Characterization of transport mechanisms and determinants critical for Na+-dependent Pisymport of the PiT family paralogs human PiT1 and PiT2

2006 ◽  
Vol 291 (6) ◽  
pp. C1377-C1387 ◽  
Author(s):  
Pernille Bøttger ◽  
Susanne E. Hede ◽  
Morten Grunnet ◽  
Boy Høyer ◽  
Dan A. Klærke ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Xenopus Laevis Oocytes ◽  
Transport Function ◽  
Knockout Mutant ◽  
Uptake Medium ◽  
The Impact ◽  
First Time

The general phosphate need in mammalian cells is accommodated by members of the Pitransport (PiT) family ( SLC20), which use either Na+or H+to mediate inorganic phosphate (Pi) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na+-dependent Pi(NaPi) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with32Pias a traceable Pisource. For PiT1, the Michaelis-Menten constant for Piwas determined as 322.5 ± 124.5 μM. PiT2 was analyzed for the first time and showed positive cooperativity in Piuptake with a half-maximal activity constant for Piof 163.5 ± 39.8 μM. PiT1- and PiT2-mediated Na+-dependent Piuptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na+dependency patterns. However, only PiT2 was capable of Na+-independent Pitransport at acidic pH. Study of the impact of divalent cations Ca2+and Mg2+revealed that Ca2+was important, but not critical, for NaPitransport function of PiT proteins. To gain insight into the NaPicotransport function, we analyzed PiT2 and a PiT2 Pitransport knockout mutant using22Na+as a traceable Na+source. Na+was transported by PiT2 even without Piin the uptake medium and also when Pitransport function was knocked out. This is the first time decoupling of Pifrom Na+transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E55and E575are responsible for linking Piimport to Na+transport in PiT2.

scholarly journals Characterization of a Cytotoxic Factor in Culture Filtrates of Serratia marcescens

2002 ◽  
Vol 70 (3) ◽  
pp. 1121-1128 ◽  
Author(s):  
Kent B. Marty ◽  
Christopher L. Williams ◽  
Linda J. Guynn ◽  
Michael J. Benedik ◽  
Steven R. Blanke
Keyword(s):  
Cytotoxic Activity ◽  
Serratia Marcescens ◽  
Mammalian Cells ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Cytotoxic Factor ◽  
Culture Filtrates ◽  
Type Strains

ABSTRACT Serratia marcescens culture filtrates have been reported to be cytotoxic to mammalian cells. Using biochemical and genetic approaches, we have identified a major source of this cytotoxic activity. Both heat and protease treatments abrogated the cytotoxicity of S. marcescens culture filtrates towards HeLa cells, suggesting the involvement of one or more protein factors. A screen for in vitro cytotoxic activity revealed that S. marcescens mutant strains that are deficient in production of a 56-kDa metalloprotease are significantly less cytotoxic to mammalian cells. Cytotoxicity was significantly reduced when culture filtrates prepared from wild-type strains were pretreated with either EDTA or 1,10-phenanthroline, which are potent inhibitors of the 56-kDa metalloprotease. Furthermore, cytotoxic activity was restored when the same culture filtrates were incubated with zinc divalent cations, which are essential for enzymatic activity of the 56-kDa metalloprotease. Finally, recombinant expression of the S. marcescens 56-kDa metalloprotease conferred a cytotoxic phenotype on the culture filtrates of a nonpathogenic Escherichia coli strain. Collectively, these data suggest that the 56-kDa metalloprotease contributes significantly to the in vitro cytotoxic activity commonly observed in S. marcescens culture filtrates.

scholarly journals The Influence of Bisphenol A (BPA) on Neuregulin 1-Like Immunoreactive Nerve Fibers in the Wall of Porcine Uterus

2018 ◽  
Vol 19 (10) ◽  
pp. 2962 ◽  
Author(s):  
Liliana Rytel
Keyword(s):  
Bisphenol A ◽  
Nerve Fibers ◽  
Double Immunofluorescence ◽  
Negative Effects ◽  
Protective Mechanisms ◽  
Neuregulin 1 ◽  
Small Doses ◽  
The Impact ◽  
First Time

