scholarly journals 2114

2017 ◽  
Vol 1 (S1) ◽  
pp. 57-57
Author(s):  
Jennifer Knudtson ◽  
Ya-Guang Liu ◽  
Marlen Tellez Santos ◽  
Rajeshwar Tekmal ◽  
Ratna Vadlamudi ◽  
...  
Keyword(s):  
Positive Staining ◽  
Test Results ◽  
Endometriotic Lesion ◽  
Vitro Assay ◽  
Epithelial Origin ◽  
Immortalized Cells ◽  
Study Population ◽  
Chromosomal Profile

OBJECTIVES/SPECIFIC AIMS: To further elucidate the role of estrogen receptor β (ER-β) in the early endometriotic lesion attachment. METHODS/STUDY POPULATION: EECs were immortalized using a telomerase vector. Immortalized cells and parental cells were characterized by genotyping, and expression of ER-β as well as other epithelial cell markers. ER-β was knocked-down in immortalized EECs using lentivirus-mediated shRNA transduction. ER-β knockdown was confirmed by RT-qPCR and Western analysis. EEC cells with or without ER-β knockdown were used to assess their attachment to PMCs in an established in vitro assay (Lucidi, 2005). Results were analyzed with Student t-test. RESULTS/ANTICIPATED RESULTS: Genotyping using karyotype assay confirmed a normal chromosomal profile. Also positive staining for cytokeratin and lack of any staining with vimentin confirms the epithelial origin of these cells. ER-β knockdown has a significant decrease in attachment compared to control (p=0.02). DISCUSSION/SIGNIFICANCE OF IMPACT: Primary and immortalized cells were 46XX, cytokeratin positive, and vimentin negative confirming their epithelial origin. ER-β knockdown has a significant decrease in attachment compared with control.

scholarly journals Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1092
Author(s):  
János András Mótyán ◽  
Márió Miczi ◽  
Stephen Oroszlan ◽  
József Tőzsér
Keyword(s):  
Amino Acid ◽  
Zinc Finger ◽  
Cleavage Site ◽  
Potential Role ◽  
Binding Mode ◽  
Interaction Energies ◽  
Vitro Assay ◽  
Hiv 1

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.

scholarly journals Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45

2013 ◽  
Vol 24 (15) ◽  
pp. 2389-2397 ◽  
Author(s):  
Jennifer Roccisana ◽  
Jessica B. A. Sadler ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Vitro Assay ◽  
Protein Receptor ◽  
Storage Vesicles ◽  
Storage Vesicle ◽  
Impaired Insulin ◽  
Endosomal Pathway

Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target–soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub­cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.

scholarly journals Rap1 controls cell adhesion and cell motility through the regulation of myosin II

2007 ◽  
Vol 176 (7) ◽  
pp. 1021-1033 ◽  
Author(s):  
Taeck J. Jeon ◽  
Dai-Jen Lee ◽  
Sylvain Merlot ◽  
Gerald Weeks ◽  
Richard A. Firtel
Keyword(s):  
Cell Adhesion ◽  
Cell Motility ◽  
Myosin Ii ◽  
Leading Edge ◽  
In Vitro Assay ◽  
Filamentous Actin ◽  
Guanosine Triphosphate ◽  
Vitro Assay

We have investigated the role of Rap1 in controlling chemotaxis and cell adhesion in Dictyostelium discoideum. Rap1 is activated rapidly in response to chemoattractant stimulation, and activated Rap1 is preferentially found at the leading edge of chemotaxing cells. Cells expressing constitutively active Rap1 are highly adhesive and exhibit strong chemotaxis defects, which are partially caused by an inability to spatially and temporally regulate myosin assembly and disassembly. We demonstrate that the kinase Phg2, a putative Rap1 effector, colocalizes with Rap1–guanosine triphosphate at the leading edge and is required in an in vitro assay for myosin II phosphorylation, which disassembles myosin II and facilitates filamentous actin–mediated leading edge protrusion. We suggest that Rap1/Phg2 plays a role in controlling leading edge myosin II disassembly while passively allowing myosin II assembly along the lateral sides and posterior of the cell.

