scholarly journals Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform

2019 ◽  
Vol 476 (10) ◽  
pp. 1521-1537 ◽  
Author(s):  
Emma J. Goldberg ◽  
Katherine A. Buddo ◽  
Kelsey L. McLaughlin ◽  
Regina F. Fernandez ◽  
Andrea S. Pereyra ◽  
...  
Keyword(s):  
Acid Oxidation ◽  
Valuable Insight ◽  
Keto Acid ◽  
Isolated Mitochondria ◽  
Mouse Tissues ◽  
Branched Chain

Abstract Alterations to branched-chain keto acid (BCKA) oxidation have been implicated in a wide variety of human diseases, ranging from diabetes to cancer. Although global shifts in BCKA metabolism—evident by gene transcription, metabolite profiling, and in vivo flux analyses have been documented across various pathological conditions, the underlying biochemical mechanism(s) within the mitochondrion remain largely unknown. In vitro experiments using isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across disciplines to shed valuable insight into mitochondrial-linked pathologies. That said, few studies have attempted to model in vitro BCKA oxidation in isolated organelles. The impetus for the present study stemmed from the knowledge that complete oxidation of each of the three BCKAs involves a reaction dependent upon bicarbonate and ATP, both of which are not typically included in respiration experiments. Based on this, it was hypothesized that the inclusion of exogenous bicarbonate and stimulation of respiration using physiological shifts in ATP-free energy, rather than excess ADP, would allow for maximal BCKA-supported respiratory flux in isolated mitochondria. This hypothesis was confirmed in mitochondria from several mouse tissues, including heart, liver and skeletal muscle. What follows is a thorough characterization and validation of a novel biochemical tool for investigating BCKA metabolism in isolated mitochondria.

In vivo and in vitro response to octreotide LAR in a TSH-secreting adenoma: characterization of somatostatin receptor expression and role of subtype 5

Pituitary ◽  
2010 ◽  
Vol 14 (2) ◽  
pp. 141-147 ◽  
Author(s):  
Federico Gatto ◽  
Federica Barbieri ◽  
Lara Castelletti ◽  
Marica Arvigo ◽  
Alessandra Pattarozzi ◽  
...  
Keyword(s):  
Somatostatin Receptor ◽  
Receptor Expression ◽  
Octreotide Lar ◽  
In Vitro Response

scholarly journals Identification and Characterization of Two Bordetella avium Gene Products Required for Hemagglutination

2010 ◽  
Vol 78 (6) ◽  
pp. 2370-2376 ◽  
Author(s):  
Louise M. Temple ◽  
David M. Miyamoto ◽  
Manju Mehta ◽  
Christian M. Capitini ◽  
Stephen Von Stetina ◽  
...  
Keyword(s):  
Whooping Cough ◽  
Bioinformatic Analysis ◽  
Tracheal Ring ◽  
Tracheal Cell ◽  
Bordetella Avium ◽  
Identification And Characterization

ABSTRACT Bordetella avium causes bordetellosis in birds, a disease similar to whooping cough caused by Bordetella pertussis in children. B. avium agglutinates guinea pig erythrocytes via an unknown mechanism. Loss of hemagglutination ability results in attenuation. We report the use of transposon mutagenesis to identify two genes required for hemagglutination. The genes (hagA and hagB) were adjacent and divergently oriented and had no orthologs in the genomes of other Bordetella species. Construction of in-frame, unmarked mutations in each gene allowed examination of the role of each in conferring erythrocyte agglutination, explanted tracheal cell adherence, and turkey poult tracheal colonization. In all of the in vitro and in vivo assays, the requirement for the trans-acting products of hagA and hagB (HagA and HagB) was readily shown. Western blotting, using antibodies to purified HagA and HagB, revealed proteins of the predicted sizes of HagA and HagB in an outer membrane-enriched fraction. Antiserum to HagB, but not HagA, blocked B. avium erythrocyte agglutination and explanted turkey tracheal ring binding. Bioinformatic analysis indicated the similarity of HagA and HagB to several two-component secretory apparatuses in which one product facilitates the exposition of the other. HagB has the potential to serve as a useful immunogen to protect turkeys against colonization and subsequent disease.

