scholarly journals Homotypic Lysosome Fusion in Macrophages: Analysis Using an In Vitro Assay

1997 ◽  
Vol 139 (3) ◽  
pp. 665-673 ◽  
Author(s):  
Diane M. Ward ◽  
Jonathan D. Leslie ◽  
Jerry Kaplan
Keyword(s):  
Fusion Reaction ◽  
In Vitro Assay ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Toxin A ◽  
Concentration Dependent Manner ◽  
Vitro Assay ◽  
Sensitive Factor

Lysosomes are dynamic structures capable of fusing with endosomes as well as other lysosomes. We examined the biochemical requirements for homotypic lysosome fusion in vitro using lysosomes obtained from rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E. The in vitro assay measures the formation of a biotinylated HRP–avidin conjugate, in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis. We determined that lysosome fusion in vitro was time- and temperature-dependent and required ATP and an N-ethylmaleimide (NEM)-sensitive factor from cytosol. The NEM-sensitive factor was NSF as purified recombinant NSF could completely replace cytosol in the fusion assay whereas a dominant-negative mutant NSF inhibited fusion. Fusion in vitro was extensive; up to 30% of purified macrophage lysosomes were capable of self-fusion. Addition of GTPγs to the in vitro assay inhibited fusion in a concentration-dependent manner. Purified GDP-dissociation inhibitor inhibited homotypic lysosome fusion suggesting the involvement of rabs. Fusion was also inhibited by the heterotrimeric G protein activator mastoparan, but not by its inactive analogue Mas-17. Pertussis toxin, a Gαi activator, inhibited in vitro lysosome fusion whereas cholera toxin, a Gαs activator did not inhibit the fusion reaction. Addition of agents that either promoted or disrupted microtubule function had little effect on either the extent or rate of lysosome fusion. The high value of homotypic fusion was supported by in vivo experiments examining lysosome fusion in heterokaryons formed between cells containing fluorescently labeled lysosomes. In both macrophages and J774E cells, almost complete mixing of the lysosome labels was observed within 1–3 h of UV sendai-mediated cell fusion. These studies provide a model system for identifying the components required for lysosome fusion.

Early mouse endoderm is patterned by soluble factors from adjacent germ layers

Development ◽  
2000 ◽  
Vol 127 (8) ◽  
pp. 1563-1572 ◽  
Author(s):  
J.M. Wells ◽  
D.A. Melton
Keyword(s):  
Primitive Streak ◽  
In Vitro Assay ◽  
Specific Gene ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Germ Layers ◽  
Vitro Assay ◽  
Organ Specific ◽  
Early Mouse

Endoderm that forms the respiratory and digestive tracts is a sheet of approximately 500–1000 cells around the distal cup of an E7.5 mouse embryo. Within 2 days, endoderm folds into a primitive gut tube from which numerous organs will bud. To characterize the signals involved in the developmental specification of this early endoderm, we have employed an in vitro assay using germ layer explants and show that adjacent germ layers provide soluble, temporally specific signals that induce organ-specific gene expression in endoderm. Furthermore, we show that FGF4 expressed in primitive streak-mesoderm can induce the differentiation of endoderm in a concentration-dependent manner. We conclude that the differentiation of gastrulation-stage endoderm is directed by adjacent mesoderm and ectoderm, one of the earliest reported patterning events in formation of the vertebrate gut tube.

scholarly journals Fgr but not Syk tyrosine kinase is a target for beta2 integrin-induced c-Cbl-mediated ubiquitination in adherent human neutrophils

2003 ◽  
Vol 370 (2) ◽  
pp. 687-694 ◽  
Author(s):  
Fredrik MELANDER ◽  
Tommy ANDERSSON ◽  
Karim DIB
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Src Kinase ◽  
E3 Ligase ◽  
In Vitro Assay ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Β2 Integrin

