scholarly journals Discrete Role for Cytosolic Phospholipase A2α in Platelets

2002 ◽  
Vol 196 (3) ◽  
pp. 349-357 ◽  
Author(s):  
Dennis A. Wong ◽  
Yoshihiro Kita ◽  
Naonori Uozumi ◽  
Takao Shimizu
Keyword(s):  
Therapeutic Potential ◽  
Genetically Engineered ◽  
Collagen Receptors ◽  
Cytosolic Phospholipase ◽  
Genetically Engineered Mice ◽  
Cytosolic Pla2 ◽  
In Vivo Effects ◽  
Deficient Mice

Among several different types of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2)α and group IIA (IIA) secretory PLA2 (sPLA2) have been studied intensively. To determine the discrete roles of cPLA2α in platelets, we generated two sets of genetically engineered mice (cPLA2α−/−/sPLA2-IIA−/− and cPLA2α−/−/sPLA2-IIA+/+) and compared their platelet function with their respective wild-type C57BL/6J mice (cPLA2α+/+/sPLA2-IIA−/−) and C3H/HeN (cPLA2α+/+/sPLA2-IIA+/+). We found that cPLA2α is needed for the production of the vast majority of thromboxane (TX)A2 with collagen stimulation of platelets. In cPLA2α-deficient mice, however, platelet aggregation in vitro is only fractionally decreased because small amounts of TX produced by redundant phospholipase enzymes sufficiently preserve aggregation. In comparison, adenosine triphosphate activation of platelets appears wholly independent of cPLA2α and sPLA2-IIA for aggregation or the production of TX, indicating that these phospholipases are specifically linked to collagen receptors. However, the lack of high levels of TX limiting vasoconstriction explains the in vivo effects seen: increased bleeding times and protection from thromboembolism. Thus, cPLA2α plays a discrete role in the collagen-stimulated production of TX and its inhibition has a therapeutic potential against thromboembolism, with potentially limited bleeding expected.

scholarly journals Generation and Characterization of Typhoid Toxin-Neutralizing Human Monoclonal Antibodies

2020 ◽  
Vol 88 (10) ◽  
Author(s):  
Xuyao Jiao ◽  
Sarah Smith ◽  
Gabrielle Stack ◽  
Qi Liang ◽  
Allan Bradley ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Typhoid Fever ◽  
Severe Disease ◽  
Genetically Engineered ◽  
Human Monoclonal Antibodies ◽  
Genetically Engineered Mice ◽  
Typhoid Toxin

ABSTRACT Typhoid toxin is a virulence factor of Salmonella enterica serovar Typhi, the causative agent of typhoid fever, and is thought to be responsible for the symptoms of severe disease. This toxin has a unique A2B5 architecture with two active subunits, the ADP ribosyl transferase PltA and the DNase CdtB, linked to a pentameric B subunit, which is alternatively made of PltB or PltC. Here, we describe the generation and characterization of typhoid toxin-neutralizing human monoclonal antibodies by immunizing genetically engineered mice that have a full set of human immunoglobulin variable region genes. We identified several monoclonal antibodies with strong in vitro and in vivo toxin-neutralizing activity and different mechanisms of toxin neutralization. These antibodies could serve as the basis for the development of novel therapeutic strategies against typhoid fever.

scholarly journals Inducers of Senescence, Toxic Compounds, and Senolytics: The Multiple Faces of Nrf2-Activating Phytochemicals in Cancer Adjuvant Therapy

2018 ◽  
Vol 2018 ◽  
pp. 1-32 ◽  
Author(s):  
Marco Malavolta ◽  
Massimo Bracci ◽  
Lory Santarelli ◽  
Md Abu Sayeed ◽  
Elisa Pierpaoli ◽  
...  
Keyword(s):  
Adjuvant Therapy ◽  
Therapeutic Potential ◽  
Epigallocatechin Gallate ◽  
Natural Compounds ◽  
Related Factor ◽  
Phenethyl Isothiocyanate ◽  
Toxic Compounds ◽  
In Vivo Effects

