scholarly journals Peripheral Hybrid CB1R and iNOS Antagonist MRI-1867 Displays Anti-Fibrotic Efficacy in Bleomycin-Induced Skin Fibrosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Charles N. Zawatsky ◽  
Joshua K. Park ◽  
Jasmina Abdalla ◽  
George Kunos ◽  
Malliga R. Iyer ◽  
...  
Keyword(s):  
Therapeutic Efficacy ◽  
Skin Fibrosis ◽  
Mouse Strains ◽  
Drug Efflux ◽  
Kidney Fibrosis ◽  
Knock Out ◽  
Drug Candidates ◽  
Target Exposure ◽  
Inos Inhibitor ◽  
Increased Activity

Scleroderma, or systemic sclerosis, is a multi-organ connective tissue disease resulting in fibrosis of the skin, heart, and lungs with no effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) and increased activity of inducible NO synthase (iNOS) promote tissue fibrosis including skin fibrosis, and joint targeting of these pathways may improve therapeutic efficacy. Recently, we showed that in mouse models of liver, lung and kidney fibrosis, treatment with a peripherally restricted hybrid CB1R/iNOS inhibitor (MRI-1867) yields greater anti-fibrotic efficacy than inhibiting either target alone. Here, we evaluated the therapeutic efficacy of MRI-1867 in bleomycin-induced skin fibrosis. Skin fibrosis was induced in C57BL/6J (B6) and Mdr1a/b-Bcrp triple knock-out (KO) mice by daily subcutaneous injections of bleomycin (2 IU/100 µL) for 28 days. Starting on day 15, mice were treated for 2 weeks with daily oral gavage of vehicle or MRI-1867. Skin levels of MRI-1867 and endocannabinoids were measured by mass spectrometry to assess target exposure and engagement by MRI-1867. Fibrosis was characterized histologically by dermal thickening and biochemically by hydroxyproline content. We also evaluated the potential increase of drug-efflux associated ABC transporters by bleomycin in skin fibrosis, which could affect target exposure to test compounds, as reported in bleomycin-induced lung fibrosis. Bleomycin-induced skin fibrosis was comparable in B6 and Mdr1a/b-Bcrp KO mice. However, the skin level of MRI-1867, an MDR1 substrate, was dramatically lower in B6 mice (0.023 µM) than in Mdr1a/b-Bcrp KO mice (8.8 µM) due to a bleomycin-induced increase in efflux activity of MDR1 in fibrotic skin. Furthermore, the endocannabinoids anandamide and 2-arachidonylglycerol were elevated 2-4-fold in the fibrotic vs. control skin in both mouse strains. MRI-1867 treatment attenuated bleomycin-induced established skin fibrosis and the associated increase in endocannabinoids in Mdr1a/b-Bcrp KO mice but not in B6 mice. We conclude that combined inhibition of CB1R and iNOS is an effective anti-fibrotic strategy for scleroderma. As bleomycin induces an artifact in testing antifibrotic drug candidates that are substrates of drug-efflux transporters, using Mdr1a/b-Bcrp KO mice for preclinical testing of such compounds avoids this pitfall.

scholarly journals Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1391
Author(s):  
Brent Race ◽  
Katie Williams ◽  
Chase Baune ◽  
James F. Striebel ◽  
Clayton W. Winkler ◽  
...  
Keyword(s):  
Prion Disease ◽  
Molecular Motors ◽  
Prion Diseases ◽  
Mouse Strains ◽  
Survival Times ◽  
Knock Out ◽  
Transport Vesicles ◽  
The Central Nervous System ◽  
Knock Out Mice ◽  
Scrapie Strains

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c−/−) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c−/− mice.

scholarly journals Therapeutic Efficacy of Antibacterial Ocellatin Peptides- A Comprehensive Review

2021 ◽  
Vol 12 (5) ◽  
pp. 6804-6814
Keyword(s):  
Therapeutic Efficacy ◽  
Innate Immune ◽  
South American ◽  
Hydrophobic Residues ◽  
Microbial Drug Resistance ◽  
Bacterial Membranes ◽  
Drug Candidates ◽  
Small Molecule Drugs ◽  
Living Organisms ◽  
Dynamic Interplay

