Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

PubMed Central

Boonyawat, Boonchai; Monsereenusorn, Chalinee; Traivaree, Chanchai

2014-01-01

Background Beta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia. Objective This study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital. Patients and methods Eighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS), direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively. Results A total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT) (37.5%), followed by codon 17 (A>T) (26.1%), IVS-I-5 (G>C) (8%), IVS-II-654 (C>T) (6.8%), IVS-I-1 (G>T) (4.5%), and codon 71/72 (+A) (2.3%), and all these six common mutations (85.2%) were detected by M-ARMS. Six uncommon mutations (10.2%) were identified by DNA sequencing including 4.5% for codon 35 (C>A) and 1.1% initiation codon mutation (ATG>AGG), codon 15 (G>A), codon 19 (A>G), codon 27/28 (+C), and codon 123/124/125 (-ACCCCACC), respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT)/codon 26 (G>A) or betaE accounting for 40%. Conclusion All of the beta-thalassemia alleles have been characterized by a combination of techniques including M-ARMS, DNA sequencing, and gap polymerase chain reaction for 3.4 kb deletion detection. Thirteen mutations account for 100% of the beta-thalassemia genes among the pediatric patients in our study. PMID:25525381

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice

PubMed Central

Kamal, Ram P.; Kumar, Amrita; Davis, Charles T.; Tzeng, Wen-Pin; Nguyen, Tung; Donis, Ruben O.; Katz, Jacqueline M.

2015-01-01

ABSTRACT Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. IMPORTANCE Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses. PMID:25787281

Mitochondrial genome of Pteronotus personatus (Chiroptera: Mormoopidae): comparison with selected bats and phylogenetic considerations.

PubMed

López-Wilchis, Ricardo; Del Río-Portilla, Miguel Ángel; Guevara-Chumacero, Luis Manuel

2017-02-01

We described the complete mitochondrial genome (mitogenome) of the Wagner's mustached bat, Pteronotus personatus, a species belonging to the family Mormoopidae, and compared it with other published mitogenomes of bats (Chiroptera). The mitogenome of P. personatus was 16,570 bp long and contained a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region (D-loop). Most of the genes were encoded on the H-strand, except for eight tRNA and the ND6 genes. The order of protein-coding and rRNA genes was highly conserved in all mitogenomes. All protein-coding genes started with an ATG codon, except for ND2, ND3, and ND5, which initiated with ATA, and terminated with the typical stop codon TAA/TAG or the codon AGA. Phylogenetic trees constructed using Maximum Parsimony, Maximum Likelihood, and Bayesian inference methods showed an identical topology and indicated the monophyly of different families of bats (Mormoopidae, Phyllostomidae, Vespertilionidae, Rhinolophidae, and Pteropopidae) and the existence of two major clades corresponding to the suborders Yangochiroptera and Yinpterochiroptera. The mitogenome sequence provided here will be useful for further phylogenetic analyses and population genetic studies in mormoopid bats.

Novel mutations of the AGXT gene causing primary hyperoxaluria type 1.

PubMed

Yuen, Yuet-Ping; Lai, Chi-Kong; Tong, Gensy Mei-Wah; Wong, Ping-Nam; Wong, Francis Kim-Ming; Mak, Siu-Ka; Lo, Kin-Yee; Wong, Andrew Kui-Man; Tong, Sui-Fan; Chan, Yan-Wo; Lam, Ching-Wan

2004-01-01

Primary hyperoxaluria type 1 (PH1), an inherited cause of nephrolithiasis, is due to a functional defect of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). A definitive PH1 diagnosis can be established by analyzing AGT activity in liver tissue or mutation analysis of the AGXT gene. The molecular basis of PH1 in three Chinese patients, two with adult-onset and one with childhood-onset recurrent nephrolithiasis, was established by analyzing the entire AGXT gene. Three novel mutations (c2T>C, c817insAG and c844C>T) and two previously reported mutations (c33insC and 679-IVS6+2delAAgt) were identified. c2T>C converts the initiation codon from ATG to ACG, which predicts significant reduction, if not complete abolition, of protein translation. c817insAG leads to a frameshift and changes the amino acid sequence after codon 274. c844C>T changes glutamine at codon 282 to a termination codon, resulting in protein truncation. This is the first report describing AGXT gene mutations in Chinese patients with PH1. AGXT genotypes cannot fully explain the clinical heterogeneity of PH1, and other factors involved in disease pathogenesis remain to be identified. Our experience emphasizes the importance of excluding PH1 in patients with recurrent nephrolithiasis to avoid delay or inappropriate management.

The complete mitochondrial genome of the Aluterus monoceros.

PubMed

Li, Wenshen; Zhang, Guoqing; Wen, Xin; Wang, Qian; Chen, Guohua

2016-07-01

The complete mitochondrial genome of Aluterus monoceros (A. monoceros) has been sequenced. The mitochondrial genome of A. monoceros is 16,429 bp in length, consisting of 22 tRNA genes, 2 rRNA genes, 13 protein-coding genes and a D-loop region (Gen Bank accession number KP637022). The base A + T of the mitochondrial genome is 63.25%, including 33.16% of A, 30.09% of T and 20.74% of C. Twelve protein-coding genes start with a standard ATG as the initiation codon, expect for the COXI, which begins with GTG. Some of the termination codons are incomplete T or TA, except for the ND1, COXI, ATP8, ND4L1, ND5 and ND6, which stop with TAA. Construction of phylogenetic trees based on the entire mitochondrial genome sequence of 14 Tetrodontiformes species constructed has suggested that A. monoceros has closer relationship with Acreichthys tomentosus and Monacanthus chinensis, and they constitute a sister group.

Complete mitochondrial genome of the mottled skate: Raja pulchra (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Kim, Sung; Kim, Choong-Gon; Myoung, Jung-Goo; Lee, Youn-Ho

2016-05-01

The complete sequence of mitochondrial DNA of a mottled skate, Raja pulchra was sequenced as being circular molecules of 16,907 bp including 2 rRNA, 22 tRNA, 13 protein-coding genes (PCGs), and an AT-rich control region. The organization of the PCGs is the same as those found in other Rajidae species. The nucleotide of L-strand is composed of 29.8% A, 28.0% C, 27.9% T, and 14.3% G with a bias toward A + T slightly. Twelve of 13 PCGs are initiated by the ATG codon while COX1 starts with GTG. Only ND4 harbors the incomplete termination codon, TA. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA with the exception of [Formula: see text] which has a reduced DHU arm. This mitogenome will provide essential information for better phylogenetic resolution and precision of the family Rajidae and the genus Raja as well as for establishment of a fish stock recovery plan of the species.

The Atg1-kinase complex tethers Atg9-vesicles to initiate autophagy

NASA Astrophysics Data System (ADS)

Rao, Yijian; Perna, Marco G.; Hofmann, Benjamin; Beier, Viola; Wollert, Thomas

2016-01-01

Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1-kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1-kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1-kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1-Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.

Complete mitochondrial genome of the Yellownose skate: Zearaja chilensis (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2016-01-01

The complete sequence of mitochondrial DNA of a Yellownose skate, Zearaja chilensis was determined for the first time. It is 16,909 bp in length covering 2 rRNA, 22 tRNA and 13 protein coding genes with the identical gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of low G (14.3%), and slightly high A + T (58.9%) nucleotides. The strong codon usage bias against the use of G (6.0%) is found at the third codon positions. Twelve of the 13 protein coding genes use ATG as the start codon while COX1 starts with GTG. As for the stop codon, only ND4 shows an incomplete stop codon TA. This is the first report of the mitogenome for a species in the genus Zearaja, providing a valuable source of genetic information on the evolution of the family Rajidae and the genus Zearaja as well as for establishment of a sustainble fishery management plan of the species.

An initiator codon mutation in SDE2 causes recessive embryonic lethality in Holstein cattle.

PubMed

Fritz, Sébastien; Hoze, Chris; Rebours, Emmanuelle; Barbat, Anne; Bizard, Méline; Chamberlain, Amanda; Escouflaire, Clémentine; Vander Jagt, Christy; Boussaha, Mekki; Grohs, Cécile; Allais-Bonnet, Aurélie; Philippe, Maëlle; Vallée, Amélie; Amigues, Yves; Hayes, Benjamin J; Boichard, Didier; Capitan, Aurélien

2018-04-18

Researching depletions in homozygous genotypes for specific haplotypes among the large cohorts of animals genotyped for genomic selection is a very efficient strategy to map recessive lethal mutations. In this study, by analyzing real or imputed Illumina BovineSNP50 (Illumina Inc., San Diego, CA) genotypes from more than 250,000 Holstein animals, we identified a new locus called HH6 showing significant negative effects on conception rate and nonreturn rate at 56 d in at-risk versus control mating. We fine-mapped this locus in a 1.1-Mb interval and analyzed genome sequence data from 12 carrier and 284 noncarrier Holstein bulls. We report the identification of a strong candidate mutation in the gene encoding SDE2 telomere maintenance homolog (SDE2), a protein essential for genomic stability in eukaryotes. This A-to-G transition changes the initiator ATG (methionine) codon to ACG because the gene is transcribed on the reverse strand. Using RNA sequencing and quantitative reverse-transcription PCR, we demonstrated that this mutation does not significantly affect SDE2 splicing and expression level in heterozygous carriers compared with control animals. Initiation of translation at the closest in-frame methionine codon would truncate the SDE2 precursor by 83 amino acids, including the cleavage site necessary for its activation. Finally, no homozygote for the G allele was observed in a large population of nearly 29,000 individuals genotyped for the mutation. The low frequency (1.3%) of the derived allele in the French population and the availability of a diagnostic test on the Illumina EuroG10K SNP chip routinely used for genomic evaluation will enable rapid and efficient selection against this deleterious mutation. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Complete mitochondrial genome of the Kwangtung skate: Dipturus kwangtungensis (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Kim, Sung; Kim, Choong-Gon; Lee, Youn-Ho

2015-01-01

The complete sequence of mitochondrial DNA of a Kwangtung skate, Dipturus kwangtungensis, was determined as being circular molecules of 16,912 bp including 2 rRNA, 22 tRNA, 13 protein coding genes (PCGs) and a control region. The arrangement of the PCGs is the same as that found in other Rajidae species. The nucleotide of L-strand which encodes most of the proteins is composed of 30.2% A, 27.4% C, 28.2% T and 14.2% G with a bias toward A+T slightly. Twelve of 13 PCGs are initiated by the ATG codon while COX1 starts with GTG. Only ND4 harbors the incomplete termination codon, TA. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA with the exception of tRNA(Ser)AGY, which has a reduced DHU arm. This mitogenome is the first report for a species of the genus Dipturus, which will become an important source of information on the phylogenetic relationship and the evolution of the genus Dipturus within the family Rajidae.

Hb Alesha [beta67(E11)Val-->Met, GTG-->ATG] in an Argentinean girl.

PubMed

Eberle, Silvia Eandi; Noguera, Nélida I; Sciuccati, Gabriela; Bonduel, Mariana; Díaz, Lilian; Staciuk, Raquel; Targovnik, Héctor M; Feliu-Torres, Aurora

2007-01-01

Hb Alesha is caused by a GTG-->ATG mutation at codon 67 of the beta-globin gene, resulting in abnormal beta-globin chains in which the normal beta67(E11) valine is changed to methionine. This hemoglobin (Hb) is also known as Hb Bristol, the first unstable Hb described, since in a fraction of the variant the methionine is modified into an aspartic acid by a posttranslational modification. This replacement disrupts the apolar bonds between the valine and the heme group, producing an unstable Hb and severe hemolysis. We have identified this rare hemoglobinopathy in an Argentinean girl with severe hemolytic anemia, splenomegaly and frequent requirement for red blood cell transfusions.

The complete mitochondrial genome of the mudsnail Cipangopaludina cathayensis (Gastropoda: Viviparidae).

PubMed

Yang, Huirong; Zhang, Jia-En; Luo, Hao; Luo, Mingzhu; Guo, Jing; Deng, Zhixin; Zhao, Benliang

2016-05-01

We present the complete mitochondrial genome of Cipangopaludina cathayensis in this study. The mitochondrial genome is 17,157 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes. All of them are encoded on the heavy strand except 7 tRNA genes on the light strand. Overall nucleotide compositions of the light strand are 44.51% of A, 26.74% of T, 20.48% of C and 8.28% of G. All the protein-coding genes start with ATG initiation codon except ATP6 with ATA and ND4 with TTG, and 2 types of termination codons are TAA (ATP6, ND2, COX1, COX2, ATP8, ND1, ND6, Cytb, COX3, ND4) and TAG (ND4L, ND5, ND3). There are 29 intergenic spacers and 5 gene overlaps. The tandem repeat sequences are observed in COX2, tRNA(Asp), ATP6, tRNA(Cys), S-rRNA, ND1, Cytb, ND4 and COX3 genes. Gene arrangement and distribution are different from the typical vertebrates. The absence of D-loop is consistent with the Gastropoda, but at least one lengthy non-coding region is essential regulatory element for the initiation of transcription and replication.

The complete mitochondrial genome of the Longnose skate: Raja rhina (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Lee, Youn-Ho

2015-02-01

The complete sequence of mitochondrial DNA of a longnose skate, Raja rhina was determined for the first time. It is 16,910 bp in length containing 2 rRNA, 22 tRNA and 13 protein coding genes with the same gene order and structure as those of other Rajidae species. The nucleotide of L-strand is composed of 30.1% A, 27.2% C, 28.5% T and 14.2% G, showing a slight A + T bias. The G is the least used base and markedly lower at the third codon position (5.4%). Twelve of the 13 protein coding genes use ATG as their start codon while the COX1 starts with GTG. As for stop codon, only ND4 shows incomplete stop codon TA. This mitogenome is the first report for a species of the genus Raja, and providing a valuable resource of genetic information for understanding the phylogenetic relationship and the evolution of the genus Raja as well as the family, Rajidae.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape

PubMed Central

Lapierre, Pascal; Mir, Mushtaq; Chase, Michael R.; Pyle, Margaret M.; Gawande, Richa; Ahmad, Rushdy; Sarracino, David A.; Ioerger, Thomas R.; Fortune, Sarah M.; Derbyshire, Keith M.; Wade, Joseph T.; Gray, Todd A.

2015-01-01

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5’ untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5’ end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5’ ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5’ UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression. PMID:26536359

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

PubMed

Shell, Scarlet S; Wang, Jing; Lapierre, Pascal; Mir, Mushtaq; Chase, Michael R; Pyle, Margaret M; Gawande, Richa; Ahmad, Rushdy; Sarracino, David A; Ioerger, Thomas R; Fortune, Sarah M; Derbyshire, Keith M; Wade, Joseph T; Gray, Todd A

2015-11-01

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5' untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5' end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5' ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5' UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

PubMed

Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

2015-10-01

Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

The complete mitochondrial genome of the Korean skate: Hongeo koreana (Rajiformes, Rajidae).

PubMed

Jeong, Dageum; Kim, Sung; Kim, Choong-Gon; Lee, Youn-Ho

2014-12-01

The complete mitochondrial genome of the Korean skate, Hongeo koreana, the sole member of its genus, is investigated for the first time. The genome consists of 16,906 bp in length including 2 rRNA, 22 tRNA and 13 protein coding genes with the same gene order and structure of the genome as those of other Rajidae species. The overall nucleotide composition of the L-strand is A = 29.8%, C = 27.9%, T = 27.9% and G = 14.3%, showing a high A + T bias. The anti-G bias (6.0%) is more significant in the third codon position. Twelve of the 13 protein-coding genes use ATG as their start codon while the COX1 gene starts with GTG. For stop codon, ND3 and ND4 genes show incomplete stop codon T. The mitogenome sequence of H. koreana will provide important information on the evolution and the phylogenetic relation of the genus Hongeo in relation to the other genera of the family Rajidae.

Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy.

PubMed

Zhou, Changqian; Ma, Kaili; Gao, Ruize; Mu, Chenglong; Chen, Linbo; Liu, Qiangqiang; Luo, Qian; Feng, Du; Zhu, Yushan; Chen, Quan

2017-02-01

Autophagy requires diverse membrane sources and involves membrane trafficking of mATG9, the only membrane protein in the ATG family. However, the molecular regulation of mATG9 trafficking for autophagy initiation remains unclear. Here we identified two conserved classic adaptor protein sorting signals within the cytosolic N-terminus of mATG9, which mediate trafficking of mATG9 from the plasma membrane and trans-Golgi network (TGN) via interaction with the AP1/2 complex. Src phosphorylates mATG9 at Tyr8 to maintain its endocytic and constitutive trafficking in unstressed conditions. In response to starvation, phosphorylation of mATG9 at Tyr8 by Src and at Ser14 by ULK1 functionally cooperate to promote interactions between mATG9 and the AP1/2 complex, leading to redistribution of mATG9 from the plasma membrane and juxta-nuclear region to the peripheral pool for autophagy initiation. Our findings uncover novel mechanisms of mATG9 trafficking and suggest a coordination of basal and stress-induced autophagy.

Structure of Atg7 Alone and its Atg8-Bound Forms

NASA Astrophysics Data System (ADS)

Noda, Nobuo

Atg7 is a noncanonical E1 enzyme that activates Atg8 and transfers it to Atg3 (E2 enzyme), thus playing an essential role in conjugating Atg8 with phosphatidylethanolamine and thus in autophagy. Atg7 protomer is comprised of two globular domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), and forms a homodimer through CTD. Atg7-Atg8 complex structures and biochemical analyses revealed that Atg8 is initially recognized by the C-terminal tail of CTD and is then transferred to the adenylation domain in CTD, where Atg8 Gly116 is adenylated and thioester-linked to the catalytic cysteine of Atg7. Atg8 is then transferred to Atg3 bound to the NTD of the opposite protomer within an Atg7 dimer via a trans mechanism.

Complete mitochondrial genome of the agarophyte red alga Gelidium vagum (Gelidiales).

PubMed

Yang, Eun Chan; Kim, Kyeong Mi; Boo, Ga Hun; Lee, Jung-Hyun; Boo, Sung Min; Yoon, Hwan Su

2014-08-01

We describe the first complete mitochondrial genome of Gelidium vagum (Gelidiales) (24,901 bp, 30.4% GC content), an agar-producing red alga. The circular mitochondrial genome contains 43 genes, including 23 protein-coding, 18 tRNA and 2 rRNA genes. All the protein-coding genes have a typical ATG start codon. No introns were found. Two genes, secY and rps12, were overlapped by 41 bp.

An Out-of-frame Overlapping Reading Frame in the Ataxin-1 Coding Sequence Encodes a Novel Ataxin-1 Interacting Protein*

PubMed Central

Bergeron, Danny; Lapointe, Catherine; Bissonnette, Cyntia; Tremblay, Guillaume; Motard, Julie; Roucou, Xavier

2013-01-01

Spinocerebellar ataxia type 1 is an autosomal dominant cerebellar ataxia associated with the expansion of a polyglutamine tract within the ataxin-1 (ATXN1) protein. Recent studies suggest that understanding the normal function of ATXN1 in cellular processes is essential to decipher the pathogenesis mechanisms in spinocerebellar ataxia type 1. We found an alternative translation initiation ATG codon in the +3 reading frame of human ATXN1 starting 30 nucleotides downstream of the initiation codon for ATXN1 and ending at nucleotide 587. This novel overlapping open reading frame (ORF) encodes a 21-kDa polypeptide termed Alt-ATXN1 (Alternative ATXN1) with a completely different amino acid sequence from ATXN1. We introduced a hemagglutinin tag in-frame with Alt-ATXN1 in ATXN1 cDNA and showed in cell culture the co-expression of both ATXN1 and Alt-ATXN1. Remarkably, Alt-ATXN1 colocalized and interacted with ATXN1 in nuclear inclusions. In contrast, in the absence of ATXN1 expression, Alt-ATXN1 displays a homogenous nucleoplasmic distribution. Alt-ATXN1 interacts with poly(A)+ RNA, and its nuclear localization is dependent on RNA transcription. Polyclonal antibodies raised against Alt-ATXN1 confirmed the expression of Alt-ATXN1 in human cerebellum expressing ATXN1. These results demonstrate that human ATXN1 gene is a dual coding sequence and that ATXN1 interacts with and controls the subcellular distribution of Alt-ATXN1. PMID:23760502

The complete mitochondrial genome of the giant African snail Achatina fulica (Mollusca: Achatinidae).

PubMed

Yang, Huirong; Zhang, Jia-En; Guo, Jing; Deng, Zhixin; Luo, Hao; Luo, Mingzhu; Zhao, Benliang

2016-05-01

We present the complete mitochondrial genome of the Achatina fulica in this study. The results show that the mitochondrial genome is 15,057 bp in length, which is comprised of 13 protein-coding genes, 2 rRNA genes, 21 tRNA genes. The nucleotide compositions of the light strand are 35.47% of A, 27.97% of T 19.46% of C, and 17.10% of G. Except the ND3, 7 tRNA, ATP6, ATP8, COX3 and 12S-rRNA on the light strand, the rest are encoded on the heavy strand. Five types of inferred initiation codons are ATA (ND1, ND5), GTG (ND6), ATG (COX3, COX2), ATT (ND4) and TTG (COX1, ND2, ND3, ND4L, ATP6, ATP8, Cytb), and 3 types of inferred termination codons are T (COX3, ND2), TAA (ND1, ND4L, ND5, ND6, ATP6), and TAG (ND3, ND4, COX1, COX2, Cytb, ATP8). There are 24 intergenic spacers and 6 gene overlaps. The tandem repeat sequence (total 52 bp) of (AATAATT)n is observed in 16S-rRNA. Gene arrangement and distribution are inconsistent with the typical vertebrates.

Xylanase from the extremely thermophilic bacterium Caldocellum saccharolyticum: Overexpression of the gene in Escherichia coli and characterization of the gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Luethi, E.; Jasmat, N.B.; Bergquist, P.L.

A xylanase encoded by the xynA gene of the extreme thermophile Caldocellum saccharolyticum was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible {lambda} P{sub R} and P{sub L} promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42{degrees}C, and the xylanase made up of 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E.coli. The temperature and pH optima were determined to be 70{degrees}C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h ifmore » incubated at 60{degrees}C, with half-lives of 8 to 9 h at 70{degrees}C and 2 to 3 min at 80{degrees}C. The enzyme had high activity on xylan and ortho-nitrophenyl {beta}-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl {beta}-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.« less

The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

PubMed

Meßling, Susanne; Matthias, Jan; Xiong, Qiuhong; Fischer, Sarah; Eichinger, Ludwig

2017-06-01

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Posttranscriptional mRNA processing as a mechanism for regulation of human A1 adenosine receptor expression.

PubMed Central

Ren, H; Stiles, G L

1994-01-01

The human A1 adenosine receptor gene contains six exons with exons 1, 2, 3, 4, and part of 5 representing 5' untranslated regions. Reverse transcription-PCR with exon-specific primers showed two distinct transcripts containing either exons 3, 5, and 6 or exons 4, 5, and 6, with exons 3 and 4 being mutually exclusive. No mature mRNAs containing exons 1 and 2 have been detected. All human tissues that express any A1 receptors contain mRNA with exons 4, 5, and 6. Tissues which express high levels of A1 receptors contain mRNA with exons 3, 5, and 6. Exon 4 contains two upstream ATG codons whereas exon 3 contains none. COS cells transfected with expression vectors containing exon 4 (exons 1-6, 3-6, or Ex4-6) express much lower levels of A1 receptors than vectors without exon 4 (exons 3, 5, and 6). Mutation of upstream ATG codons in exon 4 leads to 3- to 7-fold increased A1 receptor expression, up to the level seen with the construct containing exons 3, 5, and 6. Thus, in human tissues "basal" levels of A1 receptors can be expressed by use of mRNA containing exons 4, 5, and 6, but when high levels are needed, alternative transcripts with exons 3, 5, and 6 are produced. Images PMID:8197148

Characterization of the complete mitochondrial genome sequence of wild yak (Bos mutus).

PubMed

Chunnian, Liang; Wu, Xiaoyun; Ding, Xuezhi; Wang, Hongbo; Guo, Xian; Chu, Min; Bao, Pengjia; Yan, Ping

2016-11-01

Wild yak is a special breed in China and it is regarded as an important genetic resource for sustainably developing the animal husbandry in Tibetan area and enriching region's biodiversity. The complete mitochondrial genome of wild yak (16,322 bp in length) displayed 37 typical animal mitochondrial genes and A + T-rich (61.01%), with an overall G + C content of only 38.99%. It contained a non-coding control region (D-loop), 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. Most of the genes have ATG initiation codons, whereas ND2, ND3, and ND5 genes start with ATA and were encoded on H-strand. The gene order of wild yak mitogenome is identical to that observed in most other vertebrates. The complete mitochondrial genome sequence of wild yak reported here could provide valuable information for developing genetic markers and phylogenetic analysis in yak.

Novel Immune Modulating Cellular Vaccine for Prostate Cancer

DTIC Science & Technology

2014-10-01

restriction sites. Murine PSMA : The cDNA encoding mPSMA was purchased from Sino Biologicals and was cloned into the HindIII and BamHI sites of pSP73-Sph/A64...sequence) and reverse primer 5’-TATATAGAGCTCTCAGATGTTCCGATACACATCTC-3’ Murine PSMA no signal sequence (mPSMA-SS): Murine PSMA minus the signal sequence...contains a HindIII site for cloning and utilizes an ATG that lies downstream of the signal sequence as the start codon in PSMA -SS ( PSMA without signal

Effect of Estrogen on Mutagenesis in Human Mammary Epithelial Cells

DTIC Science & Technology

2005-06-01

instability remains undefined in most human cancers, it appears to arise from subtle, intragenic mutations of the genes , whose products play a key role in...cells and is less labor-intensive. A G-G or T-G mismatch was introduced into ATG start codon of the enhanced green fluorescent protein (EGFP) gene ...Repair of the G-G or T-G mismatch to G-C or T-A, respectively in the heteroduplex plasmid generates a functional EGFP gene expression. The heteroduplex

Congenital nephrogenic diabetes insipidus with a novel mutation in the aquaporin 2 gene.

PubMed

Park, Youn Jong; Baik, Haing Woon; Cheong, Hae Il; Kang, Ju Hyung

2014-07-01

Congenital nephrogenic diabetes insipidus (CNDI) is a rare disorder caused by mutations of the arginine vasopressin (AVP) V2 receptor or aquaporin 2 ( AQP2 ) genes. The current study presented the case of CNDI in a 1-month-old male with a novel mutation in the AQP2 gene. The patient was referred due to the occurrence of hypernatremia and mild-intermittent fever since birth. An AVP stimulation test was compatible with CNDI as there was no significant response to desmopressin. Molecular genetic analysis demonstrated two mutations in exon 1 of the AQP2 gene: C to T transition, which resulted in a missense mutation of 108 Thr (ACG) to Met (ATG); and a 127, 128 delCA, which resulted in a deletion mutation of glutamine in position 43 at codon CAG as the first affected amino acid, with the new reading frame endign in a termination codon at position 62. The molecular genetic analysis of the parents showed that the missense mutation was inherited maternally and the deletion mutation was inherited paternally. The parents showed no signs or symptoms of CNDI, indicating autosomal recessive inheritance. The 108 Thr (ACG) to Met (ATG) mutation was confirmed as a novel mutation. Therefore, the molecular identification of the AQP2 gene has clinical significance, as early recognition of CNDI in infants that show only non-specific symptoms, can be facilitated. Thus, repeated episodes of dehydration, which may cause physical and mental retardation can be avoided.

Congenital nephrogenic diabetes insipidus with a novel mutation in the aquaporin 2 gene

PubMed Central

PARK, YOUN JONG; BAIK, HAING WOON; CHEONG, HAE IL; KANG, JU HYUNG

2014-01-01

Congenital nephrogenic diabetes insipidus (CNDI) is a rare disorder caused by mutations of the arginine vasopressin (AVP) V2 receptor or aquaporin 2 (AQP2) genes. The current study presented the case of CNDI in a 1-month-old male with a novel mutation in the AQP2 gene. The patient was referred due to the occurrence of hypernatremia and mild-intermittent fever since birth. An AVP stimulation test was compatible with CNDI as there was no significant response to desmopressin. Molecular genetic analysis demonstrated two mutations in exon 1 of the AQP2 gene: C to T transition, which resulted in a missense mutation of 108Thr (ACG) to Met (ATG); and a 127, 128 delCA, which resulted in a deletion mutation of glutamine in position 43 at codon CAG as the first affected amino acid, with the new reading frame endign in a termination codon at position 62. The molecular genetic analysis of the parents showed that the missense mutation was inherited maternally and the deletion mutation was inherited paternally. The parents showed no signs or symptoms of CNDI, indicating autosomal recessive inheritance. The 108Thr (ACG) to Met (ATG) mutation was confirmed as a novel mutation. Therefore, the molecular identification of the AQP2 gene has clinical significance, as early recognition of CNDI in infants that show only non-specific symptoms, can be facilitated. Thus, repeated episodes of dehydration, which may cause physical and mental retardation can be avoided. PMID:24944815

Xylanase II from an alkaliphilic thermophilic Bacillus with a distinctly different structure from other xylanases: evolutionary relationship to alkaliphilic xylanases.

PubMed

Kulkarni, N; Lakshmikumaran, M; Rao, M

1999-10-05

A 1.0 kilobase gene fragment from the genomic DNA of an alkaliphilic thermophilic Bacillus was found to code for a functional xylanase (XynII). The complete nucleotide sequence including the structural gene and the 5' and 3' flanking sequences of the xylanase gene have been determined. An open reading frame starting from ATG initiator codon comprising 402 nucleotides gave a preprotein of 133 amino acids of calculated molecular mass 14.090 kDa. The occurrence of three potential N-glycosylation sites in XynII gene is a unique feature for a gene of bacterial origin. The stop codon was followed by hairpin loop structures indicating the presence of transcription termination signals. The secondary structure analysis of XynII predicted that the polypeptide was primarily formed of beta-sheets. XynII appeared to be a member of family G/11 of xylanases based on its molecular weight and basic pI (8.0). However, sequence homology revealed similar identity with families 10 and 11 of xylanases. The conserved triad (Val-Val-Xaa, where Xaa is Asn or Asp) was identified only in the xylanases from alkaliphilic organisms. Our results implicate for the first time the concept of convergent evolution for XynII and provide a basis for research in evolutionary relationship among the xylanases from alkaliphilic and neutrophilic organisms. Copyright 1999 Academic Press.

Co-expression of the Thermotoga neapolitana aglB gene with an upstream 3'-coding fragment of the malG gene improves enzymatic characteristics of recombinant AglB cyclomaltodextrinase.

PubMed

Lunina, Natalia A; Agafonova, Elena V; Chekanovskaya, Lyudmila A; Dvortsov, Igor A; Berezina, Oksana V; Shedova, Ekaterina N; Kostrov, Sergey V; Velikodvorskaya, Galina A

2007-07-01

A cluster of Thermotoga neapolitana genes participating in starch degradation includes the malG gene of sugar transport protein and the aglB gene of cyclomaltodextrinase. The start and stop codons of these genes share a common overlapping sequence, aTGAtg. Here, we compared properties of expression products of three different constructs with aglB from T. neapolitana. The first expression vector contained the aglB gene linked to an upstream 90-bp 3'-terminal region of the malG gene with the stop codon overlapping with the start codon of aglB. The second construct included the isolated coding sequence of aglB with two tandem potential start codons. The expression product of this construct in Escherichia coli had two tandem Met residues at its N terminus and was characterized by low thermostability and high tendency to aggregate. In contrast, co-expression of aglB and the 3'-terminal region of malG (the first construct) resulted in AglB with only one N-terminal Met residue and a much higher specific activity of cyclomaltodextrinase. Moreover, the enzyme expressed by such a construct was more thermostable and less prone to aggregation. The third construct was the same as the second one except that it contained only one ATG start codon. The product of its expression had kinetic and other properties similar to those of the enzyme with only one N-terminal Met residue.

The dynamic Atg13-free conformation of the Atg1 EAT domain is required for phagophore expansion.

PubMed

Lin, Mary G; Schöneberg, Johannes; Davies, Christopher W; Ren, Xuefeng; Hurley, James H

2018-05-15

Yeast macroautophagy begins with the de novo formation of a double-membrane phagophore at the preautophagosomal structure/phagophore assembly site (PAS), followed by its expansion into the autophagosome responsible for cargo engulfment. The kinase Atg1 is recruited to the PAS by Atg13 through interactions between the EAT domain of the former and the tMIM motif of the latter. Mass-spectrometry data have shown that, in the absence of Atg13, the EAT domain structure is strikingly dynamic, but the function of this Atg13-free dynamic state has been unclear. We used structure-based mutational analysis and quantitative and superresolution microscopy to show that Atg1 is present on autophagic puncta at, on average, twice the stoichiometry of Atg13. Moreover, Atg1 colocalizes with the expanding autophagosome in a manner dependent on Atg8 but not Atg13. We used isothermal titration calorimetry and crystal structure information to design an EAT domain mutant allele ATG1 DD that selectively perturbs the function of the Atg13-free state. Atg1 DD shows reduced PAS formation and does not support phagophore expansion, showing that the EAT domain has an essential function that is separate from its Atg13-dependent role in autophagy initiation.

ATG13

PubMed Central

Alers, Sebastian; Wesselborg, Sebastian; Stork, Björn

2014-01-01

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play. PMID:24879146

Complete mitochondrial genome of the Freshwater Whipray Himantura dalyensis.

PubMed

Feutry, Pierre; Kyne, Peter M; Peng, Zaiqing; Pan, Lianghao; Chen, Xiao

2016-05-01

The complete mitochondrial genome of the Freshwater Whipray Himantura dalyensis is presented in this study. It is 17,693 bp in length and contains 37 genes in typical gene order and transcriptional orientation observed in vertebrates. There were a total of 86 bp short intergenic spacers and 22 bp overlaps in the genome. The overall base composition was 31.4% A, 25.5% C, 13.2% G and 29.9% T. Two start codons (GTG and ATG) and two stop codons (TAG and TAA/T) were found in 13 protein-coding genes. The length of 22 tRNA genes ranged from 68 (tRNA-Cys and tRNA-Ser2) to 75 bp (tRNA-Leu1). The origin of L-strand replication (OL) was found between the tRNA-Asn and tRNA-Cys genes. The base composition of the control region (1940 bp) was similar to the whole mitogenome.

Development of new plasmid DNA vaccine vectors with R1-based replicons

PubMed Central

2012-01-01

Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production. PMID:22889338

Structure of the Human Atg13-Atg101 HORMA Heterodimer: an Interaction Hub within the ULK1 Complex.

PubMed

Qi, Shiqian; Kim, Do Jin; Stjepanovic, Goran; Hurley, James H

2015-10-06

The ULK1 complex, consisting of the ULK1 protein kinase itself, FIP200, Atg13, and Atg101, controls the initiation of autophagy in animals. We determined the structure of the complex of the human Atg13 HORMA (Hop1, Rev7, Mad2) domain in complex with the full-length HORMA domain-only protein Atg101. The two HORMA domains assemble with an architecture conserved in the Mad2 conformational heterodimer and the S. pombe Atg13-Atg101 HORMA complex. The WF finger motif that is essential for function in human Atg101 is sequestered in a hydrophobic pocket, suggesting that the exposure of this motif is regulated. Benzamidine molecules from the crystallization solution mark two hydrophobic pockets that are conserved in, and unique to, animals, and are suggestive of sites that could interact with other proteins. These features suggest that the activity of the animal Atg13-Atg101 subcomplex is regulated and that it is an interaction hub for multiple partners. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rift Valley Fever Virus: Molecular Biologic Studies of the M Segment RNA(Ribonucleic Acids) for Application in Disease Prevention

DTIC Science & Technology

1988-12-01

correctly expresses, processes , and transports all M segment proteins (virus 7; r-eferences 2,3) were pulse -labeled with 35S- methionine and subsequently...c-ranslationii~y processed to yield the mature proteins. The first ATG codon of tP’o _iinle OrF is required for production of the 78kd protein...employed for the expression of the 78kd and l4kd proteins serves to control glycosylation arnd proteolytic. processing of the resultant poI~peptidles

ATG13: just a companion, or an executor of the autophagic program?

PubMed

Alers, Sebastian; Wesselborg, Sebastian; Stork, Björn

2014-06-01

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Daohong; Chen, Xi; Children’s Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003

Highlights: ► CCCP-induced LC3 lipidation can be independent of initiation/nucleation molecules. ► Atg9-mediated trafficking is critically required for CCCP-induced LC3 lipidation. ► CCCP-induced mitophagy may thus be more dependent on Atg9-positive vesicles. -- Abstract: Treatment of cells with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial proton gradient uncoupler, can result in mitochondrial damage and autophagy activation, which in turn eliminates the injured mitochondria in a Parkin-dependent way. How CCCP mobilizes the autophagy machinery is not fully understood. By analyzing a key autophagy step, LC3 lipidation, we examined the roles of two kinase complexes typically involved in the initiation and nucleation phasesmore » of autophagy, namely the ULK kinase complex (UKC) and the Beclin 1/Atg14 complex. We found that CCCP-induced LC3 lipidation could be independent of Beclin 1 and Atg14. In addition, deletion or knockdown of the UKC component FIP200 or Atg13 only led to a partial reduction in LC3 lipidation, indicating that UKC could be also dispensable for this step during CCCP treatment. In contrast, Atg9, which is important for transporting vesicles to early autophagosomal structure, was required for CCCP-induced LC3 lipidation. Taken together, these data suggest that CCCP-induced autophagy and mitophagy depends more critically on Atg9 vesicles than on UKC and Beclin 1/Atg14 complex.« less

Complete mitochondrial genome of Skylark, Alauda arvensis (Aves: Passeriformes): the first representative of the family Alaudidae with two extensive heteroplasmic control regions.

PubMed

Qian, Chaoju; Wang, Yuanxiu; Guo, Zhichun; Yang, Jianke; Kan, Xianzhao

2013-06-01

The circular mitochondrial genome of Alauda arvensis is 17,018 bp in length, containing 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes, and 2 extensive heteroplasmic control regions. All of the genes encoded on the H-strand, with the exceptions of one PCG (nad6) and eight tRNA genes (tRNA(Gln), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), tRNA(Ser(UCN)), tRNA(Pro), and tRNA(Glu)), as found in other birds' mitochondrial genomes. All of these PCGs are initiated with ATG, while stopped by six types of stop codons. All tRNA genes have the potential to fold into typical clover-leaf structure. Two extensive heteroplasmic control regions were found, and more interestingly, a minisatellite of 37 nucleotides (5'-TCAATCCCATTGATTTCATTATATTAGTATAAAGAAA-3') with 6 tandem repeats was detected at the end of CR2.

Efficient initiation of mammalian mRNA translation at a CUG codon.

PubMed Central

Dasso, M C; Jackson, R J

1989-01-01

Nucleotide substitutions were made at the initiation codon of an influenza virus NS cDNA clone in a vector carrying the bacteriophage T7 promoter. When capped mRNA transcripts of these constructs were translated in the rabbit reticulocyte lysate, a change in the initiation codon from...AUAAUGG...to...AUACUGG...reduced the in vitro translational efficiency by only 50-60%, and resulted in only a small increase in the yield of short products presumed to be initiated at downstream sites. Synthesis of the full-length product was initiated exclusively at the mutated codon, with negligible use either of in-frame upstream CUG or GUG codons, or of an in-frame downstream GUG codon. We conclude that CUG has the potential to function as an efficient initiation codon in mammalian systems, at least in certain contexts. Images PMID:2780285

Exon sequencing of PKD1 gene in an Iranian patient with autosomal-dominant polycystic kidney disease.

PubMed

Hafizi, Atousa; Khatami, Saeid Reza; Galehdari, Hamid; Shariati, Gholamreza; Saberi, Ali Hossein; Hamid, Mohammad

2014-07-01

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic kidney disorders with the incidence of 1 in 1,000 births. ADPKD is genetically heterogeneous with two genes identified: PKD1 (16p13.3, 46 exons) and PKD2 (4q21, 15 exons). Eighty five percent of the patients with ADPKD have at least one mutation in the PKD1 gene. Genetic studies have demonstrated an important allelic variability among patients, but very few data are known about the genetic variation among Iranian populations. In this study, exon direct sequencing of PKD1 was performed in a seven-year old boy with ADPKD and in his parents. The patient's father was ADPKD who was affected without any kidney dysfunction, and the patient's mother was congenitally missing one kidney. Molecular genetic testing found a mutation in all three members of this family. It was a missense mutation GTG>ATG at position 3057 in exon 25 of PKD1. On the other hand, two novel missense mutations were reported just in the 7-year-old boy: ACA>GCA found in exon 15 at codon 2241 and CAC>AAC found in exon 38 at codon 3710. For checking the pathogenicity of these mutations, exons 15, 25, and 38 of 50 unrelated normal cases were sequenced. our findings suggested that GTG>ATG is a polymorphism with high frequency (60%) as well as ACA>GCA and CAC>AAC are polymorphisms with frequencies of 14% and 22%, respectively in the population of Southwest Iran.

A novel mutation in the albumin gene (c.1A>C) resulting in analbuminemia.

PubMed

Caridi, Gianluca; Dagnino, Monica; Lugani, Francesca; Shalev, Stavit A; Campagnoli, Monica; Galliano, Monica; Spiegel, Ronen; Minchiotti, Lorenzo

2013-01-01

Analbuminemia (OMIM # 103600) is a rare autosomal recessive disorder manifested by the absence or severe reduction of circulating serum albumin in homozygous or compound heterozygous subjects. The trait is caused by a variety of mutations within the albumin gene. We report here the clinical and molecular characterisation of two new cases of congenital analbuminemia diagnosed in two members of the Druze population living in a Galilean village (Northern Israel) on the basis of their low level of circulating albumin. The albumin gene was screened by single-strand conformation polymorphism and heteroduplex analysis, and the mutated region was submitted to DNA sequencing. Both the analbuminemic subjects resulted homozygous for a previously unreported c.1 A>C transversion, for which we suggest the name Afula from the hospital where the two cases were investigated. This mutation causes the loss of the primary start codon ATG for Met1, which is replaced by a - then untranslated - triplet CTG for Leu. (p.Met1Leu). The use of an alternative downstream ATG codon would probably give rise to a completely aberrant polypeptide chain, leading to a misrouted intracellular transport and a premature degradation. The discovery of this new ALB mutation, probably inherited from a common ancestor, sheds light on the molecular mechanism underlying the analbuminemic trait and may serve in the development of a rapid genetic test for the identification of a-symptomatic heterozygous carriers in the Druze population in the Galilee. © 2012 The Authors. European Journal of Clinical Investigation © 2012 Stichting European Society for Clinical Investigation Journal Foundation.

Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

PubMed Central

2010-01-01

Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to significant increase in the activity of GUS in the transgenic plants. Promoter deletion analysis led to the identification of a short promoter region (-436 bp to ATG) that exhibited a higher promoter strength but similar developmental expression pattern as compared with the full-length ShCYC-B promoter. Conclusion Functional characterization of the full-length ShCYC-B promoter and its deletion fragments in transient expression system in fruto as well as in stable transgenic tomato revealed that the promoter is developmentally regulated and its expression is upregulated in chromoplast-rich flowers and fruits. Our study identified a short promoter region with functional activity and developmental expression pattern similar to that of the full-length ShCYC-B promoter. This 436 bp promoter region can be used in promoter::reporter fusion molecular genetic screens to identify mutants impaired in CYC-B expression, and thus can be a valuable tool in understanding carotenoid metabolism in tomato. Moreover, this short promoter region of ShCYC-B may be useful in genetic engineering of carotenoid content and other agronomic traits in tomato fruits. PMID:20380705

Polypyrimidine tract-binding protein 1-mediated down-regulation of ATG10 facilitates metastasis of colorectal cancer cells.

PubMed

Jo, Yoon Kyung; Roh, Seon Ae; Lee, Heejin; Park, Na Yeon; Choi, Eun Sun; Oh, Ju-Hee; Park, So Jung; Shin, Ji Hyun; Suh, Young-Ah; Lee, Eun Kyung; Cho, Dong-Hyung; Kim, Jin Cheon

2017-01-28

Autophagy plays complex roles in tumor initiation and development, and the expression of autophagy-related genes (ATGs) is differentially regulated in various cancer cells, depending on their environment. In this study, we analyzed the expressional relationship between polypyrimidine tract-binding protein 1 (PTBP1) and ATG10 in metastatic colorectal cancer. PTBP1 is associated with tumor metastasis in primary colorectal tumors and colorectal cancer liver metastasis (CLM) tissues. In addition, PTPB1 directly interacts with mRNA of ATG10, and regulates ATG10 expression level in colorectal cancer cells. Ectopic expression of PTBP1 decreased ATG10 expression, whereas down-regulation of PTBP1 increased ATG10 level. In contrast to PTBP1, expression of ATG10 was decreased in CLM tissues. Knock down of ATG10 promoted cell migration and invasion of colorectal cancer cells. Moreover, depletion of ATG10 modulated epithelial-mesenchymal transition-associated proteins in colorectal cancer cells: N-cadherin, TCF-8/ZEB1, and CD44 were up-regulated, whereas E-cadherin was down-regulated. Taken together, our findings suggest that expression of ATG10 negatively regulated by PTBP1 is associated with metastasis of colorectal cancer cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Molecular Characterisation of α- and β-Thalassaemia among Indigenous Senoi Orang Asli Communities in Peninsular Malaysia.

PubMed

Koh, Danny Xuan Rong; Raja Sabudin, Raja Zahratul Azma; Mohd Yusoff, Malisa; Hussin, Noor Hamidah; Ahmad, Rahimah; Othman, Ainoon; Ismail, Endom

2017-09-01

Thalassaemia is a public health problem in Malaysia, with each ethnic group having their own common mutations. However, there is a lack on data on the prevalence and common mutations among the indigenous people. This cross-sectional study was performed to determine the common mutations of α- and β-thalassaemia among the subethnic groups of Senoi, the largest Orang Asli group in Peninsular Malaysia. Blood samples collected from six Senoi subethnic groups were analysed for full blood count and haemoglobin analysis (HbAn). Samples with abnormal findings were then screened for α- and β-globin gene mutations. Out of the 752 samples collected, 255 showed abnormal HbAn results, and 122 cases showing abnormal red cell indices with normal HbAn findings were subjected to molecular screening. DNA analysis revealed a mixture of α- and β-globin gene mutations with 25 concomitant cases. The types of gene abnormalities detected for α-thalassaemia were termination codon (T>C) Hb CS (α CS α), Cd59 (G>A) haemoglobin Adana (Hb Adana) (α Cd59 α), initiation codon (ATG>A-G) (α IniCd α), two-gene deletion (- SEA ), and single-gene 3.7-kb deletion (-α 3.7 ). For β-thalassaemia, there were Cd26 (G>A) Hb E (β E ), Cd19 (A>G) Haemoglobin Malay (Hb Malay) (β Cd19 ), and IVS 1-5 (G>C) (β IVS 1-5 ). © 2017 John Wiley & Sons Ltd/University College London.

Sirtuin1 and autophagy protect cells from fluoride-induced cell stress.

PubMed

Suzuki, Maiko; Bartlett, John D

2014-02-01

Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum (ER) stress and oxidative stress. Previously, we reported that fluoride induces ER-stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augment SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50, 100 and 125ppm) in drinking water for 6weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis. Copyright © 2013 Elsevier B.V. All rights reserved.

Sirtuin1 and autophagy protect cells from fluoride-induced cell stress

PubMed Central

Suzuki, Maiko; Bartlett, John D.

2014-01-01

Sirtuin1 (SIRT1) is an (NAD+)-dependent deacetylase functioning in the regulation of metabolism, cell survival and organismal lifespan. Active SIRT1 regulates autophagy during cell stress, including calorie restriction, endoplasmic reticulum stress and oxidative stress. Previously, we reported that fluoride induces endoplasmic reticulum (ER) stress in ameloblasts responsible for enamel formation, suggesting that ER-stress plays a role in dental fluorosis. However, the molecular mechanism of how cells respond to fluoride-induced cell stress is unclear. Here, we demonstrate that fluoride activates SIRT1 and initiates autophagy to protect cells from fluoride exposure. Fluoride treatment of ameloblast-derived cells (LS8) significantly increased Sirt1 expression and induced SIRT1 phosphorylation resulting in the augmentation of SIRT1 deacetylase activity. To demonstrate that fluoride exposure initiates autophagy, we characterized the expression of autophagy related genes (Atg); Atg5, Atg7 and Atg8/LC3 and showed that both their transcript and protein levels were significantly increased following fluoride treatment. To confirm that SIRT1 plays a protective role in fluoride toxicity, we used resveratrol (RES) to augmented SIRT1 activity in fluoride treated LS8 cells. RES increased autophagy, inhibited apoptosis, and decreased fluoride cytotoxicity. Rats treated with fluoride (0, 50 and 100 ppm) in drinking water for 6 weeks had significantly elevated expression levels of Sirt1, Atg5, Atg7 and Atg8/LC3 in their maturation stage enamel organs. Increased protein levels of p-SIRT1, ATG5 and ATG8/LC3 were present in fluoride-treated rat maturation stage ameloblasts. Therefore, the SIRT1/autophagy pathway may play a critical role as a protective response to help prevent dental fluorosis. PMID:24296261

Differential regulation of two FLNA transcripts explains some of the phenotypic heterogeneity in the loss-of-function filaminopathies.

PubMed

Jenkins, Zandra A; Macharg, Alison; Chang, Cheng-Yee; van Kogelenberg, Margriet; Morgan, Tim; Frentz, Sophia; Wei, Wenhua; Pilch, Jacek; Hannibal, Mark; Foulds, Nicola; McGillivray, George; Leventer, Richard J; García-Miñaúr, Sixto; Sugito, Stuart; Nightingale, Scott; Markie, David M; Dudding, Tracy; Kapur, Raj P; Robertson, Stephen P

2018-01-01

Loss-of-function mutations in the X-linked gene FLNA can lead to abnormal neuronal migration, vascular and cardiac defects, and congenital intestinal pseudo-obstruction (CIPO), the latter characterized by anomalous intestinal smooth muscle layering. Survival in male hemizygotes for such mutations is dependent on retention of residual FLNA function but it is unclear why a subgroup of males with mutations in the 5' end of the gene can present with CIPO alone. Here, we demonstrate evidence for the presence of two FLNA isoforms differing by 28 residues at the N-terminus initiated at ATG +1 and ATG +82 . A male with CIPO (c.18_19del) exclusively expressed FLNA ATG +82 , implicating the longer protein isoform (ATG +1 ) in smooth muscle development. In contrast, mutations leading to reduction of both isoforms are associated with compound phenotypes affecting the brain, heart, and intestine. RNA-seq data revealed three distinct transcription start sites, two of which produce a protein isoform utilizing ATG +1 while the third utilizes ATG +82 . Transcripts sponsoring translational initiation at ATG +1 predominate in intestinal smooth muscle, and are more abundant compared with the level measured in fibroblasts. Together these observations describe a new mechanism of tissue-specific regulation of FLNA that could reflect the differing mechanical requirements of these cell types during development. © 2017 Wiley Periodicals, Inc.

The complete mitochondrial genome of Pomacea canaliculata (Gastropoda: Ampullariidae).

PubMed

Zhou, Xuming; Chen, Yu; Zhu, Shanliang; Xu, Haigen; Liu, Yan; Chen, Lian

2016-01-01

The mitochondrial genome of Pomacea canaliculata (Gastropoda: Ampullariidae) is the first complete mtDNA sequence reported in the genus Pomacea. The total length of mtDNA is 15,707 bp, which containing 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a 359 bp non-coding region. The A + T content of the overall base composition of H-strand is 71.7% (T: 41%, C: 12.7%, A: 30.7%, G: 15.6%). ATP6, ATP8, CO1, CO2, ND1-3, ND5, ND6, ND4L and Cyt b genes begin with ATG as start codon, CO3 and ND4 begin with ATA. ATP8, CO2-3, ND4L, ND2-6 and Cyt b genes are terminated with TAA as stop codon, ATP6, ND1, and CO1 end with TAG. A long non-coding region is found and a 23 bp repeat unit repeat 11 times in this region.

Complete mitochondrial genome of the Yellow-spotted skate Okamejei hollandi (Rajiformes: Rajidae).

PubMed

Li, Weidong; Chen, Xiao; Liu, Wenai; Sun, Renjie; Zhou, Haolang

2016-07-01

The complete mitochondrial genome of the Yellow-spotted skate Okamejei hollandi was determined in this study. It is 16,974 bp in length and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one putative control region. The overall base composition is 30.5% A, 27.8% C, 14.0% G, and 27.8% T. There are 28 bp short intergenic spaces located in 12 gene junctions and 31 bp overlaps located in nine gene junctions in the whole mitogenome. Two start codons (ATG and GTG) and two stop codons (TAG and TAA/T) were used in the protein-coding genes. The lengths of 22 tRNA genes range from 68 (tRNA-Ser2) to 75 (tRNA-Leu1) bp. The origin of L-strand replication (OL) sequence (37 bp) was identified between the tRNA-Asn and tRNA-Cys genes. The control region is 1311 bp in length with high A + T and poor G content.

Complete mitochondrial genome sequence of Urechis caupo, a representative of the phylum Echiura

PubMed Central

Boore, Jeffrey L

2004-01-01

Background Mitochondria contain small genomes that are physically separate from those of nuclei. Their comparison serves as a model system for understanding the processes of genome evolution. Although hundreds of these genome sequences have been reported, the taxonomic sampling is highly biased toward vertebrates and arthropods, with many whole phyla remaining unstudied. This is the first description of a complete mitochondrial genome sequence of a representative of the phylum Echiura, that of the fat innkeeper worm, Urechis caupo. Results This mtDNA is 15,113 nts in length and 62% A+T. It contains the 37 genes that are typical for animal mtDNAs in an arrangement somewhat similar to that of annelid worms. All genes are encoded by the same DNA strand which is rich in A and C relative to the opposite strand. Codons ending with the dinucleotide GG are more frequent than would be expected from apparent mutational biases. The largest non-coding region is only 282 nts long, is 71% A+T, and has potential for secondary structures. Conclusions Urechis caupo mtDNA shares many features with those of the few studied annelids, including the common usage of ATG start codons, unusual among animal mtDNAs, as well as gene arrangements, tRNA structures, and codon usage biases. PMID:15369601

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of mitochondrial genome of sea cucumber Stichopus horrens: a novel gene arrangement in Holothuroidea.

PubMed

Fan, SiGang; Hu, ChaoQun; Wen, Jing; Zhang, LvPing

2011-05-01

The complete mitochondrial DNA sequence contains useful information for phylogenetic analyses of metazoa. In this study, the complete mitochondrial DNA sequence of sea cucumber Stichopus horrens (Holothuroidea: Stichopodidae: Stichopus) is presented. The complete sequence was determined using normal and long PCRs. The mitochondrial genome of Stichopus horrens is a circular molecule 16257 bps long, composed of 13 protein-coding genes, two ribosomal RNA genes and 22 transfer RNA genes. Most of these genes are coded on the heavy strand except for one protein-coding gene (nad6) and five tRNA genes (tRNA ( Ser(UCN) ), tRNA ( Gln ), tRNA ( Ala ), tRNA ( Val ), tRNA ( Asp )) which are coded on the light strand. The composition of the heavy strand is 30.8% A, 23.7% C, 16.2% G, and 29.3% T bases (AT skew=0.025; GC skew=-0.188). A non-coding region of 675 bp was identified as a putative control region because of its location and AT richness. The intergenic spacers range from 1 to 50 bp in size, totaling 227 bp. A total of 25 overlapping nucleotides, ranging from 1 to 10 bp in size, exist among 11 genes. All 13 protein-coding genes are initiated with an ATG. The TAA codon is used as the stop codon in all the protein coding genes except nad3 and nad4 that use TAG as their termination codon. The most frequently used amino acids are Leu (16.29%), Ser (10.34%) and Phe (8.37%). All of the tRNA genes have the potential to fold into typical cloverleaf secondary structures. We also compared the order of the genes in the mitochondrial DNA from the five holothurians that are now available and found a novel gene arrangement in the mitochondrial DNA of Stichopus horrens.

20-Hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body.

PubMed

Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

2013-08-01

Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcR (DN) ) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body.

20-hydroxyecdysone upregulates Atg genes to induce autophagy in the Bombyx fat body

PubMed Central

Tian, Ling; Ma, Li; Guo, Enen; Deng, Xiaojuan; Ma, Sanyuan; Xia, Qingyou; Cao, Yang; Li, Sheng

2013-01-01

Autophagy is finely regulated at multiple levels and plays crucial roles in development and disease. In the fat body of the silkworm, Bombyx mori, autophagy occurs and Atg gene expression peaks during the nonfeeding molting and pupation stages when the steroid hormone (20-hydroxyecdysone; 20E) is high. Injection of 20E into the feeding larvae upregulated Atg genes and reduced TORC1 activity resulting in autophagy induction in the fat body. Conversely, RNAi knockdown of the 20E receptor partner (USP) or targeted overexpression of a dominant negative mutant of the 20E receptor (EcRDN) in the larval fat body reduced autophagy and downregulated the Atg genes, confirming the importance of 20E-induction of Atg gene expression during pupation. Moreover, in vitro treatments of the larval fat body with 20E upregulated the Atg genes. Five Atg genes were potentially 20E primary-responsive, and a 20E response element was identified in the Atg1 (ortholog of human ULK1) promoter region. Furthermore, RNAi knockdown of 4 key genes (namely Br-C, E74, HR3 and βftz-F1) in the 20E-triggered transcriptional cascade reduced autophagy and downregulated Atg genes to different levels. Taken together, we conclude that in addition to blocking TORC1 activity for autophagosome initiation, 20E upregulates Atg genes to induce autophagy in the Bombyx fat body. PMID:23674061

Overexpression of Plasmodium berghei ATG8 by Liver Forms Leads to Cumulative Defects in Organelle Dynamics and to Generation of Noninfectious Merozoites

PubMed Central

Voss, Christiane; Ehrenman, Karen; Mlambo, Godfree; Mishra, Satish; Kumar, Kota Arun; Sacci, John B.; Sinnis, Photini

2016-01-01

ABSTRACT Plasmodium parasites undergo continuous cellular renovation to adapt to various environments in the vertebrate host and insect vector. In hepatocytes, Plasmodium berghei discards unneeded organelles for replication, such as micronemes involved in invasion. Concomitantly, intrahepatic parasites expand organelles such as the apicoplast that produce essential metabolites. We previously showed that the ATG8 conjugation system is upregulated in P. berghei liver forms and that P. berghei ATG8 (PbATG8) localizes to the membranes of the apicoplast and cytoplasmic vesicles. Here, we focus on the contribution of PbATG8 to the organellar changes that occur in intrahepatic parasites. We illustrated that micronemes colocalize with PbATG8-containing structures before expulsion from the parasite. Interference with PbATG8 function by overexpression results in poor development into late liver stages and production of small merosomes that contain immature merozoites unable to initiate a blood infection. At the cellular level, PbATG8-overexpressing P. berghei exhibits a delay in microneme compartmentalization into PbATG8-containing autophagosomes and elimination compared to parasites from the parental strain. The apicoplast, identifiable by immunostaining of the acyl carrier protein (ACP), undergoes an abnormally fast proliferation in mutant parasites. Over time, the ACP staining becomes diffuse in merosomes, indicating a collapse of the apicoplast. PbATG8 is not incorporated into the progeny of mutant parasites, in contrast to parental merozoites in which PbATG8 and ACP localize to the apicoplast. These observations reveal that Plasmodium ATG8 is a key effector in the development of merozoites by controlling microneme clearance and apicoplast proliferation and that dysregulation in ATG8 levels is detrimental for malaria infectivity. PMID:27353755

Molecular and catalytic properties of 2,4'-dihydroxyacetophenone dioxygenase from Burkholderia sp. AZ11.

PubMed

Enya, Mayu; Aoyagi, Keiko; Hishikawa, Yoshihiro; Yoshimura, Azusa; Mitsukura, Koichi; Maruyama, Kiyofumi

2012-01-01

The gene dad encoding 2,4'-dihydroxyacetophenone (DHAP) dioxygenase was cloned from Burkholderia sp. AZ11. The initiation codon GTG was converted to ATG for high-level expression of the enzyme in Escherichia coli. The enzyme was moderately thermostable, and the recombinant enzyme was briefly purified. The enzyme (M(r)=90 kDa) was a homotetramer with a subunit M(r) of 23 kDa. It contained 1.69 mol of non-heme iron, and had a dark gray color. On anaerobic incubation of it with DHAP, the absorption at around 400 nm increased due to the formation of an enzyme-DHAP complex. Multiple sequence alignment suggested that His77, His79, His115, and Glu96 in the cupin fold were possible metal ligands. The apparent K(m) for DHAP and the apparent V(max) were estimated to be 1.60 µM and 6.28 µmol/min/mg respectively. 2-Hydroxyacetophenone was a poor substrate. CuCl(2) and HgCl(2) strongly inhibited the enzyme, while FeSO(4) weakly activated it.

Hb Chile [beta28(B10)Leu-->Met]: an unstable hemoglobin associated with chronic methemoglobinemia and sulfonamide or methylene blue-induced hemolytic anemia.

PubMed

Hojas-Bernal, R; McNab-Martin, P; Fairbanks, V F; Holmes, M W; Hoyer, J D; McCormick, D J; Kubik, K S

1999-05-01

Among the causes of life-long cyanosis are congenital methemoglobinemia due to M hemoglobins, congenital methemoglobinemia due to methemoglobin reductase deficiency, a small number of low oxygen affinity hemoglobins, and a small number of unstable hemoglobins that spontaneously form methemoglobin in vivo at an accelerated rate. We report an unstable hemoglobin with these characteristics that was observed in a family of indigenous (native American) origin living near Santiago, Chile. This variant has the substitution beta28(B10)Leu-->Met, unambiguously corresponding to the DNA mutation of CTG-->ATG in beta-globin gene codon 28.

Mutational analysis in a patient with a variant form of Gaucher disease caused by SAP-2 deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rafi, M.A.; Gala, G. de; Xunling Zhang

1993-01-01

It is now clear that the lysosomal hydrolysis of sphingolipids requires both lysosomal enzymes and so-called sphingolipid activator proteins (SAPs). One gene, called prosaposin, codes for a precursor protein that is proteolytically cut into four putative SAPs. These four SAPs, of about 80 amino acids, share some structural features but differ somewhat in their specificity. Domain 3 of prosaposin mRNA contains the coding region for SAP-2, an activator of glucocerebrosidase. While most patients with Gaucher disease store glucosylceramide due to defects in glucocerebrosidase, a few patients store this lipid in the presence of normal enzyme levels. In this paper themore » authors describe the identification of a point mutation in domain 3 of a patient who died with this variant form of Gaucher disease. Polymerase chain reaction amplification was performed in the small amount of genomic DNA available using primers generated from the intronic sequence surrounding domain 3. The patient was found to have a T-to-G substitution at position 1144 (counting from the A of ATG initiation codon) in half of the M13 recombinant clones. This changes the codon for cysteine[sub 382] to glycine. His father and unaffected brother also had this mutation, but his mother did not. She was found to have half of the normal amount of mRNA for prosaposin in her cultured skin fibroblasts. Therefore, this child inherited a point mutation in domain 3 from his father and a deficiency of all four SAPs coded for by prosaposin from his mother. 29 refs., 3 figs., 1 tab.« less

Safety and Feasibility of Outpatient Rabbit Antithymocyte Globulin Induction Therapy Administration in Kidney Transplant Recipients.

PubMed

Varga, Alexandra N; Johnson, David; Sawinski, Deirdre L; Lim, Mary Ann; Bloom, Roy D; Abt, Peter L; Goral, Simin; Bleicher, Melissa; Levine, Matthew H; Naji, Ali; Nazarian, Susanna; Porrett, Paige; Trofe-Clark, Jennifer

2018-06-01

Kidney transplant induction therapy often includes inpatient administration of rabbit antithymocyte globulin (rATG) over multiple days. To reduce hospital length of stay (LOS) and drug expenditures, the rATG induction course was completed in the outpatient setting via peripheral intravenous administration. The present study assesses early readmission trends ascribable to an outpatient rATG administration protocol to ensure initial reduction in hospital LOS is sustained early after discharge. This was a retrospective study of kidney recipient outcomes for patients transplanted between January 1, 2008, and February 29, 2016, immediately following implementation of an outpatient rATG protocol. Readmission data within 7 days of outpatient rATG administration were collected. The relatedness of rATG administration to an adverse drug reaction resulting in readmission was determined by the World Health Organization-Uppsala Monitoring Centre Causality Assessment Scale and the Naranjo Adverse Drug Reaction Probability Scale. A total of 1104 patients received outpatient doses of rATG and were included. An upward trend in kidney transplant volume and outpatient rATG administrations per year was found from 2008-2015. Following protocol implementation, the percentage of overall readmissions ranged from 9% to just over 12% from 2008-2014 and remained less than 10% for 2014 through 2016. The percentage of outpatient rATG infusions that potentially led to rATG-related readmissions was less than 4% per year over the study period. A total of 1124 hospital days were saved, 125 days per year on average. Outpatient administration of rATG is feasible, safe, and did not increase readmissions in the period directly following administration. The findings of this analysis support our continued use of the outpatient rATG protocol at our institution. © 2018 Pharmacotherapy Publications, Inc.

Single-dose rATG induction at renal transplantation: superior renal function and glucoregulation with less hypomagnesemia.

PubMed

Stevens, R Brian; Lane, James T; Boerner, Brian P; Miles, Clifford D; Rigley, Theodore H; Sandoz, John P; Nielsen, Kathleen J; Skorupa, Jill Y; Skorupa, Anna J; Kaplan, Bruce; Wrenshall, Lucile E

2012-01-01

Rabbit anti-thymocyte globulin (rATG) induction reduces reperfusion injury and improves renal function in kidney recipients by means of properties unrelated to T-cell lysis. Here, we analyze intensive rATG induction (single dose, rATG(S) , vs. divided dose, rATG(D) ) for improved renal function and protection against hyperglycemia. Patients without diabetes (n = 98 of 180) in a prospective randomized trial of intensive rATG induction were followed for six months for the major secondary composite end point of impaired glucose regulation (hyperglycemia and new-onset diabetes after transplantation, NODAT). Prospectively collected data included fasting blood glucose and HbA(1c). Serum Mg(++) was routinely collected and retrospectively analyzed. Induction with rATG(S) produced less impaired glucose regulation (p = 0.05), delayed NODAT development (p = 0.02), less hyperglycemia (p = 0.02), better renal function (p = 0.04), and less hypomagnesemia (p = 0.02), a factor associated with a lower incidence of NODAT. Generalized linear modeling confirmed that rATG(S) protects against a synergistic interaction between tacrolimus and sirolimus that otherwise increased hypomagnesemia (p = 0.008) and hyperglycemia (p = 0.03). rATG(S) initiated before renal reperfusion improved early renal function and reduced impaired glucose regulation, an injury by diabetogenic maintenance agents (tacrolimus and sirolimus). © 2011 John Wiley & Sons A/S.

The complete mitochondrial genome of Plodia interpunctella (Lepidoptera: Pyralidae) and comparison with other Pyraloidea insects.

PubMed

Liu, Qiu-Ning; Chai, Xin-Yue; Bian, Dan-Dan; Zhou, Chun-Lin; Tang, Bo-Ping

2016-01-01

The mitochondrial (mt) genome can provide important information for the understanding of phylogenetic relationships. The complete mt genome of Plodia interpunctella (Lepidoptera: Pyralidae) has been sequenced. The circular genome is 15 287 bp in size, encoding 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The AT skew of this mt genome is slightly negative, and the nucleotide composition is biased toward A+T nucleotides (80.15%). All PCGs start with the typical ATN (ATA, ATC, ATG, and ATT) codons, except for the cox1 gene which may start with the CGA codon. Four of the 13 PCGs harbor the incomplete termination codon T or TA. All the tRNA genes are folded into the typical clover-leaf structure of mitochondrial tRNA, except for trnS1 (AGN) in which the DHU arm fails to form a stable stem-loop structure. The overlapping sequences are 35 bp in total and are found in seven different locations. A total of 240 bp of intergenic spacers are scattered in 16 regions. The control region of the mt genome is 327 bp in length and consisted of several features common to the sequenced lepidopteran insects. Phylogenetic analysis based on 13 PCGs using the Maximum Likelihood method shows that the placement of P. interpunctella was within the Pyralidae.

Mycobacterium tuberculosis embB codon 306 mutations confer moderately increased resistance to ethambutol in vitro and in vivo.

PubMed

Plinke, Claudia; Walter, Kerstin; Aly, Sahar; Ehlers, Stefan; Niemann, Stefan

2011-06-01

Ethambutol (EMB) is a major component of the first-line therapy of tuberculosis. Mutations in codon 306 of embB (embB306) were suggested as a major resistance mechanism in clinical isolates. To directly analyze the impact of individual embB306 mutations on EMB resistance, we used allelic exchange experiments to generate embB306 mutants of M. tuberculosis H37Rv. The level of EMB resistance conferred by particular mutations was measured in vitro and in vivo after EMB therapy by daily gavage in a mouse model of aerogenic tuberculosis. The wild-type embB306 ATG codon was replaced by embB306 ATC, ATA, or GTG, respectively. All of the obtained embB306 mutants exhibited a 2- to 4-fold increase in EMB MIC compared to the wild-type H37Rv. In vivo, the one selected embB306 GTG mutant required a higher dose of ethambutol to restrict its growth in the lung compared to wild-type H37Rv. These experiments demonstrate that embB306 point mutations enhance the EMB MIC in vitro to a moderate, but significant extent, and reduce the efficacy of EMB treatment in the animal model. We propose that conventional EMB susceptibility testing, in combination with embB306 genotyping, may guide dose adjustment to avoid clinical treatment failure in these low-level resistant strains.

A transducer for microbial sensory rhodopsin that adopts GTG as a start codon is identified in Haloarcula marismortui.

PubMed

Fu, Hsu-Yuan; Lu, Yen-Hsu; Yi, Hsiu-Ping; Yang, Chii-Shen

2013-04-05

Microbial sensory rhodopsins are known to mediate phototaxis, and all of the known sensory rhodopsins execute this function with a specific cognate transducer that has two-transmembrane (2-TM) regions. In the genome of Haloarcula marismortui, a total of six rhodopsin genes were annotated, and we previously showed three of them to be the ion type and suggested the other three as sensory type, even though the candidate transducer gene, htr, for HmSRI was missing the 2-TM region that is found in all of the other known transducers. Here we showed this htr gene featured a preceding 2-TM region when the alternative start codon GTG located 291 nucleotides upstream of the original annotated open reading frame (ORF) was introduced and it is named as htrI in this study. Overexpression of HmHtrI exhibited it existed as a membrane protein and several biophysical assays confirmed it functionally interacted with HmSRI. Together with our previous reverse-transcriptase-PCR results and phototaxis measurements, the new ORF of original predicted soluble htr gene product was a membrane protein with a 2-TM region, HmHtrI; and it serves as the cognate transducer for HmSRI. HmHtrI therefore is the first transducer for the sensory rhodopsin adopted start codon other than ATG. Copyright © 2013 Elsevier B.V. All rights reserved.

Cloning, expression analysis, and RNA interference study of a HORMA domain containing autophagy-related gene 13 (ATG13) from the coleopteran beetle, Tenebrio molitor

PubMed Central

Lee, Jung Hee; Jo, Yong Hun; Patnaik, Bharat Bhusan; Park, Ki Beom; Tindwa, Hamisi; Seo, Gi Won; Chandrasekar, Raman; Lee, Yong Seok; Han, Yeon Soo

2015-01-01

Autophagy is a process that is necessary during starvation, as it replenishes metabolic precursors by eliminating damaged organelles. Autophagy is mediated by more than 35 autophagy-related (Atg) proteins that participate in the nucleation, elongation, and curving of the autophagosome membrane. In a pursuit to address the role of autophagy during development and immune resistance of the mealworm beetle, Tenebrio molitor, we screened ATG gene sequences from the whole-larva transcriptome database. We identified a homolog of ATG13 gene in T. molitor (designated as TmATG13) that comprises a cDNA of 1176 bp open reading frame (ORF) encoding a protein of 391 amino acids. Analyses of the structure-specific features of TmAtg13 showed an intrinsically disordered middle and C-terminal region that was rich in regulatory phosphorylation sites. The N-terminal Atg13 domain had a HORMA (Hop1, Rev7, and Mad2) fold containing amino acid residues conserved across the Atg13 insect orthologs. A quantitative reverse-transcription-polymerase chain reaction analysis revealed that TmATG13 was expressed ubiquitously during all developmental stages of the insect. TmATG13 mRNA expression was high in the fat body and gut of the larval and adult stages of the insect. The TmATG13 transcripts were expressed at a high level until 6 days of ovarian development, followed by a significant decline. Silencing of ATG13 transcripts in T. molitor larvae showed a reduced survivability of 39 and 38% in response to Escherichia coli and Staphylococcus aureus infection. Furthermore, the role of TmAtg13 in initiating autophagy as a part of the host cell autophagic complex of the host cells against the intracellular pathogen Listeria monocytogenes is currently under study and will be critical to unfold the structure-function relationships. PMID:26136688

Mitochondrial genome and phylogenetic position of the tawny nurse shark (Nebrius ferrugineus).

