Effect of Maintained Hypoxic Exposure on the Crayfish Orconectes Rusticus: II. Modulation of Haemocyanin Oxygen Affinity

Haemolymph Na+, Cl−, K+, Mg2+, Ca2+, Cu2+ and protein levels, in vivo postbranchial acid-base status (total CO2, pH and PCOCO2), in vitro haemolymph buffer value, Bohr value and oxygen affinity were measured before and after a 3½-week period in which control crayfish were maintained at normoxia and experimental crayfish were maintained at an ambient oxygen tension of 50-55 torr. Analysis of haemolymph Cu2+ and protein levels in control and experimental crayfish indicated no increase in haemocyanin and therefore oxygen carrying capacity of the haemolymph. Although the Bohr value was not significantly different between control and experimental crayfish, the haemocyanin oxygen affinity was elevated in the hypoxic crayfish by two mechanisms. The first was dependent upon the haemolymph H+ concentration, i.e. a Bohr shift resulting from a respiratory alkalosis. The second mechanism was independent of haemolymph H+ concentration in that at a given pH haemolymph from experimental crayfish had a significantly higher oxygen affinity. The decrease in p50 probably cannot be attributed to a specific cation effect.

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Modulation of Haemocyanin Oxygen-Affinity by L-Lactate and Urate in the Prawn Penaeus Japonicus

Journal of Experimental Biology ◽

10.1242/jeb.147.1.133 ◽

1989 ◽

Vol 147 (1) ◽

pp. 133-146 ◽

Cited By ~ 2

Author(s):

F. LALLIER ◽

J. P. TRUCHOT

Keyword(s):

Lactate Concentration ◽

Oxygen Affinity ◽

Hypoxic Exposure ◽

Oxygen Binding ◽

Maximum Increase ◽

Penaeus Japonicus ◽

In Vitro Effects ◽

Hypoxia Acclimation

The addition of either L-lactate or urate to dialysed haemolymph from the prawn Penaeus japonicus (Bate) increased the in vitro haemocyanin oxygenaffinity. The quantitative values of these two effects, expressed as ΔlogP50/Δlog[effector], were found to be −0.077 for L-lactate and −0.032 for urate, at pH7.6 and 25°C. The normal, significant Bohr effect (ΔlogP50/ΔpH approx. −1.5 at pH7.6, 25°C) was not modified by the two effectors tested, nor was the cooperativity of haemocyanin oxygen-binding (n50 approx. 4). Hypoxic exposure of the prawns to PwOO2: =6.3 or 4.4 kPa (1 kPa=7.5 mmHg) for up to 48 h at 25°C induced only a small, less than 2.5-fold, elevation of L-lactate concentration in the haemolymph, all values remaining below 0.5 mmol I−1, but urate concentration increased to a greater extent (12-fold maximum increase from 0.01 to 0.12 mmol I−1). Haemocyanin oxygen-affinity, measured in vitro on haemolymph samples drawn from hypoxic prawns, increased slightly during the first 3h of hypoxia acclimation (ΔP50=0.8-0.9 kPa at pH 7.6), returning to near normoxic control values after a 48 h hypoxic exposure. The respective roles of L-lactate and urate in enhancing oxygen transport during hypoxia are discussed on the basis of their in vitro effects on haemocyanin oxygenaffinity and their in vivo concentration variations in haemolymph.

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Effect of Maintained Hypoxic Exposure on the Crayfish Orconectes Rusticus: I. Ventilatory, Acid-Base and Cardiovascular Adjustments

Journal of Experimental Biology ◽

10.1242/jeb.98.1.119 ◽

1982 ◽

Vol 98 (1) ◽

pp. 119-137 ◽

Cited By ~ 2

Author(s):

P.R. H. WILKES ◽

B. R. McMAHON

Keyword(s):

