Specific receptor for hydrazine: mapping the in situ release of hydrazine in live cells and in an in vitro enzymatic assay

2016 ◽  
Vol 52 (36) ◽  
pp. 6166-6169 ◽  
Author(s):  
Firoj Ali ◽  
Anila H. A. ◽  
Nandaraj Taye ◽  
Devraj G. Mogare ◽  
Samit Chattopadhyay ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Enzymatic Assay ◽  
Live Cells ◽  
Specific Receptor ◽  
Specific Detection ◽  
Physiological Conditions

New chemodosimetric reagent for the specific detection of hydrazine in physiological conditions as well as for the mapping of its in situ generation in live Hct116 and HepG2 cells by enzymatic transformations.

Absorption of beta-casomorphins from autoperfused lamb and piglet small intestine

1990 ◽  
Vol 259 (3) ◽  
pp. G443-G452 ◽  
Author(s):  
L. C. Read ◽  
A. P. Lord ◽  
V. Brantl ◽  
G. Koch
Keyword(s):  
Small Intestine ◽  
Intestinal Lumen ◽  
Physiological Conditions ◽  
Naturally Occurring ◽  
Gut Epithelium ◽  
Measured Absorption ◽  
Beta Casein ◽  
Kinetics Of

beta-Casomorphins (beta-CMs) derived from milk beta-casein may exert various opiate activities in milk-fed infants. To assess the physiological significance of beta-CMs as a source of circulating opioids in infants, we measured absorption rates of several beta-CMs under near-physiological conditions using in situ autoperfused lamb intestine. The naturally occurring beta-CMs, beta-CM-7 and beta-CM-4-amide, were absorbed readily into blood with no transfer into lymph. Uptake peaked within several minutes of the luminal infusion of peptide but then declined sharply and stopped within a further 10-15 min. The recovery in blood, intestinal contents, and tissue at the end of the 30-min experiment was less than 1% of the infused dose. The low recovery was due to rapid proteolysis based on in vitro studies that demonstrated half-lives of less than 5 min in lamb blood, luminal contents, and lymph. The synthetic dipeptidyl peptidase IV-resistant analogue beta-[D-Ala2]CM- 4-amide was stable during incubation in blood, lymph, or luminal contents and was absorbed into blood at rates that were maximal within several minutes and remained steady for the 30-min period. We conclude that although natural beta-CMs are transferred across the lamb small intestine, rapid degradation within the intestinal lumen, gut epithelium, and blood would prevent entry into the circulation under normal conditions. Val-beta-CM-7, a putative stable precursor, had similar stability and kinetics of absorption to beta-CM-7, results that exclude Val-beta-CM-7 as a stable precursor for delivery of beta-CMs to the circulation. Essentially identical results to those in lambs were obtained in 7-day-old piglets.

scholarly journals A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

2021 ◽  
Author(s):  
◽  
Sarah Cordiner
Keyword(s):  
Cell Morphology ◽  
Hepg2 Cells ◽  
Mammalian Cells ◽  
Live Cells ◽  
Shellfish Poisoning ◽  
Hl60 Cells ◽  
Apoptosis Inducer ◽  
Blood Leukocyte ◽  
Shock Proteins

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

scholarly journals A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

2021 ◽  
Author(s):  
◽  
Sarah Cordiner
Keyword(s):  
Cell Morphology ◽  
Hepg2 Cells ◽  
Mammalian Cells ◽  
Live Cells ◽  
Shellfish Poisoning ◽  
Hl60 Cells ◽  
Apoptosis Inducer ◽  
Blood Leukocyte ◽  
Shock Proteins

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

scholarly journals Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

2021 ◽  
Author(s):  
Xiaoyun Zhang ◽  
Jochen Spiegel ◽  
Sergio Martínez Cuesta ◽  
Santosh Adhikari ◽  
Shankar Balasubramanian
Keyword(s):  
Protein Interactions ◽  
Protein Profiling ◽  
Live Cells ◽  
Interacting Proteins ◽  
Chemical Profiling ◽  
G Quadruplex ◽  
Functional Classes ◽  
Chemical Strategy

AbstractDNA–protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.

scholarly journals Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands.

1991 ◽  
Vol 113 (1) ◽  
pp. 187-194 ◽  
Author(s):  
R P Mecham ◽  
L Whitehouse ◽  
M Hay ◽  
A Hinek ◽  
M P Sheetz
Keyword(s):  
Cell Surface ◽  
Laminin Receptor ◽  
Live Cells ◽  
Receptor Interaction ◽  
Ligand Complex ◽  
Lectin Domain ◽  
Receptor Complexes ◽  
Laminin Binding Protein

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand.