PubMed

Wang, Junjie; Chen, Hao; Lin, Lingling; Ai, Weiming; Chen, Xiao

2017-01-01

The complete mitochondrial genome of the tawny nurse shark (Nebrius ferrugineus) was first presented in this study. It was 16 693 bp in length with the typical gene order in vertebrates. The overall base composition was 33.6% A, 25.6% C, 12.7% G and 28.1% T. Two start (ATG and GTG) and two stop (TAG and TAA/T--) codons were found in the protein-coding genes. The size of 22 tRNA genes ranged from 67 to 75 bp. The origin of L-strand replication could form a hairpin structure. All nodes strongly supported that N. ferrugineus was placed as sister to Rhincodon typus in the Bayesian tree.

Lost in Translation: Bioinformatic Analysis of Variations Affecting the Translation Initiation Codon in the Human Genome.

PubMed

Abad, Francisco; de la Morena-Barrio, María Eugenia; Fernández-Breis, Jesualdo Tomás; Corral, Javier

2018-06-01

Translation is a key biological process controlled in eukaryotes by the initiation AUG codon. Variations affecting this codon may have pathological consequences by disturbing the correct initiation of translation. Unfortunately, there is no systematic study describing these variations in the human genome. Moreover, we aimed to develop new tools for in silico prediction of the pathogenicity of gene variations affecting AUG codons, because to date, these gene defects have been wrongly classified as missense. Whole-exome analysis revealed the mean of 12 gene variations per person affecting initiation codons, mostly with high (> 0:01) minor allele frequency (MAF). Moreover, analysis of Ensembl data (December 2017) revealed 11,261 genetic variations affecting the initiation AUG codon of 7,205 genes. Most of these variations (99.5%) have low or unknown MAF, probably reflecting deleterious consequences. Only 62 variations had high MAF. Genetic variations with high MAF had closer alternative AUG downstream codons than did those with low MAF. Besides, the high-MAF group better maintained both the signal peptide and reading frame. These differentiating elements could help to determine the pathogenicity of this kind of variation. Data and scripts in Perl and R are freely available at https://github.com/fanavarro/hemodonacion. jfernand@um.es. Supplementary data are available at Bioinformatics online.

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

PubMed Central

Williams, N P; Mueller, P P; Hinnebusch, A G

1988-01-01

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function. Images PMID:3065626

A Major Controversy in Codon-Anticodon Adaptation Resolved by a New Codon Usage Index

PubMed Central

Xia, Xuhua

2015-01-01

Two alternative hypotheses attribute different benefits to codon-anticodon adaptation. The first assumes that protein production is rate limited by both initiation and elongation and that codon-anticodon adaptation would result in higher elongation efficiency and more efficient and accurate protein production, especially for highly expressed genes. The second claims that protein production is rate limited only by initiation efficiency but that improved codon adaptation and, consequently, increased elongation efficiency have the benefit of increasing ribosomal availability for global translation. To test these hypotheses, a recent study engineered a synthetic library of 154 genes, all encoding the same protein but differing in degrees of codon adaptation, to quantify the effect of differential codon adaptation on protein production in Escherichia coli. The surprising conclusion that “codon bias did not correlate with gene expression” and that “translation initiation, not elongation, is rate-limiting for gene expression” contradicts the conclusion reached by many other empirical studies. In this paper, I resolve the contradiction by reanalyzing the data from the 154 sequences. I demonstrate that translation elongation accounts for about 17% of total variation in protein production and that the previous conclusion is due to the use of a codon adaptation index (CAI) that does not account for the mutation bias in characterizing codon adaptation. The effect of translation elongation becomes undetectable only when translation initiation is unrealistically slow. A new index of translation elongation ITE is formulated to facilitate studies on the efficiency and evolution of the translation machinery. PMID:25480780

Identification and use of the putative Bacteroides ovatus xylanase promoter for the inducible production of recombinant human proteins.

PubMed

Hamady, Zaed Z R; Farrar, Mark D; Whitehead, Terence R; Holland, Keith T; Lodge, J Peter A; Carding, Simon R

2008-10-01

The use of genetically modified bacteria to deliver biologically active molecules directly to the gut has become an increasingly attractive area of investigation. The challenge of regulation of production of the therapeutic molecule and colonization of the bowel led us to investigate Bacteroides ovatus for the production of these molecules, due to its ability to colonize the colon and xylan utilization properties. Here we have identified the putative xylanase promoter. The 5' region of the corresponding mRNA was determined by 5'RACE analysis and the transcription initiation site was identified 216 bp upstream of the ATG start codon. The putative xylanase promoter was regulated by xylan in a dose- and time-dependent manner, and repressed by glucose. This promoter was subsequently used to direct the controlled expression of a gene encoding the human intestinal trefoil factor (TFF-3) after integration as a single copy into the chromosome of B. ovatus. The resulting strain produced biologically active TFF-3 in the presence of xylan. These findings identify the B. ovatus xylanase operon promoter and show that it can be utilized to direct xylan-inducible expression of heterologous eukaryotic genes in B. ovatus.

Purification and characterization of an endoglucanase from Streptomyces lividans 66 and DNA sequence of the gene.

PubMed Central

Théberge, M; Lacaze, P; Shareck, F; Morosoli, R; Kluepfel, D

1992-01-01

The endoglucanase isolated from culture filtrates of Streptomyces lividans IAF74 was shown to have an Mr of 46,000 and a pI of 3.3. The specific enzyme activity of 539 IU/mg, determined by the reducing assay method on carboxymethyl cellulose, is among the highest reported in the literature. The cellulase showed typical endo-type activity when reacting on oligocellodextrins. Optimal enzyme activity was obtained at 50 degrees C and pH 5.5. The kinetic constants for this endoglucanase, determined with carboxymethyl cellulose as the substrate, were a Vmax of 24.9 IU/mg of enzyme and a Km of 4.2 mg/ml. Activity was found against neither methylumbelliferyl- nor p-nitrophenyl-cellobiopyranoside nor with xylan. The DNA sequence contains one possible reading frame validated by the N terminus of the mature purified protein. However, neither ATG nor GTG starting codons were identified near the ribosome-binding site. A putative TTG codon was found as a good candidate for the start codon. Comparison of the primary amino acid sequence of the endoglucanase of S. lividans revealed that the N terminus contains a bacterial cellulose-binding domain. The catalytic domain at the C terminus showed similarity to endoglucanases from a Bacillus sp. Thus, the endoglucanase CelA belongs to family A of cellulases as described before (N. R. Gilkes, B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren, Microbiol. Rev. 55:303-315, 1991. Images PMID:1575483

Problem-Solving Test: The Effect of Synonymous Codons on Gene Expression

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2009-01-01

Terms to be familiar with before you start to solve the test: the genetic code, codon, degenerate codons, protein synthesis, aminoacyl-tRNA, anticodon, antiparallel orientation, wobble, unambiguous codons, ribosomes, initiation, elongation and termination of translation, peptidyl transferase, translocation, degenerate oligonucleotides, green…

In silico modeling of the Moniliophthora perniciosa Atg8 protein.

PubMed

Pereira, A C F; Cardoso, T H S; Brendel, M; Pungartnik, C

2013-12-11

Autophagy is defined as an intracellular system of lysosomal degradation in eukaryotic cells, and the genes involved in this process are conserved from yeast to humans. Among these genes, ATG8 encodes a ubiquitin-like protein that is conjugated to a phosphatidylethanolamine (PE) membrane by the ubiquitination system. The Atg8p-PE complex is important in initiating the formation of the autophagosome and thus plays a critical role in autophagy. In silico modeling of Atg8p of Moniliophthora perniciosa revealed its three-dimensional structure and enabled comparison with its Saccharomyces cerevisiae homologue ScAtg8p. Some common and distinct features were observed between these two proteins, including the conservation of residues required to allow the interaction of α-helix1 with the ubiquitin core. However, the electrostatic potential surfaces of these helices differ, implying particular roles in selecting specific binding partners. The proposed structure was validated by the programs PROCHECK 3.4, ANOLEA, and QMEAN, which demonstrated 100% of amino acids located in favorable regions with low total energy. Our results showed that MpAtg8p contains the same functional domains (3 α-helices and 4 β-sheets) and is similar in structure as the ScAtg8p yeast. Both proteins have many conserved sequences in common, and therefore, their proposed three-dimensional models show similar configuration.

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents.

PubMed

Villahermosa, Desirée; Knapp, Karen; Fleck, Oliver

2017-12-01

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.

Nucleotide sequence and transcriptional start site of the Methylobacterium organophilum XX methanol dehydrogenase structural gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machlin, S.M.; Hanson, R.S.

The nucleotide sequence of a cloned 2.5-kilobase-pair SmaI fragment containing the methanol dehydrogenase (MDH) structural gene from Methylobacterium organophilum XX was determined. A single open reading frame with a coding capacity of 626 amino acids (molecular weight, 66,000) was identified on one stand, and N-terminal sequencing of purified MDH revealed that 27 of these residues constituted a putative signal peptide. Primer extension mapping of in vivo transcripts indicated that the start of mRNA synthesis was 160 to 170 base pairs upstream of the ATG codon. Northern (RNA) blot analysis further demonstrated that the transcript was 2.1 kilobase pairs in lengthmore » and therefore appeared to encode only MDH.« less

The mitochondrial genome of Cethosia biblis (Drury) (Lepidoptera: Nymphalidae).

PubMed

Xin, Tianrong; Li, Lei; Yao, Chengyi; Wang, Yayu; Zou, Zhiwen; Wang, Jing; Xia, Bin

2016-07-01

We present the complete mitogenome of Cethosia biblis (Drury) (Lepidoptera: Nymphalidae) in this article. The mitogenome was a circle molecular consisting of 15,286 nucleotides, 37 genes, and an A + T-rich region. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. The overall base composition of the genome is A (37.41%), T (42.80%), C (11.87%), and G (7.91%) with an A + T-rich hallmark as that of other invertebrate mitochondrial genomes. The start codon was mainly ATA in most of the mitochondrial protein-coding genes such as ND2, COI, ATP8, ND3, ND5, ND4, ND6, and ND1, but COII, ATP6, COIII, ND4L, and Cob genes employing ATG. The stop codon was TAA in all the protein-coding genes. The A + T region is located between 12S rRNA and tRNA(M)(et). The phylogenetic relationships of Lepidoptera species were constructed based on the nucleotides sequences of 13 PCGs of mitogenomes using the neighbor-joining method. The molecular-based phylogeny supported the traditional morphological classification on relationships within Lepidoptera species.

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed Central

Butler, J S; Springer, M; Grunberg-Manago, M

1987-01-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo. PMID:2954162

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed

Butler, J S; Springer, M; Grunberg-Manago, M

1987-06-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

AMPK and vacuole-associated Atg14p orchestrate μ-lipophagy for energy production and long-term survival under glucose starvation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Arnold Y.; Lau, Pick -Wei; Feliciano, Daniel

Dietary restriction increases the longevity of many organisms, but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy (µ-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger µ-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interactedmore » with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating µ-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate µ-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms.« less

AMPK and vacuole-associated Atg14p orchestrate μ-lipophagy for energy production and long-term survival under glucose starvation

DOE PAGES

Seo, Arnold Y.; Lau, Pick -Wei; Feliciano, Daniel; ...

2017-04-10

Dietary restriction increases the longevity of many organisms, but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy (µ-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger µ-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interactedmore » with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating µ-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate µ-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms.« less

Negative and Translation Termination-Dependent Positive Control of FLI-1 Protein Synthesis by Conserved Overlapping 5′ Upstream Open Reading Frames in Fli-1 mRNA

PubMed Central

Sarrazin, Sandrine; Starck, Joëlle; Gonnet, Colette; Doubeikovski, Alexandre; Melet, Fabrice; Morle, François

2000-01-01

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5′ upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG −41 and GUG −37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5′ uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5′ uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5′ uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons. PMID:10757781

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

PubMed Central

Moustakas, A; Sonstegard, T S; Hackett, P B

1993-01-01

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production. Images PMID:7685415

The peculiar Type Ia supernova iPTF14atg: Chandrasekhar-mass explosion or violent merger?

NASA Astrophysics Data System (ADS)

Kromer, M.; Fremling, C.; Pakmor, R.; Taubenberger, S.; Amanullah, R.; Cenko, S. B.; Fransson, C.; Goobar, A.; Leloudas, G.; Taddia, F.; Röpke, F. K.; Seitenzahl, I. R.; Sim, S. A.; Sollerman, J.

2016-07-01

iPTF14atg, a subluminous peculiar Type Ia supernova (SN Ia) similar to SN 2002es, is the first SN Ia for which a strong UV flash was observed in the early-time light curves. This has been interpreted as evidence for a single-degenerate (SD) progenitor system, where such a signal is expected from interactions between the SN ejecta and the non-degenerate companion star. Here, we compare synthetic observables of multidimensional state-of-the-art explosion models for different progenitor scenarios to the light curves and spectra of iPTF14atg. From our models, we have difficulties explaining the spectral evolution of iPTF14atg within the SD progenitor channel. In contrast, we find that a violent merger of two carbon-oxygen white dwarfs with 0.9 and 0.76 M⊙, respectively, provides an excellent match to the spectral evolution of iPTF14atg from 10 d before to several weeks after maximum light. Our merger model does not naturally explain the initial UV flash of iPTF14atg. We discuss several possibilities like interactions of the SN ejecta with the circumstellar medium and surface radioactivity from an He-ignited merger that may be able to account for the early UV emission in violent merger models.

Improved long-term survival after intra-operative single high-dose ATG-Fresenius induction in renal transplantation: a single centre experience.

PubMed

Kaden, Jürgen; May, Gottfried; Völp, Andreas; Wesslau, Claus

2009-01-01

In organ grafts donor-specific sensitization is initiated immediately after revascularization. Therefore, in 1990 we introduced the intra-operative single high-dose ATG-Fresenius (ATG-F) induction in addition to standard triple drug therapy (TDT) consisting of steroids, azathioprine and cyclosporin. A total of 778 first renal transplantations from deceased donors, performed between 1987 and 1998, were included in this evaluation. This retrospective analysis of clinic records and electronic databases presents data of all recipients of first kidney grafts who received two different ATG-F inductions (1(st) group: 9 mg/kg body weight as single high-dose intra-operatively, n=484; 2(nd) group: 3 mg/kg body weight on 7 or 8 consecutive days as multiple-dose starting also intra-operatively, n=78) and standard TDT alone (3(rd) group: TDT alone, n=216). The 10-year patient survival rates were 72.6+/-2.6% (TDT + ATG-F single high-dose), 79.5+/-5.1% (TDT + ATG-F multiple-dose) and 67.2+/-3.7%% (TDT alone; Kaplan-Meier estimates with standard errors; ATG-F vs TDT alone, p=0.001). The 10-year graft survival rates with censoring of patients that died with a functioning graft were 73.8+/-2.4%, 57.7+/-5.8% and 58.4+/-3.6% (Kaplan-Meier estimates with standard errors; 1(st) vs 2(nd )and 3(rd) group, respectively, p<0.001) and the 10-year graft survival rates with patient death counted as graft failure were 58.3+/-2.7%, 55.7+/-5.8% and 48.2+/-3.5% (Kaplan-Meier estimates with standard errors; ATG-F single high-dose vs TDT, p=0.023). In pre-sensitized recipients there were also significant differences in favour of ATG-F, more notably in the single high-dose ATG-F induction. A total of 69% of the patients in the two cohorts receiving ATG-F did not experience any transplant rejections compared to 56% in patients undergoing TDT alone (p=0.018). The incidence of infectious complications was comparable across all groups. According to evidence obtained from the routine documentation of 778 renal transplantations, ATG-F induction therapy administered as a part of immunosuppressive therapy significantly improves patient survival and reduces the risk of graft failure and transplant rejections.

Anti-thymocyte globulin induces neoangiogenesis and preserves cardiac function after experimental myocardial infarction.

PubMed

Lichtenauer, Michael; Mildner, Michael; Werba, Gregor; Beer, Lucian; Hoetzenecker, Konrad; Baumgartner, Andrea; Hasun, Matthias; Nickl, Stefanie; Mitterbauer, Andreas; Zimmermann, Matthias; Gyöngyösi, Mariann; Podesser, Bruno Karl; Klepetko, Walter; Ankersmit, Hendrik Jan

2012-01-01

Acute myocardial infarction (AMI) followed by ventricular remodeling is the major cause of congestive heart failure and death in western world countries. Of relevance are reports showing that infusion of apoptotic leucocytes or anti-lymphocyte serum after AMI reduces myocardial necrosis and preserves cardiac function. In order to corroborate this therapeutic mechanism, the utilization of an immunosuppressive agent with a comparable mechanism, such as anti-thymocyte globulin (ATG) was evaluated in this study. AMI was induced in rats by ligation of the left anterior descending artery. Initially after the onset of ischemia, rabbit ATG (10 mg/rat) was injected intravenously. In vitro and in vivo experiments showed that ATG induced a pronounced release of pro-angiogenic and chemotactic factors. Moreover, paracrine factors released from ATG co-incubated cell cultures conferred a down-regulation of p53 in cardiac myocytes. Rats that were injected with ATG evidenced higher numbers of CD68+ macrophages in the ischemic myocardium. Animals injected with ATG evidenced less myocardial necrosis, showed a significant reduction of infarct dimension and an improvement of post-AMI remodeling after six weeks (infarct dimension 24.9% vs. 11.4%, p<0.01). Moreover, a higher vessel density in the peri-infarct region indicated a better collateralization in rats that were injected with ATG. These data indicate that ATG, a therapeutic agent successfully applied in clinical transplant immunology, triggered cardioprotective effects after AMI that salvaged ischemic myocardium by down-regulation of p53. This might have raised the resistance against apoptotic cell death during ischemia. The combination of these mechanisms seems to be causative for improved cardiac function and less ventricular remodeling after experimental AMI.

Efficacy and Safety of ATG-Fresenius as an Induction Agent in Living-Donor Kidney Transplantation.

PubMed

Yilmaz, M; Sezer, T Ö; Günay, E; Solak, I; Çeltik, A; Hoşcoşkun, C; Töz, H

2017-04-01

Induction therapy is mostly recommended for deceased-donor transplantation, whereas it has some controversies in live-donor transplantation. In this study, we described the outcomes of live-donor renal transplant recipients who received ATG-Fresenius (ATG-F) induction. Live-donor transplantations in patients over 18 years old with ATG-F induction between 2009 and 2015 were included. All patients received quadruple immunosuppression, one of which was ATG-F induction. Biopsies after the artery anastomosis (zero hour) and protocol biopsies at the 6th month and at the 1st first year were obtained. Acute graft dysfunction was defined as a 20% to 25% increase in creatinine level from baseline. All acute rejection episodes were biopsy-confirmed. All episodes were initially treated with intravenous methyl prednisolone (MP) or ATG-F if resistant to MP. Four hundred twenty-two patients with live-donor transplantation were evaluated. The mean age was 40 ± 13 (18-73) years. The mean panel-reactive antibody levels were 42% ± 30% and 45% ± 30% for class I and II, respectively. The mean mismatch number for living unrelated donors (n = 112) was 4.6 ± 1.0. Acute rejection rate was 29.1% (123 patients) within the first year. The mean cumulative ATG-F doses for per patient and per kilogram were 344 ± 217 mg and 5.1 ± 2.7 mg, respectively. Patient survival rates were 98.3% and 96.7% for 12 months and 60 months, respectively. Death-censored graft survival rates were 97.6% and 92.1% for 12 months and 60 months, respectively. ATG-F induction provided excellent graft and patient survival rates without any significantly increased side effects. Increasing sensitized patient numbers, more unrelated donors, increasing re-transplantation numbers, and more desensitization protocols make ATG-F more favorable in an induction regimen. Copyright © 2017 Elsevier Inc. All rights reserved.

mir-30d Regulates multiple genes in the autophagy pathway and impairs autophagy process in human cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Xiaojun; Department of General Surgery, Gansu Provincial Hospital, Lanzhou, Gansu 710000; Zhong, Xiaomin

2013-02-15

Highlights: ► Gene set enrichment analysis indicated mir-30d might regulate the autophagy pathway. ► mir-30d represses the expression of BECN1, BNIP3L, ATG12, ATG5 and ATG2. ► BECN1, BNIP3L, ATG12, ATG5 and ATG2 are direct targets of mir-30d. ► mir-30d inhibits autophagosome formation and LC3B-I conversion to LC3B-II. ► mir-30d regulates the autophagy process. -- Abstract: In human epithelial cancers, the microRNA (miRNA) mir-30d is amplified with high frequency and serves as a critical oncomir by regulating metastasis, apoptosis, proliferation, and differentiation. Autophagy, a degradation pathway for long-lived protein and organelles, regulates the survival and death of many cell types. Increasingmore » evidence suggests that autophagy plays an important function in epithelial tumor initiation and progression. Using a combined bioinformatics approach, gene set enrichment analysis, and miRNA target prediction, we found that mir-30d might regulate multiple genes in the autophagy pathway including BECN1, BNIP3L, ATG12, ATG5, and ATG2. Our further functional experiments demonstrated that the expression of these core proteins in the autophagy pathway was directly suppressed by mir-30d in cancer cells. Finally, we showed that mir-30d regulated the autophagy process by inhibiting autophagosome formation and LC3B-I conversion to LC3B-II. Taken together, our results provide evidence that the oncomir mir-30d impairs the autophagy process by targeting multiple genes in the autophagy pathway. This result will contribute to understanding the molecular mechanism of mir-30d in tumorigenesis and developing novel cancer therapy strategy.« less

Hb Alesha [β67(E11)Val→Met (GTG>ATG); HBB: c.202G > A] Found in a Chinese Girl.

PubMed

Jiang, Hua; Yan, Jin-Mei; Zhou, Jian-Ying; Li, Dong-Zhi

2016-11-01

Mutations that cause destabilization of the hemoglobin (Hb) tetramer are a rare cause of hemolytic anemia. In contrast to the hemolytic anemia caused by enzyme deficiencies, a dominant mode of inheritance characterizes the unstable Hbs. Hb Alesha [β67(E11)Val→Met; HBB: c.202G>A] is caused by a G>A mutation at codon 67 of the β-globin gene, resulting in a valine to methionine substitution at helix E11. This replacement disrupts the apolar bonds between valine and the heme group, producing an unstable Hb and severe hemolysis. We report this rare hemoglobinopathy in a Chinese girl with severe hemolytic anemia, splenomegaly and frequent requirement for red blood cell (RBC) transfusions.

Suppression of autophagy impedes glioblastoma development and induces senescence.

PubMed

Gammoh, Noor; Fraser, Jane; Puente, Cindy; Syred, Heather M; Kang, Helen; Ozawa, Tatsuya; Lam, Du; Acosta, Juan Carlos; Finch, Andrew J; Holland, Eric; Jiang, Xuejun

2016-09-01

The function of macroautophagy/autophagy during tumor initiation or in established tumors can be highly distinct and context-dependent. To investigate the role of autophagy in gliomagenesis, we utilized a KRAS-driven glioblastoma mouse model in which autophagy is specifically disrupted via RNAi against Atg7, Atg13 or Ulk1. Inhibition of autophagy strongly reduced glioblastoma development, demonstrating its critical role in promoting tumor formation. Further supporting this finding is the observation that tumors originating from Atg7-shRNA injections escaped the knockdown effect and thereby still underwent functional autophagy. In vitro, autophagy inhibition suppressed the capacity of KRAS-expressing glial cells to form oncogenic colonies or to survive low serum conditions. Molecular analyses revealed that autophagy-inhibited glial cells were unable to maintain active growth signaling under growth-restrictive conditions and were prone to undergo senescence. Overall, these results demonstrate that autophagy is crucial for glioma initiation and growth, and is a promising therapeutic target for glioblastoma treatment.

Characterization and chromosomal mapping of the human TFG gene involved in thyroid carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mencinger, M.; Panagopoulos, I.; Andreasson, P.

1997-05-01

Homology searches in the Expressed Sequence Tag Database were performed using SPYGQ-rich regions as query sequences to find genes encoding protein regions similar to the N-terminal parts of the sarcoma-associated EWS and FUS proteins. Clone 22911 (T74973), encoding a SPYGQ-rich region in its 5{prime} end, and several other clones that overlapped 22911 were selected. The combined data made it possible to assemble a full-length cDNA sequence. This cDNA sequence is 1677 bp, containing an initiation codon ATG, an open reading frame of 400 amino acids, a poly(A) signal, and a poly(A) tail. We found 100% identity between the 5{prime} partmore » of the consensus sequence and the 598-bp-long sequence named TFG. The TFG sequence is fused to the 3{prime} end of NTRK1, generating the TRK-T3 fusion transcript found in papillary thyroid carcinoma. The cDNA therefore represents the full-length transcript of the TFG gene. TFG was localized to 3q11-q12 by fluorescence in situ hybridization. The 3{prime} and the 5{prime} ends of the TFG cDNA probe hybridized to a 2.2-kb band on Northern blot filters in all tissues examined. 28 refs., 5 figs., 1 tab.« less

Fifteen new earthworm mitogenomes shed new light on phylogeny within the Pheretima complex

PubMed Central

Zhang, Liangliang; Sechi, Pierfrancesco; Yuan, Minglong; Jiang, Jibao; Dong, Yan; Qiu, Jiangping

2016-01-01

The Pheretima complex within the Megascolecidae family is a major earthworm group. Recently, the systematic status of the Pheretima complex based on morphology was challenged by molecular studies. In this study, we carry out the first comparative mitogenomic study in oligochaetes. The mitogenomes of 15 earthworm species were sequenced and compared with other 9 available earthworm mitogenomes, with the main aim to explore their phylogenetic relationships and test different analytical approaches on phylogeny reconstruction. The general earthworm mitogenomic features revealed to be conservative: all genes encoded on the same strand, all the protein coding loci shared the same initiation codon (ATG), and tRNA genes showed conserved structures. The Drawida japonica mitogenome displayed the highest A + T content, reversed AT/GC-skews and the highest genetic diversity. Genetic distances among protein coding genes displayed their maximum and minimum interspecific values in the ATP8 and CO1 genes, respectively. The 22 tRNAs showed variable substitution patterns between the considered earthworm mitogenomes. The inclusion of rRNAs positively increased phylogenetic support. Furthermore, we tested different trimming tools for alignment improvement. Our analyses rejected reciprocal monophyly among Amynthas and Metaphire and indicated that the two genera should be systematically classified into one. PMID:26833286

Autophagy Sustains Mitochondrial Glutamine Metabolism and Growth of BRAFV600E–Driven Lung Tumors

PubMed Central

Strohecker, Anne M.; Guo, Jessie Yanxiang; Karsli-Uzunbas, Gizem; Price, Sandy M.; Chen, Guanghua Jim; Mathew, Robin; McMahon, Martin; White, Eileen

2013-01-01

Autophagic elimination of defective mitochondria suppresses oxidative stress and preserves mitochondrial function. Here, the essential autophagy gene Atg7 was deleted in a mouse model of BRAFV600E-induced lung cancer in the presence or absence of the tumor suppressor TRP53. Atg7 deletion initially induced oxidative stress and accelerated tumor cell proliferation in a manner indistinguishable from Nrf2 ablation. Compound deletion of Atg7 and Nrf2 had no additive effect suggesting that both genes modulate tumorigenesis by regulating oxidative stress, revealing a potential mechanism of autophagy-mediated tumor suppression. At later stages of tumorigenesis, Atg7 deficiency resulted in an accumulation of defective mitochondria, proliferative defects, reduced tumor burden, conversion of adenomas and adenocarcinomas to oncocytomas, and increased mouse lifespan. Autophagy-defective tumor-derived cell lines were impaired in their ability to respire, survive starvation and were glutamine-dependent, suggesting that autophagy-supplied substrates from protein degradation sustains BRAFV600E-tumor growth and metabolism. PMID:23965987

Complete mitochondrial genome of Bactrocera arecae (Insecta: Tephritidae) by next-generation sequencing and molecular phylogeny of Dacini tribe

PubMed Central

Yong, Hoi-Sen; Song, Sze-Looi; Lim, Phaik-Eem; Chan, Kok-Gan; Chow, Wan-Loo; Eamsobhana, Praphathip

2015-01-01

The whole mitochondrial genome of the pest fruit fly Bactrocera arecae was obtained from next-generation sequencing of genomic DNA. It had a total length of 15,900 bp, consisting of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding region (A + T-rich control region). The control region (952 bp) was flanked by rrnS and trnI genes. The start codons included 6 ATG, 3 ATT and 1 each of ATA, ATC, GTG and TCG. Eight TAA, two TAG, one incomplete TA and two incomplete T stop codons were represented in the protein-coding genes. The cloverleaf structure for trnS1 lacked the D-loop, and that of trnN and trnF lacked the TΨC-loop. Molecular phylogeny based on 13 protein-coding genes was concordant with 37 mitochondrial genes, with B. arecae having closest genetic affinity to B. tryoni. The subgenus Bactrocera of Dacini tribe and the Dacinae subfamily (Dacini and Ceratitidini tribes) were monophyletic. The whole mitogenome of B. arecae will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general. PMID:26472633

Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

PubMed

Yang, Wang-Yong; Wilson, Henry D; Velagapudi, Sai Pradeep; Disney, Matthew D

2015-04-29

One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.

Hb Koln [β98(FG5) [GTG → ATG, Val → Met]: the first report from India.

PubMed

Warang, Prashant; Nair, Sona; Nadkarni, Anita; Kedar, Prabhakar; Bhave, Abhay; Ghosh, Kanjaksha; Colah, Roshan

2014-06-01

The group of unstable hemoglobins are associated with congenital non-spherocytic hemolytic anemia due to instability of the hemoglobin molecule. They often lead to formation of the characteristic inclusion bodies or Heinz bodies. To identity the cause of mild anemia, reticulocytosis, and hepatosplenomegly in a case of non-spherocytic hemolytic anemia. A 34-year-old female patient originating from Maharashtra, western India presented with mild anemia and jaundice which had persisted since childhood. Investigations included a complete blood count, screening for red cell membrane protein defects, Hb analysis by high-performance liquid chromatography (HPLC) and cellulose acetate electrophoresis (pH 8.9), heat instability test and DNA sequencing. Hemoglobin analysis by HPLC showed an abnormal peak in the Hb C window (9.8%) with a retention time of 4.90 minutes. Cellulose acetate electrophoresis (pH 8.9) showed a slow moving band (6.15%) between Hb A2 and Hb S. The heat instability test was positive. DNA analysis of α globin genes showed absence of both deletional and non- deletional α thalassemia. DNA sequencing of the β globin gene revealed heterozygosity for a mutation at codon 98 [GTG → ATG, Val → Met], which gives rise to Hb-Koln. Hb Koln is the commonest unstable Hb variant reported from many populations in the world. However, this is the first report of this unstable Hb variant from India.

The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.

PubMed

Guenther, Ulf-Peter; Weinberg, David E; Zubradt, Meghan M; Tedeschi, Frank A; Stawicki, Brittany N; Zagore, Leah L; Brar, Gloria A; Licatalosi, Donny D; Bartel, David P; Weissman, Jonathan S; Jankowsky, Eckhard

2018-06-27

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation 1 . Mutations in DDX3 are linked to tumorigenesis 2-4 and intellectual disability 5 , and the enzyme is targeted by a range of viruses 6 . How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.

Autophagy initiation correlates with the autophagic flux in 3D models of mesothelioma and with patient outcome.

PubMed

Follo, Carlo; Barbone, Dario; Richards, William G; Bueno, Raphael; Broaddus, V Courtney

2016-07-02

Understanding the role of autophagy in cancer has been limited by the inability to measure this dynamic process in formalin-fixed tissue. We considered that 3-dimensional models including ex vivo tumor, such as we have developed for studying mesothelioma, would provide valuable insights. Using these models, in which we could use lysosomal inhibitors to measure the autophagic flux, we sought a marker of autophagy that would be valid in formalin-fixed tumor and be used to assess the role of autophagy in patient outcome. Autophagy was studied in mesothelioma cell lines, as 2-dimensional (2D) monolayers and 3-dimensional (3D) multicellular spheroids (MCS), and in tumor from 25 chemonaive patients, both as ex vivo 3D tumor fragment spheroids (TFS) and as formalin-fixed tissue. Autophagy was evaluated as autophagic flux by detection of the accumulation of LC3 after lysosomal inhibition and as autophagy initiation by detection of ATG13 puncta. We found that autophagic flux in 3D, but not in 2D, correlated with ATG13 positivity. In each TFS, ATG13 positivity was similar to that of the original tumor. When tested in tissue microarrays of 109 chemonaive patients, higher ATG13 positivity correlated with better prognosis and provided information independent of known prognostic factors. Our results show that ATG13 is a static marker of the autophagic flux in 3D models of mesothelioma and may also reflect autophagy levels in formalin-fixed tumor. If confirmed, this marker would represent a novel prognostic factor for mesothelioma, supporting the notion that autophagy plays an important role in this cancer.

Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores.

PubMed

Gómez-Sánchez, Rubén; Rose, Jaqueline; Guimarães, Rodrigo; Mari, Muriel; Papinski, Daniel; Rieter, Ester; Geerts, Willie J; Hardenberg, Ralph; Kraft, Claudine; Ungermann, Christian; Reggiori, Fulvio

2018-05-30

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion. © 2018 Gómez-Sánchez et al.

Prognostic value of autophagy related proteins ULK1, Beclin 1, ATG3, ATG5, ATG7, ATG9, ATG10, ATG12, LC3B and p62/SQSTM1 in gastric cancer

PubMed Central

Cao, Qing-Hua; Liu, Fang; Yang, Zu-Li; Fu, Xin-Hui; Yang, Zi-Huan; Liu, Quentin; Wang, Lei; Wan, Xiang-Bo; Fan, Xin-Juan

2016-01-01

Autophagy-related (ATG) genes contributed to tumorigenesis and cancer progression. This study aims to investigate the expression of ATG proteins and their clinicopathological significance in gastric cancer. Nine well-known ATG proteins, (ULK1, Beclin 1, ATG3, ATG5, ATG7, ATG9, ATG10, ATG12 and LC3B) and p62/SQSTM1, which represented key regulators that participated in whole autophagosomes stepwise processes, were detected in a large cohort of 352 primary gastric cancer patients. Among these 352 patients, 117 cases were randomly assigned to the training set to detect the clinicopathological value of ATG proteins, and another 235 patients were used as the testing set for further validation. Except for Beclin 1, ATG9 and ATG10, another six ATG proteins and p62/SQSTM1 were closely correlated with histological types for gastric cancer. Moreover, low expression of ULK1, Beclin 1 and ATG10 were associated with lymph node metastasis. In addition, down-regulation of ULK1, Beclin 1, ATG7 and ATG10, up-regulation of ATG12 correlated with advanced TNM stage. Importantly, multivariate cox analysis identified ULK1, Beclin 1, ATG3 and ATG10 as favorable independent prognostic factors for overall survival. Combination analysis of ULK1, Beclin 1, ATG3, ATG10 revealed the improved prognostic accuracy for gastric cancer. Our study showed that ATG proteins might serve as novel prognostic biomarkers in gastric cancer, and supply a new valuable insight into cancer treatment targeting autophagy for patients. PMID:27725863

Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4.

PubMed

Seo, Eunyoung; Woo, Jongchan; Park, Eunsook; Bertolani, Steven J; Siegel, Justin B; Choi, Doil; Dinesh-Kumar, Savithramma P

2016-11-01

Autophagy is important for degradation and recycling of intracellular components. In a diversity of genera and species, orthologs and paralogs of the yeast Atg4 and Atg8 proteins are crucial in the biogenesis of double-membrane autophagosomes that carry the cellular cargoes to vacuoles and lysosomes. Although many plant genome sequences are available, the ATG4 and ATG8 sequence analysis is limited to some model plants. We identified 28 ATG4 and 116 ATG8 genes from the available 18 different plant genome sequences. Gene structures and protein domain sequences of ATG4 and ATG8 are conserved in plant lineages. Phylogenetic analyses classified ATG8s into 3 subgroups suggesting divergence from the common ancestor. The ATG8 expansion in plants might be attributed to whole genome duplication, segmental and dispersed duplication, and purifying selection. Our results revealed that the yeast Atg4 processes Arabidopsis ATG8 but not human LC3A (HsLC3A). In contrast, HsATG4B can process yeast and plant ATG8s in vitro but yeast and plant ATG4s cannot process HsLC3A. Interestingly, in Nicotiana benthamiana plants the yeast Atg8 is processed compared to HsLC3A. However, HsLC3A is processed when coexpressed with HsATG4B in plants. Molecular modeling indicates that lack of processing of HsLC3A by plant and yeast ATG4 is not due to lack of interaction with HsLC3A. Our in-depth analyses of ATG4 and ATG8 in the plant lineage combined with results of cross-kingdom ATG8 processing by ATG4 further support the evolutionarily conserved maturation of ATG8. Broad ATG8 processing by HsATG4B and lack of processing of HsLC3A by yeast and plant ATG4s suggest that the cross-kingdom ATG8 processing is determined by ATG8 sequence rather than ATG4.

PSMD10/gankyrin induces autophagy to promote tumor progression through cytoplasmic interaction with ATG7 and nuclear transactivation of ATG7 expression.

PubMed

Luo, Tao; Fu, Jing; Xu, An; Su, Bo; Ren, Yibing; Li, Ning; Zhu, Junjie; Zhao, Xiaofang; Dai, Rongyang; Cao, Jie; Wang, Bibo; Qin, Wenhao; Jiang, Jinhua; Li, Juan; Wu, Mengchao; Feng, Gensheng; Chen, Yao; Wang, Hongyang

2016-08-02

Although autophagy is most critical for survival of cancer cells, especially in fast-growing tumors, the mechanism remains to be fully characterized. Herein we report that PSMD10/gankyrin promotes autophagy in hepatocellular carcinoma (HCC) in response to starvation or stress through 2 complementary routes. PSMD10 was physically associated with ATG7 in the cytoplasm, and this association was enhanced by initial nutrient deprivation. Subsequently, PSMD10 translocated into the nucleus and bound cooperatively with nuclear HSF1 (heat shock transcription factor 1) onto the ATG7 promoter, upregulated ATG7 expression in the advanced stage of starvation. Intriguingly, the type of PSMD10-mediated autophagy was independent of the proteasome system, although PSMD10 has been believed to be an indispensable chaperone for assembly of the 26S proteasome. A significant correlation between PSMD10 expression and ATG7 levels was detected in human HCC biopsies, and the combination of these 2 parameters is a powerful predictor of poor prognosis. The median survival of sorafenib-treated HCC patients with high expression of PSMD10 was much shorter than those with low expression of PSMD10. Furthermore, PSMD10 augmented autophagic flux to resist sorafenib or conventional chemotherapy, and inhibition of autophagy suppressed PSMD10-mediated resistance. We conclude that these results present a novel mechanism involving modulation of ATG7 by PSMD10 in sustaining autophagy, promoting HCC cell survival against starvation or chemotherapy. Targeting of PSMD10 might therefore be an attractive strategy in HCC treatment by suppressing autophagy and inducing HCC cell sensitivity to drugs.

Cloning, characterization, and expression of Cytochrome b ( Cytb)—a key mitochondrial gene from Prorocentrum donghaiense

NASA Astrophysics Data System (ADS)

Zhao, Liyuan; Mi, Tiezhu; Zhen, Yu; Yu, Zhigang

2012-05-01

Mitochondrial cytochrome b (Cytb), one of the few proteins encoded by the mitochondrial DNA, plays an important role in transferring electrons. As a mitochondrial gene, it has been widely used for phylogenetic analysis. Previously, a 949-bp fragment of the coding gene and mRNA editing were characterized from Prorocentrum donghaiense, which might prove useful for resolving P. donghaiense from closely related species. However, the full-length coding region has not been characterized. In this study, we used rapid amplification of cDNA ends (RACE) to obtain full-length, 1 124 bp cDNA. Cytb transcript contained a standard initiation codon ATG, but did not have a recognizable stop codon. Homology comparison showed that the P. donghaiense Cytb had a high sequence identity to Cytb sequences from other dinoflagellate species. Phylogenetic analysis placed Cytb from P. donghaiense in the clade of dinoflagellates and it clustered together strongly with that from P. minimum. Based on the full-length sequence, we inferred 32 editing events at different positions, accounting for 2.93% of the Cytb gene. 34.4% (11) of the changes were A to G, 25% (8) were T to C, and 25% (8) were C to U, with smaller proportions of G to C and G to A edits (9.4% (3) and 6.2% (2), respectively). The expression level of the Cytb transcript was quantified by real-time PCR with a TaqMan probe at different times during the whole growth phase. The average Cytb transcript was present at 39.27±7.46 copies of cDNA per cell during the whole growth cycle, and the expression of Cytb was relatively stable over the different phases. These results deepen our understanding of the structure and characteristics of Cytb in P. donghaiense, and confirmed that Cytb in P. donghaiense is a candidate reference gene for studying the expression of other genes.

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shipley, J.M.; Klinkenberg, M.; Wu, B.M.

1993-03-01

PCR of cDNA produced from patient fibroblasts allowed the authors to determine the paternal mutation in the first patient reported with [beta]-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G[r arrow]T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reducedmore » stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, the authors found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed them to amplify and characterize genomic sequences for the true [beta]-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C[r arrow]T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression. 39 refs., 5 figs., 1 tab.« less

Differential processing of Arabidopsis ubiquitin-like Atg8 autophagy proteins by Atg4 cysteine proteases

PubMed Central

Woo, Jongchan; Park, Eunsook; Dinesh-Kumar, S. P.

2014-01-01

Autophagy is a highly conserved biological process during which double membrane bound autophagosomes carry intracellular cargo material to the vacuole or lysosome for degradation and/or recycling. Autophagosome biogenesis requires Autophagy 4 (Atg4) cysteine protease-mediated processing of ubiquitin-like Atg8 proteins. Unlike single Atg4 and Atg8 genes in yeast, the Arabidopsis genome contains two Atg4 (AtAtg4a and AtAtg4b) and nine Atg8 (AtAtg8a–AtAtg8i) genes. However, we know very little about specificity of different AtAtg4s for processing of different AtAtg8s. Here, we describe a unique bioluminescence resonance energy transfer-based AtAtg8 synthetic substrate to assess AtAtg4 activity in vitro and in vivo. In addition, we developed a unique native gel assay of superhRLUC catalytic activity assay to monitor cleavage of AtAtg8s in vitro. Our results indicate that AtAtg4a is the predominant protease and that it processes AtAtg8a, AtAtg8c, AtAtg8d, and AtAtg8i better than AtAtg4b in vitro. In addition, kinetic analyses indicate that although both AtAtg4s have similar substrate affinity, AtAtg4a is more active than AtAtg4b in vitro. Activity of AtAtg4s is reversibly inhibited in vitro by reactive oxygen species such as H2O2. Our in vivo bioluminescence resonance energy transfer analyses in Arabidopsis transgenic plants indicate that the AtAtg8 synthetic substrate is efficiently processed and this is AtAtg4 dependent. These results indicate that the synthetic AtAtg8 substrate is used efficiently in the biogenesis of autophagosomes in vivo. Transgenic Arabidopsis plants expressing the AtAtg8 synthetic substrate will be a valuable tool to dissect autophagy processes and the role of autophagy during different biological processes in plants. PMID:24379391

Obatoclax induces Beclin 1- and ATG5-dependent apoptosis and autophagy in adenoid cystic carcinoma cells.

PubMed

Liang, L-Z; Ma, B; Liang, Y-J; Liu, H-C; Zhang, T-H; Zheng, G-S; Su, Y-X; Liao, G-Q

2015-05-01

Adenoid cystic carcinoma (ACC) is one of the most common salivary gland cancers. The prognosis of adenoid cystic carcinoma is poor for its high frequency of distant metastases and insensitivity to chemotherapy or molecular therapies. This study investigated the effect of Obatoclax on adenoid cystic carcinoma cells and its cytotoxic mechanism. Western blot, transmission electron microscopy, and pEGFP-LC3 plasmids transfection were carried out to detect autophagy in ACC cells treated with Obatoclax. 3-MA and RNA interference against Beclin 1 and ATG5 were used to inhibit autophagy. Then we used Western blot and Hochest 33342 staining for apoptosis assessment. Finally, cell viability was assessed by MTT assay. We found that Obatoclax induced cytoprotective autophagy which depended on ATG5 and partly on Beclin 1 in adenoid cystic carcinoma cells. Furthermore, pharmacologically inhibiting Obatoclax-induced autophagy promoted apoptosis. Downregulation of Beclin 1 or ATG5 attenuated the cytotoxicity of Obatoclax by suppressing both autophagy and apoptosis. Finally, when apoptosis was pharmacologically inhibited, autophagic cell death was initiated in adenoid cystic carcinoma cells treated with Obatoclax. In summary, Beclin 1 and ATG5 play important roles in regulating both Obatoclax-induced autophagy and apoptosis in adenoid cystic carcinoma. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

PubMed Central

Hahn, Julia; Migur, Anzhela; von Boeselager, Raphael Freiherr; Kubatova, Nina; Kubareva, Elena; Schwalbe, Harald

2017-01-01

ABSTRACT Up to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level. PMID:27834614

Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.).

PubMed

Wang, He; Fan, Mingxia; Wang, Guohong; Zhang, Chunyu; Shi, Lei; Wei, Zhengyi; Ma, Wenjuan; Chang, Jing; Huang, Senxin; Lin, Feng

2017-06-01

ZmSTK2_USP, located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2100 bp upstream from the start codon ATG has shown that it contains cis-element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In proZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetative tissues. These results show that proZmSTK2_USP is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.

The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

PubMed Central

Fletcher, Simon P; Ali, Iraj K; Kaminski, Ann; Digard, Paul; Jackson, Richard J

2002-01-01

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs. PMID:12515388

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

PubMed Central

Park, Eunsook; Woo, Jongchan; Dinesh-Kumar, SP

2014-01-01

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes. PMID:24658121

Cloning of Russian sturgeon (Acipenser gueldenstaedtii) growth hormone and insulin-like growth factor I and their expression in male and female fish during the first period of growth.

PubMed

Yom Din, S; Hurvitz, A; Goldberg, D; Jackson, K; Levavi-Sivan, B; Degani, G

2008-03-01

In this study, the GH and IGF-I of the Russian sturgeon (rs), Acipenser gueldenstaedtii, were cloned and sequenced, and their mRNA gene expression determined. In addition, to improve our understanding of the GH function, the expression of this hormone was assessed in young males and females. Moreover, IGF-I expression was quantified in young males and compared to that in older ones. The nucleotide sequence of the rsGH cDNA was 980 bp long and had an open reading frame of 642 bp, beginning with the first ATG codon at position 39 and ending with the stop codon at position 683. A putative polyadenylation signal, AATAAA, was recognized 42 bp upstream of the poly (A) tail. The position of the signal- peptide cleavage site was predicted to be at position 111, yielding a signal peptide of 24 amino-acids (aa) and a mature peptide of 190 aa. When the rsGH aa sequence was compared with other species, the highest degree of identity was found to be with mammalians (66-70% identity), followed by anguilliformes and amphibia (61%) and other fish (39-47%). The level of rsGH mRNA was discovered to be similar in pituitaries of females and males of 5 age groups (1, 2, 3, 4, and 5- yr-old). In females and males, the levels did not change dramatically during the first 5 yr of growth. The partial nucleotide sequence of the rsIGF-I was 445 bp long and had an open reading frame of 396 bp, beginning with the ATG codon at position 50. The position of the signal-peptide cleavage site was predicted to be at position 187, yielding a signal peptide of 44 aa. The highest level of IGF-I mRNA expression was recorded in the kidney of adult sturgeons. The IGF-I mRNA expression levels in the intestine, pituitary gland, and liver were not significantly different. Low levels of expression were found in the brain, heart, and muscle. In most tissues, there was no significant difference between mRNA levels of one and 5-yr-old fish. In conclusion, based on the GH-sequence analysis, A. gueldenstaedtii is genetically distant from other teleosts. The expression of the GH mRNA was similar in males and females, and its level remained constant during the first 5 yr of growth. While the IGF-I mRNA expression differed amongst various tissues, the level in each tissue was similar in 1 and 5-yr-old fish.

Non-AUG translation: a new start for protein synthesis in eukaryotes

PubMed Central

Kearse, Michael G.; Wilusz, Jeremy E.

2017-01-01

Although it was long thought that eukaryotic translation almost always initiates at an AUG start codon, recent advancements in ribosome footprint mapping have revealed that non-AUG start codons are used at an astonishing frequency. These non-AUG initiation events are not simply errors but instead are used to generate or regulate proteins with key cellular functions; for example, during development or stress. Misregulation of non-AUG initiation events contributes to multiple human diseases, including cancer and neurodegeneration, and modulation of non-AUG usage may represent a novel therapeutic strategy. It is thus becoming increasingly clear that start codon selection is regulated by many trans-acting initiation factors as well as sequence/structural elements within messenger RNAs and that non-AUG translation has a profound impact on cellular states. PMID:28982758

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila.

PubMed

Nagy, Péter; Kárpáti, Manuéla; Varga, Agnes; Pircs, Karolina; Venkei, Zsolt; Takáts, Szabolcs; Varga, Kata; Erdi, Balázs; Hegedűs, Krisztina; Juhász, Gábor

2014-03-01

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.

Identification of the initiation site of poliovirus polyprotein synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dorner, A.J.; Dorner, L.F.; Larsen, G.R.

1982-06-01

The complete nucleotide sequence of poliovirus RNA has a long open reading frame capable of encoding the precursor polyprotein NCVPOO. The first AUG codon in this reading frame is located 743 nucleotides from the 5' end of the RNA and is preceded by eight AUG codons in all three reading frames. Because all proteins that map at the amino terminus of the polyprotein (P1-1a, VPO, and VP4) are blocked at their amino termini and previous studies of ribosome binding have been inconclusive, direct identification of the initiation site of protein synthesis was difficult. We separated and identified all of themore » tryptic peptides of capsid protein VP4 and correlated these peptides with the amino acid sequence predicted to follow the AUG codon at nucleotide 743. Our data indicate that VP4 begins with a blocked glycine that is encoded immediately after the AUG codon at nucleotide 743. An S1 nuclease analysis of poliovirus mRNA failed to reveal a splice in the 5' region. We concluded that synthesis of poliovirus polyprotein is initiated at nucleotide 743, the first AUG codon in the long open reading frame.« less

Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

PubMed

Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Noh, Mi Young; Kim, Dong Hyun; Kim, Iksoo; Han, Yeon Soo; Lee, Yong Seok; Lee, Bok Luel; Kim, Nam Jung

2015-01-01

Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model. © 2014 Wiley Periodicals, Inc.

uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs

PubMed Central

Ivanov, Ivaylo P.; Loughran, Gary; Atkins, John F.

2008-01-01

In a minority of eukaryotic mRNAs, a small functional upstream ORF (uORF), often performing a regulatory role, precedes the translation start site for the main product(s). Here, conserved uORFs in numerous ornithine decarboxylase homologs are identified from yeast to mammals. Most have noncanonical evolutionarily conserved start codons, the main one being AUU, which has not been known as an initiator for eukaryotic chromosomal genes. The AUG-less uORF present in mouse antizyme inhibitor, one of the ornithine decarboxylase homologs in mammals, mediates polyamine-induced repression of the downstream main ORF. This repression is part of an autoregulatory circuit, and one of its sensors is the AUU codon, which suggests that translation initiation codon identity is likely used for regulation in eukaryotes. PMID:18626014

Foot-and-mouth disease virus infection suppresses autophagy and NF-кB antiviral responses via degradation of ATG5-ATG12 by 3Cpro

PubMed Central

Fan, Xuxu; Han, Shichong; Yan, Dan; Gao, Yuan; Wei, Yanquan; Liu, Xiangtao; Liao, Ying; Guo, Huichen; Sun, Shiqi

2017-01-01

Autophagy-related protein ATG5-ATG12 is an essential complex for the autophagophore elongation in autophagy, which has been reported to be involved in foot-and-mouth disease virus (FMDV) replication. Previous reports show that ATG5-ATG12 positively or negatively regulates type I interferon (IFN-α/β) pathway during virus infection. In this study, we found that FMDV infection rapidly induced LC3 lipidation and GFP-LC3 subcellular redistribution at the early infection stage in PK-15 cells. Along with infection time course to 2–5 h.p.i., the levels of LC3II and ATG5-ATG12 were gradually reduced. Further study showed that ATG5-ATG12 was degraded by viral protein 3Cpro, demonstrating that FMDV suppresses autophagy along with viral protein production. Depletion of ATG5-ATG12 by siRNA knock down significantly increased the FMDV yields, whereas overexpression of ATG5-ATG12 had the opposite effects, suggesting that degradation of ATG5-ATG12 benefits virus growth. Further experiment showed that overexpression of ATG5-ATG12 positively regulated NF-кB pathway during FMDV infection, marked with promotion of IKKα/β phosphorylation and IκBα degradation, inhibition of p65 degradation, and facilitation of p65 nuclear translocation. Meanwhile, ATG5-ATG12 also promoted the phosphorylation of TBK1 and activation of IRF3 via preventing TRAF3 degradation. The positive regulation of NF-кB and IRF3 pathway by ATG5-ATG12 resulted in enhanced expression of IFN-β, chemokines/cytokines, and IFN stimulated genes, including anti-viral protein PKR. Altogether, above findings suggest that ATG5-ATG12 positively regulate anti-viral NF-κB and IRF3 signaling during FMDV infection, thereby limiting FMDV proliferation. FMDV has evolved mechanisms to counteract the antiviral function of ATG5-ATG12, via degradation of them by viral protein 3Cpro. PMID:28102839

Foot-and-mouth disease virus infection suppresses autophagy and NF-кB antiviral responses via degradation of ATG5-ATG12 by 3Cpro.

PubMed

Fan, Xuxu; Han, Shichong; Yan, Dan; Gao, Yuan; Wei, Yanquan; Liu, Xiangtao; Liao, Ying; Guo, Huichen; Sun, Shiqi

2017-01-19

Autophagy-related protein ATG5-ATG12 is an essential complex for the autophagophore elongation in autophagy, which has been reported to be involved in foot-and-mouth disease virus (FMDV) replication. Previous reports show that ATG5-ATG12 positively or negatively regulates type I interferon (IFN-α/β) pathway during virus infection. In this study, we found that FMDV infection rapidly induced LC3 lipidation and GFP-LC3 subcellular redistribution at the early infection stage in PK-15 cells. Along with infection time course to 2-5 h.p.i., the levels of LC3II and ATG5-ATG12 were gradually reduced. Further study showed that ATG5-ATG12 was degraded by viral protein 3C pro , demonstrating that FMDV suppresses autophagy along with viral protein production. Depletion of ATG5-ATG12 by siRNA knock down significantly increased the FMDV yields, whereas overexpression of ATG5-ATG12 had the opposite effects, suggesting that degradation of ATG5-ATG12 benefits virus growth. Further experiment showed that overexpression of ATG5-ATG12 positively regulated NF-кB pathway during FMDV infection, marked with promotion of IKKα/β phosphorylation and IκBα degradation, inhibition of p65 degradation, and facilitation of p65 nuclear translocation. Meanwhile, ATG5-ATG12 also promoted the phosphorylation of TBK1 and activation of IRF3 via preventing TRAF3 degradation. The positive regulation of NF-кB and IRF3 pathway by ATG5-ATG12 resulted in enhanced expression of IFN-β, chemokines/cytokines, and IFN stimulated genes, including anti-viral protein PKR. Altogether, above findings suggest that ATG5-ATG12 positively regulate anti-viral NF-κB and IRF3 signaling during FMDV infection, thereby limiting FMDV proliferation. FMDV has evolved mechanisms to counteract the antiviral function of ATG5-ATG12, via degradation of them by viral protein 3C pro .

Insights into factorless translational initiation by the tRNA-like pseudoknot domain of a viral IRES.

PubMed

Au, Hilda H T; Jan, Eric

2012-01-01

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA(i)-independent IRES translation.

Insights into Factorless Translational Initiation by the tRNA-Like Pseudoknot Domain of a Viral IRES

PubMed Central

Au, Hilda H. T.; Jan, Eric

2012-01-01

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNAi-independent IRES translation. PMID:23236506

Inhibition of Atg6 and Pi3K59F autophagy genes in neurons decreases lifespan and locomotor ability in Drosophila melanogaster.

PubMed

M'Angale, P G; Staveley, B E

2016-10-24

Autophagy is a cellular mechanism implicated in the pathology of Parkinson's disease. The proteins Atg6 (Beclin 1) and Pi3K59F are involved in autophagosome formation, a key step in the initiation of autophagy. We first used the GMR-Gal4 driver to determine the effect of reducing the expression of the genes encoding these proteins on the developing Drosophila melanogaster eye. Subsequently, we inhibited their expression in D. melanogaster neurons under the direction of a Dopa decarboxylase (Ddc) transgene, and examined the effects on longevity and motor function. Decreased longevity coupled with an age-dependent loss of climbing ability was observed. In addition, we investigated the roles of these genes in the well-studied α-synuclein-induced Drosophila model of Parkinson's disease. In this context, lowered expression of Atg6 or Pi3K59F in Ddc-Gal4-expressing neurons results in decreased longevity and associated age-dependent loss of locomotor ability. Inhibition of Atg6 or Pi3K59F together with overexpression of the sole pro-survival Bcl-2 Drosophila homolog Buffy in Ddc-Gal4-expressing neurons resulted in further decrease in the survival and climbing ability of Atg6-RNAi flies, whereas these measures were ameliorated in Pi3K59F-RNAi flies.

Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress

PubMed Central

Antonucci, Laura; Fagman, Johan B.; Kim, Ju Youn; Todoric, Jelena; Gukovsky, Ilya; Mackey, Mason; Ellisman, Mark H.; Karin, Michael

2015-01-01

Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7Δpan mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7Δpan mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7Δpan mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures. PMID:26512112

The Steroid Hormone 20-Hydroxyecdysone Regulates the Conjugation of Autophagy-Related Proteins 12 and 5 in a Concentration and Time-Dependent Manner to Promote Insect Midgut Programmed Cell Death

PubMed Central

Li, Yong-Bo; Yang, Ting; Wang, Jin-Xing; Zhao, Xiao-Fan

2018-01-01

Autophagy requires the conjugation of autophagy-related protein 12 (ATG12) to autophagy-related protein 5 (ATG5) through covalent attachment. However, the signals regulating ATG12–ATG5 conjugation are unclear. The larval midgut of lepidopteran insects performs autophagy and apoptosis sequentially during the transition of larvae to pupae under regulation by the steroid hormone 20-hydroxyecdysone (20E), thus representing a model to study steroid hormone regulation of ATG12–ATG5 conjugation. In the present study, using the lepidopteran insect Helicoverpa armigera as a model, we report that 20E regulates the conjugation of ATG12–ATG5 in a concentration and time-dependent manner. The ATG12–ATG5 conjugate was abundant in the epidermis, midgut, and fat body during metamorphosis from the larvae to the pupae; however, the ATG12–ATG5 conjugate level decreased at the time of pupation. At low concentrations (2–5 µM) over a short time course (1–48 h), 20E promoted the conjugation of ATG12–ATG5; however, at 10 µM and 72 h, 20E repressed the conjugation of ATG12–ATG5. ATG12 was localized in the larval midgut during metamorphosis. Knockdown of ATG12 in larvae caused death with delayed pupation, postponed the process of midgut programmed cell death (PCD), and repressed ATG8 (also called LC3-I) transformation to LC3-II and the cleavage of caspase-3; therefore, knockdown of ATG12 in larvae blocked both autophagy and apoptosis. Knockdown of ATG12 in H. armigera epidermis cell line cells also repressed 20E-induced autophagosome formation and caspase-3 activation. The results suggested that 20E plays key role in the regulation of ATG12–ATG5 conjugation in a concentration and time-dependent manner for autophagy or apoptosis, and that ATG12 is necessary by both autophagy and apoptosis during insect midgut PCD. PMID:29467720

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guss, Adam M.; Rother, Michael; Zhang, Jun Kai

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strainsmore » that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

DOE PAGES

Guss, Adam M.; Rother, Michael; Zhang, Jun Kai; ...

2008-01-01

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri P mcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strainsmore » that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR -regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.« less

Induction Therapy With Antithymocyte Globulin in Patients Undergoing Cardiac Transplantation Is Associated With Decreased Coronary Plaque Progression as Assessed by Intravascular Ultrasound.

PubMed

Azarbal, Babak; Cheng, Richard; Vanichsarn, Christopher; Patel, Jignesh K; Czer, Lawrence S; Chang, David H; Kittleson, Michelle M; Kobashigawa, Jon A

2016-01-01

Antithymocyte globulin (ATG) is used as induction therapy after cardiac transplant for enhancing immunosuppression and delaying the initiation of nephrotoxic drugs. It is unknown if ATG induction is associated with decreased coronary plaque progression by intravascular ultrasound (IVUS). Patients transplanted between March 2010 and December 2012 with baseline and 1-year IVUS were included. All patients transplanted were included in a secondary analysis. Change in plaque progression was measured in a blinded fashion on matched coronary segments and contrasted between patients induced with ATG and those who were not. One hundred and three patients were included in IVUS arms. Mean age at transplant was 55.8 ± 12.6 years, and 33.0% were female. Patients induced with ATG were more sensitized (54.3% versus 14.3%). Plaque progression was attenuated in patients who received ATG by changes in maximal intimal area (1.0 ± 1.2 versus 2.3 ± 2.6 mm(2); P = 0.001), maximal percent stenosis (6.3 ± 7.9 versus 12.8 ± 12.3%; = 0.003), maximal intimal thickness (0.2 ± 0.2 versus 0.3 ± 0.3 mm; P = 0.035), and plaque volume (0.5 ± 0.7 versus 1.0 ± 1.3 mm(3)/mm; P = 0.016). Rapid plaque progression by maximal percent stenosis (≥ 20%) occurred less frequently in the ATG arm (4.3% versus 26.3; P = 0.003). Survival (P = 0.242) and any treated rejection (P = 0.166) were not statistically different between groups. Patients receiving ATG had a higher rate of first-year infection (P = 0.003), perhaps related to increased intravenous antibiotic use immediately postoperatively, and a trend toward more biopsy-proven rejection (P = 0.073). Induction therapy with ATG is associated with reduced first-year coronary plaque progression as assessed by IVUS, despite an increased prevalence of sensitized patients with a trend toward more rejection. © 2016 American Heart Association, Inc.

Expression of Malus xiaojinensis IRT1 (MxIRT1) protein in transgenic yeast cells leads to degradation through autophagy in the presence of excessive iron.

PubMed

Li, Shuang; Zhang, Xi; Zhang, Xiu-Yue; Xiao, Wei; Berry, James O; Li, Peng; Jin, Si; Tan, Song; Zhang, Peng; Zhao, Wei-Zhong; Yin, Li-Ping

2015-07-01

Iron is essential for plants, but highly toxic when present in excess. Consequently, iron uptake by root transporters must be finely tuned to avoid excess uptake from soil under iron excess. The iron-regulated transporter of Malus xiaojinensis (MxIRT1), induced in roots under iron deficiency, is a highly effective iron(II) transporter. Here, we investigated how the presence of excessive iron leads to MxIRT1 degradation in yeast expressing this plant iron transporter protein. To determine the relationship between iron abundance and MxIRT1 degradation, relative levels of autophagy-related gene-8 (ATG8) mRNA and the active ATG8-phosphatidylethanolamine-conjugated (PE) protein were measured in wild-type yeast and the autophagic mutant strains atg1∆, atg5∆, atg7∆, ypt7∆ and tor1∆ under normal and excessive iron conditions. The data showed that the exposure of MxIRT1-eGFP-transformed wild-type and tor1∆ strains to excessive iron led to significantly increased levels of ATG8 transcript and ATG8-PE protein, which resulted in enhanced MxIRT1 degradation. Co-localization of mCherry-ATG8 and MxIRT1-eGFP provided evidence that these proteins interact during autophagy in yeast. While inhibition of autophagic initiation, autophagosome formation and vacuole fusion all decreased MxIRT1 degradation. PMSF inhibition of autophagy prevented degradation, leading to the accumulation of MxIRT1-containing vesicles in the vacuoles. MxIRT1-vesicles were sorted into autophagosomes for iron-induced degradation in yeast, whereas the endogenous iron(II) transporter Fet4 was degraded in an autophagy-independent manner. Moreover, immunoprecipitation showed that multimono-ubiquitins provided MxIRT1 with the ubiquitination signal. Together, three factors, iron excess, autophagy and mono-ubiquitination, affect the functional activity and stability of exogenous MxIRT1 in yeast, thereby preventing iron uptake via this root transporter. Copyright © 2015 John Wiley & Sons, Ltd.

Evidence of post-transcriptional readthrough regulation in FGF5 gene of alpaca.

PubMed

Pallotti, Stefano; Pediconi, Dario; Subramanian, Dharaneedharan; Molina, María Gabriela; Antonini, Marco; Morelli, Maria Beatrice; Renieri, Carlo; La Terza, Antonietta

2018-03-20

Two different phenotypes are described in alpaca, identified as suri and huacaya, which differ in the type of fleece. The huacaya fleece is characterized by compact, soft and highly crimped fibers, while the suri fleece is longer, straight, less-crimped and lustrous. In our study, the Fibroblast growth factor 5 (FGF5) was investigated as a possible candidate gene for hair length in alpaca (Vicugna pacos). As previously identified in other mammals, our results show that the alpaca FGF5 gene gives rise to a short (FGF5S) and a long (FGF5) isoform. Interestingly, in the long isoform, we observed a point mutation (i.e., a transition C>T at position 499 downstream of the ATG codon) that is able to generate a premature termination codon (PTC). The highly conserved nucleotide and amino acid sequence after PTC suggested a readthrough event (RT) that was confirmed by western blot analysis. The analysis of cDNA sequence revealed motifs and structures of mRNA undergoing RT. In fact, the event is positively influenced by particular signals harbored by the transcript. To the best of our knowledge, this is the first case of a readthrough event on PTC reported for the FGF5 gene and the first case of this translational mechanism in alpaca. Copyright © 2018 Elsevier B.V. All rights reserved.

Outcomes of peripheral blood stem cell transplantation patients from HLA-mismatched unrelated donor with antithymocyte globulin (ATG)-Thymoglobulin versus ATG-Fresenius: a single-center study.

PubMed

Huang, Wenrong; Zhao, Xiaoli; Tian, Yamin; Cao, Tingting; Li, Yanfen; Liu, Zhanxiang; Jing, Yu; Wang, Shuhong; Gao, Chunji; Yu, Li

2015-02-01

Although antithymocyte globulin (ATG) had been widely used in hematopoietic stem cell transplantation from unrelated donor due to its ability to prevent acute and chronic graft-versus-host disease (GVHD), the comparative efficacy and safety of ATG-Thymoglobulin (ATG-T) and ATG-Fresenius (ATG-F) in patients undergoing HLA-mismatched allogeneic peripheral blood stem cell transplantation from unrelated donors (UR-PBSCT) has not been evaluated. Retrospective analysis of patients who underwent HLA-mismatched UR-PBSCT between January 2003 and December 2013 and received pre-transplant ATG-T at a total dose of 10 mg/kg or ATG-F at a total dose of 20 mg/kg was performed. Patients who received ATG-T (n = 23) or ATG-F (n = 28) had similar baseline demographic, disease, and transplant characteristics. There were no significant between-groups differences in the probability of acute GVHD (P = 0.721) and chronic GVHD (P = 0.439). ATG-F was associated with nonsignificant trends toward higher disease-free survival at 3-year follow-up compared with ATG-T (45.7 ± 11.1 vs 61.3 ± 9.7 %, respectively, P = 0.07). A significantly greater proportion of ATG-T patients experienced high fever than ATG-F patients (P < 0.01) during ATG infusion. There was no difference in the rate of infection between the two treatment groups. There were less adverse effects comparing ATG-F with ATG-T. ATG-T at a total dose of 10 mg/kg and ATG-F at a total dose of 20 mg/kg had a similar clinical outcome in the setting of HLA-mismatched UR-PBSCT.

The autophagy-related genes BbATG1 and BbATG8 have different functions in differentiation, stress resistance and virulence of mycopathogen Beauveria bassiana

PubMed Central

Ying, Sheng-Hua; Liu, Jing; Chu, Xin-Ling; Xie, Xue-Qin; Feng, Ming-Guang

2016-01-01

Autophagy-related proteins play significantly different roles in eukaryotes. In the entomopathogenic fungus Beauveria bassiana, autophagy is associated with fungal growth and development. BbATG1 (a serine/threonine protein kinase) and BbATG8 (a ubiquitin-like protein) have similar roles in autophagy, but different roles in other processes. Disruption mutants of BbATG1 and BbATG8 had impaired conidial germination under starvation stress. The mutant ΔBbATG8 exhibited enhanced sensitivity to oxidative stress, while a ΔBbATG1 mutant did not. BbATG1 and BbATG8 showed different roles in spore differentiation. The blastospore yield was reduced by 70% and 92% in ΔBbATG1 and ΔBbATG8 mutants, respectively, and the double mutant had a reduction of 95%. Conidial yield was reduced by approximately 90% and 50% in ΔBbATG1 and ΔBbATG8 mutants, respectively. A double mutant had a reduction similar to ΔBbATG1. Additionally, both BbATG1 and BbATG8 affected the levels of conidial protein BbCP15p required for conidiation. The virulence of each autophagy-deficient mutant was considerably weakened as indicated in topical and intrahemocoel injection assays, and showed a greater reduction in topical infection. However, BbATG1 and BbATG8 had different effects on fungal virulence. Our data indicate that these autophagy-related proteins have different functions in fungal stress response, asexual development and virulence. PMID:27197558

A 20-basepair duplication in the human thyroid peroxidase gene results in a total iodide organification defect and congenital hypothyroidism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bikker, H.; Hartog, M.T. den; Gons, M.H.

1994-07-01

In this study, the authors present the molecular basis of a total iodide organification defect causing severe congenital hypothyroidism. In the thyroid gland of the patient, thyroid peroxidase (TPO) activity and the iodination degree of thyroglobulin were below detection limits, and no TPO messenger ribonucleic acid was detectable by Northern blot analysis. Denaturing gradient gel electrophoretic analysis of the TPO gene of the patient revealed a homozygous mutation in exon 2. Sequence analysis showed the presence of a 20-basepair duplication, 47 basepairs down-stream of the ATG start codon. This duplication generates a frame shift, resulting in a termination signal inmore » exon 3, compatible with the complete absence of TPO. Both parents of the patient are heterozygous for the same duplication, confirming the recessive mode of inheritance of the mutation. 32 refs., 4 figs.« less

Characterization of the human peroxisome proliferator activated receptor delta gene and its expression.

PubMed

Skogsberg, J; Kannisto, K; Roshani, L; Gagne, E; Hamsten, A; Larsson, C; Ehrenborg, E

2000-07-01

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors regulating the expression of genes involved in lipid and glucose metabolism. Three different PPARs; alpha (PPARA), gamma (PPARG) and delta (PPARD) have been characterized and they are distinguished from each other by tissue distribution and cell activation. In this study, the structure and detailed chromosomal localization of the human PPARD gene was determined. Three genomic clones containing the PPARD gene was isolated from a human P1 library. The gene spans approximately 85 kb of DNA and consists of 9 exons and 8 introns with exons ranging in size from 84 bp to 2.3 kb and introns ranging from 180 bp to 50 kb. All splice acceptor and donor sites conform to the consensus sequences including the AG-GT motif. Although PPARD lacks a TATA box, the gene is transcribed from a unique start site located 380 bp upstream of the ATG initiation codon. The 5' and 3' ends were mapped by rapid amplification of cDNA ends and the mRNA size of PPARD based upon the structure of the gene is 3803 bp. In addition, the chromosomal sublocalization of PPARD was determined by radiation hybrid mapping. The PPARD gene is located at 14 cR from the colipase gene and 15 cR from the serine kinase gene at chromosomal region 6p21.2.

Bm91 is an envelope component of ODV but is dispensable for the propagation of Bombyx mori nucleopolyhedrovirus.

PubMed

Tang, Qi; Li, Guohui; Yao, Qin; Chen, Liang; Lv, Peng; Lian, Chaoqun; Chen, Keping

2013-05-01

Orf91 (Bm91) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes a predicted 105-amino-acid protein, but its function remains unknown. In the current study, 5'-RACE revealed that the transcription initiation site of Bm91 was - 12 nucleotides upstream of the start codon ATG, transcription of Bm91 was detected from 12 to 96 h postinfection (p.i.) and Bm91 protein was detected from 24 to 96 h p.i. in BmNPV-infected BmN cells. Furthermore, Western blot analysis revealed that Bm91 was in occlusion-derived virus (ODV) but not in budded virus (BV). To investigate the role of Bm91 in baculovirus life cycle, a Bm91-knockout virus was constructed by bacmid recombination in E. coli. Fluorescence and light microscopy showed that the production of BV and occlusion bodies (OBs) in Bm91-deficient-virus-infected BmN cells were similar to those in wild-type-virus-infected ones. Bioassay results showed that genetic deletion of Bm91 did not significantly affect BmNPV infectivity, but extended the median lethal time (LT50). Taken together, these results indicate that Bm91 is not essential for viral propagation in vitro, but absence of the gene may affect the virulence of ODVs in silkworm larvae. Copyright © 2013 Elsevier Inc. All rights reserved.