An early and critical event in β2 integrin signalling during neutrophil adhesion is activation of Src tyrosine kinases and Syk. In the present study, we report Src kinase-dependent β2 integrin-induced tyrosine phosphorylation of Cbl occurring in parallel with increased Cbl-associated tyrosine kinase activity. These events concurred with activation of Fgr and, surprisingly, also with dissociation of this Src tyrosine kinase from Cbl. Moreover, the presence of the Src kinase inhibitor PP1 in an in vitro assay had only a limited effect on the Cbl-associated kinase activity. These results suggest that an additional active Src-dependent tyrosine kinase associates with Cbl. The following observations imply that Syk is such a kinase: (i) β2 integrins activated Syk in a Src-dependent manner, (ii) Syk was associated with Cbl much longer than Fgr was, and (iii) the Syk inhibitor piceatannol (3,4,3′,5′-tetrahydroxy-trans-stilbene) abolished the Cbl-associated kinase activity in an in vitro assay. Effects of the mentioned interactions between these two kinases and Cbl may be related to the finding that Cbl is a ubiquitin E3 ligase. Indeed, we detected β2 integrin-induced ubiquitination of Fgr that, similar to the phosphorylation of Cbl, was abolished in cells pretreated with PP1. However, the ubiquitination of Fgr did not cause any apparent degradation of the protein. In contrast with Fgr, Syk was not modified by the E3 ligase. Thus Cbl appears to be essential in β2 integrin signalling, first by serving as a matrix for a subsequent agonist-induced signalling interaction between Fgr and Syk, and then by mediating ubiquitination of Fgr which possibly affects its interaction with Cbl.

scholarly journals Effect of Massoia (Massoia aromatica Becc.) Bark on the Phagocytic Activity of Wistar Rat Macrophages

2018 ◽  
Author(s):  
Triana Hertiani ◽  
Agustinus Yuswanto ◽  
Sylvia Utami Tunjung Pratiwi ◽  
Harlyanti Mashar
Keyword(s):  
Phagocytic Activity ◽  
Wistar Rats ◽  
In Vitro Assay ◽  
Phagocytic Index ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Macrophage Cells ◽  
In Vivo Assay

Massoia (Massoia aromatica Becc., Lauraceae) bark has been widely used as a component of traditional Indonesian medicine. The indigenous people boil or steam the bark for traditional applications. Our preliminary research revealed the potency of Massoia essential oil and its major compound, C-10 Massoialactone as potential immunomodulator in vitro. However, no scientific evidence regarding its in vivo effects is available. Therefore, this study evaluated the potential immunomodulatory effects of Massoia bark infusion on the nonspecific immune response (phagocytosis) of Wistar rats. The aqueous extract of Massoia bark was obtained by boiling pulverized bark in water, and the C-10 massoialactone content of the extract was determined through Thin Layer Chromatography (TLC) densitometry. For the in vitro assay, macrophages were treated with the freeze-dried infusion at the concentrations of 2.5, 5, 10, 20, or 40 μg/mL media. For the in vivo assay, 2-month-old male Wistar rats were divided into 5 groups. The baseline group received distilled water at the dose of 1 mL/100 g BW with the immunostimulant herbal product “X” administered as the positive control at the dose of 0.54 mL/rat. The treatment groups received the infusion at a dose of 100, 300, or 500 mg/100 g BW. Treatments were given orally every day for 14 days. The ability of macrophage cells to phagocyte latex was determined as phagocytic index (PI) and was observed under microscopy with 300 macrophages. The in vitro study revealed that the phagocytic activity of the infusion-treated macrophages significantly increased in comparison with that of the control macrophages in a concentration-dependent manner. Among all treatment concentrations, the concentration of 40 μg/ml provided the highest activity with a PI value of 70.51% ± 1.11%. The results of the in vivo assay confirmed those of the in vitro assay. The results of the present study indicate that Massoia bark can increase the phagocytic activity of rat macrophage cells. Its potential as a naturally derived immunomodulatory agent requires further study.

Epitope specificity and isotype of monoclonal anti-D antibodies dictate their ability to inhibit phagocytosis of opsonized platelets

Blood ◽  
2007 ◽  
Vol 110 (4) ◽  
pp. 1359-1361 ◽  
Author(s):  
Mimi Kjaersgaard ◽  
Rukhsana Aslam ◽  
Michael Kim ◽  
Edwin R. Speck ◽  
John Freedman ◽  
...  
Keyword(s):  
Immune Globulin ◽  
In Vitro Assay ◽  
Dependent Manner ◽  
Hemolytic Disease ◽  
Thrombocytopenic Purpura ◽  
Vitro Assay ◽  
Autoimmune Thrombocytopenic Purpura ◽  
Epitope Specificity ◽  
Primary Indication