Antimicrobial peptides (AMPs), ascribed to their decreased microbial drug resistance, can be employed as potent small-molecule drugs to treat various diseases. AMPs have been conserved in a wide variety of living organisms as a result of the evolution of the innate immune system. Notably, Ocellatin AMPs derived from South American Leptodactylus genus frogs have a higher therapeutic efficacy against infections. Inhibitory activity of Ocellatin AMPs against bacterial membranes is determined by the dynamic interplay of peptide cationic, hydrophobicity, helicity, and amphipathicity. Another advantage of using AMPs as drug candidates is their cell selectivity that is non-hemolytic to human cells. Ocellatin AMPs with optimal hydrophobic residues would therefore be a recommended therapeutic candidate. Henceforth, such AMPs could be used as an alternative strategy in curbing antimicrobial resistance. It is noteworthy that the therapeutic efficacy of Ocellatins is to be appreciated for its broad application as it has been proved to be active against several humans, animal, and plant bacterial pathogens.

Abstract 13469: Drp1 Accumulates in Mitochondria and Plays a Protective Role in the Heart in Response to Pressure Overload

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Akihiro Shirakabe ◽  
Yoshiyuki Ikeda ◽  
Peiyong Zai ◽  
Junichi Sadoshima
Keyword(s):  
Diastolic Pressure ◽  
Pressure Overload ◽  
Protective Role ◽  
Left Ventricular ◽  
Multiple Time ◽  
Lung Weight ◽  
Knock Out ◽  
Multiple Time Points ◽  
Increased Activity ◽  
Adaptive Role

Dynamin-related protein 1 (Drp1) plays an essential role in maintaining the quality control of mitochondria through mitochondrial (Mt) fission and mitophagy. We investigated how Mt function, autophagy and Drp1 are regulated in the heart during pressure overload (PO) and whether endogenous Drp1 plays an important role in regulating cardiac function. Mice were subjected to transverse aortic constriction (TAC) at multiple time points between 6 hours and 30 days. Left ventricular (LV) weight/tibial length (LVW/TL) was significantly elevated at Day 7 (TAC vs sham; 5.92 ± 0.27 vs 4.22 ± 0.12, p<0.05). Ejection fraction (EF) was maintained at Day 7, but gradually decreased thereafter (at 30 days; 65±9 vs 83±9 %, p<0.05). LC3-II was decreased (-45.7%, p<0.05) while p62 accumulated (1.17 fold, p<0.05) significantly at Day 7. Both Mt ATP content (-65.6%, p<0.05) and production (-90.3%, p<0.05) were reduced significantly at Days 7 and 14, respectively, and thereafter. Mt mass, evaluated by electron microscopy, was also reduced (-19.9%, p<0.05) at Day 7. Drp1 accumulated in mitochondria at Day 7, and S616 phosphorylation of Drp1, associated with increased activity, was increased at Day 7. Thus, PO suppresses autophagy and induces Mt dysfunction by Day 7, at which time Drp1 accumulates in mitochondria and Mt mass is decreased. To examine the functional significance of endogenous Drp1 during PO, cardiac-specific heterozygous Drp1 knock out (Drp-hetCKO) mice were subjected to TAC. At Day 7, decreases in EF (61± 2 vs 84 ± 7%, p<0.05) and increases in LVW/TL (7.22 ± 0.26 vs 5.86 ± 0.65, p<0.05) and lung weight/TL (12.01 ± 1.10 vs 6.31 ± 1.19, p<0.05) were exacerbated in Drp-hetCKO compared to in control mice. LV end diastolic pressure was significantly higher (22.0 ± 2.8 vs 5.7 ± 2.9 mmHg, p<0.05) and myocardial fibrosis (14.1 ± 2.5 vs 6.2 ± 4.3 %, p<0.05) was greater in Drp-hetCKO than in control mice. Mt mass was also significantly greater in Drp-hetCKO than in control mice (relative Mt mass, 1.21 ± 0.46 vs 1.00 ± 0.02, p<0.05). These results suggest that PO inhibits autophagy and induces mitochondrial dysfunction by Day7, which coincides with Mt accumulation of Drp1. Drp1 plays an adaptive role in this condition, mediating decreases in Mt mass and protecting the heart from dysfunction.

