invasion

OBJECTIVE: Early cervical adenocarcinoma (ECA) with a tumour depth of <3 mm has a good prognosis. To clarify the cytological features of ECAs with depth <3 mm, these were compared with those of ECA with 3-5 mm and invasive adenocarcinoma (IA) invading the cervical wall with more than 5 mm in depth. METHODS: The cervical cytological features of ECAs with depth <3 mm (14 cases) were compared with those of ECA with 3-5 mm (four cases) and IA (13 cases). Cytologically, the presence or absence of tumour diathesis, number of atypical cells, crowded cell groups, groups with glandular structures, feathering, groups with palisading borders, rosettes, clusters, cell shape and size, nuclear shape and size, nucleolar shape and size, chromatin distribution, border and character of cytoplasm, and single cell pattern were evaluated. RESULTS: A tumour diathesis was seen in five of 14 ECA <3 mm in depth (36%), all four ECA with 3-5 mm (100%) and 11 of 13 IA with more than 5 mm (85%). Single cells, macronucleoli and coarsely granular chromatin pattern were less frequent in ECA of <3 mm than that in ECA with 3-5 mm and IA. The number of atypical cells and glandular structures in ECA was significantly less than that in IA. Cell crowding, feathering, palisading and rosettes were common in both ECA and IA. CONCLUSION: The characteristic cytological features of ECA with depth <3 mm, having a good prognosis, were clean background, fewer single cells and macronucleoli, and less frequent coarsely granular chromatin pattern compared with those in ECA with 3-5 mm and IA. The number of atypical cells and glandular structures in ECA was significantly less than that in IA. Familiarity with the cytological features of ECA and its mimics is essential.     

Formation and stability of complex organic compounds in space environments.

Complex organic compounds have been found in extraterrestrial bodies such as meteorites and comets. We confirmed the formation of complex organic compounds that contained amino acid precursors from a mixture of carbon monoxide (or methanol), ammonia and water by radiation or UV. Molecular weights of the complex organics were several thousands. Stability of the complex precursors was studied. When free amino acids were irradiated with gamma rays or synchrotron radiation, they easily decomposed. The complex precursors were, however, much more stable than free amino acid against irradiation. We propose to examine the formation and alteration of amino acid precursors in space by using exposed facility of ISS.