Abstract Rh immune globulin (WinRho SDF; Cangene, Mississauga, ON, Canada) is an effective treatment for autoimmune thrombocytopenic purpura; however, maintaining a sustained supply for its use in autoimmune thrombocytopenic purpura and its primary indication, hemolytic disease of the newborn, makes the development of alternative reagents desirable. We compared Rh immune globulin and 6 human monoclonal anti-D antibodies (MoAnti-D) with differing isotypes and specificities for their ability to opsonize erythrocytes and inhibit platelet phagocytosis in an in vitro assay. Results demonstrated that opsonization of erythrocytes with Rh immune globulin significantly (P < .001) reduced phagocytosis of fluorescently labeled opsonized platelets in an Fc-dependent manner. Of the MoAnti-D that shared specificity but differed in isotype, only IgG3 antibodies could significantly (P < .001) inhibit platelet phagocytosis. In contrast, 2 MoAnti-D shared isotypes and differed in specificity; however, only one could significantly (P < .001) inhibit platelet phagocytosis. The results suggest that MoAnti-D epitope specificity and isotypes are critical requirements for optimal inhibition of opsonized platelet phagocytosis.

scholarly journals Negative Regulation of the Antimetastatic Gene Nm23-H1 by Thyroid Hormone Receptors*

Endocrinology ◽  
2000 ◽  
Vol 141 (7) ◽  
pp. 2540-2547 ◽  
Author(s):  
Kwang-huei Lin ◽  
Hsing-ying Shieh ◽  
Hai-Chu Hsu
Keyword(s):  
Thyroid Hormone ◽  
Messenger Rna ◽  
Promoter Region ◽  
Tumor Metastasis ◽  
Thyroid Hormone Receptor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Receptor Subtype ◽  
Dependent Manner ◽  
Vitro Assay

Metastasis of various malignant cells is inversely related to the abundance of the Nm23-H1 protein. The possible role of thyroid hormones in tumor metastasis has now been investigated by examining the effect of T3 on the expression of the Nm23-H1 gene. Human hepatoma HepG2 cells, in which endogenous thyroid hormone receptor subtype α1 (TRα1) is expressed at a low level, were stably transfected, either with expression plasmids encoding wild-type TRα1 or a dominant negative mutant of TRα1, or with the empty vector (yielding HepG2-Wt, HepG2-Mt, and HepG2-Neo cells, respectively). Immunoblot analysis revealed that exposure of HepG2-Wt and HepG2-Neo cells, but not HepG2-Mt cells, to T3-induced time-dependent decreases in the abundance of Nm23-H1 messenger RNA and protein, with the extent of these effects correlating with the level of expression of TRα1. An in vitro assay also revealed that T3 induced a marked increase in the invasive activity of HepG2-Wt cells; it induced a smaller increase in that of HepG2-Neo cells but had no effect on that of HepG2-Mt cells. Finally, the promoter region of Nm23-H1 spanning nucleotides −471 to− 437 (relative to the transcriptional initiation site) inhibited the expression of a downstream reporter gene, in a T3-dependent manner, in COS-1 cells also transfected with an expression plasmid encoding TRα1 or TRβ1. The DNA binding domain of TRβ1 was required for this inhibitory effect. These results indicate that T3, acting through TRs, inhibits transcription of Nm23-H1, and that this effect is mediated by a negative regulatory element in the promoter region of the gene. Thus, it is possible that T3 promotes tumor metastasis by inducing down-regulation of Nm23-H1 expression.

scholarly journals Evaluation of the anthelmintic properties of a traditional remedy based on a mixture of red algae using an in vitro assay on gastrointestinal nematodes of donkeys

2021 ◽  
Vol 4 (1) ◽  
pp. 1-7
Author(s):  
Michela Maestrini ◽  
◽  
Marcelo Beltrão Molento ◽  
Simone Mancini ◽  
Francesco Saverio Robustelli della Cuna ◽  
...  
Keyword(s):  
Red Algae ◽  
Gastrointestinal Nematode ◽  
Gastrointestinal Nematodes ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Egg Hatch ◽  
Vitro Assay ◽  
Reference Drug ◽  
Negative Controls

The anthelmintic properties and composition of an Italian traditional anthelmintic remedy based on a red algae mixture (RAE) was assessed using the egg hatch test (EHT). The ability of different dilutions \((1.0, 5.0, 50, or 100%)\) of RAE was determined and compared with the positive and negative controls against gastrointestinal nematode (GIN) of donkeys. The experiment was performed in triplicate. Data were analysed using the ANOVA and Tukey test. In the mixture, Palisada tenerrima, Laurencia intricata and Laurencia spp. red algae were identified. The \(100%\) RAE was able to totally inhibit the egg hatch, showing an efficacy comparable \((P < 0.05)\) to that of the reference drug \((98.7%)\). An egg hatch reduction of \(89.5, 43.7\), and \(23.4%\) was observed at \(50, 5\) and \(1%\) dilutions, respectively. In conclusion, RAE was able to inhibit the egg hatch of GIN of donkeys in a concentration-dependent manner with a correlation coefficient \((R2)\) of \(0.968\), corroborating with its anthelmintic effect.