scholarly journals Branched chain amino acid aminotransferase 2 Regulates Ferroptotic Cell Death in Cancer Cells

2020 ◽  
Author(s):  
Kang Wang ◽  
Zhengyang Zhang ◽  
Tsai Hsiang-i ◽  
Yanfang Liu ◽  
Ming Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cancer Cells ◽  
Therapeutic Strategy ◽  
Ectopic Expression ◽  
Branched Chain Amino Acid ◽  
Pancreatic Cancer Cells ◽  
Branched Chain

AbstractFerroptosis has been implicated as a tumor-suppressor function for cancer therapy. Recently the sensitivity to ferroptosis was tightly linked to numerous biological processes, including metabolism of amino acid. Here, using a high-throughput CRISPR/Cas9 based genetic screen in HepG2 cells to search for metabolic proteins inhibiting ferroptosis, we identified branched chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib and sulfasalazine) activated AMPK/SREBP1 signaling pathway through ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc– inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

scholarly journals Correction: Tissue-specific characterization of mitochondrial branched-chain keto acid oxidation using a multiplexed assay platform

2019 ◽  
Vol 476 (17) ◽  
pp. 2519-2519
Author(s):  
Emma J. Goldberg ◽  
Katherine A. Buddo ◽  
Kelsey L. McLaughlin ◽  
Regina F. Fernandez ◽  
Andrea S. Pereyra ◽  
...  
Keyword(s):  
Acid Oxidation ◽  
Keto Acid ◽  
Tissue Specific ◽  
Branched Chain

scholarly journals NLRP3- and AIM2-autonomy in a mouse model of MSU crystal-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1[beta] expression as relevant therapy for gout patients.

2019 ◽  
Author(s):  
Alexandre Mariotte ◽  
Aurore Decauwer ◽  
Chrystelle Po ◽  
Cherine Abou-Faycal ◽  
Angelique Pichot ◽  
...  
Keyword(s):  
Mouse Model ◽  
Acute Inflammation ◽  
Joint Inflammation ◽  
Inflammatory Action ◽  
Recognition Receptor ◽  
Msu Crystals

The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1b in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1b; secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Here, we provide an extensive clinical, biological and molecular characterization of the acute uratic inflammation mouse model induced by subcutaneous injection of MSU crystals, which accurately mimics human gout. Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities, among which the use of topical application of imiquimod to promote interferon-dependent anti-inflammatory action maybe relevant.

scholarly journals Characterization of AcMNPV with a deletion of ac69 gene

2011 ◽  
Vol 2 (1) ◽  
pp. 4
Author(s):  
Jianhao Ke ◽  
Jinwen Wang ◽  
Riqiang Deng ◽  
Lin Lin ◽  
Bei Jinlong ◽  
...  
Keyword(s):  
Spodoptera Frugiperda ◽  
Trichoplusia Ni ◽  
Virus Production ◽  
Viral Infectivity ◽  
Methyltransferase Activity ◽  
Budded Virus

<p>ORF69 (Ac69) of <em>Autographa californica</em> multiple nucleopolyhedrovirus (Ac<em>M</em>NPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in <em>Spodoptera frugiperda</em> Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an <em>ac69</em> knockout Ac<em>M</em>NPV bacmid mutant and analyses of the influence of <em>ac69</em> deletion on the viral infectivity in Sf-9 cells and <em>Trichoplusia ni</em> larvae so as to investigate the role of <em>ac69 in the viral life cycle. Results indicated that ac69</em> deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect Ac<em>M</em>NPV infectivity for <em>Trichoplusia ni</em> larvae in LD<sub>50</sub> and LT<sub>50</sub> bioassay when administered orally. These results suggest that <em>ac69</em> may be dispensable for viral infectivity both in vitro and in vivo.</p>

scholarly journals Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in vivo-like processing patterns.