Beyond Atg8 binding: The role of AIM/LIR motifs in autophagy.

PubMed

Fracchiolla, Dorotea; Sawa-Makarska, Justyna; Martens, Sascha

2017-05-04

Selective macroautophagy/autophagy mediates the selective delivery of cytoplasmic cargo material via autophagosomes into the lytic compartment for degradation. This selectivity is mediated by cargo receptor molecules that link the cargo to the phagophore (the precursor of the autophagosome) membrane via their simultaneous interaction with the cargo and Atg8 proteins on the membrane. Atg8 proteins are attached to membrane in a conjugation reaction and the cargo receptors bind them via short peptide motifs called Atg8-interacting motifs/LC3-interacting regions (AIMs/LIRs). We have recently shown for the yeast Atg19 cargo receptor that the AIM/LIR motifs also serve to recruit the Atg12-Atg5-Atg16 complex, which stimulates Atg8 conjugation, to the cargo. We could further show in a reconstituted system that the recruitment of the Atg12-Atg5-Atg16 complex is sufficient for cargo-directed Atg8 conjugation. Our results suggest that AIM/LIR motifs could have more general roles in autophagy.

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames

PubMed Central

1996-01-01

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules. PMID:8879204

Translation initiation at an upstream CUG codon regulates the expression of Hibiscus chlorotic ringspot virus coat protein.

PubMed

Koh, Dora Chin-Yen; Wang, Xiaoxing; Wong, Sek-Man; Liu, D X

2006-12-01

Viruses depend heavily on host cells for replication and exploit the host translation machinery for its gene expression using various unorthodox translation mechanisms. According to the conventional scanning model, only the 5'-proximal gene in the viral RNA is accessible to the ribosomes whereas other genes are silent. In this study, we use a model plant RNA virus, Hibiscus chlorotic ringspot virus (HCRSV), to investigate various translation mechanisms involved in regulation of the expression of internal genes. The 3'-end 1.2kb region of HCRSV genomic and subgenomic RNAs were shown to encode four polypeptides of 38, 27, 25 and 22.5kDa. Mutagenesis studies revealed that a CUG codon ((2570)CUG) is the initiation codon for p27, the longest of the three co-C-terminal products (p27, p25 and p22.5), and translation of p25 and p22.5 was initiated at (2603)AUG and (2666)AUG, respectively. Translation initiation of the p27 expression at the (2570)CUG codon regulates the expression of p38, the viral coat protein through a leaky scanning mechanism and mutational analysis of an upstream open reading frame (ORF) demonstrated that initiation of the p27 expression at this CUG codon (instead of an AUG) may play a role in maintaining the ratio of p27 and p38. In addition, a previously identified internal ribosome entry site was shown to control the expression of p27 and p38 in the subgenomic RNA 2.

Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon

PubMed Central

Jacinto-Loeza, Eva; Vivanco-Domínguez, Serafín; Guarneros, Gabriel; Hernández-Sánchez, Javier

2008-01-01

Rare AGA or AGG codons close to the initiation codon inhibit protein synthesis by a tRNA-sequestering mechanism as toxic minigenes do. To further understand this mechanism, a parallel analysis of protein synthesis and peptidyl-tRNA accumulation was performed using both a set of lacZ constructs where AGAAGA codons were moved codon by codon from +2, +3 up to +7, +8 positions and a series of 3–8 codon minigenes containing AGAAGA codons before the stop codon. β-Galactosidase synthesis from the AGAAGA lacZ constructs (in a Pth defective in vitro system without exogenous tRNA) diminished as the AGAAGA codons were closer to AUG codon. Likewise, β-galactosidase expression from the reporter +7 AGA lacZ gene (plus tRNA, 0.25 μg/μl) waned as the AGAAGAUAA minigene shortened. Pth counteracted both the length-dependent minigene effect on the expression of β-galactosidase from the +7 AGA lacZ reporter gene and the positional effect from the AGAAGA lacZ constructs. The +2, +3 AGAAGA lacZ construct and the shortest +2, +3 AGAAGAUAA minigene accumulated the highest percentage of peptidyl-tRNAArg4. These observations lead us to propose that hungry codons at early positions, albeit with less strength, inhibit protein synthesis by a minigene-like mechanism involving accumulation of peptidyl-tRNA. PMID:18583364

HMGB1 promotes the starvation-induced autophagic degradation of α-synuclein in SH-SY5Y cells Atg 5-dependently.

PubMed

Guan, Yi; Li, Yiping; Zhao, Gang; Li, Yunqian

2018-06-01

Impaired autophagic clearance of aggregated α-synuclein is considered as one of key mechanisms underlining Parkinson disease (PD). High-mobility group protein B1 (HMGB1) has recently been demonstrated to mediate persistent neuroinflammation and consequent progressive neurodegeneration by promoting multiple inflammatory and neurotoxic factors. In this study, we examined the influence of the overexpression of wild-type (WT) and mutant-type (MT, A53T and A30P) α-synuclein on the autophagy in neuroblastoma SH-SY5Y cells under starvation, and then investigated the regulation of endogenous HMGB1 on the α-synuclein degradation and on the starvation-induced autophagy in the α-synuclein-overexpressed SH-SY5Y cells. It was demonstrated that the overexpression of WT or MT α-synuclein significantly downregulated the starvation-induced conversion of LC3I to LC3II and autophagy protein (Atg) 5 expression, whereas markedly inhibited the starvation-downregulated mTOR in SH-SY5Y cells. On the other side, the lentivirus-mediated upregulation of endogenous HMGB1 promoted the degradation of WT or MT α-synuclein in SH-SY5Y cells autophagy-dependently via promoting Atg 5, but not mTOR, the Atg 5 knockdown downregulated the HMGB1-mediated promotion to α-synuclein degeneration. Thus, we concluded that α-synuclein inhibited the starvation-induced autophagy in neuroblastoma SH-SY5Y cells via inhibiting the mTOR/Atg 5 signaling. However, the endogenous HMGB1 promoted the autophagic degradation of α-synuclein via the Atg 5-dependent autophagy-initiation pathway, implying the protective role of endogenous HMGB1 in the neuroblastoma cells against the α-synuclein accumulation. Copyright © 2018. Published by Elsevier Inc.

Outcomes of Optimized over Standard Protocol of Rabbit Antithymocyte Globulin for Severe Aplastic Anemia: A Single-Center Experience

PubMed Central

Ge, Meili; Shao, Yingqi; Huang, Jinbo; Huang, Zhendong; Zhang, Jing; Nie, Neng; Zheng, Yizhou

2013-01-01

Background Previous reports showed that outcome of rabbit antithymocyte globulin (rATG) was not satisfactory as the first-line therapy for severe aplastic anemia (SAA). We explored a modifying schedule of administration of rATG. Design and Methods Outcomes of a cohort of 175 SAA patients, including 51 patients administered with standard protocol (3.55 mg/kg/d for 5 days) and 124 cases with optimized protocol (1.97 mg/kg/d for 9 days) of rATG plus cyclosporine (CSA), were analyzed retrospectively. Results Of all 175 patients, response rates at 3 and 6 months were 36.6% and 56.0%, respectively. 51 cases received standard protocol had poor responses at 3 (25.5%) and 6 months (41.2%). However, 124 patients received optimized protocol had better responses at 3 (41.1%, P = 0.14) and 6 (62.1%, P = 0.01). Higher incidences of infection (57.1% versus 37.9%, P = 0.02) and early mortality (17.9% versus 0.8%, P<0.001) occurred in patients received standard protocol compared with optimized protocol. The 5-year overall survival in favor of the optimized over standard rATG protocol (76.0% versus. 50.3%, P<0.001) was observed. By multivariate analysis, optimized protocol (RR = 2.21, P = 0.04), response at 3 months (RR = 10.31, P = 0.03) and shorter interval (<23 days) between diagnosis and initial dose of rATG (RR = 5.35, P = 0.002) were independent favorable predictors of overall survival. Conclusions Optimized instead of standard rATG protocol in combination with CSA remained efficacious as a first-line immunosuppressive regimen for SAA. PMID:23554855

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation

NASA Astrophysics Data System (ADS)

Li, Yi-Tong; Yi, Cong; Chen, Chen-Chen; Lan, Huan; Pan, Man; Zhang, Shao-Jin; Huang, Yi-Chao; Guan, Chao-Jian; Li, Yi-Ming; Yu, Li; Liu, Lei

2017-03-01

Acetylation of Atg3 regulates the lipidation of the protein Atg8 in autophagy. The molecular mechanism behind this important biochemical event remains to be elucidated. We describe the first semi-synthesis of homogeneous K19/K48-diacetylated Atg3 through sequential hydrazide-based native chemical ligation. In vitro reconstitution experiments with the semi-synthetic proteins confirm that Atg3 acetylation can promote the lipidation of Atg8. We find that acetylation of Atg3 enhances its binding to phosphatidylethanolamine-containing liposomes and to endoplasmic reticulum, through which it promotes the lipidation process.

Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.

PubMed

Kauffman, Karlina J; Yu, Shenliang; Jin, Jiaxin; Mugo, Brian; Nguyen, Nathan; O'Brien, Aidan; Nag, Shanta; Lystad, Alf Håkon; Melia, Thomas J

2018-04-10

During macroautophagy/autophagy, mammalian Atg8-family proteins undergo 2 proteolytic processing events. The first exposes a COOH-terminal glycine used in the conjugation of these proteins to lipids on the phagophore, the precursor to the autophagosome, whereas the second releases the lipid. The ATG4 family of proteases drives both cleavages, but how ATG4 proteins distinguish between soluble and lipid-anchored Atg8 proteins is not well understood. In a fully reconstituted delipidation assay, we establish that the physical anchoring of mammalian Atg8-family proteins in the membrane dramatically shifts the way ATG4 proteases recognize these substrates. Thus, while ATG4B is orders of magnitude faster at processing a soluble unprimed protein, all 4 ATG4 proteases can be activated to similar enzymatic activities on lipid-attached substrates. The recognition of lipidated but not soluble substrates is sensitive to a COOH-terminal LIR motif both in vitro and in cells. We suggest a model whereby ATG4B drives very fast priming of mammalian Atg8 proteins, whereas delipidation is inherently slow and regulated by all ATG4 homologs.

Autophagy-Related Protein ATG18 Regulates Apicoplast Biogenesis in Apicomplexan Parasites

PubMed Central

Bansal, Priyanka; Tripathi, Anuj; Thakur, Vandana; Mohmmed, Asif

2017-01-01

ABSTRACT Mechanisms by which 3′-phosphorylated phosphoinositides (3′-PIPs) regulate the development of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are poorly understood. The catabolic process of autophagy, which is dependent on autophagy-related proteins (ATGs), is one of the major targets of 3′-PIPs in yeast and mammals. In the present study, we identified autophagy-related protein ATG18 as an effector of 3′-PIPs in these parasites. P. falciparum ATG18 (PfATG18) and T. gondii ATG18 (TgATG18) interact with 3′-PIPs but exhibited differences in their specificity of interaction with the ligand PIP. The conditional knockdown of T. gondii or P. falciparum ATG18 (Tg/PfATG18) impaired replication of parasites and resulted in their delayed death. Intriguingly, ATG18 depletion resulted in the loss of the apicomplexan parasite-specific nonphotosynthetic plastid-like organelle apicoplast, which harbors the machinery for biosynthesis of key metabolites, and the interaction of ATG18 to phosphatidylinositol 3-phosphate (PI3P) was critical for apicoplast inheritance. Furthermore, ATG18 regulates membrane association and apicoplast localization of ATG8. These findings provide insights into a novel noncanonical role of ATG18 in apicoplast inheritance. This function of ATG18 in organelle biogenesis is unprecedented in any organism and may be conserved across most apicomplexan parasites. PMID:29089429

The Membrane Dynamics of Pexophagy Are Influenced by Sar1p in Pichia pastoris

PubMed Central

Schroder, Laura A.; Ortiz, Michael V.

2008-01-01

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy. PMID:18768759

Application of Natural Language Processing and Network Analysis Techniques to Post-market Reports for the Evaluation of Dose-related Anti-Thymocyte Globulin Safety Patterns.

PubMed

Botsis, Taxiarchis; Foster, Matthew; Arya, Nina; Kreimeyer, Kory; Pandey, Abhishek; Arya, Deepa

2017-04-26

To evaluate the feasibility of automated dose and adverse event information retrieval in supporting the identification of safety patterns. We extracted all rabbit Anti-Thymocyte Globulin (rATG) reports submitted to the United States Food and Drug Administration Adverse Event Reporting System (FAERS) from the product's initial licensure in April 16, 1984 through February 8, 2016. We processed the narratives using the Medication Extraction (MedEx) and the Event-based Text-mining of Health Electronic Records (ETHER) systems and retrieved the appropriate medication, clinical, and temporal information. When necessary, the extracted information was manually curated. This process resulted in a high quality dataset that was analyzed with the Pattern-based and Advanced Network Analyzer for Clinical Evaluation and Assessment (PANACEA) to explore the association of rATG dosing with post-transplant lymphoproliferative disorder (PTLD). Although manual curation was necessary to improve the data quality, MedEx and ETHER supported the extraction of the appropriate information. We created a final dataset of 1,380 cases with complete information for rATG dosing and date of administration. Analysis in PANACEA found that PTLD was associated with cumulative doses of rATG >8 mg/kg, even in periods where most of the submissions to FAERS reported low doses of rATG. We demonstrated the feasibility of investigating a dose-related safety pattern for a particular product in FAERS using a set of automated tools.

ATF4, A Novel Mediator of the Anabolic Actions of PTH on Bone

DTIC Science & Technology

2012-01-01

5-CTG CAA ATG GCA GCC CTG GTG AC-3 (reverse). For all primers the amplification was performed as follows: initial denaturation at 95 C for 10 min...rat Atf4, 5-ATG GCT TGG CCA GTG CCTCAGA-3 (forward), 5-GCTCTGGAGTGGAAGACA GAA C-3 (reverse); mouse/ratHprt, 5-GTT GAG AGA TCA TCT CCA CC-3...primers used for real-time PCR were: cyclin D1 (GenBank Accession number-NM-007631), 50 GAG GAG GGG GAA GTG GAG GA 30 (forward, þ1,049-bp), 50 CCT CTT TGC

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt.

PubMed

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-10-07

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact "nanogenome."

Effect of the nucleotides surrounding the start codon on the translation of foot-and-mouth disease virus RNA.

PubMed

Ma, X X; Feng, Y P; Gu, Y X; Zhou, J H; Ma, Z R

2016-06-01

As for the alternative AUGs in foot-and-mouth disease virus (FMDV), nucleotide bias of the context flanking the AUG(2nd) could be used as a strong signal to initiate translation. To determine the role of the specific nucleotide context, dicistronic reporter constructs were engineered to contain different versions of nucleotide context linking between internal ribosome entry site (IRES) and downstream gene. The results indicate that under FMDV IRES-dependent mechanism, the nucleotide contexts flanking start codon can influence the translation initiation efficiencies. The most optimal sequences for both start codons have proved to be UUU AUG(1st) AAC and AAG AUG(2nd) GAA.

Association of ATG5 Gene Polymorphisms With Behçet's Disease and ATG10 Gene Polymorphisms With VKH Syndrome in a Chinese Han Population.

PubMed

Zheng, Minming; Yu, Hongsong; Zhang, Lijun; Li, Hua; Liu, Yunjia; Kijlstra, Aize; Yang, Peizeng

2015-12-01

This study was conducted to explore the association of autophagy-related genes (ATGs) single nucleotide polymorphisms (SNPs) with Behçet's disease (BD) and Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. A two-stage association study was carried out in 940 BD, 1061 VKH, and 2007 healthy controls. Genotyping for genetic variants of 10 autophagy family genes (ATG5, ATG7, ATG10, ATG16L1, IRGM, LKKR2, ATG2A, DAP, ULK1, and TSC1) was performed using PCR-restriction fragment length polymorphism (PCR-RFLP) or TaqMan SNP assays. Gene expression was quantified by real-time PCR. In the cohort of BD patients, we observed that the TT genotype of rs573775/ATG5 decreased susceptibility to BD (Pc = 8.35 × 10-6, OR = 0.490). In the case of VKH patients, the AC genotype of rs4703863/ATG10 increased susceptibility to VKH syndrome (Pc = 9.94 × 10-5, OR = 1.444), whereas the A allele and AA genotype of rs4703863 (Pc = 7.06 × 10-5, OR = 0.745; Pc = 6.34 × 10-6, OR = 0.669, respectively) acted as protective factors for VKH. Functional experiments showed an increased ATG5 expression by LPS stimulated PBMCs in TT cases of rs573775 compared with controls. The level of ATG5 mRNA in active BD patients not receiving immunosuppression was significantly higher than that in healthy controls. This study demonstrated an association of ATG5 rs573775 with BD and ATG10 rs4703863 with VKH syndrome in a Chinese Han population. Furthermore, a variant of the ATG5 gene was shown to be correlated with ATG5 expression.

Molecular cloning and characterization of two novel autophagy-related genes belonging to the ATG8 family from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

PubMed

Flores Fernández, José Miguel; Gutiérrez Ortega, Abel; Rosario Cruz, Rodrigo; Padilla Camberos, Eduardo; Alvarez, Angel H; Martínez Velázquez, Moisés

2014-12-01

Rhipicephalus (Boophilus) microplus is an obligate haematophagous arthropod and the major problem for cattle industry due to economic losses it causes. The parasite shows a remarkable adaptability to changing environmental conditions as well as an exceptional ability to survive long-term starvation. This ability has been related to a process of intracellular protein degradation called autophagy. This process in ticks is still poorly understood and only few autophagy-related (ATG) genes have been characterized. The aim of the present study was to examine the ESTs database, BmiGI, of R. microplus searching for ATG homologues. We predicted five putative ATG genes, ATG3, ATG4, ATG6 and two ATG8s. Further characterization led to the identification of RmATG8a and RmATG8b, homologues of GABARAP and MAP1LC3, respectively, and both of them belonging to the ATG8 family. PCR analyses showed that the expression level of RmATG8a and RmATG8b was higher in egg and larval stages when compared to ovary and midgut from adult ticks. This up-regulation coincides with the period in which ticks are in a starvation state, suggesting that autophagy is active in R. microplus.

miR-106a suppresses tumor cells death in colorectal cancer through targeting ATG7.

PubMed

Hao, Haibin; Xia, Guangfeng; Wang, Chao; Zhong, Fuping; Liu, Laipeng; Zhang, Dong

2017-06-01

Autophagy-related gene 7 (ATG7) and miR-106a play an important role in cancer cell autophagy and apoptosis, but the outcome of ATG7 and miR-106a in colorectal cancer (CRC) still remains not clear. In this study, we found that ATG7 and miR-106a expression were mutually related with cell death and prognosis in CRC patients. In addition, we also showed that ATG7 and miR-106a expression were changeable in colorectal cancer cell lines when compared with normal cell lines, but ATG7 and miR-106a mRNA level was negatively correlated. Furthermore, ATG7 protein and mRNA levels decreased after over-expression of miR-106a, whereas the suppression of ATG7 had the opposite effect. We confirmed that miR-106a down-regulated ATG7 mRNA level by binding the specific sequence of ATG7 mRNA 3'UTR region. Moreover, the over-expression of ATG7 induced CRC cells death both in vitro and in vivo. Taken together, our study data demonstrated that ATG7 aggravated the cell death of CRC, which was inhibited by miR-106a.

eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

PubMed

Thakur, Anil; Hinnebusch, Alan G

2018-05-01

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNA i ) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "P OUT " state to a closed conformation with TC more tightly bound in a "P IN " state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the P IN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNA i D loop in the P IN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNA i We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNA i accommodation in the P IN state without influencing the P OUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNA i clash to an electrostatic attraction that stabilizes P IN and enhances selection of poor start codons in vivo.

Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites.

PubMed Central

Scieglinska, D; Widłak, W; Konopka, W; Poutanen, M; Rahman, N; Huhtaniemi, I; Krawczyk, Z

2001-01-01

The rat Hst70 gene and its mouse counterpart Hsp70.2 belong to the family of Hsp70 heat shock genes and are specifically expressed in male germ cells. Previous studies regarding the structure of the 5' region of the transcription unit of these genes as well as localization of the 'cis' elements conferring their testis-specific expression gave contradictory results [Widlak, Markkula, Krawczyk, Kananen and Huhtaniemi (1995) Biochim. Biophys. Acta 1264, 191-200; Dix, Rosario-Herrle, Gotoh, Mori, Goulding, Barret and Eddy (1996) Dev. Biol. 174, 310-321]. In the present paper we solve these controversies and show that the 5' untranslated region (UTR) of the Hst70 gene contains an intron which is localized similar to that of the mouse Hsp70.2 gene. Reverse transcriptase-mediated PCR, Northern blotting and RNase protection analysis revealed that the transcription initiation of both genes starts at two main distant sites, and one of them is localized within the intron. As a result two populations of Hst70 gene transcripts with similar sizes but different 5' UTR structures can be detected in total testicular RNA. Functional analysis of the Hst70 gene promoter in transgenic mice and transient transfection assays proved that the DNA fragment of approx. 360 bp localized upstream of the ATG transcription start codon is the minimal promoter required for testis-specific expression of the HST70/chloramphenicol acetyltransferase transgene. These experiments also suggest that the expression of the gene may depend on 'cis' regulatory elements localized within exon 1 and the intron sequences. PMID:11563976

Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter.

PubMed

Tencomnao, T; Yu, R K; Kapitonov, D

2001-02-16

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.

Defect in the GTPase activating protein (GAP) function of eIF5 causes repression of GCN4 translation.

PubMed

Antony A, Charles; Alone, Pankaj V

2017-05-13

In eukaryotes, the eIF5 protein plays an important role in translation start site selection by providing the GAP (GTPase activating protein) function. However, in yeast translation initiation fidelity defective eIF5 G31R mutant causes preferential utilization of UUG as initiation codon and is termed as Suppressor of initiation codon (Sui - ) phenotype due to its hyper GTPase activity. The eIF5 G31R mutant dominantly represses GCN4 expression and confers sensitivity to 3-Amino-1,2,4-Trizole (3AT) induced starvation. The down-regulation of the GCN4 expression (Gcn - phenotype) in the eIF5 G31R mutant was not because of leaky scanning defects; rather was due to the utilization of upUUG initiation codons at the 5' regulatory region present between uORF1 and the main GCN4 ORF. Copyright © 2017 Elsevier Inc. All rights reserved.

Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

PubMed

Suzuki, Sho W; Onodera, Jun; Ohsumi, Yoshinori

2011-02-25

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

Atg5- and Atg7-dependent autophagy in dopaminergic neurons regulates cellular and behavioral responses to morphine.

PubMed

Su, Ling-Yan; Luo, Rongcan; Liu, Qianjin; Su, Jing-Ran; Yang, Lu-Xiu; Ding, Yu-Qiang; Xu, Lin; Yao, Yong-Gang

2017-09-02

The molecular basis of chronic morphine exposure remains unknown. In this study, we hypothesized that macroautophagy/autophagy of dopaminergic neurons would mediate the alterations of neuronal dendritic morphology and behavioral responses induced by morphine. Chronic morphine exposure caused Atg5 (autophagy-related 5)- and Atg7 (autophagy-related 7)-dependent and dopaminergic neuron-specific autophagy resulting in decreased neuron dendritic spines and the onset of addictive behaviors. In cultured primary midbrain neurons, morphine treatment significantly reduced total dendritic length and complexity, and this effect could be reversed by knockdown of Atg5 or Atg7. Mice deficient for Atg5 or Atg7 specifically in the dopaminergic neurons were less sensitive to developing a morphine reward response, behavioral sensitization, analgesic tolerance and physical dependence compared to wild-type mice. Taken together, our findings suggested that the Atg5- and Atg7-dependent autophagy of dopaminergic neurons contributed to cellular and behavioral responses to morphine and may have implications for the future treatment of drug addiction.

Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy*

PubMed Central

Puente, Cindy; Hendrickson, Ronald C.; Jiang, Xuejun

2016-01-01

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation. PMID:26801615

Characterization of the Autophagy related gene-8a (Atg8a) promoter in Drosophila melanogaster.

PubMed

Bali, Arundhati; Shravage, Bhupendra V

2017-01-01

Autophagy is an evolutionarily conserved process which is upregulated under various stress conditions, including nutrient stress and oxidative stress. Amongst autophagy related genes (Atgs), Atg8a (LC3 in mammals) is induced several-fold during nutrient limitation in Drosophila. The minimal Atg8a cis-regulatory module (CRM) which mediates transcriptional upregulation under various stress conditions is not known. Here, we describe the generation and analyses of a series of Atg8a promoter deletions which drive the expression of an mCherry-Atg8a fusion cassette. Expression studies revealed that a 200 bp region of Atg8a is sufficient to drive expression of Atg8a in nutrient rich conditions in fat body and ovaries, as well as under nutrient deficient conditions in the fat body. Furthermore, this 200 bp region can mediate Atg8a upregulation during developmental histolysis of the larval fat body and under oxidative stress conditions induced by H 2 O 2 . Finally, the expression levels of Atg8a from this promoter are sufficient to rescue the lethality of the Atg8a mutant. The 200 bp promoter-fusion reporter provides a valuable tool which can be used in genetic screens to identify transcriptional and post-transcriptional regulators of Atg8a.

Translation of vph mRNA in Streptomyces lividans and Escherichia coli after removal of the 5' untranslated leader.

PubMed

Wu, C J; Janssen, G R

1996-10-01

The Streptomyces vinaceus viomycin phosphotransferase (vph) mRNA contains an untranslated leader with a conventional Shine-Dalgarno homology. The vph leader was removed by ligation of the vph coding sequence to the transcriptional start site of a Streptomyces or an Escherichia coli promoter, such that transcription would initiate at the first position of the vph start codon. Analysis of mRNA demonstrated that transcription initiated primarily at the A of the vph AUG translational start codon in both Streptomyces lividans and E. coli; cells expressing the unleadered vph mRNA were resistant to viomycin indicating that the Shine-Dalgarno sequence, or other features contained within the leader, was not necessary for vph translation. Addition of four nucleotides (5'-AUGC-3') onto the 5' end of the unleadered vph mRNA resulted in translation initiation from the vph start codon and the AUG triplet contained within the added sequence. Translational fusions of vph sequence to a Tn5 neo reporter gene indicated that the first 16 codons of vph coding sequence were sufficient to specify the translational start site and reading frame for expression of neomycin resistance in both E. coli and S. lividans.

Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions.

PubMed

Zhang, Jingji; Pavlov, Michael Y; Ehrenberg, Måns

2018-02-16

We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of 'monitoring bases' A1492, A1493 and G530 in 16S rRNA in favor of their 'activated' state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the 'intrinsic selectivity' of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome.

Novel coding, translation, and gene expression of a replicating covalently closed circular RNA of 220 nt

PubMed Central

AbouHaidar, Mounir Georges; Venkataraman, Srividhya; Golshani, Ashkan; Liu, Bolin; Ahmad, Tauqeer

2014-01-01

The highly structured (64% GC) covalently closed circular (CCC) RNA (220 nt) of the virusoid associated with rice yellow mottle virus codes for a 16-kDa highly basic protein using novel modalities for coding, translation, and gene expression. This CCC RNA is the smallest among all known viroids and virusoids and the only one that codes proteins. Its sequence possesses an internal ribosome entry site and is directly translated through two (or three) completely overlapping ORFs (shifting to a new reading frame at the end of each round). The initiation and termination codons overlap UGAUGA (underline highlights the initiation codon AUG within the combined initiation-termination sequence). Termination codons can be ignored to obtain larger read-through proteins. This circular RNA with no noncoding sequences is a unique natural supercompact “nanogenome.” PMID:25253891

Vitrification, in vitro fertilization, and development of Atg7 deficient mouse oocytes.

PubMed

Bang, Soyoung; Lee, Geun-Kyung; Shin, Hyejin; Suh, Chang Suk; Lim, Hyunjung Jade

2016-03-01

Autophagy contributes to the clearance and recycling of macromolecules and organelles in response to stress. We previously reported that vitrified mouse oocytes show acute increases in autophagy during warming. Herein, we investigate the potential role of Atg7 in oocyte vitrification by using an oocyte-specific deletion model of the Atg7 gene, a crucial upstream gene in the autophagic pathway. Oocyte-specific Atg7 deficient mice were generated by crossing Atg7 floxed mice and Zp3-Cre transgenic mice. The oocytes were vitrified-warmed and then subjected to in vitro fertilization and development. The rates of survival, fertilization, and development were assessed in the Atg7 deficient oocytes in comparison with the wildtype oocytes. Light chain 3 (LC3) immunofluorescence staining was performed to determine whether this method effectively evaluates the autophagy status of oocytes. The survival rate of vitrified-warmed Atg7(f/f) ;Zp3-Cre (Atg7(d/d) ) metaphase II (MII) oocytes was not significantly different from that of the wildtype (Atg7(f/f) ) oocytes. Fertilization and development in the Atg7(d/d) oocytes were significantly lower than the Atg7(f/f) oocytes, comparable to the Atg5(d/d) oocytes previously described. Notably, the developmental rate improved slightly in vitrified-warmed Atg7(d/d) MII oocytes when compared to fresh Atg7(d/d) oocytes. LC3 immunofluorescence staining showed that this method can be reliably used to assess autophagic activation in oocytes. We confirmed that the LC3-positive signal is nearly absent in Atg7(d/d) oocytes. While autophagy is induced during the warming process after vitrification of MII oocytes, the Atg7 gene is not essential for survival of vitrified-warmed oocytes. Thus, induction of autophagy during warming of vitrified MII oocytes seems to be a natural response to manage cold or other cellular stresses.

[Comparison of two types of antithymocyte globulin in the treatment of children with aplastic anemia].

PubMed

Xie, X T; He, W; Shi, W; Zhou, X X; Qiao, X H

2016-04-01

This study was designed to compare the effects of the anti-human T lymphocyte globulin (Fresenius, ATG-F)and rabbit anti-human thymocyte immunoglobulin (Genzyme, R-ATG)in the treatment of childhood aplastic anemia (AA) and their effects. A total of 59 children with aplastic anemia were analyzed in the present study, including 34 cases of severe aplastic anemia (SAA), 12 cases of very severe aplastic anemia (VSAA) and 13 cases of transfusion-dependent non-severe aplastic anemia (NSAA). While receiving immunosuppressive therapy (IST), 30 and 29 patients, with long-term oral supplement with cyclosporin A (CSA), androgen and Chinese traditional medicines, were treated with ATG-F and R-ATG, respectively. When it was necessary, some supportive cares such as component transfusion and infection control were also employed. Absolute counts of peripheral blood lymphocyte (ALC) at various time points were dynamically detected after ATG therapy. According to the International Aplastic Anemia Treatment and Effect standards. There were no statistically significant differences in the overall response rate (67%(20/30)vs. 69%(20/29), χ(2)=0.036, P=0.676) and the survival rate (87%(26/30)vs. 83%(24/29), χ(2)=0.173, P=0.676) between the ATG-F and R-ATG groups. There were significant and long-term ALC decrease after ATG therapy, the rate of ALC decrease in ATG-F and R-ATG group, the ALC only recovered to 47.8% (ATG-F group) and 47.4% (R-ATG group) of the pre-treatment level respectively. ATG-F 5 mg/(kg·d) and R-ATG 3.75 mg/(kg·d)could achieve similar effects in the treatment of childhood AA, through similar significant clearance of T cells. Therefore, all of these suggest that ATG-F and R-ATG might serve as the drugs of front-line choice for IST in childhood AA patients who do not have an available human leukocyte antigen identical related donor.

‘Nodophagy’

PubMed Central

Travassos, Leonardo H

2010-01-01

Autophagy is a homeostatic pathway that processes and recycles damaged organelles and other cytoplasmic contents. While studies have implicated autophagy in the immune response to infection, the understanding of how the autophagic machinery specifically targets intracellular pathogens has remained elusive. Two recent studies have uncovered an autophagy-mediated immune response to bacteria through their detection by Nod receptors. In particular, Nod1 and Nod2 recruit the autophagic protein ATG16L1 to the plasma membrane at the bacterial entry site to promote an autophagy-dependent elimination of bacteria. In addition, Nod2 and ATG16L1 synergize to initiate an adaptive immune response to bacterial invasion by enhancing major histocompatibility complex (MHC) class II antigen presentation. These findings link two Crohn disease-associated susceptibility genes and reveal that cells expressing the risk-associated variants of ATG16L1 are defective in autophagy-mediated bacterial handling and antigen presentation. This could lead to bacterial persistence and contribute to the pathogenesis of the disease. PMID:21327039

Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis

DOE PAGES

Marshall, Richard S.; Li, Faqiang; Gemperline, David C.; ...

2015-05-21

Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. In this paper, we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuingmore » proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Finally and collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.« less

The type of ATG matters -- natural killer cells are influenced differentially by Thymoglobulin, Lymphoglobulin and ATG-Fresenius.

PubMed

Penack, Olaf; Fischer, Lars; Gentilini, Chiara; Nogai, Axel; Muessig, Arne; Rieger, Kathrin; Ganepola, Susanne; Thiel, Eckhard; Uharek, Lutz

2007-11-01

Although ATG is frequently used in hematopoietic stem cell transplantation and solid organ transplantation, little is known on its effects on NK cells, which mediate important functions in post-transplantation immunology. We incubated peripheral blood lymphocytes of healthy donors with Thymoglobulin, Lymphoglobulin or ATG-Fresenius. Cell death and apoptosis of NK cells and T cells were determined by flow cytometry using propidium iodide and Annexin V. As expected, there were no significant differences between the different ATGs regarding their T cell toxicity. Surprisingly, we found profound differences between the different ATGs regarding their impact on NK cells: In clinically relevant concentrations Lymphoglobulin had less toxic effects on NK cells as compared to Thymoglobulin or ATG-Fresenius: the median percentages of apoptotic or necrotic NK cells in response to 1 mug/ml Lymphoglobulin, ATG-Fresenius and Thymoglobulin were 2%, 35% and 38%, respectively (p<0.001). This is the first report of differential effects of different ATGs on NK cells. Lymphoglobulin appears to be superior to Thymoglobulin or ATG Fresenius regarding the preservation of NK cell mediated immunity. Randomized trials addressing the impact of different ATGs on lymphocyte subpopulations in the clinical setting are urgently warranted.

Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development

PubMed Central

Szatmári, Zsuzsanna; Sass, Miklós

2014-01-01

Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, we performed a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. We show that Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, our results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation. PMID:25006588

Wheat homologs of yeast ATG6 function in autophagy and are implicated in powdery mildew immunity.

PubMed

Yue, Jieyu; Sun, Hong; Zhang, Wei; Pei, Dan; He, Yang; Wang, Huazhong

2015-04-01

Autophagy-related ATG6 proteins are pleiotropic proteins functioning in autophagy and the phosphatidylinositol 3-phosphate-signaling pathways. Arabidopsis ATG6 regulates normal plant growth, pollen development and germination, and plant responses to biotic/abiotic stresses. However, the ATG6 functions in wheat (Triticum aestivum L.), an important food crop, are lacking. We identified three members, TaATG6a-6c, of the ATG6 family from common wheat. TaATG6a, 6b and 6c were localized on homeologous chromosomes 3DL, 3BL and 3AL, respectively, of the allo-hexaploid wheat genome, and evidence was provided for their essential role in autophagy. The TaATG6a-GFP fusion protein was found in punctate pre-autophagosomal structures. The expression of each TaATG6 gene restored the accumulation of autophagic bodies in atg6-mutant yeast. Additionally, TaATG6 knockdown plants showed impaired constitutive and pathogen-induced autophagy and growth abnormalities under normal conditions. We also examined the expression patterns of wheat ATG6s for clues to their physiological roles, and found that their expression was induced by the fungus Blumeria graminis f. sp. tritici (Bgt), the causal agent of powdery mildew, and by abiotic stress factors. A role for TaATG6s in wheat immunity to powdery mildew was further implied when knockdowns of TaATG6s weakly compromised the broad-spectrum powdery mildew resistance gene Pm21-triggered resistance response and, conversely and significantly, enhanced the basal resistance of susceptible plants. In addition, leaf cell death was sometimes induced by growth-retarded small Bgt mycelia on susceptible TaATG6 knockdown plants after a long period of interaction. Thus, we provide an important extension of the previous characterization of plant ATG6 genes in wheat, and observed a role for autophagy genes in wheat immune responses to fungal pathogens. Three wheat ATG6s were identified and shown to be essential for autophagy biogenesis. Wheat ATG6s are implicated in immunity to powdery mildew, playing a weak, positive role in the Pm21-triggered resistance response and a negative role in the basal resistance of susceptible plants.

Atg7-Mediated Autophagy Is Involved in the Neural Crest Cell Generation in Chick Embryo.

PubMed

Wang, Guang; Chen, En-Ni; Liang, Chang; Liang, Jianxin; Gao, Lin-Rui; Chuai, Manli; Münsterberg, Andrea; Bao, Yongping; Cao, Liu; Yang, Xuesong

2018-04-01

Autophagy plays a very important role in numerous physiological and pathological events. However, it still remains unclear whether Atg7-induced autophagy is involved in the regulation of neural crest cell production. In this study, we found the co-location of Atg7 and Pax7 + neural crest cells in early chick embryo development. Upregulation of Atg7 with unilateral transfection of full-length Atg7 increased Pax7 + and HNK-1 + cephalic and trunk neural crest cell numbers compared to either Control-GFP transfection or opposite neural tubes, suggesting that Atg7 over-expression in neural tubes could enhance the production of neural crest cells. BMP4 in situ hybridization and p-Smad1/5/8 immunofluorescent staining demonstrated that upregulation of Atg7 in neural tubes suppressed the BMP4/Smad signaling, which is considered to promote the delamination of neural crest cells. Interestingly, upregulation of Atg7 in neural tubes could significantly accelerate cell progression into the S phase, implying that Atg7 modulates cell cycle progression. However, β-catenin expression was not significantly altered. Finally, we demonstrated that upregulation of the Atg7 gene could activate autophagy as did Atg8. We have also observed that similar phenotypes, such as more HNK-1 + neural crest cells in the unilateral Atg8 transfection side of neural tubes, and the transfection with full-length Atg8-GFP certainly promote the numbers of BrdU + neural crest cells in comparison to the GFP control. Taken together, we reveal that Atg7-induced autophagy is involved in regulating the production of neural crest cells in early chick embryos through the modification of the cell cycle.

Prospective study of rabbit antithymocyte globulin and cyclosporine for aplastic anemia from the EBMT Severe Aplastic Anaemia Working Party.

PubMed

Marsh, Judith C; Bacigalupo, Andrea; Schrezenmeier, Hubert; Tichelli, Andre; Risitano, Antonio M; Passweg, Jakob R; Killick, Sally B; Warren, Alan J; Foukaneli, Theodora; Aljurf, Mahmoud; Al-Zahrani, H A; Höchsmann, Britta; Schafhausen, Philip; Roth, Alexander; Franzke, Anke; Brummendorf, Tim H; Dufour, Carlo; Oneto, Rosi; Sedgwick, Philip; Barrois, Alain; Kordasti, Shahram; Elebute, Modupe O; Mufti, Ghulam J; Socie, Gerard

2012-06-07

Rabbit antithymocyte globulin (rATG; thymoglobulin, Genzyme) in combination with cyclosporine, as first-line immunosuppressive therapy, was evaluated prospectively in a multicenter, European, phase 2 pilot study, in 35 patients with aplastic anemia. Results were compared with 105 age- and disease severity-matched patients from the European Blood and Marrow Transplant registry, treated with horse ATG (hATG; lymphoglobulin) and cyclosporine. The primary end point was response at 6 months. At 3 months, no patients had achieved a complete response to rATG. Partial response occurred in 11 (34%). At 6 months, complete response rate was 3% and partial response rate 37%. There were 10 deaths after rATG (28.5%) and 1 after subsequent HSCT. Infections were the main cause of death in 9 of 10 patients. The best response rate was 60% for rATG and 67% for hATG. For rATG, overall survival at 2 years was 68%, compared with 86% for hATG (P = .009). Transplant-free survival was 52% for rATG and 76% for hATG (P = .002). On multivariate analysis, rATG (hazard ratio = 3.9, P = .003) and age more than 37 years (hazard ratio = 4.7, P = .0008) were independent adverse risk factors for survival. This study was registered at www.clinicaltrials.gov as NCT00471848.

Autophagy Genes Enhance Murine Gammaherpesvirus 68 Reactivation From Latency by Preventing Virus-induced Systemic Inflammation

PubMed Central

Park, Sunmin; Buck, Michael D.; Desai, Chandni; Zhang, Xin; Loginicheva, Ekaterina; Martinez, Jennifer; Freeman, Michael L.; Saitoh, Tatsuya; Akira, Shizuo; Guan, Jun-Lin; He, You-Wen; Blackman, Marcia A.; Handley, Scott A.; Levine, Beth; Green, Douglas R.; Reese, Tiffany A.; Artyomov, Maxim N.; Virgin, Herbert W.

2016-01-01

SUMMARY Host genes that regulate systemic inflammation upon chronic viral infection are incompletely understood. Murine γ-herpesvirus 68 (MHV68) infection is characterized by latency in macrophages, and reactivation is inhibited by Interferon-γ (IFN-γ). Using a Lysozyme-M-cre (LysMcre) expression system, we show that deletion of autophagy-related (Atg) genes Fip200, beclin 1, Atg14, Atg16L1, Atg7, Atg3, and Atg5, in the myeloid compartment, inhibited MHV68 reactivation in macrophages. Atg5-deficiency did not alter reactivation from B cells, and effects on reactivation from macrophages were not explained by alterations in productive viral replication or the establishment of latency. Rather, chronic MHV68 infection triggered increased systemic inflammation, increased T cell production of IFN-γ and an IFN-γ-induced transcriptional signature in macrophages from Atg gene-deficient mice. The Atg5-related reactivation defect was partially reversed by neutralization of IFN-γ. Thus Atg genes in myeloid cells dampen virus-induced systemic inflammation, creating an environment that fosters efficient MHV68 reactivation from latency. PMID:26764599

Characterization of a novel autophagy-specific gene, ATG29

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kawamata, Tomoko; Division of Molecular Cell Biology, National Institute for Basic Biology, Okazaki 444-8585; Kamada, Yoshiaki

2005-12-30

Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w.more » atg29{delta} cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.« less

Identification and characterization of the autophagy-related genes Atg12 and Atg5 in hydra.

PubMed

Dixit, Nishikant S; Shravage, Bhupendra V; Ghaskadbi, Surendra

2017-01-01

Autophagy is an evolutionarily conserved process in eukaryotic cells that is involved in the degradation of cytoplasmic contents including organelles via the lysosome. Hydra is an early metazoan which exhibits simple tissue grade organization, a primitive nervous system, and is one of the classical non-bilaterian models extensively used in evo-devo research. Here, we describe the characterization of two core autophagy genes, Atg12 and Atg5, from hydra. In silico analyses including sequence similarity, domain analysis, and phylogenetic analysis demonstrate the conservation of these genes across eukaryotes. The predicted 3D structure of hydra Atg12 showed very little variance when compared to human Atg12 and yeast Atg12, whereas the hydra Atg5 predicted 3D structure was found to be variable, when compared with its human and yeast homologs. Strikingly, whole mount in situ hybridization showed high expression of Atg12 transcripts specifically in nematoblasts, whereas Atg5 transcripts were found to be expressed strongly in budding region and growing buds. This study may provide a framework to understand the evolution of autophagy networks in higher eukaryotes.

Ulk1-mediated Atg5-independent macroautophagy mediates elimination of mitochondria from embryonic reticulocytes.

PubMed

Honda, Shinya; Arakawa, Satoko; Nishida, Yuya; Yamaguchi, Hirofumi; Ishii, Eiichi; Shimizu, Shigeomi

2014-06-04

Macroautophagy is a highly conserved intracellular process responsible for the degradation of subcellular constituents. Macroautophagy was recently suggested to be involved in the removal of mitochondria from reticulocytes during the final stage of erythrocyte differentiation. Although Atg5 and Atg7 are indispensable for macroautophagy, their role in mitochondrial clearance remains controversial. We recently discovered that mammalian cells use conventional Atg5/Atg7-dependent macroautophagy as well as an alternative Unc-51-like kinase 1 (Ulk1)-dependent Atg5/Atg7-independent macroautophagy process. We hypothesized that the latter may be involved in mitochondrial clearance from reticulocytes during erythrocyte differentiation. Here we report that fetal definitive reticulocytes from Ulk1-deficient and Ulk1/Atg5 double-deficient mice retain their mitochondria, whereas the mitochondria are engulfed and digested within autophagic structures in wild-type and Atg5-deficient mice. Mitochondrial retention by Ulk1-deficient reticulocytes is far less marked in primitive and adult definitive reticulocytes. These data indicate that Ulk1-dependent Atg5-independent macroautophagy is the dominant process of mitochondrial clearance from fetal definitive reticulocytes.

Structure-based drug design, synthesis and biological assays of P. falciparum Atg3-Atg8 protein-protein interaction inhibitors

NASA Astrophysics Data System (ADS)

Villa, Stefania; Legnani, Laura; Colombo, Diego; Gelain, Arianna; Lammi, Carmen; Bongiorno, Daniele; Ilboudo, Denise P.; McGee, Kellen E.; Bosch, Jürgen; Grazioso, Giovanni

2018-03-01

The proteins involved in the autophagy (Atg) pathway have recently been considered promising targets for the development of new antimalarial drugs. In particular, inhibitors of the protein-protein interaction (PPI) between Atg3 and Atg8 of Plasmodium falciparum retarded the blood- and liver-stages of parasite growth. In this paper, we used computational techniques to design a new class of peptidomimetics mimicking the Atg3 interaction motif, which were then synthesized by click-chemistry. Surface plasmon resonance has been employed to measure the ability of these compounds to inhibit the Atg3-Atg8 reciprocal protein-protein interaction. Moreover, P. falciparum growth inhibition in red blood cell cultures was evaluated as well as the cyto-toxicity of the compounds.

Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

PubMed Central

Chen, Augustine; Kao, Y. F.; Brown, Chris M.

2005-01-01

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins. PMID:15731337

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

PubMed

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

Application of β-glucuronidase (GusA) as an effective reporter for extremely acidophilic Acidithiobacillus ferrooxidans.

PubMed

Wang, Huiyan; Fang, Liangyan; Wen, Qing; Lin, Jianqun; Liu, Xiangmei

2017-04-01

Acidithiobacillus ferrooxidans is a model organism for investigating metal sulfide bioleaching. The regulatory mechanism of gene expression by metabolizing various substrates is critical for understanding its role in bioleaching processes. However, no reporter has been successfully employed to study gene expression in A. ferrooxidans to date. In this study, a sensitive and robust reporter system based on β-glucuronidase (GusA) was described for feasible application in A. ferrooxidans. A set of vectors, which contained the transcriptional and translational fusions of gusA, were constructed and employed to analyze promoter activity and efficiency of translation initiation in A. ferrooxidans. Ptac and P2811 were screened out from ten tested promoters and could be used as strong promoters for gene overexpression in A. ferrooxidans. Among the four translational fusions of gusA with different start codons, ATG was most active, followed by TTG and GTG, while CTG showed the least activity. The transcriptional inhibition effect of an IclR-like transcription factor was also observed on its own encoding gene AFE_1668 as well as its neighboring AFE_1667. In addition, the specific chromogenic reaction of GusA could be detected and visualized by colonies of A. ferrooxidans containing gusA expression plasmids. Generally, the established GusA reporter system would be applied not only for quantitative analysis of promoter strength and its transcriptional regulation but also for qualitative colony screening in A. ferrooxidans in the future.

Novel mutations in the GPIHBP1 gene identified in 2 patients with recurrent acute pancreatitis.

PubMed

Ariza, María José; Martínez-Hernández, Pedro Luis; Ibarretxe, Daiana; Rabacchi, Claudio; Rioja, José; Grande-Aragón, Cristina; Plana, Nuria; Tarugi, Patrizia; Olivecrona, Gunilla; Calandra, Sebastiano; Valdivielso, Pedro

2016-01-01

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) has been demonstrated to be essential for the in vivo function of lipoprotein lipase (LPL), the major triglyceride (TG)-hydrolyzing enzyme involved in the intravascular lipolysis of TG-rich lipoproteins. Recently, loss-of-function mutations of GPIHBP1 have been reported as the cause of type I hyperlipoproteinemia in several patients. Two unrelated patients were referred to our Lipid Units because of a severe hypertriglyceridemia and recurrent pancreatitis. We measured LPL activity in postheparin plasma and serum ApoCII and sequenced LPL, APOC2, and GPIHBP1. The 2 patients exhibited very low LPL activity not associated with mutations in LPL gene or with ApoCII deficiency. The sequence of GPIHBP1 revealed 2 novel point mutations. One patient (proband 1) was found to be homozygous for a C>A transversion in exon 3 resulting in the conversion of threonine to lysine at position 80 (p.Thr80Lys). The other patient (proband 2) was found to be homozygous for a G>T transversion in the third base of the ATG translation initiation codon in exon 1, resulting in the conversion of methionine to isoleucine (p.Met1Ile). In conclusion, we have identified 2 novel GPIHBP1 missense mutations in 2 unrelated patients as the cause of their severe hypertriglyceridemia. Copyright © 2016 National Lipid Association. Published by Elsevier Inc. All rights reserved.

The in vitro cleavage of the hAtg proteins by cell death proteases.

PubMed

Norman, Joanna M; Cohen, Gerald M; Bampton, Edward T W

2010-11-01

It is becoming increasingly clear that there is crosstalk between the apoptotic and autophagic pathways, with autophagy helping to contribute to cell death by providing energy to allow the energy-requiring programmed cell death process to complete, as well as degrading cellular material in its own right. Recent evidence has suggested that Atg proteins can themselves be targets of caspases, providing potential regulation of autophagy as well as uncovering novel functions for fragments derived from Atg proteins. However, to date there has not been a detailed examination of which Atg proteins may be the targets of which death proteases. We show that the majority of human Atg (hAtg) proteins can be cleaved by calpain 1, which is activated in some apoptotic paradigms, as well as other forms of death. We also show that hAtg3 is cleaved by caspases-3, -6 and -8, hAtg6 (Beclin 1) is cleaved by caspase-3 and -6, while hAtg9, hAtg7 and the hAtg4 homologues can be cleaved by caspase-3. Cleavage of Beclin 1 was also seen in apoptosis of HeLa cells induced by staurosporine and TRAIL, along with cleavage of Atg3 and Atg4C. There were subtle effects of caspase inhibition on GFP-LC3 lipidation but more marked effects on the formation of GFP-LC3 puncta (a marker of autophagosome formation) and p62 degradation, indicating that caspase cleavage of autophagy-related proteins can affect the autophagic process. Notably we show that p62 is a target for caspase-6 and -8 cleavage.

E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

PubMed Central

Gao, Wentao; Chen, Zhixia; Wang, Wei; Stang, Michael T.

2013-01-01

p62 is constitutively degraded by autophagy via its interaction with LC3. However, the interaction of p62 with LC3 species in the context of the LC3 lipidation process is not specified. Further, the p62-mediated protein aggregation’s effect on autophagy is unclear. We systemically analyzed the interactions of p62 with all known Atg proteins involved in LC3 lipidation. We find that p62 does not interact with LC3 at the stages when it is being processed by Atg4B or when it is complexed or conjugated with Atg3. p62 does interact with LC3-I and LC3-I:Atg7 complex and is preferentially recruited by LC3-II species under autophagic stimulation. Given that Atg4B, Atg3 and LC3-Atg3 are indispensable for LC3-II conversion, our study reveals a protective mechanism for Atg4B, Atg3 and LC3-Atg3 conjugate from being inappropriately sequestered into p62 aggregates. Our findings imply that p62 could potentially impair autophagy by negatively affecting LC3 lipidation and contribute to the development of protein aggregate diseases. PMID:24023838

E1-like activating enzyme Atg7 is preferentially sequestered into p62 aggregates via its interaction with LC3-I.

PubMed

Gao, Wentao; Chen, Zhixia; Wang, Wei; Stang, Michael T

2013-01-01

p62 is constitutively degraded by autophagy via its interaction with LC3. However, the interaction of p62 with LC3 species in the context of the LC3 lipidation process is not specified. Further, the p62-mediated protein aggregation's effect on autophagy is unclear. We systemically analyzed the interactions of p62 with all known Atg proteins involved in LC3 lipidation. We find that p62 does not interact with LC3 at the stages when it is being processed by Atg4B or when it is complexed or conjugated with Atg3. p62 does interact with LC3-I and LC3-I:Atg7 complex and is preferentially recruited by LC3-II species under autophagic stimulation. Given that Atg4B, Atg3 and LC3-Atg3 are indispensable for LC3-II conversion, our study reveals a protective mechanism for Atg4B, Atg3 and LC3-Atg3 conjugate from being inappropriately sequestered into p62 aggregates. Our findings imply that p62 could potentially impair autophagy by negatively affecting LC3 lipidation and contribute to the development of protein aggregate diseases.

Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase

PubMed Central

Papinski, Daniel; Schuschnig, Martina; Reiter, Wolfgang; Wilhelm, Larissa; Barnes, Christopher A.; Maiolica, Alessio; Hansmann, Isabella; Pfaffenwimmer, Thaddaeus; Kijanska, Monika; Stoffel, Ingrid; Lee, Sung Sik; Brezovich, Andrea; Lou, Jane Hua; Turk, Benjamin E.; Aebersold, Ruedi; Ammerer, Gustav; Peter, Matthias; Kraft, Claudine

2014-01-01

Summary Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes. PMID:24440502

Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila.

PubMed

Varga, Kata; Nagy, Péter; Arsikin Csordás, Katarina; Kovács, Attila L; Hegedűs, Krisztina; Juhász, Gábor

2016-10-06

Autophagy defects lead to the buildup of damaged proteins and organelles, reduced survival during starvation and infections, hypersensitivity to stress and toxic substances, and progressive neurodegeneration. Here we show that, surprisingly, Drosophila mutants lacking the core autophagy gene Atg16 are not only defective in autophagy but also exhibit increased resistance to the sedative effects of ethanol, unlike Atg7 or Atg3 null mutant flies. This mutant phenotype is rescued by the re-expression of Atg16 in Corazonin (Crz)-producing neurosecretory cells that are known to promote the sedation response during ethanol exposure, and RNAi knockdown of Atg16 specifically in these cells also delays the onset of ethanol-induced coma. We find that Atg16 and Crz colocalize within these neurosecretory cells, and both Crz protein and mRNA levels are decreased in Atg16 mutant flies. Thus, Atg16 promotes Crz production to ensure a proper organismal sedation response to ethanol.

Crystallization and preliminary crystallographic analysis of human Atg4B–LC3 complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Satoo, Kenji; Suzuki, Nobuo N.; Fujioka, Yuko

2007-02-01

Human Atg4B and LC3 were expressed, purified and crystallized as a complex. Diffraction data were collected to a resolution of 1.9 Å. The reversible modification of Atg8 with phosphatidylethanolamine (PE) is crucial for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Atg4 is a cysteine protease that is responsible for the processing and deconjugation of Atg8. Human Atg4B (HsAtg4B; a mammalian orthologue of yeast Atg4) and LC3 (a mammalian orthologue of yeast Atg8) were expressed and purified and two complexes, one consisting of HsAtg4B(1–354) and LC3(1–120) (complex I; the product complex) and the other consisting ofmore » HsAtg4B(1–354) and LC3(1–124) (complex II; the substrate complex), were crystallized using polyethylene glycol 3350 as a precipitant. In both complexes His280 of HsAtg4B was mutated to alanine. The crystals belong to the same space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 47.5, b = 91.8, c = 102.6 Å for complex I and a = 46.9, b = 90.9, c = 102.5 Å for complex II. Diffraction data were collected to a resolution of 1.9 Å from both crystals.« less

Bm65 is essential for the propagation of Bombyx mori nucleopolyhedrovirus.

PubMed

Tang, Qi; Li, Guohui; Yao, Qin; Chen, Liang; Feng, Fan; Yuan, Yi; Chen, Keping

2013-01-01

Orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene that encodes an unknown 104-amino acid protein. In the present study, we have shown the role of Bm65 in the baculovirus life cycle. 5'-RACE analysis showed that the transcription start site of Bm65 was 14 nucleotides upstream of the start codon ATG. The transcription profile of Bm65 was detected from 6 to 72 h postinfection (p. i.) by RT-PCR. A Bm65-knockout bacmid was constructed by homologous recombination to characterize the role of Bm65 in viral life cycle. Fluorescence microscopy showed that Bm65-knockout virus was unable to generate infectious budded virus in BmN cells. Furthermore, quantitative real-time PCR analysis demonstrated that Bm65 deletion did not affect the viral DNA replication. To conclude, Bm65 is essential for the propagation of BmNPV, but is unnecessary for the replication of viral DNA.

[Knockdown of ATG5 enhances the sensitivity of human renal carcinoma cells to sunitinib].

PubMed

Li, Peng; Han, Qi; Tang, Ming; Zhang, Keqin

2017-03-01

Objective To investigate the expression levels of autophagy-related gene 5 (ATG5) and microtubule-associated protein 1 light chain 3 (LC3) and their effects on sunitinib resistance in human renal carcinoma cells. Methods After clinic-pathologic feature and survival analysis, 99 renal clear cell carcinoma tissues with different histological grades were used to detect the expression of ATG5 and LC3 by immunohistochemistry. Renal carcinoma cell line A-498 was infected with lentivirus-mediated ATG5 shRNA. Western blot analysis was performed to confirm the efficiency of ATG5 knockdown. Proliferation rate of A-498 cells in control group and ATG5 low expression group was determined by flow cytometry. Finally, the survival rate was detected by MTT assay after A-498 cells were treated with different concentrations of sunitinib. Results The expression levels of ATG5 and LC3 in renal clear cell carcinoma tissues were significantly higher than those in para-tumor tissues. The expression levels of ATG5 and LC3 were associated with classification, histological grade, TNM stage and survival rate, rather than gender, age, location, tumor size. Compared with the control group, the protein expressions of ATG5 and LC3 significantly decreased in A-498 cells with ATG5 low expression. The cell proliferation rate in ATG5 downregulation group was lower than that in the control group. Compared with control group, the survival rate in ATG5 low expression group were significantly reduced in a dose-dependent manner after sunitinib treatment. Conclusion Autophagy is active in renal clear cell carcinoma, and the drug sensitivity to sunitinib in renal cancer cells can be enhanced by the downregulation of ATG5.

Detection of a novel specificity (CTLA-4) in ATG/TMG globulins and sera from ATG-treated leukemic patients.

PubMed

Pistillo, Maria Pia; Tazzari, Pier Luigi; Bonifazi, Francesca; Bandini, Giuseppe; Kato, Tomohiro; Matsui, Toshihiro; Nishioka, Kusuki; Conte, Roberto; Ferrara, Giovanni Battista

2002-04-27

T-cell costimulation has been shown to provide positive signals for T-cell activation and generation of effector activity. In this study, we analyzed the presence of antibodies (Abs) against the T-lymphocyte costimulatory molecules CD28, CTLA-4, CD80, and CD86 in anti-T-lymphocyte (ATG) and antithymocyte (TMG) globulin preparations to address their mechanism of action. We focused our attention on the role of CTLA-4-specific Abs in the immunosuppressive effect of ATG/TMG, because anti-CTLA-4 agonistic Abs may suppress T-cell proliferation and nonagonistic Abs may lead to T-cell depletion through an Ab-dependent cell cytotoxicity mechanism. ATG/TMG and patients' sera were tested for binding to recombinant human costimulatory molecules by ELISA techniques. CTLA-4 specificity was also analyzed by cytoplasmic immunofluorescence staining of a CTLA-4 transfectant by competitive inhibition immunofluorescence and by cell proliferation assay in allogeneic mixed lymphocyte reaction (MLR). Either ATG or TMG predominantly contained anti-CTLA-4 Abs, with higher reactivity in ATG followed by anti-CD86 and -CD28 Abs, whereas anti-CD80 Abs were found only in ATG. Anti-CTLA-4 Abs present in ATG/TMG recognized the native form of CTLA-4 molecule, and their removal reduced the effect of ATG in an allogeneic MLR. Kinetic studies indicated that such Abs were present in the sera of 12 ATG-treated leukemic patients up to 21 days after ATG administration. These data suggest that the novel anti-CTLA-4 Abs found in ATG may greatly contribute to its immunosuppressive effect, thus accounting for the absence of rejection and exceptionally low incidence of graft-versus-host disease in the group of patients analyzed.

A unique role for autophagy and Atg16L1 in Paneth cells in murine and human intestine

PubMed Central

Cadwell, Ken; Liu, John; Brown, Sarah L.; Miyoshi, Hiroyuki; Loh, Joy; Lennerz, Jochen; Kishi, Chieko; KC, Wumesh; Carrero, Javier A.; Hunt, Steven; Stone, Christian; Brunt, Elizabeth M.; Xavier, Ramnik J.; Sleckman, Barry P.; Li, Ellen; Mizushima, Noboru; Stappenbeck, Thaddeus S.; Virgin, Herbert W.

2008-01-01

Susceptibility to Crohn's disease (CD), a complex inflammatory disease involving the small intestine, is controlled by up to 32 loci1. One CD risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG162. It is not known how Atg16L1 or autophagy contributes to intestinal biology or CD pathogenesis. To address these questions we generated and characterized mice that are hypomorphic for Atg16L1 protein expression, and validated conclusions based on studies in these mice by analyzing intestinal tissues that we collected from CD patients carrying the CD risk allele of ATG16L1. We show that Atg16L1 is a bona fide autophagy protein. Within the ileal epithelium, both Atg16L1 and a second essential autophagy protein Atg5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell which functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment3. Atg16L1 and Atg5-deficient Paneth cells exhibited striking abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to Atg16L1-deficient Paneth cells including increased expression of genes involved in PPAR signaling and lipid metabolism, acute phase reactants, as well as two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, CD patients homozygous for the ATG16L1 CD risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy protein-deficient mice and expressed increased levels of leptin protein. Thus, Atg16L1, and likely the process of autophagy, play their role within the intestinal epithelium of mice and CD patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells. PMID:18849966

Molecular cloning and characterization of autophagy-related gene TmATG8 in Listeria-invaded hemocytes of Tenebrio molitor.

PubMed

Tindwa, Hamisi; Jo, Yong Hun; Patnaik, Bharat Bhusan; Lee, Yong Seok; Kang, Sang Sun; Han, Yeon Soo

2015-07-01

Macroautophagy (hereinafter called autophagy) is a highly regulated process used by eukaryotic cells to digest portions of the cytoplasm that remodels and recycles nutrients and disposes of unwanted cytoplasmic constituents. Currently 36 autophagy-related genes (ATG) and their homologs have been characterized in yeast and higher eukaryotes, including insects. In the present study, we identified and functionally characterized the immune function of an ATG8 homolog in a coleopteran insect, Tenebrio molitor (TmATG8). The cDNA of TmATG8 comprises of an ORF of 363 bp that encodes a protein of 120 amino acid residues. TmATG8 transcripts are detected in all the developmental stages analyzed. TmAtg8 protein contains a highly conserved C-terminal glycine residue (Gly116) and shows high amino acid sequence identity (98%) to its Tribolium castaneum homolog, TcAtg8. Loss of function of TmATG8 by RNAi led to a significant increase in the mortality rates of T. molitor larvae against Listeria monocytogenes. Unlike dsEGFP-treated control larvae, TmATG8-silenced larvae failed to turn-on autophagy in hemocytes after injection with L. monocytogenes. These data suggest that TmATG8 play a role in mediating autophagy-based clearance of Listeria in T. molitor. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of novel tubing gait on neuromuscular imbalance in cerebral palsy.

PubMed

Shin, Yoon Kyum; Lee, Dong Ryul; Kim, Do Hyun; Lee, Jae Jin; You, Sung Joshua Hyun; Yi, Chung Hwi; Jeon, Hye Seon

2014-01-01

Gait impairments from a neuromuscular imbalance are crucial issues in cerebral palsy. The purpose of our study was to compare the effects of the assistive tubing gait (ATG) and assistive-resistive tubing gait (ARTG) on improving the vasti and hamstring muscle imbalance during the initial contact to mid-stance phases in individuals with spastic diplegic cerebral palsy (CP). Fourteen age-matched individuals including seven normal individuals (11.7 years) and seven individuals with CP (12.9 years) were recruited. All participants underwent electromyography (EMG) measurement of the unilateral vasti and hamstring muscle activity during the three gait training conditions of no-tubing gait (NTG), ATG, and ARTG. A statistical one-way repeated-measure analysis of variance (ANOVA) was used to determine differences in the vasti and hamstring activity, the vasti/hamstring ratio, and the knee joint angle across the three gait training conditions for each group. The initial vasti and hamstring muscle imbalance in CP was significantly improved by applying the ARTG compared with the ATG. The vasti/hamstring ratio during the ARTG was compatible with the ratio value obtained from the NTG of normal individuals. The knee joint angle in CP was not improved in this short-term intervention. The ARTG proportionately increased the vasti activation and reciprocally inhibited the hamstring activity, subsequently improving the neuromuscular imbalance associated with the flexed-knee gait in individuals with spastic diplegic CP.

Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast.

PubMed

Matsuhara, Hirotada; Yamamoto, Ayumu

2016-01-01

Autophagy is a conserved intracellular degradation system, which contributes to development and differentiation of various organisms. Yeast cells undergo meiosis under nitrogen-starved conditions and require autophagy for meiosis initiation. However, the precise roles of autophagy in meiosis remain unclear. Here, we show that autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Autophagy-defective strains bearing a mutation in the autophagy core factor gene atg1, atg7, or atg14 exhibit deformed nuclear structures during meiosis. These mutant cells require an extracellular nitrogen supply for meiosis progression following their entry into meiosis and show delayed meiosis progression even with a nitrogen supply. In addition, they show frequent chromosome dissociation from the spindle together with spindle overextension, forming extra nuclei. Furthermore, Aurora kinase, which regulates chromosome segregation and spindle elongation, is significantly increased at the centromere and spindle in the mutant cells. Aurora kinase down-regulation eliminated delayed initiation of meiosis I and II, chromosome dissociation, and spindle overextension, indicating that increased Aurora kinase activity may cause these aberrances in the mutant cells. Our findings show a hitherto unrecognized relationship of autophagy with the nuclear structure, regulation of cell cycle progression, and chromosome segregation in meiosis. © 2015 The Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

ATG proteins: Are we always looking at autophagy?

PubMed

Mauthe, Mario; Reggiori, Fulvio

2016-12-01

Autophagy is an intracellular degradation pathway that is regulated by the autophagy-related (ATG) proteins. For a long time it has been thought that ATG proteins were exclusively required for autophagy, but recent experimental evidence has revealed that these proteins are part of other cellular pathways, individually or as a functional group. To estimate the extent of these so-called unconventional functions of the ATG proteins, we decided to perform an unbiased siRNA screen targeting the entire ATG proteome and used viral replication as the readout. Our results have uncovered that a surprisingly high number of ATG proteins (36%) have a positive or negative role in promoting virus replication outside their classical role in autophagy. With the increasing knowledge about ATG protein unconventional functions and our investigation results, the interpretations about the possible involvement of autophagy in cellular or organismal functions that solely rely on the depletion of a single ATG protein, should be considered cautiously.

Accuracy of genetic code translation and its orthogonal corruption by aminoglycosides and Mg2+ ions

PubMed Central

Zhang, Jingji

2018-01-01

Abstract We studied the effects of aminoglycosides and changing Mg2+ ion concentration on the accuracy of initial codon selection by aminoacyl-tRNA in ternary complex with elongation factor Tu and GTP (T3) on mRNA programmed ribosomes. Aminoglycosides decrease the accuracy by changing the equilibrium constants of ‘monitoring bases’ A1492, A1493 and G530 in 16S rRNA in favor of their ‘activated’ state by large, aminoglycoside-specific factors, which are the same for cognate and near-cognate codons. Increasing Mg2+ concentration decreases the accuracy by slowing dissociation of T3 from its initial codon- and aminoglycoside-independent binding state on the ribosome. The distinct accuracy-corrupting mechanisms for aminoglycosides and Mg2+ ions prompted us to re-interpret previous biochemical experiments and functional implications of existing high resolution ribosome structures. We estimate the upper thermodynamic limit to the accuracy, the ‘intrinsic selectivity’ of the ribosome. We conclude that aminoglycosides do not alter the intrinsic selectivity but reduce the fraction of it that is expressed as the accuracy of initial selection. We suggest that induced fit increases the accuracy and speed of codon reading at unaltered intrinsic selectivity of the ribosome. PMID:29267976

Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori

PubMed Central

Yang, Xiaobing; Wu, Suli; Wu, Yongpeng; Liu, Yang; Qian, Yonghua; Jiao, Feng

2015-01-01

Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus. PMID:26163666

Tuning of Recombinant Protein Expression in Escherichia coli by Manipulating Transcription, Translation Initiation Rates, and Incorporation of Noncanonical Amino Acids.

PubMed

Schlesinger, Orr; Chemla, Yonatan; Heltberg, Mathias; Ozer, Eden; Marshall, Ryan; Noireaux, Vincent; Jensen, Mogens Høgh; Alfonta, Lital

2017-06-16

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the Eschrichia coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. On the basis of the results, we developed a biophysical model, which suggests that the density of co-transcriptional-translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

[Organization and expression of poliovirus genome].

PubMed

Vevcherenko, S G

1984-01-01

In the present paper on the basis of analysis of literary data it is postulated that along with the AUG codon at N743 there exists a second initiation codon in the poliovirus RNA (the AUG codon at N586). The translation initiated at N586 can be transferred to the phase of the major reading frame by removing the small hairpin N732-N744 formed near the first initiation site, or by removing the small region N739-N745. In the first case at the boundary between the hypothetical leader peptide encoded by the 5'-terminus of the long, open reading frame of the spliced poliovirus RNA and the capsid protein VP4 must be the Gln-Gly proteolytic cleavage signal, and in the second case--the Tyr-Gly signal. In both cases the leader peptide can be chipped off by the virus specific proteinase. It is supposed that the exon-intronic structure of the poliovirus genome is needed for coordination of translation and transcription during the poliovirus reproduction cycle.

Characterization of Atg38 and NRBF2, a fifth subunit of the autophagic Vps34/PIK3C3 complex

PubMed Central

Ohashi, Yohei; Soler, Nicolas; García Ortegón, Miguel; Zhang, Lufei; Kirsten, Marie L.; Perisic, Olga; Masson, Glenn R.; Burke, John E.; Jakobi, Arjen J.; Apostolakis, Apostolos A.; Johnson, Christopher M.; Ohashi, Maki; Ktistakis, Nicholas T.; Sachse, Carsten; Williams, Roger L.

2016-01-01

ABSTRACT The phosphatidylinositol 3-kinase Vps34 is part of several protein complexes. The structural organization of heterotetrameric complexes is starting to emerge, but little is known about organization of additional accessory subunits that interact with these assemblies. Combining hydrogen-deuterium exchange mass spectrometry (HDX-MS), X-ray crystallography and electron microscopy (EM), we have characterized Atg38 and its human ortholog NRBF2, accessory components of complex I consisting of Vps15-Vps34-Vps30/Atg6-Atg14 (yeast) and PIK3R4/VPS15-PIK3C3/VPS34-BECN1/Beclin 1-ATG14 (human). HDX-MS shows that Atg38 binds the Vps30-Atg14 subcomplex of complex I, using mainly its N-terminal MIT domain and bridges the coiled-coil I regions of Atg14 and Vps30 in the base of complex I. The Atg38 C-terminal domain is important for localization to the phagophore assembly site (PAS) and homodimerization. Our 2.2 Å resolution crystal structure of the Atg38 C-terminal homodimerization domain shows 2 segments of α-helices assembling into a mushroom-like asymmetric homodimer with a 4-helix cap and a parallel coiled-coil stalk. One Atg38 homodimer engages a single complex I. This is in sharp contrast to human NRBF2, which also forms a homodimer, but this homodimer can bridge 2 complex I assemblies. PMID:27630019

Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora

PubMed Central

Werner, Antonia; Herzog, Britta; Frey, Stefan; Pöggeler, Stefanie

2016-01-01

In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12. PMID:27309377

Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

PubMed

Werner, Antonia; Herzog, Britta; Frey, Stefan; Pöggeler, Stefanie

2016-01-01

In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.

Variable expressivity of familial medullary thyroid carcinoma (FMTC) due to a RET V804M (GTG-->ATG) mutation.

