scholarly journals Hsp22 with an N-Terminal Domain Truncation Mediates a Reduction in Tau Protein Levels

2020 ◽  
Vol 21 (15) ◽  
pp. 5442
Author(s):  
Jack M. Webster ◽  
April L. Darling ◽  
Taylor A. Sanders ◽  
Danielle M. Blazier ◽  
Yamile Vidal-Aguiar ◽  
...  
Keyword(s):  
Tau Protein ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
High Ratio ◽  
Protein Levels ◽  
Terminal Domain ◽  
Domain Truncation ◽  
Shock Proteins ◽  
Induced Aggregation

Misfolding, aggregation and accumulation of proteins are toxic elements in the progression of a broad range of neurodegenerative diseases. Molecular chaperones enable a cellular defense by reducing or compartmentalizing these insults. Small heat shock proteins (sHsps) engage proteins early in the process of misfolding and can facilitate their proper folding or refolding, sequestration, or clearance. Here, we evaluate the effects of the sHsp Hsp22, as well as a pseudophosphorylated mutant and an N-terminal domain deletion (NTDΔ) variant on tau aggregation in vitro and tau accumulation and aggregation in cultured cells. Hsp22 wild-type (WT) protein had a significant inhibitory effect on heparin-induced aggregation in vitro and the pseudophosphorylated mutant Hsp22 demonstrated a similar effect. When co-expressed in a cell culture model with tau, these Hsp22 constructs significantly reduced soluble tau protein levels when transfected at a high ratio relative to tau. However, the Hsp22 NTDΔ protein drastically reduced the soluble protein expression levels of both tau WT and tau P301L/S320F even at lower transfection ratios, which resulted in a correlative reduction of the triton-insoluble tau P301L/S320F aggregates.

scholarly journals Combined Luteolin and Indole-3-Carbinol Synergistically Constrains ERα-Positive Breast Cancer by Dual Inhibiting Estrogen Receptor Alpha and Cyclin-Dependent Kinase 4/6 Pathway in Cultured Cells and Xenograft Mice

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2116
Author(s):  
Xiaoyong Wang ◽  
Lijuan Zhang ◽  
Qi Dai ◽  
Hongzong Si ◽  
Longyun Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cell ◽  
Breast Cancer Cells ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
Cyclin Dependent Kinase ◽  
Xenograft Mice ◽  
The Individual

The high concentrations of individual phytochemicals in vitro studies cannot be physiologically achieved in humans. Our solution for this concentration gap between in vitro and human studies is to combine two or more phytochemicals. We screened 12 phytochemicals by pairwise combining two compounds at a low level to select combinations exerting the synergistic inhibitory effect of breast cancer cell proliferation. A novel combination of luteolin at 30 μM (LUT30) and indole-3-carbinol 40 μM (I3C40) identified that this combination (L30I40) synergistically constrains ERα+ breast cancer cell (MCF7 and T47D) proliferation only, but not triple-negative breast cancer cells. At the same time, the individual LUT30 and I3C40 do not have this anti-proliferative effect in ERα+ breast cancer cells. Moreover, this combination L30I40 does not have toxicity on endothelial cells compared to the current commercial drugs. Similarly, the combination of LUT and I3C (LUT10 mg + I3C10 mg/kg/day) (IP injection) synergistically suppresses tumor growth in MCF7 cells-derived xenograft mice, but the individual LUT (10 mg/kg/day) and I3C (20 mg/kg/day) do not show an inhibitory effect. This combination synergistically downregulates two major therapeutic targets ERα and cyclin dependent kinase (CDK) 4/6/retinoblastoma (Rb) pathway, both in cultured cells and xenograft tumors. These results provide a solid foundation that a combination of LUT and I3C may be a practical approach to treat ERα+ breast cancer cells after clinical trials.

Effect Of Solvents On Indium-111 Oxine Transport In Human Platelets And On Platelet Function In Vitro

1981 ◽  
Author(s):  
T Tsukada
Keyword(s):  
Platelet Function ◽  
Kinetic Constant ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Polysorbate 80 ◽  
Autologous Plasma ◽  
Indium 111 ◽  
Induced Aggregation ◽  
Water Space