Bisphenol A (BPA), a substance commonly used in the manufacture of plastics, shows multidirectional negative effects on humans and animals. Due to similarities to estrogens, BPA initially leads to disorders in the reproductive system. On the other hand, it is known that neuregulin 1 (NRG-1) is an active substance which enhances the survivability of cells, inhibits apoptosis, and protects tissues against damaging factors. Because the influence of BPA on the nervous system has also been described, the aim of the present study was to investigate for the first time the influence of various doses of BPA on neuregulin 1-like immunoreactive (NRG-1-LI) nerves located in the porcine uterus using the routine single- and double-immunofluorescence technique. The obtained results have shown that BPA increases the number and affects the neurochemical characterization of NRG-1-LI in the uterus, and changes are visible even under the impact of small doses of this toxin. The character of observed changes depended on the dose of BPA and the part of the uterus studied. These observations suggest that NRG-1 in nerves supplying the uterus may play roles in adaptive and protective mechanisms under the impact of BPA.

scholarly journals The Okur-Chung Neurodevelopmental Syndrome (OCNDS) mutation CK2K198R leads to a rewiring of kinase specificity

2021 ◽  
Author(s):  
Danielle M Caefer ◽  
Nhat Q Phan ◽  
Jennifer C Liddle ◽  
Jeremy L Balsbaugh ◽  
Joseph P O’Shea ◽  
...  
Keyword(s):  
High Resolution ◽  
Substrate Specificity ◽  
Tyrosine Phosphorylation ◽  
Initial Step ◽  
Loss Of Function ◽  
Scoring Method ◽  
Disease Condition ◽  
The Impact ◽  
First Time

AbstractOkur-Chung Neurodevelopmental Syndrome (OCNDS) is caused by heterozygous mutations to the CSNK2A1 gene, which encodes the alpha subunit of casein kinase II (CK2). The most frequently occurring mutation is lysine 198 to arginine (K198R). To investigate the impact of this mutation, we first generated a high-resolution phosphorylation motif of CK2WT, including the first characterization of specificity for tyrosine phosphorylation activity. A second high resolution motif representing CK2K198R substrate specificity was also generated. Here we report for the first time the impact of the OCNDS associated CK2K198R mutation. Contrary to prior speculation, the mutation does not result in a loss of function, but rather shifts the substrate specificity of the kinase. Broadly speaking the mutation leads to 1) a decreased preference for acidic residues in the +1 position, 2) a decreased preference for threonine phosphorylation, 3) an increased preference for tyrosine phosphorylation, and 4) an alteration of the tyrosine phosphorylation specificity motif. To further investigate the result of this mutation we have developed a probability-based scoring method, allowing us to predict shifts in phosphorylation in the K198R mutant relative to the wild type kinase. As an initial step we have applied the methodology to the set of axonally localized ion channels in an effort to uncover potential alterations of the phosphoproteome associated with the OCNDS disease condition.

scholarly journals Influence of Cation Exchange on the 27Al-NMR Spectra of Zeolites

2003 ◽  
Vol 217 (12) ◽  
pp. 1613-1626 ◽  
Author(s):  
W. Masierak ◽  
T. Emmler ◽  
Gerd Buntkowsky ◽  
A. Gutsze
Keyword(s):  
Cation Exchange ◽  
Nmr Spectra ◽  
Structural Changes ◽  
Divalent Cations ◽  
Second Order ◽  
Quadrupolar Coupling ◽  
Frame Work ◽  
Ion Radius ◽  
First Time

AbstractThe influence of cation exchange on the 27Al-NMR spectra of NaA-zeolites has been studied by 27Al-MAS- and MQ-MAS-Solid State-NMR. From the 27Al-spectra a characterization of the different Al sites in the A zeolites according to their chemical environment and the structural changes on the aluminosilicate network caused by the cation exchange are obtained. It is found that the exchange with cations with smaller ion-radius cause stronger distortions of the 27Al-NMR-spectra than exchange with larger cations like Ba2+. Employing MQ-MAS spectroscopy these distortions are revealed as second order quadrupolar effects for the smaller cations and as a combination of chemical shift and second order quadrupolar interaction for the Ba cation. These changes of the quadrupolar coupling are interpreted numerically via calculations of the lowering of the symmetry of the EFG tensor. Finally it is found that the exchange with divalent cations leads to distortions of the zeolitic framework and the formation of an extra-framework aluminum. To the best of our knowledge this is for the first time that evidence for the production of extra frame work aluminum by pure cation exchange without any thermal treatment has been found in type A zeolites.

scholarly journals A chemical screen identifies a link between lipid metabolism and mRNA translation

2020 ◽  
Author(s):  
Alba Corman ◽  
Dimitris C. Kanellis ◽  
Maria Häggblad ◽  
Vanesa Lafarga ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Mammalian Cells ◽  
Current Knowledge ◽  
Mrna Translation ◽  
Protein Translation ◽  
Sphingosine Kinases ◽  
Chemical Screen ◽  
Approved Drugs ◽  
The Impact

