scholarly journals The Pitfalls of in vivo Cardiac Physiology in Genetically Modified Mice – Lessons Learnt the Hard Way in the Creatine Kinase System

2021 ◽  
Vol 12 ◽  
Author(s):  
Craig A. Lygate
Keyword(s):  
Creatine Kinase ◽  
Genetic Modification ◽  
Unintended Consequences ◽  
Cardiac Physiology ◽  
Cardiac Energetics ◽  
Integrative Physiology ◽  
Intact Organism ◽  
Dose Dependent

In order to fully understand gene function, at some point, it is necessary to study the effects in an intact organism. The creation of the first knockout mouse in the late 1980’s gave rise to a revolution in the field of integrative physiology that continues to this day. There are many complex choices when selecting a strategy for genetic modification, some of which will be touched on in this review, but the principal focus is to highlight the potential problems and pitfalls arising from the interpretation of in vivo cardiac phenotypes. As an exemplar, we will scrutinize the field of cardiac energetics and the attempts to understand the role of the creatine kinase (CK) energy buffering and transport system in the intact organism. This story highlights the confounding effects of genetic background, sex, and age, as well as the difficulties in interpreting knockout models in light of promiscuous proteins and metabolic redundancy. It will consider the dose-dependent effects and unintended consequences of transgene overexpression, and the need for experimental rigour in the context of in vivo phenotyping techniques. It is intended that this review will not only bring clarity to the field of cardiac energetics, but also aid the non-expert in evaluating and critically assessing data arising from in vivo genetic modification.

scholarly journals Inhibition of Prostate Cancer DU-145 Cells Proliferation by Anthopleura anjunae Oligopeptide (YVPGP) via PI3K/AKT/mTOR Signaling Pathway

Marine Drugs ◽  
2018 ◽  
Vol 16 (9) ◽  
pp. 325 ◽  
Author(s):  
Xiaojuan Li ◽  
Yunping Tang ◽  
Fangmiao Yu ◽  
Yu Sun ◽  
Fangfang Huang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Signaling Pathway ◽  
Caspase 3 ◽  
Mtor Signaling ◽  
Mtor Signaling Pathway ◽  
Nude Mouse Model ◽  
Dose Dependent

We investigated the antitumor mechanism of Anthopleura anjunae oligopeptide (AAP-H, YVPGP) in prostate cancer DU-145 cells in vitro and in vivo. Results indicated that AAP-H was nontoxic and exhibited antitumor activities. Cell cycle analysis indicated that AAP-H may arrest DU-145 cells in the S phase. The role of the phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein (PI3K/AKT/mTOR) signaling pathway in the antitumor mechanism of APP-H was investigated. Results showed that AAP-H treatment led to dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered compared to the untreated DU-145 cells. Inhibition of PI3K/AKT/mTOR signaling pathway in the DU-145 cells by employing inhibitor LY294002 (10 μM) or rapamycin (20 nM) effectively attenuated AAP-H-induced phosphorylation of AKT and mTOR. At the same time, inhibitor addition further elevated AAP-H-induced cleaved-caspase-3 levels. Furthermore, the effect of AAP-H on tumor growth and the role of the PI3K/AKT/mTOR signaling pathway in nude mouse model were also investigated. Immunohistochemical analysis showed that activated AKT, PI3K, and mTOR levels were reduced in DU-145 xenografts. Western blotting showed that AAP-H treatment resulted in dose-dependent reduction in p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) levels, whereas t-AKT and t-PI3K levels remained unaltered. Similarly, Bcl-xL levels decreased, whereas that of Bax increased after AAP-H treatment. AAP-H also increased initiator (caspase 8 and 9) and executor caspase (caspase 3 and 7) levels. Therefore, the antitumor mechanism of APP-H on DU-145 cells may involve regulation of the PI3K/AKT/mTOR signaling pathway, which eventually promotes apoptosis via mitochondrial and death receptor pathways. Thus, the hydrophobic oligopeptide (YVPGP) can be developed as an adjuvant for the prevention or treatment of prostate cancer in the future.

Role Of Cyclic Amp In The Inhibition Of Human Platelet Functions By Quercetin, A Flav0N0Id That Potentiates PGI2 Effect

1981 ◽  
Author(s):  
J P Cazenave ◽  
A Beretz ◽  
A Stierlé ◽  
R Anton
Keyword(s):  
Maximum Level ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Pde Inhibitors ◽  
Pde Inhibition ◽  
Induced Aggregation ◽  
Camp Levels ◽  
Dose Dependent

