scholarly journals Supramolecular methods: the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) transport assay

2021 ◽  
pp. 1-20
Author(s):  
Alexander M. Gilchrist ◽  
Patrick Wang ◽  
Israel Carreira-Barral ◽  
Daniel Alonso-Carrillo ◽  
Xin Wu ◽  
...  
Keyword(s):  
Transport Assay

scholarly journals A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

2006 ◽  
Vol 17 (11) ◽  
pp. 4720-4735 ◽  
Author(s):  
Alistair N. Hume ◽  
Abul K. Tarafder ◽  
José S. Ramalho ◽  
Elena V. Sviderskaya ◽  
Miguel C. Seabra
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Actin Binding ◽  
Binding Studies ◽  
Null Cell ◽  
Coiled Coil Region ◽  
Actin Binding Domain ◽  
Transport Assay ◽  
Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

scholarly journals A Fluorescent Glucose Transport Assay for Screening SGLT2 Inhibitors in Endogenous SGLT2-Expressing HK-2 Cells

2018 ◽  
Vol 9 (1) ◽  
pp. 13-21 ◽  
Author(s):  
Yan-Ting Lu ◽  
Xiu-Li Ma ◽  
Yu-Hui Xu ◽  
Jing Hu ◽  
Fang Wang ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Sglt2 Inhibitors ◽  
Transport Assay

scholarly journals An integrated microfluidic platform for quantifying drug permeation across biomimetic vesicle membranes

2019 ◽  
Author(s):  
Michael Schaich ◽  
Jehangir Cama ◽  
Kareem Al Nahas ◽  
Diana Sobota ◽  
Kevin Jahnke ◽  
...  
Keyword(s):  
Drug Transport ◽  
Membrane Composition ◽  
Drug Permeability ◽  
Bacterial Membranes ◽  
Drug Molecules ◽  
Total Analysis System ◽  
Transport Assay ◽  
Order Of Magnitude ◽  
Analysis System ◽  
The Individual

The low membrane permeability of candidate drug molecules is a major challenge in drug development and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging since one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological salt and pH levels, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin across biomimetic liposomes.

A facile transport assay for H+coupled membrane transport using fluorescence probes

2012 ◽  
Vol 4 (1) ◽  
pp. 44-46 ◽  
Author(s):  
Wanjun Lan ◽  
Hongliu Ren ◽  
Yu Pang ◽  
Chuseng Huang ◽  
Yufang Xu ◽  
...  
Keyword(s):  
Membrane Transport ◽  
Fluorescence Probes ◽  
Transport Assay

scholarly journals Omega-3 Polyunsaturated Fatty Acids Inhibit the Function of Human URAT1, a Renal Urate Re-Absorber

Nutrients ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1601 ◽  
Author(s):  
Hiroki Saito ◽  
Yu Toyoda ◽  
Tappei Takada ◽  
Hiroshi Hirata ◽  
Ami Ota-Kontani ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Human Health ◽  
Serum Urate ◽  
The Body ◽  
Omega 3 ◽  
Beneficial Effects ◽  
Uricosuric Agents ◽  
Transport Assay ◽  
The Impact

The beneficial effects of fatty acids (FAs) on human health have attracted widespread interest. However, little is known about the impact of FAs on the handling of urate, the end-product of human purine metabolism, in the body. Increased serum urate levels occur in hyperuricemia, a disease that can lead to gout. In humans, urate filtered by the glomerulus of the kidney is majorly re-absorbed from primary urine into the blood via the urate transporter 1 (URAT1)-mediated pathway. URAT1 inhibition, thus, contributes to decreasing serum urate concentration by increasing net renal urate excretion. Here, we investigated the URAT1-inhibitory effects of 25 FAs that are commonly contained in foods or produced in the body. For this purpose, we conducted an in vitro transport assay using cells transiently expressing URAT1. Our results showed that unsaturated FAs, especially long-chain unsaturated FAs, inhibited URAT1 more strongly than saturated FAs. Among the tested unsaturated FAs, eicosapentaenoic acid, α-linolenic acid, and docosahexaenoic acid exhibited substantial URAT1-inhibitory activities, with half maximal inhibitory concentration values of 6.0, 14.2, and 15.2 μM, respectively. Although further studies are required to investigate whether the ω-3 polyunsaturated FAs can be employed as uricosuric agents, our findings further confirm FAs as nutritionally important substances influencing human health.

scholarly journals A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack.