ABSTRACTmRNA translation is one of the most energy-demanding processes for living cells, alterations of which have been frequently documented in human disease. Using recently developed technologies that enable image-based quantitation of overall translation levels, we here conducted a chemical screen to evaluate how medically approved drugs, as well as drugs that are currently under development, influence overall translation levels. Consistent with current knowledge, inhibitors of the mTOR signaling pathway were the most represented class among translation suppresors. In addition, we identified that inhibitors of sphingosine kinases (SPHKs) also reduce mRNA translation levels independently of mTOR. Mechanistically this is explained by an effect of the compounds on the membranes of the endoplasmic reticulum, which activates the integrated stress response (ISR). Accordingly, the impact of SPHK inhibitors on translation is alleviated by the concomitant inhibition of ISR kinases. On the other hand, and despite the large number of molecules tested, our study failed to identify chemicals capable of substantially increasing mRNA translation, raising doubts on to what extent translation can be supra-physiologically stimulated in mammalian cells. In summary, our study provides the first comprehensive characterization of the effect of known drugs on protein translation and has helped to unravel a new link between lipid metabolism and mRNA translation in human cells.

scholarly journals Evolution of Differences in Transport Function in Slc11a Family Members

2007 ◽  
Vol 282 (49) ◽  
pp. 35646-35656 ◽  
Author(s):  
Michala Eichner Techau ◽  
Javier Valdez-Taubas ◽  
Jean-François Popoff ◽  
Richard Francis ◽  
Matthew Seaman ◽  
...  
Keyword(s):  
Divalent Cation ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Divalent Cations ◽  
Transport Function ◽  
Cation Uptake ◽  
Cation Homeostasis ◽  
N Terminus ◽  
Resistance To Infection ◽  
Divalent Cation Transporter

Slc11a1 (formerly Nramp1) is a proton/divalent cation transporter that regulates cation homeostasis in macrophages. Slc11a2 mediates divalent cation uptake via the gut and delivery into cells. The mode of action of the two transporters remains controversial. Heterologous expression in frog oocytes shows Slc11a2 is a symporter, whereas Slc11a1 is an antiporter fluxing divalent cations against the proton gradient. This explains why Slc11a2, but not Slc11a1, can complement EGTA sensitivity in smf1Δ/smf2Δ/smf3Δ yeast. However, some studies of transport in mammalian cells suggest Slc11a1 is a symporter. We now demonstrate that Slc11a1, but not Slc11a2, complements a divalent cation stress phenotype in bsd2Δ/rer1Δ yeast. This is the first description of a yeast complementation assay for Slc11a1 function. Given the prior demonstration in frog oocytes that Slc11a1 acts as an antiporter, the most plausible interpretation of the data is that Slc11a1 is rescuing bsd2Δ/rer1Δ yeast by exporting divalent cations. Chimaeras define the N terminus, and a segment of the protein core preceding transmembrane domain 9 through transmembrane domain 12, as important in rescuing the divalent cation stress phenotype. EGTA sensitivity and divalent cation stress phenotypes in yeast expressing Slc11a orthologues show that symport activity is ancestral. Molecular changes that mediate rescue of the divalent cation stress phenotype post-date frogs and co-evolved with Slc11a1 orthologues that regulate divalent cation homeostasis in macrophages and resistance to infection in chickens and mammals.

scholarly journals Comparison of the In Vitro and Ex Vivo Permeation of Existing Topical Formulations Used in the Treatment of Facial Angiofibroma and Characterization of the Variations Observed

Pharmaceutics ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1060
Author(s):  
Guillaume Le Guyader ◽  
Bernard Do ◽  
Victoire Vieillard ◽  
Karine Andrieux ◽  
Muriel Paul
Keyword(s):  
Ex Vivo ◽  
Thermodynamic Activity ◽  
Physico Chemical ◽  
Ex Vivo Permeation ◽  
Franz Cells ◽  
Great Heterogeneity ◽  
The Impact ◽  
First Time

Rapamycin has been used topically to treat facial angiofibromas associated with tuberous sclerosis for more than a decade. In the absence of a commercial form, a large number of formulations have been clinically tested. However, given the great heterogeneity of these studies, particularly with regard to the response criteria, it was difficult to know the impact and thus to compare the relevance of the formulations used. The objective of this work was therefore to evaluate the link between the diffusion of rapamycin and the physico-chemical characteristics of these different formulations on Strat-M® membranes as well as on human skin using Franz cells. Our results underline the importance of the type of vehicle used (hydrogel > cream > lipophilic ointment), the soluble state of rapamycin and its concentration close to saturation to ensure maximum thermodynamic activity. Thus, this is the first time that a comparative study of the different rapamycin formulations identified in the literature for the management of facial angiofibromas has been carried out using a pharmaceutical and biopharmaceutical approach. It highlights the important parameters to be considered in the development and optimization of topical rapamycin formulations with regard to cutaneous absorption for clinical efficacy.