Injury to the endothelium (END) and subsequent platelet (PLAT)interactions with the subEND are important steps in thrombosis and atherosclerosis. Thus,drugs that protect the END from injury and also inhibit PLAT function are of interest. It has been shown that some flavonoids(FLA), a group of compounds found in plants, prevent END desquamation in vivo, inhibit cyclic nucleotide phosphodiesterases(PDE)and inhibit PLAT function. We have studied the structure-activity relationships of 13 purified FLA on aggregation and secretion of 14c-5HT of prelabeled washed human PLAT induced by ADP, collagen(COLL) and thrombin(THR). All the FLA were inhibitors of the 3 agents tested. Quercetin(Q), was the second best after fisetin. It inhibited secretion and aggregation with I50 of 330µM against 0.1 U/ML.THR, 102µM against 5µM ADP and 40 µM against COLL. This inhibitory effect is in the range of that of other PDE inhibitors like dipyridamole or 3-isobutyl-l- methylxanthine. The aggregation induced by ADP, COLL and THR is at least mediated by 3 mechanisms that can be inhibited by increasing cAMP levels. We next investigated if Q, which is a PDE inhibitor of bovine aortic microsomes,raises PLAT cAMP levels. cAMP was measured by a protein-binding method. ADP- induced aggregation(5µM) was inhibited by PGI2 (0.1 and 0.5 nM) . Inhibition was further potentiated(l.7 and 3.3 times) by lOµM Q, which alone has no effect on aggregation. The basal level of cAMP(2.2 pmol/108PLAT) was not modified by Q (50 to 500µM). Using these concentrations of Q,the rise in cAMP caused by PGI2(0.1 and 0.5nM) was potentiated in a dose dependent manner. Q potentiated the effect of PGI2 on the maximum level of cAMP and retarded its breakdown. Thus Q and possibly other FLA could inhibit the interaction of PLAT with the components of the vessel wall by preventing END damage and by inhibiting PLAT function through a rise in cAMP secondary to PDE inhibition and potentiation of the effect of vascular PGI2 on PLAT adenylate cyclase.

scholarly journals Hyperinsulinemia, But Not Other Factors Associated with Insulin Resistance, Acutely Enhances Colorectal Epithelial Proliferation in Vivo

Endocrinology ◽  
2006 ◽  
Vol 147 (4) ◽  
pp. 1830-1837 ◽  
Author(s):  
Thien T. Tran ◽  
Dinaz Naigamwalla ◽  
Andrei I. Oprescu ◽  
Loretta Lam ◽  
Gail McKeown-Eyssen ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Epithelial Cells ◽  
Dependent Manner ◽  
Epithelial Proliferation ◽  
Euglycemic Clamp ◽  
The Individual ◽  
Dose Dependent ◽  
Circulating Levels

The similarity in risk factors for insulin resistance and colorectal cancer (CRC) led to the hypothesis that markers of insulin resistance, such as elevated circulating levels of insulin, glucose, fatty acids, and triglycerides, are energy sources and growth factors in the development of CRC. The objective was thus to examine the individual and combined effects of these circulating factors on colorectal epithelial proliferation in vivo. Rats were fasted overnight, randomized to six groups, infused iv with insulin, glucose, and/or Intralipid for 10 h, and assessed for 5-bromo-2-deoxyuridine labeling of replicating DNA in colorectal epithelial cells. Intravenous infusion of insulin, during a 10-h euglycemic clamp, increased colorectal epithelial proliferation in a dose-dependent manner. The addition of hyperglycemia to hyperinsulinemia did not further increase proliferation. Intralipid infusion alone did not affect proliferation; however, the combination of insulin, glucose, and Intralipid infusion resulted in greater hyperinsulinemia than the infusion of insulin alone and further increased proliferation. Insulin infusion during a 10-h euglycemic clamp decreased total IGF-I levels and did not affect insulin sensitivity. These results provide evidence for an acute role of insulin, at levels observed in insulin resistance, in the proliferation of colorectal epithelial cells in vivo.

Safflower Yellow Prevents Pulmonary Metastasis of Breast Cancer by Inhibiting Tumor Cell Invadopodia

2016 ◽  
Vol 44 (07) ◽  
pp. 1491-1506 ◽  
Author(s):  
Huiying Fu ◽  
Renjie Wu ◽  
Yuanyuan Li ◽  
Lizong Zhang ◽  
Xiaofang Tang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Pulmonary Metastasis ◽  
Breast Cancer Cells ◽  
Dependent Cell ◽  
Response Profiles ◽  
Dose Dependent