Roles of GABA Signaling in Directing Basket Cell Axonal Projections Toward Purkinje Cells in the Cerebellum

2020 ◽  
Author(s):  
Hideki Miwa ◽  
Ken Kobayashi ◽  
Shinobu Hirai ◽  
Mitsuhiko Yamada ◽  
Masahiko Watanabe ◽  
...  
Keyword(s):  
Purkinje Cells ◽  
Motor Coordination ◽  
Basket Cell ◽  
K Channel ◽  
Mouse Strains ◽  
Gamma Aminobutyric Acid ◽  
Knock Out ◽  
Inhibitory Neurotransmitter ◽  
Basket Cells ◽  
Gaba Signaling

Abstract Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD67. GABA may act as a trophic factor during brain development, but its contribution to the development and maturation of cerebellar neural circuits is not known. To understand the roles of GABA in cerebellar development and associated functions in motor coordination and balance, we examined GAD65 conventional knock out (KO) mice and mice in which GAD67 was eliminated in parvalbumin-expressing neurons ( PV-Cre ; GAD67 flox/flox mice). We found aberrant subcellular localization of the Shaker-type K channel Kv1.1 in basket cell collaterals of PV-Cre ; GAD67 flox/flox mice and abnormal projections from basket cells to Purkinje cells in both mouse strains. Furthermore, PV-Cre ; GAD67 flox/flox mice exhibited abnormal motor coordination in the rotarod test. These results indicate that GABA signaling in the cerebellum during development is critical for establishing appropriate connections between basket cells and Purkinje cells and is associated with motor coordination in mice.

scholarly journals Attenuated apoptotic BAX expression as a xenobiotic reporter in Saccharomyces cerevisiae

2019 ◽  
Vol 19 (5) ◽  
Author(s):  
Mikhail V Keniya ◽  
Brian C Monk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fungal Infections ◽  
Pathogenic Fungi ◽  
Cross Resistance ◽  
Drug Efflux ◽  
Drug Candidates ◽  
Drug Efflux Pumps ◽  
Drug Dependent ◽  
Prophylactic Use ◽  
Translation Coupling

ABSTRACT Fungal infections are a major challenge to medicine and agriculture. Repeated and prophylactic use of antifungals can lead to pathogen cross-resistance to different classes of drugs. The early development of multidrug resistance in pathogenic fungi includes drug tolerance mediated by drug-dependent activation of drug efflux. In Saccharomyces cerevisiae and the fungal pathogen Candida glabrata, xenobiotic sensing motifs in transcription factors upregulate expression of several ATP-binding cassette (ABC) drug efflux pumps. We have therefore considered how drug candidates that trigger or prevent drug resistance could be identified and evaluated during drug discovery. We report a robust and sensitive, S. cerevisiae-based xenobiotic sensing system using the Pdr1 protein as a sensor and an attenuated version of the apoptotic murine BCL2-associated X (BAX) gene as a reporter. A molecular mechanism of attenuation that involves frameshift reversal may be associated with translation coupling and requires further investigation.

Factor IX: Insights from knock-out and genetically engineered mice

2008 ◽  
Vol 100 (10) ◽  
pp. 563-575 ◽  
Author(s):  
Paul E. Monahan
Keyword(s):  
Mouse Models ◽  
Positional Cloning ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Factor Ix ◽  
Mouse Strains ◽  
Knock Out ◽  
Haemophilia B ◽  
Genetically Engineered Mice ◽  
Knock Out Mice

SummaryThe study of coagulation factors has been rapidly advanced by studies performed in genetically engineered mouse strains. Investigation of factor IX (FIX) has benefited from excellent genedeleted mouse models that recapitulate many of the features of human haemophilia B. Moreover, advanced positional cloning techniques and availability of technology to allow not only knock-out mice, but also knock-in and knock-down mice, provide new opportunities to observe genotype-phenotype and structure-function correlations regarding FIX, as well as the interaction of FIX with inflammatory, immune, and tissue repair systems. In this paper, available FIX knock-out mice and additional haemophilia B mouse models are reviewed specifically in regards to observations these models have facilitated concerning: factor IX gene expression and factor IX protein pharmacokinetics; the role of FIX in haemostasis, thrombosis and wound healing; insights into coagulation FIX arising out of gene therapy applications in haemophilia mouse models; immunology of tolerance or loss of tolerance of FIX and inhibitor antibody formation.

