scholarly journals Effects of cross-linked dimers of ribonuclease A or of lysozyme on the processing of endocytosed peroxidase by hepatoma cells

1982 ◽  
Vol 202 (2) ◽  
pp. 543-550
Author(s):  
J Bartholeyns ◽  
P Baudhuin
Keyword(s):  
Mechanism Of Action ◽  
Binding Sites ◽  
Culture Medium ◽  
Basic Protein ◽  
Intracellular Localization ◽  
Ribonuclease A ◽  
Protein Processing ◽  
Intracellular Protein ◽  
Intracellular Degradation ◽  
Htc Cells

Cross-linked dimers of ribonuclease, added at a concentration of 0.05 mg/ml to the culture medium of hepatoma (HTC) cells, were previously shown to inhibit intracellular degradation of peroxidase taken up by endocytosis. Intracellular localization showed that endocytosed peroxidase does not reach lysosomes in dimer-treated cells. The present study shows that preloading of lysosomes with fluorescent anti-peroxidase IgG, obtained by exposing HTC cells for 48 h to 0.1 mg of antibody/ml, restores intracellular degradation of endocytosed peroxidase. Moreover, accumulation of peroxidase into lysosomes, which no longer occurs in dimer-treated cells, occurs again under these conditions. We conclude that inhibition of transfer of peroxidase from phagosomes to lysosomes is most likely to be the alteration resulting from the exposure of the cells to ribonuclease dimer, rather than inhibition of fusion between phagosomes and lysosomes. The dimer of another basic protein, lysozyme added at a concentration of 0.2 mg/ml to the culture medium, is shown to induce the same type of effects as does the dimer of ribonuclease; the half-life of endocytosed peroxidase increased from 5 to 15 h after 2 h exposure of HTC cells to dimerized lysozyme. The effect of both dimers on intracellular protein processing can be reversed by addition of 100 mm-galactose to the culture medium, up to 5 h after pretreatment of the cells. The dimers of ribonuclease A or of lysozyme have thus probably the same mechanism of action. Evidence that the two dimers share the same binding sites on the cells is presented.

Nuclear location of PLA2-I in proliferative cells

1998 ◽  
Vol 111 (7) ◽  
pp. 985-994 ◽  
Author(s):  
J.M. Fayard ◽  
C. Tessier ◽  
J.F. Pageaux ◽  
M. Lagarde ◽  
C. Laugier
Keyword(s):  
Binding Sites ◽  
Culture Medium ◽  
Aristolochic Acid ◽  
Direct Interaction ◽  
Nuclear Level ◽  
Proliferative Effect ◽  
Stromal Cell Line ◽  
Normal Rat ◽  
Nuclear Location ◽  
Insight Into

We have previously demonstrated that pancreatic PLA2 (PLA2-I) stimulates the proliferation of UIII cells, a stromal cell line derived from normal rat uterus. In order to gain further insight into the mechanism of action of PLA2-I, we have investigated the intracellular processing of PLA2-I. Either highly proliferative or growth arrested UIII cells were analyzed. Growth arrested cells were obtained from a contact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, western blotting, immunocytochemistry and confocal microscopy, we demonstrate that endogenous PLA2-I was mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmic in non proliferative cells. When non confluent UIII cells were incubated with nanomolar amounts of exogenous PLA2-I, the enzyme was internalized and, in the majority of cells, appeared within the nucleus. Both internalization and nuclear location of exogenous PLA2-I were suppressed by the addition of aristolochic acid to the culture medium. Binding experiments performed on purified nuclear preparations showed the presence of specific cooperative binding sites for PLA2-I. Collectively our data suggest that the proliferative effect exerted by pancreatic PLA2 in UIII cells is mediated by a direct interaction of the enzyme at the nuclear level. Putative mechanisms and targets are discussed.

scholarly journals Analyses of Homing Endonucleases and Mechanism of Action of CRISPR-Cas9 HNH Endonucleases

2020 ◽  
pp. 1-25
Author(s):  
Peramachi Palanivelu
Keyword(s):  
Amino Acids ◽  
Mechanism Of Action ◽  
Metal Binding ◽  
Binding Sites ◽  
Proton Acceptor ◽  
Homing Endonucleases ◽  
Multiple Sequence ◽  
Conserved Motifs ◽  
X Ray ◽  
Metal Binding Sites