R412 – Role of KITENIN in Progression and Metastasis of SCC

2008 ◽  
Vol 139 (2_suppl) ◽  
pp. P181-P181
Author(s):  
Mark Zarrella ◽  
Joon Kyoo Lee MD ◽  
Sang-Chul Lim
Keyword(s):  
Squamous Cell Carcinoma ◽  
Lymph Nodes ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Vitro Assay ◽  
Transfected Cells ◽  
Metastatic Lymph Nodes ◽  
Metastatic Lymph

Problem The expression of KAI1 (a metastatic suppressor gene) in cancer cells results in reduced cell motility and invasiveness. A cDNA clone of VANGL1, a member of the tetraspanin protein family that specifically interacts with the COOH-terminal cytoplasm domain of KAI1, was isolated and renamed KITENIN (KAI1 COOH-terminal interacting tetraspanin). The purpose of this study was to investigate the role of KITENIN on the progression and metastasis of transfected squamous cell carcinoma using in vivo and in vitro experiments. Methods Locally advanced squamous cell carcinoma tissues from five patients were obtained for investigation of KITENIN expression. Malignant tumors, normal adjacent mucosa tissues, metastatic lymph nodes, and non-metastatic lymph nodes were collected. KITENIN or vector only (control) was transfected into SCC (squamous cell carcinoma) VII, a mouse squamous cell carcinoma cell line, using FuGENE 6. An in vitro assay (invasion, migration, and proliferation) for KITENIN and the vector-transfected group was studied. The KITENIN or vector-transfected SCC VII cells were injected subcutaneously into 12 C3H/HeJ syngeneic mice (6 mice for each group). The tumor size was measured daily for 4 weeks. During the fifth week after injection, the presence of metastasis in the lung and liver tissue was evaluated for each mouse with a tumor mass on the back; the tissues were assessed by gross and microscopic examination. Results KITENIN was highly expressed in tumors and metastatic lymph nodes from patients. KITENIN-transfected cells showed significantly increased invasion, migration, and proliferation compared with the vector-transfected cells. Tumor volume was more increased in the KITENIN-transfected cells-injected mice. Lung metastasis was found in the KITENIN-group (3/6 mice), while no metastasis in the vector-group. Conclusion KITENIN participates in the progression and metastasis of SCC. Significance An antisense KITENIN strategy may be a useful method to inhibit progression and metastasis in squamous cell carcinoma. Support This study was financially supported by Chonnam National University, 2006.

scholarly journals gamma-tubulin is a minus end-specific microtubule binding protein.

1995 ◽  
Vol 131 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Q Li ◽  
H C Joshi
Keyword(s):  
Chromosome Segregation ◽  
Structural Integrity ◽  
Cell Periphery ◽  
Vitro Assay ◽  
Interphase Cells ◽  
Gamma Tubulin ◽  
Slow Growing ◽  
Directional Transport

The role of microtubules in mediating chromosome segregation during mitosis is well-recognized. In addition, interphase cells depend upon a radial and uniform orientation of microtubules, which are intrinsically asymmetric polymers, for the directional transport of many cytoplasmic components and for the maintenance of the structural integrity of certain organelles. The slow growing minus ends of microtubules are linked to the centrosome ensuring extension of the fast growing plus ends toward the cell periphery. However, the molecular mechanism of this linkage is not clear. One hypothesis is that gamma-tubulin, located at the centrosome, binds to the minus ends of microtubules. To test this model, we synthesized radiolabeled gamma-tubulin in vitro. We demonstrate here biochemically a specific, saturable, and tight (Kd = 10(-10) M) interaction of gamma-tubulin and microtubule ends with a stoichiometry of 12.6 +/- 4.9 molecules of gamma-tubulin per microtubule. In addition, we designed an in vitro assay to visualize gamma-tubulin at the minus ends of axonemal microtubules. These data show that gamma-tubulin represents the first protein to bind microtubule minus ends and might be responsible for mediating the link between microtubules and the centrosome.

scholarly journals Biochemical and Mutational Analyses of AcuA, the Acetyltransferase Enzyme That Controls the Activity of the Acetyl Coenzyme A Synthetase (AcsA) in Bacillus subtilis