The reactivation of senescence in cancer and the subsequent clearance of senescent cells are suggested as therapeutic intervention in the eradication of cancer. Several natural compounds that activate Nrf2 (nuclear factor erythroid-derived 2-related factor 2) pathway, which is involved in complex cytoprotective responses, have been paradoxically shown to induce cell death or senescence in cancer. Promoting the cytoprotective Nrf2 pathway may be desirable for chemoprevention, but it might be detrimental in later stages and advanced cancers. However, senolytic activity shown by some Nrf2-activating compounds could be used to target senescent cancer cells (particularly in aged immune-depressed organisms) that escape immunosurveillance. We herein describe in vitro and in vivo effects of fifteen Nrf2-interacting natural compounds (tocotrienols, curcumin, epigallocatechin gallate, quercetin, genistein, resveratrol, silybin, phenethyl isothiocyanate, sulforaphane, triptolide, allicin, berberine, piperlongumine, fisetin, and phloretin) on cellular senescence and discuss their use in adjuvant cancer therapy. In light of available literature, it can be concluded that the meaning and the potential of adjuvant therapy with natural compounds in humans remain unclear, also taking into account the existence of few clinical trials mostly characterized by uncertain results. Further studies are needed to investigate the therapeutic potential of those compounds that display senolytic activity.

scholarly journals Blocking gephyrin phosphorylation or microglia BDNF signaling prevents synapse loss and reduces infarct volume after ischemia

2020 ◽  
Author(s):  
Teresa Cramer ◽  
Raminder Gill ◽  
Zahra S Thirouin ◽  
Markus Vaas ◽  
Suchita Sampath ◽  
...  
Keyword(s):  
Hippocampal Formation ◽  
Infarct Volume ◽  
Point Mutations ◽  
Null Point ◽  
Genetically Engineered ◽  
Infarct Area ◽  
Synapse Plasticity ◽  
Genetically Engineered Mice

AbstractMicroglia interact with neurons to facilitate synapse plasticity; however, signal transducers between microglia and neuron remain unknown. Here, using in vitro organotypic hippocampal slice cultures and transient MCAO in genetically-engineered mice in vivo, we report that at 24 h post-ischemia microglia release BDNF to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the CA1 hippocampal formation in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75NTR and TrkB receptors respectively. Post-MCAO, we report that in the peri- infarct area and in the corresponding contralateral hemisphere similar neuroplasticity occur through microglia activation and gephyrin phosphorylation at Ser268, Ser270 in vivo. Targeted deletion of the Bdnf gene in microglia or GphnS268A/S270A (phospho-null) point-mutations protect against ischemic brain damage, neuroinflamation and synapse downregulation normally seen post-MCAO. Collectively, we report that gephyrin phosphorylation and microglia derived BDNF faciliate synapse plasticity after transient ischemia.

scholarly journals Inhibition of human cytosolic phospholipase A2 by human annexin V

1998 ◽  
Vol 329 (2) ◽  
pp. 369-372 ◽  
Author(s):  
G. Andrew BUCKLAND ◽  
C. David WILTON
Keyword(s):  
Phospholipase A2 ◽  
Annexin V ◽  
High Molecular Mass ◽  
Anionic Phospholipid ◽  
Annexin 1 ◽  
Cytosolic Phospholipase ◽  
Cytosolic Pla2 ◽  
Hydrolysis Of

The ability of annexins, particularly annexin 1 (lipocortin 1), to inhibit phospholipase A2 (PLA2) is well known and a substrate depletion mechanism is now widely accepted as the explanation for most inhibitory studies. However, there are only a very limited number of reported studies involving annexins and the high-molecular-mass cytosolic PLA2 (cPLA2). In this study we have examined the effect of human recombinant annexin V, a potentially abundant cytosolic protein, on the ability of human recombinant cPLA2 to hydrolyse a variety of phospholipid substrates. The results show clearly that, under the conditions of our study, annexin V can inhibit cPLA2 activity by a mechanism of substrate depletion and that this inhibition is dependent on the nature of the phospholipids and the concentration of Ca2+ ions in the assay. The hydrolysis of 1-stearoyl 2-arachidonyl phosphatidylcholine by cPLA2 was not significantly affected by annexin V over a range of Ca2+ concentrations (1 μM-2.5 mM), a result that presumably reflects the zwitterionic nature of the phospholipid and the known inability of annexins to bind to such interfaces. In contrast, the hydrolysis of dioleoyl phosphatidylglycerol, which is an effective anionic phospholipid substrate for this enzyme, and more significantly that of 1-stearoyl 2-arachidonyl phosphatidic acid, were readily inhibited by annexin V, although these effects were Ca2+-dependent. The Ca2+ concentrations required for inhibition in the assay system in vitro are greater than those associated with Ca2+-stimulated events within the cell, suggesting that a role for annexin V in regulating cPLA2 activity might not involve a substrate depletion mechanism in vivo unless factors in addition to Ca2+ and phospholipids contribute to the binding of annexin V to cell membranes.