PubMed

Feldman, G L; Edmonds, M W; Ainsworth, P J; Schuffenecker, I; Lenoir, G M; Saxe, A W; Talpos, G B; Roberson, J; Petrucelli, N; Jackson, C E

2000-07-01

Multiple endocrine neoplasia type 2 (MEN 2) and familial medullary thyroid carcinoma (FMTC) are autosomal dominantly inherited cancer syndromes that predispose to C-cell hyperplasia and MTC. MEN 2A and FMTC are caused by mutations in the RET proto-oncogene. We used a multiplex polymerase chain reaction-based assay to screen exons 10, 11, 13, and 14 of RET for mutations in 2 families with FMTC. We correlated mutation status with calcitonin and pathologic studies to determine genotype-phenotype correlations. We identified a mutation in codon 804 in exon 14 (GTG-->ATG; V804M) in both families. An 86-year-old person who was a gene carrier and other individuals over age 70 who were suspected by pedigree analysis to be gene carriers had no overt clinical evidence of MTC. Four of 21 patients who underwent a thyroidectomy also had papillary thyroid cancer. One individual in each family had metastatic MTC at age 30 and 32 years, and all 26 people having thyroidectomies had either MTC or C-cell hyperplasia, leading us to continue to recommend prophylactic thyroidectomy for all identified patients who were gene carriers. Because of active MTC in younger members of these families, including metastases, we have continued to advocate thyroid surgery in mutation-positive individuals. While DNA diagnosis of gene carriers and subsequent genetic counseling was relatively straightforward, the acceptance of surgical recommendations was more difficult for some individuals. These families demonstrate that the search for RET mutations should include exons 13, 14, 15, and 16 in patients whose studies in exons 10 and 11 are negative.

Atg1 kinase regulates early and late steps during autophagy

PubMed Central

Kijanska, Monika; Peter, Matthias

2013-01-01

The notion that phosphorylation constitutes a major mechanism to induce autophagy was established 15 years ago when a conserved Atg1/ULK kinase family was identified as an essential component of the autophagy machinery. The key observation was that starved atg1Δ cells lack autophagosomes in the cytosol and fail to accumulate autophagic bodies in the vacuole. Although many studies have revealed important details of Atg1 activation and function, a cohesive model for how Atg1 regulates the autophagic machinery is lacking. Our recent findings identified conserved steps of temporal and spatial regulation of Atg1/ULK1 kinase at both the PAS and autophagosomal membranes, suggesting that Atg1 not only promotes autophagy induction, but may also facilitate late stages of autophagosome biogenesis. PMID:23108207

Polyclonal antithymocyte globulin and cardiovascular disease in kidney transplant recipients.

PubMed

Ducloux, Didier; Courivaud, Cécile; Bamoulid, Jamal; Crepin, Thomas; Chalopin, Jean-Marc; Tiberghien, Pierre; Saas, Philippe

2014-06-01

T-lymphocyte activation may contribute to atherosclerosis, the prevalence of which is increased in transplant patients. However, the cardiovascular consequences of polyclonal antithymocyte globulin (ATG)-induced immune modifications, which include alterations in T-cell subsets, are unknown. We conducted a retrospective single-center study to assess whether ATG associates with an increased incidence of atherosclerotic events (CVEs) in kidney transplant patients. Propensity score analysis was performed to address potential confounding by indication. We also tested whether ATG use induces a proatherogenic immune status. Sixty-nine (12.2%) CVEs occurred during follow-up (87±31 months). The cumulative incidence of CVEs was higher in ATG-treated patients (14.7% versus 8.2%; P=0.03). Cox regression analysis revealed that ATG use was an independent risk factor for CVEs (hazard ratio [HR], 2.36; 95% confidence interval [95% CI], 1.35 to 4.13; P=0.003). Results obtained in the propensity score match analysis recapitulated those obtained from the overall cohort (HR, 2.09; 95% CI, 1.11 to 3.98; P=0.02). Late-stage differentiated CD8(+) T cells increased 1 year after transplantation only in ATG-treated patients. More generally, ATG associated with features of immune activation. These modifications increased markedly in patients exposed to cytomegalovirus (CMV). Subanalyses suggest that the effect of ATG on CVEs is restricted to CMV-exposed patients. However, CMV infection associated significantly with CVEs only in ATG-treated patients (HR, 2.07; 95% CI, 1.16 to 3.70; P=0.01). In conclusion, ATG associated with both immune activation and post-transplant CVEs in this cohort. Further studies should precisely determine whether ATG-induced immune activation is the causal link between ATG and CVEs. Copyright © 2014 by the American Society of Nephrology.

Polyclonal Antithymocyte Globulin and Cardiovascular Disease in Kidney Transplant Recipients

PubMed Central

Courivaud, Cécile; Bamoulid, Jamal; Crepin, Thomas; Chalopin, Jean-Marc; Tiberghien, Pierre; Saas, Philippe

2014-01-01

T-lymphocyte activation may contribute to atherosclerosis, the prevalence of which is increased in transplant patients. However, the cardiovascular consequences of polyclonal antithymocyte globulin (ATG)–induced immune modifications, which include alterations in T-cell subsets, are unknown. We conducted a retrospective single-center study to assess whether ATG associates with an increased incidence of atherosclerotic events (CVEs) in kidney transplant patients. Propensity score analysis was performed to address potential confounding by indication. We also tested whether ATG use induces a proatherogenic immune status. Sixty-nine (12.2%) CVEs occurred during follow-up (87±31 months). The cumulative incidence of CVEs was higher in ATG-treated patients (14.7% versus 8.2%; P=0.03). Cox regression analysis revealed that ATG use was an independent risk factor for CVEs (hazard ratio [HR], 2.36; 95% confidence interval [95% CI], 1.35 to 4.13; P=0.003). Results obtained in the propensity score match analysis recapitulated those obtained from the overall cohort (HR, 2.09; 95% CI, 1.11 to 3.98; P=0.02). Late-stage differentiated CD8+ T cells increased 1 year after transplantation only in ATG-treated patients. More generally, ATG associated with features of immune activation. These modifications increased markedly in patients exposed to cytomegalovirus (CMV). Subanalyses suggest that the effect of ATG on CVEs is restricted to CMV-exposed patients. However, CMV infection associated significantly with CVEs only in ATG-treated patients (HR, 2.07; 95% CI, 1.16 to 3.70; P=0.01). In conclusion, ATG associated with both immune activation and post-transplant CVEs in this cohort. Further studies should precisely determine whether ATG-induced immune activation is the causal link between ATG and CVEs. PMID:24511120

A small-molecule activator induces ULK1-modulating autophagy-associated cell death in triple negative breast cancer.

PubMed

Ouyang, Liang; Zhang, Lan; Fu, Leilei; Liu, Bo

2017-04-03

ULK1 (unc-51 like autophagy activating kinase 1) is well known to be required to initiate the macroautophagy/autophagy process, and thus activation of ULK1-modulating autophagy/autophagy-associated cell death (ACD) may be a possible therapeutic strategy in triple negative breast cancer (TNBC). Here, our integrated The Cancer Genome Atlas (TCGA) data set, tissue microarray-based analyses and multiple biologic evaluations together demonstrate a new small-molecule activator of ULK1 for better understanding of how ULK1, the mammalian homolog of yeast Atg1, as a potential drug target can regulate ACD by the ULK complex (ULK1-ATG13-RB1CC1/FIP200-ATG101), as well as other possible ULK1 interactors, including ATF3, RAD21 and CASP3/caspase3 in TNBC. Moreover, such new inspiring findings may help us discover that this activator of ULK1 (LYN-1604) with its anti-tumor activity and ACD-modulating mechanisms can be further exploited as a small-molecule candidate drug for future TNBC therapy.

RNA editing makes mistakes in plant mitochondria: editing loses sense in transcripts of a rps19 pseudogene and in creating stop codons in coxI and rps3 mRNAs of Oenothera.

PubMed Central

Schuster, W; Brennicke, A

1991-01-01

An intact gene for the ribosomal protein S19 (rps19) is absent from Oenothera mitochondria. The conserved rps19 reading frame found in the mitochondrial genome is interrupted by a termination codon. This rps19 pseudogene is cotranscribed with the downstream rps3 gene and is edited on both sides of the translational stop. Editing, however, changes the amino acid sequence at positions that were well conserved before editing. Other strange editings create translational stops in open reading frames coding for functional proteins. In coxI and rps3 mRNAs CGA codons are edited to UGA stop codons only five and three codons, respectively, downstream to the initiation codon. These aberrant editings in essential open reading frames and in the rps19 pseudogene appear to have been shifted to these positions from other editing sites. These observations suggest a requirement for a continuous evolutionary constraint on the editing specificities in plant mitochondria. Images PMID:1762921

The Andes hantavirus NSs protein is expressed from the viral small mRNA by a leaky scanning mechanism.

PubMed

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P; Pino, Karla; Tischler, Nicole D; Ohlmann, Théophile; Darlix, Jean-Luc; López-Lastra, Marcelo

2012-02-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism.

The Andes Hantavirus NSs Protein Is Expressed from the Viral Small mRNA by a Leaky Scanning Mechanism

PubMed Central

Vera-Otarola, Jorge; Solis, Loretto; Soto-Rifo, Ricardo; Ricci, Emiliano P.; Pino, Karla; Tischler, Nicole D.; Ohlmann, Théophile; Darlix, Jean-Luc

2012-01-01

The small mRNA (SmRNA) of all Bunyaviridae encodes the nucleocapsid (N) protein. In 4 out of 5 genera in the Bunyaviridae, the smRNA encodes an additional nonstructural protein denominated NSs. In this study, we show that Andes hantavirus (ANDV) SmRNA encodes an NSs protein. Data show that the NSs protein is expressed in the context of an ANDV infection. Additionally, our results suggest that translation initiation from the NSs initiation codon is mediated by ribosomal subunits that have bypassed the upstream N protein initiation codon through a leaky scanning mechanism. PMID:22156529

A Mutation in the Start Codon of γ-Crystallin D Leads to Nuclear Cataracts in the Dahl SS/Jr-Ctr Strain

PubMed Central

Johnson, Ashley C.; Lee, Jonathan W.; Harmon, Ashlyn C.; Morris, Zaliya; Wang, Xuexiang; Fratkin, Jonathan; Rapp, John P.; Gomez-Sanchez, Elise; Garrett, Michael R.

2013-01-01

Cataracts are a major cause of blindness. The most common forms of cataracts are age and UV related and develops mostly in the elderly, while congenital cataracts appear at birth or in early childhood. The Dahl salt-sensitive (SS/Jr) rat is an extensively used model of salt-sensitive hypertension that exhibits concomitant renal disease. In the mid 1980’s, cataracts appeared in a few animals in the Dahl S colony, presumably the result of a spontaneous mutation. The mutation was fixed and bred to establish the SS/Jr-Ctr substrain. The SS/Jr-Ctr substrain has been exclusively used by a single investigator to study the role of steroids and hypertension. Using a classical positional cloning approach, we localized the cataract gene with high-resolution to a less than 1 Mbp region on chromosome 9 using an F1 (SS/Jr-Ctr X SHR) X SHR backcross population. The 1 Mbp region contained only 13 genes, including 4 genes from the γ-crystallins (Cryg) gene family which are known to play a role in cataract formation. All of the γ-crystallins were sequenced and a novel point mutation in the start codon (ATG → GTG) of the Crygd gene was identified which led to the complete absence of CRYGD protein in the eyes of the SS/Jr-Ctr strain. In summary, the identification of the genetic cause in this novel cataract model may provide an opportunity to better understand the development of cataracts, particularly in the context of hypertension. PMID:23404175

Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

PubMed

Vogt, Michael; Haas, Sabine; Polen, Tino; van Ooyen, Jan; Bott, Michael

2015-03-01

2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1)  h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Atg22p, a vacuolar membrane protein involved in the amino acid compartmentalization of Schizosaccharomyces pombe.

PubMed

Sugimoto, Naoko; Iwaki, Tomoko; Chardwiriyapreecha, Soracom; Shimazu, Masamitsu; Kawano, Miyuki; Sekito, Takayuki; Takegawa, Kaoru; Kakinuma, Yoshimi

2011-01-01

The fission yeast Schizosaccharomyces pombe has a homolog of the budding yeast Atg22p, which is involved in spore formation (Mukaiyama H. et al., Microbiology, 155, 3816-3826 (2009)). GFP-tagged Atg22p in the fission yeast was localized to the vacuolar membrane. Upon disruption of atg22, the amino acid levels of the cellular fraction as well as the vacuolar fraction decreased. The uptake of several amino acids, such as lysine, histidine, and arginine, was impaired in atg22Δ cells. S. pombe Atg22p plays an important role in the compartmentalization of amino acids.

Gas exchange and lactate anaerobic thresholds: inter- and intraevaluator agreement.

PubMed

Gladden, L B; Yates, J W; Stremel, R W; Stamford, B A

1985-06-01

Twenty-four coded graph sets of gas exchange variables and blood lactate concentration (LA) plotted against time at 15-s intervals were analyzed by nine evaluators who determined the gas exchange (ATGE) and LA (ATLA) anaerobic thresholds. In addition, ATGE and ATLA were determined by a linear regression computer program. Agreement between ATGE and ATLA was poor; the median intraclass correlation coefficient (ri) was 0.53. Among evaluators, ATLA agreement (median ri = 0.81) was better than ATGE agreement (median ri = 0.70). In general, the ability of any evaluator to choose similar values from duplicate plots for either ATGE (median ri = 0.97) or ATLA (median ri = 0.995) was good. There was better agreement between the mean ATLA of the evaluators and the computer ATLA (ComLA) (ri = 0.88) than between the mean ATGE of the evaluators and the computer ATGE (ComVE), (ri = 0.58). Agreement between ComVE and ComLA was poor (ri = 0.29). These results suggest that ATGE does not accurately predict ATLA and that different evaluators choose different thresholds from the same data. Further assessment of the validity and precision of ATGE based on breath-by-breath and minute-by-minute data is needed.

Lack of Connection Between Midgut Cell Autophagy Gene Expression and BmCPV Infection in the Midgut of Bombyx mori.

PubMed

Yang, Xiaobing; Wu, Suli; Wu, Yongpeng; Liu, Yang; Qian, Yonghua; Jiao, Feng

2015-01-01

Autophagy is associated with multiple biological processes and has protective and defensive functions with respect to immunity, inflammation, and resistance to microbial infection. In this experiment, we wished to investigate whether autophagy is a factor in the midgut cell response of Bombyx mori to infection by the B. mori cytoplasmic polyhedrosis virus (BmCPV). Our results indicated that the expression of three autophagy-related genes (BmAtg8, BmAtg5, and BmAtg7) in the midgut did not change greatly after BmCPV infection in B. mori. Basal ATG8/ATG8PE protein expression was detected in different B. mori tissues by using western blot analysis. Immunohistochemistry showed that the ATG8/ATG8PE proteins were located mainly in the cytoplasm. ATG8/ATG8PE protein levels decreased at 12 and 16 h after BmCPV infection. Our results indicate that autophagy responded slightly to BmCPV infection, but could not prevent the invasion and replication of the virus. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.

The Autophagy Gene BcATG8 Regulates the Vegetative Differentiation and Pathogenicity of Botrytis cinerea.

PubMed

Ren, Weichao; Liu, Na; Sang, Chengwei; Shi, Dongya; Zhou, Mingguo; Chen, Changjun; Qin, Qingming; Chen, Wenchan

2018-06-01

Autophagy is a conserved degradation process that maintains intracellular homeostasis to ensure normal cell differentiation and development in eukaryotes. ATG8 is one of the key molecular components of the autophagy pathway. In this study, we identified and characterized BcATG8 , a homologue of Saccharomyces cerevisiae (yeast) ATG8 in the necrotrophic plant pathogen Botrytis cinerea Yeast complementation experiments demonstrated that BcATG8 can functionally complement the defects of the yeast ATG8 null mutant. Direct physical interaction between BcAtg8 and BcAtg4 was detected in the yeast two-hybrid system. Subcellular localization assays showed that green fluorescent protein-tagged BcAtg8 (GFP-BcAtg8) localized in the cytoplasm as preautophagosomal structures (PAS) under general conditions but mainly accumulated in the lumen of vacuoles in the case of autophagy induction. Deletion of BcATG8 (Δ BcAtg8 mutant) blocked autophagy and significantly impaired mycelial growth, conidiation, sclerotial formation, and virulence. In addition, the conidia of the Δ BcAtg8 mutant contained fewer lipid droplets (LDs), and quantitative real-time PCR (qRT-PCR) assays revealed that the basal expression levels of the LD metabolism-related genes in the mutant were significantly different from those in the wild-type (WT) strain. All of these phenotypic defects were restored by gene complementation. These results indicate that BcATG8 is essential for autophagy to regulate fungal development, pathogenesis, and lipid metabolism in B. cinerea IMPORTANCE The gray mold fungus Botrytis cinerea is an economically important plant pathogen with a broad host range. Although there are fungicides for its control, many classes of fungicides have failed due to its genetic plasticity. Exploring the fundamental biology of B. cinerea can provide the theoretical basis for sustainable and long-term disease management. Autophagy is an intracellular process for degradation and recycling of cytosolic materials in eukaryotes and is now known to be vital for fungal life. Here, we report studies of the biological role of the autophagy gene BcATG8 in B. cinerea The results suggest that autophagy plays a crucial role in vegetative differentiation and virulence of B. cinerea . Copyright © 2018 American Society for Microbiology.

Mitochondrial genome of the sweet potato hornworm, Agrius convolvuli (Lepidoptera: Sphingidae), and comparison with other Lepidoptera species.

PubMed

Dai, Li-Shang; Li, Sheng; Yu, Hui-Min; Wei, Guo-Qing; Wang, Lei; Qian, Cen; Zhang, Cong-Fen; Li, Jun; Sun, Yu; Zhao, Yue; Zhu, Bao-Jian; Liu, Chao-Liang

2017-02-01

In the present study, we sequenced the complete mitochondrial genome (mitogenome) of Agrius convolvuli (Lepidoptera: Sphingidae) and compared it with previously sequenced mitogenomes of lepidopteran species. The mitogenome was a circular molecule, 15 349 base pairs (bp) long, containing 37 genes. The order and orientation of genes in the A. convolvuli mitogenome were similar to those in sequenced mitogenomes of other lepidopterans. All 13 protein-coding genes (PCGs) were initiated by ATN codons, except for the cytochrome c oxidase subunit 1 (cox1) gene, which seemed to be initiated by the codon CGA, as observed in other lepidopterans. Three of the 13 PCGs had the incomplete termination codon T, while the remainder terminated with TAA. Additionally, the codon distributions of the 13 PCGs revealed that Asn, Ile, Leu2, Lys, Phe, and Tyr were the most frequently used codon families. All transfer RNAs were folded into the expected cloverleaf structure except for tRNA Ser (AGN), which lacked a stable dihydrouridine arm. The length of the adenine (A) + thymine (T)-rich region was 331 bp. This region included the motif ATAGA followed by a 19-bp poly-T stretch and a microsatellite-like (TA) 8 element next to the motif ATTTA. Phylogenetic analyses (maximum likelihood and Bayesian methods) showed that A. convolvuli belongs to the family Sphingidae.

Complete mitochondrial genome of Chocolate Pansy, Junonia iphita (Lepidoptera: Nymphalidae: Nymphalinae).

PubMed

Vanlalruati, Catherine; Mandal, Surajit De; Gurusubramanian, Guruswami; Senthil Kumar, Nachimuthu

2016-07-01

The complete mitochondrial genome of Junonia iphita was determined to be 15,433 bp in length, including 37 typical mitochondrial genes and an AT-rich region. All the protein coding genes (PCGs) are initiated by typical ATN codons, except cox1 gene that is by CGA codon. Eight genes use complete termination codon (TAA), whereas the cox1, cox2 and nad5 genes end with single T; nad4 and nad1 ends with stop codon TA. All the tRNA show secondary cloverleaf structures except trnS1 (AGN). The A + T rich region is 546 bp in length containing ATAGA motif followed by a 18 bp poly-T stretch, two microsatellite-like (TA)9 elements and 8 bp poly-A stretch immediately upstream of trnM gene.

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

Mutation in ATG5 reduces autophagy and leads to ataxia with developmental delay.

PubMed

Kim, Myungjin; Sandford, Erin; Gatica, Damian; Qiu, Yu; Liu, Xu; Zheng, Yumei; Schulman, Brenda A; Xu, Jishu; Semple, Ian; Ro, Seung-Hyun; Kim, Boyoung; Mavioglu, R Nehir; Tolun, Aslıhan; Jipa, Andras; Takats, Szabolcs; Karpati, Manuela; Li, Jun Z; Yapici, Zuhal; Juhasz, Gabor; Lee, Jun Hee; Klionsky, Daniel J; Burmeister, Margit

2016-01-26

Autophagy is required for the homeostasis of cellular material and is proposed to be involved in many aspects of health. Defects in the autophagy pathway have been observed in neurodegenerative disorders; however, no genetically-inherited pathogenic mutations in any of the core autophagy-related (ATG) genes have been reported in human patients to date. We identified a homozygous missense mutation, changing a conserved amino acid, in ATG5 in two siblings with congenital ataxia, mental retardation, and developmental delay. The subjects' cells display a decrease in autophagy flux and defects in conjugation of ATG12 to ATG5. The homologous mutation in yeast demonstrates a 30-50% reduction of induced autophagy. Flies in which Atg5 is substituted with the mutant human ATG5 exhibit severe movement disorder, in contrast to flies expressing the wild-type human protein. Our results demonstrate the critical role of autophagy in preventing neurological diseases and maintaining neuronal health.

Zonda is a novel early component of the autophagy pathway in Drosophila

PubMed Central

Melani, Mariana; Valko, Ayelén; Romero, Nuria M.; Aguilera, Milton O.; Acevedo, Julieta M.; Bhujabal, Zambarlal; Perez-Perri, Joel; de la Riva-Carrasco, Rocío V.; Katz, Maximiliano J.; Sorianello, Eleonora; D’Alessio, Cecilia; Juhász, Gabor; Johansen, Terje; Colombo, María I.; Wappner, Pablo

2017-01-01

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum–associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation. PMID:28904211

Induction of Autophagy interferes the tachyzoite to bradyzoite transformation of Toxoplasma gondii.

PubMed

Li, Xiangzhi; Chen, Di; Hua, Qianqian; Wan, Yujing; Zheng, Lina; Liu, Yangyang; Lin, Jiaxin; Pan, Changwang; Hu, Xin; Tan, Feng

2016-04-01

Autophagy process in Toxoplasma gondii plays a vital role in regulating parasite survival or death. Thus, once having an understanding of certain effects of autophagy on the transformation of tachyzoite to bradyzoite this will allow us to elucidate the function of autophagy during parasite development. Herein, we used three TgAtg proteins involved in Atg8 conjugation system, TgAtg3, TgAtg7 and TgAtg8 to evaluate the autophagy level in tachyzoite and bradyzoite of Toxoplasma in vitro based on Pru TgAtg7-HA transgenic strains. We showed that both TgAtg3 and TgAtg8 were expressed at a significantly lower level in bradyzoites than in tachyzoites. Importantly, the number of parasites containing fluorescence-labelled TgAtg8 puncta was significantly reduced in bradyzoites than in tachyzoites, suggesting that autophagy is downregulated in Toxoplasma bradyzoite in vitro. Moreover, after treatment with drugs, bradyzoite-specific gene BAG1 levels decreased significantly in rapamycin-treated bradyzoites and increased significantly in 3-MA-treated bradyzoites in comparison with control bradyzoites, indicating that Toxoplasma autophagy is involved in the transformation of tachyzoite to bradyzoite in vitro. Together, it is suggested that autophagy may serve as a potential strategy to regulate the transformation.

Wheat Gene TaATG8j Contributes to Stripe Rust Resistance.

PubMed

Mamun, Md Abdullah-Al; Tang, Chunlei; Sun, Yingchao; Islam, Md Nazrul; Liu, Peng; Wang, Xiaojie; Kang, Zhensheng

2018-06-05

Autophagy-related 8 (ATG8) protein has been reported to be involved in plant's innate immune response, but it is not clear whether such genes play a similar role in cereal crops against obligate biotrophic fungal pathogens. Here, we reported an ATG8 gene from wheat ( Triticum aestivum ), designated TaATG8j . This gene has three copies located in chromosomes 2AS, 2BS, and 2DS. The transcriptions of all three copies were upregulated in plants of the wheat cultivar Suwon 11, inoculated with an avirulent race (CYR23) of Puccinia striiformis f. sp. tritici ( Pst ), the causal fungal pathogen of stripe rust. The transient expression of TaATG8j in Nicotiana benthamiana showed that TaATG8j proteins were distributed throughout the cytoplasm, but mainly in the nucleus and plasma membrane. The overexpression of TaATG8j in N. benthamiana slightly delayed the cell death caused by the mouse apoptotic protein BAX (BCL2-associated X protein). However, the expression of TaATG8j in yeast ( Schizosaccharomyces pombe ) induced cell death. The virus-induced gene silencing of all TaATG8j copies rendered Suwon 11 susceptible to the avirulent Pst race CYR23, accompanied by an increased fungal biomass and a decreased necrotic area per infection site. These results indicate that TaATG8j contributes to wheat resistance against stripe rust fungus by regulating cell death, providing information for the understanding of the mechanisms of wheat resistance to the stripe rust pathogen.

Identification of breast cancer cell subtypes sensitive to ATG4B inhibition.

PubMed

Bortnik, Svetlana; Choutka, Courtney; Horlings, Hugo M; Leung, Samuel; Baker, Jennifer H; Lebovitz, Chandra; Dragowska, Wieslawa H; Go, Nancy E; Bally, Marcel B; Minchinton, Andrew I; Gelmon, Karen A; Gorski, Sharon M

2016-10-11

Autophagy, a lysosome-mediated degradation and recycling process, functions in advanced malignancies to promote cancer cell survival and contribute to cancer progression and drug resistance. While various autophagy inhibition strategies are under investigation for cancer treatment, corresponding patient selection criteria for these autophagy inhibitors need to be developed. Due to its central roles in the autophagy process, the cysteine protease ATG4B is one of the autophagy proteins being pursued as a potential therapeutic target. In this study, we investigated the expression of ATG4B in breast cancer, a heterogeneous disease comprised of several molecular subtypes. We examined a panel of breast cancer cell lines, xenograft tumors, and breast cancer patient specimens for the protein expression of ATG4B, and found a positive association between HER2 and ATG4B protein expression. We showed that HER2-positive cells, but not HER2-negative breast cancer cells, require ATG4B to survive under stress. In HER2-positive cells, cytoprotective autophagy was dependent on ATG4B under both starvation and HER2 inhibition conditions. Combined knockdown of ATG4B and HER2 by siRNA resulted in a significant decrease in cell viability, and the combination of ATG4B knockdown with trastuzumab resulted in a greater reduction in cell viability compared to trastuzumab treatment alone, in both trastuzumab-sensitive and -resistant HER2 overexpressing breast cancer cells. Together these results demonstrate a novel association of ATG4B positive expression with HER2 positive breast cancers and indicate that this subtype is suitable for emerging ATG4B inhibition strategies.

Heterologous Expression of ATG8c from Soybean Confers Tolerance to Nitrogen Deficiency and Increases Yield in Arabidopsis

PubMed Central

Liu, Dong; Chai, Wenting; Gong, Qingqiu; Wang, Ning Ning

2012-01-01

Nitrogen is an essential element for plant growth and yield. Improving Nitrogen Use Efficiency (NUE) of crops could potentially reduce the application of chemical fertilizer and alleviate environmental damage. To identify new NUE genes is therefore an important task in molecular breeding. Macroautophagy (autophagy) is an intracellular process in which damaged or obsolete cytoplasmic components are encapsulated in double membraned vesicles termed autophagosomes, then delivered to the vacuole for degradation and nutrient recycling. One of the core components of autophagosome formation, ATG8, has been shown to directly mediate autophagosome expansion, and the transcript of which is highly inducible upon starvation. Therefore, we postulated that certain homologs of Saccharomyces cerevisiae ATG8 (ScATG8) from crop species could have potential for NUE crop breeding. A soybean (Glycine max, cv. Zhonghuang-13) ATG8, GmATG8c, was selected from the 11 family members based on transcript analysis upon nitrogen deprivation. GmATG8c could partially complement the yeast atg8 mutant. Constitutive expression of GmATG8c in soybean callus cells not only enhanced nitrogen starvation tolerance of the cells but accelerated the growth of the calli. Transgenic Arabidopsis over-expressing GmATG8c performed better under extended nitrogen and carbon starvation conditions. Meanwhile, under optimum growth conditions, the transgenic plants grew faster, bolted earlier, produced larger primary and axillary inflorescences, eventually produced more seeds than the wild-type. In average, the yield was improved by 12.9%. We conclude that GmATG8c may serve as an excellent candidate for breeding crops with enhanced NUE and better yield. PMID:22629371

Hematologic adverse effects of 2 different polyclonal antilymphocyte preparations in de novo kidney transplant patients.

PubMed

Rostaing, Lionel; Lavayssière, Laurence; Kamar, Nassim

2010-06-01

To evaluate the hematologic adverse effects of polyclonal antilymphocyte globulins within the first month after surgery in kidney transplant recipients. In this prospective, randomized trial, we included 16 adult-sensitized (panel-reactive antibodies > 30%) recipients of a kidney from a deceased donor. Eight patients received therapy with Genzyme (Thymoglobulin: ATG-G; 6.2 +/- 2.9 mg/kg for 7 days), and 8 patients received Fresenius (Lymphoglobulin: ATG-F; 22.6 +/- 7.9 mg/kg for 6 days). Other immunosuppressants included mycophenolate mofetil, tacrolimus, and steroids. Platelet counts were normal before transplant and significantly reduced after transplant; however, this was more pronounced in ATG-F patients, and had normalized by day 7 in the ATG-G and by day 10 in the ATG-F groups. Mean leukocyte/polymorphonuclear cell counts remained within the normal range in both groups through follow-up. Hemoglobin levels were similar at approximately 10 g/dL for both groups, up to day 10. However, erythropoietin-stimulating-agent therapy had been given to more patients in the ATG-F group than patients in the ATG-G group. Reticulocyte counts were significantly lower in ATG-F patients by days 3, 5, 7, and 10. From day 14 onwards, reticulocyte counts were similar in both groups. With regard to lymphocyte counts, these were normal in both groups before transplant and then significantly decreased afterward. No patient presented with acute rejection or serum-sickness disease. Reduced platelet and reticulocyte counts occur more frequently immediately after transplant when using ATG-F compared with ATG-G therapy. Consequently, erythropoietin-stimulating-agent therapy was needed more often for ATG-F patients.

Characterization of the Autophagy Marker Protein Atg8 Reveals Atypical Features of Autophagy in Plasmodium falciparum

PubMed Central

Allanki, Aparna Devi; Sijwali, Puran Singh

2014-01-01

Conventional autophagy is a lysosome-dependent degradation process that has crucial homeostatic and regulatory functions in eukaryotic organisms. As malaria parasites must dispose a number of self and host cellular contents, we investigated if autophagy in malaria parasites is similar to the conventional autophagy. Genome wide analysis revealed a partial autophagy repertoire in Plasmodium, as homologs for only 15 of the 33 yeast autophagy proteins could be identified, including the autophagy marker Atg8. To gain insights into autophagy in malaria parasites, we investigated Plasmodium falciparum Atg8 (PfAtg8) employing techniques and conditions that are routinely used to study autophagy. Atg8 was similarly expressed and showed punctate localization throughout the parasite in both asexual and sexual stages; it was exclusively found in the pellet fraction as an integral membrane protein, which is in contrast to the yeast or mammalian Atg8 that is distributed among cytosolic and membrane fractions, and suggests for a constitutive autophagy. Starvation, the best known autophagy inducer, decreased PfAtg8 level by almost 3-fold compared to the normally growing parasites. Neither the Atg8-associated puncta nor the Atg8 expression level was significantly altered by treatment of parasites with routinely used autophagy inhibitors (cysteine (E64) and aspartic (pepstatin) protease inhibitors, the kinase inhibitor 3-methyladenine, and the lysosomotropic agent chloroquine), indicating an atypical feature of autophagy. Furthermore, prolonged inhibition of the major food vacuole protease activity by E64 and pepstatin did not cause accumulation of the Atg8-associated puncta in the food vacuole, suggesting that autophagy is primarily not meant for degradative function in malaria parasites. Atg8 showed partial colocalization with the apicoplast; doxycycline treatment, which disrupts apicoplast, did not affect Atg8 localization, suggesting a role, but not exclusive, in apicoplast biogenesis. Collectively, our results reveal several atypical features of autophagy in malaria parasites, which may be largely associated with non-degradative processes. PMID:25426852

Non-Thermal Treatment of Hanford Site Low-Level Mixed Waste

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-09-01

DOE proposes to transport contact-handled LLMW from the Hanford Site to the Allied Technology Group (ATG) Mixed Waste Facility (MWF) in Richland, Washington, for non-thermal treatment and to return the treated waste to the Hanford Site for eventual land disposal. Over a 3-year period the waste would be staged to the ATG MWF, and treated waste would be returned to the Hanford Site. The ATG MWF would be located on an 18 hectare (ha) (45 acre [at]) ATG Site adjacent to ATG's licensed low-level waste processing facility at 2025 Battelle Boulevard. The ATG MWF is located approximately 0.8 kilometers (km)more » (0.5 miles [mi]) south of Horn Rapids Road and 1.6 km (1 mi) west of Stevens Drive. The property is located within the Horn Rapids triangle in northern Richland (Figure 2.1). The ATG MWF is to be located on the existing ATG Site, near the DOE Hanford Site, in an industrial area in the City of Richland. The effects of siting, construction, and overall operation of the MWF have been evaluated in a separate State Environmental Policy Act (SEPA) EIS (City of Richland 1998). The proposed action includes transporting the LLMW from the Hanford Site to the ATG Facility, non-thermal treatment of the LLMW at the ATG MWF, and transporting the waste from ATG back to the Hanford Site. Impacts fi-om waste treatment operations would be bounded by the ATG SEPA EIS, which included an evaluation of the impacts associated with operating the non-thermal portion of the MWF at maximum design capacity (8,500 metric tons per year) (City of Richland 1998). Up to 50 employees would be required for non-thermal treatment portion of the MWF. This includes 40 employees that would perform waste treatment operations and 10 support staff. Similar numbers were projected for the thermal treatment portion of the MWF (City of Richland 1998).« less

Analysis of Autophagy Genes in Microalgae: Chlorella as a Potential Model to Study Mechanism of Autophagy

PubMed Central

Jiang, Qiao; Zhao, Li; Dai, Junbiao; Wu, Qingyu

2012-01-01

Background Microalgae, with the ability to mitigate CO2 emission and produce carbohydrates and lipids, are considered one of the most promising resources for producing bioenergy. Recently, we discovered that autophagy plays a critical role in the metabolism of photosynthetic system and lipids production. So far, more than 30-autophagy related (ATG) genes in all subtypes of autophagy have been identified. However, compared with yeast and mammals, in silico and experimental research of autophagy pathways in microalgae remained limited and fragmentary. Principal Findings In this article, we performed a genome-wide analysis of ATG genes in 7 microalgae species and explored their distributions, domain structures and evolution. Eighteen “core autophagy machinery” proteins, four mammalian-specific ATG proteins and more than 30 additional proteins (including “receptor-adaptor” complexes) in all subtypes of autophagy were analyzed. Data revealed that receptor proteins in cytoplasm-to-vacuole targeting and mitophagy seem to be absent in microalgae. However, most of the “core autophagy machinery” and mammalian-specific proteins are conserved among microalgae, except for the ATG9-cycling system in Chlamydomonas reinhardtii and the second ubiquitin-like protein conjugation complex in several algal species. The catalytic and binding residues in ATG3, ATG5, ATG7, ATG8, ATG10 and ATG12 are also conserved and the phylogenetic tree of ATG8 coincides well with the phylogenies. Chlorella contains the entire set of the core autophagy machinery. In addition, RT-PCR analysis verified that all crucial ATG genes tested are expressed during autophagy in both Chlorella and Chlamydomonas reinhardtii. Finally, we discovered that addition of 3-Methyladenine (a PI3K specific inhibitor) could suppress the formation of autophagic vacuoles in Chlorella. Conclusions Taken together, Chlorella may represent a potential model organism to investigate autophagy pathways in photosynthetic eukaryotes. The study will not only promote understanding of the general features of autophagic pathways, but also benefit the production of Chlorella-derived biofuel with future commercial applications. PMID:22848622

Genome-wide analysis of autophagy-associated genes in foxtail millet (Setaria italica L.) and characterization of the function of SiATG8a in conferring tolerance to nitrogen starvation in rice.

PubMed

Li, Weiwei; Chen, Ming; Wang, Erhui; Hu, Liqin; Hawkesford, Malcolm J; Zhong, Li; Chen, Zhu; Xu, Zhaoshi; Li, Liancheng; Zhou, Yongbin; Guo, Changhong; Ma, Youzhi

2016-10-12

Autophagy is a cellular degradation process that is highly evolutionarily-conserved in yeast, plants, and animals. In plants, autophagy plays important roles in regulating intracellular degradation and recycling of amino acids in response to nutrient starvation, senescence, and other environmental stresses. Foxtail millet (Setaria italica) has strong resistance to stresses and has been proposed as an ideal material for use in the study of the physiological mechanisms of abiotic stress tolerance in plants. Although the genome sequence of foxtail millet (Setaria italica) is available, the characteristics and functions of abiotic stress-related genes remain largely unknown for this species. A total of 37 putative ATG (autophagy-associated genes) genes in the foxtail millet genome were identified. Gene duplication analysis revealed that both segmental and tandem duplication events have played significant roles in the expansion of the ATG gene family in foxtail millet. Comparative synteny mapping between the genomes of foxtail millet and rice suggested that the ATG genes in both species have common ancestors, as their ATG genes were primarily located in similar syntenic regions. Gene expression analysis revealed the induced expression of 31 SiATG genes by one or more phytohormone treatments, 26 SiATG genes by drought, salt and cold, 24 SiATG genes by darkness and 25 SiATG genes by nitrogen starvation. Results of qRT-PCR showing that among 37 SiATG genes, the expression level of SiATG8a was the highest after nitrogen starvation treatment 24 h, suggesting its potential role in tolerance to nutrient starvation. Moreover, the heterologous expression of SiATG8a in rice improved nitrogen starvation tolerance. Compared to wild type rice, the transgenic rice performed better and had higher aboveground total nitrogen content when the plants were grown under nitrogen starvation conditions. Our results deepen understanding about the characteristics and functions of ATG genes in foxtail millet and also identify promising new genetic resources that should be of use in future efforts to develop varieties of foxtail millet and other crop species that have resistance to nitrogen deficiency stress.

Aplastic crisis revealing the diagnosis of Hb Evans [alpha62(E11)Val-->Met, GTG-->ATG (alpha2)] in a Hispanic kindred: case report and review.

PubMed

Steiner, Laurie A; Van Hoff, Jack; Kutlar, Ferdane; Gallagher, Patrick G

2007-01-01

This report describes a Hispanic girl who presented in aplastic crisis due to parvovirus infection. She was subsequently found to have a chronic hemolytic anemia secondary to an unstable hemoglobinopathy. Genetic testing revealed that the girl and two symptomatic family members had Hb Evans [alpha62(E11)Val-->Met, GTG-->ATG (alpha2)], an unstable hemoglobin (Hb) variant due to a mutation in the alpha2-globin chain. Hb Evans has been described only once previously, in a Caucasian kindred. Literature review indicates aplastic crisis is a rare initial presentation of unstable hemoglobinopathies and that these disorders are infrequent but important causes of hemolytic anemia in Hispanic patients.

High resolution melting analytical platform for rapid prenatal and postnatal diagnosis of β-thalassemia common among Southeast Asian population.

PubMed

Prajantasen, Thanet; Fucharoen, Supan; Fucharoen, Goonnapa

2015-02-20

High resolution melting (HRM) analysis is a powerful technology for scanning sequence alteration. We have applied this HRM assay to screen common β-thalassemia mutations found among Southeast Asian population. Known DNA samples with 8 common mutations were used in initial development of the methods including -28 A-G, codon 17 A-T, IVSI-1G-T, IVSI-5G-C, codon 26G-A (Hb E), codons 41/42 -TTCT, codons 71/72+A and IVSII-654 C-T. Further validation was done on 60 postnatal and 6 prenatal diagnoses of β-thalassemia. Each mutation has specific HRM profile which could be used in rapid screening. Apart from those with DNA deletions, the results of HRM assay matched 100% with those of routine diagnosis made by routine allele specific PCR. In addition, the HRM assay could initially recognize three unknown mutations including a hitherto un-described one in Thai population. The established HRM assay should prove useful for rapid and high throughput platform for screening and prenatal diagnosis of β-thalassemia common in the region. Copyright © 2014 Elsevier B.V. All rights reserved.

LeCTR1, a Tomato CTR1-Like Gene, Demonstrates Ethylene Signaling Ability in Arabidopsis and Novel Expression Patterns in Tomato1

PubMed Central

Leclercq, Julie; Adams-Phillips, Lori C.; Zegzouti, Hicham; Jones, Brian; Latché, Alain; Giovannoni, James J.; Pech, Jean-Claude; Bouzayen, Mondher

2002-01-01

LeCTR1 was initially isolated by both differential display reverse transcriptase-polymerase chain reaction screening for tomato (Lycopersicon esculentum) fruit ethylene-inducible genes and through homology with the Arabidopsis CTR1 cDNA. LeCTR1 shares strong nucleotide sequence homology with Arabidopsis CTR1, a gene acting downstream of the ethylene receptor and showing similarity to the Raf family of serine/threonine protein kinases. The length of the LeCTR1 transcribed region from ATG to stop codon (12,000 bp) is more than twice that of Arabidopsis CTR1 (4,700 bp). Structural analysis reveals perfect conservation of both the number and position of introns and exons in LeCTR1 and Arabidopsis CTR1. The introns in LeCTR1 are much longer, however. To address whether this structural conservation is indicative of functional conservation of the corresponding proteins, we expressed LeCTR1 in the Arabidopsis ctr1-1 (constitutive triple response 1) mutant under the direction of the 35S promoter. Our data clearly show that ectopic expression of LeCTR1 in the Arabidopsis ctr1-1 mutant can restore normal ethylene signaling. The recovery of normal ethylene sensitivity upon heterologous expression of LeCTR1 was also confirmed by restored glucose sensitivity absent in the Arabidopsis ctr1-1 mutant. Expression studies confirm ethylene responsiveness of LeCTR1 in various tissues, including ripening fruit, and may suggest the evolution of alternate regulatory mechanisms in tomato versus Arabidopsis. PMID:12427980

Evidence for a functional genetic polymorphism of the human thiosulfate sulfurtransferase (Rhodanese), a cyanide and H2S detoxification enzyme.

PubMed

Billaut-Laden, Ingrid; Allorge, Delphine; Crunelle-Thibaut, Aurélie; Rat, Emmanuel; Cauffiez, Christelle; Chevalier, Dany; Houdret, Nicole; Lo-Guidice, Jean-Marc; Broly, Franck

2006-08-01

Rhodanese or thiosulfate sulfurtransferase (TST) is a mitochondrial matrix enzyme that plays roles in cyanide detoxification, the formation of iron-sulfur proteins and the modification of sulfur-containing enzymes. Transsulfuration reaction catalyzed by TST is also involved in H(2)S detoxification. To date, no polymorphism of the human TST gene had been reported. We developed a screening strategy based on a PCR-SSCP method to search for mutations in the 3 exons of TST and their proximal flanking regions. This strategy has been applied to DNA samples from 50 unrelated French individuals of Caucasian origin. Eleven polymorphisms consisting in seven nucleotide substitutions in non-coding regions, two silent mutations and two missense mutations were characterized. The functional consequences of the identified mutations were assessed in vivo by measurement of erythrocyte TST activity and/or in vitro using heterologous expression in Saccharomyces cerevisiae or transient transfection assay in HT29 and Caco-2 cell lines. The P(285)A variant appears to encode a protein with a 50% decrease of in vitro intrinsic clearance compared to the wild-type enzyme. Additionally, the six polymorphisms located upstream the ATG initiation codon are responsible for a significant decrease (ranging from 40% to 73%) in promoter activity of a reporter gene compared to the corresponding wild-type sequence. This work constitutes the first report of the existence of a functional genetic polymorphism affecting TST activity and should be of great help to investigate certain disorders for which impairment of CN(-) or H(2)S detoxification have been suggested to be involved.

Genetic analysis of the dsz promoter and associated regulatory regions of Rhodococcus erythropolis IGTS8.

PubMed Central

Li, M Z; Squires, C H; Monticello, D J; Childs, J D

1996-01-01

The dsz gene cluster of Rhodococcus erythropolis IGTS8 comprises three genes, dszA, dszB, and dszC, whose products are involved in the conversion of dibenzothiophene (DBT) to 2-hydroxybiphenyl and sulfite. This organism can use DBT as the sole sulfur source but not as a carbon source. Dsz activity is repressed by methionine, cysteine, Casamino Acids, and sulfate but not by DBT or dimethyl sulfoxide. We cloned 385 bp of the DNA immediately 5' to dszA in front of the reporter gene lacZ of Escherichia coli. We showed that this region contains a Rhodococcus promoter and at least three dsz regulatory regions. After hydrazine mutagenesis of this DNA, colonies that were able to express beta-galactosidase in the presence of Casamino Acids were isolated. Sequencing of these mutants revealed two possible regulatory regions. One is at -263 to -244, and the other is at -93 to -38, where -1 is the base preceding the A of the initiation codon ATG of dszA. An S1 nuclease protection assay showed that the start of the dsz promoter is the G at -46 and that transcription is repressed by sulfate and cysteine but not by dimethyl sulfoxide. The promoter encompasses a region of potential diad symmetry that may contain an operator. Immediately upstream of the promoter is a protein-binding domain between -146 and -121. Deletion of this region did not affect repression, but promoter activity appeared to be reduced by threefold. Thus, it could be an activator binding site or an enhancer region. PMID:8932295

An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum

PubMed Central

López-Estraño, Carlos; Gopalakrishnan, Anusha M.; Semblat, Jean-Philippe; Fergus, M. Ross; Mazier, Dominique; Haldar, Kasturi

2008-01-01

The asexual blood stage of Plasmodium falciparum is comprised of morphologically distinct ring, trophozoite and schizont stages. Each of these developmental stages possesses a distinct pattern of gene expression. Regulation of P. falciparum gene expression is thought to occur, at least in part, at the promoter level. Previously, we have found that although the RNA of the P. falciparum hrp3 gene is only seen in ring-stage parasites, deletion of a specific sequensce in the 5’ end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage parasites. In order to investigate this stage specific regulation of gene expression, we employed a series of nested deletions of the 1.7-kb hrp3 promoter. Firefly luciferase gene was used as a reporter to evaluate the role of promoter sequences in gene regulation. Using this approach, we identified a ring-stage specific regulatory region on the hrp3 promoter located between -1.7-kb and -1.1-kb from the ATG initiation codon. Small 100–150 bp truncations on this enhancer-like region failed to uncover discrete regulatory sequences, suggesting the multipartite nature of this element. The data presented in this study demonstrates that stage specific promoter activity of the hrp3 gene in P. falciparum blood stage parasites is supported, at least in-part, by a small promoter region that can function in the absence of a larger chromosomal context. PMID:17570541

Carcinogen-induced mutations in the mouse c-Ha-ras gene provide evidence of multiple pathways for tumor progression.

PubMed Central

Brown, K; Buchmann, A; Balmain, A

1990-01-01

A number of mouse skin tumors initiated by the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylnitrosourea (MNU), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz[a]anthracene (DMBA) have been shown to contain activated Ha-ras genes. In each case, the point mutations responsible for activation have been characterized. Results presented demonstrate the carcinogen-specific nature of these ras mutations. For each initiating agent, a distinct spectrum of mutations is observed. Most importantly, the distribution of ras gene mutations is found to differ between benign papillomas and carcinomas, suggesting that molecular events occurring at the time of initiation influence the probability with which papillomas progress to malignancy. This study provides molecular evidence in support of the existence of subsets of papillomas with differing progression frequencies. Thus, the alkylating agents MNNG and MNU induced exclusively G ---- A transitions at codon 12, with this mutation being found predominantly in papillomas. MCA initiation produced both codon 13 G ---- T and codon 61 A ---- T transversions in papillomas; only the G ---- T mutation, however, was found in carcinomas. These findings provide strong evidence that the mutational activation of Ha-ras occurs as a result of the initiation process and that the nature of the initiating event can affect the probability of progression to malignancy. Images PMID:2105486

Codon influence on protein expression in E. coli correlates with mRNA levels

PubMed Central

Boël, Grégory; Wong, Kam-Ho; Su, Min; Luff, Jon; Valecha, Mayank; Everett, John K.; Acton, Thomas B.; Xiao, Rong; Montelione, Gaetano T.; Aalberts, Daniel P.; Hunt, John F.

2016-01-01

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli. PMID:26760206

Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

PubMed Central

Chen, Song; Wang, Chenran; Yeo, Syn; Liang, Chun-Chi; Okamoto, Takako; Sun, Shaogang; Wen, Jian; Guan, Jun-Lin

2016-01-01

Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582–585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo. PMID:27013233

Chronic graft-versus-host disease: long-term results from a randomized trial on graft-versus-host disease prophylaxis with or without anti-T-cell globulin ATG-Fresenius.

PubMed

Socié, Gérard; Schmoor, Claudia; Bethge, Wolfgang A; Ottinger, Hellmut D; Stelljes, Matthias; Zander, Axel R; Volin, Liisa; Ruutu, Tapani; Heim, Dominik A; Schwerdtfeger, Rainer; Kolbe, Karin; Mayer, Jiri; Maertens, Johan A; Linkesch, Werner; Holler, Ernst; Koza, Vladimir; Bornhäuser, Martin; Einsele, Hermann; Kolb, Hans-Jochem; Bertz, Hartmut; Egger, Matthias; Grishina, Olga; Finke, Jürgen

2011-06-09

Previous randomized graft-versus-host disease (GVHD)-prophylaxis trials have failed to demonstrate reduced incidence and severity of chronic GVHD (cGVHD). Here we reanalyzed and updated a randomized phase 3 trial comparing standard GVHD prophylaxis with or without pretransplantation ATG-Fresenius (ATG-F) in 201 adult patients receiving myeloablative conditioning before transplantation from unrelated donors. The cumulative incidence of extensive cGVHD after 3 years was 12.2% in the ATG-F group versus 45.0% in the control group (P < .0001). The 3-year cumulative incidence of relapse and of nonrelapse mortality was 32.6% and 19.4% in the ATG-F group and 28.2% and 33.5% in the control group (hazard ratio [HR] = 1.21, P = .47, and HR = 0.68, P = .18), respectively. This nonsignificant reduction in nonrelapse mortality without increased relapse risk led to an overall survival rate after 3 years of 55.2% in the ATG-F group and 43.3% in the control group (HR = 0.84, P = .39, nonsignificant). The HR for receiving immunosuppressive therapy (IST) was 0.31 after ATG-F (P < .0001), and the 3-year probability of survival free of IST was 52.9% and 16.9% in the ATG-F versus control, respectively. The addition of ATG-F to standard cyclosporine, methotrexate GVHD prophylaxis lowers the incidence and severity of cGVHD, and the risk of receiving IST without raising the relapse rate. ATG-F prophylaxis reduces cGVHD morbidity.

Effective prevention of GVHD using in vivo T-cell depletion with anti-lymphocyte globulin in HLA-identical or -mismatched sibling peripheral blood stem cell transplantation.

PubMed

Wolschke, C; Zabelina, T; Ayuk, F; Alchalby, H; Berger, J; Klyuchnikov, E; Pein, U-M; Schumacher, S; Amtsfeld, G; Adjallé, R; Wortmann, F; Lellek, H; Randenborgh, A; Zander, A; Kröger, N

2014-01-01

To investigate the impact of anti-lymphocyte globulin (ATG-Fresenius) as part of the HLA-sibling transplantation, we evaluated 238 patients (median age 48 years) with different diagnoses (AML, ALL, CML and lymphoproliferative disorders). A total of 79 patients received ATG and 159 patients did not. In the ATG group, there were more HLA-mismatched donors (6% vs 1%, p=0.02), bad risk patients (70% vs 55%, P=0.04), reduced intensity conditioning (RIC) regimens (65% vs 34%, P=<0.001) and older patients (median age 51 vs 48 years, P=0.002). The median time to leukocyte engraftment was significantly faster in the non-ATG group (13 vs 15 days, P < 0.001). EBV reactivation was more often seen in the ATG group (9% vs 2%, P=0.05). Cumulative incidence of acute and chronic GVHD was less observed in the ATG group (27% vs 40%, P=0.004, and 33% vs 54%, P=0.002). The cumulative incidence rates of non-relapse mortality and of relapse at 5 years were 20 and 34%, respectively, for ATG and 34 and 29%, respectively, for non-ATG (P=0.06 and P=0.3). ATG can prevent GVHD without an obvious risk of relapse but should be confirmed in a randomized study.

Cytoprotective role of autophagy against BH3 mimetic gossypol in ATG5 knockout cells generated by CRISPR-Cas9 endonuclease.

PubMed

Kim, Na-Yeon; Han, Byeal-I; Lee, Michael

2016-01-01

Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the ROS/p38 MAPK pathway and epigenetic regulation.

PubMed

Hsieh, Chia-Jung; Kuo, Po-Lin; Hsu, Ying-Chan; Huang, Ya-Fang; Tsai, Eing-Mei; Hsu, Ya-Ling

2014-02-01

This study investigates the anticancer effect of arctigenin (ATG), a natural lignan product of Arctium lappa L., in human breast cancer MDA-MB-231 cells. Results indicate that ATG inhibits MDA-MB-231 cell growth by inducing apoptosis in vitro and in vivo. ATG triggers the mitochondrial caspase-independent pathways, as indicated by changes in Bax/Bcl-2 ratio, resulting in AIF and EndoG nuclear translocation. ATG increased cellular reactive oxygen species (ROS) production by increasing p22(phox)/NADPH oxidase 1 interaction and decreasing glutathione level. ATG clearly increases the activation of p38 MAPK, but not JNK and ERK1/2. Antioxidant EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, significantly decreases ATG-mediated p38 activation and apoptosis. Blocking p38 with a specific inhibitor suppresses ATG-mediated Bcl-2 downregulation and apoptosis. Moreover, ATG activates ATF-2, a transcription factor activated by p38, and then upregulates histone H3K9 trimethylation in the Bcl-2 gene promoter region, resulting in Bcl-2 downregulation. Taken together, the results demonstrate that ATG induces apoptosis of MDA-MB-231 cells via the ROS/p38 MAPK pathway and epigenetic regulation of Bcl-2 by upregulation of histone H3K9 trimethylation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

Antithymocyte Globulin Induces a Tolerogenic Phenotype in Human Dendritic Cells

PubMed Central

Roider, Tobias; Katzfuß, Michael; Matos, Carina; Singer, Katrin; Renner, Kathrin; Oefner, Peter J.; Dettmer-Wilde, Katja; Herr, Wolfgang; Holler, Ernst; Kreutz, Marina; Peter, Katrin

2016-01-01

Antithymocyte globulin (ATG) is used in the prevention of graft-versus-host disease during allogeneic hematopoietic stem cell transplantation. It is generally accepted that ATG mediates its immunosuppressive effect primarily via depletion of T cells. Here, we analyzed the impact of ATG-Fresenius (now Grafalon®) on human monocyte-derived dendritic cells (DC). ATG induced a semi-mature phenotype in DC with significantly reduced expression of CD14, increased expression of HLA-DR, and intermediate expression of CD54, CD80, CD83, and CD86. ATG-DC showed an increase in IL-10 secretion but no IL-12 production. In line with this tolerogenic phenotype, ATG caused a significant induction of indoleamine 2,3-dioxygenase expression and a concomitant increase in levels of tryptophan metabolites in the supernatants of DC. Further, ATG-DC did not induce the proliferation of allogeneic T cells in a mixed lymphocyte reaction but actively suppressed the T cell proliferation induced by mature DC. These data suggest that besides its well-known effect on T cells, ATG modulates the phenotype of DC in a tolerogenic way, which might constitute an essential part of its immunosuppressive action in vivo. PMID:27973435

Preferences of AAA/AAG codon recognition by modified nucleosides, τm5s2U34 and t6A37 present in tRNALys.

PubMed

Sonawane, Kailas D; Kamble, Asmita S; Fandilolu, Prayagraj M

2017-12-27

Deficiency of 5-taurinomethyl-2-thiouridine, τm 5 s 2 U at the 34th 'wobble' position in tRNA Lys causes MERRF (Myoclonic Epilepsy with Ragged Red Fibers), a neuromuscular disease. This modified nucleoside of mt tRNA Lys , recognizes AAA/AAG codons during protein biosynthesis process. Its preference to identify cognate codons has not been studied at the atomic level. Hence, multiple MD simulations of various molecular models of anticodon stem loop (ASL) of mt tRNA Lys in presence and absence of τm 5 s 2 U 34 and N 6 -threonylcarbamoyl adenosine (t 6 A 37 ) along with AAA and AAG codons have been accomplished. Additional four MD simulations of multiple ASL mt tRNA Lys models in the context of ribosomal A-site residues have also been performed to investigate the role of A-site in recognition of AAA/AAG codons. MD simulation results show that, ASL models in presence of τm 5 s 2 U 34 and t 6 A 37 with codons AAA/AAG are more stable than the ASL lacking these modified bases. MD trajectories suggest that τm 5 s 2 U recognizes the codons initially by 'wobble' hydrogen bonding interactions, and then tRNA Lys might leave the explicit codon by a novel 'single' hydrogen bonding interaction in order to run the protein biosynthesis process smoothly. We propose this model as the 'Foot-Step Model' for codon recognition, in which the single hydrogen bond plays a crucial role. MD simulation results suggest that, tRNA Lys with τm 5 s 2 U and t 6 A recognizes AAA codon more preferably than AAG. Thus, these results reveal the consequences of τm 5 s 2 U and t 6 A in recognition of AAA/AAG codons in mitochondrial disease, MERRF.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Minfei; Li, Yue; Wyborny, Shane

ATG14 binding to BECN/Beclin homologs is essential for autophagy, a critical catabolic homeostasis pathway. Here, we show that the α-helical, coiled-coil domain (CCD) of BECN2, a recently identified mammalian BECN1 paralog, forms an antiparallel, curved homodimer with seven pairs of nonideal packing interactions, while the BECN2 CCD and ATG14 CCD form a parallel, curved heterodimer stabilized by multiple, conserved polar interactions. Compared to BECN1, the BECN2 CCD forms a weaker homodimer, but binds more tightly to the ATG14 CCD. Mutation of nonideal BECN2 interface residues to more ideal pairs improves homodimer self-association and thermal stability. Unlike BECN1, all BECN2 CCDmore » mutants bind ATG14, although more weakly than wild type. Thus, polar BECN2 CCD interface residues result in a metastable homodimer, facilitating dissociation, but enable better interactions with polar ATG14 residues stabilizing the BECN2:ATG14 heterodimer. These structure-based mechanistic differences in BECN1 and BECN2 homodimerization and heterodimerization likely dictate competitive ATG14 recruitment.« less

Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage.

PubMed

Nicolao, María Celeste; Loos, Julia A; Rodriguez Rodrigues, Christian; Beas, Viviana; Cumino, Andrea C

2017-01-01

Cystic echinococcosis (CE) is a worldwide distributed helminthic zoonosis caused by Echinococcus granulosus. Benzimidazole derivatives are currently the only drugs for chemotherapeutic treatment of CE. However, their low efficacy and the adverse effects encourage the search for new therapeutic targets. We evaluated the in vitro efficacy of Bortezomib (Bz), a proteasome inhibitor, in the larval stage of the parasite. After 96 h, Bz showed potent deleterious effects at a concentration of 5 μM and 0.5 μM in protoscoleces and metacestodes, respectively (P < 0.05). After 48 h of exposure to this drug, it was triggered a mRNA overexpression of chaperones (Eg-grp78 and Eg-calnexin) and of Eg-ire2/Eg-xbp1 (the conserved UPR pathway branch) in protoscoleces. No changes were detected in the transcriptional expression of chaperones in Bz-treated metacestodes, thus allowing ER stress to be evident and viability to highly decrease in comparison with protoscoleces. We also found that Bz treatment activated the autophagic process in both larval forms. These facts were evidenced by the increase in the amount of transcripts of the autophagy related genes (Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16) together with the increase in Eg-Atg8-II detected by western blot and by in toto immunofluorescence labeling. It was further confirmed by direct observation of autophagic structures by electronic microscopy. Finally, in order to determine the impact of autophagy induction on Echinococcus cell viability, we evaluated the efficacy of Bz in combination with rapamycin and a synergistic cytotoxic effect on protoscolex viability was observed when both drugs were used together. In conclusion, our findings demonstrated that Bz induced endoplasmic reticulum stress, autophagy and subsequent death allowing to identify unstudied parasite-host pathways that could provide a new insight for control of parasitic diseases.

Bortezomib initiates endoplasmic reticulum stress, elicits autophagy and death in Echinococcus granulosus larval stage

PubMed Central

Nicolao, María Celeste; Loos, Julia A.; Rodriguez Rodrigues, Christian; Beas, Viviana

2017-01-01

Cystic echinococcosis (CE) is a worldwide distributed helminthic zoonosis caused by Echinococcus granulosus. Benzimidazole derivatives are currently the only drugs for chemotherapeutic treatment of CE. However, their low efficacy and the adverse effects encourage the search for new therapeutic targets. We evaluated the in vitro efficacy of Bortezomib (Bz), a proteasome inhibitor, in the larval stage of the parasite. After 96 h, Bz showed potent deleterious effects at a concentration of 5 μM and 0.5 μM in protoscoleces and metacestodes, respectively (P < 0.05). After 48 h of exposure to this drug, it was triggered a mRNA overexpression of chaperones (Eg-grp78 and Eg-calnexin) and of Eg-ire2/Eg-xbp1 (the conserved UPR pathway branch) in protoscoleces. No changes were detected in the transcriptional expression of chaperones in Bz-treated metacestodes, thus allowing ER stress to be evident and viability to highly decrease in comparison with protoscoleces. We also found that Bz treatment activated the autophagic process in both larval forms. These facts were evidenced by the increase in the amount of transcripts of the autophagy related genes (Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16) together with the increase in Eg-Atg8-II detected by western blot and by in toto immunofluorescence labeling. It was further confirmed by direct observation of autophagic structures by electronic microscopy. Finally, in order to determine the impact of autophagy induction on Echinococcus cell viability, we evaluated the efficacy of Bz in combination with rapamycin and a synergistic cytotoxic effect on protoscolex viability was observed when both drugs were used together. In conclusion, our findings demonstrated that Bz induced endoplasmic reticulum stress, autophagy and subsequent death allowing to identify unstudied parasite-host pathways that could provide a new insight for control of parasitic diseases. PMID:28817601

A Non-Cell-Autonomous Role of BEC-1/BECN1/Beclin1 in Coordinating Cell-Cycle Progression and Stem Cell Proliferation during Germline Development.

PubMed

Ames, Kristina; Da Cunha, Dayse S; Gonzalez, Brenda; Konta, Marina; Lin, Feng; Shechter, Gabriel; Starikov, Lev; Wong, Sara; Bülow, Hannes E; Meléndez, Alicia

2017-03-20

The decision of stem cells to proliferate and differentiate is finely controlled. The Caenorhabditis elegans germline provides a tractable system for studying the mechanisms that control stem cell proliferation and homeostasis [1-4]. Autophagy is a conserved cellular recycling process crucial for cellular homeostasis in many different contexts [5], but its function in germline stem cell proliferation remains poorly understood. Here, we describe a function for autophagy in germline stem cell proliferation. We found that autophagy genes such as bec-1/BECN1/Beclin1, atg-16.2/ATG16L, atg-18/WIPI1/2, and atg-7/ATG7 are required for the late larval expansion of germline stem cell progenitors in the C. elegans gonad. We further show that BEC-1/BECN1/Beclin1 acts independently of the GLP-1/Notch or DAF-7/TGF-β pathways but together with the DAF-2/insulin IGF-1 receptor (IIR) signaling pathway to promote germline stem cell proliferation. Similar to DAF-2/IIR, BEC-1/BECN1/Beclin1, ATG-18/WIPI1/2, and ATG-16.2/ATG16L all promote cell-cycle progression and are negatively regulated by the phosphatase and tensin homolog DAF-18/PTEN. However, whereas BEC-1/BECN1/Beclin1 acts through the transcriptional regulator SKN-1/Nrf1, ATG-18/WIPI1/2 and ATG-16.2/ATG16L exert their function through the DAF-16/FOXO transcription factor. In contrast, ATG-7 functions in concert with the DAF-7/TGF-β pathway to promote germline proliferation and is not required for cell-cycle progression. Finally, we report that BEC-1/BECN1/Beclin1 functions non-cell-autonomously to facilitate cell-cycle progression and stem cell proliferation. Our findings demonstrate a novel non-autonomous role for BEC-1/BECN1/Beclin1 in the control of stem cell proliferation and cell-cycle progression, which may have implications for the understanding and development of therapies against malignant cell growth in the future. Copyright © 2017 Elsevier Ltd. All rights reserved.

The inositol trisphosphate receptor in the control of autophagy.

PubMed

Criollo, Alfredo; Vicencio, José Miguel; Tasdemir, Ezgi; Maiuri, M Chiara; Lavandero, Sergio; Kroemer, Guido

2007-01-01

The second messenger myo-inositol-1,4,5-trisphosphate (IP(3)) acts on the IP(3) receptor (IP(3)R), an IP(3)-activated Ca(2+) channel of the endoplasmic reticulum (ER). The IP(3)R agonist IP(3) inhibits starvation-induced autophagy. The IP(3)R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-XL, two proteins that physically interact with IP(3)R. Autophagy can also be induced by depletion of the IP(3)R by small interfering RNAs. Autophagy induction by IP(3)R blockade cannot be explained by changes in steady state levels of Ca(2+) in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP(3)R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

ATG-Fresenius inhibits blood circulating cell proliferation in a dose-dependent manner: an experimental study.

PubMed

Werner, I; Seitz-Merwald, I; Kiessling, A H; Kur, F; Beiras-Fernandez, A

2014-11-01

Antithymocyte globulin (ATG)-Fresenius (Neovii-Biotech, Graefelfing, Germany), a highly purified rabbit polyclonal antihuman T-lymphocyte immunoglobulin resulting from immunization of rabbits with the Jurkat T-lymphoblast cell line, is currently used for the prevention of acute rejection in patients receiving solid organ transplants. Our aim was to investigate the in vitro activity of ATG-Fresenius regarding the proliferation of peripheral blood mononuclear cells (PBMCs), an important mechanism of rejection after solid organ transplantation. PBMCs were isolated from 6 healthy donors. Proliferation was assayed using [(3)H] thymidine incorporation. For analysis of mitogen-stimulated proliferation, the PBMCs were incubated at 37°C with various concentrations of ATG-Fresenius in the absence/presence of 40 μg/mL phytohemagglutinin. For analysis of the mixed lymphocyte reaction, PBMCs were incubated at 37°C with various concentrations of ATG-Fresenius for 3 days. On day 3, PBMCs (stimulator cells) from allogeneic donors were incubated with 25 μg/mL mitomycin C. The responder cells (preincubated with ATG-Fresenius) were then cultured at 37°C with the stimulator cells for 6 days. Groups were compared using ANOVA and the Tukey-Kramer multiple comparison test. Preincubation of PBMCs with ATG results in concentration-dependent inhibition of phytohemagglutinin-stimulated proliferation. The effect was more pronounced after 2 and 3 days of treatment with ATG compared with 1 day. There was a concentration-dependent decrease in the mixed lymphocyte reaction-induced proliferation (up to 80%) at ATG-Fresenius concentrations as low as 0.05 to 0.5 μg/mL. No further effect on proliferation at ATG-Fresenius concentrations of 0.5 to 50 μg/mL was seen, and higher concentrations (>100 μg/mL) totally inhibited proliferation. Our in vitro results provide more evidence of the beneficial effect of ATGs in the early phase of solid organ transplantation, by reducing effector cell proliferation.

Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori.

PubMed

Casati, Barbara; Terova, Genciana; Cattaneo, Anna Giulia; Rimoldi, Simona; Franzetti, Eleonora; de Eguileor, Magda; Tettamanti, Gianluca

2012-12-15

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase. Copyright © 2012 Elsevier B.V. All rights reserved.

Arctigenin suppresses renal interstitial fibrosis in a rat model of obstructive nephropathy.

PubMed

Li, Ao; Zhang, Xiaoxun; Shu, Mao; Wu, Mingjun; Wang, Jun; Zhang, Jingyao; Wang, Rui; Li, Peng; Wang, Yitao

2017-07-01

Renal tubulointerstitial fibrosis (TIF) is commonly the final result of a variety of progressive injuries and leads to end-stage renal disease. There are few therapeutic agents currently available for retarding the development of renal TIF. The aim of the present study is to evaluate the role of arctigenin (ATG), a lignan component derived from dried burdock (Arctium lappa L.) fruits, in protecting the kidney against injury by unilateral ureteral obstruction (UUO) in rats. Rats were subjected to UUO and then administered with vehicle, ATG (1 and 3mg/kg/d), or losartan (20mg/kg/d) for 11 consecutive days. The renoprotective effects of ATG were evaluated by histological examination and multiple biochemical assays. Our results suggest that ATG significantly protected the kidney from injury by reducing tubular dilatation, epithelial atrophy, collagen deposition, and tubulointerstitial compartment expansion. ATG administration dramatically decreased macrophage (CD68-positive cell) infiltration. Meanwhile, ATG down-regulated the mRNA levels of pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1) and cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ), in the obstructed kidneys. This was associated with decreased activation of nuclear factor κB (NF-κB). ATG attenuated UUO-induced oxidative stress by increasing the activity of renal manganese superoxide dismutase (SOD2), leading to reduced levels of lipid peroxidation. Furthermore, ATG inhibited the epithelial-mesenchymal transition (EMT) of renal tubules by reducing the abundance of transforming growth factor-β1 (TGF-β1) and its type I receptor, suppressing Smad2/3 phosphorylation and nuclear translocation, and up-regulating Smad7 expression. Notably, the efficacy of ATG in renal protection was comparable or even superior to losartan. ATG could protect the kidney from UUO-induced injury and fibrogenesis by suppressing inflammation, oxidative stress, and tubular EMT, thus supporting the potential role of ATG in renal fibrosis treatment. Copyright © 2017 Elsevier GmbH. All rights reserved.

Predictors of early mortality after rabbit antithymocyte globulin as first-line treatment in severe aplastic anemia.

PubMed

Atta, Elias H; Lima, Carlos B L; Dias, Danielle S P; Clé, Diego V; Bonduel, Mariana M; Sciuccati, Gabriela B; Medeiros, Larissa A; Oliveira, Michel M; Salvino, Marco A; Garanito, Marlene P; Blum Fonseca, Patricia B; Saad, Sara Teresinha O; Calado, Rodrigo T; Scheinberg, Phillip

2017-11-01

Despite being recommended as first-line immunosuppressive therapy in severe aplastic anemia (SAA), horse antithymocyte globulin (ATG) is still unavailable in many countries outside the USA. Rabbit ATG is more lymphocytoxic than horse ATG, and this might result in a higher incidence of severe infections and early mortality. This study was designed to identify the risk factors for early mortality and overall survival (OS) after rabbit ATG in patients with SAA. We retrospectively reviewed 185 patients with SAA who underwent rabbit ATG and cyclosporine. The incidence of death in 3 months following rabbit ATG therapy was 15.1% (28/185). Early mortality was mainly related to infectious complications, despite adequate antibiotic and/or antifungal treatment. Age > 35 years (odds ratio [OR] 5.06, P = 0.001) and baseline absolute neutrophil count (ANC) ≤ 0.1 × 10 9 /L (OR 7.64, P < 0.001) were independent risk factors for early mortality after immunosuppressive therapy with this agent. Hematological response at 6 months was observed in only 29.7% of all patients. OS at 1 year after rabbit ATG was 75.3%; and age > 35 years (OR 1.88, P = 0.03), baseline ANC ≤ 0.1 × 10 9 /L (OR 2.65, P < 0.001), and lack of response to rabbit ATG (OR 11.40, P < 0.001) were independently associated with mortality. Alternative strategies are needed for the treatment of SAA patients in countries were horse ATG is unavailable, particularly for those at high risk for early mortality after rabbit ATG due to a higher age and very low pre-treatment neutrophil count.

Functional analysis of AoAtg11 in selective autophagy in the filamentous fungus Aspergillus oryzae.

PubMed

Tadokoro, Takayuki; Kikuma, Takashi; Kitamoto, Katsuhiko

2015-07-01

Autophagy is a highly conserved cellular degradation process in eukaryotes and consists of both non-selective and selective types. Selective autophagic processes include pexophagy, mitophagy, and the cytoplasm-to-vacuole targeting (Cvt) pathway of yeast, in which particular vacuolar proteins, such as aminopeptidase I (Ape1), are selectively transported to vacuoles. Although selective autophagy has been mainly studied in the yeasts Saccharomyces cerevisiae and Pichia pastoris, there is evidence for selective autophagy in filamentous fungi; however, the details are poorly understood. In S. cerevisiae, Atg11 is a selective autophagy-specific protein that recognizes and transports substrates to the pre-autophagosomal structure (PAS). Here, we first identified an ATG11 homologue in the filamentous fungus Aspergillus oryzae and analyzed the localization of the corresponding protein, designated AoAtg11, fused to enhanced green fluorescent protein (EGFP). Imaging analysis revealed that AoAtg11-EGFP was localized to PAS-like structures. We next constructed an Aoatg11 disruptant of A. oryzae and showed that AoAtg11 is involved in pexophagy and mitophagy. In addition, AoAtg11 was found to be dispensable for non-selective autophagy and for transporting AoApe1 to vacuoles. Taken together, these results suggest that AoAtg11 is a selective autophagy-specific protein in A. oryzae, and has distinct molecular functions from that of S. cerevisiae Atg11. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

In Vivo T Cell Depletion with Myeloablative Regimens on Outcomes after Cord Blood Transplantation for Acute Lymphoblastic Leukemia in Children.