Cardiac Output ◽

Oxygen Affinity ◽

Fold Increase ◽

Respiratory Alkalosis ◽

Hypoxic Exposure ◽

Orconectes Rusticus ◽

Reduced Pressure ◽

Dissolved Carbon ◽

Ambient Oxygen ◽

Dissolved Carbon Dioxide

Oxygen uptake in the crayfish Orconectes rusticus was maintained at normoxic levels throughout 6 days exposure to an ambient oxygen tension of 45-55 torr. This was attributed to compensatory responses of the ventilatory and cardiovascular pumps. The first 72 h of hypoxic exposure were characterized by a transitory 3-fold increase in both scaphognathite rate and cardiac output above initial normoxic values. During the latter 72 h of hypoxic exposure both scaphognathite pumping and cardiac output were significantly below maximum values but were maintained above initial normoxic levels. Thus, as reflected by the increased convection requirements for both water and haemolymph, the increased scaphognathite and heart activity served to increase oxygen delivery to both the branchial chambers and the tissues. Additionally, the increased branchial ventilation and gill perfusion facilitated removal of dissolved carbon dioxide from the haemolymph, effecting a respiratory alkalosis. The increased haemolymph pH elevated the oxygen affinity of the haemocyanin via the Bohr affect, enabling a greater volume of oxygen to be picked up at the gills by the haemocyanin despite the reduced pressure gradient.

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Radiolabeled r-Hirudin as a Measure of Thrombin Activity at, or within, the Rabbit Aorta Wall In Vitro and In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642467 ◽

1994 ◽

Vol 71 (04) ◽

pp. 499-506 ◽

Cited By ~ 10

Author(s):

Mark W C Hatton ◽

Bonnie Ross-Ouellet

Keyword(s):

Rabbit Aorta ◽

Balloon Injury ◽

Recombinant Hirudin ◽

Aorta Wall ◽

Thrombin Activity ◽

Before And After ◽

The Matrix

SummaryThe behavior of 125I-labeled recombinant hirudin towards the uninjured and de-endothelialized rabbit aorta wall has been studied in vitro and in vivo to determine its usefulness as an indicator of thrombin activity associated with the aorta wall. Thrombin adsorbed to either sulfopropyl-Sephadex or heparin-Sepharose bound >95% of 125I-r-hirudin and the complex remained bound to the matrix. Binding of 125I-r-hirudin to the exposed aorta subendothelium (intima-media) in vitro was increased substantially if the tissue was pre-treated with thrombin; the quantity of l25I-r-hirudin bound to the de-endothelialized intima-media (i.e. balloon-injured in vitro) correlated positively with the quantity of bound 131I-thrombin (p <0.01). Aortas balloon-injured in vivo were measured for thrombin release from, and binding of 125I-r-hirudin to, the de-endothelialized intimal surface in vitro; 125I-r-hirudin binding correlated with the amount of active thrombin released (p <0.001). Uptake of 125I-r-hirudin by the aorta wall in vivo was proportional to the uptake of 131I-fibrinogen (as an indicator of thrombin activity) before and after balloon injury. After 30 min in the circulation, specific 125I-r-hirudin binding to the uninjured and de-endo- thelialized (at 1.5 h after injury) aorta wall was equivalent to 3.4 (± 2.5) and 25.6 (±18.1) fmol of thrombin/cm2 of intima-media, respectively. Possibly, only hirudin-accessible, glycosaminoglycan-bound thrombin is measured in this way.

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All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽

10.3390/cells10051202 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1202

Author(s):

Bojjibabu Chidipi ◽

Syed Islamuddin Shah ◽

Michelle Reiser ◽

Manasa Kanithi ◽

Amanda Garces ◽

...

Keyword(s):

Retinoic Acid ◽

Mitochondrial Fission ◽

Normal Function ◽

Mitochondrial Network ◽

Protein Levels ◽

Trans Retinoic Acid ◽

All Trans Retinoic Acid ◽

Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

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TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽

10.1186/s12885-021-08562-5 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Chengwu Xiao ◽

Wei Zhang ◽

Meimian Hua ◽

Huan Chen ◽

Bin Yang ◽

...

