scholarly journals Uridine Diphosphate–Glucose Transport into the Endoplasmic Reticulum ofSaccharomyces cerevisiae:In Vivo and In Vitro Evidence

1999 ◽  
Vol 10 (4) ◽  
pp. 1019-1030 ◽  
Author(s):  
Olga Castro ◽  
Ling Yun Chen ◽  
Armando J. Parodi ◽  
Claudia Abeijón
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Wall ◽  
Glucose Transport ◽  
Subcellular Location ◽  
Functional Expression ◽  
Uridine Diphosphate ◽  
Gene Encoding ◽  
Sugar Nucleotide

It has been proposed that synthesis of β-1,6-glucan, one ofSaccharomyces cerevisiae cell wall components, is initiated by a uridine diphosphate (UDP)-glucose–dependent reaction in the lumen of the endoplasmic reticulum (ER). Because this sugar nucleotide is not synthesized in the lumen of the ER, we have examined whether or not UDP–glucose can be transported across the ER membrane. We have detected transport of this sugar nucleotide into the ER in vivo and into ER–containing microsomes in vitro. Experiments with ER-containing microsomes showed that transport of UDP–glucose was temperature dependent and saturable with an apparentKmof 46 μM and a Vmaxof 200 pmol/mg protein/3 min. Transport was substrate specific because UDP–N-acetylglucosamine did not enter these vesicles. Demonstration of UDP–glucose transport into the ER lumen in vivo was accomplished by functional expression of Schizosaccharomyces pombe UDP–glucose:glycoprotein glucosyltransferase (GT) inS. cerevisiae, which is devoid of this activity. Monoglucosylated protein-linked oligosaccharides were detected inalg6 or alg5 mutant cells, which transfer Man9GlcNAc2to protein; glucosylation was dependent on the inhibition of glucosidase II or the disruption of the gene encoding this enzyme. Although S. cerevisiae lacks GT, it contains Kre5p, a protein with significant homology and the same size and subcellular location as GT. Deletion mutants, kre5Δ, lack cell wall β-1,6 glucan and grow very slowly. Expression of S. pombe GT in kre5Δ mutants did not complement the slow-growth phenotype, indicating that both proteins have different functions in spite of their similarities.

scholarly journals Antigen 84, an Effector of Pleiomorphism in Mycobacterium smegmatis

2007 ◽  
Vol 189 (21) ◽  
pp. 7896-7910 ◽  
Author(s):  
Liem Nguyen ◽  
Nicole Scherr ◽  
John Gatfield ◽  
Anne Walburger ◽  
Jean Pieters ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Smegmatis ◽  
Cell Shape ◽  
Gene Disruption ◽  
Gene Encoding ◽  
Peptidoglycan Biosynthesis ◽  
Asymmetrically Distributed ◽  
Cell Wall Expansion

ABSTRACT While in most rod-shaped bacteria, morphology is based on MreB-like proteins that form an actin-like cytoskeletal scaffold for cell wall biosynthesis, the factors that determine the more flexible rod-like shape in actinobacteria such as Mycobacterium species are unknown. Here we show that a Mycobacterium smegmatis protein homologous to eubacterial DivIVA-like proteins, including M. tuberculosis antigen 84 (Ag84), localized symmetrically to centers of peptidoglycan biosynthesis at the poles and septa. Controlled gene disruption experiments indicated that the gene encoding Ag84, wag31, was essential; when overexpressed, cells became longer and wider, with Ag84 asymmetrically distributed at one pole. Many became grossly enlarged, bowling-pin-shaped cells having up to 80-fold-increased volume. In these cells, Ag84 accumulated predominantly at a bulbous pole that was apparently generated by uncontrolled cell wall expansion. In some cells, Ag84 was associated with exceptional sites of cell wall expansion (buds) that evolved into branches. M. bovis BCG Ag84 was able to form oligomers in vitro, perhaps reflecting its superstructure in vivo. These data suggested a role for Ag84 in cell division and modulating cell shape in pleiomorphic actinobacteria.

scholarly journals AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Steffen Preissler ◽  
Cláudia Rato ◽  
Ruming Chen ◽  
Robin Antrobus ◽  
Shujing Ding ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Covalent Modification ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Unfolded Protein ◽  
Peptide Dissociation

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

How Flies Find Fungal Foes

2007 ◽  
Vol 2007 (369) ◽  
pp. tw21-tw21
Author(s):  
L. Bryan Ray
Keyword(s):  
Cell Wall ◽  
Virulence Factors ◽  
Defense Mechanism ◽  
Fungal Infections ◽  
Toll Pathway ◽  
Online Journal ◽  
Gene Encoding ◽  
Fungal Virulence

