Pasqua T.,University of California at San Diego |
Mahata S.,California Institute of Technology |
Bandyopadhyay G.K.,University of California at San Diego |
Biswas A.,University of California at San Diego |
And 6 more authors.
Cell and Tissue Research
Chromogranin A (CgA) is a prohormone and granulogenic factor in neuroendocrine tissues with a regulated secretory pathway. The impact of CgA depletion on secretory granule formation has been previously demonstrated in cell culture. However, studies linking the structural effects of CgA deficiency with secretory performance and cell metabolism in the adrenomedullary chromaffin cells in vivo have not previously been reported. Adrenomedullary content of the secreted adrenal catecholamines norepinephrine (NE) and epinephrine (EPI) was decreased 30–40 % in Chga-KO mice. Quantification of NE and EPI-storing dense core (DC) vesicles (DCV) revealed decreased DCV numbers in chromaffin cells in Chga-KO mice. For both cell types, the DCV diameter in Chga-KO mice was less (100–200 nm) than in WT mice (200–350 nm). The volume density of the vesicle and vesicle number was also lower in Chga-KO mice. Chga-KO mice showed an ~47 % increase in DCV/DC ratio, implying vesicle swelling due to increased osmotically active free catecholamines. Upon challenge with 2 U/kg insulin, there was a diminution in adrenomedullary EPI, no change in NE and a very large increase in the EPI and NE precursor dopamine (DA), consistent with increased catecholamine biosynthesis during prolonged secretion. We found dilated mitochondrial cristae, endoplasmic reticulum and Golgi complex, as well as increased synaptic mitochondria, synaptic vesicles and glycogen granules in Chga-KO mice compared to WT mice, suggesting that decreased granulogenesis and catecholamine storage in CgA-deficient mouse adrenal medulla is compensated by increased VMAT-dependent catecholamine update into storage vesicles, at the expense of enhanced energy expenditure by the chromaffin cell. © 2015, Springer-Verlag Berlin Heidelberg. Source
Ambrosi C.,National Center for Microscopy and Imaging Research |
Boassa D.,National Center for Microscopy and Imaging Research |
Pranskevich J.,National Center for Microscopy and Imaging Research |
Smock A.,National Center for Microscopy and Imaging Research |
And 5 more authors.
Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), C×26T135A in M3 produces a closed channel; 2), C×26M34A in M1 severely decreases channel activity; 3), C×26P87L in M2 has been implicated in defective channel gating; and 4), C×26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP3 transfer. These mutations do not affect C×26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, C×26M34A and C×26V84L are stable as hexamers or dodecamers, but C×26T135A and C×26P87L oligomers are not. This instability is also found in C×26T135A and C×26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type C×26 and C×26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of C×26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating. © 2010 by the Biophysical Society. Source
Darshi M.,Howard Hughes Medical Institute |
Perkins G.A.,National Center for Microscopy and Imaging Research |
Patel H.H.,University of California at San Diego |
Ellisman M.H.,National Center for Microscopy and Imaging Research |
Taylor S.S.,Howard Hughes Medical Institute
Molecular and Cellular Proteomics
Insulin resistance plays a major role in the development of type 2 diabetes and obesity and affects a number of biological processes such as mitochondrial biogenesis. Though mitochondrial dysfunction has been linked to the development of insulin resistance and pathogenesis of type 2 diabetes, the precise mechanism linking the two is not well understood. We used high fat diet (HFD)-induced obesity dependent diabetes mouse models to gain insight into the potential pathways altered with metabolic disease, and carried out quantitative proteomic analysis of liver mitochondria. As previously reported, proteins involved in fatty acid oxidation, branched chain amino acid degradation, tricarboxylic acid cycle, and oxidative phosphorylation were uniformly up-regulated in the liver of HFD fed mice compared with that of normal diet. Further, our studies revealed that retinol metabolism is distinctly down-regulated and the mitochondrial structural proteins-components of mitochondrial intermembrane space bridging (MIB) complex (Mitofilin, Sam50, and ChChd3), and Tim proteins-essential for protein import, are significantly up-regulated in HFD fed mice. Structural and functional studies on HFD and normal diet liver mitochondria revealed remodeling of HFD mitochondria to a more condensed form with increased respiratory capacity and higher ATP levels compared with normal diet mitochondria. Thus, it is likely that the structural remodeling is essential to accommodate the increased protein content in presence of HFD: the mechanism could be through the MIB complex promoting contact site and crista junction formation and in turn facilitating the lipid and protein uptake. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source
Crawled News Article
