scholarly journals MIF is among the proinflammatory cytokines increased by LPS in the human trophoblast line

2016 ◽  
Vol 68 (4) ◽  
pp. 715-722 ◽  
Author(s):  
Milica Jovanovic-Krivokuca ◽  
Ivana Stefanoska ◽  
Abu Rabi ◽  
Aleksandra Vilotic ◽  
Milos Petronijevic ◽  
...  
Keyword(s):  
Gelatin Zymography ◽  
Strong Immune Response ◽  
Human Trophoblast ◽  
E Coli ◽  
Cell Wall Constituent ◽  
A Cell ◽  
First Time ◽  
Trophoblast Cell Line

Infection is increasingly considered to contribute to pathological conditions in pregnancy. The placenta acts as a protective immunological fetomaternal barrier which recognizes microbes by pattern recognition receptors on the trophoblast. Lipopolysaccharide (LPS) is a cell wall constituent of Gram-negative bacteria that elicits a strong immune response. In this study, LPS from E. coli was used to treat the HTR-8/SVneo trophoblast cell line and examine its influence on cytokines IL-6, IL-8 and MIF using real-time PCR, metalloproteinases (MMP)-2 and -9 by gelatin zymography, and Western analysis of integrin subunits ?1 and ?1, all known to contribute to migration of human trophoblasts in vitro. The results described herein for the first time, show that MIF mRNA and secreted MIF protein were significantly elevated (2.5-3- and 2-fold, respectively) in LPS-treated cells. MMP-2 and MMP-9 levels were increased, as well as cell migration, as judged by a wound-healing test, however, no changes in the studied integrin subunits, cell viability or cell numbers were observed. The data obtained furthers our understanding of LPS actions on the trophoblast in vitro, additionally implicate MIF, and suggest that infection in vivo could indeed alter the functional characteristics of the trophoblast.

scholarly journals In Vivo Remodeling of Altered Autophagy-Lysosomal Pathway by a Phosphopeptide in Lupus

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2328
Author(s):  
Fengjuan Wang ◽  
Inmaculada Tasset ◽  
Ana Maria Cuervo ◽  
Sylviane Muller
Keyword(s):  
Metabolic Disorders ◽  
Inflammatory Diseases ◽  
Cellular Mechanism ◽  
Major Protein ◽  
Recovery Effect ◽  
A Cell ◽  
First Time ◽  
Chaperone Mediated Autophagy

The phosphopeptide P140/Lupuzor, which improves the course of lupus disease in mice and patients, targets chaperone-mediated autophagy (CMA), a selective form of autophagy that is abnormally upregulated in lupus-prone MRL/lpr mice. Administered intravenously to diseased mice, P140 reduces the expression level of two major protein players of CMA, LAMP2A and HSPA8, and inhibits CMA in vitro in a cell line that stably expresses a CMA reporter. Here, we aimed to demonstrate that P140 also affects CMA in vivo and to unravel the precise cellular mechanism of how P140 interacts with the CMA process. MRL/lpr mice and CBA/J mice used as control received P140 or control peptides intravenously. Lysosome-enriched fractions of spleen or liver were prepared to examine lysosomal function. Highly purified lysosomes were further isolated and left to incubate with the CMA substrate to study at which cellular step P140 interacts with the CMA process. The data show that P140 effectively regulates CMA in vivo in MRL/lpr mice at the step of substrate lysosomal uptake and restores some alterations of defective lysosomes. For the first time, it is demonstrated that by occluding the intralysosome uptake of CMA substrates, a therapeutic molecule can attenuate excessive CMA activity in a pathological pro-inflammatory context and protect against hyperinflammation. This recovery effect of P140 on hyperactivated CMA is not only important for lupus therapy but potentially also for treating other (auto)inflammatory diseases, including neurologic and metabolic disorders, where CMA modulation would be highly beneficial.

scholarly journals Development of a Cell-Based Assay Probing the Specific Interaction between the Human Immunodeficiency Virus Type 1 Nucleocapsid and Psi RNA In Vivo

2007 ◽  
Vol 81 (11) ◽  
pp. 6151-6155 ◽  
Author(s):  
Soo In Jang ◽  
Young Ho Kim ◽  
Soon Young Paik ◽  
Ji Chang You
Keyword(s):  
Human Immunodeficiency Virus ◽  
Specific Interaction ◽  
Deletion Mapping ◽  
Packaging Signal ◽  
Immunodeficiency Virus ◽  
A Cell ◽  
First Time