1984 ◽  
Vol 4 (8) ◽  
pp. 1605-1617 ◽  
Author(s):  
G Gaines ◽  
G Attardi
Keyword(s):  
Transcription Initiation ◽  
Ribosomal Proteins ◽  
High Efficiency ◽  
Rrna Synthesis ◽  
Isolated Mitochondria ◽  
Highly Efficient ◽  
Light Strand

A highly efficient RNA-synthesizing system with isolated HeLa cell mitochondria has been developed and characterized regarding its requirements and its products. In this system, transcription is initiated and the transcripts are processed in a way which closely reproduces the in vivo patterns. Total RNA labeling in isolated mitochondria proceeds at a constant rate for about 30 min at 37 degrees C; the estimated rate of synthesis is at least 10 to 15% of the in vivo rate. Polyadenylation of the mRNAs is less extensive in this system than in vivo. Furthermore, compared with the in vivo situation, rRNA synthesis in vitro is less efficient than mRNA synthesis. This is apparently due to a decreased rate of transcription initiation at the rRNA promoter and probably a tendency also for premature termination of the nascent rRNA chains. The 5'-end processing of rRNA also appears to be slowed down, and it is very sensitive to the incubation conditions, in contrast to mRNA processing. It is suggested that the lower efficiency and the lability of rRNA synthesis and processing in isolated mitochondria may be due to cessation of import from the cytoplasm of ribosomal proteins that play a crucial role in these processes. The formation of the light-strand-coded RNA 18 (7S RNA) is affected by high pH or high ATP concentration differently from the overall light-strand transcription. The dissociation of the two processes may have important implications for the mechanism of formation and the functional role of this unusual RNA species. The high efficiency, initiation capacity, and processing fidelity of the in vitro RNA-synthesizing system described here make it a valuable tool for the analysis of the role of nucleocytoplasmic-mitochondrial interactions in organelle gene expression.

scholarly journals Antigenic and genetic characterization of a divergent African virus, Ikoma lyssavirus

2014 ◽  
Vol 95 (5) ◽  
pp. 1025-1032 ◽  
Author(s):  
Daniel L. Horton ◽  
Ashley C. Banyard ◽  
Denise A. Marston ◽  
Emma Wise ◽  
David Selden ◽  
...  
Keyword(s):  
Laboratory Mouse ◽  
Rabies Vaccine ◽  
Molecular Techniques ◽  
Phenotypic Characteristics ◽  
Nucleotide Divergence ◽  
The Brain

In 2009, a novel lyssavirus (subsequently named Ikoma lyssavirus, IKOV) was detected in the brain of an African civet (Civettictis civetta) with clinical rabies in the Serengeti National Park of Tanzania. The degree of nucleotide divergence between the genome of IKOV and those of other lyssaviruses predicted antigenic distinction from, and lack of protection provided by, available rabies vaccines. In addition, the index case was considered likely to be an incidental spillover event, and therefore the true reservoir of IKOV remained to be identified. The advent of sensitive molecular techniques has led to a rapid increase in the discovery of novel viruses. Detecting viral sequence alone, however, only allows for prediction of phenotypic characteristics and not their measurement. In the present study we describe the in vitro and in vivo characterization of IKOV, demonstrating that it is (1) pathogenic by peripheral inoculation in an animal model, (2) antigenically distinct from current rabies vaccine strains and (3) poorly neutralized by sera from humans and animals immunized against rabies. In a laboratory mouse model, no protection was elicited by a licensed rabies vaccine. We also investigated the role of bats as reservoirs of IKOV. We found no evidence for infection among 483 individuals of at least 13 bat species sampled across sites in the Serengeti and Southern Kenya.

In Vitro and In Vivo Characterization of Scleral Implant of Indomethacin: Role of Plasticizer and Cross-Linking Time

Drug Delivery ◽  
2003 ◽  
Vol 10 (4) ◽  
pp. 269-275 ◽  
Author(s):  
M. Thilek Kumar ◽  
C. Rajeswari ◽  
J. Balasubramaniam ◽  
J. K. Pandit ◽  
S. Kant
Keyword(s):  
Cross Linking ◽  
In Vivo Characterization
Sign in / Sign up

Export Citation Format

Share Document