Aim: To analyze different HNH endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 proteins for their conserved motifs, metal-binding sites and catalytic amino acids and propose a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 as the model enzyme. Study Design: Multiple sequence analysis (MSA) of homing endonucleases including the CRISPR-Cas9 using Clustal Omega was studied. Other biochemical, Site-directed mutagenesis (SDM) and X-ray crystallographic data were also analyzed. Place and Duration of Study: School of Biotechnology, Madurai Kamaraj University, Madurai, India, between 2007 and 2013. Methodology: Bioinformatics, Biochemical, SDM and X-ray crystallographic data of the HNH endonucleases from different organisms including CRISPR-Cas9 enzymes were analyzed. The advanced version of Clustal Omega was used for protein sequence analysis of different HNH endonucleases from various sources. The conserved motifs identified by the bioinformatics analysis were analyzed further with the data already available from biochemical and SDM and X-ray crystallographic analyses of this group of enzymes and to confirm the possible amino acids involved in the active sites and catalysis. Results: Different types of homing endonucleases from various sources including the HNH endonuclease regions of CRISPR-Cas9 enzymes exhibit different catalytic regions and metal-binding sites. However, the catalytic amino acid, i.e., the proton acceptor histidine (His), is completely conserved in all homing endonucleases analyzed. From these data, a plausible mechanism of action for HNH endonucleases, using CRISPR-Cas9 from Streptococcus pyogenes, as the model enzyme is proposed. Furthermore, multiple sequence alignment (MSA) of various homing endonucleases from different organisms showed many highly conserved motifs also among them. However, some of the HNH endonucleases showed consensus only around the active site regions. Possible catalytic amino acids identified among them belong to either -DH---N or -HH--N types. There are at least two types of metal-binding sites and bind Mg2+ or Zn2+ or both. The CRISPR-Cas9 enzyme from S. pyogenes belongs to the -DH- based HNH endonucleases and possesses –DxD- type metal-binding site where it possibly binds to a Mg2+ ion. The other HNH enzymes possess one or two invariant Zn binding CxxC/ CxxxC motifs. Conclusions: The CRISPR-Cas9 enzymes are found to be -DH- type where the first D is likely to involve in metal-binding and the second invariant H acts as the proton acceptor and the N in –HNH- Cas9 confers specificity by interacting with the nucleotide near the catalytic region. In this communication, a metal-bound water molecule is shown as the nucleophile initiating catalysis. Homing endonucleases may be used as novel DNA binding and cleaving reagents for a variety of genome editing applications and Zinc finger nucleases have already found applications in genome editing.

Morphological and Histochemical Changes in Macrophage Activity After Novel Anti-Neoplastic Platinum Agents

1997 ◽  
Vol 3 (S2) ◽  
pp. 11-12
Author(s):  
H. J. Muenchen ◽  
S.K. Aggarwal ◽  
H.K. Misra ◽  
P. J. Andrulis
Keyword(s):  
Confocal Microscopy ◽  
Mechanism Of Action ◽  
Culture Medium ◽  
Second Generation ◽  
Peritoneal Macrophages ◽  
Immunocytochemical Analysis ◽  
Macrophage Activity ◽  
Murine Peritoneal Macrophages ◽  
Histochemical Changes ◽  
Platinum Agents

Poly-[(trans-1,2-diaminocyclohexane) platinumj-carboxyamylose (“poly-plat”), 5-sulfosalicylato-trans -(1,2-diaminocyclohexane) platinum (SSP), and 4-hydroxy-∝-sulfonylphenylacetato (trans 1,2-diaminocyclohexane) platinum (II) (SAP) are second generation analogs of cisplatin (CDDP) with higher efficacy and potency than cisplatin. This is particularly true of “poly-plat” which contains 1/5 the platinum of CDDP. In order to understand the mechanism of action of these compounds, isolated murine peritoneal macrophages in culture medium were treated with “poly-plat”, SSP, or SAP (5 μg/ml) for 2 h. Drug containing medium was then replaced with fresh medium and the cells were allowed to incubate at 37° C (5% CO2) for 24 h. Supernatants were collected at 0.5, 1, 2, and 24 h post-treatment for immunocytochemical analysis. Confocal microscopy studies demonstrated an increase in the number of lysosomes in the treated macrophages, but only “poly-plat” and SSP treated macrophages were stimulated to form cytoplasmic extensions at 2 h and 24 h.

Effects of glucocorticoids on lymphocyte metabolism

1993 ◽  
Vol 264 (1) ◽  
pp. E24-E28
Author(s):  
M. A. Serrano ◽  
R. Curi ◽  
M. Parry-Billings ◽  
J. F. Williams ◽  
E. A. Newsholme
Keyword(s):  
Lymph Node ◽  
Immune System ◽  
Mechanism Of Action ◽  
Pyruvate Dehydrogenase ◽  
Incubation Medium ◽  
Culture Medium ◽  
Immunosuppressive Effect ◽  
Glutamine Metabolism ◽  
Pyruvate Metabolism