Carthamus tinctorius L. is a traditional Chinese medicine that activates blood circulation and dissipates blood stasis, and has been extensively used as antitumor treatment in a clinical setting in single or in compound preparation form. However, empirical evidence and a better understanding of the possible mechanisms involved are still required. Here, we investigated the role of safflower yellow (SY), the active ingredient of C. tinctorius, in the pulmonary metastasis of breast cancer, and the underlying mechanism of action. EGF-meditated time- and dose-dependent cell response profiles were applied to screen for the activity of SY in vitro, while orthotopic lung metastasis and intravenous injection were used to evaluate the antimetastatic role of SY in vivo. SY could dose-dependently inhibit EGF-mediated time- and dose-dependent cell response profiles by inhibiting cytoskeletal rearrangement. We also found that SY significantly inhibited the migration of breast cancer cells in vitro and pulmonary metastasis of breast cancer cells in vivo. Consistent with these phenotypes, formation of invadopodia and the expression of MMP-9 and p-Src proteins were decreased after EGF stimulation in MBA-MD-231 cells treat with SY, as well as in lung metastatic foci. Additionally, circulating tumor cells retained in lung capillaries were also reduced. These results suggest that the antimetastatic effect of SY is due to its inhibition of invadopodia formation, which occurs mainly through Src-dependent cytoskeleton rearrangement. We suggest that SY should be considered as a potential novel therapeutic agent for the treatment of breast cancer.

scholarly journals SMAD1/5 Signaling in the Early Equine Placenta Regulates Trophoblast Differentiation and Chorionic Gonadotropin Secretion

Endocrinology ◽  
2014 ◽  
Vol 155 (8) ◽  
pp. 3054-3064 ◽  
Author(s):  
Victoria Cabrera-Sharp ◽  
Jordan E. Read ◽  
Stephanie Richardson ◽  
Alycia A. Kowalski ◽  
Douglas F. Antczak ◽  
...  
Keyword(s):  
Chorionic Gonadotropin ◽  
Trophoblast Cells ◽  
Rt Pcr ◽  
Trophoblast Differentiation ◽  
Gonadotropin Secretion ◽  
Morphogenetic Protein ◽  
Dose Dependent

TGFβ superfamily proteins, acting via SMAD (Sma- and Mad-related protein)2/3 pathways, regulate placental function; however, the role of SMAD1/5/8 pathway in the placenta is unknown. This study investigated the functional role of bone morphogenetic protein (BMP)4 signaling through SMAD1/5 in terminal differentiation of primary chorionic gonadotropin (CG)-secreting trophoblast. Primary equine trophoblast cells or placental tissues were isolated from day 27–34 equine conceptuses. Detected by microarray, RT-PCR, and quantitative RT-PCR, equine chorionic girdle trophoblast showed increased gene expression of receptors that bind BMP4. BMP4 mRNA expression was 20- to 60-fold higher in placental tissues adjacent to the chorionic girdle compared with chorionic girdle itself, suggesting BMP4 acts primarily in a paracrine manner on the chorionic girdle. Stimulation of chorionic girdle-trophoblast cells with BMP4 resulted in a dose-dependent and developmental stage-dependent increase in total number and proportion of terminally differentiated binucleate cells. Furthermore, BMP4 treatment induced non-CG-secreting day 31 chorionic girdle trophoblast cells to secrete CG, confirming a specific functional response to BMP4 stimulation. Inhibition of SMAD2/3 signaling combined with BMP4 treatment further enhanced differentiation of trophoblast cells. Phospho-SMAD1/5, but not phospho-SMAD2, expression as determined by Western blotting was tightly regulated during chorionic girdle trophoblast differentiation in vivo, with peak expression of phospho-SMAD1/5 in vivo noted at day 31 corresponding to maximal differentiation response of trophoblast in vitro. Collectively, these experiments demonstrate the involvement of BMP4-dependent pathways in the regulation of equine trophoblast differentiation in vivo and primary trophoblast differentiation in vitro via activation of SMAD1/5 pathway, a previously unreported mechanism of TGFβ signaling in the mammalian placenta.

scholarly journals Wnt/beta-catenin signalling is dispensable for adult neural stem cell homeostasis and activation

2020 ◽  
Author(s):  
Sophie H. L. Austin ◽  
Lachlan Harris ◽  
Oana Paun ◽  
Piero Rigo ◽  
François Guillemot ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Catenin ◽  
Dependent Manner ◽  
Direct Role ◽  
Adult Nscs ◽  
Hippocampal Networks ◽  
Dose Dependent

AbstractAdult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/β-catenin signalling acts at different steps along the hippocampal neurogenic lineage and has been shown to promote the proliferation of intermediate progenitor cells. However, whether it has a direct role in the regulation of NSCs still remains unclear. Here we used Wnt/β-catenin reporters and transcriptomic data from in vivo and in vitro models to show that both active and quiescent adult NSCs respond to Wnt/β-catenin signalling. Wnt/β-catenin stimulation instructed neuronal differentiation of active NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, we found that inhibiting NSCs response to Wnt, by conditionally deleting β-catenin, did not affect their activation or maintenance of their stem cell characteristics. Together, our results indicate that whilst NSCs do respond to Wnt/β-catenin stimulation in a dose-dependent and state-specific manner, Wnt/β-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which could reconcile some of the contradictions in the literature as to the role of Wnt/β-catenin signalling in adult hippocampal NSCs.