« SLAC, U Toronto team develops new highly efficient ternary OER catalyst for water-splitting using earth-abundant metals; >3x TOF prior record-holder | Main | Six automated truck platoons to compete in European Truck Platooning Challenge » Researchers from the J. Craig Venter Institute (JCVI) and Synthetic Genomics, Inc. (SGI) have designed and constructed of the first minimal synthetic bacterial cell, JCVI-syn3.0. Using the first synthetic cell, Mycoplasma mycoides JCVI-syn1.0 (created by this same team in 2010, earlier post), JCVI-syn3.0 was developed through a design, build, and test process using genes from JCVI-syn1.0. The new minimal synthetic cell contains 531,560 base pairs and just 473 genes, making it the smallest genome of any organism that can be grown in laboratory media. Of these genes 149 are of unknown biological function. By comparison the first synthetic cell, M. mycoides JCVI-syn1.0 has 1.08 million base pairs and 901 genes. A paper describing this research is being published in the journal Science by lead authors Clyde A. Hutchison, III, Ph.D. and Ray-Yuan Chuang, Ph.D., senior author J. Craig Venter, Ph.D., and senior team of Hamilton O. Smith, MD, Daniel G. Gibson, Ph.D., and John I. Glass, Ph.D. Our attempt to design and create a new species, while ultimately successful, revealed that 32% of the genes essential for life in this cell are of unknown function, and showed that many are highly conserved in numerous species. All the bioinformatics studies over the past 20 years have underestimated the number of essential genes by focusing only on the known world. This is an important observation that we are carrying forward into the study of the human genome. The research to construct the first minimal synthetic cell at JCVI was the culmination of 20 years of research that began in 1995 after the genome sequencing of the first free-living organism, Haemophilus influenza, followed by the sequencing of Mycoplasma genitalium. A comparison of these two genomes revealed a common set of 256 genes which the team thought could be a minimal set of genes needed for viability. In 1999 Dr. Hutchison led a team who published a paper describing the use of global transposon mutagenesis techniques to identify the nonessential genes in M. genitalium. Over the last 50 years more than 2,000 publications have contemplated minimal cells and their use in elucidating first principals of biology. From the start, the goal of the JCVI team was similar—build a minimal operating system of a cell to understand biology but to also have a desirable chassis for use in industrial applications. The creation of the first synthetic cell in 2010 did not inform new genome design principles since the M. mycoides genome was mostly recapitulated as in nature. Rather, it established a work flow for building and testing whole genome designs, including a minimal cell, from the bottom up starting from a genome sequence. To create JCVI-syn3.0, the team used an approach of whole genome design and chemical synthesis followed by genome transplantation to test if the cell was viable. Their first attempt to minimize the genome began with a simple approach using information in the biochemical literature and some limited transposon mutagenesis work, but this did not result in a viable genome. After improving transposon methods, they discovered a set of quasi-essential genes that are necessary for robust growth which explained the failure of their first attempt. To facilitate debugging of non-functional reduced genome segments, the team built the genome in eight segments at a time so that each could be tested separately before combining them to generate a minimal genome. The team also explored gene order and how that affects cell growth and viability, noting that gene content was more critical to cell viability than gene order. They went through three cycles of designing, building, and testing ensuring that the quasi-essential genes remained, which in the end resulted in a viable, self-replicating minimal synthetic cell that contained just 473 genes, 35 of which are RNA-coding. In addition, the cell contains a unique 16S gene sequence. The team was able to assign biological function to the majority of the genes with 41% of them responsible for genome expression information, 18% related to cell membrane structure and function, 17% related to cytosolic metabolism, and 7% preservation of genome information. However, a surprising 149 genes could not be assigned a specific biological function despite intensive study. This remains an area of continued work for the researchers. The team concludes that a major outcome of this minimal cell program are new tools and semi-automated processes for whole genome synthesis. Many of these synthetic biology tools and services are commercially available through SGI and SGI-DNA including a synthetic DNA construction service specializing in building large and complex DNA fragments including combinatorial gene libraries, Archetype genomics software, Gibson Assembly kits, and the BioXp, which is a benchtop instrument for producing accurate synthetic DNA fragments. Other authors on the paper are: Thomas J. Deerinck and Mark H. Ellisman, Ph.D., University of California, San Diego National Center for Microscopy and Imaging Research; James F. Pelletier, Center for Bits and Atoms and Department of Physics, Massachusetts Institute of Technology; Elizabeth A. Strychalski, National Institute of Standards and Technology. This work was funded by SGI, the JCVI endowment and the Defense Advanced Research Projects Agency’s Living Foundries program, HR0011-12-C-0063.
Inokuchi-Shimizu S.,University of California at San Diego |
Park E.J.,University of California at San Diego |
Roh Y.S.,University of California at San Diego |
Yang L.,University of California at San Diego |
And 17 more authors.
Journal of Clinical Investigation
The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity. Source