Mechanism of Indium-111 oxine(In) transport in human platelets in buffered saline and the effect of In-labeling on platelet function were studied using In dissolved in 25% of ethanol in saline (In-ES) or 0.01% of polysorbate 80 in HEPES buffer(In-PH). Increase in temperature up to 37° C progressively enhanced the transport of In-ES, while transport of In-PH reached to plateau at 15°C. A states of equilibrium was not reached during 2 hr incubation at 22°C in In-ES. Uptake of In-PH reached to plateau after only 15 min of incubation. Distribution of In taken up by platelets in InES was 57% in cytosol and 27% in stroma, while in In-PH 69% in stroma and 22% in cytosol. 88% of In in cytosol was bound to lipids(46% in cholesterol and 27% in PS+PI). 82% of In in stroma was found in PS+PI fraction.The fact that the ratio of free In between the platelet water space and the outside medium after 30 min of incubation at up to 0.1 uM of In exceeded unity, suggests satura- , ble component of In transport prevails at this concentration in In-ES and In-PH. Kinetic constant could be calculated, Kt= 2nM, Vmax= 2.5 pmol/min/ml in In-ES, and Kt= InM, Vmax=0.7 pmol/min/ml in In-PH.Elution of In from radiolableled platelets in autologous plasma incubated at 37°C for 5 hr was less than 10% in the case of In-ES and 56% in the case of In-PH. Less than 3% of labeled-In was eluated from platelets in collagen-induced aggregation and 4-7% of In was eluated in thrombin-induced aggregation.Although 0.3% of ethanol and/or 6nM of oxine have no inhibitory effect of platelet aggregation, collagen-induced aggregation and release reaction of In-labeled platelet was impaired. 0.003% of polysorbate 80 itself abolished completely the aggregability of platelets by collagen or thrombin.It is concluded In-PH is unsuitable for platelet labeling. In-111 oxine also seems to have problems which Cr-51 has, i.e. inhomogenous distribution of In in a platelet population, elution of In from labeled platelets in circulation.

scholarly journals Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Encarnación Medina-Carmona ◽  
Rogelio J. Palomino-Morales ◽  
Julian E. Fuchs ◽  
Esperanza Padín-Gonzalez ◽  
Noel Mesa-Torres ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Loss Of Function ◽  
Quinone Oxidoreductase ◽  
Protein Levels ◽  
Terminal Domain ◽  
Directional Preference

Abstract Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S and to develop new pharmacological therapies to rescue this function.

scholarly journals Overexpression of IRS-4 Correlates with Procaspase 3 Levels in Tumoural Tissue of Patients with Colorectal Cancer

2018 ◽  
Vol 2018 ◽  
pp. 1-14
Author(s):  
Patricia Sanmartín-Salinas ◽  
Luis G. Guijarro
Keyword(s):  
Colorectal Cancer ◽  
Caspase 3 ◽  
Cultured Cells ◽  
Cyclin Dependent Kinase ◽  
Kinase Activation ◽  
Tnm System ◽  
Protein Levels ◽  
Receptor Signalling ◽  
T Values

We reported that insulin receptor substrate 4 (IRS-4) levels increased in tissue from colorectal cancer (CRC) patients and promoted retinoblastoma-cyclin-dependent kinase activation. The aim of the present study was to evaluate the effect of IRS-4 on IGF-1 receptor pathway and its impact on procaspase 3 and PARP expression in RKO and HepG2 cancer cell lines. The results obtained in vitro were compared with those obtained from biopsies of patients with CRC (n = 18), tubulovillous adenomas (TA) (n = 2) and in matched adjacent normal colorectal (MANC) tissue (n = 20). IRS-4 overexpression in cultured cells induced the overactivation of IGF-1/BRK/AKT/GSK-3/β-catenin/cyclin D1 pathways, which led to increased expression of procaspase 3 and PARP protein levels. Studies carried out on CRC and TA tissues revealed the overactivation of the IGF-1 receptor signalling pathway, as well as the overexpression of procaspase 3 and PARP in tumoural tissue with respect to MANC tissue. The upregulation of IRS-4 in tumoural samples correlated significantly with the increase in pIGF-1 receptor (Tyr 1165/1166) (r = 0.84; p < 0.0001), procaspase 3 (r = 0. 77; p < 0. 0005) and PARP (r = 0. 89; p < 0. 0005). Similarly, we observed an increase in the proteolysis of procaspase 3 in tumoural tissue with respect to MANC tissue, which correlated significantly with the degradation of PARP (r = 0.86; p < 0.0001), p53 (r = 0.84; p < 0.0001), and GSK-3 (r = 0.78; p < 0.0001). The stratification of patient samples using the TNM system revealed that procaspase 3 and caspase 3 increased gradually with T values, which suggests their involvement in the size and local invasion of primary tumours. Taken together, our findings suggest that IRS-4 overexpression promotes the activation of the IGF-1 receptor pathway, which leads to the increase in procaspase 3 levels in CRC.