Responses of the immune systems of plants and animals show what appears to be evidence of ancient attacks and counterattacks by pathogens and their hosts in the battle for survival. Drosophila have developed receptors that recognize constituents of bacterial cell walls and mount an immune response that causes proteolytic cleavage of the cytokine Spätzle. The Spätzle fragment then activates Toll receptors and leads to production of antimicrobial peptides. Gottar et al. explored the response of Drosophila to fungal infections and found a similar defense mechanism but also unveiled a second signaling pathway that detects a virulence factor produced by the fungus. The authors infected flies by pricking them with a needle dipped in fungus-containing solution and monitored survival or Toll-dependent expression of the gene encoding an antifungal peptide. They found that the receptor GNBP3 (Gram-negative binding protein 3) was required for detection of cell wall components of the fungi and consequent activation of Toll receptors. However, cells with a mutated GNB3 protein could still respond to fungi and activate Toll, but in this case cell wall-derived components were not the trigger. This response depended on the presence of live fungi and, presumably, the production of virulence factors. One such factor is the protease PR1, and the authors showed that expression of PR1 alone led to activation of the Toll pathway. Knowing that a fly protease PSH (Persephone), which is thought to participate in a cascade of proteases that lead to Spätzle cleavage and activation of the Toll pathway in response to fungi, itself requires proteolytic removal of a prodomain for activity, the authors tested whether PR1 might activate PSH. Indeed, studies in vitro and in vivo indicated that PSH appears to be a direct substrate of PR1. The fungi use the PR1 protease to break down the protective cuticle of the insect and allow infection. The authors propose that PSH acts like a sensor to monitor the status of the cuticle. If the presence of PR1 shows that the defense barrier is being broken, PSH is cleaved and the antimicrobial signaling is initiated. Whether humans have such a sensor system to recognize fungal virulence factors remains unknown.M. Gottar, V. Gobert, A. A. Matskevich, J.-M. Reichhart, C. Wang, T. M. Butt, M. Belvin, J. A. Hoffmann, D. Ferrandon, Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell127, 1425-1437 (2006). [Online Journal]

scholarly journals CELL WALL COMPOSITION AND VIRULENCE IN ESCHERICHIA COLI

1968 ◽  
Vol 128 (3) ◽  
pp. 399-414 ◽  
Author(s):  
Donald N. Medearis ◽  
Bruce M. Camitta ◽  
Edward C. Heath
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Uridine Diphosphate ◽  
E Coli ◽  
Endotoxin Activity ◽  
A Cell ◽  
The Difference ◽  
Definition Of

Uridine diphosphate galactose 4-epimerase and phosphomannose isomerase-deficient mutants of Escherichia coli O111:B4 were studied to test the hypothesis that in E. coli a specific relationship exists between O antigenicity, virulence, and capacity to resist phagocytosis. The first mutant, designated J-5, produces a cell wall lipopolysaccharide, the side chains of which do not contain galactose, glucose, N-acetylglucosamine, or colitose. The second mutant produces a cell wall lipopolysaccharide which lacks only colitose. The capacity of these various organisms to kill mice was strikingly different. E. coli O111 was 1000 times as virulent as J-5, and 100 times as virulent as L-2. The capacity of the organisms to kill mice was correlated with their ability to resist phagocytosis and to persist in the peritoneal cavity. The parent strain of O111 resisted phagocytosis by macrophages in vivo and polymorphonuclear leukocytes in vitro. The mutants did not, and the organism most deficient in the saccharide component of its LPS was most susceptible to phagocytosis and least virulent. These results were corroborated by growing the mutants in appropriately supplemented media which permitted the synthesis of complete LPS, reversed the susceptibility to phagocytosis, and restored virulence. Finally, serological reactivity was consistent with previous observations which had demonstrated that the O antigenicity of E. coli is determined by the saccharide composition of its cell wall lipopolysaccharide. Despite the difference in the capacity of the various log-phase organisms to kill mice when injected intraperitoneally, purified lipopolysaccharides extracted from them did not differ significantly in their capacity to kill or produce fever. Thus virulence was shown to be independent of endotoxin activity which in turn seemed to be unrelated to the saccharide composition of the cell wall LPS. Collectively, these data provide at least a partial molecular definition of virulence in E. coli by demonstrating that the presence or absence of specific sugars in its cell wall lipopolysaccharide is a determinant of its antiphagocytic capacity and its virulence.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

scholarly journals Ibudilast Mitigates Delayed Bone Healing Caused by Lipopolysaccharide by Altering Osteoblast and Osteoclast Activity

2021 ◽  
Vol 22 (3) ◽  
pp. 1169
Author(s):  
Yuhan Chang ◽  
Chih-Chien Hu ◽  
Ying-Yu Wu ◽  
Steve W. N. Ueng ◽  
Chih-Hsiang Chang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bone Formation ◽  
Cancellous Bone ◽  
Bone Healing ◽  
Bone Bridge ◽  
Cell Wall Components ◽  
Osteoclast Activity ◽  
Wall Components

