ResearchPad - fluorescence-recovery-after-photobleaching https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Flavonoids and antioxidant activity of rare and endangered fern: <i>Isoetes sinensis</i>]]> https://www.researchpad.co/article/elastic_article_7844 Isoetes sinensis Palmer is a critically endangered, first-class protected plant in China. Until now, researchers have primarily focused on the ultrastructure, phylogeny, and transcriptomes of the plant. However, flavonoid profiles and bioactivity of I. sinensis have not been extensively investigated. To develop the endangered I. sinensis for edible and medicinal purposes, flavonoid content, chemical constitution, and antioxidant activities were investigated in this study. Results revealed the following. 1) The total flavonoid content was determined as 10.74 ± 0.25 mg/g., 2) Antioxidant activities were stronger than most ferns, especially ABTS free radical scavenging activities. 3) Four flavones, containing apigenin, apigenin-7-glucuronide, acacetin-7-O-glcopyranoside, and homoplantageninisoetin; four flavonols, namely, isoetin, kaempferol-3-O-glucoside, quercetin-3-O-[6”-O-(3-hydroxy-3-methylglutaryl)-β-D-glucopyranoside], and limocitrin-Neo; one prodelphinidin (procyanidins;) and one nothofagin (dihydrochalcone) were tentatively identified in the mass spectrometry-DAD (254nm) chromatograms. This study was the first to report on flavonoid content and antioxidant activities of I. sinensis. Stronger antioxidant activity and flavonoid content suggests that the endangered I. sinensis is an important and potentially edible and medicinal plant.

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<![CDATA[NAP (davunetide) preferential interaction with dynamic 3-repeat Tau explains differential protection in selected tauopathies]]> https://www.researchpad.co/article/5c92b379d5eed0c4843a4107

The microtubule (MT) associated protein Tau is instrumental for the regulation of MT assembly and dynamic instability, orchestrating MT-dependent cellular processes. Aberration in Tau post-translational modifications ratio deviation of spliced Tau isoforms 3 or 4 MT binding repeats (3R/4R) have been implicated in neurodegenerative tauopathies. Activity-dependent neuroprotective protein (ADNP) is vital for brain formation and cognitive function. ADNP deficiency in mice causes pathological Tau hyperphosphorylation and aggregation, correlated with impaired cognitive functions. It has been previously shown that the ADNP-derived peptide NAP protects against ADNP deficiency, exhibiting neuroprotection, MT interaction and memory protection. NAP prevents MT degradation by recruitment of Tau and end-binding proteins to MTs and expression of these proteins is required for NAP activity. Clinically, NAP (davunetide, CP201) exhibited efficacy in prodromal Alzheimer’s disease patients (Tau3R/4R tauopathy) but not in progressive supranuclear palsy (increased Tau4R tauopathy). Here, we examined the potential preferential interaction of NAP with 3R vs. 4R Tau, toward personalized treatment of tauopathies. Affinity-chromatography showed that NAP preferentially interacted with Tau3R protein from rat brain extracts and fluorescence recovery after photobleaching assay indicated that NAP induced increased recruitment of human Tau3R to MTs under zinc intoxication, in comparison to Tau4R. Furthermore, we showed that NAP interaction with tubulin (MTs) was inhibited by obstruction of Tau-binding sites on MTs, confirming the requirement of Tau-MT interaction for NAP activity. The preferential interaction of NAP with Tau3R may explain clinical efficacy in mixed vs. Tau4R pathologies, and suggest effectiveness in Tau3R neurodevelopmental disorders.

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<![CDATA[The seven transmembrane domain protein MoRgs7 functions in surface perception and undergoes coronin MoCrn1-dependent endocytosis in complex with Gα subunit MoMagA to promote cAMP signaling and appressorium formation in Magnaporthe oryzae]]> https://www.researchpad.co/article/5c7d95f6d5eed0c484735053

