The use of 1-deoxymannojirimycin to evaluate the role of various alpha-mannosidases in oligosaccharide processing in intact cells.

Mechanical force is found to be increasingly important during development and for proper homeostatic maintenance of cells and tissues. The nucleus occupies a large volume fraction of the cell and is interconnected with the cytoskeleton. Here, to determine the direct role of the nucleus itself in converting forces to changes in gene expression, also known as, mechanotransduction, we examine changes in nuclear mechanics and gene reorganization associated with cell fate and with extracellular force. We measure mechanics of nuclei in many model cell systems using micropipette aspiration to show changes in nuclear mechanics. In intact cells we characterize the rheological changes induced in the genome organization with live cell imaging and particle tracking, and we suggest how these changes relate to gene expression.

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Biochemical Studies on Active Transport

The Journal of General Physiology ◽

10.1085/jgp.52.1.279 ◽

1968 ◽

Vol 52 (1) ◽

pp. 279-295 ◽

Cited By ~ 12

Author(s):

Arthur B. Pardee

Keyword(s):

Active Transport ◽

Cell Function ◽

Specific Protein ◽

Transport Systems ◽

Initial State ◽

Intact Cells ◽

The Past ◽

Biochemical Studies ◽

Active Transport Process

The active transport process, so important in cell function, has been studied in the past with intact cells. Models which have arisen from this work all depend on: first, a specific protein to recognize the substrate; second, translocation of the substrate across the cell membrane; third, release of substrate within the cell and restoration of the system to its initial state. These steps are adequate for facilitated transport, but in active transport an energy input is required to maintain a concentration gradient. Parts of transport systems have been isolated recently. A protein which specifically recognizes ß-galactosides has been partially purified. In another case, a protein that appears to be the recognition part of the sulfate transport system of Salmonella typhimurium has been crystallized, and many of its properties have been described. The role of this protein in recognition and in translocation is discussed. Also proteins that phosphorylate a variety of sugars as they enter the cell's interior provide a mechanism for concentrating sugars as their phosphates, against a gradient.

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Effects of brefeldin A on oligosaccharide processing. Evidence for decreased branching of complex-type glycans and increased formation of hybrid-type glycans

Biochemical Journal ◽

10.1042/bj2790159 ◽

1991 ◽

Vol 279 (1) ◽

pp. 159-165 ◽

Cited By ~ 7

Author(s):

D Chawla ◽

R C Hughes

Keyword(s):

Brefeldin A ◽

Kidney Cells ◽

Lectin Affinity Chromatography ◽

Complex Type ◽

Hybrid Type ◽

Oligosaccharide Processing ◽

Fast Processing ◽

Subcellular Compartmentalization ◽

Baby Hamster Kidney Cells

Brefeldin A (BFA), a drug that induces redistribution of Golgi-apparatus proteins into the endoplasmic reticulum, was used to determine the role of subcellular compartmentalization in the processing of asparagine-linked oligosaccharides. Baby-hamster kidney cells were pulse-labelled with [3H]mannose for 30-60 min and chased for up to several hours in the presence or in the absence of BFA or labelled continuously for several hours with and without the drug. Cellular glycoproteins were digested to glycopeptides with Pronase and either fractionated into glycan classes by lectin affinity chromatography or digested further by endoglycosidase H and endoglycosidase D. Released oligosaccharides obtained in the latter procedure were then separated from each other and from endoglycosidase-resistant glycopeptides by paper chromatography. The results show that BFA induces a very fast processing of protein-linked Glc3Man9GlcNAc2 oligosaccharide down to man5GlcNAc2 and conversion into complex-type and hybrid-type glycans. The major difference between untreated and BFA-treated cells is a large increase in bi-antennary and hybrid-type glycans in the latter cells. These results indicate that galactosylation of a mono-antennary GlcNAcMan5GlcNAc2 hybrid blocks subsequent action by mannosidase II and N-acetylglucosaminyl transferase II, producing galactosylated hybrid-type glycans. Similarly, galactosylation of the product of N-acetylglucosaminyltransferases I and II, i.e. a Man3GlcNAc2 core substituted with GlcNAc beta 1→2 on both alpha 1→3- and alpha 1→6-linked mannose residues, blocks branching N-acetylglucosaminyltransferases IV and V, thereby causing an increase in bi-antennary glycans and a decrease in tri- and tetra-antennary glycans.

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Attenuated Endocytosis and Toxicity of a Mutant Cholera Toxin with Decreased Ability To Cluster Ganglioside GM1 Molecules

Infection and Immunity ◽

10.1128/iai.01286-07 ◽

2008 ◽

Vol 76 (4) ◽

pp. 1476-1484 ◽

Cited By ~ 39

Author(s):

Anne A. Wolf ◽

Michael G. Jobling ◽

David E. Saslowsky ◽

Eli Kern ◽

Kimberly R. Drake ◽

...