PubMed

Ponce, Doris M; Eapen, Mary; Sparapani, Rodney; O'Brien, Tracey A; Chan, Ka Wah; Chen, Junfang; Craddock, John; Schultz, Kirk R; Wagner, John E; Perales, Miguel-Angel; Barker, Juliet N

2015-12-01

The inclusion of antithymocyte globulin (ATG) in cord blood transplantation is controversial. We evaluated outcomes according to ATG inclusion in 297 children and adolescents with acute lymphoblastic leukemia (ALL) who received myeloablative total body irradiation-based conditioning and either single-unit (74%) or double-unit (26%) grafts. Ninety-two patients (31%) received ATG and 205 (69%) did not. ATG recipients were more likely to be cytomegalovirus seronegative. The incidences of day 100 grades II to IV acute graft-versus-host disease (GVHD; 30% versus 54%, P = .0002) and chronic GVHD (22% versus 43%, P = .0008) were lower with ATG compared with non-ATG regimens. However, day 100 grades III to IV acute GVHD was comparable (11% versus 17%, P = .15). The 3-year incidences of transplant-related mortality (16% versus 17%, P = .98), relapse (17% versus 27%, P = .12), and leukemia-free survival (66% versus 55%, P = .23) in ATG and non-ATG recipients were similar. There were no differences in viral reactivation between treatment groups (60% versus 58%, P = .83). Therefore, the data suggest that incorporation of ATG with myeloablative conditioning regimens may be useful in reducing the risk of acute and chronic GVHD without any deleterious effect on transplant-related mortality, relapse, or leukemia-free survival in children and adolescents with ALL. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12.

PubMed

Claverie-Martin, F; Diaz-Torres, M R; Kushner, S R

1987-01-01

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

MeWRKY20 and its interacting and activating autophagy-related protein 8 (MeATG8) regulate plant disease resistance in cassava.

PubMed

Yan, Yu; Wang, Peng; He, Chaozu; Shi, Haitao

2017-12-09

As a highly conserved mechanism, autophagy is responsible for the transport of cytoplasmic constituents in the vacuoles or lysosomes. Moreover, autophagy is essential for plant development and various stress responses. In this study, 34 MeATGs were systematically identified in cassava, and their transcripts were commonly regulated by Xanthomonas axonopodis pv manihotis (Xam). Through transient expression in Nicotiana benthamiana, the subcellular locations of 4 MeATG8s were revealed. Notably, MeWRKY20 was identified as physical interacting protein of MeATG8a/8f/8h and upstream transcriptional activator of MeATG8a. Through virus-induced gene silencing (VIGS) in cassava, we found that MeATG8-silenced and MeWRKY20-silenced plants resulted in disease sensitive, with less callose depositions and lower autophagic activity. This study may facilitate our understanding of the upstream MeWRKY20 and underlying target as well as interacting proteins of MeATG8s in immune response. Taken together, MeWRKY20 and MeATG8a/8f/8h are essential for disease resistance against bacterial blight by forming various transcriptional modules and interacting complex in cassava. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular characterization and expression analysis of three novel autophagy-related genes from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

PubMed

Flores Fernández, José Miguel; Barragán Álvarez, Carla Patricia; Sánchez Hernández, Carla Vanessa; Padilla Camberos, Eduardo; González Castillo, Celia; Ortuño Sahagún, Daniel; Martínez Velázquez, Moisés

2016-11-01

The cattle tick Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite of major importance for the livestock industry. It shows a remarkable ability to survive over long periods without feeding. However, the mechanisms used to endure long-term starvation are poorly understood. It is believed that autophagy, a process of intracellular protein degradation, may play a significant role to confront adverse environmental conditions. To advance our understanding of autophagy in R. microplus, in the present study we report the molecular characterization of three autophagy-related (ATG) genes, namely, RmATG3, RmATG4 and RmATG6, as well as their expression profiles in different developmental stages and organs of the parasite. The deduced amino acid sequences derived from the characterized gene sequences were subjected to Basic Local Alignment Search Tool analysis. The testing produced significant alignments with respective ATG proteins from Haemaphysalis longicornis and Ixodes scapularis ticks. Real-time polymerase chain reaction assays revealed that RmATG4 and RmATG6 transcripts were elevated in egg and ovary tissue, when compared with larva and midgut samples, while RmATG3 expression in midgut was 2-fold higher than in egg, larva and ovary samples.

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove

PubMed Central

Filbin, Megan E.; Kieft, Jeffrey S.

2011-01-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem–loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem–loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis. PMID:21606179

HCV IRES domain IIb affects the configuration of coding RNA in the 40S subunit's decoding groove.

PubMed

Filbin, Megan E; Kieft, Jeffrey S

2011-07-01

Hepatitis C virus (HCV) uses a structured internal ribosome entry site (IRES) RNA to recruit the translation machinery to the viral RNA and begin protein synthesis without the ribosomal scanning process required for canonical translation initiation. Different IRES structural domains are used in this process, which begins with direct binding of the 40S ribosomal subunit to the IRES RNA and involves specific manipulation of the translational machinery. We have found that upon initial 40S subunit binding, the stem-loop domain of the IRES that contains the start codon unwinds and adopts a stable configuration within the subunit's decoding groove. This configuration depends on the sequence and structure of a different stem-loop domain (domain IIb) located far from the start codon in sequence, but spatially proximal in the IRES•40S complex. Mutation of domain IIb results in misconfiguration of the HCV RNA in the decoding groove that includes changes in the placement of the AUG start codon, and a substantial decrease in the ability of the IRES to initiate translation. Our results show that two distal regions of the IRES are structurally communicating at the initial step of 40S subunit binding and suggest that this is an important step in driving protein synthesis.

The CUG-initiated larger form coat protein of Chinese wheat mosaic virus binds to the cysteine-rich RNA silencing suppressor.

PubMed

Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Ratti, Claudio; Chen, Jianping

2013-10-01

Some viruses use alternative translation initiation at non-AUG codons as a strategy to produce multiple proteins during gene expression. Here we show that, using this strategy, Chinese wheat mosaic virus (CWMV; Furovirus) expresses a larger form of coat protein (N-ext/CP) in infected plants. Site-directed mutagenesis and transient expression analysis confirmed that CWMV N-ext/CP is initiated at an upstream in-frame CUG codon at nucleotide position 207-209 of RNA 2, which adds a 39 amino acid (aa) N-terminal extension to the major CP. Interestingly, in planta and in vitro analyses indicated that CWMV N-ext/CP but not CP interacts with the CWMV cysteine-rich protein (CRP), an RNA silencing suppressor. We further determined that the N-terminal 39 aa extension, particularly the 10 aa region immediately upstream of the major CP coding region is responsible for the interaction of N-ext/CP with CRP. In an Agrobacterium co-infiltration assay, co-expression with N-ext/CP did not affect CRP silencing suppression activity. Thus the alternative translation initiation at a CUG codon provides the CWMV N-ext/CP with the ability to bind to the viral silencing suppressor. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular cloning and evolutionary analysis of captive forest musk deer bitter taste receptor gene T2R16.

PubMed

Zhao, G J; Wu, N; Li, D Y; Zeng, D J; Chen, Q; Lu, L; Feng, X L; Zhang, C L; Zheng, C L; Jie, H

2015-12-08

Sensing bitter tastes is crucial for most animals because it can prevent them from ingesting harmful food. This process is mainly mediated by the bitter taste receptors (T2R) that are largely expressed in the taste buds. Previous studies have identified some T2R gene repertoires. Marked variation in repertoire size has been noted among species. However, research on T2Rs is still limited and the mechanisms underlying the evolution of vertebrate T2Rs remain poorly understood. In the present study, we analyzed the structure and features of the protein encoded by the forest musk deer (Moschus berezovskii) T2R16 and submitted the gene sequence to NCBI GenBank. The results showed that the full coding DNA sequence (CDS) of musk deer T2R16 (GenBank accession No. KP677279) was 906 bp, encoding 301 amino acids, which contained ATG start codon and TGA stop codon, with a calculated molecular weight of 35.03 kDa and an isoelectric point of 9.56. The T2R16 protein receptor had seven conserved transmembrane regions. Hydrophobicity analysis showed that most amino acid residues in T2R16 protein were hydrophobic, and the grand average of hydrophobicity (GRAVY) was 0.657. Phylogenetic analysis based on this gene revealed that forest musk deer had the closest association with sheep (Ovis aries), as compared to cow (Bos taurus), Tursiops truncatus, and other species, whereas it was genetically farthest from humans (Homo sapiens). We hope these results would complement the existing data on T2R16 and encourage further research in this respect.

Stress-response transcription factors Msn2 and Msn4 couple TORC2-Ypk1 signaling and mitochondrial respiration to ATG8 gene expression and autophagy.

PubMed

Vlahakis, Ariadne; Lopez Muniozguren, Nerea; Powers, Ted

2017-01-01

Macroautophagy/autophagy is a starvation and stress-induced catabolic process critical for cellular homeostasis and adaptation. Several Atg proteins are involved in the formation of the autophagosome and subsequent degradation of cytoplasmic components, a process termed autophagy flux. Additionally, the expression of several Atg proteins, in particular Atg8, is modulated transcriptionally, yet the regulatory mechanisms involved remain poorly understood. Here we demonstrate that the AGC kinase Ypk1, target of the rapamycin-insensitive TORC2 signaling pathway, controls ATG8 expression by repressing the heterodimeric Zinc-finger transcription factors Msn2 and Msn4. We find that Msn2 and Msn4 promote ATG8 expression downstream of the histone deacetylase complex (HDAC) subunit Ume6, a previously identified negative regulator of ATG8 expression. Moreover, we demonstrate that TORC2-Ypk1 signaling is functionally linked to distinct mitochondrial respiratory complexes. Surprisingly, we find that autophagy flux during amino acid starvation is also dependent upon Msn2-Msn4 activity, revealing a broad role for these transcription factors in the autophagy response.

Comparative mitochondrial genome analysis of Daphnis nerii and other lepidopteran insects reveals conserved mitochondrial genome organization and phylogenetic relationships

PubMed Central

Sun, Yu; Chen, Chen; Gao, Jin; Abbas, Muhammad Nadeem; Kausar, Saima; Qian, Cen; Wang, Lei; Wei, Guoqing; Zhu, Bao-Jian

2017-01-01

In the present study, the complete sequence of the mitochondrial genome (mitogenome) of Daphnis nerii (Lepidoptera: Sphingidae) is described. The mitogenome (15,247 bp) of D.nerii encodes13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and an adenine (A) + thymine (T)-rich region. Its gene complement and order is similar to that of other sequenced lepidopterans. The 12 PCGs initiated by ATN codons except for cytochrome c oxidase subunit 1 (cox1) gene that is seemingly initiated by the CGA codon as documented in other insect mitogenomes. Four of the 13 PCGs have the incomplete termination codon T, while the remainder terminated with the canonical stop codon. This mitogenome has six major intergenic spacers, with the exception of A+T-rich region, spanning at least 10 bp. The A+T-rich region is 351 bp long, and contains some conserved regions, including ‘ATAGA’ motif followed by a 17 bp poly-T stretch, a microsatellite-like element (AT)9 and also a poly-A element. Phylogenetic analyses based on 13 PCGs using maximum likelihood (ML) and Bayesian inference (BI) revealed that D. nerii resides in the Sphingidae family. PMID:28598968

A Non-Canonical Initiation Site Is Required for Efficient Translation of the Dendritically Localized Shank1 mRNA

PubMed Central

Studtmann, Katrin; Ölschläger-Schütt, Janin; Buck, Friedrich; Richter, Dietmar; Sala, Carlo; Bockmann, Jürgen; Kindler, Stefan; Kreienkamp, Hans-Jürgen

2014-01-01

Local protein synthesis in dendrites enables neurons to selectively change the protein complement of individual postsynaptic sites. Though it is generally assumed that this mechanism requires tight translational control of dendritically transported mRNAs, it is unclear how translation of dendritic mRNAs is regulated. We have analyzed here translational control elements of the dendritically localized mRNA coding for the postsynaptic scaffold protein Shank1. In its 5′ region, the human Shank1 mRNA exhibits two alternative translation initiation sites (AUG+1 and AUG+214), three canonical upstream open reading frames (uORFs1-3) and a high GC content. In reporter assays, fragments of the 5′UTR with high GC content inhibit translation, suggesting a contribution of secondary structures. uORF3 is most relevant to translation control as it overlaps with the first in frame start codon (AUG+1), directing translation initiation to the second in frame start codon (AUG+214). Surprisingly, our analysis points to an additional uORF initiated at a non-canonical ACG start codon. Mutation of this start site leads to an almost complete loss of translation initiation at AUG+1, demonstrating that this unconventional uORF is required for Shank1 synthesis. Our data identify a novel mechanism whereby initiation at a non-canonical site allows for translation of the main Shank1 ORF despite a highly structured 5′UTR. PMID:24533096

Pharmacokinetic and pharmacodynamic studies of two different rabbit antithymocyte globulin dosing regimens: results of a randomized trial.

PubMed

Büchler, Matthias; Longuet, Hélène; Lemoine, Roxane; Herr, Florence; Gatault, Philippe; Thibault, Gilles; Ternant, David; Foulon, Christine; Pilorge, Bernadette; Lemay, Djamila; Sung, Crystal; Halimi, Jean-Michel; Baron, Christophe; Lebranchu, Yvon

2013-03-01

Rabbit antithymocyte globulin (rATG; Thymoglobulin(®)) is currently used to prevent acute rejection in kidney transplantation. The dose and regimen of rATG have not been optimized. Moreover, the impact of different treatment regimens on T-cell phenotype reconstitution remains unknown. We conducted a prospective randomized study of 17 renal transplant patients to determine the pharmacokinetics of total and active (bound to human cells) rATG and T-cell phenotype reconstitution after rATG administration. Patients received rATG at a total dose of 6mg/kg, administered either as 1.5mg/kg/day on days 0-3 (Group 1, n=8) or 3mg/kg on days 0 and 3 (Group 2, n=9). All patients received tacrolimus, mycophenolate mofetil and steroids. Blood samples were assayed for total rATG by enzyme linked immunosorbent assay and active rATG by flow cytometry. Maximum concentrations and terminal half-lives were similar between the two groups but at month 3 Group 1 had significantly lower values for total rATG (concentration was 6.2±1.1μg/mL versus 10.2±2.9μg/mL in Group 2, p=0.027) and total rATG dose-normalized AUC (374±83dayg/mL versus 508±149dayg/mL in Group 2, p=0.046). Time to sub-therapeutic levels (<1μg/mL) of active rATG was significantly shorter in Group 1 (18.75±6.9days versus 20±7.5days in Group 2, p<0.001). rATG induced significant depletion followed by slow reconstitution of CD3(+), CD4(+) and CD8(+) cells, with no marked differences between groups. B-cell count was unaffected, whereas CD3(-)CD56(+) NK-cell depletion was observed in both groups. rATG induced a significant decrease in the proportion of naïve CD4(+) T-cells, which plateaued after month 1 in Group 1 and after month 6 in Group 2. The proportion of central memory CD4(+) T-cells increased to a similar extent in both groups (Group 1: 38±18% at baseline, 74±23% at one year, p=0.009; Group 2: 32±14% at baseline, 65±14% at one year, p=0.001). In conclusion, our results suggest that the dosing regimen for rATG induction influences pharmacokinetic parameters without affecting the quality of immune reconstitution. Copyright © 2013 Elsevier B.V. All rights reserved.

Arctigenin: A two-edged sword in ischemia/reperfusion induced acute kidney injury.

PubMed

Han, Feng; Xia, Xin-Xin; Dou, Meng; Wang, Yu-Xiang; Xue, Wu-Jun; Ding, Xiao-Ming; Zheng, Jin; Ding, Chen-Guang; Tian, Pu-Xun

2018-07-01

Arctigenin (ATG) is one of the main active substances in fruit derived from Arctium lappa L. Previous studies have reported that ATG have antitumor, neuroprotective, antioxidant, antifibrosis and anti-inflammatory functions. However, the actions of ATG in kidney with acute injury following ischemia/ reperfusion (I/R) is still uncertain. In our study, mice were subjected to kidney I/R by having the kidney pedicles clamped and administered with vehicle or ATG (1, 3 or 9 mg/kg/d) via oral gavage for 7 consecutive days prior to I/R. Notably, ATG aggravated kidney I/R injury with the concentration increases. Multiple biochemical assays and histological examination showed ATG significantly alleviated the inflammatory response as reflected by a decreased expression of proinflammatory cytokine, TLR4/MyD88, and NF-κB, along with the infiltration of CD68 + macrophage and CD11b + Gr1 + neutrophil in the kidneys. Meanwhile, ATG alleviated I/R-induced oxidative stress proved by increasing kidney manganese superoxide dismutase and glutathione peroxidase activity but reducing levels of malonaldehyde and inducible nitric oxide synthase. On the contrary, apoptosis was significantly increased in kidneys of ATG-treated mice compared with vehicle-treated controls, especially in tubular cells. There were increased numbers of TUNEL positive cells and increased Bcl-2, Bax, cleaved-caspase-3, and cleaved-caspase-9 expression. The current study demonstrates that pretreatment of ATG aggravates I/R induced acute kidney injury by increasing apoptosis of tubular cells despite reducing infiltrating inflammatory cells and proinflammatory cytokine. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Up-regulation of autophagy-related gene 5 (ATG5) protects dopaminergic neurons in a zebrafish model of Parkinson's disease.

PubMed

Hu, Zhan-Ying; Chen, Bo; Zhang, Jing-Pu; Ma, Yuan-Yuan

2017-11-03

Parkinson's disease (PD) is one of the most epidemic neurodegenerative diseases and is characterized by movement disorders arising from loss of midbrain dopaminergic (DA) neurons. Recently, the relationship between PD and autophagy has received considerable attention, but information about the mechanisms involved is lacking. Here, we report that autophagy-related gene 5 ( ATG5 ) is potentially important in protecting dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in zebrafish. Using analyses of zebrafish swimming behavior, in situ hybridization, immunofluorescence, and expressions of genes and proteins related to PD and autophagy, we found that the ATG5 expression level was decreased and autophagy flux was blocked in this model. The ATG5 down-regulation led to the upgrade of PD-associated proteins, such as β-synuclein, Parkin, and PINK1, aggravation of MPTP-induced PD-mimicking pathological locomotor behavior, DA neuron loss labeled by tyrosine hydroxylase (TH) or dopamine transporter (DAT), and blocked autophagy flux in the zebrafish model. ATG5 overexpression alleviated or reversed these PD pathological features, rescued DA neuron cells as indicated by elevated TH/DAT levels, and restored autophagy flux. The role of ATG5 in protecting DA neurons was confirmed by expression of the human atg5 gene in the zebrafish model. Our findings reveal that ATG5 has a role in neuroprotection, and up-regulation of ATG5 may serve as a goal in the development of drugs for PD prevention and management. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Retrospective study of alemtuzumab vs ATG-based conditioning without irradiation for unrelated and matched sibling donor transplants in acquired severe aplastic anemia: a study from the British Society for Blood and Marrow Transplantation.

PubMed

Marsh, J C; Pearce, R M; Koh, M B C; Lim, Z; Pagliuca, A; Mufti, G J; Perry, J; Snowden, J A; Vora, A J; Wynn, R T; Russell, N; Gibson, B; Gilleece, M; Milligan, D; Veys, P; Samarasinghe, S; McMullin, M; Kirkland, K; Cook, G

2014-01-01

This retrospective national study compared the use of alemtuzumab-based conditioning regimens for hematopoietic SCT (HSCT) in acquired severe aplastic anemia with antithymocyte globulin (ATG)-based regimens. One hundred patients received alemtuzumab and 55 ATG-based regimens. A matched sibling donor (MSD) was used in 87 (56%), matched unrelated donor (MUD) in 60 (39%) and other related or mismatched unrelated donor (UD) in 8 (5%) patients. Engraftment failure occurred in 9% of the alemtuzumab group and 11% of the ATG group. Five-year OS was 90% for the alemtuzumab and 79% for the ATG groups, P=0.11. For UD HSCT, OS of patients was better when using alemtuzumab (88%) compared with ATG (57%), P=0.026, although smaller numbers of patients received ATG. Similar outcomes for MSD HSCT using alemtuzumab or ATG were seen (91% vs 85%, respectively, P=0.562). A lower risk of chronic GVHD (cGVHD) was observed in the alemtuzumab group (11% vs 26%, P=0.031). On multivariate analysis, use of BM as stem cell source was associated with better OS and EFS, and less acute and cGVHD; young age was associated with better EFS and lower risk of graft failure. This large study confirms successful avoidance of irradiation in the conditioning regimens for MUD HSCT patients.

Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora.

PubMed

Voigt, Oliver; Pöggeler, Stefanie

2013-01-01

Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions.

Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora

PubMed Central

Voigt, Oliver; Pöggeler, Stefanie

2013-01-01

Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions. PMID:23064313

Ribosome stalling and peptidyl-tRNA drop-off during translational delay at AGA codons

PubMed Central

Cruz-Vera, Luis Rogelio; Magos-Castro, Marco Antonio; Zamora-Romo, Efraín; Guarneros, Gabriel

2004-01-01

Minigenes encoding the peptide Met–Arg–Arg have been used to study the mechanism of toxicity of AGA codons proximal to the start codon or prior to the termination codon in bacteria. The codon sequences of the ‘mini-ORFs’ employed were initiator, combinations of AGA and CGA, and terminator. Both, AGA and CGA are low-usage Arg codons in ORFs of Escherichia coli but, whilst AGA is translated by the scarce tRNAArg4, CGA is recognized by the abundant tRNAArg2. Overexpression of minigenes harbouring AGA in the third position, next to a termination codon, was deleterious to the cell and led to the accumulation of peptidyl-tRNAArg4 and of the peptidyl-tRNA cognate to the preceding CGA or AGA Arg triplet. The minigenes carrying CGA in the third position were not toxic. Minigene-mediated toxicity and peptidyl-tRNA accumulation were suppressed by overproduction of tRNAArg4 but not by overproduction of peptidyl-tRNA hydrolase, an enzyme that is only active on substrates that have been released from the ribosome. Consistent with these findings, peptidyl-tRNAArg4 was identified to be mainly associated with ribosomes in a stand-by complex. These and previous results support the hypothesis that the primary mechanism of inhibition of protein synthesis by AGA triplets in pth+ cells involves sequestration of tRNAs as peptidyl-tRNA on the stalled ribosome. PMID:15317870

PI(5)P Regulates Autophagosome Biogenesis

PubMed Central

Vicinanza, Mariella; Korolchuk, Viktor I.; Ashkenazi, Avraham; Puri, Claudia; Menzies, Fiona M.; Clarke, Jonathan H.; Rubinsztein, David C.

2015-01-01

Summary Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles. PMID:25578879

Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase-Dependent Cell Death and Leads to Mild Growth Retardation.

PubMed

Vuppalapati, Karuna K; Bouderlique, Thibault; Newton, Phillip T; Kaminskyy, Vitaliy O; Wehtje, Henrik; Ohlsson, Claes; Zhivotovsky, Boris; Chagin, Andrei S

2015-12-01

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes. © 2015 American Society for Bone and Mineral Research.

Translational Control in Bone Marrow Failure

DTIC Science & Technology

2014-04-01

expressing the ATG2 ORF and the ATG3 ORF, we also evaluated the ATG> GTG mutation, which produces all 3 internal ORFs. (We did not test ORF4...expressing shortened forms of NE reduced clonogenic capacity (Fig. 4 of Tidwell et al.). When comparing the shorter isoforms of NE to each other, the GTG ...mutated from ATG to either ATA or GTG , shortened forms of NE were evident (Figure 2, c.3G.A, c.1A.G). When the highly conserved nucleotide in the Kozak

Methods for Studying Interactions Between Atg8/LC3/GABARAP and LIR-Containing Proteins.

PubMed

Johansen, T; Birgisdottir, Å B; Huber, J; Kniss, A; Dötsch, V; Kirkin, V; Rogov, V V

2017-01-01

LC3/GABARAP proteins (LC3/GABARAPs) are mammalian orthologues of yeast Atg8, small ubiquitin (Ub)-like proteins (UBLs) whose covalent attachment to lipid membranes is crucial for the growth and closure of the double membrane vesicle called the autophagosome. In the past decade, it was demonstrated that Atg8/LC3/GABARAPs are also required for autophagic degradation of cargos in a selective fashion. Cargo selectivity is ensured by receptor proteins, such as p62/SQSTM1, NBR1, Cue5, Atg19, NIX, Atg32, NCOA4, and FAM134B, which simultaneously bind Atg8/LC3/GABARAPs and the cargo together, thereby linking the core autophagic machinery to the target structure: a protein, an organelle, or a pathogen. LC3-interacting regions (LIRs) are short linear motifs within selective autophagy receptors and some other structural and signaling proteins (e.g., ULK1, ATG13, FIP200, and Dvl2), which mediate binding to Atg8/LC3/GABARAPs. Identification and characterization of LIR-containing proteins have provided important insights into the biology of the autophagy pathway, and studying their interactions with the core autophagy machinery represents a growing area of autophagy research. Here, we present protocols for the identification of LIR-containing proteins, i.e., by yeast-two-hybrid screening, glutathione S-transferase (GST) pulldown experiments, and peptide arrays. The use of two-dimensional peptide arrays also represents a powerful method to identify the residues of the LIR motif that are critical for binding. We also describe a biophysical method for studying interactions between Atg8/LC3/GABARAP and LIR-containing proteins and a protocol for preparation and purification of LIR peptides. © 2017 Elsevier Inc. All rights reserved.

A Double-Blind, Double-Dummy, Flexible-Design Randomized Multicenter Trial: Early Safety of Single- Versus Divided-Dose Rabbit Anti-Thymocyte Globulin Induction in Renal Transplantation.

PubMed

Stevens, R B; Wrenshall, L E; Miles, C D; Farney, A C; Jie, T; Sandoz, J P; Rigley, T H; Osama Gaber, A

2016-06-01

A previous nonblinded, randomized, single-center renal transplantation trial of single-dose rabbit anti-thymocyte globulin induction (SD-rATG) showed improved efficacy compared with conventional divided-dose (DD-rATG) administration. The present multicenter, double-blind/double-dummy STAT trial (Single dose vs. Traditional Administration of Thymoglobulin) evaluated SD-rATG versus DD-rATG induction for noninferiority in early (7-day) safety and tolerability. Ninety-five patients (randomized 1:1) received 6 mg/kg SD-rATG or 1.5 mg/kg/dose DD-rATG, with tacrolimus-mycophenolate maintenance immunosuppression. The primary end point was a composite of fever, hypoxia, hypotension, cardiac complications, and delayed graft function. Secondary end points included 12-month patient survival, graft survival, and rejection. Target enrollment was 165 patients with an interim analysis scheduled after 80 patients. Interim analysis showed primary end point noninferiority of SD-rATG induction (p = 0.6), and a conditional probability of <1.73% of continued enrollment producing a significant difference (futility analysis), leading to early trial termination. Final analysis (95 patients) showed no differences in occurrence of primary end point events (p = 0.58) or patients with no, one, or more than one event (p = 0.81), or rejection, graft, or patient survival (p = 0.78, 0.47, and 0.35, respectively). In this rigorously blinded trial in adult renal transplantation, we have shown SD-rATG induction to be noninferior to DD-rATG induction in early tolerability and equivalent in 12-month safety. (Clinical Trials.gov #NCT00906204.). © Copyright 2016 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of the American Society of Transplantation and the American Society of Transplant Surgeons.

Vitamin B6 status, immune response and inflammation markers in kidney transplant recipients treated with polyclonal anti-thymocyte globulin.

PubMed

Jankowska, M; Trzonkowski, P; Dębska-Ślizień, A; Marszałł, M; Rutkowski, B

2014-10-01

Vitamin B6 status has an impact on the body's inflammatory and immune responses. Immunosuppressive therapy may influence vitamin B6 metabolism in kidney transplant recipients. Treatment with polyclonal anti-thymocyte globulin (ATG) is associated with long-term changes in inflammatory and immune parameters. It is not known if ATG therapy also may have an impact on vitamin B6 status in kidney transplant recipients. We aimed to analyze the impact of therapy with ATG on vitamin B6 status, immune response, and the profile of inflammatory cytokines. This was a retrospective, observational study that included 44 kidney allograft recipients. Twenty patients received induction therapy with ATG (6 to 24 months before enrollment). Twenty-four patients constituted the control group, matched with respect to time since transplantation. The B6 vitamers, total lymphocyte count, CD3 percentage, interleukin (IL)-6, -7, and -10, transforming growth factor β, interferon γ, and chemokine ligand 21 were analyzed in a study group. All indicators of vitamin B6 status were lower in the ATG group than in the control group. There were also significant differences with respect to immune response (significantly lower total lymphocyte count and CD3 in the ATG group) and inflammatory status (significantly higher IL-6 and IL-10 in the ATG group). Vitamin B6 vitamers and derivatives were not related to lymphocyte count and cytokine levels or to estimated glomerular filtration rate and age of the study population. Vitamin B6 stores and active forms are lower in kidney transplant recipients treated with ATG. ATG therapy promotes CD3 and total lymphocyte depletion and increases indicators of inflammation. We found no associations between vitamers of B6, immune response cells, and inflammatory cytokines in study population.

Mammalian autophagy and the plasma membrane.

PubMed

Pavel, Mariana; Rubinsztein, David C

2017-03-01

Autophagy (literally 'self-eating') is an evolutionarily conserved degradation process where cytoplasmic components are engulfed by vesicles called autophagosomes, which are then delivered to lysosomes, where their contents are degraded. Under stress conditions, such as starvation or oxidative stress, autophagy is upregulated in order to degrade macromolecules and restore the nutrient balance. The source of membranes that participate in the initial formation of phagophores is still incompletely understood and many intracellular structures have been shown to act as lipid donors, including the endoplasmic reticulum, Golgi, nucleus, mitochondria and the plasma membrane. Here, we focus on the contributions of the plasma membrane to autophagosome biogenesis governed by ATG16L1 and ATG9A trafficking, and summarize the physiological and pathological implications of this macroautophagy route, from development and stem cell fate to neurodegeneration and cancer. © 2016 Federation of European Biochemical Societies.

Structure of a human cap-dependent 48S translation pre-initiation complex

PubMed Central

Eliseev, Boris; Yeramala, Lahari; Leitner, Alexander; Karuppasamy, Manikandan; Raimondeau, Etienne; Huard, Karine; Alkalaeva, Elena; Aebersold, Ruedi

2018-01-01

Abstract Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition. PMID:29401259

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

PubMed Central

Liu, Kun; Zhao, Qian; Liu, Pinglei; Cao, Jiani; Gong, Jiaqi; Wang, Chaoqun; Wang, Weixu; Li, Xiaoyan; Sun, Hongyan; Zhang, Chao; Li, Yufei; Jiang, Minggui; Zhu, Shaohua; Sun, Qingyuan; Jiao, Jianwei; Hu, Baoyang; Zhao, Xiaoyang; Li, Wei; Chen, Quan; Zhou, Qi; Zhao, Tongbiao

2016-01-01

ABSTRACT Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production.1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance. PMID:27575019

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance.

PubMed

Liu, Kun; Zhao, Qian; Liu, Pinglei; Cao, Jiani; Gong, Jiaqi; Wang, Chaoqun; Wang, Weixu; Li, Xiaoyan; Sun, Hongyan; Zhang, Chao; Li, Yufei; Jiang, Minggui; Zhu, Shaohua; Sun, Qingyuan; Jiao, Jianwei; Hu, Baoyang; Zhao, Xiaoyang; Li, Wei; Chen, Quan; Zhou, Qi; Zhao, Tongbiao

2016-11-01

Pluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production. 1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance.

Development of the automatic test pattern generation for NPP digital electronic circuits using the degree of freedom concept

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, D.S.; Seong, P.H.

1995-08-01

In this paper, an improved algorithm for automatic test pattern generation (ATG) for nuclear power plant digital electronic circuits--the combinational type of logic circuits is presented. For accelerating and improving the ATG process for combinational circuits the presented ATG algorithm has the new concept--the degree of freedom (DF). The DF, directly computed from the system descriptions such as types of gates and their interconnections, is the criterion to decide which among several alternate lines` logic values required along each path promises to be the most effective in order to accelerate and improve the ATG process. Based on the DF themore » proposed ATG algorithm is implemented in the automatic fault diagnosis system (AFDS) which incorporates the advanced fault diagnosis method of artificial intelligence technique, it is shown that the AFDS using the ATG algorithm makes Universal Card (UV Card) testing much faster than the present testing practice or by using exhaustive testing sets.« less

Autophagy proteins are not universally required for phagosome maturation.

PubMed

Cemma, Marija; Grinstein, Sergio; Brumell, John H

2016-09-01

Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcγR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in Atg5-deficient mouse embryonic fibroblasts, or in Atg5- or Atg7-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG proteins may be required for phagosome maturation under some conditions, but are not universally required for this process.

Formation of a Snf1-Mec1-Atg1 Module on Mitochondria Governs Energy Deprivation-Induced Autophagy by Regulating Mitochondrial Respiration.

PubMed

Yi, Cong; Tong, Jingjing; Lu, Puzhong; Wang, Yizheng; Zhang, Jinxie; Sun, Chen; Yuan, Kangning; Xue, Renyu; Zou, Bing; Li, Nianzhong; Xiao, Shuhua; Dai, Chong; Huang, Yuwei; Xu, Liling; Li, Lin; Chen, She; Miao, Di; Deng, Haiteng; Li, Hongliang; Yu, Li

2017-04-10

Autophagy is essential for maintaining glucose homeostasis, but the mechanism by which energy deprivation activates autophagy is not fully understood. We show that Mec1/ATR, a member of the DNA damage response pathway, is essential for glucose starvation-induced autophagy. Mec1, Atg13, Atg1, and the energy-sensing kinase Snf1 are recruited to mitochondria shortly after glucose starvation. Mec1 is recruited through the adaptor protein Ggc1. Snf1 phosphorylates Mec1 on the mitochondrial surface, leading to recruitment of Atg1 to mitochondria. Furthermore, the Snf1-mediated Mec1 phosphorylation and mitochondrial recruitment of Atg1 are essential for maintaining mitochondrial respiration during glucose starvation, and active mitochondrial respiration is required for energy deprivation-activated autophagy. Thus, formation of a Snf1-Mec1-Atg1 module on mitochondria governs energy deprivation-induced autophagy by regulating mitochondrial respiration. Copyright © 2017 Elsevier Inc. All rights reserved.

Conditional inhibition of autophagy genes in adult Drosophila impairs immunity without compromising longevity.

PubMed

Ren, Chunli; Finkel, Steven E; Tower, John

2009-03-01

Immune function declines with age in Drosophila and humans, and autophagy is implicated in immune function. In addition, autophagy genes are required for life span extension caused by reduced insulin/IGF1-like signaling and dietary restriction in Caenorhabditiselegans. To test if the autophagy pathway might be limiting for immunity and/or life span in adult Drosophila, the Geneswitch system was used to cause conditional inactivation of the autophagy genes Atg5, Atg7 and Atg12 by RNAi. Conditional inhibition of Atg genes in adult flies reduced lysotracker staining of adult tissues, and reduced resistance to injected Escherichia coli, as evidenced by increased bacterial titers and reduced fly survival. However, survival of uninjected flies was unaffected by Atg gene inactivation. The data indicate that Atg gene activity is required for normal immune function in adult flies, and suggest that neither autophagy nor immune function are limiting for adult life span under typical laboratory conditions.

Genome-wide analysis of autophagy-related genes in banana highlights MaATG8s in cell death and autophagy in immune response to Fusarium wilt.

PubMed

Wei, Yunxie; Liu, Wen; Hu, Wei; Liu, Guoyin; Wu, Chunjie; Liu, Wei; Zeng, Hongqiu; He, Chaozu; Shi, Haitao

2017-08-01

MaATG8s play important roles in hypersensitive-like cell death and immune response, and autophagy is essential for disease resistance against Foc in banana. Autophagy is responsible for the degradation of damaged cytoplasmic constituents in the lysosomes or vacuoles. Although the effects of autophagy have been extensively revealed in model plants, the possible roles of autophagy-related gene in banana remain unknown. In this study, 32 MaATGs were identified in the draft genome, and the profiles of several MaATGs in response to fungal pathogen Fusarium oxysporum f. sp. cubense (Foc) were also revealled. We found that seven MaATG8s were commonly regulated by Foc. Through transient expression in Nicotiana benthamiana leaves, we highlight the novel roles of MaATG8s in conferring hypersensitive-like cell death, and MaATG8s-mediated hypersensitive response-like cell death is dependent on autophagy. Notablly, autophagy inhibitor 3-methyladenine (3-MA) treatment resulted in decreased disease resistance in response to Foc4, and the effect of 3-MA treatment could be rescued by exogenous salicylic acid, jasmonic acid and ethylene, indicating the involvement of autophagy-mediated plant hormones in banana resistance to Fusarium wilt. Taken together, this study may extend our understanding the putative role of MaATG8s in hypersensitive-like cell death and the essential role of autophagy in immune response against Foc in banana.

In vitro effects of ATG-Fresenius on immune cell adhesion.

PubMed

Kanzler, I; Seitz-Merwald, I; Schleger, S; Kaczmarek, I; Kur, F; Beiras-Fernandez, A

2013-06-01

ATG-Fresenius, a purified rabbit polyclonal anti-human T-lymphocyte immunoglobulin is used for induction immunosuppression as well as prevention and treatment of acute rejection episodes among patients receiving solid organ transplants. The aim of this study was to investigate the in vitro activity of ATG-Fresenius upon immune cell adhesion, which may explain its activity to mitigate ischemia-reperfusion injury. Human vascular endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBMCs) isolated from umbilical vein or peripheral blood were incubated 20 to 24 hours before analysis. HUVEC were incubated with 10 and 100 μg/mL ATG-Fresenius or reference polyclonal rabbit immunoglobulin G. Analysis of immune cell adhesion to endothelial cells was studied in cocultures of PBMCs and adherent HUVEC. Endothelial cell expression of adhesion molecules CD62E and CD54 was determined by flow cytometry. The numbers of T-, B- and natural killer cells attached to HUVEC were also determined by flow cytometry. Groups were compared using one-way analysis of variance. We showed that ATG-Fresenius binds to endothelial cells particularly activated ones expressing increased levels of E-selectin and ICAM-1. The increased binding of ATG-Fresenius to activated endothelial cells was consistent with its known binding to Intercellular Adhesion Molecule 1 (ICAM-1) and selectins. We also showed that ATG-Fresenius inhibited adhesion of prestimulated immune cells to activated endothelium. We demonstrated dose-dependent binding of ATG-Fresenius to activated endothelial cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Efficacy of induction therapy with ATG and intravenous immunoglobulins in patients with low-level donor-specific HLA-antibodies.

PubMed

Bächler, K; Amico, P; Hönger, G; Bielmann, D; Hopfer, H; Mihatsch, M J; Steiger, J; Schaub, S

2010-05-01

Low-level donor-specific HLA-antibodies (HLA-DSA) (i.e. detectable by single-antigen flow beads, but negative by complement-dependent cytotoxicity crossmatch) represent a risk factor for early allograft rejection. The short-term efficacy of an induction regimen consisting of polyclonal anti-T-lymphocyte globulin (ATG) and intravenous immunoglobulins (IvIg) in patients with low-level HLA-DSA is unknown. In this study, we compared 67 patients with low-level HLA-DSA not having received ATG/IvIg induction (historic control) with 37 patients, who received ATG/IvIg induction. The two groups were equal regarding retransplants, HLA-matches, number and class of HLA-DSA. The overall incidence of clinical/subclinical antibody-mediated rejection (AMR) was lower in the ATG/IvIg than in the historic control group (38% vs. 55%; p = 0.03). This was driven by a significantly lower rate of clinical AMR (11% vs. 46%; p = 0.0002). Clinical T-cell-mediated rejection (TCR) was significantly lower in the ATG/IvIg than in the historic control group (0% vs. 50%; p < 0.0001). Within the first year, allograft loss due to AMR occurred in 7.5% in the historic control and in 0% in the ATG/IvIg group. We conclude that in patients with low-level HLA-DSA, ATG/IvIg induction significantly reduces TCR and the severity of AMR, but the high rate of subclinical AMR suggests an insufficient control of the humoral immune response.

The mouse Gtl2 gene is differentially expressed during embryonic development, encodes multiple alternatively spliced transcripts, and may act as an RNA.

PubMed

Schuster-Gossler, K; Bilinski, P; Sado, T; Ferguson-Smith, A; Gossler, A

1998-06-01

We have isolated a novel mouse gene (Gtl2) from the site of a gene trap integration (Gtl2lacZ) that gave rise to developmentally regulated lacZ expression, and a dominant parental-origin-dependent phenotype. Heterozygous Gtl2lacZ mice that inherited the transgene from the father showed a proportionate dwarfism phenotype, whereas the penetrance and expressivity of the phenotype was strongly reduced in Gtl2lacZ mice that inherited the transgene from the mother. Gtl2 expression is highly similar to the beta-galactosidase staining pattern, and is down-regulated but not abolished in mice carrying the Gtl2lacZ insertion. In early postimplantation embryos, Gtl2 is expressed in the visceral yolk sac and embryonic ectoderm. During subsequent development and organogenesis, Gtl2 transcripts are abundant in the paraxial mesoderm closely correlated with myogenic differentiation, in parts of the central nervous system, and in the epithelial ducts of developing excretory organs. The Gtl2 gene gives rise to various differentially spliced transcripts, which contain multiple small open reading frames (ORF). However, none of the ATG codons of these ORFs is in the context of a strong Kozak consensus sequence for initiation of translation, suggesting that Gtl2 might function as an RNA. Nuclear Gtl2 RNA was detected in a temporally and spatially regulated manner, and partially processed Gtl2 transcripts were readily detected in Northern blot hybridizations of polyadenylated RNA, suggesting that primary Gtl2 transcripts are differently processed in various cell types during development. Gtl2 transcript levels are present in parthenogenic embryos but may be reduced, consistent with the pattern of inheritance of the Gtl2lacZ phenotype.

Transforming Growth Factor-β/SMAD Target Gene SKIL Is Negatively Regulated by the Transcriptional Cofactor Complex SNON-SMAD4*

PubMed Central

Tecalco-Cruz, Angeles C.; Sosa-Garrocho, Marcela; Vázquez-Victorio, Genaro; Ortiz-García, Layla; Domínguez-Hüttinger, Elisa; Macías-Silva, Marina

2012-01-01

The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified. PMID:22674574

Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

PubMed Central

Dong, Yewei; Wang, Shuqi; Chen, Junliang; Zhang, Qinghao; Liu, Yang; You, Cuihong; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

2016-01-01

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis. PMID:27472219

Transforming growth factor-β/SMAD Target gene SKIL is negatively regulated by the transcriptional cofactor complex SNON-SMAD4.

PubMed

Tecalco-Cruz, Angeles C; Sosa-Garrocho, Marcela; Vázquez-Victorio, Genaro; Ortiz-García, Layla; Domínguez-Hüttinger, Elisa; Macías-Silva, Marina

2012-08-03

The human SKI-like (SKIL) gene encodes the SMAD transcriptional corepressor SNON that antagonizes TGF-β signaling. SNON protein levels are tightly regulated by the TGF-β pathway: whereas a short stimulation with TGF-β decreases SNON levels by its degradation via the proteasome, longer TGF-β treatment increases SNON levels by inducing SKIL gene expression. Here, we investigated the molecular mechanisms involved in the self-regulation of SKIL gene expression by SNON. Bioinformatics analysis showed that the human SKIL gene proximal promoter contains a TGF-β response element (TRE) bearing four groups of SMAD-binding elements that are also conserved in mouse. Two regions of 408 and 648 bp of the human SKIL gene (∼2.4 kb upstream of the ATG initiation codon) containing the core promoter, transcription start site, and the TRE were cloned for functional analysis. Binding of SMAD and SNON proteins to the TRE region of the SKIL gene promoter after TGF-β treatment was demonstrated by ChIP and sequential ChIP assays. Interestingly, the SNON-SMAD4 complex negatively regulated basal SKIL gene expression through binding the promoter and recruiting histone deacetylases. In response to TGF-β signal, SNON is removed from the SKIL gene promoter, and then the activated SMAD complexes bind the promoter to induce SKIL gene expression. Subsequently, the up-regulated SNON protein in complex with SMAD4 represses its own expression as part of the negative feedback loop regulating the TGF-β pathway. Accordingly, when the SNON-SMAD4 complex is absent as in some cancer cells lacking SMAD4 the regulation of some TGF-β target genes is modified.

Elements in the murine c-mos messenger RNA 5'-untranslated region repress translation of downstream coding sequences.

PubMed

Steel, L F; Telly, D L; Leonard, J; Rice, B A; Monks, B; Sawicki, J A

1996-10-01

Murine c-mos transcripts isolated from testes have 5'-untranslated regions (5'UTRs) of approximately 300 nucleotides with a series of four overlapping open reading frames (ORFs) upstream of the AUG codon that initiates the Mos ORF. Ovarian c-mos transcripts have shorter 5'UTRs (70-80 nucleotides) and contain only 1-2 of the upstream ORFs (uORFs). To test whether these 5'UTRs affect translational efficiency, we have constructed plasmids for the expression of chimeric transcripts with a mos-derived 5'UTR fused to the Escherichia coli beta-galactosidase coding region. Translational efficiency has been evaluated by measuring beta-galactosidase activity NIH3T3 cells transiently transfected with these plasmids and with plasmids where various mutations have been introduced into the 5'UTR. We show that the 5'UTR characteristic of testis-specific c-mos mRNA strongly represses translation relative to the translation of transcripts that contain a 5'UTR derived from beta-globin mRNA, and this is mainly due to the four uORFs. Each of the four upstream AUG triplets can be recognized as a start site for translation, and no single uAUG dominates the repressive effect. The uORFs repress translation by a mechanism that is not affected by the amino acid sequence in the COOH-terminal region of the uORF-encoded peptides. The very short uORF (AUGUGA) present in ovary-specific transcripts does not repress translation. Staining of testis sections from transgenic mice carrying chimeric beta-galactosidase transgene constructs, which contain a mos 5'UTR with or without the uATGs, suggests that the uORFs can dramatically change the pattern of expression in spermatogenic cells.

Delayed degradation of chlorophylls and photosynthetic proteins in Arabidopsis autophagy mutants during stress-induced leaf yellowing

PubMed Central

Sakuraba, Yasuhito; Lee, Sang-Hwa; Kim, Ye-Sol; Park, Ohkmae K.; Hörtensteiner, Stefan; Paek, Nam-Chon

2014-01-01

Plant autophagy, one of the essential proteolysis systems, balances proteome and nutrient levels in cells of the whole plant. Autophagy has been studied by analysing Arabidopsis thaliana autophagy-defective atg mutants, but the relationship between autophagy and chlorophyll (Chl) breakdown during stress-induced leaf yellowing remains unclear. During natural senescence or under abiotic-stress conditions, extensive cell death and early yellowing occurs in the leaves of atg mutants. A new finding is revealed that atg5 and atg7 mutants exhibit a functional stay-green phenotype under mild abiotic-stress conditions, but leaf yellowing proceeds normally in wild-type leaves under these conditions. Under mild salt stress, atg5 leaves retained high levels of Chls and all photosystem proteins and maintained a normal chloroplast structure. Furthermore, a double mutant of atg5 and non-functional stay-green nonyellowing1-1 (atg5 nye1-1) showed a much stronger stay-green phenotype than either single mutant. Taking these results together, it is proposed that autophagy functions in the non-selective catabolism of Chls and photosynthetic proteins during stress-induced leaf yellowing, in addition to the selective degradation of Chl–apoprotein complexes in the chloroplasts through the senescence-induced STAY-GREEN1/NYE1 and Chl catabolic enzymes. PMID:24510943

Zonda is a novel early component of the autophagy pathway in Drosophila.

PubMed

Melani, Mariana; Valko, Ayelén; Romero, Nuria M; Aguilera, Milton O; Acevedo, Julieta M; Bhujabal, Zambarlal; Perez-Perri, Joel; de la Riva-Carrasco, Rocío V; Katz, Maximiliano J; Sorianello, Eleonora; D'Alessio, Cecilia; Juhász, Gabor; Johansen, Terje; Colombo, María I; Wappner, Pablo

2017-11-01

Autophagy is an evolutionary conserved process by which eukaryotic cells undergo self-digestion of cytoplasmic components. Here we report that a novel Drosophila immunophilin, which we have named Zonda, is critically required for starvation-induced autophagy. We show that Zonda operates at early stages of the process, specifically for Vps34-mediated phosphatidylinositol 3-phosphate (PI3P) deposition. Zonda displays an even distribution under basal conditions and, soon after starvation, nucleates in endoplasmic reticulum-associated foci that colocalize with omegasome markers. Zonda nucleation depends on Atg1, Atg13, and Atg17 but does not require Vps34, Vps15, Atg6, or Atg14. Zonda interacts physically with Atg1 through its kinase domain, as well as with Atg6 and Vps34. We propose that Zonda is an early component of the autophagy cascade necessary for Vps34-dependent PI3P deposition and omegasome formation. © 2017 Melani, Valko, Romero, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome.

PubMed

Liu, Rong; Zhi, Xiaoyong; Zhong, Qing

2015-01-01

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.

Genes for cytochrome c oxidase subunit I, URF2, and three tRNAs in Drosophila mitochondrial DNA.

PubMed Central

Clary, D O; Wolstenholme, D R

1983-01-01

Genes for URF2, tRNAtrp, tRNAcys, tRNAtyr and cytochrome c oxidase subunit I (COI) have been identified within a sequenced segment of the Drosophila yakuba mtDNA molecule. The five genes are arranged in the order given. Transcription of the tRNAcys and tRNAtyr genes is in the same direction as replication, while transcription of the URF2, tRNAtrp and COI genes is in the opposite direction. A similar arrangement of these genes is found in mammalian mtDNA except that in the latter, the tRNAala and tRNAasn genes are located between the tRNAtrp and tRNAcys genes. Also, a sequence found between the tRNAasn and tRNAcys genes in mammalian mtDNA, which is associated with the initiation of second strand DNA synthesis, is not found in this region of the D. yakuba mtDNA molecule. As the D. yakuba COI gene lacks a standard translation initiation codon, we consider the possibility that the quadruplet ATAA may serve this function. As in other D. yakuba mitochondrial polypeptide genes, AGA codons in the URF2 and COI genes do not correspond in position to arginine-specifying codons in the equivalent genes of mouse and yeast mtDNAs, but do most frequently correspond to serine-specifying codons. PMID:6314262

The complete mitochondrial genome of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

PubMed

Dai, Li-Shang; Zhu, Bao-Jian; Qian, Cen; Zhang, Cong-Fen; Li, Jun; Wang, Lei; Wei, Guo-Qing; Liu, Chao-Liang

2016-01-01

The complete mitochondrial genome (mitogenome) of Plutella xylostella (Lepidoptera: Plutellidae) was determined (GenBank accession No. KM023645). The length of this mitogenome is 16,014 bp with 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes and an A + T-rich region. It presents the typical gene organization and order for completely sequenced lepidopteran mitogenomes. The nucleotide composition of the genome is highly A + T biased, accounting for 81.48%, with a slightly positive AT skewness (0.005). All PCGs are initiated by typical ATN codons, except for the gene cox1, which uses CGA as its start codon. Some PCGs harbor TA (nad5) or incomplete termination codon T (cox1, cox2, nad2 and nad4), while others use TAA as their termination codons. The A + T-rich region is located between rrnS and trnM with a length of 888 bp.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosatelli, M.C.; Faa, V.; Sardu, R.

This study reports the molecular characterization of [beta]-thalassemia in the Sardinian population. Three thousand [beta]-thalassemia chromosomes from prospective parents presenting at the genetic service were initially analyzed by dot blot analysis with oligonucleotide probes complementary to the most common [beta]-thalassemia mutations in the Mediterranean at-risk populations. The mutation which remained uncharacterized by this approach were defined by denaturing gradient gel electrophoresis (DGGE) followed by direct sequence analysis on amplified DNA. The authors reconfirmed that the predominant mutation in the Sardinian population is the codon 39 nonsense mutation, which accounts for 95.7% of the [beta]-thalassemia chromosomes. The other two relatively commonmore » mutations are frameshifts at codon 6 (2.1%) and at codon 76 (0.7%), relatively uncommon in other Mediterranean-origin populations. In this study they have detected a novel [beta]-thalassemia mutation, i.e., a frameshift at codon 1, in three [beta]-thalassemia chromosomes. The DGGE procedure followed by direct sequencing on amplified DNA is a powerful approach for the characterization of unknown mutations in this genetic system.« less

Impaired autophagy induces chronic atrophic pancreatitis in mice via sex- and nutrition-dependent processes.

PubMed

Diakopoulos, Kalliope N; Lesina, Marina; Wörmann, Sonja; Song, Liang; Aichler, Michaela; Schild, Lorenz; Artati, Anna; Römisch-Margl, Werner; Wartmann, Thomas; Fischer, Robert; Kabiri, Yashar; Zischka, Hans; Halangk, Walter; Demir, Ihsan Ekin; Pilsak, Claudia; Walch, Axel; Mantzoros, Christos S; Steiner, Jörg M; Erkan, Mert; Schmid, Roland M; Witt, Heiko; Adamski, Jerzy; Algül, Hana

2015-03-01

Little is known about the mechanisms of the progressive tissue destruction, inflammation, and fibrosis that occur during development of chronic pancreatitis. Autophagy is involved in multiple degenerative and inflammatory diseases, including pancreatitis, and requires the protein autophagy related 5 (ATG5). We created mice with defects in autophagy to determine its role in pancreatitis. We created mice with pancreas-specific disruption of Atg5 (Ptf1aCreex1;Atg5F/F mice) and compared them to control mice. Pancreata were collected and histology, immunohistochemistry, transcriptome, and metabolome analyses were performed. ATG5-deficient mice were placed on diets containing 25% palm oil and compared with those on a standard diet. Another set of mice received the antioxidant N-acetylcysteine. Pancreatic tissues were collected from 8 patients with chronic pancreatitis (CP) and compared with pancreata from ATG5-deficient mice. Mice with pancreas-specific disruption of Atg5 developed atrophic CP, independent of β-cell function; a greater proportion of male mice developed CP than female mice. Pancreata from ATG5-deficient mice had signs of inflammation, necrosis, acinar-to-ductal metaplasia, and acinar-cell hypertrophy; this led to tissue atrophy and degeneration. Based on transcriptome and metabolome analyses, ATG5-deficient mice produced higher levels of reactive oxygen species than control mice, and had insufficient activation of glutamate-dependent metabolism. Pancreata from these mice had reduced autophagy, increased levels of p62, and increases in endoplasmic reticulum stress and mitochondrial damage, compared with tissues from control mice; p62 signaling to Nqo1 and p53 was also activated. Dietary antioxidants, especially in combination with palm oil-derived fatty acids, blocked progression to CP and pancreatic acinar atrophy. Tissues from patients with CP had many histologic similarities to those from ATG5-deficient mice. Mice with pancreas-specific disruption of Atg5 develop a form of CP similar to that of humans. CP development appears to involve defects in autophagy, glutamate-dependent metabolism, and increased production of reactive oxygen species. These mice might be used to identify therapeutic targets for CP. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Molecular identification of GAPDHs in cassava highlights the antagonism of MeGAPCs and MeATG8s in plant disease resistance against cassava bacterial blight.

PubMed

Zeng, Hongqiu; Xie, Yanwei; Liu, Guoyin; Lin, Daozhe; He, Chaozu; Shi, Haitao

2018-06-01

MeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response. As an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.

ATG-Fresenius or daclizumab induction therapy in immunologically high risk kidney recipients: a prospective randomized pilot trial.

PubMed

Kim, Min Jeong; Tsinalis, Dimitrios; Franz, Stefan; Binet, Isabelle; Gürke, Lorenz; Mihatsch, Michael J; Steiger, Jürg; Thiel, Gilbert; Dickenmann, Michael

2008-01-01

Despite all the advantages in the immunosuppressive therapy, kidney transplantation in immunologically high risk patients remains a challenge. Ideally, an induction therapy should provide maximal graft protection, while adverse events rate and costs remain as low as possible. Immunologically high risk kidney recipients with CDC-PRA ł 25% within the last 3 years, a positive B-cell CDC-crossmatch or graft loss due to rejection within 3 years following a prior transplantation, were randomized 1:1 to receive ATG-Fresenius (ATG-F) (9 mg/kg day 0; 3 mg/kg day 1-4) or Daclizumab therapy (1 mg/kg day 0, 14, 28, 42, 56) in a pilot study. Additional immunosuppression consisted of cyclosporine, mycophenolate mofetil, and steroids. 11 patients were included in each group. The patient (90% in ATG-F; 100% in Daclizumab) and graft survival (censored for death) (100% in ATG-F; 90% in Daclizumab) and the mean creatinine concentration at 24 months (139+/-68 mol/l in ATG-F; 176+/-103 mol/l in Daclizumab) were similar in both groups. More severe graft rejections (3 vascular rejections in Daclizumab) and adverse events (5.3/patient in ATG-F; 6.7/patient in Daclizumab) were observed in the Daclizumab group. The costs for hospitalization/ day within 24 months were lower in ATG-F (2.32+/-3.51 USD vs. 12.25+/-9.75 USD; p=0.02) resulting in an average cost-difference of more than 10'435 USD /patient. In this pilot trial, both treatments were comparably successful regarding graft and patient outcome.

Horse versus rabbit antithymocyte globulin in immunosuppressive therapy of treatment-naïve aplastic anemia: a systematic review and meta-analysis.

PubMed

Yang, Nan; Chen, Jinqiu; Zhang, Hui; Dai, Zhiming; Yao, Huan; Ma, Xiaorong; Bai, Ju; Zhang, Yilin; Zhang, Wanggang

2017-12-01

The first-line formulation of antithymocyte globulin (ATG) remains unknown. We aimed to systematically review evidence to compare the efficacy and safety profiles of different ATGs. We did a systematic review and meta-analysis of randomized controlled trials (RCTs) and cohort controlled studies comparing horse and rabbit ATG in immunosuppressive therapy of treatment-naïve aplastic anemia. We searched The Cochrane Library, PubMed, EMBASE, ClinicalTrials.gov , and conference proceedings of American Society of Hematology and European Society for Blood and Marrow Transplantation annual meetings. The outcomes were 3-, 6-, and 12-month response; early mortality; relapse; and evolution. We pooled hazard ratios for relapse and odds ratios (ORs) for other outcomes using fixed-effect or random-effect models based on the heterogeneity. This study was registered with PROSPERO, number CRD42016036945. We included 1636 participants from three RCTs and 11 cohort controlled studies. Allocation to horse ATG increased 6-month response events by 86% compared with rabbit ATG. The benefit of horse ATG was mainly driven by increase in studies with non-Asian (OR 95% CI = 2.39 (1.54-3.69), p < 0.0001) and good partial response criterion (OR 95% CI = 2.73 (1.53-4.89), p = 0.0007). The early mortality and evolution were similar between groups. Compared with rabbit ATG, horse ATG had superior remission by 6 months and equivalent safety profiles in patients with treatment-naïve AA. Evidence for further responses beyond 6 to 12 months was limited.

Atg5 is Essential for Cellular Immunity in vivo and recruitment of a p47 GTPase to the Toxoplasma gondii Parasitophorous Vacuole in Macrophages

PubMed Central

Zhao, Zijiang; Fux, Blima; Goodwin, Megan; Dunay, Ildiko R.; Strong, David; Miller, Brian C.; Cadwell, Ken; Delgado-Vargas, Monica; Ponpuak, Marisa; Green, Karen G.; Schmidt, Robert E.; Mizushima, Noboru; Deretic, Vojo; Sibley, L. David; Virgin, Herbert W.

2008-01-01

SUMMARY The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. We show that expression of the essential autophagy protein Atg5 in granulocytes and macrophages is required for in vivo resistance to infection with L. monocytogenes and T. gondii. In primary macrophages, Atg5 was not required for IFNγ/LPS-mediated transcription, induction of nitric oxide, or inhibition of T. gondii replication. However, Atg5 was required for IFNγ/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect autophagosomes enveloping T. gondii, Atg5 was required for recruitment of the IFNγ-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking. PMID:18996346

Autophagy is required for the activation of NFκB.

PubMed

Criollo, Alfredo; Chereau, Fanny; Malik, Shoaib Ahmad; Niso-Santano, Mireia; Mariño, Guillermo; Galluzzi, Lorenzo; Maiuri, Maria Chiara; Baud, Véronique; Kroemer, Guido

2012-01-01

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.

Apigenin sensitizes hepatocellular carcinoma cells to doxorubic through regulating miR-520b/ATG7 axis.

PubMed

Gao, Ai-Mei; Zhang, Xiao-Yu; Hu, Juan-Ni; Ke, Zun-Ping

2018-01-25

Chemo-resistance is a serious obstacle for successful treatment of cancer. Apigenin, a dietary flavonoid, has been reported as an anticancer drug in various malignant cancers. This study aimed to investigate the potential chemo-sensitization effect of apigenin in doxorubicin-resistant hepatocellular carcinoma cell line BEL-7402/ADM. We observed that apigenin significantly enhanced doxorubicin sensitivity, induced miR-520b expression and inhibited ATG7-dependent autophagy in BEL-7402/ADM cells. In addition, we also showed that miR-520b mimics increased doxorubicin sensitivity and inhibited ATG7-dependent autophagy. Meanwhile, we indicated that ATG7 was a potential target of miR-520b. Furthermore, APG inhibited the growth of hepatocellar carcinoma xenografts in nude mice by up-regulating miR-520b and inhibiting ATG7. Our finding provides evidence that apigenin sensitizes BEL-7402/ADM cells to doxorubicin through miR-520b/ATG7 pathway, which furtherly supports apigenin as a potential chemo-sensitizer for hepatocellular carcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Peretinoin, an acyclic retinoid, suppresses steatohepatitis and tumorigenesis by activating autophagy in mice fed an atherogenic high-fat diet

PubMed Central

Honda, Masao; Takegoshi, Kai; Yamashita, Taro; Nakamura, Mikiko; Shirasaki, Takayoshi; Sakai, Yoshio; Shimakami, Tetsuro; Nagata, Naoto; Takamura, Toshinari; Tanaka, Takuji; Kaneko, Shuichi

2017-01-01

The pathogenesis of non-alcoholic steatohepatitis (NASH) is still unclear and the prevention of the development of hepatocellular carcinoma (HCC) has not been established. We established an atherogenic and high-fat diet mouse model that develops hepatic steatosis, inflammation, fibrosis, and liver tumors at a high frequency. Using two NASH-HCC mouse models, we showed that peretinoin, an acyclic retinoid, significantly improved liver histology and reduced the incidence of liver tumors. Interestingly, we found that peretinoin induced autophagy in the liver of mice, which was characterized by the increased co-localized expression of microtubule-associated protein light chain 3B-II and lysosome-associated membrane protein 2, and increased autophagosome formation and autophagy flux in the liver. These findings were confirmed using primary mouse hepatocytes. Among representative autophagy pathways, the autophagy related (Atg) 5-Atg12-Atg16L1 pathway was impaired; especially, Atg16L1 was repressed at both the mRNA and protein level. Decreased Atg16L1 mRNA expression was also found in the liver of patients with NASH according to disease progression. Promoter analysis revealed that peretinoin activated the promoter of Atg16L1 by increasing the expression of CCAAT/enhancer-binding-protein-alpha. Interestingly, Atg16L1 overexpression in HepG2 cells inhibited palmitate-induced NF-kB activation and interleukin-6-induced STAT3 activation. We showed that Atg16L1 induced the de-phosphorylation of Gp130, a receptor subunit of interleukin-6 family cytokines, which subsequently repressed phosphorylated-STAT3 (Tyr705) levels, and this process might be independent of autophagy function. Thus, peretinoin prevents the progression of NASH and the development of HCC through activating the autophagy pathway by increased Atg16L1 expression, which is an essential regulator of autophagy and anti-inflammatory proteins. PMID:28591717

miR-34a Modulates Angiotensin II-Induced Myocardial Hypertrophy by Direct Inhibition of ATG9A Expression and Autophagic Activity

PubMed Central

Huang, He; Ye, Jing; Pan, Wei; Zhong, Yun; Cheng, Chuanfang; You, Xiangyu; Liu, Benrong; Xiong, Longgen; Liu, Shiming

2014-01-01

Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3′-UTR, but not to the mutated 3′-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity. PMID:24728149

Use of ATG-Fresenius as an Induction Agent in Deceased-Donor Kidney Transplantation.

PubMed

Yilmaz, M; Sezer, T Ö; Kir, O; Öztürk, A; Hoşcoşkun, C; Töz, H

2017-04-01

Anti-T-lymphocyte globulins (ATG) are most commonly used as induction agents in kidney transplantation (KT). In this study, we investigated the use of ATG as induction therapy in deceased-donor KT. Among 152 deceased-donor KT recipients transplanted between January 2009 and December 2003, 147 with exact data were enrolled in this study. Delayed graft function was defined as dialysis requirement after KT. Greater than 10% panel-reactive antibody (PRA) was considered as positive. Total ATG (rATG-Fresenius) dosage and induction duration was evaluated. Mean age was 45 ± 10 years; 91 patients were male and 56 patients were female. Class I and class II PRA-positive patient numbers were 20 (13.6%) and 17 (11.5%), respectively. Pre-transplant dialysis vintage was 108 ± 63 months. Mean donor age was 42 ± 17, and cold ischemia time was 16 ± 5 hours. Eighty-nine patients (60%) had delayed graft function and needed at least one session of hemodialysis after transplantation. Cumulative ATG-F dosage was 676 ± 274 mg. The mean ATG-F cumulative dosage was 10.6 ± 3.8 mg/kg. At the end of first year, mean creatinine and proteinuria levels were 1.4 ± 1.0 mg/dL and 0.3 ± 0.4 g/d, respectively. Mean follow-up time was 32 ± 20 months. During follow-up, there were 14 graft failures and 11 patients died. Patient survival for 1 and 2 years were 93% and 92.3%, respectively. Death-censored graft survival rates for 1 and 2 years were 94.8% and 90.8%, respectively. ATG-F induction provides acceptable graft and patient survival in deceased-donor KT. ATG-F infusion is well tolerated. Infection rates seem to be acceptable compared with all transplantation populations. Copyright © 2017 Elsevier Inc. All rights reserved.

Autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia.

PubMed

Li, Yazi; Zhang, Yuying; Wang, Lei; Wang, Ping; Xue, Yanhong; Li, Xiaopeng; Qiao, Xinhua; Zhang, Xu; Xu, Tao; Liu, Guanghui; Li, Peng; Chen, Chang

2017-07-03

The majority of diabetic patients develop neuropathy and there is an increasing prevalence of neurodegeneration in the central nervous system (CNS). However, the mechanism behind this is poorly understood. Here we first observed that macroautophagy/autophagy was suppressed in the hippocampus of diabetic GK rats with hyperglycemia, whereas it was unchanged in ob/ob mice without hyperglycemia. Autophagy could be directly inhibited by high glucose in mouse primary hippocampal neurons. Moreover, autophagy was protective in high-glucose-induced neurotoxicity. Further studies revealed that autophagic flux was suppressed by high glucose due to impaired autophagosome synthesis illustrated by mRFP-GFP-LC3 puncta analysis. We showed that decreased autophagy was dependent on NO produced under high glucose conditions. Therefore, (LC-MS/MS)-based quantitative proteomic analysis of protein S-nitrosation was performed and a core autophagy protein, ATG4B was found to be S-nitrosated in the hippocampus of GK rats. ATG4B was also verified to be S-nitrosated in neuronal cells cultured with high glucose. The activities of ATG4B in the processing of unmodified, precursor Atg8-family proteins and in the deconjugation of PE from lipidated Atg8-family proteins, which are essential for efficient autophagosome biogenesis were both compromised by S-nitrosation at Cys189 and Cys292 sites. In addition, ATG4B processing of the GABARAPL1 precursor was affected the least by S-nitrosation compared with other substrates. Finally, ATG4B S-nitrosation was verified to be responsible for decreased autophagy and neurotoxicity in response to high glucose. In conclusion, autophagy impairment mediated by S-nitrosation of ATG4B leads to neurotoxicity in response to hyperglycemia. Our research reveals a novel mechanism linking hyperglycemia with CNS neurotoxicity and shows that S-nitrosation is a novel post-transcriptional modification of the core autophagy machinery.

Development of Fluorescent Substrates and Assays for the Key Autophagy-Related Cysteine Protease Enzyme, ATG4B

PubMed Central

Nguyen, Thanh G.; Honson, Nicolette S.; Arns, Steven; Davis, Tara L.; Dhe-Paganon, Sirano; Kovacic, Suzana; Kumar, Nag S.; Pfeifer, Tom A.

2014-01-01

Abstract The cysteine protease ATG4B plays a role in key steps of the autophagy process and is of interest as a potential therapeutic target. At an early step, ATG4B cleaves proLC3 isoforms to form LC3-I for subsequent lipidation to form LC3-II and autophagosome membrane insertion. ATG4B also cleaves phosphatidylethanolamine (PE) from LC3-II to regenerate LC3-I, enabling its recycling for further membrane biogenesis. Here, we report several novel assays for monitoring the enzymatic activity of ATG4B. An assay based on mass spectrometric analysis and quantification of cleavage of the substrate protein LC3-B was developed and, while useful for mechanistic studies, was not suitable for high throughput screening (HTS). A doubly fluorescent fluorescence resonance energy transfer (FRET) ligand YFP-LC3B-EmGFP (FRET-LC3) was constructed and shown to be an excellent substrate for ATG4B with rates of cleavage similar to that for LC3B itself. A HTS assay to identify candidate inhibitors of ATG4B utilizing FRET-LC3 as a substrate was developed and validated with a satisfactory Z′ factor and high signal-to-noise ratio suitable for screening small molecule libraries. Pilot screens of the 1,280-member library of pharmacologically active compounds (LOPAC™) and a 3,481-member library of known drugs (KD2) gave hit rates of 0.6% and 0.5% respectively, and subsequent titrations confirmed ATG4B inhibitory activity for three compounds, both in the FRET and mass spectrometry assays. The FRET- and mass spectrometry–based assays we have developed will allow for both HTS for inhibitors of ATG4B and mechanistic approaches to study inhibition of a major component of the autophagy pathway. PMID:24735444

Tomato HsfA1a plays a critical role in plant drought tolerance by activating ATG genes and inducing autophagy

PubMed Central

Wang, Yu; Cai, Shuyu; Yin, Lingling; Shi, Kai; Xia, Xiaojian; Zhou, Yanhong; Yu, Jingquan; Zhou, Jie

2015-01-01

Autophagy plays critical roles in plant responses to stress. In contrast to the wealth of information concerning the core process of plant autophagosome assembly, our understanding of the regulation of autophagy is limited. In this study, we demonstrated that transcription factor HsfA1a played a critical role in tomato tolerance to drought stress, in part through its positive role in induction of autophagy under drought stress. HsfA1a expression was induced by drought stress. Virus-induced HsfA1a gene silencing reduced while its overexpression increased plant drought tolerance based on both symptoms and membrane integrity. HsfA1a-silenced plants were more sensitive to endogenous ABA-mediated stomatal closure, while its overexpression lines were resistant under drought stress, indicating that phytohormone ABA did not play a major role in HsfA1a-induced drought tolerance. On the other hand, HsfA1a-silenced plants increased while its overexpression decreased the levels of insoluble proteins which were highly ubiquitinated under drought stress. Furthermore, drought stress induced numerous ATGs expression and autophagosome formation in wild-type plants. The expression of ATG10 and ATG18f, and the formation of autophagosomes were compromised in HsfA1a-silenced plants but were enhanced in HsfA1a-overexpressing plants. Both electrophoretic mobility shift assay and chromatin immunoprecipitation coupled with qPCR analysis revealed that HsfA1a bound to ATG10 and ATG18f gene promoters. Silencing of ATG10 and ATG18f reduced HsfA1a-induced drought tolerance and autophagosome formation in plants overexpressing HsfA1a. These results demonstrate that HsfA1a induces drought tolerance by activating ATG genes and inducing autophagy, which may promote plant survival by degrading ubiquitinated protein aggregates under drought stress. PMID:26649940

The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.

PubMed

Bellantuono, I; Lashford, L S; Rafferty, J A; Fairbairn, L J

2000-05-01

As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.

Analysis of infusion-site reactions in renal transplant recipients receiving peripherally administered rabbit antithymocyte globulin as compared with basiliximab.