scholarly journals Evolutionarily ancient role of cholecystokinin-type neuropeptide signalling as an inhibitory regulator of feeding-related processes revealed in an echinoderm

2020 ◽  
Author(s):  
Ana B. Tinoco ◽  
Antón Barreiro-Iglesias ◽  
Luis Alfonso Yañez-Guerra ◽  
Jérôme Delroisse ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Body Wall ◽  
Oral Feeding ◽  
Cardiac Stomach ◽  
First Time ◽  
Dose Dependent ◽  
Type Receptor ◽  
Tube Feet

AbstractCholecystokinin (CCK) / sulfakinin (SK)-type neuropeptides regulate feeding and digestion in chordates and protostomes (e.g. insects). Here we characterised CCK/SK-type signalling for the first time in a non-chordate deuterostome - the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArCCK1, ArCCK2) derived from the precursor protein ArCCKP act as ligands for a CCK/SK-type receptor (ArCCKR) and are expressed in the nervous system, digestive system, tube feet and body wall. Furthermore, ArCCK1 and ArCCK2 cause dose-dependent contraction of cardiac stomach, tube foot and body wall apical muscle preparations in vitro and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of CCK/SK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

scholarly journals Ancient role of sulfakinin/cholecystokinin-type signalling in inhibitory regulation of feeding processes revealed in an echinoderm

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ana B Tinoco ◽  
Antón Barreiro-Iglesias ◽  
Luis Alfonso Yañez Guerra ◽  
Jérôme Delroisse ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Oral Feeding ◽  
Cardiac Stomach ◽  
First Time ◽  
Dose Dependent ◽  
Type Receptor ◽  
Tube Feet ◽  
Inhibitory Regulation

Sulfakinin (SK)/cholecystokinin (CCK)-type neuropeptides regulate feeding and digestion in protostomes (e.g. insects) and chordates. Here, we characterised SK/CCK-type signalling for the first time in a non-chordate deuterostome – the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArSK/CCK1, ArSK/CCK2) derived from the precursor protein ArSK/CCKP act as ligands for an SK/CCK-type receptor (ArSK/CCKR) and these peptides/proteins are expressed in the nervous system, digestive system, tube feet, and body wall. Furthermore, ArSK/CCK1 and ArSK/CCK2 cause dose-dependent contraction of cardiac stomach, tube foot, and apical muscle preparations in vitro, and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of SK/CCK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

scholarly journals Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Ki-Seo Yoo ◽  
Kina Lee ◽  
Jun-Young Oh ◽  
Hyoeun Lee ◽  
Hyungju Park ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Postsynaptic Density ◽  
Scaffolding Protein ◽  
Synaptic Localization ◽  
Conventional Kinesin ◽  
Dose Dependent ◽  
Postsynaptic Density Protein

AbstractPostsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.

Oxygen metabolite effects on creatine kinase and cardiac energetics after reperfusion

1991 ◽  
Vol 261 (2) ◽  
pp. H590-H597 ◽  
Author(s):  
A. Banerjee ◽  
M. A. Grosso ◽  
J. M. Brown ◽  
K. B. Rogers ◽  
G. J. Whitman
Keyword(s):  
Creatine Kinase ◽  
Myocardial Tissue ◽  
H2o2 Production ◽  
Cardiac Energetics ◽  
Excitable Tissue ◽  
Reperfusion Period ◽  
Tissue Contents ◽  
Reperfusion Phase

Noninvasive 31P nuclear magnetic resonance measurements indicate that during the initial reperfusion phase myocardial tissue contents of phosphocreatine (PCr) recover rapidly, while ATP levels remain low and recover slowly. There is also a burst of H2O2 during the first 10 min of reperfusion, as indicated by the in vivo inactivation of catalase that occurs only when H2O2, and the inactivator 3-aminotriazole (AMT), are simultaneously present. Neither H2O2 production nor CK inactivation was discernable after ischemia alone. In excitable tissue the PCr and ATP pools are equilibrated by the enzyme creatine kinase (CK), but myocardial CK activity is decreased by 20% after reperfusion, though not by simple washout. Extrapolating from the well-known air sensitivity of CK, we find that limited exposure in vitro to small concentrations of H2O2 can markedly diminish CK activity. We postulate that failure of certain CK isoenzymes at energy-using termini may decouple the relative rates of PCr production and ATP regeneration and hence cause elevated PCr-to-ATP ratios. The assumptions of 1) CK equilibrium during the reperfusion period to calculate free ADP levels and 2) cardiac recovery deduced from the elevation of PCr levels may require reexamination.

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