scholarly journals Insulin-Induced Phosphorylation and Activation of Cyclic Nucleotide Phosphodiesterase 3B by the Serine-Threonine Kinase Akt

1999 ◽  
Vol 19 (9) ◽  
pp. 6286-6296 ◽  
Author(s):  
Tadahiro Kitamura ◽  
Yukari Kitamura ◽  
Shoji Kuroda ◽  
Yasuhisa Hino ◽  
Miwa Ando ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Second Messengers ◽  
Dominant Negative ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Serine Threonine Kinase ◽  
Intact Cells ◽  
Threonine Kinase

ABSTRACT Cyclic nucleotide phosphodiesterase (PDE) is an important regulator of the cellular concentrations of the second messengers cyclic AMP (cAMP) and cGMP. Insulin activates the 3B isoform of PDE in adipocytes in a phosphoinositide 3-kinase-dependent manner; however, downstream effectors that mediate signaling to PDE3B remain unknown. Insulin-induced phosphorylation and activation of endogenous or recombinant PDE3B in 3T3-L1 adipocytes have now been shown to be inhibited by a dominant-negative mutant of the serine-threonine kinase Akt, suggesting that Akt is necessary for insulin-induced phosphorylation and activation of PDE3B. Serine-273 of mouse PDE3B is located within a motif (RXRXXS) that is preferentially phosphorylated by Akt. A mutant PDE3B in which serine-273 was replaced by alanine was not phosphorylated either in response to insulin in intact cells or by purified Akt in vitro. In contrast, PDE3B mutants in which alanine was substituted for either serine-296 or serine-421, each of which lies within a sequence (RRXS) preferentially phosphorylated by cAMP-dependent protein kinase, were phosphorylated by Akt in vitro or in response to insulin in intact cells. Moreover, the serine-273 mutant of PDE3B was not activated by insulin when expressed in adipocytes. These results suggest that PDE3B is a physiological substrate of Akt and that Akt-mediated phosphorylation of PDE3B on serine-273 is important for insulin-induced activation of PDE3B.

scholarly journals Monocytes Are Highly Sensitive to Clostridium difficile Toxin A-Induced Apoptotic and Nonapoptotic Cell Death

2005 ◽  
Vol 73 (3) ◽  
pp. 1625-1634 ◽  
Author(s):  
K. Solomon ◽  
J. Webb ◽  
N. Ali ◽  
R. A. Robins ◽  
Y. R. Mahida
Keyword(s):  
Cell Death ◽  
Clostridium Difficile ◽  
Mitochondrial Permeability Transition ◽  
Specific Effect ◽  
Permeability Transition ◽  
Dependent Manner ◽  
Toxin A ◽  
Concentration Dependent Manner ◽  
Peripheral Blood Mononuclear

ABSTRACT In this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes to Clostridium difficile toxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time- and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to >100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochrome c was released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 μg/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated that C. difficile toxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.

scholarly journals Evaluation of Anti-Mycobacterial Compounds in a Silkworm Infection Model with Mycobacteroides abscessus

Molecules ◽  
2020 ◽  
Vol 25 (21) ◽  
pp. 4971
Author(s):  
Kanji Hosoda ◽  
Nobuhiro Koyama ◽  
Hiroshi Hamamoto ◽  
Akiho Yagi ◽  
Ryuji Uchida ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
In Vitro Assay ◽  
Infection Model ◽  
Therapeutic Effects ◽  
Dependent Manner ◽  
Vitro Assay ◽  
Infection Assay ◽  
Microbial Products ◽  
Dose Dependent

Among four mycobacteria, Mycobacterium avium, M. intracellulare, M. bovis BCG and Mycobacteroides (My.) abscessus, we established a silkworm infection assay with My. abscessus. When silkworms (fifth-instar larvae, n = 5) were infected through the hemolymph with My. abscessus (7.5 × 107 CFU/larva) and bred at 37 °C, they all died around 40 h after injection. Under the conditions, clarithromycin and amikacin, clinically used antimicrobial agents, exhibited therapeutic effects in a dose-dependent manner. Furthermore, five kinds of microbial compounds, lariatin A, nosiheptide, ohmyungsamycins A and B, quinomycin and steffimycin, screened in an in vitro assay to observe anti-My. abscessus activity from 400 microbial products were evaluated in this silkworm infection assay. Lariatin A and nosiheptide exhibited therapeutic efficacy. The silkworm infection model with My. abscessus is useful to screen for therapeutically effective anti-My. abscessus antibiotics.

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