PubMed

Erickson, Abbie L; Roberts, Keri; Malek, Sayeed K; Chandraker, Anil K; Tullius, Stefan G; Gabardi, Steven

2010-06-01

Antithymocyte globulin rabbit (r-ATG) has been used for the treatment and prevention of acute rejection in renal transplant recipients (RTR). Current manufacturer recommendations for r-ATG dictate the need for administration through a high-flow vein (central line). Previous studies have shown peripheral administration of r-ATG to be safe; however, these studies suggest the co-administration of heparin and hydrocortisone and did not compare the infusion-site reaction rates to a control group. A retrospective analysis was conducted of adult RTR receiving r-ATG or basiliximab between January 2004 and October 2006. Each agent was administered through a dedicated peripheral line. The primary endpoint was the incidence of infusion-site reactions. Other endpoints included the need to replace the intravenous catheter and the incidence of systemic thrombosis within 1 month of transplantation. During the study period, 152 peripheral infusions of r-ATG and 92 peripheral infusions of basiliximab were administered. No difference in infusion-site reactions was noted between the groups. There was also no difference either in the need for peripheral line replacement or the rates of systemic thrombosis. Peripheral administration of r-ATG is safe and can be infused without concomitant heparin and hydrocortisone. This method of r-ATG infusion was shown to be as safe as peripherally administered basiliximab.

The WD40 domain of ATG16L1 is required for its non-canonical role in lipidation of LC3 at single membranes.

PubMed

Fletcher, Katherine; Ulferts, Rachel; Jacquin, Elise; Veith, Talitha; Gammoh, Noor; Arasteh, Julia M; Mayer, Ulrike; Carding, Simon R; Wileman, Thomas; Beale, Rupert; Florey, Oliver

2018-02-15

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double-membrane phagophore, which is driven by the ATG16L1/ATG5-ATG12 complex. In contrast, non-canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3-associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non-canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat-containing C-terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non-canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD Further, we demonstrate activation of non-canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non-canonical use of autophagy machinery. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Complete mitogenome of the semi-aquatic grasshopper Oxya intricate (Stål.) (Insecta: Orthoptera: Catantopidae).

PubMed

Dong, Jia-Jia; Guan, De-Long; Xu, Sheng-Quan

2016-09-01

The complete mitogenome of Oxya intricate (Stål.) has been reconstructed from whole-genome Illumina sequencing data with an average coverage of 294×. The circular genome is 15,466 bp in length, and consists of 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs) and 1 D-loop region. All PCGs are initiated with ATN codons, and are terminated with TAR codons except for ND5 with the incomplete stop codon T. The nucleotide composition is asymmetric (42.5%A, 14.6%C, 10.6%G, 32.3%T) with an overall GC content of 25.2%. These data would contribute to the design of novel molecular markers for population and evolutionary studies of this and related orthopteran species.

PreTIS: A Tool to Predict Non-canonical 5’ UTR Translational Initiation Sites in Human and Mouse

PubMed Central

Reuter, Kerstin; Helms, Volkhard

2016-01-01

Translation of mRNA sequences into proteins typically starts at an AUG triplet. In rare cases, translation may also start at alternative non–AUG codons located in the annotated 5’ UTR which leads to an increased regulatory complexity. Since ribosome profiling detects translational start sites at the nucleotide level, the properties of these start sites can then be used for the statistical evaluation of functional open reading frames. We developed a linear regression approach to predict in–frame and out–of–frame translational start sites within the 5’ UTR from mRNA sequence information together with their translation initiation confidence. Predicted start codons comprise AUG as well as near–cognate codons. The underlying datasets are based on published translational start sites for human HEK293 and mouse embryonic stem cells that were derived by the original authors from ribosome profiling data. The average prediction accuracy of true vs. false start sites for HEK293 cells was 80%. When applied to mouse mRNA sequences, the same model predicted translation initiation sites observed in mouse ES cells with an accuracy of 76%. Moreover, we illustrate the effect of in silico mutations in the flanking sequence context of a start site on the predicted initiation confidence. Our new webservice PreTIS visualizes alternative start sites and their respective ORFs and predicts their ability to initiate translation. Solely, the mRNA sequence is required as input. PreTIS is accessible at http://service.bioinformatik.uni-saarland.de/pretis. PMID:27768687

Effects of two doses of anti-T lymphocyte globulin-Fresenius given after full-match sibling stem cell transplantation in acute myeloblastic leukemia patients who underwent myeloablative fludarabine/busulfan conditioning.

PubMed

Boga, Can; Yeral, Mahmut; Gereklioglu, Ciğdem; Asma, Suheyl; Maytalman, Erkan; Aytan, Pelin; Kozanoglu, Ilknur; Sariturk, Cagla; Ozdogu, Hakan

2018-02-20

Anti-T lymphocyte globulin Fresenius (rATG-F; ATG-Fresenius) and antithymocyte globulin (thymoglobulin), which are included in transplant protocols, are used to reduce the risk of chronic graft-versus-host disease (cGVHD) or suppress allograft rejection. Available clinical studies have been conducted in heterogenous patient populations and with different administration protocols including stem cell sources. Additionally, the pharmacokinetics of ATG is variable, and the clinically effective dose of rATG-F, in particular, is not exactly known. The aim of the study was to investigate the clinical outcomes of acute myeloid leukemia (AML) patients who underwent hemopoietic peripheral stem cell transplantation from full-matched sibling donors and given two different doses of r-ATG-F. This was a single-center, retrospective chart review conducted between July 2005 and July 2016. Sixty-nine consecutive AML patients who underwent transplant with fludarabine- and busulfan-based conditioning were included in the study. Patients in Group 1 received 15 mg/kg body weight rATG-F to 2013 (n = 46), and Group 2 received 30 mg/kg of rATG-F dose begining in 2013 to reduce to cGVHD (n = 23). Cyclosporine and methotrexate were used to treat acute GVHD (aGVHD) prophylaxis. Outcome parameters were compared between the groups. Although the recommended dose r-ATG-F had led to a decrease in the cumulative incidence of cGVHD (27 [58.7%] vs. 8 [34.8%]; p = .03), it also increased the infection rate at 1 year (3 [6.5%] vs. 4 [17.4%]; p = .02). The two groups were similar in terms of engraftment time, aGVHD, relapse, nonrelapse mortality, and rATG-F-related toxicity. A Cox regression model revealed that aGVHD III-IV was associated with increased nonrelapse mortality at 1 year (hazard ratio = 18.2; 95% confidence interval, 1.667-199.255; p = <.02). No patients developed rATG-F-related severe adverse events (Common Terminology Criteria grade 4 or 5). Dose difference of rATG-F did not influence survival parameters; however, increasing the dose to 30 mg/kg seems to be effective for reducing cGVHD with an increase in infection rate requiring close monitoring of infections in AML patients who received myeloablative fludarabine/busulfan conditioning. Copyright © 2018 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

Autophagy-related genes in Helicobacter pylori infection.

PubMed

Tanaka, Shingo; Nagashima, Hiroyuki; Uotani, Takahiro; Graham, David Y; Yamaoka, Yoshio

2017-06-01

In vitro studies have shown that Helicobacter pylori (H. pylori) infection induces autophagy in gastric epithelial cells. However, prolonged exposure to H. pylori reduces autophagy by preventing maturation of the autolysosome. The alterations of the autophagy-related genes in H. pylori infection are not yet fully understood. We analyzed autophagy-related gene expression in H. pylori-infected gastric mucosa compared with uninfected gastric mucosa obtained from 136 Bhutanese volunteers with mild dyspeptic symptoms. We also studied single nucleotide polymorphisms (SNPs) of autophagy-related gene in 283 Bhutanese participants to identify the influence on susceptibility to H. pylori infection. Microarray analysis of 226 autophagy-related genes showed that 16 genes were upregulated (7%) and nine were downregulated (4%). We used quantitative reverse transcriptase polymerase chain reaction to measure mRNA levels of the downregulated genes (ATG16L1, ATG5, ATG4D, and ATG9A) that were core molecules of autophagy. ATG16L1 and ATG5 mRNA levels in H. pylori-positive specimens (n=86) were significantly less than those in H. pylori-negative specimens (n=50). ATG16L1 mRNA levels were inversely related to H. pylori density. We also compared SNPs of ATG16L1 (rs2241880) among 206 H. pylori-positive and 77 H. pylori-negative subjects. The odds ratio for the presence of H. pylori in the GG genotype was 0.40 (95% CI: 0.18-0.91) relative to the AA/AG genotypes. Autophagy-related gene expression profiling using high-throughput microarray analysis indicated that downregulation of core autophagy machinery genes may depress autophagy functions and possibly provide a better intracellular habit for H. pylori in gastric epithelial cells. © 2017 John Wiley & Sons Ltd.

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

PubMed

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes.

PubMed

Diao, Jiajie; Liu, Rong; Rong, Yueguang; Zhao, Minglei; Zhang, Jing; Lai, Ying; Zhou, Qiangjun; Wilz, Livia M; Li, Jianxu; Vivona, Sandro; Pfuetzner, Richard A; Brunger, Axel T; Zhong, Qing

2015-04-23

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.

Randomized trial of single-dose versus divided-dose rabbit anti-thymocyte globulin induction in renal transplantation: an interim report.

PubMed

Stevens, R Brian; Mercer, David F; Grant, Wendy J; Freifeld, Alison G; Lane, James T; Groggel, Gerald C; Rigley, Theodore H; Nielsen, Kathleen J; Henning, Megan E; Skorupa, Jill Y; Skorupa, Anna J; Christensen, Kecia A; Sandoz, John P; Kellogg, Anna M; Langnas, Alan N; Wrenshall, Lucile E

2008-05-27

The optimal dosing protocol for rabbit anti-thymocyte globulin (rATG) induction in renal transplantation has not been determined, but evidence exists that rATG infusion before renal allograft reperfusion improves early graft function. Infusing a large rATG dose over a short interval has not previously been evaluated for its effect on renal function and allograft nephropathy in a prospective, randomized comparison against conventional rATG induction. Between April 20, 2004 and December 26, 2007 we enrolled renal transplant patients into a prospective, randomized, nonblinded trial of two rATG dosing protocols (single dose, 6 mg/kg vs. divided doses, 1.5 mg/kg every other day x 4; target enrollment=160) followed after 6 months by calcineurin-inhibitor withdrawal. Primary endpoints are renal function by calculated glomerular filtration rate (GFR) and chronic allograft nephropathy at protocol biopsy. We now present the early GFR data of all 160 patients and safety and efficacy data of the first 142 patients with 6 months follow up and before calcineurin inhibitor withdrawal (average follow up=23.3+/-11.6 months). There were no differences between groups in rATG-related adverse events, patient and graft survival, acute rejection, or chronic allograft nephropathy rate at 6 months. Calculated DeltaGFR (POD 1-4) was significantly better in the single-dose group (P=0.02), with a trend toward improved renal function from months 2 to 6 in recipients of deceased donor kidneys (P=0.08). This study demonstrates that administering 6 mg/kg of rATG over 24 hr is safe and is associated with improved early renal function compared with administering rATG in alternate-day doses.

Targeted deletion of Atg5 in chondrocytes promotes age-related osteoarthritis.

PubMed

Bouderlique, Thibault; Vuppalapati, Karuna K; Newton, Phillip T; Li, Lei; Barenius, Björn; Chagin, Andrei S

2016-03-01

It has been suggested that the lysosomal recycling process called macro-autophagy plays a role in osteoarthritis development. We thus decided to genetically ablate the autophagy-indispensable Atg5 gene specifically in chondrocytes and analyse the development of osteoarthritis upon aging and in a post-traumatic model. Mice lacking the Atg5 gene in their chondrocytes (Atg5cKO) were generated by crossing Atg5-floxed mice with transgenic mice that expressed cre recombinase driven by the collagen type 2 promoter. Animals were analysed at the age of 2, 6 and 12 months for age-related osteoarthritis or underwent mini-open partial medial meniscectomy at 2 months of age and were analysed 1 or 2 months after surgery. We evaluated osteoarthritis using the Osteoarthritis Research Society International (OARSI) scoring on safranin-O-stained samples. Cell death was evaluated by terminal deoxy-nucleotidyl-transferase-mediated deoxy-UTP nick end labelling (TUNEL) and by immunostaining of cleaved caspases. We observed the development of osteoarthritis in Atg5cKO mice with aging including fibrillation and loss of proteoglycans, which was particularly severe in males. The ablation of Atg5 was associated with an increased cell death as assessed by TUNEL, cleaved caspase 3 and cleaved caspase 9. Surprisingly, no difference in the development of post-traumatic osteoarthritis was observed between Atg5cKO and control mice. Autophagy protects from age-related osteoarthritis by facilitating chondrocyte survival. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis.

PubMed

Lee, Hae-Youn; Kim, Jinyoung; Quan, Wenying; Lee, June-Chul; Kim, Min-Soo; Kim, Seok-Hyung; Bae, Jin-Woo; Hur, Kyu Yeon; Lee, Myung-Shik

2016-08-02

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD(+):NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.

Arctigenin improves vascular tone and decreases inflammation in human saphenous vein.

PubMed

Daci, Armond; Neziri, Burim; Krasniqi, Shaip; Cavolli, Raif; Alaj, Rame; Norata, Giuseppe Danilo; Beretta, Giangiacomo

2017-09-05

The goal of this study was to test the effects of bioactive phenylpropanoid dibenzylbutyrolactone lignan arctigenin (ATG) in vascular tone. Human bypass graft vessel, from a saphenous vein (SV), were set up in organ bath system and contracted with potassium chloride (KCl, 40mM). Two concentration-response curves of noradrenaline (NE) (10nM-100μM) separated with an incubation period of 30min without (Control) or with ATG (3-100μM) were established. Inhibitors of nitric oxide, prostaglandins, K + related channels or calcium influx were used to delineate the molecular mechanisms beyond ATG effects. To investigate anti-inflammatory actions, SV were treated with 10μM or 100μM ATG and incubated for 18h in the absence or presence of both interleukin-1beta (IL-1β) and lipopolysaccharide (LPS) to mimic the physiological or inflamed tissue conditions. Proatherogenic and inflammatory mediators İnterleukine-1 beta (IL-1β), Monocyte Chemoattractant Proteine-1 (MCP-1), Tumor Necrosis Factor- α (TNF-α), İnterleukine-6 (IL-6), Prostaglandin E 2 (PGE 2 ) and İnterleukine-8 (IL-8) in the supernatant were measured. ATG significantly decreased vascular contractile response to NE. Moreover, it reduced contractions induced by KCl and cumulative addition of CaCl 2. The mediators were significantly increased in inflammatory conditions compared to normal conditions, an effect which was inhibited by ATG (10 and 100µM). ATG reduces contractions in SV and decreases the production of proinflammatory-proatherogenic mediators, setting the stage for further evaluating the effect of ATG in cardiovascular diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Interaction of Hb South Florida (codon 1; GTG-->ATG) and HbE, with beta-thalassemia (IVS1-1; G-->A): expression of different clinical phenotypes.

PubMed

Tan, Jin-Ai Mary Anne; Tan, Kim-Lian; Omar, Khairul Zaman; Chan, Lee-Lee; Wee, Yong-Chui; George, Elizabeth

2009-09-01

Interactions of different hemoglobin variants with thalassemia alleles can result in various clinical phenotypes. HbE-beta-thalassemia generally manifests with severe anemia where individuals exhibit beta-thalassemia major with regular blood transfusions or beta-thalassemia intermedia with periodic blood transfusions. This study presents a unique Malay family with three beta-globin gene defects-HbE, Hb South Florida, and IVS1-1 (G-->A). HbE activates a cryptic splice site that produces non-functional mRNAs. Hb South Florida is a rare beta-hemoglobin variant, and its interactions with other beta-thalassemia alleles have not been reported. IVS1-1 is a Mediterranean mutation that affects mRNA processing giving rise to beta(o)-thalassemia. Fifteen mutations along the beta-globin gene complex were analyzed using the amplification refractory mutation system. Hb South Florida was identified by direct sequencing using genomic DNA. The affected child with HbE/IVS1-1 produced a beta-thalassemia major phenotype. Compound heterozygosity for Hb South Florida/IVS1-1 produced a beta-thalassemia carrier phenotype in the mother.

Structure and evolution of the mitochondrial genome of Exorista sorbillans: the Tachinidae (Diptera: Calyptratae) perspective.

PubMed

Shao, Yuan-jun; Hu, Xian-qiong; Peng, Guang-da; Wang, Rui-xian; Gao, Rui-na; Lin, Chao; Shen, Wei-de; Li, Rui; Li, Bing

2012-12-01

The first complete mitochondrial genome (mitogenome) of Tachinidae Exorista sorbillans (Diptera) is sequenced by PCR-based approach. The circular mitogenome is 14,960 bp long and has the representative mitochondrial gene (mt gene) organization and order of Diptera. All protein-coding sequences are initiated with ATN codon; however, the only exception is Cox I gene, which has a 4-bp ATCG putative start codon. Ten of the thirteen protein-coding genes have a complete termination codon (TAA), but the rest are seated on the H strand with incomplete codons. The mitogenome of E. sorbillans is biased toward A+T content at 78.4 %, and the strand-specific bias is in reflection of the third codon positions of mt genes, and their T/C ratios as strand indictor are higher on the H strand more than those on the L strand pointing at any strain of seven Diptera flies. The length of the A+T-rich region of E. sorbillans is 106 bp, including a tandem triple copies of a13-bp fragment. Compared to Haematobia irritans, E. sorbillans holds distant relationship with Drosophila. Phylogenetic topologies based on the amino acid sequences, supporting that E. sorbillans (Tachinidae) is clustered with strains of Calliphoridae and Oestridae, and superfamily Oestroidea are polyphyletic groups with Muscidae in a clade.

Codon 13 KRAS mutation predicts patterns of recurrence in patients undergoing hepatectomy for colorectal liver metastases.

PubMed

Margonis, Georgios A; Kim, Yuhree; Sasaki, Kazunari; Samaha, Mario; Amini, Neda; Pawlik, Timothy M

2016-09-01

Investigations regarding the impact of tumor biology after surgical management of colorectal liver metastasis have focused largely on overall survival. We investigated the impact of codon-specific KRAS mutations on the rates and patterns of recurrence in patients after surgery for colorectal liver metastasis (CRLM). All patients who underwent curative-intent surgery for CRLM between 2002 and 2015 at Johns Hopkins who had available data on KRAS mutation status were identified. Clinico-pathologic data, recurrence patterns, and recurrence-free survival (RFS) were assessed using univariable and multivariable analyses. A total of 512 patients underwent resection only (83.2%) or resection plus radiofrequency ablation (16.8%). Although 5-year overall survival was 64.6%, 284 (55.5%) patients recurred with a median RFS time of 18.1 months. The liver was the initial recurrence site for 181 patients, whereas extrahepatic recurrence was observed in 162 patients. Among patients with an extrahepatic recurrence, 102 (63%) had a lung recurrence. Although overall KRAS mutation was not associated with overall RFS (P = 0.186), it was independently associated with a worse extrahepatic (P = 0.004) and lung RFS (P = 0.007). Among patients with known KRAS codon-specific mutations, patients with codon 13 KRAS mutation had a worse 5-year extrahepatic RFS (P = 0.01), whereas codon 12 mutations were not associated with extrahepatic (P = 0.11) or lung-specific recurrence rate (P = 0.24). On multivariable analysis, only codon 13 mutation independently predicted worse overall extrahepatic RFS (P = 0.004) and lung-specific RFS (P = 0.023). Among patients undergoing resection of CRLM, overall KRAS mutation was not associated with RFS. KRAS codon 13 mutations, but not codon 12 mutations, were associated with a higher risk for overall extrahepatic recurrence and lung-specific recurrence. Cancer 2016. © 2016 American Cancer Society. Cancer 2016;122:2698-2707. © 2016 American Cancer Society. © 2016 American Cancer Society.

Bcl-2-like protein 13 is a mammalian Atg32 homologue that mediates mitophagy and mitochondrial fragmentation

PubMed Central

Murakawa, Tomokazu; Yamaguchi, Osamu; Hashimoto, Ayako; Hikoso, Shungo; Takeda, Toshihiro; Oka, Takafumi; Yasui, Hiroki; Ueda, Hiromichi; Akazawa, Yasuhiro; Nakayama, Hiroyuki; Taneike, Manabu; Misaka, Tomofumi; Omiya, Shigemiki; Shah, Ajay M.; Yamamoto, Akitsugu; Nishida, Kazuhiko; Ohsumi, Yoshinori; Okamoto, Koji; Sakata, Yasushi; Otsu, Kinya

2015-01-01

Damaged mitochondria are removed by mitophagy. Although Atg32 is essential for mitophagy in yeast, no Atg32 homologue has been identified in mammalian cells. Here, we show that Bcl-2-like protein 13 (Bcl2-L-13) induces mitochondrial fragmentation and mitophagy in mammalian cells. First, we hypothesized that unidentified mammalian mitophagy receptors would share molecular features of Atg32. By screening the public protein database for Atg32 homologues, we identify Bcl2-L-13. Bcl2-L-13 binds to LC3 through the WXXI motif and induces mitochondrial fragmentation and mitophagy in HEK293 cells. In Bcl2-L-13, the BH domains are important for the fragmentation, while the WXXI motif facilitates mitophagy. Bcl2-L-13 induces mitochondrial fragmentation in the absence of Drp1, while it induces mitophagy in Parkin-deficient cells. Knockdown of Bcl2-L-13 attenuates mitochondrial damage-induced fragmentation and mitophagy. Bcl2-L-13 induces mitophagy in Atg32-deficient yeast cells. Induction and/or phosphorylation of Bcl2-L-13 may regulate its activity. Our findings offer insights into mitochondrial quality control in mammalian cells. PMID:26146385

Biodiversity of autolytic ability in flocculent Saccharomyces cerevisiae strains suitable for traditional sparkling wine fermentation.

PubMed

Perpetuini, Giorgia; Di Gianvito, Paola; Arfelli, Giuseppe; Schirone, Maria; Corsetti, Aldo; Tofalo, Rosanna; Suzzi, Giovanna

2016-07-01

Yeasts involved in secondary fermentation of traditional sparkling wines should show specific characteristics, such as flocculation capacity and autolysis. Recently it has been postulated that autophagy may contribute to the outcome of autolysis. In this study, 28 flocculent wine Saccahromyces cerevisiae strains characterized by different flocculation degrees were studied for their autolytic and autophagic activities. Autolysis was monitored in synthetic medium through the determination of amino acid nitrogen and total proteins released. At the same time, novel primer sets were developed to determine the expression of the genes ATG1, ATG17 and ATG29. Twelve strains were selected on the basis of their autolytic rate and ATG gene expressions in synthetic medium and were inoculated in a base wine. After 30, 60 and 180 days the autolytic process and ATG gene expressions were evaluated. The obtained data showed that autolysis and ATG gene expressions differed among strains and were independent of the degree of flocculation. This biodiversity could be exploited to select new starter stains to improve sparkling wine production. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

PubMed

Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism

PubMed Central

Rodríguez, Andrea E.; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

ABSTRACT Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells. PMID:26654586

Identification of Atg3 as an intrinsically disordered polypeptide yields insights into the molecular dynamics of autophagy-related proteins in yeast.

PubMed

Popelka, Hana; Uversky, Vladimir N; Klionsky, Daniel J

2014-06-01

The mechanism of autophagy relies on complex cell signaling and regulatory processes. Each cell contains many proteins that lack a rigid 3-dimensional structure under physiological conditions. These dynamic proteins, called intrinsically disordered proteins (IDPs) and protein regions (IDPRs), are predominantly involved in cell signaling and regulation. Yet, very little is known about their presence among proteins of the core autophagy machinery. In this work, we characterized the autophagy protein Atg3 from yeast and human along with 2 variants to show that Atg3 is an IDPRs-containing protein and that disorder/order predicted for these proteins from their amino acid sequence corresponds to their experimental characteristics. Based on this consensus, we applied the same prediction methods to all known Atg proteins from Saccharomyces cerevisiae. The data presented here provide an insight into the structural dynamics of each Atg protein. They also show that intrinsic disorder at various levels has to be taken into consideration for about half of the Atg proteins. This work should become a useful tool that will facilitate and encourage exploration of protein intrinsic disorder in autophagy.

Inhibition of autophagy exerts anti-colon cancer effects via apoptosis induced by p53 activation and ER stress.

PubMed

Sakitani, Kosuke; Hirata, Yoshihiro; Hikiba, Yohko; Hayakawa, Yoku; Ihara, Sozaburo; Suzuki, Hirobumi; Suzuki, Nobumi; Serizawa, Takako; Kinoshita, Hiroto; Sakamoto, Kei; Nakagawa, Hayato; Tateishi, Keisuke; Maeda, Shin; Ikenoue, Tsuneo; Kawazu, Shoji; Koike, Kazuhiko

2015-10-24

Although some molecularly targeted drugs for colorectal cancer are used clinically and contribute to a better prognosis, the current median survival of advanced colorectal cancer patients is not sufficient. Autophagy, a basic cell survival mechanism mediated by recycling of cellular amino acids, plays an important role in cancer. Recently, autophagy has been highlighted as a promising new molecular target. The unfolded protein response (UPR) reportedly act in complementary fashion with autophagy in intestinal homeostasis. However, the roles of UPR in colon cancer under autophagic inhibition remain to be elucidated. We aim to clarify the inhibitory effect of autophagy on colon cancer. We crossed K19 (CreERT) and Atg5 (flox/flox) mice to generate Atg5 (flox/flox)/K19 (CreERT) mice. Atg5 (flox/flox)/K19 (CreERT) mice were first treated with azoxymethane/dextran sodium sulfate and then injected with tamoxifen to inhibit autophagy in CK19-positive epithelial cells. To examine the anti-cancer mechanisms of autophagic inhibition, we used colon cancer cell lines harboring different p53 gene statuses, as well as small interfering RNAs (siRNAs) targeting Atg5 and immunoglobulin heavy-chain binding protein (BiP), a chaperone to aid folding of unfolded proteins. Colon tumors in Atg5 (flox/flox)/K19 (CreERT) mice showed loss of autophagic activity and decreased tumor size (the total tumor diameter was 28.1 mm in the control and 20.7 mm in Atg5 (flox/flox)/K19 (CreERT) mice, p = 0.036). We found that p53 and UPR/endoplasmic reticulum (ER) stress-related proteins, such as cleaved caspase 3, and CAAT/enhancer-binding protein homologous protein, are up-regulated in colon tumors of Atg5 (flox/flox)/K19 (CreERT) mice. Although Atg5 and BiP silencing, respectively, increased apoptosis in p53 wild type cells, Atg5 silencing alone did not show the same effect on apoptosis in p53 mutant cells. However, co-transfection of Atg5 and BiP siRNAs led to increased apoptosis in p53 mutant cells. Blocking autophagy has potential in the treatment of colon cancer by inducing apoptosis via p53 and ER stress, and suppressing the UPR pathway is a valid strategy to overcome resistance to autophagic inhibition.

Autophagy-deficient breast cancer shows early tumor recurrence and escape from dormancy

PubMed Central

Aqbi, Hussein F.; Tyutyunyk-Massey, Liliya; Keim, Rebecca C.; Butler, Savannah E.; Thekkudan, Theresa; Joshi, Supriya; Smith, Timothy M.; Bandyopadhyay, Dipankar; Idowu, Michael O.; Bear, Harry D.; Payne, Kyle K.; Gewirtz, David A.; Manjili, Masoud H.

2018-01-01

Breast cancer patients who initially respond to cancer therapies often succumb to distant recurrence of the disease. It is not clear why people with the same type of breast cancer respond to treatments differently; some escape from dormancy and relapse earlier than others. In addition, some tumor clones respond to immunotherapy while others do not. We investigated how autophagy plays a role in accelerating or delaying recurrence of neu-overexpressing mouse mammary carcinoma (MMC) following adriamycin (ADR) treatment, and in affecting response to immunotherapy. We explored two strategies: 1) transient blockade of autophagy with chloroquine (CQ), which blocks fusion of autophagosomes and lysosomes during ADR treatment, and 2) permanent inhibition of autophagy by a stable knockdown of ATG5 (ATG5KD), which inhibits the formation of autophagosomes in MMC during and after ADR treatment. We found that while CQ prolonged tumor dormancy, but that stable knockdown of autophagy resulted in early escape from dormancy and recurrence. Interestingly, ATG5KD MMC contained an increased frequency of ADR-induced polyploid-like cells and rendered MMC resistant to immunotherapy. On the other hand, a transient blockade of autophagy did not affect the sensitivity of MMC to immunotherapy. Our observations suggest that while chemotherapy-induced autophagy may facilitate tumor relapse, cell-intrinsic autophagy delays tumor relapse, in part, by inhibiting the formation of polyploid-like tumor dormancy. PMID:29774126

Beclin-1 Expression in Normal Bladder and in Cd+2 and As+3 Exposed and Transformed Human Urothelial Cells (UROtsa)

PubMed Central

Larson, Jennifer L.; Somji, Seema; Zhou, Xu Dong; Sens, Mary Ann; Garrett, Scott H.; Sens, Donald A.; Dunlevy, Jane R.

2010-01-01

The expression of beclin-1 in normal human bladder and in Cd+2 and As+3 exposed and transformed urothelial cells (UROtsa) was examined in this study. It was shown using a combination of real time PCR, western analysis and immunohistochemistry that beclin-1 was expressed in the urothelial cells of the normal bladder. It was also demonstrated that the parental UROtsa cell line expressed beclin-1 mRNA and protein at levels similar to that of the in situ urothelium. The level of beclin-1 expression underwent only modest alterations when the UROtsa cells were malignantly transformed by Cd+2 or As+3 or when the parental cells were exposed acutely to Cd+2 or As+3. While there were instances of significant alterations at individual time points and within cell line-to-cell line comparisons there was no evidence of a dose response relationship or correlations to the phenotypic properties of the cell lines. Similar results were obtained for the expression of the Atg-5, Atg-7, Atg-12 and LC3B autophagy-related proteins. The findings provide initial evidence for beclin-1 expression in normal bladder and that large alterations in the expression of beclin-1 and associated proteins do not occur when human urothelial cells are malignantly transformed with, or exposed to, either Cd+2 or As+3. PMID:20206246

GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes

PubMed Central

Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

2014-01-01

Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12–ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex. PMID:25046113

Immunohistochemical assessment of ATG7, LC3, and p62 in ameloblastomas.

PubMed

Okada, Miwa; Oikawa, Mariko; Miki, Yasuhiro; Shimizu, Yoshinaka; Echigo, Seishi; Takahashi, Tetsu; Kumamoto, Hiroyuki

2014-09-01

To investigate the roles of autophagy in tumorigenesis, cytodifferentiation, and prognosis of odontogenic tumors, we analyzed the immunohistochemical expression of ATG7, LC3, and p62 in odontogenic tissues. Tissue specimens of nine dental follicles and 69 ameloblastomas were immunohistochemically examined with antibodies against ATG7, LC3, and p62. Immunohistochemical reactivity for ATG7, LC3, and p62 was detected in many odontogenic epithelial cells and several endothelial cells and fibroblasts in dental follicles and ameloblastomas. ATG7 reactivity in ameloblatomas was significantly higher than that in dental follicles. Expression of ATG7, LC3, and p62 was found markedly in neoplastic cells near the basement membrane rather than central polyhedral cells in ameloblastomas. Reactivity for these molecules was significantly higher in unicystic ameloblastomas than in solid ameloblastomas. Granular cells in granular cell ameloblastomas showed obvious reactivity for the autophagy- related molecules, and LC3 reactivity in granular cell ameloblastomas was significantly higher than in other ameloblastoma variations. Recurrent ameloblastomas showed significantly lower reactivity of LC3 and p62 than primary ameloblastomas. Expression of ATG7, LC3, and p62 in dental follicles and ameloblastomas suggests that autophagy regulation might be affected by microenvironment alterations during tumorigenesis. The molecular machinery for autophagy is possibly involved in tissue architecture, neoplastic cell differentiation, and prognosis of the benign epithelial odontogenic tumor. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A Functional ATG16L1 (T300A) Variant is Associated with Necrotizing Enterocolitis in Premature Infants

PubMed Central

Sampath, Venkatesh; Bhandari, Vineet; Berger, Jessica; Merchant, Daniel; Zhang, Liyun; Ladd, Mihoko; Menden, Heather; Garland, Jeffery; Ambalavanan, Namasivayam; Mulrooney, Neil; Quasney, Michael; Dagle, John; Lavoie, Pascal M; Simpson, Pippa; Dahmer, Mary

2017-01-01

Background The genetic basis of dysfunctional immune responses in necrotizing enterocolitis (NEC) remains unknown. We hypothesized that variants in Nucleotide binding and Oligomerization Domain (NOD)-Like Receptors (NLRs) and Autophagy (ATG) genes modulate vulnerability to NEC. Methods We genotyped a multi-center cohort of premature infants with and without NEC for NOD1, NOD2, ATG16L1, CARD8 and NLRP3 variants. Chi-square tests and logistic regression were used for statistical analysis. Results In our primary cohort (n=1015), 86 (8.5%) infants developed NEC. The A allele of the ATG16L1 (Thr300Ala) variant was associated with increased NEC (AA vs. AG vs. GG; 11.3% vs. 8.4% vs. 4.8%, p=0.009). In regression models for NEC that adjusted for epidemiological confounders, GA (p=0.033) and the AA genotype (p=0.038) of ATG16L1 variant were associated with NEC. The association between the A allele of the ATG16L1 variant and NEC remained significant among Caucasian infants (p=0.02). In a replication cohort (n=259), NEC rates were highest among infants with the AA genotype but did not reach statistical significance. Conclusion We report a novel association between a hypomorphic variant in an autophagy gene (ATG16L1) and NEC in premature infants. Our data suggest that decreased autophagy arising from genetic variants may confer protection against NEC. PMID:27893720

Ribosome hijacking: a role for small protein B during trans-translation.

PubMed

Nonin-Lecomte, Sylvie; Germain-Amiot, Noella; Gillet, Reynald; Hallier, Marc; Ponchon, Luc; Dardel, Frédéric; Felden, Brice

2009-02-01

Tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the 'tmRNA-SmpB' system (transfer messenger RNA-small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon-anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon-anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for DeltasmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.

4-Acetylantroquinonol B suppresses autophagic flux and improves cisplatin sensitivity in highly aggressive epithelial cancer through the PI3K/Akt/mTOR/p70S6K signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Mingche

Targeting residual self-renewing, chemoresistant cancerous cells may represent the key to overcoming therapy resistance. The entry of these quiescent cells into an activated state is associated with high metabolic demand and autophagic flux. Therefore, modulating the autophagy pathway in aggressive carcinomas may be beneficial as a therapeutic modality. In this study, we evaluated the anti-tumor activities of 4-acetylantroquinonol B (4-AAQB) in chemoresistant ovarian cancer cells, particularly its ability to modulate autophagy through autophagy-related genes (Atg). Atg-5 was overexpressed in invasive ovarian cancer cell lines and tissue (OR: 5.133; P = 0.027) and depleting Atg-5 in ES-2 cell lines significantly inducedmore » apoptosis. 4-AAQB effectively suppressed viability of various subtypes of ovarian cancer. Cells with higher cisplatin-resistance were more responsive to 4-AAQB. For the first time, we demonstrate that 4-AAQB significantly suppress Atg-5 and Atg-7 expression with decreased autophagic flux in ovarian cancer cells via inhibition of the PI3K/Akt/mTOR/p70S6K signaling pathway. Similar to Atg-5 silencing, 4-AAQB-induced autophagy inhibition significantly enhanced cell death in vitro. These results are comparable to those of hydroxychloroquine (HCQ). In addition, 4-AAQB/cisplatin synergistically induced apoptosis in ovarian cancer cells. In vivo, 4-AAQB/cisplatin also significantly induced apoptosis and autophagy in an ES-2 mouse xenografts model. This is the first report demonstrating the efficacy of 4-AAQB alone or in combination with cisplatin on the suppression of ovarian cancer via Atg-5-dependent autophagy. We believe these findings will be beneficial in the development of a novel anti-ovarian cancer therapeutic strategy. - Highlights: • Atg-5 is overexpressed in ovarian cancer and silencing Atg-5 induces apoptosis. • 4-AAQB suppresses autophagy and PI3K/Akt/mTOR pathway. • 4-AAQB + cisplatin synergistically suppresses ovarian cancer via autophagy.« less

Outcomes in African-Americans vs. Caucasians using thymoglobulin or interleukin-2 receptor inhibitor induction: analysis of USRDS database.

PubMed

Jindal, Rahul M; Das, Neal P; Neff, Robert T; Hurst, Frank P; Falta, Edward M; Elster, Eric A; Abbott, Kevin C

2009-01-01

We used the USRDS database to test the hypothesis that graft survival was similar using either rabbit antithymocyte globulin (rATG) vs. interleukin-2 receptor inhibitor (IL2i) in the Prograf era. We further explored the variable of race in the two groups of patients. We conducted a retrospective cohort study of kidney transplant patients in the USRDS from 2000 through 2005 to compare graft survival (including death) using rATG vs. IL2i with particular reference to outcomes between African-Americans vs. Caucasians. Kaplan-Meier analysis was performed to assess patient and graft survival after transplantation, stratified by recipient induction with rATG versus IL2i. Cox regression analysis was performed to assess adjusted survival after transplantation, assessing whether induction rATG (vs. IL2i) was significant as an interaction term (i.e. an effect modifier) with black race for graft survival. Propensity score analysis was used to address potential confounding by indication. In stratified Cox Regression analysis limited to IL2i, black race was significantly associated with graft loss (adjusted hazard ratio (AHR) 1.17, 95% CI, 1.09-1.26). In analysis limited to rATG induction, black race was not significant (AHR 1.00, 95% CI, 0.92-1.10). We detected a significant interaction between rATG and black race (in comparison with non-black race) for the development of graft loss (AHR, 0.86, 95% CI, 0.76-0.97). Analysis limited to black recipients showed that while use of rATG was not significantly different from IL2i (AHR 0.95, 95% CI 0.87-1.04), the direction of this association was in the opposite direction of non-blacks. Patient and graft survival were similar in African-American and Caucasian recipients of kidney transplantation using either rATG or IL2i. Limitations of the study are the retrospective nature of USRDS data, center-bias in using rATG vs. IL2i and lack of data on steroid dosage. Results of the present study call for a critical review of induction practices. (c) 2008 S. Karger AG, Basel.

ATG16L1 and IL23R are associated with inflammatory bowel diseases but not with celiac disease in the Netherlands.

PubMed

Weersma, Rinse K; Zhernakova, Alexandra; Nolte, Ilja M; Lefebvre, Céline; Rioux, John D; Mulder, Flip; van Dullemen, Hendrik M; Kleibeuker, Jan H; Wijmenga, Cisca; Dijkstra, Gerard

2008-03-01

Inflammatory bowel disease (IBD)--Crohn's disease (CD) and ulcerative colitis (UC)--and celiac disease are intestinal inflammatory disorders with a complex genetic background. Recently, two novel genes were found to be associated with IBD susceptibility. One, an uncommon coding variant (rs11209026) in the gene encoding for the interleukin-23 receptor (IL23R), conferred strong protection against CD. The other, rs2241880 in the autophagy-related 16-like 1 gene (ATG16L1), was associated with CD. We performed a case-control study for the association of IBD with IL23R and ATG16L1 in a Dutch cohort. We also looked at the association of IL23R and ATG16L1 with celiac disease. Five hundred eighteen Dutch white IBD patients (311 CD and 207 UC, including 176 trios of patients with both parents), 508 celiac disease patients, and 893 healthy controls were studied for association with the rs11209026 (IL23R) and rs2241880 (ATG16L1) single nucleotide polymorphisms (SNP). The rs11209026 SNP in IL23R had a protective effect for IBD in the case-control analysis (odds ratio [OR] 0.19, 95% confidence interval [CI] 0.10-0.37, P= 6.6E-09). Both CD (OR 0.14, CI 0.06-0.37, P= 3.9E-07) and UC (OR 0.33, CI 0.15-0.73, P= 1.4E-03) were associated with IL23R. For ATG16L1, the rs2241880 SNP was associated with CD susceptibility (OR 1.36, CI 1.12-1.66, P= 0.0017). The population-attributable risk of carrying allele G is 0.24 and is 0.19 for homozygosity for allele G in CD. No association was found between IL23R or ATG16L1 and celiac disease. We confirmed the association of IL23R and ATG16L1 with CD susceptibility and also the association of IL23R with UC. We found IL23R and ATG16L1 were not associated with celiac disease susceptibility.

Genetic and Hormonal Risk Factors for Cancer in African American Men

DTIC Science & Technology

2006-05-01

rs2740574 CYP3A4 7q21.1 5’ UTR (-293) G to A 49.6 46.8 rs700519 CYP19 15q21.1 Exon 6 (codon 264) C to T 30.1 30.8 rs9282858 SDR5A2 2p23 Exon 1...HSD17B2 Months 12-24 CYP19 CYP3A4 IGF1 We had initially planned to perform our SNP assays using either ABI PRISM® 7700 Sequence Detection...31.4 rs6163 CYP17 10q24.3 Exon 1 (Codon 65) G to T 57.9 66.4 rs6162 CYP17 10q24.3 Exon 1 (Codon 46) C to T 58.7 66.9 rs743572 CYP17 10q24.3 5’-UTR

Web-Based Naval Fleet Logistics Information System

DTIC Science & Technology

2007-12-01

Ship AOR Area of Responsibility ATG Assistant Training Group B2B Business to business Beach DET Beach Detachment CHT Collection and Holding...operationalize supply domain. While the initiatives did not specify a Web-based information system concept, business to business ( B2B ) information...cost more per pound to deliver and frequently cause delays compared to the commercial freight companies . Military personnel traveling overseas are

The observation of mitochondrial movement and ATG5 position in Arabidopsis during the process of infection with virulent and avirulent P. syringae strains

NASA Astrophysics Data System (ADS)

Yang, Liu; Ma, Chao; Chen, Wen li

2012-03-01

Infection of plants with pathogens leads to programmed cell death (PCD) associated with the pathogen-triggered hypersensitive response (HR) during plant innate immunity. In this study, the effects of infection by virulent Pseudomonas syringae pv. tomato (Pst) DC3000 and strains harboring avirulence factors AvrRps4 on the induction of HR-PCD were compared. We used Arabidopsis thaliana plants as materials, which expressed green fluorescent protein labeled mitochondria (mito-GFP) and green fluorescent protein tagged ATG5 (ATG5-GFP), these GFP are instantaneous expression. We found both Pst DC3000 and Pst-avrRps4 could induce mitochondria to assemble, the effect of Pst DC3000 was more obvious. ATG5 was located in chloroplasts after infection with Pst DC3000 or Pst-avrRps4. Under the condition of Pst-avrRps4, the expression of ATG5 was stronger than Pst DC3000 treatment.

Biochemical Analysis of Autophagy in Algae and Plants by Monitoring the Electrophoretic Mobility of ATG8.

PubMed

Pérez-Pérez, María Esther; Andrés-Garrido, Ascensión; Crespo, José L

2016-01-01

Identification of specific autophagy markers has been fundamental to investigate autophagy as catabolic process. Among them, the ATG8 protein turned out to be one of the most widely used and specific molecular markers of autophagy both in higher and lower eukaryotes. Here, we describe how ATG8 can be used to monitor autophagy in Chlamydomonas and Arabidopsis by western blot analysis.

Control of Autophagy in Chlamydomonas Is Mediated through Redox-Dependent Inactivation of the ATG4 Protease.

PubMed

Pérez-Pérez, María Esther; Lemaire, Stéphane D; Crespo, José L

2016-12-01

Autophagy is a major catabolic pathway by which eukaryotic cells deliver unnecessary or damaged cytoplasmic material to the vacuole for its degradation and recycling in order to maintain cellular homeostasis. Control of autophagy has been associated with the production of reactive oxygen species in several organisms, including plants and algae, but the precise regulatory molecular mechanisms remain unclear. Here, we show that the ATG4 protease, an essential protein for autophagosome biogenesis, plays a central role for the redox regulation of autophagy in the model green alga Chlamydomonas reinhardtii Our results indicate that the activity of C. reinhardtii ATG4 is regulated by the formation of a single disulfide bond with a low redox potential that can be efficiently reduced by the NADPH/thioredoxin system. Moreover, we found that treatment of C. reinhardtii cells with norflurazon, an inhibitor of carotenoid biosynthesis that generates reactive oxygen species and triggers autophagy in this alga, promotes the oxidation and aggregation of ATG4. We propose that the activity of the ATG4 protease is finely regulated by the intracellular redox state, and it is inhibited under stress conditions to ensure lipidation of ATG8 and thus autophagy progression in C. reinhardtii. © 2016 American Society of Plant Biologists. All Rights Reserved.

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A.

PubMed

Abdul Rahim, Siti Aminah; Dirkse, Anne; Oudin, Anais; Schuster, Anne; Bohler, Jill; Barthelemy, Vanessa; Muller, Arnaud; Vallar, Laurent; Janji, Bassam; Golebiewska, Anna; Niclou, Simone P

2017-09-05

Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM. Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells. We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion. This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors.

Natural dietary compound naringin prevents azoxymethane/dextran sodium sulfate-induced chronic colorectal inflammation and carcinogenesis in mice.

PubMed

Zhang, Yu-Sheng; Wang, Feng; Cui, Shu-Xiang; Qu, Xian-Jun

2018-03-26

Naringin, a natural occurring flavonoid compound, enriches in citrus fruits. We aimed to evaluate the inhibitory effect of naringin on colitis and chronic inflammation-driven carcinogenesis. Male C57BL/6 mice were exposed to AOM/DSS to induce colorectal inflammation and carcinogenesis. Naringin by oral administration prevented AOM/DSS-induced ulcerative colitis and carcinogenesis without significant side effects. Naringin attenuated the severity of colitis and colorectal adenomas through inhibiting myeloid-derived suppressor cells (MDSCs), pro-inflammatory mediators GM-CSF/M-CSF, IL-6 and TNF-α and the NF-κB/IL-6/STAT3 cascades in colorectal tissues. Naringin-treated mice exhibited normalized structures of colorectal tissues. Electron microscopy analysis showed the suppression of robust endoplasmic reticulum (ER) stress-induced autophagy. Naringin inhibited the secretion of the ER-spanning transmembrane proteins, such as GRP78 ATF6, IRE1α and activated PERK phosphorylated eIF-2α and complex of autophagosomes ATG3, ATG5, ATG7, ATG12, ATG16 and ATG16L1 in the colorectal mucosal cells. Naringin prevented colitis and colorectal carcinogenesis through suppressing robust ER stress-induced autophagy in colorectal mucosal cells. Naringin could develop a promising therapeutic agent for the prevention of ulcerative colitis and colorectal tumor.

ATG4B inhibitors with a benzotropolone core structure block autophagy and augment efficiency of chemotherapy in mice.

PubMed

Kurdi, Ammar; Cleenewerck, Matthias; Vangestel, Christel; Lyssens, Sophie; Declercq, Wim; Timmermans, Jean-Pierre; Stroobants, Sigrid; Augustyns, Koen; De Meyer, Guido R Y; Van Der Veken, Pieter; Martinet, Wim

2017-08-15

Autophagy is a cell survival mechanism hijacked by advanced tumors to endure a rough microenvironment. Late autophagy inhibitors such as (hydroxy)chloroquine have been used clinically to halt tumor progression with modest success. However, given the toxic nature of these compounds and their lack of specificity, novel targets should be considered. We recently identified a benzotropolone derivative that significantly inhibited the essential autophagy protein ATG4B. Therefore, we synthesized and tested additional benzotropolone compounds to identify a promising ATG4B inhibitor that impairs autophagy both in vitro and in vivo. A compound library containing 27 molecules with a benzotropolone backbone was synthesized and screened for inhibition of recombinant ATG4B. Depending on the benzotropolone compound, inhibition of recombinant ATG4B ranged from 3 to 82%. Active compounds were evaluated in cellular assays to confirm inhibition of ATG4B and suppression of autophagy. Seven compounds inhibited processing of the autophagy protein LC3 and autophagosome formation. Compound UAMC-2526 was selected for further in vivo use because of its fair plasma stability. This compound abolished autophagy both in nutrient-deprived GFP-LC3 mice and in CD1 -/- Foxn1nu mice bearing HT29 colorectal tumor xenografts. Moreover, addition of UAMC-2526 to the chemotherapy drug oxaliplatin significantly improved inhibition of tumor growth. Our data indicate that suppression of autophagy via ATG4B inhibition is a feasible strategy to augment existing chemotherapy efficacy and to halt tumor progression. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptomic analysis of the autophagy machinery in crustaceans.

PubMed

Suwansa-Ard, Saowaros; Kankuan, Wilairat; Thongbuakaew, Tipsuda; Saetan, Jirawat; Kornthong, Napamanee; Kruangkum, Thanapong; Khornchatri, Kanjana; Cummins, Scott F; Isidoro, Ciro; Sobhon, Prasert

2016-08-09

The giant freshwater prawn, Macrobrachium rosenbergii, is a decapod crustacean that is commercially important as a food source. Farming of commercial crustaceans requires an efficient management strategy because the animals are easily subjected to stress and diseases during the culture. Autophagy, a stress response process, is well-documented and conserved in most animals, yet it is poorly studied in crustaceans. In this study, we have performed an in silico search for transcripts encoding autophagy-related (Atg) proteins within various tissue transcriptomes of M. rosenbergii. Basic Local Alignment Search Tool (BLAST) search using previously known Atg proteins as queries revealed 41 transcripts encoding homologous M. rosenbergii Atg proteins. Among these Atg proteins, we selected commonly used autophagy markers, including Beclin 1, vacuolar protein sorting (Vps) 34, microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), p62/sequestosome 1 (SQSTM1), and lysosomal-associated membrane protein 1 (Lamp-1) for further sequence analyses using comparative alignment and protein structural prediction. We found that crustacean autophagy marker proteins contain conserved motifs typical of other animal Atg proteins. Western blotting using commercial antibodies raised against human Atg marker proteins indicated their presence in various M. rosenbergii tissues, while immunohistochemistry localized Atg marker proteins within ovarian tissue, specifically late stage oocytes. This study demonstrates that the molecular components of autophagic process are conserved in crustaceans, which is comparable to autophagic process in mammals. Furthermore, it provides a foundation for further studies of autophagy in crustaceans that may lead to more understanding of the reproduction- and stress-related autophagy, which will enable the efficient aquaculture practices.

A Crohn's disease variant in Atg16l1 enhances its degradation by caspase 3

NASA Astrophysics Data System (ADS)

Murthy, Aditya; Li, Yun; Peng, Ivan; Reichelt, Mike; Katakam, Anand Kumar; Noubade, Rajkumar; Roose-Girma, Merone; Devoss, Jason; Diehl, Lauri; Graham, Robert R.; van Lookeren Campagne, Menno

2014-02-01

Crohn's disease is a debilitating inflammatory bowel disease (IBD) that can involve the entire digestive tract. A single-nucleotide polymorphism (SNP) encoding a missense variant in the autophagy gene ATG16L1 (rs2241880, Thr300Ala) is strongly associated with the incidence of Crohn's disease. Numerous studies have demonstrated the effect of ATG16L1 deletion or deficiency; however, the molecular consequences of the Thr300Ala (T300A) variant remains unknown. Here we show that amino acids 296-299 constitute a caspase cleavage motif in ATG16L1 and that the T300A variant (T316A in mice) significantly increases ATG16L1 sensitization to caspase-3-mediated processing. We observed that death-receptor activation or starvation-induced metabolic stress in human and murine macrophages increased degradation of the T300A or T316A variants of ATG16L1, respectively, resulting in diminished autophagy. Knock-in mice harbouring the T316A variant showed defective clearance of the ileal pathogen Yersinia enterocolitica and an elevated inflammatory cytokine response. In turn, deletion of the caspase-3-encoding gene, Casp3, or elimination of the caspase cleavage site by site-directed mutagenesis rescued starvation-induced autophagy and pathogen clearance, respectively. These findings demonstrate that caspase 3 activation in the presence of a common risk allele leads to accelerated degradation of ATG16L1, placing cellular stress, apoptotic stimuli and impaired autophagy in a unified pathway that predisposes to Crohn's disease.

Elucidating the composition and conservation of the autophagy pathway in photosynthetic eukaryotes

PubMed Central

Shemi, Adva; Ben-Dor, Shifra; Vardi, Assaf

2015-01-01

Aquatic photosynthetic eukaryotes represent highly diverse groups (green, red, and chromalveolate algae) derived from multiple endosymbiosis events, covering a wide spectrum of the tree of life. They are responsible for about 50% of the global photosynthesis and serve as the foundation for oceanic and fresh water food webs. Although the ecophysiology and molecular ecology of some algal species are extensively studied, some basic aspects of algal cell biology are still underexplored. The recent wealth of genomic resources from algae has opened new frontiers to decipher the role of cell signaling pathways and their function in an ecological and biotechnological context. Here, we took a bioinformatic approach to explore the distribution and conservation of TOR and autophagy-related (ATG) proteins (Atg in yeast) in diverse algal groups. Our genomic analysis demonstrates conservation of TOR and ATG proteins in green algae. In contrast, in all 5 available red algal genomes, we could not detect the sequences that encode for any of the 17 core ATG proteins examined, albeit TOR and its interacting proteins are conserved. This intriguing data suggests that the autophagy pathway is not conserved in red algae as it is in the entire eukaryote domain. In contrast, chromalveolates, despite being derived from the red-plastid lineage, retain and express ATG genes, which raises a fundamental question regarding the acquisition of ATG genes during algal evolution. Among chromalveolates, Emiliania huxleyi (Haptophyta), a bloom-forming coccolithophore, possesses the most complete set of ATG genes, and may serve as a model organism to study autophagy in marine protists with great ecological significance. PMID:25915714

Arctigenin alleviates ER stress via activating AMPK

PubMed Central

Gu, Yuan; Sun, Xiao-xiao; Ye, Ji-ming; He, Li; Yan, Shou-sheng; Zhang, Hao-hao; Hu, Li-hong; Yuan, Jun-ying; Yu, Qiang

2012-01-01

Aim: To investigate the protective effects of arctigenin (ATG), a phenylpropanoid dibenzylbutyrolactone lignan from Arctium lappa L (Compositae), against ER stress in vitro and the underlying mechanisms. Methods: A cell-based screening assay for ER stress regulators was established. Cell viability was measured using MTT assay. PCR and Western blotting were used to analyze gene and protein expression. Silencing of the CaMKKβ, LKB1, and AMPKα1 genes was achieved by RNA interference (RNAi). An ATP bioluminescent assay kit was employed to measure the intracellular ATP levels. Results: ATG (2.5, 5 and 10 μmol/L) inhibited cell death and unfolded protein response (UPR) in a concentration-dependent manner in cells treated with the ER stress inducer brefeldin A (100 nmol/L). ATG (1, 5 and 10 μmol/L) significantly attenuated protein synthesis in cells through inhibiting mTOR-p70S6K signaling and eEF2 activity, which were partially reversed by silencing AMPKα1 with RNAi. ATG (1-50 μmol/L) reduced intracellular ATP level and activated AMPK through inhibiting complex I-mediated respiration. Pretreatment of cells with the AMPK inhibitor compound C (25 μmol/L) rescued the inhibitory effects of ATG on ER stress. Furthermore, ATG (2.5 and 5 μmol/L) efficiently activated AMPK and reduced the ER stress and cell death induced by palmitate (2 mmol/L) in INS-1 β cells. Conclusion: ATG is an effective ER stress alleviator, which protects cells against ER stress through activating AMPK, thus attenuating protein translation and reducing ER load. PMID:22705729

ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells.

PubMed

Lv, Wei; Sui, Linlin; Yan, Xiaona; Xie, Huaying; Jiang, Liping; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Kong, Ying; Cao, Jun

2018-01-05

Cadmium (Cd) is a toxic heavy metal that is widely used in industry and agriculture. In this study the role of autophagy in Cd-induced proliferation, migration and invasion was investigated in A549 cells. Exposure to Cd (2 μM) significantly increased reactive oxygen species (ROS) production, induced autophagy and enhanced cell growth, migration and invasion in A549 cells. Western blot analysis showed that the expression of autophagy-related proteins, LC3-II, Beclin-1 and Atg4 and invasion-related protein MMP-9 were upregulated in Cd-treated cells. N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of A549 cells and the increasing protein level of LC3-II and Atg4. Blocking Atg4 expression by siRNA strongly reduced Beclin-1 and LC3-II protein expression and the number of autophagosome positive cells induced by Cd. Furthermore, Atg4 siRNA increased the number of cells at G0/G1 phase, reduced the number of S and G2/M phase cells, and inhibited Cd-induced cell growth significantly compared with that of Cd-treated Control siRNA cells. 3-MA pretreatment increased the percentage of G0/G1 phase cells, decreased S phase and G2/M phase percentage, and inhibited Cd-induced cell growth remarkably compared with that of only Cd-treated cells. Knocking down Atg4 reduced the number of cells that migrated and invaded through the Matrigel matrix significantly and led to a significant decrease of MMP-9 expression. In addition, in lung tissues of Cd-treated BALB/c mice, the increased expression of LC3-II, Beclin-1 and Atg4 were observed. Taken together, our results demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced cell growth, migration and invasion in A549 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification and pharmacological induction of autophagy in the larval stages of Echinococcus granulosus: an active catabolic process in calcareous corpuscles.

PubMed

Loos, Julia A; Caparros, Pedro A; Nicolao, María Celeste; Denegri, Guillermo M; Cumino, Andrea C

2014-06-01

Autophagy is a fundamental catabolic pathway conserved from yeast to mammals, but which remains unknown in parasite cestodes. In this work, the pharmacological induction of autophagy was cellularly and molecularly analysed in the larval stages of Echinococcus granulosus. Metacestode sensitivity to rapamycin and TORC1 expression in protoscoleces and metacestodes were shown. Ultrastructural studies showed that treated parasites had an isolation membrane, autophagosomes and autolysosomes, all of which evidenced the autophagic flux. Genes coding for key autophagy-related proteins were also identified in the Echinococcus genome. These genes were involved in autophagosome formation and transcriptional over-expression of Eg-atg5, Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 and Eg-atg18 was shown in presence of rapamycin or arsenic trioxide. Thus, Echinococcus autophagy could be regulated by non-transcriptional inhibition through TOR and by transcription-dependent up-regulation via FoxO-like transcription factors and/or TFEB proteins. An increase in the punctate pattern and Eg-Atg8 polypeptide level in the tegument, parenchyma cells and excretory system of protoscoleces and in vesicularised parasites was detected after rapamycin treatment. This suggests the occurrence of basal autophagy in the larval stages and during vesicular development. In arsenic-treated protoscoleces, high Eg-Atg8 polypeptide levels within the free cytoplasmic matrix of calcareous corpuscles were observed, thus verifying the occurrence of autophagic events. These experiments also confirmed that the calcareous corpuscles are sites of arsenic trioxide accumulation. The detection of the autophagic machinery in this parasite represents a basic starting point to unravel the role of autophagy under both physiological and stress conditions which will allow identification of new strategies for drug discovery against neglected parasitic diseases caused by cestodes. Copyright © 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

A successful desensitization protocol for horse-derived antithymocyte globulin in severe aplastic anemia.

PubMed

Demir, Esen; Cigerci Günaydın, Nurşen; Karadaş, Nihal; Gülen, Figen; Tanac, Remziye; Yılmaz, Deniz

2015-03-01

Horse antithymocyte globulin (h-ATG) (ATGAM(®) ) is the first choice of treatment in very severe patients with aplastic anemia who do not have any HLA matched sibling donor. h-ATG is a heterologous serum that may cause anaphylaxis. Alternative treatment strategies must be planned in case of hypersensitivity. Desensitization must be considered in patients without an alternative treatment of choice. We aimed to present the h-ATG desensitization protocol and consider its effectiveness in patients with aplastic anemia who are hypersensitized with h-ATG and do not have an alternative treatment of choice. Skin prick tests were performed with non-diluted solution in eight very severe patients with aplastic anemia who are followed up in Ege University Children's Hospital. Although skin prick test was found negative in these eight patients, different dilution h-ATG intradermal tests were performed and found positive in all patients. h-ATG desensitization program was started to these hypersensitized patients. Desensitization program was started to six male and two female very severe patients with aplastic anemia whose ages were between seven and 19 yr (median: 12.9 yr). All of the patients completed the desensitization program. While local reaction was seen in two patients, systemic reaction was seen in one patient and late reaction was seen in one patient during and after desensitization program. A successful desensitization program with h-ATG in children with aplastic anemia is presented. Even though there is not an exposure before to such high allergy potential heterologous serum, skin tests should be performed and desensitization must be started to patients who are hypersensitized to h-ATG. As the expected effectiveness of the treatment is so much, the desensitization protocol can be carried out safely and effectively with trained stuff although allergic reactions can be seen. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Rabbit antithymocyte globulin–induced serum sickness disease and human kidney graft survival

PubMed Central

Couvrat-Desvergnes, Grégoire; Salama, Apolline; Le Berre, Ludmilla; Evanno, Gwénaëlle; Viklicky, Ondrej; Hruba, Petra; Vesely, Pavel; Guerif, Pierrick; Dejoie, Thomas; Rousse, Juliette; Nicot, Arnaud; Bach, Jean-Marie; Ang, Evelyn; Foucher, Yohann; Brouard, Sophie; Castagnet, Stéphanie; Giral, Magali; Harb, Jean; Perreault, Hélène; Charreau, Béatrice; Lorent, Marine; Soulillou, Jean-Paul

2015-01-01

BACKGROUND. Rabbit-generated antithymocyte globulins (ATGs), which target human T cells, are widely used as immunosuppressive agents during treatment of kidney allograft recipients. However, ATGs can induce immune complex diseases, including serum sickness disease (SSD). Rabbit and human IgGs have various antigenic differences, including expression of the sialic acid Neu5Gc and α-1-3-Gal (Gal), which are not synthesized by human beings. Moreover, anti-Neu5Gc antibodies have been shown to preexist and be elicited by immunization in human subjects. This study aimed to assess the effect of SSD on long-term kidney allograft outcome and to compare the immunization status of grafted patients presenting with SSD following ATG induction treatment. METHODS. We analyzed data from a cohort of 889 first kidney graft recipients with ATG induction (86 with SSD [SSD+] and 803 without SSD [SSD–]) from the Données Informatisées et Validées en Transplantation data bank. Two subgroups of SSD+ and SSD– patients that had received ATG induction treatment were then assessed for total anti-ATG, anti-Neu5Gc, and anti-Gal antibodies using ELISA assays on sera before and after transplantation. RESULTS. SSD was significantly associated with long-term graft loss (>10 years, P = 0.02). Moreover, SSD+ patients exhibited significantly elevated titers of anti-ATG (P = 0.043) and anti-Neu5Gc (P = 0.007) IgGs in late post-graft samples compared with SSD– recipients. CONCLUSION. In conclusion, our data indicate that SSD is a major contributing factor of late graft loss following ATG induction and that anti-Neu5Gc antibodies increase over time in SSD+ patients. FUNDING. This study was funded by Société d’Accélération du Transfert de Technologies Ouest Valorisation, the European FP7 “Translink” research program, the French National Agency of Research, Labex Transplantex, the Natural Science and Engineering Research Council of Canada, and the Canadian Foundation for Innovation. PMID:26551683

Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress

PubMed Central

Gonzalez, Yanira; Aryal, Baikuntha; Chehab, Leena; Rao, V. Ashutosh

2014-01-01

The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ. PMID:24681637

Atg7- and Keap1-dependent autophagy protects breast cancer cell lines against mitoquinone-induced oxidative stress.

PubMed

Gonzalez, Yanira; Aryal, Baikuntha; Chehab, Leena; Rao, V Ashutosh

2014-03-30

The interplay between oxidative stress and autophagy is critical for determining the fate of cancer cells exposed to redox-active and cytotoxic chemotherapeutic agents. Mitoquinone (MitoQ), a mitochondrially-targeted redox-active ubiquinone conjugate, selectively kills breast cancer cells over healthy mammary epithelial cells. We reported previously that MitoQ, although a derivative of the antioxidant ubiquinone, can generate excess ROS and trigger the Keap1-Nrf2 antioxidant response in the MDA-MB-231 cell line. Following MitoQ treatment, a greater number of cells underwent autophagy than apoptosis. However, the relationship between MitoQ-induced oxidative stress and autophagy as a primary cellular response was unclear. In this report, we demonstrate that MitoQ induces autophagy related gene 7 (Atg7)-dependent, yet Beclin-1-independent, autophagy marked by an increase in LC3-II. Both the ATG7-deficient human MDA-MB-231 cells and Atg7-knockout mouse embryonic fibroblasts exhibited lower levels of autophagy following MitoQ treatment than their respective wild-type counterparts. Increased apoptosis was confirmed in these autophagy-deficient isogenic cell line pairs, indicating that autophagy was attempted for survival in wild type cell lines. Furthermore, we observed higher levels of ROS in Atg7-deficient cells, as measured by hydroethidine oxidation. In Atg7-deficient cells, redox-sensitive Keap1 degradation was decreased, suggesting autophagy- and Atg7-dependent degradation of Keap1. Conversely, downregulation of Keap1 decreased autophagy levels, increased Nrf2 activation, upregulated cytoprotective antioxidant gene expression, and caused accumulation of p62, suggesting a feedback loop between ROS-regulated Keap1-Nrf2 and Atg7-regulated autophagy. Our data indicate that excessive ROS causes the upregulation of autophagy, and autophagy acts as an antioxidant feedback response triggered by cytotoxic levels of MitoQ.

Genome-wide analysis of autophagy-related genes (ARGs) in grapevine and plant tolerance to copper stress.

PubMed

Shangguan, Lingfei; Fang, Xiang; Chen, Lide; Cui, Liwen; Fang, Jinggui

2018-06-01

Grapevine autophagy-related genes (ARGs) include 35 members that have unique evolutionary backgrounds and expression patterns, with some of them responding to abiotic stresses, including copper stress. Autophagy is one of the most crucial self-regulating phenomena in livings organisms, including animals, plants, yeasts, etc. In the genomes of plants, like Arabidopsis, rice, tobacco, and barley, more than 30 autophagy-related genes (ARGs) have been found. These ARGs are involved in plant development, programed cell death, and the stress response process. In plants, and particularly in grapevine, high copper stress results from the application of the Bordeaux mixture, a widely used fungicide. However, the function of autophagy in plant tolerance to copper stress is unknown. Accordingly, in this study, a genome-wide analysis was performed to identify Vitis vinifera ARGs (VvARGs), and 35 VvARGs were detected. A gene family analysis revealed that the tandem and segmental duplication events played significant roles in the VvARG gene family expansion. Moreover, there was more intense signature of purifying selection for the comparison between grape and rice than between grape and Arabidopsis. In response to copper treatment, both the autophagosome number and malondialdehyde concentration increased during the initial 4 h post-treatment, and reached maximal values at 24 h. An expression analysis indicated that most VvARGs responded to copper stress at 4 h post-treatment, and some VvARGs (e.g., VvATG6, VvATG8i, and VvATG18h) exhibited responses to most abiotic stresses. These results provide a detailed overview of the ARGs in grapevine and indicate multiple functions of autophagy in fruit development and abiotic stresses in grapevine. The key ARG (e.g., ATG8i) should be investigated in more detail in grapevine and other plant species.

Autophagy and Apoptosis Act as Partners to Induce Germ Cell Death after Heat Stress in Mice

PubMed Central

Zhang, Mianqiu; Jiang, Min; Bi, Ye; Zhu, Hui; Zhou, Zuomin; Sha, Jiahao

2012-01-01

Testicular heating suppresses spermatogenesis which is marked by germ cell loss via apoptotic pathways. Recently, it is reported that autophagy also can be induced by heat treatment in somatic cells. In this study, the status of autophagy in germ cells after heat treatment, as well as the partnership between autophagy and apoptosis in these cells was investigated. The results demonstrated that besides initiating apoptotic pathways, heat also induced autophagic pathways in germ cells. Exposure of germ cells to hyperthermia resulted in several specific features of the autophagic process, including autophagosome formation and the conversion of LC3-I to LC3-II. Furthermore, the ubiquitin-like protein conjugation system was implicated as being likely responsible for heat-induced autophagy in germ cells since all genes involving this system were found to be expressed in the testes. In addition, the upstream protein in this system, Atg7 (Autophagy-related gene 7), was found to be expressed in all types of spermatogenic cells, and its expression level was positively correlated with the level of autophagy in germ cells. As a result, Atg7 was selected as the investigative target to further analyze the role of autophagy in heat-induced germ cell death. It was shown that down expression of Atg7 protein resulted in the notable decrease in the level of autophagy in heat-treated germ cells, and this down-regulation of autophagy caused by Atg7 knockdown further reduced the apoptotic rate of germ cells. These results suggest that autophagy plays a positive role in the process of germ cell apoptosis after heat treatment. In conclusion, this study demonstrates that heat triggers autophagy and apoptosis in germ cells. These two mechanisms might act as partners, not antagonist, to induce cell death and lead to eventual destruction of spermatogenesis. PMID:22848486

The prediction of human exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solovyev, V.V.; Salamov, A.A.; Lawrence, C.B.

1994-12-31

Discriminant analysis is applied to the problem of recognition 5`-, internal and 3`-exons in human DNA sequences. Specific recognition functions were developed for revealing exons of particular types. The method based on a splice site prediction algorithm that uses the linear Fisher discriminant to combine the information about significant triplet frequencies of various functional parts of splice site regions and preferences of oligonucleotide in protein coding and nation regions. The accuracy of our splice site recognition function is about 97%. A discriminant function for 5`-exon prediction includes hexanucleotide composition of upstream region, triplet composition around the ATG codon, ORF codingmore » potential, donor splice site potential and composition of downstream introit region. For internal exon prediction, we combine in a discriminant function the characteristics describing the 5`- intron region, donor splice site, coding region, acceptor splice site and Y-intron region for each open reading frame flanked by GT and AG base pairs. The accuracy of precise internal exon recognition on a test set of 451 exon and 246693 pseudoexon sequences is 77% with a specificity of 79% and a level of pseudoexon ORF prediction of 99.96%. The recognition quality computed at the level of individual nucleotides is 89%, for exon sequences and 98% for intron sequences. A discriminant function for 3`-exon prediction includes octanucleolide composition of upstream nation region, triplet composition around the stop codon, ORF coding potential, acceptor splice site potential and hexanucleotide composition of downstream region. We unite these three discriminant functions in exon predicting program FEX (find exons). FEX exactly predicts 70% of 1016 exons from the test of 181 complete genes with specificity 73%, and 89% exons are exactly or partially predicted. On the average, 85% of nucleotides were predicted accurately with specificity 91%.« less

Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2',3'-dideoxyinosine and 2',3'-dideoxycytidine.

PubMed Central

Gu, Z; Gao, Q; Li, X; Parniak, M A; Wainberg, M A

1992-01-01

We have used the technique of in vitro selection to generate variants of human immunodeficiency virus type 1 (HIV-1) that are resistant to 2',3'-dideoxyinosine (ddI) and cross-resistant to 2',3'-dideoxycytidine (ddC). The complete reverse transcriptase (RT)-coding regions, plus portions of flanking sequences, of viruses possessing a ddI-resistant phenotype were cloned and sequenced by polymerase chain reaction (PCR)-based methods. We observed that several of these viruses possessed mutations at amino acid sites 184 (Met-->Val; ATG-->GTG) and 294 (Pro-->Ser; CCA-->TCA). These mutations were introduced in the pol gene of infectious, cloned HXB2-D DNA by site-directed mutagenesis. Viral replication assays confirmed the importance of site 184 with regard to resistance to ddI. The recombinant viruses thus generated displayed more than fivefold-greater resistance to ddI than parental HXB2-D did. Moreover, more than fivefold-greater resistance to ddC was also documented; however, the recombinant viruses continued to be inhibited by zidovudine (AZT). No resistance to ddI, ddC, or AZT was introduced by inclusion of mutation site 294 in the pol gene of HXB2-D. PCR analysis performed on viral samples obtained from patients receiving long-term ddI therapy confirmed the presence of mutation site 184 in five of seven cases tested. In three of these five positive cases, the wild-type codon was also detected, indicating that mixtures of viral quasispecies were apparently present. Viruses possessing a ddI resistance phenotype were isolated from both subjects whose viruses contained only the mutated rather than wild-type codon at position 184 as well as from a third individual, whose viruses appeared to be mostly of the mutated variety. Images PMID:1279198

Evolutionary Silence of the Acid Chaperone Protein HdeB in Enterohemorrhagic Escherichia coli O157:H7

PubMed Central

Louie, Jacqueline W.; Fagerquist, Clifton K.; Sultan, Omar; Miller, William G.; Mandrell, Robert E.