ABSTRACT Here, we describe a cell-based in vivo assay that probes the specific interaction between nucleocapsid (NC) protein and Psi (Ψ) RNA, the human immunodeficiency virus (HIV) packaging signal. The results demonstrate for the first time a specific NC-Ψ interaction within living cells. The specificity and applicability of the assay were confirmed by mutational studies of NC and deletion-mapping analyses of Ψ-RNA as well as by testing the in vivo NC-binding effects of NC-aptamer RNAs identified previously in vitro. This assay system would facilitate further detailed studies of the NC-Ψ interaction in vivo and the screening of various anti-HIV molecules targeting NC and the specific interaction.

scholarly journals Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

2012 ◽  
Vol 287 (42) ◽  
pp. 35747-35755 ◽  
Author(s):  
Tanmay Dutta ◽  
Arun Malhotra ◽  
Murray P. Deutscher
Keyword(s):  
Potential Role ◽  
Wild Type ◽  
X Ray ◽  
E Coli ◽  
Phage T4 ◽  
A Cell ◽  
Rnase Z

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.

scholarly journals Optimization of a bacterial three-hybrid assay through in vivo titration of an RNA-DNA adapter-protein

2020 ◽  
Author(s):  
Clara D. Wang ◽  
Rachel Mansky ◽  
Hannah LeBlanc ◽  
Chandra M. Gravel ◽  
Katherine E. Berry
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Regulate Gene Expression ◽  
E Coli ◽  
Binding Energetics ◽  
Response To Stress ◽  
First Time ◽  
Regulate Gene

ABSTRACTNon-coding RNAs regulate gene expression in every domain of life. In bacteria, small RNAs (sRNAs) regulate gene expression in response to stress and are often assisted by RNA-chaperone proteins, such as Hfq. We have recently developed a bacterial three-hybrid (B3H) assay that detects the strong binding interactions of certain E. coli sRNAs with proteins Hfq and ProQ. Despite the promise of this system, the signal-to-noise has made it challenging to detect weaker interactions. In this work, we use Hfq-sRNA interactions as a model system to optimize the B3H assay, so that weaker RNA-protein interactions can be more reliably detected. We find that the concentration of the RNA-DNA adapter is an important parameter in determining the signal in the system, and have modified the plasmid expressing this component to tune its concentration to optimal levels. In addition, we have systematically perturbed the binding affinity of Hfq-RNA interactions to define, for the first time, the relationship between B3H signal and in vitro binding energetics. The new pAdapter construct presented here substantially expands the range of detectable interactions in the B3H assay, broadening its utility. This improved assay will increase the likelihood of identifying novel protein-RNA interactions with the B3H system, and will facilitate exploration of the binding mechanisms of these interactions.

scholarly journals CELL WALL COMPOSITION AND VIRULENCE IN ESCHERICHIA COLI

1968 ◽  
Vol 128 (3) ◽  
pp. 399-414 ◽  
Author(s):  
Donald N. Medearis ◽  
Bruce M. Camitta ◽  
Edward C. Heath
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Uridine Diphosphate ◽  
E Coli ◽  
Endotoxin Activity ◽  
A Cell ◽  
The Difference ◽  
Definition Of

Uridine diphosphate galactose 4-epimerase and phosphomannose isomerase-deficient mutants of Escherichia coli O111:B4 were studied to test the hypothesis that in E. coli a specific relationship exists between O antigenicity, virulence, and capacity to resist phagocytosis. The first mutant, designated J-5, produces a cell wall lipopolysaccharide, the side chains of which do not contain galactose, glucose, N-acetylglucosamine, or colitose. The second mutant produces a cell wall lipopolysaccharide which lacks only colitose. The capacity of these various organisms to kill mice was strikingly different. E. coli O111 was 1000 times as virulent as J-5, and 100 times as virulent as L-2. The capacity of the organisms to kill mice was correlated with their ability to resist phagocytosis and to persist in the peritoneal cavity. The parent strain of O111 resisted phagocytosis by macrophages in vivo and polymorphonuclear leukocytes in vitro. The mutants did not, and the organism most deficient in the saccharide component of its LPS was most susceptible to phagocytosis and least virulent. These results were corroborated by growing the mutants in appropriately supplemented media which permitted the synthesis of complete LPS, reversed the susceptibility to phagocytosis, and restored virulence. Finally, serological reactivity was consistent with previous observations which had demonstrated that the O antigenicity of E. coli is determined by the saccharide composition of its cell wall lipopolysaccharide. Despite the difference in the capacity of the various log-phase organisms to kill mice when injected intraperitoneally, purified lipopolysaccharides extracted from them did not differ significantly in their capacity to kill or produce fever. Thus virulence was shown to be independent of endotoxin activity which in turn seemed to be unrelated to the saccharide composition of the cell wall LPS. Collectively, these data provide at least a partial molecular definition of virulence in E. coli by demonstrating that the presence or absence of specific sugars in its cell wall lipopolysaccharide is a determinant of its antiphagocytic capacity and its virulence.