Keyword(s):

Cholera Toxin ◽

Host Cells ◽

Wild Type ◽

Intact Cells ◽

B Subunit ◽

Binding Pockets ◽

Detergent Resistant Membranes ◽

Toxin Uptake ◽

Chimeric Toxins

ABSTRACT Cholera toxin (CT) moves from the plasma membrane (PM) of host cells to the endoplasmic reticulum (ER) by binding to the lipid raft ganglioside GM1. The homopentomeric B-subunit of the toxin can bind up to five GM1 molecules at once. Here, we examined the role of polyvalent binding of GM1 in CT action by producing chimeric CTs that had B-subunits with only one or two normal binding pockets for GM1. The chimeric toxins had attenuated affinity for binding to host cell PM, as expected. Nevertheless, like wild-type (wt) CT, the CT chimeras induced toxicity, fractionated with detergent-resistant membranes extracted from toxin-treated cells, displayed restricted diffusion in the plane of the PM in intact cells, and remained bound to GM1 when they were immunoprecipitated. Thus, binding normally to two or perhaps only one GM1 molecule is sufficient for association with lipid rafts in the PM and toxin action. The chimeric toxins, however, were much less potent than wt toxin, and they entered the cell by endocytosis more slowly, suggesting that clustering of GM1 molecules by the B-subunit enhances the efficiency of toxin uptake and perhaps also trafficking to the ER.

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Role of the NF-κB Signaling Pathway and κB cis-Regulatory Elements on the IRF-1 and iNOS Promoter Regions in Mycobacterial Lipoarabinomannan Induction of Nitric Oxide

Infection and Immunity ◽

10.1128/iai.71.3.1442-1452.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1442-1452 ◽

Cited By ~ 56

Author(s):

Kristin R. Morris ◽

Ryan D. Lutz ◽

Hyung-Seok Choi ◽

Tetsu Kamitani ◽

Kathryn Chmura ◽

...

Keyword(s):

Nitric Oxide ◽

Signaling Pathway ◽

Host Defense ◽

Regulatory Elements ◽

Mononuclear Phagocytes ◽

Intact Cells ◽

Ifn Γ ◽

Flanking Region ◽

Mycobacterial Cell Wall

ABSTRACT Nitric oxide (NO·) produced by inducible nitric oxide synthase (iNOS) is an important host defense molecule against Mycobacterium tuberculosis in mononuclear phagocytes. The objective of this study was to determine the role of the IκBα kinase-nuclear factor κB (IKK-NF-κB) signaling pathway in the induction of iNOS and NO· by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM) in mouse macrophages costimulated with gamma interferon (IFN-γ). NF-κB was activated by ManLAM as shown by electrophoretic mobility shift assay, by immunofluorescence of translocated NF-κB in intact cells, and by a reporter gene driven by four NF-κB-binding elements. Transduction of an IκBα mutant (Ser32/36Ala) significantly inhibited NO· expression induced by IFN-γ plus ManLAM. An activated SCF complex, a heterotetramer (Skp1, Cul-1, β-TrCP [F-box protein], and ROC1) involved with ubiquitination, is also required for iNOS-NO· induction. Two NF-κB-binding sites (κBI and κBII) present on the 5′-flanking region of the iNOS promoter bound ManLAM-induced NF-κB similarly. By use of reporter constructs in which one or both sites are mutated, both NF-κB-binding positions were essential in iNOS induction by IFN-γ plus ManLAM. IFN-γ-induced activation of the IRF-1 transcriptional complex is a necessary component in host defense against tuberculosis. Although the 5′-flanking region of the IRF-1 promoter contains an NF-κB-binding site and ManLAM-induced NF-κB also binds to this site, ManLAM was unable to induce IRF-1 expression. The influence of mitogen-activated protein kinases on IFN-γ plus ManLAM induction of iNOS-NO· is not due to any effects on ManLAM induction of NF-κB.

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Role of adenosine deaminase, ecto-(5'-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes

Biochemical Journal ◽

10.1042/bj1860907 ◽

1980 ◽

Vol 186 (3) ◽

pp. 907-918 ◽

Cited By ~ 46

Author(s):

A C Newby

Keyword(s):

Adenosine Deaminase ◽

Adenine Nucleotides ◽

Specific Inhibitor ◽

Adenosine Monophosphate ◽

Polymorphonuclear Leucocytes ◽

Intact Cells ◽

Endogenous Substrates ◽

A Minor

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable (less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.