scholarly journals Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Marine Drugs ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. 562
Author(s):  
Marlene Vuillemin ◽  
Artem S. Silchenko ◽  
Hang Thi Thuy Cao ◽  
Maxim S. Kokoulin ◽  
Vo Thi Dieu Trang ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Divalent Cations ◽  
Recombinant Expression ◽  
Biological Activities ◽  
Functional Characterization ◽  
Maximal Activity ◽  
Brown Macroalgae ◽  
Pharmaceutical Uses ◽  
Enzymatic Depolymerization

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

scholarly journals Environmental sources along natural cave ripening shape the microbiome and metabolome of artisanal blue-veined cheeses

2021 ◽  
Author(s):  
Elena Alexa ◽  
José F Cobo-Diaz ◽  
Erica Renes ◽  
Tom F O´Callaghan ◽  
Kieran Kilcawley ◽  
...  
Keyword(s):  
Longitudinal Study ◽  
Source Tracking ◽  
Fermented Foods ◽  
Quality And Safety ◽  
Microbial Succession ◽  
Quality Markers ◽  
Food Quality And Safety ◽  
The Impact ◽  
First Time

Abstract Microorganisms colonising processing environments can significantly impact food quality and safety. Here we describe a detailed longitudinal study assessing the impact of cave ripening on the microbial succession and quality markers across different producers of blue-veined cheese. Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome and metabolome. The cheese microbiome was significantly determined by the microbiome of caves, which were a source of Brevibacterium, Corynebacterium, Staphylococcus, Tetragenococcus and Yaniella, among others, as demonstrated through source tracking and the characterization of 613 metagenome assembled genomes. Tetragenococcus koreensis and T. halophilus were detected at high abundance in cheese for the first time, associated with the occurrence of various metabolites, and showed high levels of horizontal gene transfer with other members of the cheese microbiome. Overall, we demonstrated that processing environments can be a source of non-starter microorganisms of relevance to ripening of artisanal fermented foods.

scholarly journals Functional differences between flounder and rat thiazide-sensitive Na-Cl cotransporter

2002 ◽  
Vol 282 (4) ◽  
pp. F599-F607 ◽  
Author(s):  
Norma Vázquez ◽  
Adriana Monroy ◽  
Elisa Dorantes ◽  
Rosario A. Muñoz-Clares ◽  
Gerardo Gamba
Keyword(s):  
Phorbol Esters ◽  
Xenopus Laevis Oocytes ◽  
Kinase C ◽  
Functional Differences ◽  
Low Concentrations ◽  
Biphasic Effect ◽  
Uptake Medium ◽  
Regulatory Properties ◽  
Menten Constant

The purpose of the present study was to determine the major functional, pharmacological, and regulatory properties of the flounder thiazide-sensitive Na-Cl cotransporter (flTSC) to make a direct comparison with our recent characterization of the rat TSC (rTSC; Monroy A, Plata C, Hebert SC, and Gamba G. Am J Physiol Renal Physiol 279: F161–F169, 2000). When expressed in Xenopus laevis oocytes, flTSC exhibits lower affinity for Na+ than for Cl−, with apparent Michaelis-Menten constant ( K m) values of 58.2 ± 7.1 and 22.1 ± 4.2 mM, respectively. These K m values are significantly higher than those observed in rTSC. The Na+ and Cl− affinities decreased when the concentration of the counterion was lowered, suggesting that the binding of one ion increases the affinity of the transporter for the other. The effect of several thiazides on flTSC function was biphasic. Low concentrations of thiazides (10−9 to 10−7 M) resulted in activation of the cotransporter, whereas higher concentrations (10−6 to 10−4 M) were inhibitory. In rTSC, this biphasic effect was observed only with chlorthalidone. The affinity for thiazides in flTSC was lower than in rTSC, but the affinity in flTSC was not affected by the Na+ or the Cl− concentration in the uptake medium. In addition to thiazides, flTSC and rTSC were inhibited by Hg2+, with an apparent higher affinity for rTSC. Finally, flTSC function was decreased by activation of protein kinase C with phorbol esters and by hypertonicity. In summary, we have found significant regulatory, kinetic, and pharmacological differences between flTSC and rTSC orthologues.

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