Phenethyl-4-ANPP: A Marginally Active Byproduct Suggesting a Switch in Illicit Fentanyl Synthesis Routes

2021 ◽  
Author(s):  
Marthe M Vandeputte ◽  
Alex J Krotulski ◽  
Fabian Hulpia ◽  
Serge Van Calenbergh ◽  
Christophe P Stove
Keyword(s):  
Valuable Insight ◽  
Unknown Compound ◽  
The Usa ◽  
A Cell ◽  
Synthesis Routes ◽  
And Control ◽  
First Time ◽  
Single Reaction

Abstract Profiling of the illicit fentanyl supply is invaluable from surveillance and intelligence perspectives. An important strategy includes the study of chemical attribution signatures (e.g., trace amounts of synthesis precursors, impurities/byproducts in seized material and metabolites in biological samples). This information provides valuable insight into the employed synthesis routes at the heart of illicit fentanyl manufacture (previously mainly the so-called Janssen or Siegfried methods), allowing to track and ultimately regulate crucial precursors. This report focuses on phenethyl-4-anilino-N-phenethylpiperidine (phenethyl-4-ANPP), a formerly unknown compound that was identified for the first time in a fentanyl powder sample seized in April 2019, followed by its identification in a biological sample in December 2019. Between 2019-Q4 and 2020-Q3, phenethyl-4-ANPP was detected in 25/1,054 fentanyl cases in the USA. There are currently no reports on how this compound may have ended up in illicit drug preparations and whether its presence may have potential in vivo relevance. We propose three possible fentanyl synthesis routes that, when badly executed in a single reaction vessel, may involve the formation of phenethyl-4-ANPP. We hypothesize that the presence of the latter is the result of a shift in fentanyl synthesis routes in an attempt to circumvent restrictions on previously used precursors. Using a cell-based µ-opioid receptor recruitment assay, we show that the extent of MOR activation caused by 100 µM phenethyl-4-ANPP is comparable to that exerted by a roughly 100,000-fold lower concentration of fentanyl (0.001 µM or 0.336 ng/mL). Negligible in vitro opioid activity, combined with its low abundance in fentanyl preparations, most likely renders phenethyl-4-ANPP biologically irrelevant in vivo. However, as clandestine operations are constantly changing shape, monitoring of fentanyl attributions remains pivotal in our understanding and control of illicit fentanyl manufacture and supply.

scholarly journals Line-FRAP, a versatile method based on fluorescence recovery after photobleaching to measure diffusion rates in vitro and in vivo

2020 ◽  
Author(s):  
Debabrata Dey ◽  
Shir Marciano ◽  
Gideon Schreiber
Keyword(s):  
Diffusion Coefficients ◽  
Protein Concentration ◽  
Confocal Microscope ◽  
Standard Equipment ◽  
E Coli ◽  
A Cell ◽  
Diffusion Rates ◽  
Classical Mode

AbstractA cell is a densely packed conglomerate of macromolecules, where diffusion is essential for their function. The crowded conditions may affect diffusion both through hard (occluded space) and soft (weak, non-specific) interactions. Multiple-methods have been developed to measure diffusion rates at physiological protein concentrations within cells, however, each of them has its limitations. Here, we introduce Line-FRAP, a method based on measuring recovery of photobleaching under a confocal microscope that allows diffusion rate measurements for fast diffusing molecules to be measured in versatile environments using standard equipment. Implementation of Line mode to the classical FRAP technique greatly improves the time resolution in data acquisition, from 20-50 Hz in the classical mode to 800 Hz in the line mode. We also introduce an updated method for data analysis to obtain diffusion coefficients in various environments, with the number of pixels bleached at the first frame after bleaching being a critical parameter. We evaluated the method using different proteins either chemically labelled or by fusion to YFP. The calculated diffusion rates were comparable to literature data as measured in vitro, in HeLa cells and in E.coli. Diffusion coefficients in HeLa was ~2.5-fold slower and in E. coli 15-fold slower than measured in buffer. Moreover, we show that increasing the osmotic pressure on E.coli further decreases diffusion, till a point where proteins stop to move. The method presented here is easy to apply on a standard confocal microscope, fits a large range of molecules with different sizes and provides robust results in any conceivable environment and protein concentration for fast diffusing molecules.