A highly selective fluorescent nanoprobe based on AIE and ESIPT for imaging hydrogen sulfide in live cells and zebrafish

2017 ◽  
Vol 1 (5) ◽  
pp. 838-845 ◽  
Author(s):  
Peisheng Zhang ◽  
Xuezheng Nie ◽  
Meng Gao ◽  
Fang Zeng ◽  
Anjun Qin ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Live Cells ◽  
Specific Detection ◽  
Fluorescent Nanoprobe

An AIE/ESIPT-based nanoprobe has been developed for the specific detection and imaging of H2Sin vitroandin vivo.

scholarly journals Regulation of heart rate and the pacemaker current by phosphoinositide 3-kinase signaling

2019 ◽  
Vol 151 (8) ◽  
pp. 1051-1058 ◽  
Author(s):  
Richard Z. Lin ◽  
Zhongju Lu ◽  
Evgeny P. Anyukhovsky ◽  
Ya-Ping Jiang ◽  
Hong Zhan Wang ◽  
...  
Keyword(s):  
Heart Rate ◽  
Sinoatrial Node ◽  
Cardiac Pacing ◽  
Pacemaker Current ◽  
Physiological Conditions ◽  
Diastolic Depolarization ◽  
Important Regulator ◽  
Phosphoinositide 3 Kinase

Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, IF, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of IF are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias.

scholarly journals Bioluminescent Mammalian Cells Grown in Sponge Matrices to Monitor Immune Rejection

2007 ◽  
Vol 6 (5) ◽  
pp. 7290.2007.00026
Author(s):  
Okechukwu Ojogho ◽  
Alice Li ◽  
Craig W. Zuppan ◽  
Alan Escher
Keyword(s):  
Mammalian Cells ◽  
Light Emission ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Renilla Luciferase ◽  
Live Cells ◽  
Gelatin Sponge ◽  
Immune Rejection

The growth and bioluminescence of cells seeded in collagen and gelatin sponge matrices were compared in vitro under different conditions, and immune rejection was quantified and visualized directly in situ based on loss of bioluminescence activity. Mammalian cells expressing a Renilla luciferase complementary deoxyribonucleic acid (cDNA) were used to seed collagen and gelatin sponge matrices soaked in either polylysine or gelatin to determine optimal growth conditions in vitro. The sponges were incubated in tissue culture plates for 3 weeks and received 2, 9, or 15 injections of coelenterazine. Measurements of bioluminescence activity indicated that gelatin sponges soaked in gelatin emitted the highest levels of light emission, multiple injections of coelenterazine did not affect light emission significantly, and light emission from live cells grown in sponges could be measured qualitatively but not quantitatively. Histologic analysis of sponge matrices cultured in vitro showed that cells grew best in gelatin matrices. Visualization of subcutaneously implanted sponges in mice showed accelerated loss of light emission in immunocompetent BALB/c mice compared with immunodeficient BALB/c- scid mice, which was associated with increased cell infiltration. Our results indicate that sponge matrices carrying bioluminescent mammalian cells are a valid model system to study immune rejection in situ.

Two-photon fluorescent turn-on probes for highly efficient detection and profiling of thiols in live cells and tissues

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Congzhen Shen ◽  
Duoteng Zhang ◽  
Fang Xu ◽  
Yang Yang ◽  
Yi Tan ◽  
...  
Keyword(s):  
Live Cells ◽  
Dual Purpose ◽  
Two Photon ◽  
Simultaneous Imaging ◽  
Turn On ◽  
Efficient Detection ◽  
Tumor Tissues ◽  
Cancer Diagnosis And Therapy

Abstract Thiols are important units in amino acids such as cysteine and peptides like glutathione. Development of chemical sensors capable of precise detection of thiols is important in cancer diagnosis and therapy. We have developed novel two-photon fluorescent turn-on probes for selective detection of thiols. The probes displayed excellent sensitivity and low detection limits. The dual-purpose probes have been demonstrated to be suitable for simultaneous imaging and proteome profiling in live cells and tumor tissues. The unique turn-on design endows the probes with excellent selectivity toward thiols in vitro and in situ, and can be further developed to support a thiol-quantification assay.

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