2012-01-01

The periplasmic chaperones HdeA and HdeB are known to be important for cell survival at low pH (pH < 3) in Escherichia coli and Shigella spp. Here we investigated the roles of HdeA and HdeB in the survival of various enterohemorrhagic E. coli (EHEC) following exposure to pH 2.0. Similar to K-12 strains, the acid protections conferred by HdeA and HdeB in EHEC O145 were significant: loss of HdeA and HdeB led to over 100- to 1,000-fold reductions in acid survival, depending on the growth condition of prechallenge cells. However, this protection was much less in E. coli O157:H7 strains. Deletion of hdeB did not affect the acid survival of cells, and deletion of hdeA led to less than a 5-fold decrease in survival. Sequence analysis of the hdeAB operon revealed a point mutation at the putative start codon of the hdeB gene in all 26 E. coli O157:H7 strains analyzed, which shifted the ATG start codon to ATA. This mutation correlated with the lack of HdeB in E. coli O157:H7; however, the plasmid-borne O157-hdeB was able to restore partially the acid resistance in an E. coli O145ΔhdeAB mutant, suggesting the potential function of O157-HdeB as an acid chaperone. We conclude that E. coli O157:H7 strains have evolved acid survival strategies independent of the HdeA/B chaperones and are more acid resistant than nonpathogenic K-12 for cells grown under nonfavorable culturing conditions such as in Luria-Bertani no-salt broth at 28°C. These results suggest a divergent evolution of acid resistance mechanisms within E. coli. PMID:22179243

Eliminating Late Recurrence to Eradicate Breast Cancer

DTIC Science & Technology

2014-09-01

Chen GC, Lee JY, Tang HW, Debnath J, Thomas SM, Settleman J. Genetic interactions between Drosophila melanogaster Atg1 and paxillin reveal a role...and the generation of cell lines expressing shATGs have been described (1). The target sequences for hairpins directed against human ATG7 (NM_006395...are the Page 80 Lock et. al., CD-13-0841R, Supplemental Materials, page 4 primer sequences used: GAPDH; For: 5’-CATGTTCGTCATGGGTGTGAACCA-3’ Rev: 5

Transfection and heat-inducible expression of molluscan promoter-luciferase reporter gene constructs in the Biomphalaria glabrata embryonic snail cell line.

PubMed

Yoshino, T P; Wu, X J; Liu, H D

1998-09-01

Studies were initiated to begin developing a genetic transformation system for cells derived from the freshwater gastropod, Biomphalaria glabrata, an intermediate host of the human blood fluke Schistosoma mansoni. Using a 70-kD heat-shock protein (HSP70) cDNA probe obtained from the B. glabrata embryonic (Bge) cell line, we cloned from Bge cells a complete HSP70 gene including a 1-kb genomic DNA fragment in its 5'-flanking region containing sequences indicative of a HSP promoter. Identified in the 5'-half (416 nucleotides) of this genomic fragment were TATA and CAAT boxes, two putative transcription initiation sites, and a series of palindromic DNA repeats with shared homology to the heat-shock element consensus sequence (Bge HSP70(0.5k) promoter). The 3'-half of this upstream flanking region was comprised of a 508-base intron located immediately 5' of the ATG start codon. To determine the functionality of the putative snail promoter sequence, Bge HSP promoter/luciferase (Luc) reporter gene constructs were introduced into Bge cells by N-(1-(2,3-dioleoyloxy) propyl)-N,N,N-trimethylammonium methylsulfate (DOTAP)-mediated transfection methods, and assayed for Luc activity 48 hr following a 1.5-hr heat-shock treatment (40 degrees C). Compared with control vectors or the Bge HSP70(0.5k/1.0k) promoter constructs at 26 degrees C, a 10- to 300-fold increase in Luc expression was obtained only in the Bge HSP70 promoter/Luc-transfected cells following heat-shock. Results of transfection experiments demonstrate that the Bge HSP70(0.5k) DNA segment contains appropriate promoter sequences for driving temperature-inducible gene expression in the Bge snail cell line. This report represents the first isolation and functional characterization of an inducible promoter from a freshwater gastropod mollusc. Successful transient expression of a foreign reporter gene in Bge cells using a homologous, inducible promoter sequence now paves the way for development of methods for stable integration and expression of snail genes of interest into the Bge cell line.

Ribosomal protein S14 transcripts are edited in Oenothera mitochondria.

PubMed Central

Schuster, W; Unseld, M; Wissinger, B; Brennicke, A

1990-01-01

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci. Images PMID:2326162

Effects of Fok-I polymorphism in vitamin D receptor gene on serum 25-hydroxyvitamin D, bone-specific alkaline phosphatase and calcaneal quantitative ultrasound parameters in young adults.

PubMed

Tanabe, Rieko; Kawamura, Yuka; Tsugawa, Naoko; Haraikawa, Mayu; Sogabe, Natsuko; Okano, Toshio; Hosoi, Takayuki; Goseki-Sone, Masae

2015-01-01

Several genes have been implicated as genetic determinants of osteoporosis. Vitamin D receptor (VDR) is an intracellular hormone receptor that specifically binds to the biologically active form of vitamin D, 1-alpha, 25- dihydroxyvitamin D3 [1, 25(OH)2D], and mediates its effects. One of the most frequently studied single nucleotide polymorphisms is the restriction fragment length polymorphism (RFLP) Fok-I (rs2228570). The presence of a Fok-I site, designated f, allows protein translation to initiate from the first ATG. An allele lacking the site (ATG>ACG: designated F), initiates from a second ATG site. In the present study, we explored the effect of the VDR Fok-I genotype on associations among serum bone-specific alkaline phosphatase (ALP), 25- hydroxyvitamin D3 [25(OH)D], 1, 25(OH)2D, and the dietary nutrient intake in healthy young Japanese subjects (n=193). Dietary nutrient intakes were calculated based on 3-day food records before the day of blood examinations. Quantitative ultrasound (QUS) parameters at the right calcaneus (heel bone) were measured. The allele frequencies were 0.622 for the F allele and 0.378 for the f allele in all subjects. Grouped by the VDR genotype, a significant positive correlation between the levels of serum bone-specific ALP and 25(OH)D was observed in the FF-type (p=0.005), but not in the ff-type. In addition, there was a significant positive correlation between the level of serum 25(OH)D and osteo-sono assessment index (OSI) in the FF-type (p=0.008), but not in the ff-type. These results suggest that the level of circulating 25(OH)D is an important factor when assessing the VDR Fok-I polymorphism to prevent osteoporosis.

Epstein-Barr virus-associated posttransplantation lymphoproliferative disorder after high-dose immunosuppressive therapy and autologous CD34-selected hematopoietic stem cell transplantation for severe autoimmune diseases.

PubMed

Nash, Richard A; Dansey, Roger; Storek, Jan; Georges, George E; Bowen, James D; Holmberg, Leona A; Kraft, George H; Mayes, Maureen D; McDonagh, Kevin T; Chen, Chien-Shing; Dipersio, John; Lemaistre, C Fred; Pavletic, Steven; Sullivan, Keith M; Sunderhaus, Julie; Furst, Daniel E; McSweeney, Peter A

2003-09-01

High-dose immunosuppressive therapy followed by autologous hematopoietic stem cell transplantation (HSCT) is currently being evaluated for the control of severe autoimmune diseases. The addition of antithymocyte globulin (ATG) to high-dose chemoradiotherapy in the high-dose immunosuppressive therapy regimen and CD34 selection of the autologous graft may induce a higher degree of immunosuppression compared with conventional autologous HSCT for malignant diseases. Patients may be at higher risk of transplant-related complications secondary to the immunosuppressed state, including Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disorder (PTLD), but this is an unusual complication after autologous HSCT. Fifty-six patients (median age, 42 years; range, 23-61 years) with either multiple sclerosis (n = 26) or systemic sclerosis (n = 30) have been treated. The median follow-up has been 24 months (range, 2-60 months). Two patients (multiple sclerosis, n = 1; systemic sclerosis, n = 1) had significant reactivations of herpesvirus infections early after HSCT and then developed aggressive EBV-PTLD and died on days +53 and +64. Multiorgan clonal B-cell infiltrates that were EBV positive by molecular studies or immunohistology were identified at both autopsies. Both patients had positive screening skin tests for equine ATG (Atgam) and had been converted to rabbit ATG (Thymoglobulin) from the first dose. Of the other 54 patients, 2 of whom had partial courses of rabbit ATG because of a reaction to the intravenous infusion of equine ATG, only 1 patient had a significant clinical reactivation of a herpesvirus infection (herpes simplex virus 2) early after HSCT, and none developed EBV-PTLD. The T-cell count in the peripheral blood on day 28 was 0/microL in all 4 patients who received rabbit ATG; this was significantly less than in patients who received equine ATG (median, 174/microL; P =.001; Mann-Whitney ranked sum test). Although the numbers are limited, the time course and similarity of the 2 cases of EBV-PTLD and the effect on day 28 T-cell counts support a relationship between the development of EBV-PTLD and the administration of rabbit ATG. The differences between equine and rabbit ATG are not yet clearly defined, and they should not be considered interchangeable in this regimen without further study.

Sensitivity factor of the axial-symmetric transmission gauge: Deviation and long-term variation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, N.; Tuzi, Y.; Arakawa, I.

2007-07-15

The deviation of the sensitivity factors for about 50 sensors of the axial-symmetric transmission gauge (ATG) was examined. The deviation has been compared with that for about 30 sensors of the B-A gauge and for about 30 sensors of the extractor gauge. Almost the same deviations are found for the above three types of gauges. The long-term variations of the sensitivity factors for two sensors of the ATG were also measured during four years. After the initial change with operating time, the sensitivity factor stabilized after long-term operation. The room temperature effect of the sensitivity factor plays an important rolemore » on the total pressure measurement by an ionization gauge, but the change of the sensitivity factor was larger than the room temperature effect. The reason for the change of the factor is estimated in connection with the annealing and the alignment of the filament and with the deviation of the place of electron emission on the filament.« less

Role and regulation of autophagy in heat stress responses of tomato plants

PubMed Central

Zhou, Jie; Wang, Jian; Yu, Jing-Quan; Chen, Zhixiang

2014-01-01

As sessile organisms, plants are constantly exposed to a wide spectrum of stress conditions such as high temperature, which causes protein misfolding. Misfolded proteins are highly toxic and must be efficiently removed to reduce cellular proteotoxic stress if restoration of native conformations is unsuccessful. Although selective autophagy is known to function in protein quality control by targeting degradation of misfolded and potentially toxic proteins, its role and regulation in heat stress responses have not been analyzed in crop plants. In the present study, we found that heat stress induced expression of autophagy-related (ATG) genes and accumulation of autophagosomes in tomato plants. Virus-induced gene silencing (VIGS) of tomato ATG5 and ATG7 genes resulted in increased sensitivity of tomato plants to heat stress based on both increased development of heat stress symptoms and compromised photosynthetic parameters of heat-stressed leaf tissues. Silencing of tomato homologs for the selective autophagy receptor NBR1, which targets ubiquitinated protein aggregates, also compromised tomato heat tolerance. To better understand the regulation of heat-induced autophagy, we found that silencing of tomato ATG5, ATG7, or NBR1 compromised heat-induced expression of not only the targeted genes but also other autophagy-related genes. Furthermore, we identified two tomato genes encoding proteins highly homologous to Arabidopsis WRKY33 transcription factor, which has been previously shown to interact physically with an autophagy protein. Silencing of tomato WRKY33 genes compromised tomato heat tolerance and reduced heat-induced ATG gene expression and autophagosome accumulation. Based on these results, we propose that heat-induced autophagy in tomato is subject to cooperative regulation by both WRKY33 and ATG proteins and plays a critical role in tomato heat tolerance, mostly likely through selective removal of heat-induced protein aggregates. PMID:24817875

Targeted deletion of Atg5 reveals differential roles of autophagy in keratin K5-expressing epithelia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sukseree, Supawadee; Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok; Rossiter, Heidemarie

2013-01-11

Highlights: Black-Right-Pointing-Pointer We generated mice lacking Atg5 and autophagy in keratin K5-positive epithelia. Black-Right-Pointing-Pointer Suppression of autophagy in thymic epithelium was not associated with signs of autoimmunity. Black-Right-Pointing-Pointer Autophagy was required for normal terminal differentiation of preputial gland cells. Black-Right-Pointing-Pointer Autophagy-deficient cells of the preputial glands degraded nuclear DNA prematurely. -- Abstract: Autophagy contributes to the homeostasis of many tissues, yet its role in epithelia is incompletely understood. A recent report proposed that Atg5-dependent autophagy in thymic epithelial cells is essential for their function in the negative selection of self-reactive T-cells and, thus, for the suppression of tissue inflammation. Heremore » we crossed mice carrying floxed alleles of the Atg5 gene with mice expressing the Cre recombinase under the control of the keratin K5 promoter to suppress autophagy in all K5-positive epithelia. The efficiency of autophagy abrogation was confirmed by immunoanalyses of LC3, which was converted to the autophagy-associated LC3-II form in normal but not Atg5-deficient cells, and of p62, which accumulated in Atg5-deficient cells. Mice carrying the epithelium-specific deletion of Atg5 showed normal weight gain, absence of tissue inflammation, and a normal morphology of the thymic epithelium. By contrast, autophagy-deficient epithelial cells of the preputial gland showed aberrant eosinophilic staining in histology and premature degradation of nuclear DNA during terminal differentiation. Taken together, the results of this study suggest that autophagy is dispensable for the suppression of autoimmunity by thymic epithelial cells but essential for normal differentiation of the preputial gland in mice.« less

Intraoperative anti-thymocyte globulin-Fresenius (ATG-F) administration as induction immunosuppressive therapy in kidney transplantation.

PubMed

Abou-Jaoude, Maroun M; Almawi, Wassim Y

2003-07-01

We reviewed 43 adult kidney transplant patients (32 males and 11 females, 14-68 years of age) performed at our center between July 1999 and February 2002. Donors (39 males and 4 females) comprised two cadaverics, five living-related and 36 living-unrelated; age 18-44 years. Indications for kidney transplantation (KT) were: chronic glomerulonephritis (8), re-transplantation (4) and chronic pyelonephritis (3); kidney disease was unknown in 15 cases. ATG-F was given as a single intra-operative bolus induction therapy in 26 patients; extended ATG-F dose was given in 17 patients because of a high sensitization status, slow graft function (SGF) or development of calcineurin inhibitors toxicity. ATG-F was stopped in seven out of 17 patients because of thrombocytopenia or severe anemia. ATG-F-related fever occurred in six patients. Acute rejection (AR) occurred in eight patients (18%) 5-11 days post-KT. ATG-F was given in three steroid-resistant AR. Infection occurred in 19 patients (44%) for a total of 32 infectious episodes comprising 24 bacterial infections (nine urinary, seven catheter-related and three respiratory), six viral infections (five CMV and one herpes) and two fungal infections (one pulmonary aspergillosis and one catheter-related candidiasis). The hospital stay was 8-75 days for a median of 13 days. The mean serum creatinine upon discharge, at 1 and 6 months after KT were: 2.04+/-0.37, 1.43+/-0.16 and 1.29+/-0.08, respectively. One patient lost his graft on day 9 because of graft microthrombi related to Factor V-Leiden mutation. The 6 months actuarial patient and graft survival were 100 and 97.6%, respectively. ATG-F as a bolus therapy is an effective and safe induction treatment in KT.

Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis.

PubMed

Li, Wei-wei; Chen, Ming; Zhong, Li; Liu, Jia-ming; Xu, Zhao-shi; Li, Lian-cheng; Zhou, Yong-Bin; Guo, Chang-Hong; Ma, You-Zhi

2015-12-25

Autophagy is an evolutionarily conserved biological process in all eukaryotes for the degradation of intracellular components for nutrient recycling. Autophagy is known to be involved in responses to low nitrogen stress in Arabidopsis. Foxtail millet has strong abiotic stress resistance to both low nutrient and drought stress. However, to date, there have only been a few genes reported to be related with abiotic stress resistance in foxtail millet. In this study, we identified an autophagy-related gene, SiATG8a, from foxtail millet. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments. SiATG8a was localized in the membrane and cytoplasm of foxtail millet. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants. Taken together, our results represent the first identified case where overexpression of autophagy related gene can simultaneously improve plant resistance to low nitrogen and drought stresses. These findings implicate plant autophagy in plant stress responses to low nitrogen and drought and should be helpful in efforts to improve stresses resistance to nitrogen starvation and drought of crops by genetic transformation. Copyright © 2015 Elsevier Inc. All rights reserved.

The V471A Polymorphism in Autophagy-Related Gene ATG7 Modifies Age at Onset Specifically in Italian Huntington Disease Patients

PubMed Central

Metzger, Silke; Walter, Carolin; Riess, Olaf; Roos, Raymund A. C.; Nielsen, Jørgen E.; Craufurd, David; Nguyen, Huu Phuc

2013-01-01

The cause of Huntington disease (HD) is a polyglutamine repeat expansion of more than 36 units in the huntingtin protein, which is inversely correlated with the age at onset of the disease. However, additional genetic factors are believed to modify the course and the age at onset of HD. Recently, we identified the V471A polymorphism in the autophagy-related gene ATG7, a key component of the autophagy pathway that plays an important role in HD pathogenesis, to be associated with the age at onset in a large group of European Huntington disease patients. To confirm this association in a second independent patient cohort, we analysed the ATG7 V471A polymorphism in additional 1,464 European HD patients of the “REGISTRY” cohort from the European Huntington Disease Network (EHDN). In the entire REGISTRY cohort we could not confirm a modifying effect of the ATG7 V471A polymorphism. However, analysing a modifying effect of ATG7 in these REGISTRY patients and in patients of our previous HD cohort according to their ethnic origin, we identified a significant effect of the ATG7 V471A polymorphism on the HD age at onset only in the Italian population (327 patients). In these Italian patients, the polymorphism is associated with a 6-years earlier disease onset and thus seems to have an aggravating effect. We could specify the role of ATG7 as a genetic modifier for HD particularly in the Italian population. This result affirms the modifying influence of the autophagic pathway on the course of HD, but also suggests population-specific modifying mechanisms in HD pathogenesis. PMID:23894380

Midgut morphological changes and autophagy during metamorphosis in sand flies.

PubMed

Malta, Juliana; Heerman, Matthew; Weng, Ju Lin; Fernandes, Kenner M; Martins, Gustavo Ferreira; Ramalho-Ortigão, Marcelo

2017-06-01

During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector.

Unrelated cord blood transplantation vs. related transplantation with HLA 1-antigen mismatch in the GVH direction.

PubMed

Kanda, Junya

2015-05-01

The donor selection superiority of HLA 1-antigen mismatched related donor versus unrelated cord blood (UCB) is an important issue for patients without an HLA-matched related or unrelated donor. Using Japanese registry data, we analyzed patients with leukemia and myelodysplastic syndrome who received transplantation using UCB or from a related donor with 1-antigen mismatch in the graft-versus-host (GVH) direction (RD/1AG-MM-GVH). Compared to the UCB group, neutrophil engraftment was significantly faster, and the incidences of acute and chronic GVHD were significantly higher in the RD/1AG-MM-GVH group. As a result, there was no significant difference in overall survival between transplantation using the RD/1AG-MM-GVH and UCB. However, the HLA-B-antigen mismatched group showed significantly inferior overall survival. The RD/1AG-MM-GVH group using anti-thymocyte globulin (ATG) showed neutrophil engraftment comparable to that of the non-ATG group and a GVHD incidence similar to that of the UCB group, which resulted in a better overall survival rate in the ATG than in the UCB group. In particular, the adverse effects of HLA-B mismatch were not observed in the ATG group. RD/1AGMM-GVH transplantation using ATG could potentially improve outcomes, and a prospective study of RD/1AGMM-GVH transplantation using low-dose ATG is currently ongoing.

Problem-Based Test: An "In Vitro" Experiment to Analyze the Genetic Code

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2010-01-01

Terms to be familiar with before you start to solve the test: genetic code, translation, synthetic polynucleotide, leucine, serine, filter precipitation, radioactivity measurement, template, mRNA, tRNA, rRNA, aminoacyl-tRNA synthesis, ribosomes, degeneration of the code, wobble, initiation, and elongation of protein synthesis, initiation codon.…

AoAtg26, a putative sterol glucosyltransferase, is required for autophagic degradation of peroxisomes, mitochondria, and nuclei in the filamentous fungus Aspergillus oryzae.

PubMed

Kikuma, Takashi; Tadokoro, Takayuki; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2017-02-01

Autophagy is a conserved process in eukaryotic cells for degradation of cellular proteins and organelles. In filamentous fungi, autophagic degradation of organelles such as peroxisomes, mitochondria, and nuclei occurs in basal cells after the prolonged culture, but its mechanism is not well understood. Here, we functionally analyzed the filamentous fungus Aspergillus oryzae AoAtg26, an ortholog of the sterol glucosyltransferase PpAtg26 involved in pexophagy in the yeast Pichia pastoris. Deletion of Aoatg26 caused a severe decrease in conidiation and aerial hyphae formation, which is typically observed in the autophagy-deficient A. oryzae strains. In addition, cup-shaped AoAtg8-positive membrane structures were accumulated in the Aoatg26 deletion strain, indicating that autophagic process is impaired. Indeed, the Aoatg26 deletion strain was defective in the degradation of peroxisomes, mitochondria, and nuclei. Taken together, AoAtg26 plays an important role for autophagic degradation of organelles in A. oryzae, which may physiologically contribute to the differentiation in filamentous fungi.

The cell non-autonomous function of ATG-18 is essential for neuroendocrine regulation of Caenorhabditis elegans lifespan

PubMed Central

Minnerly, Justin; Zhang, Jiuli; Parker, Thomas

2017-01-01

Dietary restriction (DR) and reduced insulin growth factor (IGF) signaling extend lifespan in Caenorhabditis elegans and other eukaryotic organisms. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has emerged as a central pathway regulated by various longevity signals including DR and IGF signaling in promoting longevity in a variety of eukaryotic organisms. However, the mechanism remains unclear. Here we show that the autophagy protein ATG-18 acts cell non-autonomously in neuronal and intestinal tissues to maintain C. elegans wildtype lifespan and to respond to DR and IGF-mediated longevity signaling. Moreover, ATG-18 activity in chemosensory neurons that are involved in food detection sufficiently mediates the effect of these longevity pathways. Additionally, ATG-18-mediated cell non-autonomous signaling depends on the release of neurotransmitters and neuropeptides. Interestingly, our data suggest that neuronal and intestinal ATG-18 acts in parallel and converges on unidentified neurons that secrete neuropeptides to regulate C. elegans lifespan through the transcription factor DAF-16/FOXO in response to reduced IGF signaling. PMID:28557996

Self-organizing approach for meta-genomes.

PubMed

Zhu, Jianfeng; Zheng, Wei-Mou

2014-12-01

We extend the self-organizing approach for annotation of a bacterial genome to analyze the raw sequencing data of the human gut metagenome without sequence assembling. The original approach divides the genomic sequence of a bacterium into non-overlapping segments of equal length and assigns to each segment one of seven 'phases', among which one is for the noncoding regions, three for the direct coding regions to indicate the three possible codon positions of the segment starting site, and three for the reverse coding regions. The noncoding phase and the six coding phases are described by two frequency tables of the 64 triplet types or 'codon usages'. A set of codon usages can be used to update the phase assignment and vice versa. An iteration after an initialization leads to a convergent phase assignment to give an annotation of the genome. In the extension of the approach to a metagenome, we consider a mixture model of a number of categories described by different codon usages. The Illumina Genome Analyzer sequencing data of the total DNA from faecal samples are then examined to understand the diversity of the human gut microbiome. Copyright © 2014 Elsevier Ltd. All rights reserved.

Heterozygous genotype at codon 129 correlates with prolonged disease course in Heidenhain variant sporadic CJD: case report.

PubMed

Townley, Ryan A; Dawson, Elliot T; Drubach, Daniel A

2018-02-01

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rapid and fatal neurodegenerative disease defined by misfolded prion proteins accumulating in the brain. A minority of cases initially present with posterior cortical atrophy (PCA) phenotype, also known as Heidenhain variant or visual variant CJD. This case provides further evidence of sCJD presenting as PCA. The case also provides evidence for early DWI changes and cortical atrophy over 30 months before neurologic decline and subsequent death. The prolonged disease course correlates with prion protein codon 129 heterozygosity and coexistence of multiple prion strains.

A randomized 2×2 factorial trial, part 1: single-dose rabbit antithymocyte globulin induction may improve renal transplantation outcomes.

PubMed

Stevens, R Brian; Foster, Kirk W; Miles, Clifford D; Lane, James T; Kalil, Andre C; Florescu, Diana F; Sandoz, John P; Rigley, Theodore H; Nielsen, Kathleen J; Skorupa, Jill Y; Kellogg, Anna M; Malik, Tamer; Wrenshall, Lucile E

2015-01-01

We conducted a randomized and unblinded 2 × 2 sequential-factorial trial, composed of an induction arm (part 1) comparing single-dose (SD) versus divided-dose rabbit antithymocyte globulin (rATG), and a maintenance arm (part 2) comparing tacrolimus minimization versus withdrawal. We report the long-term safety and efficacy of SD-rATG induction in the context of early steroid withdrawal and tacrolimus minimization or withdrawal. Patients (n=180) received 6 mg/kg rATG, SD or four alternate-day doses (1.5 mg/kg/dose), with early steroid withdrawal and tacrolimus or sirolimus maintenance. After 6 months targeted maintenance levels were tacrolimus, 2 to 4 ng/mL and sirolimus, 4 to 6 ng/mL or, if calcineurin inhibitor-withdrawn, sirolimus 8 to 12 ng/mL with mycophenolate mofetil 2 g two times per day. Primary endpoints were renal function (abbreviated modification of diet in renal disease) and chronic graft histopathology (Banff). Secondary endpoints included patient survival, graft survival, biopsy-proven rejection, and infectious or noninfectious complications. Follow-up averaged longer than 4 years. Tacrolimus or sirolimus and mycophenolate mofetil exposure was identical between groups. The SD-rATG associated with improved renal function (2-36 months; P<0.001) in deceased donor recipients. The SD-rATG associated with quicker lymphocyte, CD4 T cell, and CD4-CD8 recovery and fewer infections. Cox multivariate hazard modeling showed divided-dose-rATG (P=0.019), deceased donor (P=0.003), serious infection (P=0.0.018), and lower lymphocyte count (P=0.001) associated with increased mortality. Patients with all four covariates showed a 27-fold increased likelihood of death (P=0.00002). Chronic graft histopathology, rejection rates, and death-censored graft survival were not significantly different between groups. The SD-rATG induction improves the 3-year renal function in recipients of deceased donor kidneys. This benefit, along with possibly improved patient survival and fewer infections suggest that how rATG is administered may impact its efficacy and safety.

A Randomized 2×2 Factorial Trial, Part 1: Single-Dose Rabbit Antithymocyte Globulin Induction May Improve Renal Transplantation Outcomes

PubMed Central

Stevens, R. Brian; Foster, Kirk W.; Miles, Clifford D.; Lane, James T.; Kalil, Andre C.; Florescu, Diana F.; Sandoz, John P.; Rigley, Theodore H.; Nielsen, Kathleen J.; Skorupa, Jill Y.; Kellogg, Anna M.; Malik, Tamer; Wrenshall, Lucile E.

2015-01-01

Background We conducted a randomized and unblinded 2×2 sequential-factorial trial, composed of an induction arm (part 1) comparing single-dose (SD) versus divided-dose rabbit antithymocyte globulin (rATG), and a maintenance arm (part 2) comparing tacrolimus minimization versus withdrawal. We report the long-term safety and efficacy of SD-rATG induction in the context of early steroid withdrawal and tacrolimus minimization or withdrawal. Methods Patients (n=180) received 6 mg/kg rATG, SD or four alternate-day doses (1.5 mg/kg/dose), with early steroid withdrawal and tacrolimus or sirolimus maintenance. After 6 months targeted maintenance levels were tacrolimus, 2 to 4 ng/mL and sirolimus, 4 to 6 ng/mL or, if calcineurin inhibitor–withdrawn, sirolimus 8 to 12 ng/mL with mycophenolate mofetil 2 g two times per day. Primary endpoints were renal function (abbreviated modification of diet in renal disease) and chronic graft histopathology (Banff). Secondary endpoints included patient survival, graft survival, biopsy-proven rejection, and infectious or noninfectious complications. Results Follow-up averaged longer than 4 years. Tacrolimus or sirolimus and mycophenolate mofetil exposure was identical between groups. The SD-rATG associated with improved renal function (2-36 months; P<0.001) in deceased donor recipients. The SD-rATG associated with quicker lymphocyte, CD4 T cell, and CD4-CD8 recovery and fewer infections. Cox multivariate hazard modeling showed divided-dose–rATG (P=0.019), deceased donor (P=0.003), serious infection (P=0.0.018), and lower lymphocyte count (P=0.001) associated with increased mortality. Patients with all four covariates showed a 27-fold increased likelihood of death (P=0.00002). Chronic graft histopathology, rejection rates, and death-censored graft survival were not significantly different between groups. Conclusion The SD-rATG induction improves the 3-year renal function in recipients of deceased donor kidneys. This benefit, along with possibly improved patient survival and fewer infections suggest that how rATG is administered may impact its efficacy and safety. PMID:25083614

Mucopolysaccharidosis IVA: Four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomatsu, Shunji; Fukuda, Seiji; Yamagishi, Atsushi

1996-05-01

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations: V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resultedmore » in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation. 48 refs., 5 figs., 1 tab.« less

Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hipp, Katharina, E-mail: katharina.hipp@bio.uni-st

Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysismore » of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential. -- Highlights: •The ACMV AC4 may be translated from one or the other in-frame start codon. •Both AC4 variants are translated in fission yeast. •The long AC4 protein localizes to the cytoplasm, the short to the plasma membrane. •The short variant is myristoylated in yeast and may promote membrane localization. •Only the shorter AC4 variant has an impact on viral infections in plants.« less

Thymic function, anti-thymocytes globulins, and cancer after renal transplantation.

PubMed

Ducloux, Didier; Bamoulid, Jamal; Courivaud, Cécile; Gaugler, Béatrice; Rebibou, Jean-Michel; Ferrand, Christophe; Chalopin, Jean-Marc; Borg, Christophe; Tiberghien, Pierre; Saas, Philippe

2011-07-01

Prolonged CD4 T cell lymphopenia after polyclonal antithymocyte globulins (ATG) is associated with an increased rate of cancers. Here, we examined whether pre-transplant thymic function estimated by TREC levels is predictive of cancer occurrence following ATG treatment. The impact of TREC on cancer occurrence was analyzed in 115 consecutive incident renal transplant recipients having received ATG. Mean follow-up was 7.5±2.6years. After ATG induction, patients with the lowest pre-transplant TREC values had lower post-transplant CD4(+) and CD4(+) CD45RA(+) CD45RO(-) T cell counts, and a higher frequency of T cells with a regulatory phenotype (CD127(+)CD4(+)CD25(+)Foxp3(+)). Log-transformed pre-transplant TREC values were significantly lower in patients who developed cancer after transplantation (p<0.0001). The cumulative incidence of cancer was higher in patients having the lowest pre-transplant TREC values (T1 [low]: 47.4%, T2 [medium]: 12.5%, and T3 [high]: 2.7%; p<0.0001). In multivariate analysis, pre-transplant TREC value was the only predictive factor of cancer (HR, 0.39; 95% CI, 0.16 to 0.97, for one log (TREC/10(6) PBMC); p=0.046). Pre-transplant thymic function is associated with an increased rate of post-transplant cancer in patients having received ATG. Omitting ATG in recipients with low pre-transplant TREC values should be considered. Copyright © 2011 Elsevier B.V. All rights reserved.

ATG-Fresenius S combined with cyclosporine a: an effective immunosuppressive therapy for children with aplastic anemia.

PubMed

Luo, Cheng-Juan; Gao, Yi-Jin; Tang, Jing-Yan; Zhu, Xiao-Hua; Xue, Hui-Liang; Lu, Feng-Juan; Pan, Ci; Jiang, Hua; Luo, Chang-Ying; Ye, Qi-Dong; Zhou, Min; Chen, Jing

2014-07-01

For the first time, we conducted a 2-center retrospective study to show the efficacy of antithymocyte globulin (ATG)-Fresenius S plus cyclosporine treatment of children with severe aplastic anemia. From March 1997 to May 2011, a total of 124 patients (median age, 7.5 y; range, 1.5 to 16 y) from 2 centers with acquired AA treated with an immunosuppressive therapy (IST) regimen, consisting of ATG-Fresenius S (5 mg/kg per day for 5 d) and cyclosporine, were enrolled. The response rate was 55.6%. The median time between IST and response was 6 (0.5 to 18) months. After a median follow-up time of 29 (6 to 153) months, the rates of relapse and clonal evolution were 3.2% and 0.8%, respectively. Overall, 17 patients (13.7%) died in this study: 14 resulted from sepsis, 1 resulted from intracranial hemorrhage, 1 occurred after hematopoietic stem cell transplantation, and 1 resulted from clonal disease progression. The 5-year overall survival rate for the entire cohort was 74.7%. IST responders had a better survival rate (100%) than nonresponders (70.7%). The use of ATG-Fresenius S plus cyclosporine as a first-line immunosuppressive treatment appeared to be effective for children with severe aplastic anemia in our study. ATG-Fresenius S could be another option in the treatment arsenal, especially in countries where the other ATG products are harder to acquire.

ATG-Fresenius treatment and low-dose tacrolimus: results of a randomized controlled trial in liver transplantation.

PubMed

Benítez, C E; Puig-Pey, I; López, M; Martínez-Llordella, M; Lozano, J J; Bohne, F; Londoño, M C; García-Valdecasas, J C; Bruguera, M; Navasa, M; Rimola, A; Sánchez-Fueyo, A

2010-10-01

We report the results of a prospective randomized controlled trial in liver transplantation assessing the efficacy and safety of antithymocyte globulin (ATG-Fresenius) plus tacrolimus monotherapy at gradually decreasing doses. Patients were randomized to either: (a) standard-dose tacrolimus plus steroids;or (b) peritransplant ATG-Fresenius plus reduced-dose tacrolimus monotherapy followed by weaning of tacrolimus starting 3 months after transplantation. The primary end-point was the achievement of very low-dose tacrolimus (every-other-day or once daily dose with <5 ng/mL trough levels) at 12 months after transplantation. Acute rejection occurring during the first 3 months after transplantation was more frequent in the ATG group (52.4% vs. 25%). Moreover, late acute rejection episodes occurred in all recipients in whom weaning was attempted and no recipients reached the primary end-point. This motivated the premature termination of the trial. Tacrolimus trough levels were lower in the ATG-Fresenius group but no benefits in terms of improved renal function, lower metabolic complications or increased prevalence of tolerance-related biomarkers were observed. In conclusion, the use of ATG-Fresenius and tacrolimus at gradually decreasing doses was associated with a high rate of rejection, did not allow for the administration of very low doses of tacrolimus and failed to provide detectable clinical benefits. ClinicalTrials.gov identifier: NCT00436722. © 2010 The Authors Journal compilation © 2010 The American Society of Transplantation and the American Society of Transplant Surgeons.

Protective effect of arctigenin on ethanol-induced neurotoxicity in PC12 cells.

PubMed

Huang, Jia; Xiao, Lan; Wei, Jing-Xiang; Shu, Ya-Hai; Fang, Shi-Qi; Wang, Yong-Tang; Lu, Xiu-Min

2017-04-01

As a neurotropic substance, ethanol can damage nerve cells through an increase in the production of free radicals, interference of neurotrophic factor signaling pathways, activation of endogenous apoptotic signals and other molecular mechanisms. Previous studies have revealed that a number of natural drugs extracted from plants offer protection of nerve cells from damage. Among these, arctigenin (ATG) is a lignine extracted from Arctium lappa (L.), which has been found to exert a neuroprotective effect on scopolamine‑induced memory deficits in mice with Alzheimer's disease and glutamate-induced neurotoxicity in primary neurons. As a result, it may offer beneficial effects on ethanol-induced neurotoxicity. However, the effects of ATG on ethanol‑induced nerve damage remain to be elucidated. To address this issue, the present study used rat pheochromocytoma PC12 cells to investigate the neuroprotective effects of ATG on ethanol-induced cell damage by performing an MTT reduction assay, cell cycle analysis, Hoechst33342/propidium iodide fluorescence staining and flow cytometry to examine apoptosis. The results showed that 10 µM ATG effectively promoted the proliferation of damaged cells, and increased the distribution ratio of the cells at the G2/M and S phases (P<0.05). In addition, the apoptosis and necrosis of the PC12 cells were significantly decreased following treatment with ATG. Therefore, it was concluded that 10 µM ATG had a protective effect on ethanol‑induced injury in PC12 cells.

Stabilization of Clostridium perfringens collagenase mRNA by VR-RNA-dependent cleavage in 5' leader sequence.

PubMed

Obana, Nozomu; Shirahama, Yu; Abe, Kimihiro; Nakamura, Kouji

2010-09-01

The small RNA (sRNA), VR-RNA that is directly regulated by the VirR/VirS two-component system, regulates many genes including toxin genes such as collagenase (colA) and phospholipase C (plc) in Clostridium perfringens. Although the VR-RNA 3' region is sufficient to regulate the colA and plc genes, the molecular mechanism of toxin gene regulation by VR-RNA remains unclear. Here, we found that colA mRNA is cleaved at position -79 and -78 from the A of the first codon (ATG) in the presence of VR-RNA. The processed transcripts were stable compared with longer intact transcripts. On the other hand, colA mRNA was labile in a VR-RNA-deficient strain, and processed transcripts were undetectable. The stability and processing of colA mRNA were restored by transformation of the 3' region of VR-RNA-expression vector. The 3' region of VR-RNA and colA mRNA had significant complementation and interacted in vitro. These results show that VR-RNA base pairs with colA mRNA and induces cleavage in the 5' untranslated region (UTR) of colA mRNA, which leads to the stabilization of colA mRNA and the activation of colA expression. © 2010 Blackwell Publishing Ltd.

Complete mitochondrial DNA genome of Bemisia tabaci cryptic pest species complex Asia I (Hemiptera: Aleyrodidae).

PubMed

Tay, W T; Elfekih, S; Court, L; Gordon, K H; De Barro, P J

2016-01-01

The complete length of the Asia I member of the Bemisia tabaci species complex mitochondrial DNA genome (mitogenome) is 15,210 bp (GenBank accession no. KJ778614) with an A-T biased nucleotide composition (A: 32.7%; T: 42.4%; G: 14.0%; C: 10.8%). The mitogenome consists of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), 2 ribosomal RNA (rRNAs) and a 467 bp putative control region which also includes the A+T rich repeat region. All PCGs have an ATA (n = 8) or ATG (n = 5) start codon. Gene synteny of Asia I is overall similar to B. afer and two other members of the B. tabaci species complex Mediterranean and New World 1, and contains the tRNA-Ser2 located between the Cytb and ND1 genes found in Mediterranean and New World 1, but which is absent in B. afer. The orientation of the tRNA-Arg in Asia I is on the "plus" strand and differed from Mediterranean which is found on the "minus" strand. The Asia I mitogenome size is currently ranked the second smallest after B. afer (14,968 bp) followed by New World 1 (15,322 bp) and Mediterranean (15,632 bp).

Isolation of a polyphenol oxidase (PPO) cDNA from artichoke and expression analysis in wounded artichoke heads.

PubMed

Quarta, Angela; Mita, Giovanni; Durante, Miriana; Arlorio, Marco; De Paolis, Angelo

2013-07-01

The polyphenol oxidase (PPO) enzyme, which can catalyze the oxidation of phenolics to quinones, has been reported to be involved in undesirable browning in many plant foods. This phenomenon is particularly severe in artichoke heads wounded during the manufacturing process. A full-length cDNA encoding for a putative polyphenol oxidase (designated as CsPPO) along with a 1432 bp sequence upstream of the starting ATG codon was characterized for the first time from [Cynara cardunculus var. scolymus (L.) Fiori]. The 1764 bp CsPPO sequence encodes a putative protein of 587 amino acids with a calculated molecular mass of 65,327 Da and an isoelectric point of 5.50. Analysis of the promoter region revealed the presence of cis-acting elements, some of which are putatively involved in the response to light and wounds. Expression analysis of the gene in wounded capitula indicated that CsPPO was significantly induced after 48 h, even though the browning process had started earlier. This suggests that the early browning event observed in artichoke heads was not directly related to de novo mRNA synthesis. Finally, we provide the complete gene sequence encoding for polyphenol oxidase and the upstream regulative region in artichoke. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

PubMed Central

Uemura, Takeshi; Mori, Takuma; Kurihara, Taiga; Kawase, Shiori; Koike, Rie; Satoga, Michiru; Cao, Xueshan; Li, Xue; Yanagawa, Toru; Sakurai, Takayuki; Shindo, Takayuki; Tabuchi, Katsuhiko

2016-01-01

Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons. PMID:27782168

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques.

PubMed

Uemura, Takeshi; Mori, Takuma; Kurihara, Taiga; Kawase, Shiori; Koike, Rie; Satoga, Michiru; Cao, Xueshan; Li, Xue; Yanagawa, Toru; Sakurai, Takayuki; Shindo, Takayuki; Tabuchi, Katsuhiko

2016-10-26

Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons.

An ATF4-ATG5 signaling in hypothalamic POMC neurons regulates obesity.

PubMed

Xiao, Yuzhong; Deng, Yalan; Yuan, Feixiang; Xia, Tingting; Liu, Hao; Li, Zhigang; Chen, Shanghai; Liu, Zhixue; Ying, Hao; Liu, Yi; Zhai, Qiwei; Guo, Feifan

2017-06-03

ATF4 (activating transcription factor 4) is an important transcription factor that has many biological functions, while its role in hypothalamic POMC (pro-opiomelanocortin-α) neurons in the regulation of energy homeostasis has not been explored. We recently discovered that mice with an Atf4 deletion specific to POMC neurons (PAKO mice) are lean and have higher energy expenditure. Furthermore, these mice are resistant to high-fat diet (HFD)-induced obesity and obesity-related metabolic disorders. Mechanistically, we found the expression of ATG5 (autophagy-related 5) is upregulated in POMC neurons of PAKO mice, and ATF4 regulates ATG5 expression by binding directly to its promoter. Mice with Atf4 and Atg5 double knockout in POMC neurons have reduced energy expenditure and gain more fat mass compared with PAKO mice under a HFD. Finally, the effect of Atf4 knockout in POMC neurons is possibly mediated by enhanced ATG5-dependent macroautophagy/autophagy and α-melanocyte-stimulating hormone (α-MSH) production in the hypothalamus. Together, this work not only identifies a beneficial role for ATF4 in hypothalamic POMC neurons in the regulation of obesity, but also provides a new potential therapeutic target for obesity and obesity-related metabolic diseases.

A high-level prokaryotic expression system: synthesis of human interleukin 1 alpha and its receptor antagonist.

PubMed

Birikh, K R; Lebedenko, E N; Boni, I V; Berlin, Y A

1995-10-27

Synthetic intronless genes, coding for human interleukin 1 alpha (IL 1 alpha) and interleukin 1 receptor antagonist (IL1ra), have been expressed efficiently in a specially designed prokaryotic vector, pGMCE (a pGEM1 derivative), where the target gene forms the second part of a two-cistron system. The first part of the system is a translation enhancer-containing mini-cistron, whose termination codon overlaps the start codon of the target gene. In the case of the IL1 alpha gene, the high expression level is largely due to the direct efficient translation initiation at the second cistron, whereas with the IL1ra gene in the same system, the proximal translation initiation region (TIR) provides a high level of coupled expression of the target gene. Thus, pGMCE is a potentially versatile vector for direct prokaryotic expression.

MicroRNA-1 overexpression increases chemosensitivity of non-small cell lung cancer cells by inhibiting autophagy related 3-mediated autophagy.

PubMed

Hua, Li; Zhu, Guirong; Wei, Jianguo

2018-05-30

Non-small cell lung cancer (NSCLC) is a major type of lung cancer. Drug resistance is a enormous obstacle for cancer treatment. Copious microRNAs (miRNAs) have been demonstrated to be implicated in drug resistance in NSCLC. In the present study, RT-qPCR assay revealed that microRNA-1 (miR-1) expression was downregulated in DDP resistant NSCLC tissues and cells. Western blot assay presented a remarkable increase of LC3B-II/LC3B-I ratio and a notable decline of p62 level in DDP resistant NSCLC cells, while these effects were weakened by miR-1. GFP-LC3 puncta experiment showed that ectopic expression of miR-1 induced a noticeable downregulation of GFP-LC3 positive cell percentage in DDP resistant NSCLC cells. Bioinformatical analysis and luciferase assay revealed that autophagy related 3 (ATG3) was a target of miR-1. Also, western blot and RT-qPCR assays manifested that ATG3 was highly expressed in DDP resistant NSCLC tissues and cells. Additionally, miR-1 inhibited ATG3 expression and ATG3 upregulation abolished miR-1-meidated autophagy inhibition in DDP resistant NSCLC cells. Cell Counting Kit-8 (CCK-8) assay showed that the half maximal inhibitory concentration (IC 50 ) of cisplatin (DDP) was reduced in miR-1-enforced DDP resistant NSCLC cells, but was restored following the overexpression of ATG3. Flow cytometry experiments further showed that miR-1 overexpression induced a significant upregulation of apoptotic rate and ATG3 restoration weakened miR-1-induced apoptosis in DDP resistant NSCLC cells. Collectively, our study validated that miR-1 overexpression improved DDP sensitivity of NSCLC cells by inhibiting ATG3-mediated autophagy, providing a potential therapeutic target for easing chemoresistance of anti-tumor drugs. This article is protected by copyright. All rights reserved.

MiR-29b Downregulation Induces Phenotypic Modulation of Vascular Smooth Muscle Cells: Implication for Intracranial Aneurysm Formation and Progression to Rupture.

PubMed

Sun, Liqian; Zhao, Manman; Zhang, Jingbo; Lv, Ming; Li, Youxiang; Yang, Xinjian; Liu, Aihua; Wu, Zhongxue

2017-01-01

Our previous microarray results identified numerous microRNAs (miRNAs), including miR-29b, that were differentially expressed in the serum of intracranial aneurysm (IA) patients. The current study aimed to investigate whether miR-29b downregulation in IA could promote the phenotypic modulation of vascular smooth muscle cells (VSMCs) involved in the pathogenesis of aneurysm by activating ATG14-mediated autophagy. First, the levels of miR-29b and autophagy related genes (ATGs) between IA patients and normal subjects were compared. Next, we modified the level of miR-29b via lentivirus particles in the VSMCs and examined the effects of miR-29b on proliferation, migration, and phenotypic modulation of VSMCs from a contractile phenotype to a synthetic phenotype, as well as the levels of autophagy. Finally, the binding of miR-29b to the 3'UTR of ATG14 mRNA and its effects on ATG14 expression were analysed by a luciferase reporter assay and Western blot, respectively. The level of miR-29b was decreased, and autophagy markers were increased in the IA patients compared to that of the normal subjects. Knockdown of miR-29b significantly promoted VSMCs proliferation and migration and, more importantly, induced the phenotypic modulation associated with autophagy activation, whereas miR-29b overexpression showed the opposite effects. The luciferase reporter assay demonstrated that ATG14 was a functional target gene of miR-29b. Notably, knockdown of ATG14 by siRNA apparently abrogated miR-29b inhibition-mediated phenotypic modulation. Downregulation of miR-29b induced VSMCs phenotypic modulation by directly activating ATG14-mediated autophagy, which is associated with the formation, growth and rupture of IAs. © 2017 The Author(s) Published by S. Karger AG, Basel.

Single-shot antithymocyte globulin (ATG) induction for pancreas/kidney transplantation: ATG-Fresenius versus Thymoglobulin.

PubMed

Schulz, T; Papapostolou, G; Schenker, P; Kapischke, M

2005-03-01

Single-shot antithymocyte globulin (ATG) prior to reperfusion followed by tacrolimus (TAC), mycophenolate mofetil (MMF), and prednisolone (PRD) is an established induction therapy in simultaneous pancreas kidney transplant (SPK) recipients. We retrospectively analyzed 6-month data from 105 patients who received their first SPK. From January 1996 to December 2000, ATG-Fresenius was used. Since January 2001, Thymoglobulin has been administered. In the first group, 58 patients were treated with ATG-Fresenius (4-6 mg/kg body weight). In the second group, 47 patients received Thymoglobulin (1.5-2.5 mg/kg body weight). HLA-mismatch was comparable. After an observation period of 6 months, patients, kidney, and pancreas graft survival is 98.3%, 96.6%, and 93.1% in group I and 97.9%, 97.9%, and 85.1% in group II, respectively. In each group, one death with functioning graft (DWFG) was observed. Twenty (34.5%) acute rejection episodes (AR) were observed (18 patients) in group I. They were treated with steroids (n = 16) or steroids/OKT3 (n = 4). One kidney graft failure was observed due to rejection and one due to DWFG. Four pancreas grafts were lost (thrombosis, n = 2; AR, n = 1; DWFG, n = 1). In group II, 15 AR (31.9%) were seen in 12 patients and were treated with steroids (n = 12), steroids/ATG (n = 1), or steroids/OKT3 (n = 2). Seven pancreas (thrombosis, n = 5; rejection, n = 1; DWFG, n = 1) and one kidney (DWFG, n = 1) graft losses occurred. These data clearly establish that single-shot ATG prior to reperfusion, followed by TAC, MMF, and PRD results in a low incidence of AR (34.5% in group I and 31.9% in group II) after SPK. Only 6.9% (group I) and 6.4% (group II) of the patients received antibodies for rejection treatment.

Effect of a single intraoperative high-dose ATG-Fresenius on delayed graft function in donation after cardiac-death donor renal allograft recipients: a randomized study.

PubMed

van den Hoogen, Martijn W F; Kho, Marcia M L; Abrahams, Alferso C; van Zuilen, Arjan D; Sanders, Jan-Stephan; van Dijk, Marja; Hilbrands, Luuk B; Weimar, Willem; Hoitsma, Andries J

2013-04-01

Reducing the incidence of delayed graft function after transplant with donation after cardiac death donor renal allografts would facilitate managing recipients during their first weeks after a transplant. To reduce this incidence, in most studies, induction therapy with depleting anti-T-lymphocyte antibodies is coupled with a reduction of the dosage of the calcineurin inhibitor. The separate effect of anti-T-cell therapy on the incidence and duration of delayed graft function is therefore difficult to assess. We performed a randomized study to evaluate the effect of a single intraoperative high-dose of anti-T-lymphocyte immunoglobulin (ATG)-Fresenius (9 mg/kg body weight) on the incidence of delayed graft function. Eligible adult recipients of a first donation after cardiac death donor renal allograft were randomly assigned to ATG-Fresenius or no induction therapy. Maintenance immunosuppression consisted of tacrolimus, in an unadjusted dose, mycophenolate mofetil, and steroids. The study was prematurely terminated because of a lower-than-anticipated inclusion rate. Baseline characteristics were comparable in the ATG-Fresenius group (n=28) and the control group (n=24). Twenty-two patients in the ATG-Fresenius group (79%) had delayed graft function, compared with 13 in the control group (54%; P = .06). Allograft and patient survival were comparable in both groups. Serious adverse events occurred more frequently in the ATG-Fresenius group than they did in the control group (57% vs 29%; P < .05). Intraoperative administration of a single high-dose of ATG-Fresenius in donation after cardiac death donor renal allograft recipients, followed by triple immunosuppression with an unadjusted tacrolimus dose, seems ineffective to reduce the incidence of delayed graft function. Moreover, this was associated with a higher rate of serious adverse events (EudraCT-number, 2007-000210-36.).

Atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle G2/M arrest and renal fibrosis.

PubMed

Li, Huiyan; Peng, Xuan; Wang, Yating; Cao, Shirong; Xiong, Liping; Fan, Jinjin; Wang, Yihan; Zhuang, Shougang; Yu, Xueqing; Mao, Haiping

2016-09-01

Macroautophagy/autophagy protects against cellular stress. Renal sublethal injury-triggered tubular epithelial cell cycle arrest at G2/M is associated with interstitial fibrosis. However, the role of autophagy in renal fibrosis is elusive. Here, we hypothesized that autophagy activity in tubular epithelial cells is pivotal for inhibition of cell cycle G2/M arrest and subsequent fibrogenic response. In both renal epithelial cells stimulated by angiotensin II (AGT II) and the murine kidney after unilateral ureteral obstruction (UUO), we observed that occurrence of autophagy preceded increased production of COL1 (collagen, type I). Pharmacological enhancement of autophagy by rapamycin suppressed COL1 accumulation and renal fibrosis. In contrast, genetic ablation of autophagy by proximal tubular epithelial cell-specific deletion of Atg5, with reduction of the LC3-II protein level and degradation of SQSTM1/p62, showed marked cell cycle arrest at the G2/M phase, robust COL1 deposition, and severe interstitial fibrosis in a UUO model, as compared with wild-type mice. In vitro, AGT II exposure triggered autophagy preferentially in the G1/S phase, and increased COL1 expression in the G2/M phase in renal epithelial cells. Stimulation of Atg5-deficient primary proximal tubular cells with AGT II also resulted in elevated G2/M arrest and COL1 production. Pharmacological or genetic inhibition of autophagy increased AGT II-mediated G2/M arrest. Enhanced expression of ATG5, but not the autophagy-deficient ATG5 mutant K130R, rescued the G2/M arrest, suggesting the regulation of cell cycle progression by ATG5 is autophagy dependent. In conclusion, Atg5-mediated autophagy in proximal epithelial cells is a critical host-defense mechanism that prevents renal fibrosis by blocking G2/M arrest.

A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells

PubMed Central

Kim, A D; Kang, K A; Kim, H S; Kim, D H; Choi, Y H; Lee, S J; Kim, H S; Hyun, J W

2013-01-01

Compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol) is an active metabolite of ginsenosides and induces apoptosis in various types of cancer cells. This study investigated the role of autophagy in compound K-induced cell death of human HCT-116 colon cancer cells. Compound K activated an autophagy pathway characterized by the accumulation of vesicles, the increased positive acridine orange-stained cells, the accumulation of LC3-II, and the elevation of autophagic flux. Whereas blockade of compound K-induced autophagy by 3-methyladenein and bafilomycin A1 significantly increased cell viability. In addition, compound K augmented the time-dependent expression of the autophagy-related proteins Atg5, Atg6, and Atg7. However, knockdown of Atg5, Atg6, and Atg7 markedly inhibited the detrimental impact of compound K on LC3-II accumulation and cell vitality. Compound K-provoked autophagy was also linked to the generation of intracellular reactive oxygen species (ROS); both of these processes were mitigated by the pre-treatment of cells with the antioxidant N-acetylcysteine. Moreover, compound K activated the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas downregulation of JNK by its specific inhibitor SP600125 or by small interfering RNA against JNK attenuated autophagy-mediated cell death in response to compound K. Compound K also provoked apoptosis, as evidenced by an increased number of apoptotic bodies and sub-G1 hypodiploid cells, enhanced activation of caspase-3 and caspase-9, and modulation of Bcl-2 and Bcl-2-associated X protein expression. Notably, compound K-stimulated autophagy as well as apoptosis was induced by disrupting the interaction between Atg6 and Bcl-2. Taken together, these results indicate that the induction of autophagy and apoptosis by compound K is mediated through ROS generation and JNK activation in human colon cancer cells. PMID:23907464

Let-7i-Induced Atg4B Suppression Is Essential for Autophagy of Placental Trophoblast in Preeclampsia.

PubMed

Xu, Yinyan; Huang, Xinyan; Xie, Juan; Chen, Yanni; Fu, Jing; Wang, Li

2017-09-01

Autophagy, identified as type II programmed cell death, has already been known to be involved in the pathophysiology of preeclampsia (PE), which is a gestational disease with high morbidity. The present study aims to investigate the functional role of let-7i, a miRNA, in trophoblastic autophagy. Placental tissue used in this study was collected from patients with severe preeclampsia (SPE) or normal pregnant women. A decreased level of let-7i was found in placenta of SPE. In addition, autophagic vacuoles were observed in SPE and the expression of microtubule associated protein 1 light chain 3 (LC3) II/I was elevated. In vitro, let-7i mimics suppressed the autophagic activities in human HTR-8/SVneo trophoblast cell line (HTR-8) and human placental choriocarcinoma cell line JEG-3, whereas let-7i inhibitor enhanced the activities. As a potential target of let-7i, autophagy-related 4B cysteine peptidase (Atg4B) had an increased expression level in SPE. As expected, the increased expression of Atg4B was negatively regulated by let-7i using dual luciferase reporter assay. Furthermore, these trophoblast-like cells transfected with the let-7i mimic or inhibitors resulted in a significant change of Atg4B in both mRNA and protein level. More importantly, Atg4B overexpression could partly reverse let-7i mimic-reduced LC3II/I levels; whereas Atg4B silencing partly attenuated let-7i inhibitor-induced the level of LC3II/I expression. Taken together, these findings suggest that let-7i is able to regulate autophagic activity via regulating Atg4B expression, which might contribute to the pathogenesis of PE. J. Cell. Physiol. 232: 2581-2589, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The inhibition of activated hepatic stellate cells proliferation by arctigenin through G0/G1 phase cell cycle arrest: persistent p27(Kip1) induction by interfering with PI3K/Akt/FOXO3a signaling pathway.

PubMed

Li, Ao; Wang, Jun; Wu, Mingjun; Zhang, Xiaoxun; Zhang, Hongzhi

2015-01-15

Proliferation of hepatic stellate cells (HSCs) is vital for the development of fibrosis during liver injury. In this study, we describe that arctigenin (ATG), a major bioactive component of Fructus Arctii, exhibited selective cytotoxic activity via inhibiting platelet-derived growth factor-BB (PDGF-BB)-activated HSCs proliferation and arrested cell cycle at G0/G1 phase, which could not be observed in normal human hepatocytes in vitro. The cyclin-dependent kinase (CDK) 4/6 activities could be strongly inhibited by ATG through down-regulation of cyclin D1 and CDK4/6 expression in early G1 phase arrest. In the ATG-treated HSCs, the expression level of p27(Kip1) and the formation of CDK2-p27(Kip1) complex were also increased. p27(Kip1) silencing significantly attenuated the effect of ATG, including cell cycle arrest and suppression of proliferation in activated HSCs. We also found that ATG suppressed PDGF-BB-induced phosphorylation of Akt and its downstream transcription factor Forkhead box O 3a (FOXO3a), decreased binding of FOXO3a to 14-3-3 protein, and stimulated nuclear translocation of FOXO3a in activated HSCs. Furthermore, knockdown of FOXO3a expression by FOXO3a siRNA attenuated ATG-induced up-regulation of p27(Kip1) in activated HSCs. All the above findings suggested that ATG could increase the levels of p27(Kip1) protein through inhibition of Akt and improvement of FOXO3a activity, in turn inhibited the CDK2 kinase activity, and eventually caused an overall inhibition of HSCs proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

Enraf Series 854 advanced technology gauge (ATG) acceptance test procedure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huber, J.H.

1996-09-11

This Acceptance Test Procedure was written to test the Enraf Series 854 Advanced Technology Gauge (ATG) prior to installation in the Tank Farms. The procedure sets various parameters and verifies that the gauge is functional.

Disruption of microtubules in plants suppresses macroautophagy and triggers starch excess-associated chloroplast autophagy

PubMed Central

Wang, Yan; Zheng, Xiyin; Yu, Bingjie; Han, Shaojie; Guo, Jiangbo; Tang, Haiping; Yu, Alice Yunzi L; Deng, Haiteng; Hong, Yiguo; Liu, Yule

2015-01-01

Microtubules, the major components of cytoskeleton, are involved in various fundamental biological processes in plants. Recent studies in mammalian cells have revealed the importance of microtubule cytoskeleton in autophagy. However, little is known about the roles of microtubules in plant autophagy. Here, we found that ATG6 interacts with TUB8/β-tubulin 8 and colocalizes with microtubules in Nicotiana benthamiana. Disruption of microtubules by either silencing of tubulin genes or treatment with microtubule-depolymerizing agents in N. benthamiana reduces autophagosome formation during upregulation of nocturnal or oxidation-induced macroautophagy. Furthermore, a blockage of leaf starch degradation occurred in microtubule-disrupted cells and triggered a distinct ATG6-, ATG5- and ATG7-independent autophagic pathway termed starch excess-associated chloroplast autophagy (SEX chlorophagy) for clearance of dysfunctional chloroplasts. Our findings reveal that an intact microtubule network is important for efficient macroautophagy and leaf starch degradation. PMID:26566764

Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

NASA Astrophysics Data System (ADS)

Qi, Fei; Guo, Huarong; Wang, Jian

2008-02-01

Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

PubMed Central

Shulami, Smadar; Gat, Orit; Sonenshein, Abraham L.; Shoham, Yuval

1999-01-01

A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α-d-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular α-glucuronidase (aguA) and a β-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer. PMID:10368143

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability*

PubMed Central

Howard, Michael T.; Carlson, Bradley A.; Anderson, Christine B.; Hatfield, Dolph L.

2013-01-01

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins. PMID:23696641

Beneficial Role of Low-Dose Antithymocyte Globulin in Unrelated Stem Cell Transplantation for Adult Patients with Acquired Severe Aplastic Anemia: Reduction of Graft-versus-Host Disease and Improvement of Graft-versus-Host Disease-Free, Failure-Free Survival Rate.

PubMed

Park, Sung-Soo; Kwak, Dae Hun; Jeon, Young-Woo; Yoon, Jae-Ho; Lee, Sung-Eun; Cho, Byung-Sik; Eom, Ki-Seong; Kim, Yoo-Jin; Kim, Hee-Je; Lee, Seok; Min, Chang-Ki; Cho, Seok-Goo; Kim, Dong-Wook; Min, Woo-Sung; Lee, Jong Wook

2017-09-01

Stem cell transplantation (SCT) from an unrelated donor (URD) is often considered in patients with severe aplastic anemia (SAA) whom immunosuppressive therapy failed and matched sibling donor is not available. To reduce the incidence of graft-versus-host disease (GVHD) in URD SCT, introducting antithymocyte globulin (ATG) into the conditioning regimen has been proposed. Although ATG was shown to play a role in reducing GVHD in a cohort with diverse hematologic diseases, its role in SAA remains uncertain. The aim of this study was to determine the efficacy and toxicity of ATG in URD SCT for adult patients with SAA. We investigated 83 adult patients with SAA who underwent URD SCT between 2003 and 2014. The transplantation strategy consisted of total body irradiation (total 800 cGy) and cyclophosphamide (total 100 mg/kg to 120 mg/kg), followed by tacrolimus and a short-term methotrexate. We divided patients into 2 groups: group 1 (n = 25), which received HLA-matched (8/8) bone marrow (BM) without ATG, and group 2 (n = 58), which received SCT from either an HLA-mismatched donor or peripheral blood (PB). Thereafter, group 2 was subdivided according to ATG use into group 2A (without ATG, n = 26), which served as a historical cohort, and group 2B (with ATG, n = 32). Rabbit ATG (Thymoglobulin; Genzyme-Sanofi, Lyon, France) was used in group 2B at a dose of 2.5 mg/kg. The median age of all patients was 30 years (range, 17 to 59 years). The incidence of GVHD was significantly lower in group 2B than group 2A, as demonstrated by the rate of grade II to IV acute GVHD at day 100 (31.2% versus 61.5%, P = .003) and the rate of chronic GVHD at 3 years (21.9% versus 65.4%, P = .002). The overall survival rates of the 3 groups were similar. However, GVHD-free, failure-free survival (GFFS) was significantly higher in group 2B than group 2A (P = .034). A multivariable model identified use of ATG as an independent factor affecting grades II to IV acute GVHD (hazard ratio [HR], 2.902; 95% confidence interval [CI], 1.417 to 5.942; P = .004), chronic GVHD (HR , 3.005; 95% CI, 1.279 to 7.059; P = .012), and GFFS (HR, 2.363; 95% CI, 1.162 to 4.805; P = .014). Toxicities, including infectious complications, were not different among the 3 groups. In conclusion, low-dose ATG (2.5 mg/kg) can reduce the incidence of acute and chronic GVHD and improve the quality of life in patients with SAA who receive stem cells from either an HLA-mismatched donor or PB; importantly, these benefits are achieved without increased toxicity. Furthermore, ATG can be considered in URD SCT from HLA-matched BM cells. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Divergence and codon usage bias of Betanodavirus, a neurotropic pathogen in fish.

PubMed

He, Mei; Teng, Chun-Bo

2015-02-01

Betanodavirus is a small bipartite RNA virus of global economical significance that can cause severe neurological disorders to an increasing number of marine fish species. Herein, to further the understanding of the evolution of betanodavirus, Bayesian coalescent analyses were conducted to the time-stamped entire coding sequences of their RNA polymerase and coat protein genes. Similar moderate nucleotide substitution rates were then estimated for the two genes. According to age calculations, the divergence of the two genes into the four genotypes initiated nearly simultaneously at ∼700 years ago, despite the different scenarios, whereas the seven analyzed chimeric isolates might be the outcomes of a single genetic reassortment event taking place in the early 1980s in Southern Europe. Furthermore, codon usage bias analyses indicated that each gene had influences in addition to mutational bias and codon choice of betanodavirus was not completely complied with that of fish host. Copyright © 2014 Elsevier Inc. All rights reserved.

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

PubMed

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Nucleation versus instability race in strained films

NASA Astrophysics Data System (ADS)

Liu, Kailang; Berbezier, Isabelle; David, Thomas; Favre, Luc; Ronda, Antoine; Abbarchi, Marco; Voorhees, Peter; Aqua, Jean-Noël

2017-10-01

Under the generic term "Stranski-Krastanov" are grouped two different growth mechanisms of SiGe quantum dots. They result from the self-organized Asaro-Tiller-Grinfel'd (ATG) instability at low strain, while at high strain, from a stochastic nucleation. While these regimes are well known, we elucidate here the origin of the transition between these two pathways thanks to a joint theoretical and experimental work. Nucleation is described within the master equation framework. By comparing the time scales for ATG instability development and three-dimensional (3D) nucleation onset, we demonstrate that the transition between these two regimes is simply explained by the crossover between their divergent evolutions. Nucleation exhibits a strong exponential deviation at low strain while ATG behaves only algebraically. The associated time scale varies with exp(1 /x4) for nucleation, while it only behaves as 1 /x8 for the ATG instability. Consequently, at high (low) strain, nucleation (instability) occurs faster and inhibits the alternate evolution. It is then this different kinetic evolution which explains the transition from one regime to the other. Such a kinetic view of the transition between these two 3D growth regimes was not provided before. The crossover between nucleation and ATG instability is found to occur both experimentally and theoretically at a Ge composition around 50% in the experimental conditions used here. Varying the experimental conditions and/or the system parameters does not allow us to suppress the transition. This means that the SiGe quantum dots always grow via ATG instability at low strain and nucleation at high strain. This result is important for the self-organization of quantum dots.

Autophagy Deficiency Compromises Alternative Pathways of Respiration following Energy Deprivation in Arabidopsis thaliana.

PubMed

Barros, Jessica A S; Cavalcanti, João Henrique F; Medeiros, David B; Nunes-Nesi, Adriano; Avin-Wittenberg, Tamar; Fernie, Alisdair R; Araújo, Wagner L

2017-09-01

Under heterotrophic conditions, carbohydrate oxidation inside the mitochondrion is the primary energy source for cellular metabolism. However, during energy-limited conditions, alternative substrates are required to support respiration. Amino acid oxidation in plant cells plays a key role in this by generating electrons that can be transferred to the mitochondrial electron transport chain via the electron transfer flavoprotein/ubiquinone oxidoreductase system. Autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid pools and nutrient remobilization. Although the association between autophagy and alternative respiratory substrates has been suggested, the extent to which autophagy and primary metabolism interact to support plant respiration remains unclear. To investigate the metabolic importance of autophagy during development and under extended darkness, Arabidopsis ( Arabidopsis thaliana ) mutants with disruption of autophagy ( atg mutants) were used. Under normal growth conditions, atg mutants showed lower growth and seed production with no impact on photosynthesis. Following extended darkness, atg mutants were characterized by signatures of early senescence, including decreased chlorophyll content and maximum photochemical efficiency of photosystem II coupled with increases in dark respiration. Transcript levels of genes involved in alternative pathways of respiration and amino acid catabolism were up-regulated in atg mutants. The metabolite profiles of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acid levels were partially compromised in atg mutants. Although an enhanced respiration in atg mutants was observed during extended darkness, autophagy deficiency compromises protein degradation and the generation of amino acids used as alternative substrates to the respiration. © 2017 American Society of Plant Biologists. All Rights Reserved.

Hematology: ATG and Newton's third law of motion.

PubMed

Brunstein, Claudio G

2010-01-01

Patients with hematological malignancies have a risk of developing graft-versus-host disease (GVHD) following allogeneic hematopoietic stem-cell transplantation. The addition of ATG to prophylaxis regimens decreases the incidence of GVHD without compromising overall survival in these patients.

Knockdown of long non-coding RNA HOTAIR increases miR-454-3p by targeting Stat3 and Atg12 to inhibit chondrosarcoma growth.

PubMed

Bao, Xing; Ren, Tingting; Huang, Yi; Sun, Kunkun; Wang, Shidong; Liu, Kuisheng; Zheng, Bingxin; Guo, Wei

2017-02-09

Current practices for the therapy of chondrosarcoma, including wide-margin surgical resection and chemotherapy, are less than satisfactory. Recently, emerging evidence has demonstrated that long non-coding RNAs (lncRNAs) have an essential role in the initiation and progression of tumors. As a typical lncRNA, HOTAIR is significantly overexpressed in various tumors. However, the function and potential biological mechanisms of HOTAIR in human chondrosarcoma remain unknown. Quantitative RT-PCR demonstrated that HOTAIR expression was upregulated in chondrosarcoma tissues and cell lines. High HOTAIR expression is correlated with tumor stage and poor prognosis. Functional experiments reveal that HOTAIR knockdown leads to growth inhibition of human chondrosarcoma cells in vitro and in vivo. In addition to cycle arrest and apoptosis, knockdown of HOTAIR inhibits autophagy, which favors cell death. Mechanistically, we demonstrated that HOTAIR induced DNA methylation of miR-454-3p by recruiting EZH2 and DNMT1 to the miR-454-3p promoter regions, which markedly silences miR-454-3p expression. Further analysis revealed that STAT3 and ATG12 are targets of miR-454-3p, initiate HOTAIR deficiency-induced apoptosis and reduce autophagy. Collectively, our data reveal the roles and functional mechanisms of HOTAIR in human chondrosarcoma and suggest that HOTAIR may act as a prognostic biomarker and potential therapeutic target for chondrosarcoma.

Translation efficiency is determined by both codon bias and folding energy

PubMed Central

Tuller, Tamir; Waldman, Yedael Y.; Kupiec, Martin; Ruppin, Eytan

2010-01-01

Synonymous mutations do not alter the protein produced yet can have a significant effect on protein levels. The mechanisms by which this effect is achieved are controversial; although some previous studies have suggested that codon bias is the most important determinant of translation efficiency, a recent study suggested that mRNA folding at the beginning of genes is the dominant factor via its effect on translation initiation. Using the Escherichia coli and Saccharomyces cerevisiae transcriptomes, we conducted a genome-scale study aiming at dissecting the determinants of translation efficiency. There is a significant association between codon bias and translation efficiency across all endogenous genes in E. coli and S. cerevisiae but no association between folding energy and translation efficiency, demonstrating the role of codon bias as an important determinant of translation efficiency. However, folding energy does modulate the strength of association between codon bias and translation efficiency, which is maximized at very weak mRNA folding (i.e., high folding energy) levels. We find a strong correlation between the genomic profiles of ribosomal density and genomic profiles of folding energy across mRNA, suggesting that lower folding energies slow down the ribosomes and decrease translation efficiency. Accordingly, we find that selection forces act near uniformly to decrease the folding energy at the beginning of genes. In summary, these findings testify that in endogenous genes, folding energy affects translation efficiency in a global manner that is not related to the expression levels of individual genes, and thus cannot be detected by correlation with their expression levels. PMID:20133581

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

PubMed Central

Llácer, Jose L.; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

2015-01-01

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition. PMID:26212456

Phototrophy and starvation-based induction of autophagy upon removal of Gcn5-catalyzed acetylation of Atg7 in Magnaporthe oryzae.

PubMed

Zhang, Shulin; Liang, Meiling; Naqvi, Naweed I; Lin, Chaoxiang; Qian, Wanqiang; Zhang, Lian-Hui; Deng, Yi Zhen

2017-08-03

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.

Brain cDNA clone for human cholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

McTiernan, C.; Adkins, S.; Chatonnet, A.

1987-10-01

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum.more » The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase.« less

A comprehensive, genome-wide analysis of autophagy-related genes identified in tobacco suggests a central role of autophagy in plant response to various environmental cues

PubMed Central

Zhou, Xue-mei; Zhao, Peng; Wang, Wei; Zou, Jie; Cheng, Tian-he; Peng, Xiong-bo; Sun, Meng-xiang

2015-01-01

Autophagy is an evolutionarily conserved mechanism in both animals and plants, which has been shown to be involved in various essential developmental processes in plants. Nicotiana tabacum is considered to be an ideal model plant and has been widely used for the study of the roles of autophagy in the processes of plant development and in the response to various stresses. However, only a few autophagy-related genes (ATGs) have been identified in tobacco up to now. Here, we identified 30 ATGs belonging to 16 different groups in tobacco through a genome-wide survey. Comprehensive expression profile analysis reveals an abroad expression pattern of these ATGs, which could be detected in all tissues tested under normal growth conditions. Our series tests further reveal that majority of ATGs are sensitive and responsive to different stresses including nutrient starvation, plant hormones, heavy metal and other abiotic stresses, suggesting a central role of autophagy, likely as an effector, in plant response to various environmental cues. This work offers a detailed survey of all ATGs in tobacco and also suggests manifold functions of autophagy in both normal plant growth and plant response to environmental stresses. PMID:26205094

Elevation of autophagy markers in Sjögren syndrome dry eye.