scholarly journals Cross Kingdom Functional Conservation of the Core Universally Conserved Threonylcarbamoyladenosine tRNA Synthesis Enzymes

2014 ◽  
Vol 13 (9) ◽  
pp. 1222-1231 ◽  
Author(s):  
Patrick C. Thiaville ◽  
Basma El Yacoubi ◽  
Ludovic Perrochia ◽  
Arnaud Hecker ◽  
Magali Prigent ◽  
...  
Keyword(s):  
Mitochondrial Pathway ◽  
Stem Loop ◽  
Content Type ◽  
E Coli ◽  
Functional Conservation ◽  
The Core ◽  
First Time ◽  
Keops Complex

ABSTRACT Threonylcarbamoyladenosine (t 6 A) is a universal modification located in the anticodon stem-loop of tRNAs. In yeast, both cytoplasmic and mitochondrial tRNAs are modified. The cytoplasmic t 6 A synthesis pathway was elucidated and requires Sua5p, Kae1p, and four other KEOPS complex proteins. Recent in vitro work suggested that the mitochondrial t 6 A machinery of Saccharomyces cerevisiae is composed of only two proteins, Sua5p and Qri7p, a member of the Kae1p/TsaD family (L. C. K. Wan et al., Nucleic Acids Res. 41:6332–6346, 2013, http://dx.doi.org/10.1093/nar/gkt322 ). Sua5p catalyzes the first step leading to the threonyl-carbamoyl-AMP intermediate (TC-AMP), while Qri7 transfers the threonyl-carbamoyl moiety from TC-AMP to tRNA to form t 6 A. Qri7p localizes to the mitochondria, but Sua5p was reported to be cytoplasmic. We show that Sua5p is targeted to both the cytoplasm and the mitochondria through the use of alternative start sites. The import of Sua5p into the mitochondria is required for this organelle to be functional, since the TC-AMP intermediate produced by Sua5p in the cytoplasm is not transported into the mitochondria in sufficient amounts. This minimal t 6 A pathway was characterized in vitro and, for the first time, in vivo by heterologous complementation studies in Escherichia coli . The data revealed a potential for TC-AMP channeling in the t 6 A pathway, as the coexpression of Qri7p and Sua5p is required to complement the essentiality of the E. coli tsaD mutant. Our results firmly established that Qri7p and Sua5p constitute the mitochondrial pathway for the biosynthesis of t 6 A and bring additional advancement in our understanding of the reaction mechanism.

scholarly journals Distinct metabolic states of a cell guide alternate fates of mutational buffering through altered proteostasis

2019 ◽  
Author(s):  
Kanika Verma ◽  
Kanika Saxena ◽  
Rajashekar Donaka ◽  
Aseem Chaphalkar ◽  
Manish Kumar Rai ◽  
...  
Keyword(s):  
Cellular Metabolism ◽  
Negative Regulation ◽  
Metabolic Alterations ◽  
E Coli ◽  
Metabolic States ◽  
Metabolite Concentrations ◽  
A Cell ◽  
Bona Fide

SummaryChanges in metabolism can alter the cellular milieu; can this also change intracellular proteostasis? Since proteostasis can modulate mutational buffering, if change in metabolism has the ability to change proteostasis, arguably, it should also alter mutational buffering. Building on this, we find that altered cellular metabolic states in E. coli buffer distinct mutations. Buffered-mutants had folding problems in vivo and were differently chaperoned in different metabolic states. Notably, this assistance was dependent upon the metabolites and not on the increase in canonical chaperone machineries. Additionally, we were able to reconstitute the folding assistance afforded by metabolites in vitro and propose that changes in metabolite concentrations have the potential to alter proteostasis. Collectively, we unravel that the metabolite pools are bona fide members of proteostasis and aid in mutational buffering. Given the plasticity in cellular metabolism, we posit that metabolic alterations may play an important role in the positive or negative regulation of proteostasis.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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