Keyword(s):

Cell Lines ◽

Prognostic Indicator ◽

The Cancer Genome Atlas ◽

Mrna Levels ◽

Loss Of Function ◽

Protein Levels ◽

Kaplan Meier ◽

Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

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Long noncoding RNA SNHG14 promotes hepatocellular carcinoma progression by regulating miR-876-5p/SSR2 axis

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01838-5 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Zhibin Liao ◽

Hongwei Zhang ◽

Chen Su ◽

Furong Liu ◽

Yachong Liu ◽

...

Keyword(s):

Noncoding Rna ◽

Animal Experiments ◽

Luciferase Reporter ◽

Western Blot Assay ◽

Downstream Target ◽

Protein Levels ◽

Elevated Expression ◽

Rna Immunoprecipitation

Abstract Background Aberrant expressions of long noncoding RNAs (lncRNAs) have been demonstrated to be related to the progress of HCC. The mechanisms that SNHG14 has participated in the development of HCC are obscure. Methods Quantitative real-time PCR (qRT-PCR) was used to measure the lncRNA, microRNA and mRNA expression level. Cell migration, invasion and proliferation ability were evaluated by transwell and CCK8 assays. The ceRNA regulatory mechanism of SNHG14 was evaluated by RNA immunoprecipitation (RIP) and dual luciferase reporter assay. Tumorigenesis mouse model was used to explore the roles of miR-876-5p in vivo. The protein levels of SSR2 were measured by western blot assay. Results In this study, we demonstrated that SNHG14 was highly expressed in HCC tissues, meanwhile, the elevated expression of SNHG14 predicted poor prognosis in patients with HCC. SNHG14 promoted proliferation and metastasis of HCC cells. We further revealed that SNHG14 functioned as a competing endogenous RNA (ceRNA) for miR-876-5p and that SSR2 was a downstream target of miR-876-5p in HCC. Transwell, CCK8 and animal experiments exhibited miR-876-5p inhibited HCC progression in vitro and in vivo. By conducting rescue experiments, we found the overexpression of SSR2 or knocking down the level of miR-876-5p could reverse the suppressive roles of SNHG14 depletion in HCC. Conclusion SNHG14 promotes HCC progress by acting as a sponge of miR-876-5p to regulate the expression of SSR2 in HCC.

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miR-146a-5p Promotes Chondrocyte Apoptosis and Inhibits Autophagy of Osteoarthritis by Targeting NUMB

Cartilage ◽

10.1177/19476035211023550 ◽

2021 ◽

pp. 194760352110235

Author(s):

Hongjun Zhang ◽

Wendi Zheng ◽

Du Li ◽

Jia Zheng

Keyword(s):

Caspase 3 ◽

Cartilage Tissue ◽

Luciferase Reporter ◽

Chondrocyte Apoptosis ◽

Knee Cartilage ◽

Before And After ◽

Related Proteins ◽

Human And Mouse

Objective miR-146a-5p was found to be significantly upregulated in cartilage tissue of patients with osteoarthritis (OA). NUMB was shown to be involved in the autophagy regulation process of cells. We aimed to learn whether NUMB was involved in the apoptosis or autophagy process of chondrocytes in OA and related with miR-146a-5p. Methods QRT-PCR was used to detect miR-146a-5p level in 22 OA cartilage tissues and 22 controls. The targets of miR-146a-5p were analyzed using software and the luciferase reporter experiment. The apoptosis and autophagy, and related proteins were detected in chondrocytes treated with miR-146a-5p mimic/inhibitor or pcDNA3.1-NUMB/si-NUMB and IL-1β, respectively. In vivo experiment, intra-articular injection of miR-146a-5p antagomir/NC was administered at the knee of OA male mice before and after model construction. Chondrocyte apoptosis and the expression of apoptosis and autophagy-related proteins were also detected. Results miR-146a-5p was highly expressed in knee cartilage tissue of patients with OA, while NUMB was lowly expressed and negatively regulated by miR-146a-5p. Upregulation of miR-146a-5p can promote cell apoptosis and reduce autophagy of human and mouse chondrocytes by modulating the levels of cleaved caspase-3, cleaved PARP, Bax, Beclin 1, ATG5, p62, LC3-I, and LC3-II. Increasing the low level of NUMB reversed the effects of miR-146a-5p on chondrocyte apoptosis and autophagy. Intra-articular injection of miR-146a-5p antagomir can also reverse the effects of miR-146a-5p on the apoptosis and autophagy of knee joint chondrocytes in OA mice. Conclusion Downregulation of miR-146a-5p suppresses the apoptosis and promotes autophagy of chondrocytes by targeting NUMB in vivo and in vitro.