Regulator of G-protein signaling (RGS) proteins primarily function as GTPase-accelerating proteins (GAPs) to promote GTP hydrolysis of Gα subunits, thereby regulating G-protein mediated signal transduction. RGS proteins could also contain additional domains such as GoLoco to inhibit GDP dissociation. The rice blast fungus Magnaporthe oryzae encodes eight RGS and RGS-like proteins (MoRgs1 to MoRgs8) that have shared and distinct functions in growth, appressorium formation and pathogenicity. Interestingly, MoRgs7 and MoRgs8 contain a C-terminal seven-transmembrane domain (7-TM) motif typical of G-protein coupled receptor (GPCR) proteins, in addition to the conserved RGS domain. We found that MoRgs7, but not MoRgs8, couples with Gα MoMagA to undergo endocytic transport from the plasma membrane to the endosome upon sensing of surface hydrophobicity. We also found that MoRgs7 can interact with hydrophobic surfaces via a hydrophobic interaction, leading to the perception of environmental hydrophobiccues. Moreover, we found that MoRgs7-MoMagA endocytosis is regulated by actin patch-associated protein MoCrn1, linking it to cAMP signaling. Our studies provided evidence suggesting that MoRgs7 could also function in a GPCR-like manner to sense environmental signals and it, together with additional proteins of diverse functions, promotes cAMP signaling required for developmental processes underlying appressorium function and pathogenicity.

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<![CDATA[Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation]]> https://www.researchpad.co/article/5c4a3091d5eed0c4844c0568

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.

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<![CDATA[The brown algal mode of tip growth: Keeping stress under control]]> https://www.researchpad.co/article/5c466523d5eed0c4845179cd

Tip growth has been studied in pollen tubes, root hairs, and fungal and oomycete hyphae and is the most widely distributed unidirectional growth process on the planet. It ensures spatial colonization, nutrient predation, fertilization, and symbiosis with growth speeds of up to 800 μm h−1. Although turgor-driven growth is intuitively conceivable, a closer examination of the physical processes at work in tip growth raises a paradox: growth occurs where biophysical forces are low, because of the increase in curvature in the tip. All tip-growing cells studied so far rely on the modulation of cell wall extensibility via the polarized excretion of cell wall–loosening compounds at the tip. Here, we used a series of quantitative measurements at the cellular level and a biophysical simulation approach to show that the brown alga Ectocarpus has an original tip-growth mechanism. In this alga, the establishment of a steep gradient in cell wall thickness can compensate for the variation in tip curvature, thereby modulating wall stress within the tip cell. Bootstrap analyses support the robustness of the process, and experiments with fluorescence recovery after photobleaching (FRAP) confirmed the active vesicle trafficking in the shanks of the apical cell, as inferred from the model. In response to auxin, biophysical measurements change in agreement with the model. Although we cannot strictly exclude the involvement of a gradient in mechanical properties in Ectocarpus morphogenesis, the viscoplastic model of cell wall mechanics strongly suggests that brown algae have evolved an alternative strategy of tip growth. This strategy is largely based on the control of cell wall thickness rather than fluctuations in cell wall mechanical properties.

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<![CDATA[Effects of different pretreatments on flavonoids and antioxidant activity of Dryopteris erythrosora leave]]> https://www.researchpad.co/article/5c3667dad5eed0c4841a66be

Flavonoids are secondary metabolites of plants that often have medical applications. The influences of different sample drying pretreatments on flavonoids and antioxidant activity of ferns have not studies. Dryopteris erythrosora leaves used to analyze flavonoid alterations resulting from drying pretreatments. The total flavonoid content of D. erythrosora leaves exposed to different pretreatments was significantly different. The total flavonoid content of samples initially air-dried in shade and then oven-dried at 75°C were the highest (7.6%), while samples initially dried at 75°C had the lowest content (2.17%). Antioxidant activities of D. erythrosora leaves with different pretreatments varied. Group B first air-dried in the shade and then oven-dried at 75°C and group C first air-dried in the sun and then oven-dried at 75°C, both showed relatively stronger antioxidant activity. The best pretreatment for preserving the flavonoids was to first dry the plant material in the shade and then complete the drying process in an oven at 75°C. It was tentatively identified 22 flavonoids among the four different pretreatments by HPLC-ESI-TOF-MS.