Bacterial infection in orthopedic surgery is challenging because cell wall components released after bactericidal treatment can alter osteoblast and osteoclast activity and impair fracture stability. However, the precise effects and mechanisms whereby cell wall components impair bone healing are unclear. In this study, we characterized the effects of lipopolysaccharide (LPS) on bone healing and osteoclast and osteoblast activity in vitro and in vivo and evaluated the effects of ibudilast, an antagonist of toll-like receptor 4 (TLR4), on LPS-induced changes. In particular, micro-computed tomography was used to reconstruct femoral morphology and analyze callus bone content in a femoral defect mouse model. In the sham-treated group, significant bone bridge and cancellous bone formation were observed after surgery, however, LPS treatment delayed bone bridge and cancellous bone formation. LPS inhibited osteogenic factor-induced MC3T3-E1 cell differentiation, alkaline phosphatase (ALP) levels, calcium deposition, and osteopontin secretion and increased the activity of osteoclast-associated molecules, including cathepsin K and tartrate-resistant acid phosphatase in vitro. Finally, ibudilast blocked the LPS-induced inhibition of osteoblast activation and activation of osteoclast in vitro and attenuated LPS-induced delayed callus bone formation in vivo. Our results provide a basis for the development of a novel strategy for the treatment of bone infection.

scholarly journals Dehydrodiisoeugenol inhibits colorectal cancer growth by endoplasmic reticulum stress-induced autophagic pathways

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Changhong Li ◽  
Kui Zhang ◽  
Guangzhao Pan ◽  
Haoyan Ji ◽  
Chongyang Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Endoplasmic Reticulum ◽  
Cell Growth ◽  
Er Stress ◽  
Cancer Cells ◽  
Tumor Xenograft ◽  
Anticancer Activities ◽  
Colorectal Cancer Cells

Abstract Background Dehydrodiisoeugenol (DEH), a novel lignan component extracted from nutmeg, which is the seed of Myristica fragrans Houtt, displays noticeable anti-inflammatory and anti-allergic effects in digestive system diseases. However, the mechanism of its anticancer activity in gastrointestinal cancer remains to be investigated. Methods In this study, the anticancer effect of DEH on human colorectal cancer and its underlying mechanism were evaluated. Assays including MTT, EdU, Plate clone formation, Soft agar, Flow cytometry, Electron microscopy, Immunofluorescence and Western blotting were used in vitro. The CDX and PDX tumor xenograft models were used in vivo. Results Our findings indicated that treatment with DEH arrested the cell cycle of colorectal cancer cells at the G1/S phase, leading to significant inhibition in cell growth. Moreover, DEH induced strong cellular autophagy, which could be inhibited through autophagic inhibitors, with a rction in the DEH-induced inhibition of cell growth in colorectal cancer cells. Further analysis indicated that DEH also induced endoplasmic reticulum (ER) stress and subsequently stimulated autophagy through the activation of PERK/eIF2α and IRE1α/XBP-1 s/CHOP pathways. Knockdown of PERK or IRE1α significantly decreased DEH-induced autophagy and retrieved cell viability in cells treated with DEH. Furthermore, DEH also exhibited significant anticancer activities in the CDX- and PDX-models. Conclusions Collectively, our studies strongly suggest that DEH might be a potential anticancer agent against colorectal cancer by activating ER stress-induced inhibition of autophagy.

Dietary (-)-epicatechin mitigates high fat-induced apoptosis, oxidative and endoplasmic reticulum stress in pancreatic β-cells both in vivo and in vitro

2021 ◽  
Vol 165 ◽  
pp. 44
Author(s):  
Eleonora Cremonini ◽  
Maëlys Rouget ◽  
Solenne Arredi ◽  
Charlotte Devulder-Mercier ◽  
Robin Cellier ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
High Fat ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Induced Apoptosis

Hypoxic Stress Upregulates the Expression of Slc38a1 in Brown Adipocytes via Hypoxia-Inducible Factor-1α

Pharmacology ◽  
2017 ◽  
Vol 101 (1-2) ◽  
pp. 64-71 ◽  
Author(s):  
Tetsuhiro Horie ◽  
Kazuya Fukasawa ◽  
Takashi Iezaki ◽  
Gyujin Park ◽  
Yuki Onishi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hypoxia Inducible Factor ◽  
Amino Acid Transporters ◽  
Hypoxic Stress ◽  
Gene Encoding ◽  
Brown Adipocytes ◽  
Hypoxia Inducible Factor 1Α ◽  
Brown Adipose

The availability of amino acid in the brown adipose tissue (BAT) has been shown to be altered under various conditions; however, little is known about the possible expression and pivotal role of amino acid transporters in BAT under physiological and pathological conditions. The present study comprehensively investigated whether amino acid transporters are regulated by obesogenic conditions in BAT in vivo. Moreover, we investigated the mechanism underlying the regulation of the expression of amino acid transporters by various stressors in brown adipocytes in vitro. The expression of solute carrier family 38 member 1 (Slc38a1; gene encoding sodium-coupled neutral amino acid transporter 1) was preferentially upregulated in the BAT of both genetic and acquired obesity mice in vivo. Moreover, the expression of Slc38a1 was induced by hypoxic stress through hypoxia-inducible factor-1α, which is a master transcription factor of the adaptive response to hypoxic stress, in brown adipocytes in vitro. These results indicate that Slc38a1 is an obesity-associated gene in BAT and a hypoxia-responsive gene in brown adipocytes.

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