2008 ◽  
Vol 190 (14) ◽  
pp. 5132-5136 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Effective Means ◽  
Wild Type ◽  
Modification System ◽  
Vitro Assay ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme

ABSTRACT The acuABC genes of Bacillus subtilis comprise a putative posttranslational modification system. The AcuA protein is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, the AcuC protein is a class I histone deacetylase, and the role of the AcuB protein is not known. AcuA controls the activity of acetyl coenzyme A synthetase (AcsA; EC 6.2.1.1) in this bacterium by acetylating residue Lys549. Here we report the kinetic analysis of wild-type and variant AcuA proteins. We contrived a genetic scheme for the identification of AcuA residues critical for activity. Changes at residues H177 and G187 completely inactivated AcuA and led to its rapid turnover. Changes at residues R42 and T169 were less severe. In vitro assay conditions were optimized, and an effective means of inactivating the enzyme was found. The basic kinetic parameters of wild-type and variant AcuA proteins were obtained and compared to those of eukaryotic GNATs. Insights into how the isolated mutations may exert their deleterious effect were investigated by using the crystal structure of an AcuA homolog.

Regulation of cholesterol metabolism in fetal rabbit aorta: role of amniotic fluid factors

1988 ◽  
Vol 255 (1) ◽  
pp. H160-H168
Author(s):  
Z. Rymaszewski ◽  
R. L. Yunker ◽  
M. Ashraf ◽  
M. Park ◽  
M. T. Subbiah
Keyword(s):  
Amniotic Fluid ◽  
Arterial Wall ◽  
Cholesterol Metabolism ◽  
Rabbit Aorta ◽  
Cholesterol Esterification ◽  
Cholesteryl Esters ◽  
Vitro Assay ◽  
Stimulating Factor

This study shows that amniotic fluid enhances cholesterol esterification in arterial wall, as measured by in vitro assay of acyl-CoA:cholesterol acyltransferase (ACAT) activity and by incorporation of oleic acid to cholesteryl esters in cultured fetal aortas and smooth muscle cells. This property is mostly evident in the fraction of molecular weight greater than 100,000, and it is abolished by delipidation, indicating that stimulating factor is probably lipoprotein in nature. Despite an increased cholesterol esterification by the presence of amniotic fluid in medium of cultured fetal aortas, the content of cholesterol and cholesteryl esters was much lower. The cellular structures are better preserved in explants cultured with amniotic fluid than in control animals. This study indicates that amniotic fluid contains factors that may have a pronounced effect on arterial wall during development.

Defining the role of albumin infusion in cirrhosis-associated hyponatremia

2014 ◽  
Vol 307 (2) ◽  
pp. G229-G232 ◽  
Author(s):  
Minhtri K. Nguyen ◽  
Vahram Ornekian ◽  
Liyo Kao ◽  
Anthony W. Butch ◽  
Ira Kurtz
Keyword(s):  
Sodium Concentration ◽  
Albumin Concentration ◽  
Intravascular Volume ◽  
Volume Depletion ◽  
Vitro Assay ◽  
Albumin Infusion ◽  
Donnan Equilibrium ◽  
Intravascular Volume Depletion

The presence of negatively charged, impermeant proteins in the plasma space alters the distribution of diffusible ions in the plasma and interstitial fluid (ISF) compartments to preserve electroneutrality and is known as Gibbs-Donnan equilibrium. In patients with hypoalbuminemia due to underlying cirrhosis, the decrease in the plasma water albumin concentration ([Alb−]pw) would be expected to result in a decrease in the plasma water sodium concentration ([Na+]pw) due to an alteration in the distribution of Na+ between the plasma and ISF. In addition, cirrhosis-associated hyponatremia may be due to the renal diluting defect resulting from the intravascular volume depletion due to gastrointestinal losses and overdiuresis and/or decreased effective circulatory volume secondary to splanchnic vasodilatation. Therefore, albumin infusion may result in correction of the hyponatremia in cirrhotic patients either by modulating the Gibbs-Donnan effect due to hypoalbuminemia or by restoring intravascular volume in patients with intravascular volume depletion due to gastrointestinal losses and overdiuresis. However, the differential role of albumin infusion in modulating the [Na+]pw in these patients has not previously been analyzed quantitatively. In the present study, we developed an in vitro assay system to examine for the first time the quantitative effect of changes in albumin concentration on the distribution of Na+ between two compartments separated by a membrane that allows the free diffusion of Na+. Our findings demonstrated that changes in [Alb−]pw are linearly related to changes in [Na+]pw as predicted by Gibbs-Donnan equilibrium. However, based on our findings, we predict that the improvement in cirrhosis-associated hyponatremia due to intravascular volume depletion results predominantly from the restoration of intravascular volume rather than alterations in Gibbs-Donnan equilibrium.