Abstract 168: Drp1 Accumulates in Mitochondria and Plays a Protective Role in the Heart in Response to Pressure Overload

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Akihiro Shirakabe ◽  
Yoshiyuki Ikeda ◽  
Toshiro Saito ◽  
Peiyong Zhai ◽  
Junichi Sadoshima
Keyword(s):  
Diastolic Pressure ◽  
Pressure Overload ◽  
Protective Role ◽  
Left Ventricular ◽  
Multiple Time ◽  
Lung Weight ◽  
Knock Out ◽  
Multiple Time Points ◽  
Increased Activity ◽  
Adaptive Role

Dynamin-related protein 1 (Drp1) plays an essential role in maintaining the quality control of mitochondria through mitochondrial (Mt) fission and mitophagy. We investigated how Mt function, autophagy and Drp1 are regulated in the heart during pressure overload (PO) and whether endogenous Drp1 plays an important role in regulating cardiac function. Mice were subjected to transverse aortic constriction (TAC) at multiple time points between 6 hours and 30 days. Left ventricular (LV) weight/tibial length (LVW/TL) was significantly elevated at Day 5 (TAC vs Baseline; 6.21 ± 0.28 vs 4.59 ± 0.36, p<0.05). Ejection fraction (EF) was maintained at Day 5 (79±5 vs 82±7%), but gradually decreased thereafter (30 days; 51±12%, p<0.05). LC3-II was decreased (-40.0%, p<0.05) while p62 accumulated (1.84 fold, p<0.05) significantly at Day 5. Both Mt ATP content (-65.6%, p<0.05) and production (-90.3%, p<0.05) were reduced significantly at Days 7 and 14, respectively, and thereafter. Mt mass, evaluated by electron microscopy, was also reduced (-19.9%, p<0.05) at Day 7. Drp1 accumulated in mitochondria at Day 7, and S616 phosphorylation of Drp1, associated with increased activity, was increased at Day 7. Thus, PO suppresses autophagy and induces Mt dysfunction by Day 7, at which time Drp1 accumulates in mitochondria and Mt mass is decreased. To examine the functional significance of endogenous Drp1 during PO, cardiac-specific heterozygous Drp1 knock out (Drp-hetCKO) mice were subjected to TAC. At Day 7, decreases in EF (57± 11 vs 80 ± 7%, p<0.05) and increases in LVW/TL (7.22 ± 0.26 vs 5.86 ± 0.65, p<0.05) and lung weight/TL (13.03 ± 1.09 vs 7.00 ± 1.31, p<0.05) were exacerbated in Drp-hetCKO compared to in control mice. LV end diastolic pressure was significantly higher (20.0 ± 5.7 vs 7.4 ± 3.1 mmHg, p<0.05) and myocardial fibrosis (14.1 ± 2.5 vs 6.2 ± 4.3 %, p<0.05) was greater, and Mt mass was also significantly greater in Drp-hetCKO than in control mice (relative Mt mass, 1.21 ± 0.46 vs 1.00 ± 0.02, p<0.05). These results suggest that PO inhibits autophagy and induces mitochondrial dysfunction by Day7, which coincides with Mt accumulation of Drp1. Drp1 plays an adaptive role in this condition, mediating decreases in Mt mass and protecting the heart from dysfunction.

scholarly journals Acetyl:Succinate CoA-transferase in ProcyclicTrypanosoma brucei

2004 ◽  
Vol 279 (44) ◽  
pp. 45337-45346 ◽  
Author(s):  
Loïc Rivière ◽  
Susanne W. H. van Weelden ◽  
Patricia Glass ◽  
Patricia Vegh ◽  
Virginie Coustou ◽  
...  
Keyword(s):  
Protozoan Parasite ◽  
Glucose Consumption ◽  
Population Level ◽  
Gene Encoding ◽  
Knock Out ◽  
Genetic Ablation ◽  
Quantitative Correlation ◽  
Acetate Excretion ◽  
Coa Transferase ◽  
Increased Activity

Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasiteTrypanosoma bruceiwas identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Δasct/Δasctknock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of β-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of13C-enriched and14C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate inT. brucei.

scholarly journals C57BL/6 mice require a higher dose of cisplatin to induce renal fibrosis and CCL2 correlates with cisplatin-induced kidney injury