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<![CDATA[Anisotropic Crb accumulation, modulated by Src42A, is coupled to polarised epithelial tube growth in Drosophila]]> https://www.researchpad.co/article/5c059de3d5eed0c4849c965b

The control of the size of internal tubular organs, such as the lungs or vascular system, is critical for proper physiological activity and to prevent disease or malformations. This control incorporates the intrinsic physical anisotropy of tubes to generate proportionate organs that match their function. The exact mechanisms underlying tube size control and how tubular anisotropy is translated at the cellular level are still not fully understood. Here we investigate these mechanisms using the Drosophila tracheal system. We show that the apical polarity protein Crumbs transiently accumulates anisotropically at longitudinal cell junctions during tube elongation. We provide evidence indicating that the accumulation of Crumbs in specific apical domains correlates with apical surface expansion, suggesting a link between the anisotropic accumulation of Crumbs at the cellular level and membrane expansion. We find that Src42A is required for the anisotropic accumulation of Crumbs, thereby identifying the first polarised cell behaviour downstream of Src42A. Our results indicate that Src42A regulates a mechanism that increases the fraction of Crb protein at longitudinal junctions, and genetic interaction experiments are consistent with Crb acting downstream of Src42A in controlling tube size. Collectively, our results suggest a model in which Src42A would sense the inherent anisotropic mechanical tension of the tube and translate it into a polarised Crumbs accumulation, which may promote a bias towards longitudinal membrane expansion, orienting cell elongation and, as a consequence, longitudinal growth at the tissue level. This work provides new insights into the key question of how organ growth is controlled and polarised and unveils the function of two conserved proteins, Crumbs and Src42A, with important roles in development and homeostasis as well as in disease, in this biological process.

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<![CDATA[Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231]]> https://www.researchpad.co/article/5989dac6ab0ee8fa60bb2606

Golgi phosphoprotein 3 (GOLPH3) has been implicated in the development of carcinomas in many human tissues, and is currently considered a bona fide oncoprotein. Importantly, several tumor types show overexpression of GOLPH3, which is associated with tumor progress and poor prognosis. However, the underlying molecular mechanisms that connect GOLPH3 function with tumorigenicity are poorly understood. Experimental evidence shows that depletion of GOLPH3 abolishes transformation and proliferation of tumor cells in GOLPH3-overexpressing cell lines. Conversely, GOLPH3 overexpression drives transformation of primary cell lines and enhances mouse xenograft tumor growth in vivo. This evidence suggests that overexpression of GOLPH3 could result in distinct features of GOLPH3 in tumor cells compared to that of non-tumorigenic cells. GOLPH3 is a peripheral membrane protein mostly localized at the trans-Golgi network, and its association with Golgi membranes depends on binding to phosphatidylinositol-4-phosphate. GOLPH3 is also contained in a large cytosolic pool that rapidly exchanges with Golgi-associated pools. GOLPH3 has also been observed associated with vesicles and tubules arising from the Golgi, as well as other cellular compartments, and hence it has been implicated in several membrane trafficking events. Whether these and other features are typical to all different types of cells is unknown. Moreover, it remains undetermined how GOLPH3 acts as an oncoprotein at the Golgi. Therefore, to better understand the roles of GOLPH3 in cancer cells, we sought to compare some of its biochemical and cellular properties in the human breast cancer cell lines MCF7 and MDA-MB-231 with that of the non-tumorigenic breast human cell line MCF 10A. We found unexpected differences that support the notion that in different cancer cells, overexpression of GOLPH3 functions in diverse fashions, which may influence specific tumorigenic phenotypes.

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<![CDATA[Spatial-Temporal Study of Rab1b Dynamics and Function at the ER-Golgi Interface]]> https://www.researchpad.co/article/5989db17ab0ee8fa60bcd51b

The GTPase Rab1b is involved in ER to Golgi transport, with multiple Rab1b effectors (located at ERES, VTCs and the Golgi complex) being required for its function. In this study, we performed live-cell dual-expression studies to analyze the dynamics of Rab1b and some effectors located at the ERES-Golgi interface. Rab1b occupied widely distributed mobile punctate and tubular structures, displaying a transient overlaps with its effectors and showing that these overlaps occurred at the same time in spatially distinct steps of ER to Golgi transport. In addition, we assessed Rab1b dynamics during cargo sorting by analyzing the concentration at ERES of a Golgi protein (SialT2-CFP) during Brefeldin A washout (BFA WO). Rab1b was associated to most of the ERES structures, but at different times during BFA WO, and recurrently SialT2-CFP was sorted in the ERES-Rab1b positive structures. Furthermore, we reveal for first time that Rab1b localization time at ERES depended on GBF1, a Rab1b effector that acts as the guanine nucleotide exchange factor of Arf1, and that Rab1b membrane association/dissociation dynamics at ERES was dependent on the GBF1 membrane association and activity, which strongly suggests that GBF1 activity modulates Rab1b membrane cycling dynamic.