scholarly journals Simple and large-scale chromosomal engineering of mouse zygotes via in vitro and in vivo electroporation

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Satoru Iwata ◽  
Hitomi Nakadai ◽  
Daisuke Fukushi ◽  
Mami Jose ◽  
Miki Nagahara ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Genetically Engineered ◽  
Guide Rna ◽  
Chromosomal Inversions ◽  
Genetically Engineered Mice ◽  
Cas9 Protein ◽  
Mouse Zygotes

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has facilitated dramatic progress in the field of genome engineering. Whilst microinjection of the Cas9 protein and a single guide RNA (sgRNA) into mouse zygotes is a widespread method for producing genetically engineered mice, in vitro and in vivo electroporation (which are much more convenient strategies) have recently been developed. However, it remains unknown whether these electroporation methods are able to manipulate genomes at the chromosome level. In the present study, we used these techniques to introduce chromosomal inversions of several megabases (Mb) in length in mouse zygotes. Using in vitro electroporation, we successfully introduced a 7.67 Mb inversion, which is longer than any previously reported inversion produced using microinjection-based methods. Additionally, using in vivo electroporation, we also introduced a long chromosomal inversion by targeting an allele in F1 hybrid mice. To our knowledge, the present study is the first report of target-specific chromosomal inversions in mammalian zygotes using electroporation.

In vitro and in vivo effects of 17β-N-(4-phenylcarbamoyl) androst-4-en-3-one derivatives as 5a-reductase inhibitors and on androgen-dependent glands

2020 ◽  
Vol 16 ◽  
Author(s):  
Marisa Cabeza ◽  
Lucero Bautista ◽  
Eugene Bratoeff ◽  
Juan Soriano ◽  
Yvonne Heuze
Keyword(s):  
Therapeutic Potential ◽  
Hamster Model ◽  
Toxicological Effects ◽  
Reductase Inhibitors ◽  
Ic50 Values ◽  
Prostate Diseases ◽  
In Vivo Effects ◽  
Vitro Effect

Background: 5α-Reductase inhibitors have proven useful for the treatment of prostate diseases, which can result from the unregulated activity of the 5α-reductase enzyme. This study was focused on determining the activity of four different derivatives of 17β-phenyl carbamoyl-androst-4-en-3-one 1–4 as inhibitors of 5α-reductase (5RD5A), to improve on the effect of current drugs. Methods: In vitro effect of compounds 1-4 on the activity of the human prostate enzyme, 5α-reductase, was determined measuring IC50 values, the concentration of a compound that inhibits the activity of 5RD5A2 by 50%. In vivo, the pharmacological effects of compounds 1-4 were identified in a hamster model of prostate hypertrophy. Results: The steroidal 17β-carboxamides 1, 3, and 4 (IC50 = 5±0.5, 0.112±0.045, 0.167±0.056 nM) significantly inhibited the in vitro activity of the 5RD5A2 enzyme with higher potency than finasteride, which is a drug known as a specific 5RD5A2 inhibitor (IC50 = 8.5±0.3 nM). Compounds 1, 3, and 4 were more potent than finasteride to decrease the size of hamster flank organs in castrated animals treated with testosterone. Also, compounds 1–4 were more effective than finasteride itself to reduce the weight of the prostate in the hamster model, without producing toxicological effects during the six days of treatment. Conclusion: In conclusion, the steroidal 17β-carboxamides 1–4 were suitable inhibitors of human 5RD5A2 activity, in addition to being able to reduce prostate weight without causing toxicity. These steroids could, therefore, have promising therapeutic potential for the treatment of benign prostatic hyperplasia.

scholarly journals Precision modeling of gall bladder cancer patients in mice based on orthotopic implantation of organoid-derived tumor buds

Oncogenesis ◽  
2021 ◽  
Vol 10 (4) ◽  
Author(s):  
Shingo Kato ◽  
Kentaro Fushimi ◽  
Yuichiro Yabuki ◽  
Yoshiaki Maru ◽  
Sho Hasegawa ◽  
...  
Keyword(s):  
Gall Bladder ◽  
Ex Vivo ◽  
Tumor Development ◽  
Genetically Engineered ◽  
Intact Cells ◽  
Genetically Engineered Mice ◽  
Carcinogenesis Models ◽  
Subcutaneous Inoculation