PubMed

Byun, Yong-Soo; Lee, Hyun Jung; Shin, Soojung; Chung, So-Hyang

2017-12-08

Autophagy is known to be implicated in the pathogenesis of Sjögren syndrome (SS), but evidences are limited. We aimed to examine the levels of autophagy markers in tear film and conjunctival epithelial cells from SS dry eye patients, and analyze their correlations with clinical features. Patients with SS dry eye exhibited lower Schirmer values, lower tear breakup time, and higher ocular staining scores. In tears, ATG5 and LC3B-II/I levels were significantly higher in SS dry eye. ATG5 and LC3B-II mRNA in the conjunctiva were also elevated in SS dry eye compared with non-SS dry eye. The immunostaining of conjunctival epithelium showed a punctate pattern of ATG5 and LC3B-II in SS dry eye. These staining patterns were also observed in the lacrimal gland of SS animal models. ATG5 levels in tears and the conjunctival epithelium strongly correlated with ocular staining scores, and one month of topical corticosteroid treatment reduced both ATG5 and LC3B-II/I levels in tear film and the conjunctival epithelium of patients with SS dry eye. Our results suggest that autophagy is enhanced or dysregulated in SS and autophagy markers may be serve as both diagnostic and therapeutic biomarkers in SS dry eye.

Meta-analysis of treatment with rabbit and horse antithymocyte globulin for aplastic anemia.

PubMed

Hayakawa, Jin; Kanda, Junya; Akahoshi, Yu; Harada, Naonori; Kameda, Kazuaki; Ugai, Tomotaka; Wada, Hidenori; Ishihara, Yuko; Kawamura, Koji; Sakamoto, Kana; Ashizawa, Masahiro; Sato, Miki; Terasako-Saito, Kiriko; Kimura, Shun-Ichi; Kikuchi, Misato; Yamazaki, Rie; Kako, Shinichi; Kanda, Yoshinobu

2017-05-01

Aplastic anemia patients who received rabbit antithymocyte globulin exhibited response and survival rates inferior to those who received horse antithymocyte globulin in several studies. Therefore, we conducted a meta-analysis to compare rabbit and horse antithymocyte globulin as immunosuppressive therapy for aplastic anemia. We searched online databases for studies that compared antithymocyte globulin regimens as first-line treatment for aplastic anemia, including both randomized and non-randomized controlled trials. The early mortality rate at 3 months and overall response rate at 6 months were evaluated. Thirteen studies were included in the analysis. The risk ratio (RR) of early mortality for rabbit vs. horse antithymocyte globulin was 1.33 [95% confidence interval (CI) 0.69-2.57; P = 0.39], with significant heterogeneity. A sensitivity analysis suggested higher early mortality rate in patients who received rabbit antithymocyte globulin. The overall response rate was significantly higher in patients who received horse antithymocyte globulin (RR 1.27; 95% CI 1.05-1.54; P = 0.015). In conclusion, in aplastic anemia patients treated with ATG, early mortality rate was not significantly different in patients receiving horse or rabbit ATG, although a sensitivity analysis showed higher early mortality in the rabbit ATG group. Horse ATG was associated with significantly higher response rate than rabbit ATG.

A review of the evidence for use of thymoglobulin induction in renal transplantation.

PubMed

Gaber, A Osama; Knight, R J; Patel, S; Gaber, L W

2010-06-01

Depleting antilymphocyte, or antithymocyte antibodies, have long been an integral part of induction regimens and continue today to be used in the management of patients at risk of early rejection or those in whom the introduction of calcineurins or other immune suppressants must be delayed. Registry data demonstrate that the most commonly used depleting antibody, rabbit anti-human thymocyte globulin (rATG), is associated with improved outcomes following renal transplantation in high-risk patients, particularly in conjunction with steroid-avoidance regimens. Two prospective randomized trials in high-risk renal allograft patients have also demonstrated an advantage of r-ATG induction compared to the nondepleting interleukin receptor (IL2RA) antibodies. In low-immunologic-risk patients, however, r-ATG induction and IL2RA induction appear to be equivalent in terms of rejection prophylaxis and long-term function. Other studies have shown that sequential rATG-containing regimens were superior to no induction and allowed for successful late introduction of calcineurin inhibitors. The side effect profile of the depleting antibody included increased incidence of fever, hematologic abnormalities, cytomegalovirus infections when prophylaxis was not employed, and in some studies, increased incidence of posttransplant lymphoproliferative disease. This review describes the evidence supporting the use of depleting ATGs in kidney transplantation.

Analysis of synonymous codon usage patterns in the genus Rhizobium.

PubMed

Wang, Xinxin; Wu, Liang; Zhou, Ping; Zhu, Shengfeng; An, Wei; Chen, Yu; Zhao, Lin

2013-11-01

The codon usage patterns of rhizobia have received increasing attention. However, little information is available regarding the conserved features of the codon usage patterns in a typical rhizobial genus. The codon usage patterns of six completely sequenced strains belonging to the genus Rhizobium were analysed as model rhizobia in the present study. The relative neutrality plot showed that selection pressure played a role in codon usage in the genus Rhizobium. Spearman's rank correlation analysis combined with correspondence analysis (COA) showed that the codon adaptation index and the effective number of codons (ENC) had strong correlation with the first axis of the COA, which indicated the important role of gene expression level and the ENC in the codon usage patterns in this genus. The relative synonymous codon usage of Cys codons had the strongest correlation with the second axis of the COA. Accordingly, the usage of Cys codons was another important factor that shaped the codon usage patterns in Rhizobium genomes and was a conserved feature of the genus. Moreover, the comparison of codon usage between highly and lowly expressed genes showed that 20 unique preferred codons were shared among Rhizobium genomes, revealing another conserved feature of the genus. This is the first report of the codon usage patterns in the genus Rhizobium.

Genome-wide comparative analysis of codon usage bias and codon context patterns among cyanobacterial genomes.

PubMed

Prabha, Ratna; Singh, Dhananjaya P; Sinha, Swati; Ahmad, Khurshid; Rai, Anil

2017-04-01

With the increasing accumulation of genomic sequence information of prokaryotes, the study of codon usage bias has gained renewed attention. The purpose of this study was to examine codon selection pattern within and across cyanobacterial species belonging to diverse taxonomic orders and habitats. We performed detailed comparative analysis of cyanobacterial genomes with respect to codon bias. Our analysis reflects that in cyanobacterial genomes, A- and/or T-ending codons were used predominantly in the genes whereas G- and/or C-ending codons were largely avoided. Variation in the codon context usage of cyanobacterial genes corresponded to the clustering of cyanobacteria as per their GC content. Analysis of codon adaptation index (CAI) and synonymous codon usage order (SCUO) revealed that majority of genes are associated with low codon bias. Codon selection pattern in cyanobacterial genomes reflected compositional constraints as major influencing factor. It is also identified that although, mutational constraint may play some role in affecting codon usage bias in cyanobacteria, compositional constraint in terms of genomic GC composition coupled with environmental factors affected codon selection pattern in cyanobacterial genomes. Copyright © 2016 Elsevier B.V. All rights reserved.

Codon usage bias in prokaryotic pyrimidine-ending codons is associated with the degeneracy of the encoded amino acids

PubMed Central

Wald, Naama; Alroy, Maya; Botzman, Maya; Margalit, Hanah

2012-01-01

Synonymous codons are unevenly distributed among genes, a phenomenon termed codon usage bias. Understanding the patterns of codon bias and the forces shaping them is a major step towards elucidating the adaptive advantage codon choice can confer at the level of individual genes and organisms. Here, we perform a large-scale analysis to assess codon usage bias pattern of pyrimidine-ending codons in highly expressed genes in prokaryotes. We find a bias pattern linked to the degeneracy of the encoded amino acid. Specifically, we show that codon-pairs that encode two- and three-fold degenerate amino acids are biased towards the C-ending codon while codons encoding four-fold degenerate amino acids are biased towards the U-ending codon. This codon usage pattern is widespread in prokaryotes, and its strength is correlated with translational selection both within and between organisms. We show that this bias is associated with an improved correspondence with the tRNA pool, avoidance of mis-incorporation errors during translation and moderate stability of codon–anticodon interaction, all consistent with more efficient translation. PMID:22581775

Induction treatment with rabbit antithymocyte globulin versus basiliximab in renal transplant recipients with planned early steroid withdrawal.

PubMed

Martin, Spencer T; Roberts, Keri L; Malek, Sayeed K; Tullius, Stefan G; Vadivel, Nidyanandh; De Serres, Sacha; Grafals, Monica; Elsanjak, Abdelaziz; Filkins, Beth Anne; Chandraker, Anil; Gabardi, Steven

2011-06-01

To compare the safety and efficacy of rabbit antithymocyte globulin (r-ATG) with basiliximab in renal transplant recipients for whom an early steroid withdrawal (ESW) regimen was planned. Single-center, retrospective, cohort study. Tertiary care medical center, including inpatient hospital stays and outpatient nephrology clinics. Ninety-nine consecutive adult recipients of living- or deceased-donor renal transplants between January 1, 2004, and December 31, 2007, in whom ESW was planned and who received either r-ATG or basiliximab; patients receiving an extended-criteria kidney donation or a donation after cardiac death were excluded. All patients received mycophenolate mofetil and tacrolimus as maintenance therapy with planned ESW. Induction therapy was either r-ATG 1.5 mg/kg/day for 4 days (68 patients) or basiliximab 20 mg on postoperative days 0 and 4 (31 patients). The primary composite end point of biopsy-proven acute rejection (BPAR), graft loss, and death occurred in 6 patients (9%) and 9 patients (29%) in the r-ATG and basiliximab groups at 1 year after transplantation, respectively (p=0.01), with rates of 7% (5/68 patients) and 26% (8/31 patients) for BPAR (p=0.02), 0% and 3% (1/31 patients) for graft loss (p=0.31), and 2% (1/68 patients) and 0% for patient death (p>0.99). Average time to first BPAR was significantly longer in the r-ATG group (mean ± SD 151.4 ± 82.9 vs 53.6 ± 68.4 days, p<0.01). Kidney function at 12 months was similar between the two groups. Rabbit-ATG was associated with a lower frequency and delayed onset of BPAR compared with basiliximab in renal transplant recipients who received an ESW regimen.

Nationwide survey on the use of horse antithymocyte globulins (ATGAM) in patients with acquired aplastic anemia: A report on behalf of the French Reference Center for Aplastic Anemia.

PubMed

Peffault de Latour, Régis; Tabrizi, Reza; Marcais, Ambroise; Leblanc, Thierry; Lamy, Thierry; Mohty, Mohamad; Tavitian, Suzanne; Jubert, Charlotte; Pasquet, Marlène; Galambrun, Claire; Nguyen, Stéphanie; Cahn, Jean Yves; Braun, Thorsten; Deconinck, Eric; Bay, Jacques Olivier; Sicre de Fontbrune, Flore; Barraco, Fiorenza; Socié, Gérard

2018-05-01

Antithymocyte globulins (ATG) plus cyclosporine (CSA) is the gold standard immunosuppressive treatment (IST) for patients with aplastic anemia. A prospective randomized trial showed in 2011 that hATG was superior to rabbit ATG for first-line treatment of severe AA. The French Health Agency (ANSM) permitted a patient-named authorization for temporary use (ATU) program of hATG (ATGAM, Pfizer) in patients with AA in 2011 since commercial access to hATG is not approved. We took advantage of this program to analyze the outcomes of 465 patients who received antithymocyte globulins (ATGAM) plus CSA as first line treatment (n = 379; 81.5%), or for refractory (n = 26) or relapsed disease (n = 33), from September 2011 to March 2017. In the entire cohort one year, 72% of the patients had partial and 13% had complete response, with worse response for patients with severe AA and a longer interval between diagnosis and IST (more than 6 months). Severe adverse events were mainly linked to infections (24%), hemorrhages (6%), and elevated liver function tests (5%). Overall at 12 months, 9.7% of patients required second line IST and 15.6% received transplantation. Fifty-five patients died during the study mainly because of infections (53%). Factors predicting independently worse survival were age over 40 years, neutrophils less than 0.5 × 10 9 /L, male gender and longer delay between diagnosis and hATG (>6 months period). This study does illustrate the results of ATGAM with CSA in a true-life perspective and confirms ATGAM as standard of care IST to treat patients with AA not eligible for HSCT. © 2018 Wiley Periodicals, Inc.

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells.

PubMed

Hanamata, Shigeru; Kurusu, Takamitsu; Okada, Masaaki; Suda, Akiko; Kawamura, Koki; Tsukada, Emi; Kuchitsu, Kazuyuki

2013-01-01

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli.

In vivo imaging and quantitative monitoring of autophagic flux in tobacco BY-2 cells

PubMed Central

Hanamata, Shigeru; Kurusu, Takamitsu; Okada, Masaaki; Suda, Akiko; Kawamura, Koki; Tsukada, Emi; Kuchitsu, Kazuyuki

2013-01-01

Autophagy has been shown to play essential roles in the growth, development and survival of eukaryotic cells. However, simple methods for quantification and visualization of autophagic flux remain to be developed in living plant cells. Here, we analyzed the autophagic flux in transgenic tobacco BY-2 cell lines expressing fluorescence-tagged NtATG8a as a marker for autophagosome formation. Under sucrose-starved conditions, the number of punctate signals of YFP-NtATG8a increased, and the fluorescence intensity of the cytoplasm and nucleoplasm decreased. Conversely, these changes were not observed in BY-2 cells expressing a C-terminal glycine deletion mutant of the NtATG8a protein (NtATG8aΔG). To monitor the autophagic flux more easily, we generated a transgenic BY-2 cell line expressing NtATG8a fused to a pH-sensitive fluorescent tag, a tandem fusion of the acid-insensitive RFP and the acid-sensitive YFP. In sucrose-rich conditions, both fluorescent signals were detected in the cytoplasm and only weakly in the vacuole. In contrast, under sucrose-starved conditions, the fluorescence intensity of the cytoplasm decreased, and the RFP signal clearly increased in the vacuole, corresponding to the fusion of the autophagosome to the vacuole and translocation of ATG8 from the cytoplasm to the vacuole. Moreover, we introduce a novel simple easy way to monitor the autophagic flux non-invasively by only measuring the ratio of fluorescence of RFP and YFP in the cell suspension using a fluorescent image analyzer without microscopy. The present in vivo quantitative monitoring system for the autophagic flux offers a powerful tool for determining the physiological functions and molecular mechanisms of plant autophagy induced by environmental stimuli. PMID:23123450

TOR signaling pathway and autophagy are involved in the regulation of circadian rhythms in behavior and plasticity of L2 interneurons in the brain of Drosophila melanogaster.

PubMed

Kijak, Ewelina; Pyza, Elżbieta

2017-01-01

Drosophila melanogaster is a common model used to study circadian rhythms in behavior and circadian clocks. However, numerous circadian rhythms have also been detected in non-clock neurons, especially in the first optic neuropil (lamina) of the fly's visual system. Such rhythms have been observed in the number of synapses and in the structure of interneurons, which exhibit changes in size and shape in a circadian manner. Although the patterns of these changes are known, the mechanism remains unclear. In the present study, we investigated the role of the TOR signaling pathway and autophagy in regulating circadian rhythms based on the behavior and structural plasticity of the lamina L2 monopolar cell dendritic trees. In addition, we examined the cyclic expression of the TOR signaling pathway (Tor, Pi3K class 1, Akt1) and autophagy (Atg5 and Atg7) genes in the fly's brain. We observed that Tor, Atg5 and Atg7 exhibit rhythmic expressions in the brain of wild-type flies in day/night conditions (LD 12:12) that are abolished in per01 clock mutants. The silencing of Tor in per expressing cells shortens a period of the locomotor activity rhythm of flies. In addition, silencing of the Tor and Atg5 genes in L2 cells disrupts the circadian plasticity of the L2 cell dendritic trees measured in the distal lamina. In turn, silencing of the Atg7 gene in L2 cells changes the pattern of this rhythm. Our results indicate that the TOR signaling pathway and autophagy are involved in the regulation of circadian rhythms in the behavior and plasticity of neurons in the brain of adult flies.

Factors impacting short and long-term kidney graft survival: modification by single intra-operative -high-dose induction with ATG-Fresenius.

PubMed

Kaden, Jürgen; May, Gottfried; Völp, Andreas; Wesslau, Claus

2011-01-01

A majority of recipients benefited from the intra-operative single high-dose induction (HDI) with ATG-Fresenius (ATG-F) still leaving a group of recipients who did not profit from this kind of induction. Therefore the aim of this retrospective analysis was 1st to identify the risk factors impacting short and long-term graft survival, and 2nd to assess the efficacy of this type of induction in kidney graft recipients with or without these risk factors. A total of 606 recipients receiving two different immunosuppressive treatment regimens (1st: Triple drug therapy [TDT, n=196] consisting mainly of steroids, azathioprine and cyclosporine; 2nd: TDT + 9 mg/kg ATG-F intra-operatively [HDI, n=410]) were included in this analysis and grouped according to their kidney graft survival time (short GST: ≤1 yr, n=100 and long GST: >5 yrs, n=506). The main risk factors associated with a shortened graft survival were pre-transplant sensitization, re-transplantation, rejections (in particular vascular or mixed ones) and the necessity of a long-term anti-rejection therapy. Adding ATG-F single high dose induction to TDT was more efficient in prolonging kidney graft survival than TDT alone not only in recipients without any risk factors (p<0.005) but also in recipients with at least one risk factor (p<0.021). Only in 4.6% of recipients having two or more risk factors this effect could not be demonstrated. The intra-operative single high-dose induction with ATG-F significantly improves the kidney graft survival in recipients with or without risk factors and can therefore be recommended.

TOR signaling pathway and autophagy are involved in the regulation of circadian rhythms in behavior and plasticity of L2 interneurons in the brain of Drosophila melanogaster

PubMed Central

Kijak, Ewelina; Pyza, Elżbieta

2017-01-01

Drosophila melanogaster is a common model used to study circadian rhythms in behavior and circadian clocks. However, numerous circadian rhythms have also been detected in non-clock neurons, especially in the first optic neuropil (lamina) of the fly’s visual system. Such rhythms have been observed in the number of synapses and in the structure of interneurons, which exhibit changes in size and shape in a circadian manner. Although the patterns of these changes are known, the mechanism remains unclear. In the present study, we investigated the role of the TOR signaling pathway and autophagy in regulating circadian rhythms based on the behavior and structural plasticity of the lamina L2 monopolar cell dendritic trees. In addition, we examined the cyclic expression of the TOR signaling pathway (Tor, Pi3K class 1, Akt1) and autophagy (Atg5 and Atg7) genes in the fly’s brain. We observed that Tor, Atg5 and Atg7 exhibit rhythmic expressions in the brain of wild-type flies in day/night conditions (LD 12:12) that are abolished in per01 clock mutants. The silencing of Tor in per expressing cells shortens a period of the locomotor activity rhythm of flies. In addition, silencing of the Tor and Atg5 genes in L2 cells disrupts the circadian plasticity of the L2 cell dendritic trees measured in the distal lamina. In turn, silencing of the Atg7 gene in L2 cells changes the pattern of this rhythm. Our results indicate that the TOR signaling pathway and autophagy are involved in the regulation of circadian rhythms in the behavior and plasticity of neurons in the brain of adult flies. PMID:28196106

Poor Patient and Graft Outcome After Induction Treatment by Antithymocyte Globulin in Recipients of a Kidney Graft After Nonrenal Organ Transplantation.

PubMed

Mai, Hoa Le; Treilhaud, Michèle; Ben-Arye, Shani Leviatan; Yu, Hai; Perreault, Hélène; Ang, Evelyn; Trébern-Launay, Katy; Laurent, Julie; Malard-Castagnet, Stéphanie; Cesbron, Anne; Nguyen, Thi Van Ha; Brouard, Sophie; Rostaing, Lionel; Houssel-Debry, Pauline; Legendre, Christophe; Girerd, Sophie; Kessler, Michèle; Morelon, Emmanuel; Sicard, Antoine; Garrigue, Valérie; Karam, Georges; Chen, Xi; Giral, Magali; Padler-Karavani, Vered; Soulillou, Jean Paul

2018-04-01

End-stage renal failure occurs in a substantial number of patients having received a nonrenal transplantation (NRT), for whom a kidney transplantation is needed. The medical strategy regarding the use of immunosuppression (IS) for a kidney graft in patients after an NRT is not well established. The prekidney grafts long-term IS advocates for a mild induction, such as using anti-IL-2R antibodies, whereas addition of new incompatibilities and anti-HLA preimmunization may suggest using stronger IS such as induction by polyclonal antithymocyte globulins (ATG). We performed Cox multivariate and propensity score analysis of our validated transplant database to study the impact of the type of induction therapy on kidney graft survival of recipients of a kidney graft after NRT. We report here that kidney transplantation after NRT treated with an ATG induction has a poorer outcome (kidney and recipient survival) than that with an anti-IL-2R induction. After accounting for potential baseline differences with a multivariate Cox model, or by adjusting on a propensity score, we found that despite patients having received ATG cumulate more risk factors, ATG appears independently involved. As animal-derived biotherapeutics induce antiglycan antibodies and particularly anti-N-glycolylneuraminic acid (Neu5Gc) IgGs which may activate endothelial cells in patients and grafts, we also investigated the magnitude and the nature of the anti-Neu5Gc elicited by the induction and showed that induction was associated with a shift in anti-Neu5Gc IgG repertoire. Possible reasons and mechanisms of a deleterious ATG usage in these patients are discussed. Our study suggests that ATG induction after a kidney transplantation in recipients already under maintenance IS for a NRT should be used cautiously.

Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design

PubMed Central

Villada, Juan C.; Brustolini, Otávio José Bernardes

2017-01-01

Abstract Gene codon optimization may be impaired by the misinterpretation of frequency and optimality of codons. Although recent studies have revealed the effects of codon usage bias (CUB) on protein biosynthesis, an integrated perspective of the biological role of individual codons remains unknown. Unlike other previous studies, we show, through an integrated framework that attributes of codons such as frequency, optimality and positional dependency should be combined to unveil individual codon contribution for protein biosynthesis. We designed a codon quantification method for assessing CUB as a function of position within genes with a novel constraint: the relativity of position-dependent codon usage shaped by coding sequence length. Thus, we propose a new way of identifying the enrichment, depletion and non-uniform positional distribution of codons in different regions of yeast genes. We clustered codons that shared attributes of frequency and optimality. The cluster of non-optimal codons with rare occurrence displayed two remarkable characteristics: higher codon decoding time than frequent–non-optimal cluster and enrichment at the 5′-end region, where optimal codons with the highest frequency are depleted. Interestingly, frequent codons with non-optimal adaptation to tRNAs are uniformly distributed in the Saccharomyces cerevisiae genes, suggesting their determinant role as a speed regulator in protein elongation. PMID:28449100

Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design.

PubMed

Villada, Juan C; Brustolini, Otávio José Bernardes; Batista da Silveira, Wendel

2017-08-01

Gene codon optimization may be impaired by the misinterpretation of frequency and optimality of codons. Although recent studies have revealed the effects of codon usage bias (CUB) on protein biosynthesis, an integrated perspective of the biological role of individual codons remains unknown. Unlike other previous studies, we show, through an integrated framework that attributes of codons such as frequency, optimality and positional dependency should be combined to unveil individual codon contribution for protein biosynthesis. We designed a codon quantification method for assessing CUB as a function of position within genes with a novel constraint: the relativity of position-dependent codon usage shaped by coding sequence length. Thus, we propose a new way of identifying the enrichment, depletion and non-uniform positional distribution of codons in different regions of yeast genes. We clustered codons that shared attributes of frequency and optimality. The cluster of non-optimal codons with rare occurrence displayed two remarkable characteristics: higher codon decoding time than frequent-non-optimal cluster and enrichment at the 5'-end region, where optimal codons with the highest frequency are depleted. Interestingly, frequent codons with non-optimal adaptation to tRNAs are uniformly distributed in the Saccharomyces cerevisiae genes, suggesting their determinant role as a speed regulator in protein elongation. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

RIP3 attenuates the pancreatic damage induced by deletion of ATG7.

PubMed

Zhou, Xiaodong; Xie, Li; Xia, Leizhou; Bergmann, Frank; Büchler, Markus W; Kroemer, Guido; Hackert, Thilo; Fortunato, Franco

2017-07-13

Invalidation of pancreatic autophagy entails pancreatic atrophy, endocrine and exocrine insufficiency and pancreatitis. The aim of this study was to investigate whether depletion of Rip3, which is involved in necroptotic signaling, may attenuate the pancreatic atrophy and pancreatitis resulting from autophagy inhibition. Autophagy and necroptosis signaling were evaluated in mice lacking expression of Rip3 in all organs and Atg7 in the pancreas. Acinar cell death, inflammation and fibrosis were evaluated by using of a compendium of immunofluorescence methods and immunoblots. Mice deficient for pancreatic Atg7 developed acute pancreatitis, which progressed to chronic pancreatitis. This phenotype reduces autophagy, increase apoptosis and necroptosis, inflammation and fibrosis, as well as premature death of the animals. Knockout of Rip3 exacerbated the apoptotic death of acinar cells, increased tissue damage, reduced macrophage infiltration and further accelerated the death of the mice with Atg7-deficient pancreas. The pancreatic degeneration induced by autophagy inhibition was exacerbated by Rip3 deletion.

Cytoplastic Glyceraldehyde-3-Phosphate Dehydrogenases Interact with ATG3 to Negatively Regulate Autophagy and Immunity in Nicotiana benthamiana

PubMed Central

Han, Shaojie; Wang, Yan; Zheng, Xiyin; Jia, Qi; Zhao, Jinping; Bai, Fan; Hong, Yiguo; Liu, Yule

2015-01-01

Autophagy as a conserved catabolic pathway can respond to reactive oxygen species (ROS) and plays an important role in degrading oxidized proteins in plants under various stress conditions. However, how ROS regulates autophagy in response to oxidative stresses is largely unknown. Here, we show that autophagy-related protein 3 (ATG3) interacts with the cytosolic glyceraldehyde-3-phosphate dehydrogenases (GAPCs) to regulate autophagy in Nicotiana benthamiana plants. We found that oxidative stress inhibits the interaction of ATG3 with GAPCs. Silencing of GAPCs significantly activates ATG3-dependent autophagy, while overexpression of GAPCs suppresses autophagy in N. benthamiana plants. Moreover, silencing of GAPCs enhances N gene-mediated cell death and plant resistance against both incompatible pathogens Tobacco mosaic virus and Pseudomonas syringae pv tomato DC3000, as well as compatible pathogen P. syringae pv tabaci. These results indicate that GAPCs have multiple functions in the regulation of autophagy, hypersensitive response, and plant innate immunity. PMID:25829441

ATG16L1: A multifunctional susceptibility factor in Crohn disease

PubMed Central

Salem, Mohammad; Ammitzboell, Mette; Nys, Kris; Seidelin, Jakob Benedict; Nielsen, Ole Haagen

2015-01-01

Genetic variations in the autophagic pathway influence genetic predispositions to Crohn disease. Autophagy, the major lysosomal pathway for degrading and recycling cytoplasmic material, constitutes an important homeostatic cellular process. Of interest, single-nucleotide polymorphisms in ATG16L1 (autophagy-related 16-like 1 [S. cerevisiae]), a key component in the autophagic response to invading pathogens, have been associated with an increased risk of developing Crohn disease. The most common and well-studied genetic variant of ATG16L1 (rs2241880; leading to a T300A conversion) exhibits a strong association with risk for developing Crohn disease. The rs2241880 variant plays a crucial role in pathogen clearance, resulting in imbalanced cytokine production, and is linked to other biological processes, such as the endoplasmic reticulum stress/unfolded protein response. In this review, we focus on the importance of ATG16L1 and its genetic variant (T300A) within the elementary biological processes linked to Crohn disease. PMID:25906181

ATG16L1: A multifunctional susceptibility factor in Crohn disease.

PubMed

Salem, Mohammad; Ammitzboell, Mette; Nys, Kris; Seidelin, Jakob Benedict; Nielsen, Ole Haagen

2015-04-03

Genetic variations in the autophagic pathway influence genetic predispositions to Crohn disease. Autophagy, the major lysosomal pathway for degrading and recycling cytoplasmic material, constitutes an important homeostatic cellular process. Of interest, single-nucleotide polymorphisms in ATG16L1 (autophagy-related 16-like 1 [S. cerevisiae]), a key component in the autophagic response to invading pathogens, have been associated with an increased risk of developing Crohn disease. The most common and well-studied genetic variant of ATG16L1 (rs2241880; leading to a T300A conversion) exhibits a strong association with risk for developing Crohn disease. The rs2241880 variant plays a crucial role in pathogen clearance, resulting in imbalanced cytokine production, and is linked to other biological processes, such as the endoplasmic reticulum stress/unfolded protein response. In this review, we focus on the importance of ATG16L1 and its genetic variant (T300A) within the elementary biological processes linked to Crohn disease.

APF lncRNA regulates autophagy and myocardial infarction by targeting miR-188-3p.

PubMed

Wang, Kun; Liu, Cui-Yun; Zhou, Lu-Yu; Wang, Jian-Xun; Wang, Man; Zhao, Bing; Zhao, Wen-Ke; Xu, Shi-Jun; Fan, Li-Hua; Zhang, Xiao-Jie; Feng, Chang; Wang, Chao-Qun; Zhao, Yan-Fang; Li, Pei-Feng

2015-04-10

The abnormal autophagy is associated with a variety of cardiovascular diseases. Long noncoding RNAs (lncRNAs) are emerging as new factors in gene regulation, but how lncRNAs operate in the regulation of autophagy in the heart is unclear. Here we report that a long noncoding RNA, named autophagy promoting factor (APF), can regulate autophagic cell death by targeting miR-188-3p and ATG7. The results show that miR-188-3p suppresses autophagy and myocardial infarction by targeting ATG7. Further, we find that APF lncRNA regulates miR-188-3p, and thus affects ATG7 expression, autophagic cell death and myocardial infarction. Our present study reveals a novel regulating model of autophagic programme, which comprises APF, miR-188-3p and ATG7 in the heart. Modulation of their levels may serve as potential targets and diagnostic tools for novel therapeutic strategies of myocardial infarction and heart failure.

Posttranslational modification of autophagy-related proteins in macroautophagy

PubMed Central

Xie, Yangchun; Kang, Rui; Sun, Xiaofang; Zhong, Meizuo; Huang, Jin; Klionsky, Daniel J.; Tang, Daolin

2014-01-01

Macroautophagy is an intracellular catabolic process involved in the formation of multiple membrane structures ranging from phagophores to autophagosomes and autolysosomes. Dysfunction of macroautophagy is implicated in both physiological and pathological conditions. To date, 38 autophagy-related (ATG) genes have been identified as controlling these complicated membrane dynamics during macroautophagy in yeast; approximately half of these genes are clearly conserved up to human, and there are additional genes whose products function in autophagy in higher eukaryotes that are not found in yeast. The function of the ATG proteins, in particular their ability to interact with a number of macroautophagic regulators, is modulated by posttranslational modifications (PTMs) such as phosphorylation, glycosylation, ubiquitination, acetylation, lipidation, and proteolysis. In this review, we summarize our current knowledge of the role of ATG protein PTMs and their functional relevance in macroautophagy. Unraveling how these PTMs regulate ATG protein function during macroautophagy will not only reveal fundamental mechanistic insights into the regulatory process, but also provide new therapeutic targets for the treatment of autophagy-associated diseases. PMID:25484070

Yin and Yang of Heparanase in Breast Tumor Initiation

DTIC Science & Technology

2013-04-01

of heparanase in cancer metastasis and angiogenesis. Int J Biochem Cell Biol 38: 2018 -2039, 2006 2. Gotte M, Yip GW: Heparanase, hyaluronan, and...into a RCAS vector digested with a PacI and Cla I. The plasmid was designated RCAS-C. An oligonucleotide containing a Myc tag sequence (atg gaa...ligated into Not I/PacI- digested RCAS-C. The following plasmid designated as RCAS-8C was used to transfected DF-1 cells to generate RCAS-8C virus

Partial attenuation of Marek's disease virus by manipulation of Di-codon bias

USDA-ARS?s Scientific Manuscript database

All species studied to date demonstrate a preference for certain codons over other synonymous codons (codon bias), a preference which is also observed for pairs of codons (di-codon bias). Previous studies using poliovirus and influenza virus as models have demonstrated the ability to cause attenuat...

Interaction of E3 Ubiquitin Ligase MARCH7 with Long Noncoding RNA MALAT1 and Autophagy-Related Protein ATG7 Promotes Autophagy and Invasion in Ovarian Cancer.

PubMed

Hu, Jianguo; Zhang, Luo; Mei, Zhiqiang; Jiang, Yuan; Yi, Yuan; Liu, Li; Meng, Ying; Zhou, Lili; Zeng, Jianhua; Wu, Huan; Jiang, Xingwei

2018-05-22

Ubiquitin E3 ligase MARCH7 plays an important role in T cell proliferation and neuronal development. But its role in ovarian cancer remains unclear. This study aimed to investigate the role of Ubiquitin E3 ligase MARCH7 in ovarian cancer. Real-time PCR, immunohistochemistry and western blotting analysis were performed to determine the expression of MARCH7, MALAT1 and ATG7 in ovarian cancer cell lines and clinical specimens. The role of MARCH7 in maintaining ovarian cancer malignant phenotype was examined by Wound healing assay, Matrigel invasion assays and Mouse orthotopic xenograft model. Luciferase reporter assay, western blot analysis and ChIP assay were used to determine whether MARCH7 activates TGF-β-smad2/3 pathway by interacting with TGFβR2. MARCH7 interacted with MALAT1 by miR-200a (microRNA-200a). MARCH7 may function as a competing endogenous RNA (ceRNA) to regulate the expression of ATG7 by competing with miR-200a. MARCH7 regulated TGF-β-smad2/3 pathway by interacting with TGFβR2. Inhibition of TGF-β-smad2/3 pathway downregulated MARCH7, MALAT1 and ATG7. MiR-200a regulated TGF-β induced autophagy, invasion and metastasis of SKOV3 cells by targeting MARCH7. MARCH7 silencing inhibited autophagy invasion and metastasis of SKOV3 cells both in vitro and in vivo. In contrast, MARCH7 overexpression promoted TGF-β induced autophagy, invasion and metastasis of A2780 cells in vitro by depending on MALAT1 and ATG7. We also found that TGF-β-smad2/3 pathway regulated MARCH7 and ATG7 through MALAT1. These findings suggested that TGFβR2-Smad2/3-MALAT1/MARCH7/ATG7 feedback loop mediated autophagy, migration and invasion in ovarian cancer. © 2018 The Author(s). Published by S. Karger AG, Basel.

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards "GC" Rich Codons.

PubMed

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-04-27

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen "core" dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression.

Polyclonal and monoclonal antibodies for induction therapy in kidney transplant recipients.

PubMed

Hill, Penny; Cross, Nicholas B; Barnett, A Nicholas R; Palmer, Suetonia C; Webster, Angela C

2017-01-11

Prolonging kidney transplant survival is an important clinical priority. Induction immunosuppression with antibody therapy is recommended at transplantation and non-depleting interleukin-2 receptor monoclonal antibodies (IL2Ra) are considered first line. It is suggested that recipients at high risk of rejection should receive lymphocyte-depleting antibodies but the relative benefits and harms of the available agents are uncertain. We aimed to: evaluate the relative and absolute effects of different antibody preparations (except IL2Ra) when used as induction therapy in kidney transplant recipients; determine how the benefits and adverse events vary for each antibody preparation; determine how the benefits and harms vary for different formulations of antibody preparation; and determine whether the benefits and harms vary in specific subgroups of recipients (e.g. children and sensitised recipients). Randomised controlled trials (RCTs) comparing monoclonal or polyclonal antibodies with placebo, no treatment, or other antibody therapy in adults and children who had received a kidney transplant. Randomised controlled trials (RCTs) comparing monoclonal or polyclonal antibodies with placebo, no treatment, or other antibody therapy in adults and children who had received a kidney transplant. Two authors independently extracted data and assessed risk of bias. Dichotomous outcomes are reported as relative risk (RR) and continuous outcomes as mean difference (MD) together with their 95% confidence intervals (CI). We included 99 studies (269 records; 8956 participants; 33 with contemporary agents). Methodology was incompletely reported in most studies leading to lower confidence in the treatment estimates.Antithymocyte globulin (ATG) prevented acute graft rejection (17 studies: RR 0.63, 95% CI 0.51 to 0.78). The benefits of ATG on graft rejection were similar when used with (12 studies: RR 0.61, 0.49 to 0.76) or without (5 studies: RR 0.65, 0.43 to 0.98) calcineurin inhibitor (CNI) treatment. ATG (with CNI therapy) had uncertain effects on death (3 to 6 months, 3 studies: RR 0.41, 0.13 to 1.22; 1 to 2 years, 5 studies: RR 0.75, 0.27 to 2.06; 5 years, 2 studies: RR 0.94, 0.11 to 7.81) and graft loss (3 to 6 months, 4 studies: RR 0.60, 0.34 to 1.05; 1 to 2 years, 3 studies: RR 0.65, 0.36 to 1.19). The effect of ATG on death-censored graft loss was uncertain at 1 to 2 years and 5 years. In non-CNI studies, ATG had uncertain effects on death but reduced death-censored graft loss (6 studies: RR 0.55, 0.38 to 0.78). When CNI and older non-CNI studies were combined, a benefit was seen with ATG at 1 to 2 years for both all-cause graft loss (7 studies: RR 0.71, 0.53 to 0.95) and death-censored graft loss (8 studies: RR 0.55, 0.39 to 0.77) but not sustained longer term. ATG increased cytomegalovirus (CMV) infection (6 studies: RR 1.55, 1.24 to 1.95), leucopenia (4 studies: RR 3.86, 2.79 to 5.34) and thrombocytopenia (4 studies: RR 2.41, 1.61 to 3.61) but had uncertain effects on delayed graft function, malignancy, post-transplant lymphoproliferative disorder (PTLD), and new onset diabetes after transplantation (NODAT).Alemtuzumab was compared to ATG in six studies (446 patients) with early steroid withdrawal (ESW) or steroid minimisation. Alemtuzumab plus steroid minimisation reduced acute rejection compared to ATG at one year (4 studies: RR 0.57, 0.35 to 0.93). In the two studies with ESW only in the alemtuzumab arm, the effect of alemtuzumab on acute rejection at 1 year was uncertain compared to ATG (RR 1.27, 0.50 to 3.19). Alemtuzumab had uncertain effects on death (1 year, 2 studies: RR 0.39, 0.06 to 2.42; 2 to 3 years, 3 studies: RR 0.67, 95% CI 0.15 to 2.95), graft loss (1 year, 2 studies: RR 0.39, 0.13 to 1.30; 2 to 3 years, 3 studies: RR 0.98, 95% CI 0.47 to 2.06), and death-censored graft loss (1 year, 2 studies: RR 0.38, 0.08 to 1.81; 2 to 3 years, 3 studies: RR 2.45, 95% CI 0.67 to 8.97) compared to ATG. Creatinine clearance was lower with alemtuzumab plus ESW at 6 months (2 studies: MD -13.35 mL/min, -23.91 to -2.80) and 2 years (2 studies: MD -12.86 mL/min, -23.73 to -2.00) compared to ATG plus triple maintenance. Across all 6 studies, the effect of alemtuzumab versus ATG was uncertain on all-cause infection, CMV infection, BK virus infection, malignancy, and PTLD. The effect of alemtuzumab with steroid minimisation on NODAT was uncertain, compared to ATG with steroid maintenance.Alemtuzumab plus ESW compared with triple maintenance without induction therapy had uncertain effects on death and all-cause graft loss at 1 year, acute rejection at 6 months and 1 year. CMV infection was increased (2 studies: RR 2.28, 1.18 to 4.40). Treatment effects were uncertain for NODAT, thrombocytopenia, and malignancy or PTLD.Rituximab had uncertain effects on death, graft loss, acute rejection and all other adverse outcomes compared to placebo. ATG reduces acute rejection but has uncertain effects on death, graft survival, malignancy and NODAT, and increases CMV infection, thrombocytopenia and leucopenia. Given a 45% acute rejection risk without ATG induction, seven patients would need treatment to prevent one having rejection, while incurring an additional patient experiencing CMV disease for every 12 treated. Excluding non-CNI studies, the risk of rejection was 37% without induction with six patients needing treatment to prevent one having rejection.In the context of steroid minimisation, alemtuzumab prevents acute rejection at 1 year compared to ATG. Eleven patients would require treatment with alemtuzumab to prevent 1 having rejection, assuming a 21% rejection risk with ATG.Triple maintenance without induction therapy compared to alemtuzumab combined with ESW had similar rates of acute rejection but adverse effects including NODAT were poorly documented. Alemtuzumab plus steroid withdrawal would cause one additional patient experiencing CMV disease for every six patients treated compared to no induction and triple maintenance, in the absence of any clinical benefit. Overall, ATG and alemtuzumab decrease acute rejection at a cost of increased CMV disease while patient-centred outcomes (reduced death or lower toxicity) do not appear to be improved.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome.

PubMed

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-02-24

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts.

Multiple Evolutionary Selections Involved in Synonymous Codon Usages in the Streptococcus agalactiae Genome

PubMed Central

Ma, Yan-Ping; Ke, Hao; Liang, Zhi-Ling; Liu, Zhen-Xing; Hao, Le; Ma, Jiang-Yao; Li, Yu-Gu

2016-01-01

Streptococcus agalactiae is an important human and animal pathogen. To better understand the genetic features and evolution of S. agalactiae, multiple factors influencing synonymous codon usage patterns in S. agalactiae were analyzed in this study. A- and U-ending rich codons were used in S. agalactiae function genes through the overall codon usage analysis, indicating that Adenine (A)/Thymine (T) compositional constraints might contribute an important role to the synonymous codon usage pattern. The GC3% against the effective number of codon (ENC) value suggested that translational selection was the important factor for codon bias in the microorganism. Principal component analysis (PCA) showed that (i) mutational pressure was the most important factor in shaping codon usage of all open reading frames (ORFs) in the S. agalactiae genome; (ii) strand specific mutational bias was not capable of influencing the codon usage bias in the leading and lagging strands; and (iii) gene length was not the important factor in synonymous codon usage pattern in this organism. Additionally, the high correlation between tRNA adaptation index (tAI) value and codon adaptation index (CAI), frequency of optimal codons (Fop) value, reinforced the role of natural selection for efficient translation in S. agalactiae. Comparison of synonymous codon usage pattern between S. agalactiae and susceptible hosts (human and tilapia) showed that synonymous codon usage of S. agalactiae was independent of the synonymous codon usage of susceptible hosts. The study of codon usage in S. agalactiae may provide evidence about the molecular evolution of the bacterium and a greater understanding of evolutionary relationships between S. agalactiae and its hosts. PMID:26927064

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts.

PubMed

Nakamura, Masayuki; Sugiura, Masahiro

2007-01-01

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

ENRAF Series 854 Advanced Technology Gauge (ATG) with SPU II card for Leak Detector Use Acceptance Test Procedure

DOE Office of Scientific and Technical Information (OSTI.GOV)

SMITH, S.G.

1999-10-21

The following Acceptance Test Procedure was written to test the ENRAF series 854 ATG with SPU II card prior to installation in the Tank Farms. The procedure sets various parameters and verifies the gauge and alarms functionality.

The requirement of sterol glucoside for pexophagy in yeast is dependent on the species and nature of peroxisome inducers.

PubMed

Nazarko, Taras Y; Polupanov, Andriy S; Manjithaya, Ravi R; Subramani, Suresh; Sibirny, Andriy A

2007-01-01

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy. Surprisingly, Atg26 was also not essential for macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris, suggesting that the function of sterol glucoside (SG) in pexophagy is both species and peroxisome inducer specific. However, the rates of degradation of oleate- and amine-induced peroxisomes in P. pastoris were reduced in the absence of SG, indicating that P. pastoris specifically uses sterol conversion by Atg26 to enhance selective degradation of peroxisomes. However, methanol-induced peroxisomes apparently have lost the redundant ability to be degraded without SG. We also show that the P. pastoris Vac8 armadillo repeat protein is not essential for macropexophagy of methanol-, oleate-, or amine-induced peroxisomes, which makes PpVac8 the first known protein required for the micropexophagy, but not for the macropexophagy, machinery. The uniqueness of Atg26 and Vac8 functions under different pexophagy conditions demonstrates that not only pexophagy inducers, such as glucose or ethanol, but also the inducers of peroxisomes, such as methanol, oleate, or primary amines, determine the requirements for subsequent pexophagy in yeast.

ATG7 regulates energy metabolism, differentiation and survival of Philadelphia-chromosome-positive cells

PubMed Central

Karvela, Maria; Baquero, Pablo; Kuntz, Elodie M.; Mukhopadhyay, Arunima; Mitchell, Rebecca; Allan, Elaine K.; Chan, Edmond; Kranc, Kamil R.; Calabretta, Bruno; Salomoni, Paolo; Gottlieb, Eyal; Holyoake, Tessa L.; Helgason, G. Vignir

2016-01-01

ABSTRACT A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34+ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease. PMID:27168493

Autophagy Facilitates IFN-γ-induced Jak2-STAT1 Activation and Cellular Inflammation*

PubMed Central

Chang, Yu-Ping; Tsai, Cheng-Chieh; Huang, Wei-Ching; Wang, Chi-Yun; Chen, Chia-Ling; Lin, Yee-Shin; Kai, Jui-In; Hsieh, Chia-Yuan; Cheng, Yi-Lin; Choi, Pui-Ching; Chen, Shun-Hua; Chang, Shih-Ping; Liu, Hsiao-Sheng; Lin, Chiou-Feng

2010-01-01

Autophagy is regulated for IFN-γ-mediated antimicrobial efficacy; however, its molecular effects for IFN-γ signaling are largely unknown. Here, we show that autophagy facilitates IFN-γ-activated Jak2-STAT1. IFN-γ induces autophagy in wild-type but not in autophagy protein 5 (Atg5−/−)-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-γ induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5−/− or Atg7−/− MEFs are, independent of changes in IFN-γ receptor expression, resistant to IFN-γ-activated Jak2-STAT1, which suggests that autophagy is important for IFN-γ signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-γ-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-γ-induced activation of STAT1 in Atg5−/− MEFs. Our study provides evidence that there is a link between autophagy and both IFN-γ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation. PMID:20592027

Selective endosomal microautophagy is starvation-inducible in Drosophila.

PubMed

Mukherjee, Anindita; Patel, Bindi; Koga, Hiroshi; Cuervo, Ana Maria; Jenny, Andreas

2016-11-01

Autophagy delivers cytosolic components to lysosomes for degradation and is thus essential for cellular homeostasis and to cope with different stressors. As such, autophagy counteracts various human diseases and its reduction leads to aging-like phenotypes. Macroautophagy (MA) can selectively degrade organelles or aggregated proteins, whereas selective degradation of single proteins has only been described for chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI). These 2 autophagic pathways are specific for proteins containing KFERQ-related targeting motifs. Using a KFERQ-tagged fluorescent biosensor, we have identified an eMI-like pathway in Drosophila melanogaster. We show that this biosensor localizes to late endosomes and lysosomes upon prolonged starvation in a KFERQ- and Hsc70-4- dependent manner. Furthermore, fly eMI requires endosomal multivesicular body formation mediated by ESCRT complex components. Importantly, induction of Drosophila eMI requires longer starvation than the induction of MA and is independent of the critical MA genes atg5, atg7, and atg12. Furthermore, inhibition of Tor signaling induces eMI in flies under nutrient rich conditions, and, as eMI in Drosophila also requires atg1 and atg13, our data suggest that these genes may have a novel, additional role in regulating eMI in flies. Overall, our data provide the first evidence for a novel, starvation-inducible, catabolic process resembling endosomal microautophagy in the Drosophila fat body.

The Role of Snx41-Based Pexophagy in Magnaporthe Development

PubMed Central

Deng, Yizhen; Qu, Ziwei; Naqvi, Naweed I.

2013-01-01

Pexophagy, the degradation of peroxisomes via selective autophagy, depends on Atg20/Snx42 function in Saccharomyces cerevisiae. Besides its role in selective autophagy, Atg20/Snx42 is also involved in an autophagy-independent endosomal retrieval trafficking, in cooperation with two other sorting nexins, Snx41 and Snx4. Recently, we reported that the sorting nexin MoSnx41, which showed high sequence similarity to yeast Snx41 and Snx42/Atg20 proteins, regulates the gamma-glutamyl cycle and GSH production and is essential for conidiation and pathogenicity in Magnaporthe oryzae. Pexophagy was also found to be defective in Mosnx41Δ mutant. These findings indicate that MoSnx41 likely serves combined functions of Snx42/Atg20 and Snx41 in M. oryzae.. In this study, we performed complementation analyses and demonstrate that MoSnx41 alone serves the dual function of protein sorting (ScSnx41) and pexophagy (ScSnx42/Atg20). To study the potential biological function of pexophagy in fungal pathogenic life cycle, we created deletion mutants of potential pexophagy-specific genes, and characterized them in terms of pexophagy, conidiation and pathogenesis. We identified Pex14 as an essential protein for pexophagy in M. oryzae. Overall, our results show that pexophagy per se is not essential for asexual development or virulence in M. oryzae. PMID:24302988

Recent advances in treatment of aplastic anemia

PubMed Central

Shin, Seung Hwan; Lee, Sung Eun

2014-01-01

Recent advances in the treatment of aplastic anemia (AA) made most of patients to expect to achieve a long-term survival. Allogeneic stem cell transplantation (SCT) from HLA-matched sibling donor (MSD-SCT) is a preferred first-line treatment option for younger patients with severe or very severe AA, whereas immunosuppressive treatment (IST) is an alternative option for others. Horse anti-thymocyte globuline (ATG) with cyclosporin A (CsA) had been a standard IST regimen with acceptable response rate. Recently, horse ATG had been not available and replaced with rabbit ATG in most countries. Subsequently, recent comparative studies showed that the outcomes of patients who received rabbit ATG/CsA were similar or inferior compared to those who received horse ATG/CsA. Therefore, further studies to improve the outcomes of IST, including additional eltrombopag, are necessary. On the other hand, the upper age limit of patients who are able to receive MSD-SCT as first-line treatment is a current issue because of favorable outcomes of MSD-SCT of older patients using fludarabine-based conditioning. In addition, further studies to improve the outcomes of patients who receive allogeneic SCT from alternative donors are needed. In this review, current issues and the newly emerging trends that may improve their outcomes in near futures will be discussed focusing the management of patients with AA. PMID:25378968

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori.

PubMed

Ji, Ming-Ming; Lee, Jae Man; Mon, Hiroaki; Iiyama, Kazuhiro; Tatsuke, Tsuneyuki; Morokuma, Daisuke; Hino, Masato; Yamashita, Mami; Hirata, Kazuma; Kusakabe, Takahiro

2017-10-01

p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon

PubMed Central

Feeney, Morgan A.; Chandra, Govind; Findlay, Kim C.; Paget, Mark S. B.

2017-01-01

ABSTRACT The major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry. PMID:28611250

The Complete Mitochondrial Genome of the Rice Moth, Corcyra cephalonica

PubMed Central

Wu, Yu-Peng; Li, Jie; Zhao, Jin-Liang; Su, Tian-Juan; Luo, A-Rong; Fan, Ren-Jun; Chen, Ming-Chang; Wu, Chun-Sheng; Zhu, Chao-Dong

2012-01-01

The complete mitochondrial genome (mitogenome) of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) was determined as a circular molecular of 15,273 bp in size. The mitogenome composition (37 genes) and gene order are the same as the other lepidopterans. Nucleotide composition of the C. cephalonica mitogenome is highly A+T biased (80.43%) like other insects. Twelve protein-coding genes start with a typical ATN codon, with the exception of coxl gene, which uses CGA as the initial codon. Nine protein-coding genes have the common stop codon TAA, and the nad2, cox1, cox2, and nad4 have single T as the incomplete stop codon. 22 tRNA genes demonstrated cloverleaf secondary structure. The mitogenome has several large intergenic spacer regions, the spacer1 between trnQ gene and nad2 gene, which is common in Lepidoptera. The spacer 3 between trnE and trnF includes microsatellite-like repeat regions (AT)18 and (TTAT)3. The spacer 4 (16 bp) between trnS2 gene and nad1 gene has a motif ATACTAT; another species, Sesamia inferens encodes ATCATAT at the same position, while other lepidopteran insects encode a similar ATACTAA motif. The spacer 6 is A+T rich region, include motif ATAGA and a 20-bp poly(T) stretch and two microsatellite (AT)9, (AT)8 elements. PMID:23413968

The complete mitochondrial genome of the rice moth, Corcyra cephalonica.

PubMed

Wu, Yu-Peng; Li, Jie; Zhao, Jin-Liang; Su, Tian-Juan; Luo, A-Rong; Fan, Ren-Jun; Chen, Ming-Chang; Wu, Chun-Sheng; Zhu, Chao-Dong

2012-01-01

The complete mitochondrial genome (mitogenome) of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae) was determined as a circular molecular of 15,273 bp in size. The mitogenome composition (37 genes) and gene order are the same as the other lepidopterans. Nucleotide composition of the C. cephalonica mitogenome is highly A+T biased (80.43%) like other insects. Twelve protein-coding genes start with a typical ATN codon, with the exception of coxl gene, which uses CGA as the initial codon. Nine protein-coding genes have the common stop codon TAA, and the nad2, cox1, cox2, and nad4 have single T as the incomplete stop codon. 22 tRNA genes demonstrated cloverleaf secondary structure. The mitogenome has several large intergenic spacer regions, the spacer1 between trnQ gene and nad2 gene, which is common in Lepidoptera. The spacer 3 between trnE and trnF includes microsatellite-like repeat regions (AT)18 and (TTAT)(3). The spacer 4 (16 bp) between trnS2 gene and nad1 gene has a motif ATACTAT; another species, Sesamia inferens encodes ATCATAT at the same position, while other lepidopteran insects encode a similar ATACTAA motif. The spacer 6 is A+T rich region, include motif ATAGA and a 20-bp poly(T) stretch and two microsatellite (AT)(9), (AT)(8) elements.

Functional Versatility of AGY Serine Codons in Immunoglobulin Variable Region Genes

PubMed Central

Detanico, Thiago; Phillips, Matthew; Wysocki, Lawrence J.

2016-01-01

In systemic autoimmunity, autoantibodies directed against nuclear antigens (Ags) often arise by somatic hypermutation (SHM) that converts AGT and AGC (AGY) Ser codons into Arg codons. This can occur by three different single-base changes. Curiously, AGY Ser codons are far more abundant in complementarity-determining regions (CDRs) of IgV-region genes than expected for random codon use or from species-specific codon frequency data. CDR AGY codons are also more abundant than TCN Ser codons. We show that these trends hold even in cartilaginous fishes. Because AGC is a preferred target for SHM by activation-induced cytidine deaminase, we asked whether the AGY abundance was solely due to a selection pressure to conserve high mutability in CDRs regardless of codon context but found that this was not the case. Instead, AGY triplets were selectively enriched in the Ser codon reading frame. Motivated by reports implicating a functional role for poly/autoreactive specificities in antiviral antibodies, we also analyzed mutations at AGY in antibodies directed against a number of different viruses and found that mutations producing Arg codons in antiviral antibodies were indeed frequent. Unexpectedly, however, we also found that AGY codons mutated often to encode nearly all of the amino acids that are reported to provide the most frequent contacts with Ag. In many cases, mutations producing codons for these alternative amino acids in antiviral antibodies were more frequent than those producing Arg codons. Mutations producing each of these key amino acids required only single-base changes in AGY. AGY is the only codon group in which two-thirds of random mutations generate codons for these key residues. Finally, by directly analyzing X-ray structures of immune complexes from the RCSB protein database, we found that Ag-contact residues generated via SHM occurred more often at AGY than at any other codon group. Thus, preservation of AGY codons in antibody genes appears to have been driven by their exceptional functional versatility, despite potential autoreactive consequences. PMID:27920779

Neither eosinophils nor neutrophils require ATG5-dependent autophagy for extracellular DNA trap formation.

PubMed

Germic, Nina; Stojkov, Darko; Oberson, Kevin; Yousefi, Shida; Simon, Hans-Uwe

2017-11-01

The importance of extracellular traps (ETs) in innate immunity is well established, but the molecular mechanisms responsible for their formation remain unclear and in scientific dispute. ETs have been defined as extracellular DNA scaffolds associated with the granule proteins of eosinophils or neutrophils. They are capable of killing bacteria extracellularly. Based mainly on results with phosphoinositide 3-kinase (PI3K) inhibitors such as 3-methyladenine (3-MA) and wortmannin, which are commonly used to inhibit autophagy, several groups have reported that autophagy is required for neutrophil extracellular trap (NET) formation. We decided to investigate this apparent dependence on autophagy for ET release and generated genetically modified mice that lack, specifically in eosinophils or neutrophils, autophagy-related 5 (Atg5), a gene encoding a protein essential for autophagosome formation. Interestingly, neither eosinophils nor neutrophils from Atg5-deficient mice exhibited abnormalities in ET formation upon physiological activation or exposure to low concentrations of PMA, although we could confirm that human and mouse eosinophils and neutrophils, after pre-treatment with inhibitors of class III PI3K, show a block both in reactive oxygen species (ROS) production and in ET formation. The so-called late autophagy inhibitors bafilomycin A1 and chloroquine, on the other hand, were without effect. These data indicate that ET formation occurs independently of autophagy and that the inhibition of ROS production and ET formation in the presence of 3-MA and wortmannin is probably owing to their additional ability to block the class I PI3Ks, which are involved in signalling cascades initiated by triggers of ET formation. © 2017 John Wiley & Sons Ltd.

Complete Mitochondrial Genome of Three Bactrocera Fruit Flies of Subgenus Bactrocera (Diptera: Tephritidae) and Their Phylogenetic Implications

PubMed Central

Yong, Hoi-Sen; Song, Sze-Looi; Lim, Phaik-Eem; Eamsobhana, Praphathip; Suana, I. Wayan

2016-01-01

Bactrocera latifrons is a serious pest of solanaceous fruits and Bactrocera umbrosa is a pest of Artocarpus fruits, while Bactrocera melastomatos infests the fruit of Melastomataceae. They are members of the subgenus Bactrocera. We report here the complete mitochondrial genome of these fruit flies determined by next-generation sequencing and their phylogeny with other taxa of the subgenus Bactrocera. The whole mitogenomes of these three species possessed 37 genes namely, 13 protein-coding genes (PCGs), 2 rRNA and 22 tRNA genes. The mitogenome of B. latifrons (15,977 bp) was longer than those of B. melastomatos (15,954 bp) and B. umbrosa (15,898 bp). This difference can be attributed to the size of the intergenic spacers (283 bp in B. latifrons, 261 bp in B. melastomatos, and 211 bp in B. umbrosa). Most of the PCGs in the three species have an identical start codon, except for atp8 (adenosine triphosphate synthase protein 8), which had an ATG instead of GTG in B. umbrosa, whilst the nad3 (NADH dehydrogenase subunit 3) and nad6 (NADH dehydrogenase subunit 6) genes were characterized by an ATC instead of ATT in B. melastomatos. The three species had identical stop codon for the respective PCGs. In B. latifrons and B. melastomatos, the TΨC (thymidine-pseudouridine-cytidine)-loop was absent in trnF (phenylalanine) and DHU (dihydrouracil)-loop was absent in trnS1 (serine S1). In B. umbrosa, trnN (asparagine), trnC (cysteine) and trnF lacked the TψC-loop, while trnS1 lacked the DHU-stem. Molecular phylogeny based on 13 PCGs was in general concordant with 15 mitochondrial genes (13 PCGs and 2 rRNA genes), with B. latifrons and B. umbrosa forming a sister group basal to the other species of the subgenus Bactrocera which was monophyletic. The whole mitogenomes will serve as a useful dataset for studying the genetics, systematics and phylogenetic relationships of the many species of Bactrocera genus in particular, and tephritid fruit flies in general. PMID:26840430

RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundaram, Kumaran; Nishimura, Riko; Senn, Joseph

2007-01-01

Osteoclast differentiation is tightly regulated by receptor activator of NF-{kappa}B ligand (RANKL) signaling. Matrix metalloproteinase-9 (MMP-9), a type IV collagenase is highly expressed in osteoclast cells and plays an important role in degradation of extracellular matrix; however, the molecular mechanisms that regulate MMP-9 gene expression are unknown. In this study, we demonstrate that RANKL signaling induces MMP-9 gene expression in osteoclast precursor cells. We further show that RANKL regulates MMP-9 gene expression through TRAF6 but not TRAF2. Interestingly, blockade of p38 MAPK activity by pharmacological inhibitor, SB203580 increases MMP-9 activity whereas ERK1/2 inhibitor, PD98059 decreases RANKL induced MMP-9 activity inmore » RAW264.7 cells. These data suggest that RANKL differentially regulates MMP-9 expression through p38 and ERK signaling pathways during osteoclast differentiation. Transient expression of MMP-9 gene (+ 1 to - 1174 bp relative to ATG start codon) promoter-luciferase reporter plasmids in RAW264.7 cells and RANKL stimulation showed significant increase (20-fold) of MMP-9 gene promoter activity; however, there is no significant change with respect to + 1 bp to - 446 bp promoter region and empty vector transfected cells. These results indicated that MMP-9 promoter sequence from - 446 bp to - 1174 bp relative to start codon is responsive to RANKL stimulation. Sequence analysis of the mouse MMP-9 gene promoter region further identified the presence of binding motif (- 1123 bp to - 1153 bp) for the nuclear factor of activated T cells 1 (NFATc1) transcription factor. Inhibition of NFATc1 using siRNA and VIVIT peptide inhibitor significantly decreased RANKL stimulation of MMP-9 activity. We further confirm by oligonucleotide pull-down assay that RANKL stimuli enhanced NFATc1 binding to MMP-9 gene promoter element. In addition, over-expression of constitutively active NFAT in RAW264.7 cells markedly increased (5-fold) MMP-9 gene promoter activity in the absence of RANKL. Taken together, our results suggest that RANKL signals through TRAF6 and that NFATc1 is a downstream effector of RANKL signaling to modulate MMP-9 gene expression during osteoclast differentiation.« less

Identification and characterization of a catechol-o-methyltransferase cDNA in the catfish Heteropneustes fossilis: Tissue, sex and seasonal variations, and effects of gonadotropin and 2-hydroxyestradiol-17β on mRNA expression.

PubMed

Chaube, R; Rawat, A; Inbaraj, R M; Bobe, J; Guiguen, Y; Fostier, A; Joy, K P

2017-05-15

Catechol-O-methyltransferase (COMT) is involved in the methylation and inactivation of endogenous and xenobiotic catechol compounds, and serves as a common biochemical link in the catecholamine and catecholestrogen metabolism. Studies on cloning, sequencing and function characterization comt gene in lower vertebrates like fish are fewer. In the present study, a full-length comt cDNA of 1442bp with an open-reading frame (ORF) of 792bp, and start codon (ATG) at nucleotide 162 and stop codon (TAG) at nucleotide 953 was isolated and characterized in the stinging catfish Heteropneustes fossilis (accession No. KT597925). The ORF codes for a protein of 263 amino acid residues, which is also validated by the catfish transcriptome data analysis. The catfish Comt shared conserved putative structural regions important for S-adenosyl methionine (AdoMet)- and catechol-binding, transmembrane regions, two glycosylation sites (N-65 and N-91) at the N-terminus and two phosphorylation sites (Ser-235 and Thr-240) at the C-terminus. The gene was expressed in all tissues examined and the expression showed significant sex dimorphic distribution with high levels in females. The transcript was abundant in the liver, brain and gonads and low in muscles. The transcripts showed significant seasonal variations in the brain and ovary, increased progressively to the peak levels in spawning phase and then declined. The brain and ovarian comt mRNA levels showed periovulatory changes after in vivo and in vitro human chorionic gonadotropin (hCG) treatments with high fold increases at 16 and 24h in the brain and at 16h in the ovary. The catecholestrogen 2-hydroxyE 2 up regulated ovarian comt expression in vitro with the highest fold increase at 16h. The mRNA and protein was localized in the follicular layer of the vitellogenic follicles and in the cytoplasm of primary follicles. The data were discussed in relation to catecholamine and catecholestrogen-mediated functions in the brain and ovary of the stinging catfish. Copyright © 2016 Elsevier Inc. All rights reserved.

Chloroplast DNA codon use: evidence for selection at the psb A locus based on tRNA availability.

PubMed

Morton, B R

1993-09-01

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.

Stop Codon Reassignment in the Wild

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ivanova, Natalia; Schwientek, Patrick; Tripp, H. James

Since the discovery of the genetic code and protein translation mechanisms (1), a limited number of variations of the standard assignment between unique base triplets (codons) and their encoded amino acids and translational stop signals have been found in bacteria and phages (2-3). Given the apparent ubiquity of the canonical genetic code, the design of genomically recoded organisms with non-canonical codes has been suggested as a means to prevent horizontal gene transfer between laboratory and environmental organisms (4). It is also predicted that genomically recoded organisms are immune to infection by viruses, under the assumption that phages and their hostsmore » must share a common genetic code (5). This paradigm is supported by the observation of increased resistance of genomically recoded bacteria to phages with a canonical code (4). Despite these assumptions and accompanying lines of evidence, it remains unclear whether differential and non-canonical codon usage represents an absolute barrier to phage infection and genetic exchange between organisms. Our knowledge of the diversity of genetic codes and their use by viruses and their hosts is primarily derived from the analysis of cultivated organisms. Advances in single-cell sequencing and metagenome assembly technologies have enabled the reconstruction of genomes of uncultivated bacterial and archaeal lineages (6). These initial findings suggest that large scale systematic studies of uncultivated microorganisms and viruses may reveal the extent and modes of divergence from the canonical genetic code operating in nature. To explore alternative genetic codes, we carried out a systematic analysis of stop codon reassignments from the canonical TAG amber, TGA opal, and TAA ochre codons in assembled metagenomes from environmental and host-associated samples, single-cell genomes of uncultivated bacteria and archaea, and a collection of phage sequences« less

Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors

PubMed Central

Suzuki, Nobuhiro; Geletka, Lynn M.; Nuss, Donald L.

2000-01-01

We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences. PMID:10906211

Expression of chicken parvovirus VP2 in chicken embryo fibroblasts requires codon optimization for production of naked DNA and vectored meleagrid herpesvirus type 1 vaccines.

PubMed

Spatz, Stephen J; Volkening, Jeremy D; Mullis, Robert; Li, Fenglan; Mercado, John; Zsak, Laszlo

2013-10-01

Meleagrid herpesvirus type 1 (MeHV-1) is an ideal vector for the expression of antigens from pathogenic avian organisms in order to generate vaccines. Chicken parvovirus (ChPV) is a widespread infectious virus that causes serious disease in chickens. It is one of the etiological agents largely suspected in causing Runting Stunting Syndrome (RSS) in chickens. Initial attempts to express the wild-type gene encoding the capsid protein VP2 of ChPV by insertion into the thymidine kinase gene of MeHV-1 were unsuccessful. However, transient expression of a codon-optimized synthetic VP2 gene cloned into the bicistronic vector pIRES2-Ds-Red2, could be demonstrated by immunocytochemical staining of transfected chicken embryo fibroblasts (CEFs). Red fluorescence could also be detected in these transfected cells since the red fluorescent protein gene is downstream from the internal ribosome entry site (IRES). Strikingly, fluorescence could not be demonstrated in cells transiently transfected with the bicistronic vector containing the wild-type or non-codon-optimized VP2 gene. Immunocytochemical staining of these cells also failed to demonstrate expression of wild-type VP2, indicating that the lack of expression was at the RNA level and the VP2 protein was not toxic to CEFs. Chickens vaccinated with a DNA vaccine consisting of the bicistronic vector containing the codon-optimized VP2 elicited a humoral immune response as measured by a VP2-specific ELISA. This VP2 codon-optimized bicistronic cassette was rescued into the MeHV-1 genome generating a vectored vaccine against ChPV disease.

Genomic analysis of codon usage shows influence of mutation pressure, natural selection, and host features on Marburg virus evolution.

PubMed

Nasrullah, Izza; Butt, Azeem M; Tahir, Shifa; Idrees, Muhammad; Tong, Yigang

2015-08-26

The Marburg virus (MARV) has a negative-sense single-stranded RNA genome, belongs to the family Filoviridae, and is responsible for several outbreaks of highly fatal hemorrhagic fever. Codon usage patterns of viruses reflect a series of evolutionary changes that enable viruses to shape their survival rates and fitness toward the external environment and, most importantly, their hosts. To understand the evolution of MARV at the codon level, we report a comprehensive analysis of synonymous codon usage patterns in MARV genomes. Multiple codon analysis approaches and statistical methods were performed to determine overall codon usage patterns, biases in codon usage, and influence of various factors, including mutation pressure, natural selection, and its two hosts, Homo sapiens and Rousettus aegyptiacus. Nucleotide composition and relative synonymous codon usage (RSCU) analysis revealed that MARV shows mutation bias and prefers U- and A-ended codons to code amino acids. Effective number of codons analysis indicated that overall codon usage among MARV genomes is slightly biased. The Parity Rule 2 plot analysis showed that GC and AU nucleotides were not used proportionally which accounts for the presence of natural selection. Codon usage patterns of MARV were also found to be influenced by its hosts. This indicates that MARV have evolved codon usage patterns that are specific to both of its hosts. Moreover, selection pressure from R. aegyptiacus on the MARV RSCU patterns was found to be dominant compared with that from H. sapiens. Overall, mutation pressure was found to be the most important and dominant force that shapes codon usage patterns in MARV. To our knowledge, this is the first detailed codon usage analysis of MARV and extends our understanding of the mechanisms that contribute to codon usage and evolution of MARV.

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards “GC” Rich Codons

PubMed Central

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-01-01

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen “core” dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression. PMID:28448468

Complete DNA sequence of the mitochondrial genome of the treehopper Leptobelus gazella (Membracoidea: Hemiptera).

PubMed

Zhao, Xing; Liang, Ai-Ping

2016-09-01

The first complete DNA sequence of the mitochondrial genome (mitogenome) of Leptobelus gazelle (Membracoidea: Hemiptera) is determined in this study. The circular molecule is 16,007 bp in its full length, which encodes a set of 37 genes, including 13 proteins, 2 ribosomal RNAs, 22 transfer RNAs, and contains an A + T-rich region (CR). The gene numbers, content, and organization of L. gazelle are similar to other typical metazoan mitogenomes. Twelve of the 13 PCGs are initiated with ATR methionine or ATT isoleucine codons, except the atp8 gene that uses the ATC isoleucine as start signal. Ten of the 13 PCGs have complete termination codons, either TAA (nine genes) or TAG (cytb). The remaining 3 PCGs (cox1, cox2 and nad5) have incomplete termination codons T (AA). All of the 22 tRNAs can be folded in the form of a typical clover-leaf structure. The complete mitogenome sequence data of L. gazelle is useful for the phylogenetic and biogeographic studies of the Membracoidea and Hemiptera.

New insights into the pathways initiating and driving pancreatitis

PubMed Central

Gukovskaya, Anna S.; Pandol, Stephen J.; Gukovsky, Ilya

2016-01-01

Purpose of review In this article, we discuss recent studies that advance our understanding of molecular and cellular factors initiating and driving pancreatitis, with the emphasis on the role of acinar cell organelle disorders. Recent findings The central physiologic function of the pancreatic acinar cell – to synthesize, store, and secrete digestive enzymes – critically relies on coordinated actions of the endoplasmic reticulum (ER), the endolysosomal system, mitochondria, and autophagy. Recent studies begin to unravel the roles of these organelles’ disordering in the mechanism of pancreatitis. Mice deficient in key autophagy mediators Atg5 or Atg7, or lysosome-associated membrane protein-2, exhibit dysregulation of multiple signaling and metabolic pathways in pancreatic acinar cells and develop spontaneous pancreatitis. Mitochondrial dysfunction caused by sustained opening of the permeability transition pore is shown to mediate pancreatitis in several clinically relevant experimental models, and its inhibition by pharmacologic or genetic means greatly reduces local and systemic pathologic responses. Experimental pancreatitis is also alleviated with inhibitors of ORAI1, a key component of the plasma membrane channel mediating pathologic rise in acinar cell cytosolic Ca2+. Pancreatitis-promoting mutations are increasingly associated with the ER stress. These findings suggest novel pathways and drug targets for pancreatitis treatment. In addition, the recent studies identify new mediators (e.g., neutrophil extracellular traps) of the inflammatory and other responses of pancreatitis. Summary The recent findings illuminate a critical role of organelles regulating the autophagic, endolysosomal, mitochondrial, and ER pathways in maintaining pancreatic acinar cell homeostasis and secretory function; provide compelling evidence that organelle disordering is a key pathogenic mechanism initiating and driving pancreatitis; and identify molecular and cellular factors that could be targeted to restore organellar functions and thus alleviate or treat pancreatitis. PMID:27428704