2020 ◽  
Vol 319 (4) ◽  
pp. F674-F685 ◽  
Author(s):  
Sophia M. Sears ◽  
Cierra N. Sharp ◽  
Austin Krueger ◽  
Gabrielle B. Oropilla ◽  
Douglas Saforo ◽  
...  
Keyword(s):  
Low Dose ◽  
Immune Cell ◽  
Interstitial Fibrosis ◽  
Kidney Injury ◽  
Mouse Strains ◽  
Dosing Regimen ◽  
Littermate Control ◽  
Kidney Fibrosis ◽  
Chemokine Ligand ◽  
Multiple Strains

C57BL/6 mice are one of the most commonly used mouse strains in research, especially in kidney injury studies. However, C57BL/6 mice are resistant to chronic kidney disease-associated pathologies, particularly the development of glomerulosclerosis and interstitial fibrosis. Our laboratory and others developed a more clinically relevant dosing regimen of cisplatin (7 mg/kg cisplatin once a week for 4 wk and mice euthanized at day 24) that leads to the development of progressive kidney fibrosis in FVB/n mice. However, we found that treating C57BL/6 mice with this same dosing regimen does not result in kidney fibrosis. In this study, we demonstrated that increasing the dose of cisplatin to 9 mg/kg once a week for 4 wk is sufficient to consistently induce fibrosis in C57BL/6 mice while maintaining animal survival. In addition, we present that cohorts of C57BL/6 mice purchased from Jackson 1 yr apart and mice bred in-house display variability in renal outcomes following repeated low-dose cisplatin treatment. Indepth analyses of this intra-animal variability revealed C-C motif chemokine ligand 2 as a marker of cisplatin-induced kidney injury through correlation studies. In addition, significant immune cell infiltration was observed in the kidney after four doses of 9 mg/kg cisplatin, contrary to what has been previously reported. These results indicate that multiple strains of mice can be used with our repeated low-dose cisplatin model with dose optimization. Results also indicate that littermate control mice should be used with this model to account for population variability.

scholarly journals Expression of Matrix Metalloproteinases Subsequent to Urogenital Chlamydia muridarum Infection of Mice

2005 ◽  
Vol 73 (10) ◽  
pp. 6962-6973 ◽  
Author(s):  
K.H. Ramsey ◽  
I.M. Sigar ◽  
J. H. Schripsema ◽  
N. Shaba ◽  
K. P. Cohoon
Keyword(s):  
Matrix Metalloproteinases ◽  
Gelatin Zymography ◽  
Mouse Strains ◽  
Post Translational Modification ◽  
Chlamydia Muridarum ◽  
Central Hypothesis ◽  
Casein Zymography ◽  
Increased Activity ◽  
Mmp Genes ◽  
Chronic Sequelae

ABSTRACT The central hypothesis of this study was that matrix metalloproteinases (MMPs) would be enhanced following murine chlamydial infection and that their expression would vary in mouse strains that differ in their susceptibility to chronic chlamydia-induced disease. To address this hypothesis, female C3H/HeN and C57BL/6 mice were infected intravaginally with Chlamydia muridarum. Uterine and oviduct tissues were assessed for transcription of MMP genes and their tissue inhibitors. An increased activity of MMP genes relative to preinfection tissues was observed in the C3H/HeN mice when compared to C57BL/6 mice. Using gelatin zymography, we detected constitutive MMP-2 activity in both strains of mice but an increase in MMP-9. Casein zymography indicated the presence of two elastase-like activities consistent with MMP-12 and possibly MMP-7. Western blotting and antigen capture enzyme-linked immunoassay also confirmed an increase in MMP-9 but constitutive MMP-2 expression subsequent to the infection in both strains of mice. In C57BL/6 mice, MMP-9 was present in monomer and dimer form throughout the 56-day monitoring period. C3H/HeN mice produced dimeric MMP-9, but increases in the monomer form were also observed through day 14. Post-translational modification of MMP-9 between the two strains also differed. Immunohistochemistry revealed neutrophils as a prominent source for MMP-9 in both strains of mice. We conclude that differences in the relative expression and activity of MMPs, particularly MMP-9, occur in mice differing in their susceptibility to the development of chronic chlamydial disease. These differences may account for disparate outcomes with regard to chronic sequelae of the disease.

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