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<![CDATA[Carboxyl-Terminal SSLKG Motif of the Human Cystinosin-LKG Plays an Important Role in Plasma Membrane Sorting]]> https://www.researchpad.co/article/5989da3eab0ee8fa60b88d88

Cystinosin mediates an ATP-dependent cystine efflux from lysosomes and causes, if mutated, nephropathic cystinosis, a rare inherited lysosomal storage disease. Alternative splicing of the last exon of the cystinosin sequence produces the cystinosin-LKG isoform that is characterized by a different C-terminal region causing changes in the subcellular distribution of the protein. We have constructed RFP-tagged proteins and demonstrated by site-directed mutagenesis that the carboxyl-terminal SSLKG sequence of cystinosin-LKG is an important sorting motif that is required for efficient targeting the protein to the plasma membrane, where it can mediate H+ coupled cystine transport. Deletion of the SSLKG sequence reduced cystinosin-LKG expression in the plasma membrane and cystine transport by approximately 30%, and induced significant accumulation of the protein in the Golgi apparatus and in lysosomes. Cystinosin-LKG, unlike the canonical isoform, also moves to the lysosomes by the indirect pathway, after endocytic retrieval from the plasma membrane, mainly by a clathrin-mediated endocytosis. Nevertheless, silencing of AP-2 triggers the clathrin-independent endocytosis, showing the complex adaptability of cystinosin-LKG trafficking.

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<![CDATA[Diffusion and Binding of Mismatch Repair Protein, MSH2, in Breast Cancer Cells at Different Stages of Neoplastic Transformation]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdbba5

The interior of cells is a highly complex medium, containing numerous organelles, a matrix of different fibers and a viscous, aqueous fluid of proteins and small molecules. The interior of cells is also a highly dynamic medium, in which many components move, either by active transport or passive diffusion. The mobility and localization of proteins inside cells can provide important insights into protein function and also general cellular properties, such as viscosity. Neoplastic transformation affects numerous cellular properties, and our goal was to investigate the diffusional and binding behavior of the important mismatch repair (MMR) protein MSH2 in live human cells at various stages of neoplastic transformation. Toward this end, noncancerous, immortal, tumorigenic, and metastatic mammary epithelial cells were transfected with EGFP and EGFP-tagged MSH2. MSH2 forms two MMR proteins (MutSα and MutSβ) and we assume MSH2 is in the complex MutSα, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutSα-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14–24 μm2/s for EGFP and 3–7 μm2/s for MutSα-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutSα produces an effective diffusion coefficient that, coupled with the free EGFP diffusion measurements, can be used to extract a pure diffusion coefficient and a pseudo-equilibrium constant K*. The MutSα nuclear K* increased sixfold in the first stage of cancer and then decreased in the more advanced stages. The ratio of nuclear to cytoplasmic K*for MutSα increased almost two orders of magnitude in going from noncancerous to immortal cells, suggesting that this quantity may be a sensitive metric for recognizing the onset of cancer.

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<![CDATA[Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network]]> https://www.researchpad.co/article/5989daabab0ee8fa60ba94f9

A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization.

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<![CDATA[Membrane Topology and Cellular Dynamics of Foot-and-Mouth Disease Virus 3A Protein]]> https://www.researchpad.co/article/5989d9d5ab0ee8fa60b65a03