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Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

Biochemical Journal ◽

10.1042/bj3600707 ◽

2001 ◽

Vol 360 (3) ◽

pp. 707-715 ◽

Cited By ~ 35

Author(s):

Trevor R. PETTITT ◽

Mark McDERMOTT ◽

Khalid M. SAQIB ◽

Neil SHIMWELL ◽

Michael J. O. WAKELAM

Keyword(s):

Liquid Chromatography ◽

Phosphatidic Acid ◽

Phospholipase D ◽

Mammalian Cells ◽

Inducible Promoter ◽

In Vitro Assays ◽

Protein Levels ◽

Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

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BIOM-55. DGKζ-TARGETED REGULATION OF MIR-34A IN THE PROLIFERATION AND TUMORIGENICITY OF HUMAN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.052 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii13-ii13

Author(s):

Wangxian Gu ◽

Guoqing Wan ◽

Yanjun Zheng ◽

Xintong Yang ◽

Peng Zhang ◽

...

Keyword(s):

Cancer Cells ◽

Precancerous Lesions ◽

Lipid Kinase ◽

Human Glioblastoma ◽

Kinase Substrate ◽

Downstream Target ◽

Protein Levels ◽

Type Iv

Abstract Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.

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EXTH-51. ANTI-INVASIVE EFFICACY AND SURVIVAL BENEFIT OF THE YAP-TEAD INHIBITOR VERTEPORFIN IN PRECLINICAL GLIOBLASTOMA MODELS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.405 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii98-ii98

Author(s):

Anne Marie Barrette ◽

Alexandros Bouras ◽

German Nudelman ◽

Zarmeen Mussa ◽

Elena Zaslavsky ◽

...

Keyword(s):

Survival Benefit ◽

De Novo ◽

Epithelial Mesenchymal Transition ◽

Intraperitoneal Administration ◽

Standard Of Care ◽

Tumor Burden ◽

Mesenchymal Transition ◽

Protein Levels

Abstract Glioblastoma (GBM) remains an incurable disease, in large part due to its malignant infiltrative spread, and current clinical therapy fails to target the invasive nature of tumor cells in disease progression and recurrence. Here, we use the YAP-TEAD inhibitor Verteporfin to target a convergence point for regulating tumor invasion/metastasis and establish the robust anti-invasive therapeutic efficacy of this FDA-approved drug and its survival benefit across several preclinical glioma models. Using patient-derived GBM cells and orthotopic xenograft models (PDX), we show that Verteporfin treatment disrupts YAP/TAZ-TEAD activity and processes related to cell adhesion, migration and epithelial-mesenchymal transition. In-vitro, Verteporfin impairs tumor migration, invasion and motility dynamics. In-vivo, intraperitoneal administration of Verteporfin in mice with orthotopic PDX tumors shows consistent drug accumulation within the brain and decreased infiltrative tumor burden, across three independent experiments. Interestingly, PDX tumors with impaired invasion after Verteporfin treatment downregulate CDH2 and ITGB1 adhesion protein levels within the tumor microenvironment. Finally, Verteporfin treatment confers survival benefit in two independent PDX models: as monotherapy in de-novo GBM and in combination with standard-of-care chemoradiation in recurrent GBM. These findings indicate potential therapeutic value of this FDA-approved drug if repurposed for GBM patients.

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