The immunosuppressive effect of glucocorticoids has been widely reported; however, the mechanism of action of these hormones on the immune system has not been fully established. In the present study, the effect of glucocorticoids on glucose, glutamine, and pyruvate metabolism in lymph node lymphocytes was investigated. Addition of dexamethasone to the incubation medium did not alter glucose and glutamine metabolism but inhibited pyruvate utilization by 40%. This latter effect took 1 h to occur and remained for up to 6 h, even after removal of dexamethasone from the culture medium. Measurements of the activity of pyruvate dehydrogenase in lymphocytes and the rate of [1–14C]-pyruvate conversion into 14CO2 in incubated lymphocyte mitochondria demonstrated that glucocorticoids decrease pyruvate utilization by inhibiting the activity of this key regulatory enzyme. The effect of such an inhibition of pyruvate utilization on the function of cells of the immune system remains to be clarified.

C/EBPβ and GATA-1 Synergistically Regulate Activity of the Eosinophil Granule Major Basic Protein Promoter: Implication for C/EBPβ Activity in Eosinophil Gene Expression

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1429-1439 ◽  
Author(s):  
Yuji Yamaguchi ◽  
Hitoshi Nishio ◽  
Kenji Kishi ◽  
Steven J. Ackerman ◽  
Toshio Suda
Keyword(s):  
Binding Site ◽  
Cell Line ◽  
Binding Sites ◽  
Basic Protein ◽  
Regulatory Role ◽  
Expression Vectors ◽  
Regulatory Function ◽  
Dependent Manner ◽  
Major Basic Protein ◽  
Eosinophil Granule

Abstract Eosinophil granule major basic protein (MBP) is expressed exclusively in eosinophils and basophils in hematopoietic cells. In our previous study, we demonstrated a major positive regulatory role for GATA-1 and a negative regulatory role for GATA-2 in MBP gene transcription. Further analysis of the MBP promoter region identified a C/EBP (CCAAT/enhancer-binding protein) consensus binding site 6 bp upstream of the functional GATA-binding site in the MBP gene. In the cell line HT93A, which is capable of differentiating towards both the eosinophil and neutrophil lineages in response to retinoic acid (RA), C/EBP mRNA expression decreased significantly concomitant with eosinophilic and neutrophilic differentiation, whereas C/EBPβ expression was markedly increased. Electrophoretic mobility shift assays (EMSAs) showed that recombinant C/EBPβ protein could bind to the potential C/EBP-binding site (bp −90 to −82) in the MBP promoter. Furthermore, we have demonstrated that both C/EBPβ and GATA-1 can bind simultaneously to the C/EBP- and GATA-binding sites in the MBP promoter. To determine the functionality of both the C/EBP- and GATA-binding sites, we analyzed whether C/EBPβ and GATA-1 can stimulate the MBP promoter in the C/EBPβ and GATA-1 negative Jurkat T-cell line. Cotransfection with C/EBPβ and GATA-1 expression vectors produced a 5-fold increase compared with cotransfection with the C/EBPβ or GATA-1 expression vectors individually. In addition, GST pull-down experiments demonstrated a physical interaction between human GATA-1 and C/EBPβ. Expression of FOG (F̲riendo̲fG̲ATA), which binds to GATA-1 and acts as a cofactor for GATA-binding proteins, decreased transactivation activity of GATA-1 for the MBP promoter in a dose-dependent manner. Our results provide the first evidence that both GATA-1 and C/EBPβ synergistically transactivate the promoter of an eosinophil-specific granule protein gene and that FOG may act as a negative cofactor for the eosinophil lineage, unlike its positively regulatory function for the erythroid and megakaryocyte lineages.

Down-regulation of neutrophil functions by the ELR+CXC chemokine platelet basic protein

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 2965-2972 ◽  
Author(s):  
Jan E. Ehlert ◽  
Andreas Ludwig ◽  
Tobias A. Grimm ◽  
Buko Lindner ◽  
Hans-Dieter Flad ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Chemokine Receptors ◽  
Cell Activation ◽  
Basic Protein ◽  
Cathepsin G ◽  
Terminal Part ◽  
Down Regulation ◽  
Cxc Chemokine ◽  
Continuous Accumulation

Abstract The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR+ CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2– but not CXCR-1– dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

Down-regulation of neutrophil functions by the ELR+CXC chemokine platelet basic protein

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 2965-2972 ◽  
Author(s):  
Jan E. Ehlert ◽  
Andreas Ludwig ◽  
Tobias A. Grimm ◽  
Buko Lindner ◽  
Hans-Dieter Flad ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Sites ◽  
Chemokine Receptors ◽  
Cell Activation ◽  
Basic Protein ◽  
Cathepsin G ◽  
Terminal Part ◽  
Down Regulation ◽  
Cxc Chemokine ◽  
Continuous Accumulation

The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR+ CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2– but not CXCR-1– dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

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