Foot-and-mouth disease virus non-structural protein 3A plays important roles in virus replication, virulence and host-range; nevertheless little is known on the interactions that this protein can establish with different cell components. In this work, we have performed in vivo dynamic studies from cells transiently expressing the green fluorescent protein (GFP) fused to the complete 3A (GFP3A) and versions including different 3A mutations. The results revealed the presence of a mobile fraction of GFP3A, which was found increased in most of the mutants analyzed, and the location of 3A in a continuous compartment in the cytoplasm. A dual behavior was also observed for GFP3A upon cell fractionation, being the protein equally recovered from the cytosolic and membrane fractions, a ratio that was also observed when the insoluble fraction was further fractioned, even in the presence of detergent. Similar results were observed in the fractionation of GFP3ABBB, a 3A protein precursor required for initiating RNA replication. A nonintegral membrane protein topology of FMDV 3A was supported by the lack of glycosylation of versions of 3A in which each of the protein termini was fused to a glycosylation acceptor tag, as well as by their accessibility to degradation by proteases. According to this model 3A would interact with membranes through its central hydrophobic region exposing its N- and C- termini to the cytosol, where interactions between viral and cellular proteins required for virus replication are expected to occur.

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<![CDATA[Chemical characterization and extractives composition of heartwood and sapwood from Quercus faginea]]> https://www.researchpad.co/article/5989db5fab0ee8fa60be107a

Heartwood and sapwood of Quercus faginea were evaluated in relation to summative chemical composition and non-polar and polar extracts composition, including an assessment of antioxidant properties (DPPH and FRAP). Twenty trees from two sites in Portugal were analysed. Heartwood had approximately two times more solvent extractible compounds than sapwood (on average 19.0% and 9.5%). The lipophilic extractible compounds were below 1%, and most of them were polar e.g. ethanol-soluble compounds corresponded to 65% of total extractives in heartwood and 43% in sapwood. Lignin content was similar in sapwood and heartwood (28.1% and 28.6% of extractive-free wood respectively) as well as the sugar composition. Site did not influence the chemical composition. The lipophilic extractible compounds from both sapwood and heartwood included mainly saturated fatty acids (23.0% and 36.9% respectively) and aromatic compounds were also abundant in sapwood (22.9%). The ethanol-water extractibles had a high content of phenolic substances (558.0 and 319.4 mg GAE/g extract, respectively of heartwood and sapwood). The polyphenolic composition was similar in heartwood and sapwood with higher content of ellagitannins (168.9 and 153.5 mg tannic acid/g of extract in sapwood and heartwood respectively) and very low content of condensed tannins. The antioxidant activity was very high with IC50 of 2.6 μg/ml and 3.3 μg/ml for sapwood and heartwood respectively, as compared to standard antioxidants (IC50 of 3.8 μg/ml for Trolox). The ferric reducing ability was 2.8 and 2.0 mMol Trolox equivalents/g extract of heartwood and sapwood respectively. The variability between trees was low and no differences between the two sites were found. Q. faginea showed a very good potential for cooperage and other applications for which a source of compounds with antioxidant properties is desirable.

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<![CDATA[Chemical and Cellular Assays Combined with In Vitro Digestion to Determine the Antioxidant Activity of Flavonoids from Chinese Bayberry (Myrica rubra Sieb. et Zucc.) Leaves]]> https://www.researchpad.co/article/5989d9edab0ee8fa60b6d013

Oxidative stress is highly associated with the development of cardiovascular diseases and cancer and has drawn great attention. Natural products suggest a potential role in prevention of these disorders. The aim of this study was to investigate the antioxidant and anti-cancer properties of Chinese bayberry leaves (Myrica rubra Sieb. et Zucc.) flavonoids (BLF) comprehensively through the combination of in vitro digestion, chemical and cellular antioxidant assays. Based on the LC/MS data, the major flavonoids of BLF were myricitrin and quercetin 3-rhamnoside. BLF owned strong chemical and cellular antioxidant activity (CAA) with its CAA value at 4253.884 ± 435.366 μmol of QE/100 g DW. After the in vitro digestion, the total flavonoids content, myricitrin and quercetin 3-rhamnoside decreased significantly (P < 0.05). Lower levels of the total flavonoid content and cellular uptake of myricitrin and quercetin 3-rhamnoside might contribute to the lower CAA value of digested BLF (DBLF). However, DBLF still owns considerable chemical antioxidant activities and CAA compared with many plants. Furthermore, both BLF and DBLF exhibited dose-dependent relationship against HepG2 proliferation. Taken together, BLF has a great potential to be developed as a natural antioxidant for promoting public health.

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<![CDATA[Structure-function analysis of human stomatin: A mutation study]]> https://www.researchpad.co/article/5989db5cab0ee8fa60be0292

Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic “hairpin” structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.