Inhibition of Human Platelet Aggregation by the Proteolytic Effect of Streptokinase (SK). Role of the Factor VIII Related Antigen

1975 ◽  
Author(s):  
R. Castillo ◽  
S. Maragall ◽  
J. A. Guisasola ◽  
F. Casals ◽  
C. Ruiz ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Factor Viii ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Factor Viii Related Antigen ◽  
Human Factor Viii ◽  
Induced Aggregation

Defective ADP-induced platelet aggregation has been observed in patients treated with streptokinase. This same effect appears “in vitro” when adding SK to platelet rich plasma (PRP). Classic hemophilia and normal platelet poor plasmas (PPP) treated with SK inhibit the aggregation of washed platelets; plasmin-treated normal human serum also shows an inhibitory effect on platelet aggregation. However, von Willebrand SK-treated plasmas do not inhibit the aggregation of washed platelets. The same results appear when plasmas are previously treated with a rabbit antibody to human factor VIII.This confirms that the antiaggregating effect is mainly linked to the digested factor VIII related antigen.The inhibition of ADP-induced platelet aggregation has been proved in gel filtration-isolated and washed platelets from SK-treated PRP.Defective ristocetin-induced platelet aggregation has also been observed- This action does not appear in washed platelets from SK-treated PRP in presence of normal PPP, but it does in presence of SK-treated PPP, which suggests that the inhibition of the ristocetin-induced aggregation is due to the lack of factor VIII and not to the factor VIII-related products.Heparin, either “in vivo” or “in vitro”, has corrected the antiaggregating effect of SK.

scholarly journals IMMUNIZATION OF DISSOCIATED SPLEEN CELL CULTURES FROM NORMAL MICE

1967 ◽  
Vol 126 (3) ◽  
pp. 423-442 ◽  
Author(s):  
Robert I. Mishell ◽  
Richard W. Dutton
Keyword(s):  
Spleen Cell ◽  
Cell Cultures ◽  
Culture System ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
Initial Exposure ◽  
In Vitro Immunization ◽  
In Vitro Response

A culture system for cell suspensions from mouse spleens has been described. The system provides adequate conditions for in vitro immunization on initial exposure to heterologous erythrocytes. The in vitro response closely parallels that observed in vivo with respect to size, early kinetics, antigen dose, and the inhibitory effect of passive antibody. The response of cultured cells differs in two respects from that seen in vivo. There is an increase in the ability to discriminate between different varieties of homologous erythrocytes and the in vitro response does not appear to be limited by whatever mechanisms regulate the in vivo response.

scholarly journals Detection of Genistein in Soy Protein Isolate and Soymilk Powder by Spectrophotometric and Chromatographic Method

2019 ◽  
Vol 11 ◽  
pp. 69-73
Author(s):  
Md. Anisur Rahman Mazumder ◽  
Parichat Hongsprabhas
Keyword(s):  
Soy Protein ◽  
Cultured Cells ◽  
Soy Protein Isolate ◽  
Milk Powder ◽  
Density Lipoprotein ◽  
Inhibitory Effect ◽  
Protein Isolate ◽  
Hplc Method ◽  
Spectrophotometric Methods

Genistein proposed as a treatment for osteoporosis for postmenopausal women, elderly men, lowering cardiovascular disease and reduces hormone dependent cancers. Genistein also exerted inhibitory effect on lipid peroxidation induced in vitro by pro-oxidant agents on model and natural membranes on cultured cells and on low density lipoprotein. Genistein detection in soy products is very much important for Food Scientist. Gensitein can be detected by UV-Visible spectrophotometric and HPLC method. This study focused on the detection of genistein by HPLC and spectrophotometric methods. Genistein content of both soy protein isolate (SPI) and spray dried soy milk powder (SMP) was determined by spectrophotometry (93.12±1.15 and 74.78±0.75 mg/100g, respectively) were slightly higher but not significantly differ than HPLC analysis (89.67±5.16 and 72.34±0.27 mg/100g, respectively). This study suggested that genistein and its glycoside could be detected by spectrophotometric methods with high accuracy.