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Role of PBP1 in Cell Division of Staphylococcus aureus

Journal of Bacteriology ◽

10.1128/jb.00044-07 ◽

2007 ◽

Vol 189 (9) ◽

pp. 3525-3531 ◽

Cited By ~ 62

Author(s):

S. F. F. Pereira ◽

A. O. Henriques ◽

M. G. Pinho ◽

H. de Lencastre ◽

A. Tomasz

Keyword(s):

Cell Division ◽

Cell Mass ◽

Nile Red ◽

Inducible Promoter ◽

Wall Structure ◽

Intact Cells ◽

Thin Sections ◽

Methicillin Resistant ◽

Essential Function

ABSTRACT We constructed a conditional mutant of pbpA in which transcription of the gene was placed under the control of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible promoter in order to explore the role of PBP1 in growth, cell wall structure, and cell division. A methicillin-resistant strain and an isogenic methicillin-susceptible strain, each carrying the pbpA mutation, were unable to grow in the absence of the inducer. Conditional mutants of pbpA transferred into IPTG-free medium underwent a four- to fivefold increase in cell mass, which was not accompanied by a proportional increase in viable titer. Examination of thin sections of such cells by transmission electron microscopy or fluorescence microscopy of intact cells with Nile red-stained membranes showed a morphologically heterogeneous population of bacteria with abnormally increased sizes, distorted axial ratios, and a deficit in the number of cells with completed septa. Immunofluorescence with an antibody specific for PBP1 localized the protein to sites of cell division. No alteration in the composition of peptidoglycan was detectable in pbpA conditional mutants grown in the presence of a suboptimal concentration of IPTG, which severely restricted the rate of growth, and the essential function of PBP1 could not be replaced by PBP2A present in methicillin-resistant cells. These observations suggest that PBP1 is not a major contributor to the cross-linking of peptidoglycan and that its essential function must be intimately integrated into the mechanism of cell division.

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The Molecular Information About Deadwood Bacteriomes Partly Depends on the Targeted Environmental DNA

Frontiers in Microbiology ◽

10.3389/fmicb.2021.640386 ◽

2021 ◽

Vol 12 ◽

Author(s):

Maraike Probst ◽

Judith Ascher-Jenull ◽

Heribert Insam ◽

María Gómez-Brandón

Keyword(s):

Environmental Dna ◽

Extracellular Dna ◽

Masking Effect ◽

Differential Analysis ◽

Intact Cells ◽

Total Dna ◽

Dna Types ◽

Dna Pool ◽

First Time

Microbiome studies mostly rely on total DNA extracts obtained directly from environmental samples. The total DNA consists of both intra- and extracellular DNA, which differ in terms of their ecological interpretation. In the present study, we have investigated for the first time the differences among the three DNA types using microbiome sequencing of Picea abies deadwood logs (Hunter decay classes I, III, and V). While the bacterial compositions of all DNA types were comparable in terms of more abundant organisms and mainly depended on the decay class, we found substantial differences between DNA types with regard to less abundant amplicon sequence variants (ASVs). The analysis of the sequentially extracted intra- and extracellular DNA fraction, respectively, increased the ecological depth of analysis compared to the directly extracted total DNA pool. Both DNA fractions were comparable in proportions and the extracellular DNA appeared to persist in the P. abies deadwood logs, thereby causing its masking effect. Indeed, the extracellular DNA masked the compositional dynamics of intact cells in the total DNA pool. Our results provide evidence that the choice of DNA type for analysis might benefit a study’s answer to its respective ecological question. In the deadwood environment researched here, the differential analysis of the DNA types underlined the relevance of Burkholderiales, Rhizobiales and other taxa for P. abies deadwood decomposition and revealed that the role of Acidobacteriota under this scenario might be underestimated, especially compared to Actinobacteriota.

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Phorbol ester-induced changes of cytoplasmic pH in neutrophils: role of exocytosis in Na+-H+ exchange

AJP Cell Physiology ◽

10.1152/ajpcell.1985.248.3.c379 ◽

1985 ◽

Vol 248 (3) ◽

pp. C379-C386 ◽

Cited By ~ 46

Author(s):

S. Grinstein ◽

B. Elder ◽

W. Furuya

Keyword(s):

Phorbol Ester ◽

Secretory Granules ◽

Cytoplasmic Ph ◽

Ph Homeostasis ◽

Intact Cells ◽

Acid Load ◽

Induced Changes ◽

Acid Extrusion ◽

Acid Loading

Cytoplasmic pH homeostasis was studied in intact and granule-free porcine neutrophils following activation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In intact cells, TPA activated at least two separate processes: a Na+-independent and amiloride-insensitive acidification, and a compensatory acid extrusion. The latter is Na+ dependent and blocked by amiloride and is likely to represent Na+-H+ exchange. Enucleated and degranulated neutrophils (cytoplasts) were prepared by sedimentation of cytochalasin B-treated neutrophils through a discontinuous density gradient. Cytoplasts responded to an artificially imposed acid load with activation of an amiloride-sensitive Na+-H+ exchange. TPA also activated both acid production and Na+-dependent acid extrusion in cytoplasts. The magnitude of the responses was comparable in intact neutrophils and in cytoplasts. These data suggest that 1) the nucleus and the secretory granules are not involved in the acidifying response to TPA, and 2) exocytosis of secretory vesicles is not required for activation of Na+-H+ exchange during acid loading or following treatment with the phorbol ester TPA.

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