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<![CDATA[Vinculin association with actin cytoskeleton is necessary for stiffness-dependent regulation of vinculin behavior]]> https://www.researchpad.co/article/5989db53ab0ee8fa60bdcabb

The extracellular matrix (ECM) is a major regulator of cell behavior. Recent studies have indicated the importance of the physical properties of the ECM, including its stiffness, for cell migration and differentiation. Using actomyosin-generated forces, cells pull the ECM and sense stiffness via cell-ECM adhesion structures called focal adhesions (FAs). Vinculin, an actin-binding FA protein, has emerged as a major player in FA-mediated mechanotransduction. Although vinculin is important for sensing ECM stiffness, the role of vinculin binding to actin in the ECM stiffness-mediated regulation of vinculin behavior remains unknown. Here, we show that an actin binding-deficient mutation disrupts the ECM stiffness-dependent regulation of CSB (cytoskeleton stabilization buffer) resistance and the stable localization of vinculin. These results suggest that the vinculin-actin interaction participates in FA-mediated mechanotransduction.

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<![CDATA[Antioxidant Activities of 4-Methylumbelliferone Derivatives]]> https://www.researchpad.co/article/5989db18ab0ee8fa60bcd867

The synthesis of derivatives of 4-Methylumbelliferone (4-MUs), which are structurally interesting antioxidants, was performed in this study. The modification of 4-Methylumbelliferone (4-MU) by different reaction steps was performed to yield the target compounds, the 4-MUs. The 4-MUs were characterized by different spectroscopic techniques (Fourier transform infrared; FT-IR and Nuclear magnetic resonance; NMR) and micro-elemental analysis (CHNS). The in vitro antioxidant activity of the 4-MUs was evaluated in terms of their free radical scavenging activities against 2,2-diphenyl-1-picrylhydrazyl (DPPH), Nitric oxide radical scavenging activity assay, chelating activity and their (FRAP) ferric-reducing antioxidant power, which were compared with a standard antioxidant. Our results reveal that the 4-MUs exhibit excellent radical scavenging activities. The antioxidant mechanisms of the 4-MUs were also studied. Density Function Theory (DFT)-based quantum chemical studies were performed with the basis set at 3-21G. Molecular models of the synthesized compounds were studied to understand the antioxidant activity. The electron levels, namely HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital), for these synthesized antioxidants were also studied.

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<![CDATA[Static Clathrin Assemblies at the Peripheral Vacuole—Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia]]> https://www.researchpad.co/article/5989da2aab0ee8fa60b820bc

Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain (GlCHC), dynamin-related protein (GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of endocytosis in this species.

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<![CDATA[The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells]]> https://www.researchpad.co/article/5989da16ab0ee8fa60b7b398

Modulating key dynamics of plant growth and development, the effects of the plant hormone cytokinin on animal cells gained much attention recently. Most previous studies on cytokinin effects on mammalian cells have been conducted with elevated cytokinin concentration (in the μM range). However, to examine physiologically relevant dose effects of cytokinins on animal cells, we systematically analyzed the impact of kinetin in cultured cells at low and high concentrations (1nM-10μM) and examined cytotoxic and genotoxic conditions. We furthermore measured the intrinsic antioxidant activity of kinetin in a cell-free system using the Ferric Reducing Antioxidant Power assay and in cells using the dihydroethidium staining method. Monitoring viability, we looked at kinetin effects in mammalian cells such as HL60 cells, HaCaT human keratinocyte cells, NRK rat epithelial kidney cells and human peripheral lymphocytes. Kinetin manifests no antioxidant activity in the cell free system and high doses of kinetin (500 nM and higher) reduce cell viability and mediate DNA damage in vitro. In contrast, low doses (concentrations up to 100 nM) of kinetin confer protection in cells against oxidative stress. Moreover, our results show that pretreatment of the cells with kinetin significantly reduces 4-nitroquinoline 1-oxide mediated reactive oxygen species production. Also, pretreatment with kinetin retains cellular GSH levels when they are also treated with the GSH-depleting agent patulin. Our results explicitly show that low kinetin doses reduce apoptosis and protect cells from oxidative stress mediated cell death. Future studies on the interaction between cytokinins and human cellular pathway targets will be intriguing.

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