BHLHE40 plays a pathological role in pre-eclampsia through upregulating SNX16 by transcriptional inhibition of miR-196a-5p

2020 ◽  
Vol 26 (7) ◽  
pp. 532-548 ◽  
Author(s):  
Chunmei Mi ◽  
Bin Ye ◽  
Zhou Gao ◽  
Jinzhi Du ◽  
Ruizhen Li ◽  
...  
Keyword(s):  
Cell Migration ◽  
Neonatal Morbidity ◽  
Inhibitory Effect ◽  
Abnormal Development ◽  
Luciferase Reporter ◽  
Protein Levels ◽  
Transcriptional Inhibition ◽  
Uterine Perfusion

Abstract Pre-eclampsia (PE), which results from abnormal placentation, is a primary cause of maternal and neonatal morbidity and mortality. However, the causes of abnormal development of the placenta remain poorly understood. BHLHE40 is a transcriptional repressor in response to hypoxia. Bioinformatics analysis demonstrated that BHLHE40 negatively regulates miR-196a-5p expression, which may decrease miR-196a-5p to target SNX16. Since SNX16 exerts an inhibitory effect on cell migration, it may disrupt trophoblast cell migration in placentation. Therefore, the objective of this study was to explore a possible role of the BHLHE40/miR-196a-5p/SNX16 axis in PE pathogenesis. BHLHE40, miR-196a-5p and SNX16 mRNA and/or protein levels were detected in PE and normal placenta tissues. PE models in vitro and in vivo were constructed by culturing trophoblasts under hypoxia and reducing the uterine perfusion pressure in pregnant C57/BL6N mice, respectively. BHLHE40 and SNX16 were upregulated in PE placenta, while miR-196a-5p was downregulated. Knockdown of BHLHE40 reversed miR-196a-5p expression in trophoblasts under hypoxia, and upregulation of miR-196a-5p inhibited SNX16 expression. As indicated by ChIP assay, BHLHE40 bound to the promoter of the miR-196a-5p gene; luciferase reporter analysis showed that miR-196a-5p could bind to the 3ʹ-untranslated region of SNX16 mRNA. Knockdown of either BHLHE40 or SNX16, or an increase in miR-196a-5p, restored cell viability, migration, invasion and matrix metalloprotein (MMP)-2 and MMP-9 expression under hypoxia. BHLHE40 knockdown also alleviated PE symptoms in pregnant C57/BL6N mice. This study supports involvement of the BHLHE40/miR-196a-5p/SNX16 axis in PE pathogenesis; Proper adjustment of the BHLHE40/miR-196a-5p/SNX16 axis is able to attenuate PE symptoms.

Inhibition of cytochrome P-450 C17 enzyme by a GnRH agonist in ovarian follicles from gonadotropin-stimulated rats

2007 ◽  
Vol 292 (5) ◽  
pp. E1456-E1464 ◽  
Author(s):  
Griselda Irusta ◽  
Fernanda Parborell ◽  
Marta Tesone
Keyword(s):  
Direct Action ◽  
Regulatory Protein ◽  
Follicular Development ◽  
Inhibitory Effect ◽  
Female Rats ◽  
Serum Progesterone ◽  
Protein Levels ◽  
Androgen Synthesis ◽  
Cytochrome P 450

Our objective was to study the direct action of a GnRH-I agonist, leuprolide acetate (LA), on ovarian steroidogenesis in preovulatory follicles obtained from equine chorionic gonadotropin (eCG)-treated rats. Previously, we have demonstrated an inhibitory effect of LA on steroidogenesis and follicular development. In this study, we tested the hypothesis that gonadotropin-releasing hormone (GnRH) exerts its negative effect on follicular development by inhibiting thecal cytochrome P-450 C17 (P450C17) α-hydroxylase expression and, consequently, androgen synthesis. Studies were carried out in prepubertal female rats injected with either eCG (control) or eCG plus LA (LA) and killed at different time points. Immunohistochemical studies indicated that LA induced steroidogenic acute regulatory protein (StAR) expression mainly in theca cells of preantral and antral follicles. In addition, serum progesterone levels increased significantly ( P < 0.05), whereas those of androsterone decreased ( P < 0.05) after 8 h of LA treatment. This inhibition caused by LA seemed to be a consequence of the decreased expression of follicular P450C17 α-hydroxylase, as demonstrated by Western blot and RT-PCR techniques. In vitro studies using follicles isolated from 48-h-eCG-treated rats and cultured with LA showed a significant ( P < 0.05) inhibition of FSH-induced androsterone follicular content as well as P450C17 α-hydroxylase protein levels, as determined by Western analysis. However, LA increased StAR protein expression in these follicles without significant changes in P450scc enzyme levels. Taking all these findings into account, we suggest that GnRH-I exerts a direct inhibitory action on gonadotropin-induced follicular development by decreasing the temporal expression of the P450C17 enzyme and, consequently, androgen production, thus reducing the supply of estrogens available to developing follicles.

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