1992 ◽  
Vol 118 (5) ◽  
pp. 1015-1026 ◽  
Author(s):  
M G Waters ◽  
D O Clary ◽  
J E Rothman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protein Transport ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Peptide Sequence ◽  
Golgi Stack ◽  
Transport Assay ◽  
Weight Protein

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.

scholarly journals Hepatocanalicular organic-anion transport is regulated by protein kinase C

1991 ◽  
Vol 278 (3) ◽  
pp. 637-641 ◽  
Author(s):  
H Roelofsen ◽  
R Ottenhoff ◽  
R P J Oude Elferink ◽  
P L M Jansen
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Organic Anion ◽  
Phosphodiesterase Inhibitor ◽  
Second Messenger ◽  
Anion Transport ◽  
Organic Anion Transport ◽  
Kinase C ◽  
Transport Assay ◽  
Messenger System

In order to investigate the regulation of canalicular organic-anion transport, we used a hepatocyte transport assay in which canalicular secretion of a model organic anion, dinitrophenyl-glutathione (GS-DNP), was measured in the presence of stimulators and inhibitors of the Ca2+/protein kinase C (PKC) second-messenger system and of the cyclic AMP (cAMP) second-messenger system. Vasopressin (24 nM) and the phorbol ester phorbol 12-myristate 13-acetate (1 microgram/ml), both stimulators of PKC, stimulated GS-DNP efflux by 65 +/- 36% and 55 +/- 28% respectively, whereas staurosporine (10 microM), an inhibitor of PKC, inhibited efflux by 53 +/- 13%. Glucagon and forskolin, both stimulators of the cAMP second-messenger system, as well as the cAMP analogue dibutyryl cAMP and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, did not significantly influence the GS-DNP efflux. It can be concluded that canalicular organic-anion transport in hepatocytes is either directly or indirectly regulated by PKC.

MEK inhibitors impair insulin-stimulated glucose uptake in 3T3-L1 adipocytes

2004 ◽  
Vol 287 (4) ◽  
pp. E758-E766 ◽  
Author(s):  
Anne W. Harmon ◽  
David S. Paul ◽  
Yashomati M. Patel
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Pi3k Pathway ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Mek Inhibitors ◽  
Transport Assay

In 3T3-L1 adipocytes, insulin activates three major signaling cascades, the phosphoinositide 3-kinase (PI3K) pathway, the Cbl pathway, and the mitogen-activated protein kinase (MAPK) pathway. Although PI3K and Cbl mediate insulin-stimulated glucose uptake by promoting the translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane, the MAPK pathway does not have an established role in insulin-stimulated glucose uptake. We demonstrate in this report that PI3K inhibitors also inhibit the MAPK pathway. To investigate the role of the MAPK pathway separately from that of the PI3K pathway in insulin-stimulated glucose uptake, we used two specific inhibitors of MAPK kinase (MEK) activity, PD-98059 and U-0126, which reduced insulin-stimulated glucose uptake by ∼33 and 50%, respectively. Neither MEK inhibitor affected the activation of Akt or PKCζ/λ, downstream signaling molecules in the PI3K pathway. Inhibition of MEK with U-0126 did not prevent GLUT4 from translocating to the plasma membrane, nor did it inhibit the subsequent docking and fusion of GLUT4- myc with the plasma membrane. MEK inhibitors affected glucose transport mediated by GLUT4 but not GLUT1. Importantly, the presence of MEK inhibitors only at the time of the transport assay markedly impaired both insulin-stimulated glucose uptake and MAPK signaling. Conversely, removal of MEK inhibitors before the transport assay restored glucose uptake and MAPK signaling. Collectively, our studies suggest a possible role for MEK in the activation of GLUT4.

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