scholarly journals Atropine Differentially Modulates ECM Production by Ocular Fibroblasts, and Its Ocular Surface Toxicity Is Blunted by Colostrum

Biomedicines ◽  
2020 ◽  
Vol 8 (4) ◽  
pp. 78
Author(s):  
Martina Cristaldi ◽  
Melania Olivieri ◽  
Salvatore Pezzino ◽  
Giorgia Spampinato ◽  
Gabriella Lupo ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Test Results ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Atropine Treatment ◽  
Progressive Myopia ◽  
Methyl Xanthine

Background: The etiology and the mechanism behind atropine treatment of progressive myopia are still poorly understood. Our study addressed the role of scleral and choroidal fibroblasts in myopia development and atropine function. Methods: Fibroblasts treated in vitro with atropine or 7-methylxanthine were tested for ECM production by Western blotting. Corneal epithelial cells were treated with atropine in the presence or absence of colostrum or fucosyl-lactose, and cell survival was evaluated by the MTT metabolic test. Results: Atropine and 7-methyl-xanthine stimulated collagen I and fibronectin production in scleral fibroblasts, while they inhibited their production in choroidal fibroblasts. Four days of treatment with atropine of corneal epithelial cells significantly decreased cell viability, which could be prevented by the presence of colostrum or fucosyl-lactose. Conclusions: Our results show that atropine may function in different ways in different eye districts, strengthening the scleral ECM and increasing permeability in the choroid. The finding that colostrum or fucosyl-lactose attenuate the corneal epithelial toxicity after long-term atropine treatment suggests the possibility that both compounds can efficiently blunt its toxicity in children subjected to chronic atropine treatment.

scholarly journals Immunopurified Small Nucleolar Ribonucleoprotein Particles Pseudouridylate rRNA Independently of Their Association with Phosphorylated Nopp140

2002 ◽  
Vol 22 (24) ◽  
pp. 8457-8466 ◽  
Author(s):  
Chen Wang ◽  
Charles C. Query ◽  
U. Thomas Meier
Keyword(s):  
Cajal Body ◽  
Small Nucleolar Rnas ◽  
Ribonucleoprotein Particle ◽  
Vitro Assay ◽  
Body Protein ◽  
Core Proteins ◽  
Layer Chromatography ◽  
Nucleolar Rnas

ABSTRACT The isomerization of up to 100 uridines to pseudouridines (Ψs) in eukaryotic rRNA is guided by a similar number of box H/ACA small nucleolar RNAs (snoRNAs), each forming a unique small nucleolar ribonucleoprotein particle (snoRNP) with the same four core proteins, NAP57 (also known as dyskerin or Cbf5p), GAR1, NHP2, and NOP10. Additionally, the nucleolar and Cajal body protein Nopp140 (Srp40p) associates with the snoRNPs. To understand the role of these factors in pseudouridylation, we established an in vitro assay system. Short site-specifically 32P-labeled rRNA substrates were incubated with subcellular fractions, and the conversion of uridine to Ψ was monitored by thin-layer chromatography after digestion to single nucleotides. Immunopurified box H/ACA core particles were sufficient for the reaction. SnoRNPs associated quantitatively and reversibly with Nopp140. However, pseudouridylation activity was independent of Nopp140, consistent with a chaperoning role for this highly phosphorylated protein. Although up to 14 bp between the snoRNA and rRNA were required for the in vitro reaction, rRNA pseudouridylation and release occurred in the absence of ATP and magnesium. These data suggest that substrate release takes place without RNA helicase activity but may be aided by the snoRNP core proteins.

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