AbstractGenetically engineered mice (GEM) are the gold standard for cancer modeling. However, strict recapitulation of stepwise carcinogenesis from a single tumor-initiating epithelial cell among genetically intact cells in adults is not feasible with the currently available techniques using GEM. In previous studies, we partially overcame this challenge by physically isolating organs from adult animals, followed by genetic engineering in organoids and subcutaneous inoculation in nude mice. Despite the establishment of suitable ex vivo carcinogenesis models for diverse tissues, tumor development remained ectopic and occurred under immunodeficient conditions. Further refinement was, therefore, necessary to establish ideal models. Given the poor prognosis and few models owing to the lack of gall bladder (GB)-specific Cre strain, we assumed that the development of authentic models would considerably benefit GB cancer research. Here, we established a novel model using GB organoids with mutant Kras and Trp53 loss generated in vitro by lentiviral Cre transduction and CRISPR/Cas9 gene editing, respectively. Organoid-derived subcutaneous tumor fragments were sutured to the outer surface of the GB in syngeneic mice, which developed orthotopic tumors that resembled human GB cancer in histological and transcriptional features. This model revealed the infiltration of similar subsets of immune cells in both subcutaneous and orthotopic tumors, confirming the appropriate immune environment during carcinogenesis. In addition, we accurately validated the in vivo efficacy of gemcitabine, a common therapeutic agent for GB cancer, in large cohorts. Taken together, this model may serve as a promising avatar of patients with GB cancer in drug discovery and precision medicine.

Multinucleated Osteoclast Formation In Vivo and In Vitro by P2X7 Receptor-Deficient Mice

2003 ◽  
Vol 13 (2-4) ◽  
pp. 12 ◽  
Author(s):  
Alison Gartland ◽  
Katherine A. Buckley ◽  
Robert A. Hipskind ◽  
M. J. Perry ◽  
J. H. Tobias ◽  
...  
Keyword(s):  
P2x7 Receptor ◽  
Osteoclast Formation ◽  
Deficient Mice

Phytosomal Curcumin Elicits Anti-tumor Properties Through Suppression of Angiogenesis, Cell Proliferation and Induction of Oxidative Stress in Colorectal Cancer

2019 ◽  
Vol 24 (39) ◽  
pp. 4626-4638 ◽  
Author(s):  
Reyhaneh Moradi-Marjaneh ◽  
Seyed M. Hassanian ◽  
Farzad Rahmani ◽  
Seyed H. Aghaee-Bakhtiari ◽  
Amir Avan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Colorectal Cancer ◽  
Therapeutic Potential ◽  
Vegf Signaling ◽  
Anticancer Effects ◽  
Inhibition Of Angiogenesis ◽  
Underlying Mechanisms ◽  
Apoptotic Activity

Background: Colorectal cancer (CRC) is one of the most common causes of cancer-associated mortality in the world. Anti-tumor effect of curcumin has been shown in different cancers; however, the therapeutic potential of novel phytosomal curcumin, as well as the underlying molecular mechanism in CRC, has not yet been explored. Methods: The anti-proliferative, anti-migratory and apoptotic activity of phytosomal curcumin in CT26 cells was assessed by MTT assay, wound healing assay and Flow cytometry, respectively. Phytosomal curcumin was also tested for its in-vivo activity in a xenograft mouse model of CRC. In addition, oxidant/antioxidant activity was examined by DCFH-DA assay in vitro, measurement of malondialdehyde (MDA), Thiol and superoxidedismutase (SOD) and catalase (CAT) activity and also evaluation of expression levels of Nrf2 and GCLM by qRT-PCR in tumor tissues. In addition, the effect of phytosomal curcumin on angiogenesis was assessed by the measurement of VEGF-A and VEGFR-1 and VEGF signaling regulatory microRNAs (miRNAs) in tumor tissue. Results: Phytosomal curcumin exerts anti-proliferative, anti-migratory and apoptotic activity in-vitro. It also decreases tumor growth and augmented 5-fluorouracil (5-FU) anti-tumor effect in-vivo. In addition, our data showed that induction of oxidative stress and inhibition of angiogenesis through modulation of VEGF signaling regulatory miRNAs might be underlying mechanisms by which phytosomal curcumin exerted its antitumor effect. Conclusion: Our data confirmed this notion that phytosomal curcumin administrates anticancer effects and can be used as a complementary treatment in clinical settings.

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