scholarly journals Proteolytic processing of endogenous and recombinant beta 4 integrin subunit.

1992 ◽  
Vol 118 (4) ◽  
pp. 951-959 ◽  
Author(s):  
F G Giancotti ◽  
M A Stepp ◽  
S Suzuki ◽  
E Engvall ◽  
E Ruoslahti
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Cytoplasmic Domain ◽  
Basement Membranes ◽  
Proteolytic Processing ◽  
In Vitro Assays ◽  
Integrin Subunit ◽  
Calcium Dependent

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.

scholarly journals Ultrastructure of blebbing and phagocytosis of blebs by hyperplastic thyroid epithelial cells in vivo.

1977 ◽  
Vol 72 (3) ◽  
pp. 584-594 ◽  
Author(s):  
J D Zeligs ◽  
S H Wollman
Keyword(s):  
Thyroid Gland ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
Ultrastructural Organization ◽  
Contractile Ring ◽  
Phagocytic Process ◽  
Different Types ◽  
Rat Thyroid

In addition to pseudopods, somewhat pleomorphic blebs were consistently found protruding from the apical surfaces of hyperplastic rat thyroid epithelial cells into the follicular lumens in vivo. Many blebs were knobby, roughly hemispherical protrusions, with a diameter of 2-3 mum. Such blebs had a densely packed microfilamentous core and contained numerous apparent ribosomes. They were morphologically similar to blebs that have been observed in a variety of cultured cells. Other blebs were larger, more elongate, and less knobby, but had a similar ultrastructural organization. Blebs of all sizes appeared to be phagocytosed on some occasions by nearby epithelial cells. The phagocytic process involved partial engulfment of the bleb by a typical epithelial pseudopod, followed by an apparent pinching-off process, presumably resulting in the separation of the bleb from its cells or origin. The pinching-off process was associated with a band of approx. 6-nm diameter microfilaments that developed within the pseudopod cytoplasm surrounding the base of the bleb and is postulated to function as a contractile ring. The finding of blebbing is an intact tissue in vivo indicates that this phenomenon is not restricted to cultured cells, and thus tends to extend the significance of in vitro observations of the process. In relation to their occurrence in the hyperplastic thyroid gland in vivo, possible interconversions are considered between different types of blebs, and between blebs and pseudopods.

Functions of the cytoplasmic domain of the beta PS integrin subunit during Drosophila development

Development ◽  
1994 ◽  
Vol 120 (1) ◽  
pp. 91-102 ◽  
Author(s):  
Y. Grinblat ◽  
S. Zusman ◽  
G. Yee ◽  
R.O. Hynes ◽  
F.C. Kafatos
Keyword(s):  
Germ Line ◽  
Cytoplasmic Tail ◽  
Cytoplasmic Domain ◽  
P Element ◽  
Integrin Subunit ◽  
Wild Type ◽  
Mutant Proteins ◽  
Adhesive Interactions

Integrins constitute a family of membrane-spanning, heterodimeric proteins that mediate adhesive interactions between cells and surrounding extracellular matrices (or other cells) and participate in signal transduction. We are interested in assessing integrin functions in the context of developing Drosophila melanogaster. This report, using mutants of the beta PS subunit encoded by the myospheroid (mys) locus, analyzes the relationships between integrin protein structure and developmental functions in an intact organism. As a first step in this analysis, we demonstrated the ability of a fragment of wild-type mys genomic DNA, introduced into the germ line in a P-element vector P[mys+], to rescue phenotypes attributed to lack of (or defects in) the endogenous beta PS during several discrete morphogenetic events. We then produced in vitro a series of modifications of the wild-type P[mys+] transposon, which encode beta PS derivatives with mutations within the small and highly conserved cytoplasmic domain. In vivo analysis of these mutant transposons led to the following conclusions. (1) The cytoplasmic tail of beta PS is essential for all developmental functions of the protein that were assayed. (2) An intron at a conserved position in the DNA sequence encoding the cytoplasmic tail is thought to participate in important alternative splicing events in vertebrate beta integrin subunit genes, but is not required for the developmental functions of the mys gene assayed here. (3) Phosphorylation on two conserved tyrosines found in the C terminus of the beta PS cytoplasmic tail is not necessary for the tested developmental functions. (4) Four highly conserved amino acid residues found in the N-terminal portion of the cytoplasmic tail are important but not critical for the developmental functions of beta PS; furthermore, the efficiencies with which these mutant proteins function during different morphogenetic processes vary greatly, strongly suggesting that the cytoplasmic interactions involving PS integrins are developmentally modulated.

The VSL#3 probiotic formula induces mucin gene expression and secretion in colonic epithelial cells

2007 ◽  
Vol 292 (1) ◽  
pp. G315-G322 ◽  
Author(s):  
C. Caballero-Franco ◽  
K. Keller ◽  
C. De Simone ◽  
K. Chadee
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
In Vivo Studies ◽  
Lactobacillus Species ◽  
Mucin Secretion ◽  
Colonic Epithelial Cells ◽  
Dnase Treatment

Several studies have stressed the importance of the microbiota in the maintenance of the gastrointestinal epithelium. Administration of probiotic bacteria, supplements composed of microbiota constituents, was previously shown to diminish symptoms in patients suffering from inflammatory bowel diseases. This raises the possibility that probiotics may play an active role in enhancing the intestinal barrier at the mucosal surface. In this study, we investigated whether the clinically tested VSL#3 probiotic formula and/or its secreted components can augment the protective mucus layer in vivo and in vitro. For in vivo studies, Wistar rats were orally administered the probiotic mixture VSL#3 on a daily basis for seven days. After treatment, basal luminal mucin content increased by 60%. In addition, we exposed isolated rat colonic loops to the VSL#3 probiotic formula, which significantly stimulated colonic mucin (MUC) secretion and MUC2 gene expression; however, MUC1 and MUC3 gene expression were only slightly elevated. The effect of the VSL#3 mucin secretagogue was also tested in vitro by use of LS 174T colonic epithelial cells. In contrast to the animal studies, cultured cells incubated with VSL#3 bacteria did not exhibit increased mucin secretion. However, the bacterial secreted products contained in the conditioned media stimulated a remarkable mucin secretion effect. Among the three bacterial groups ( Lactobacilli, Bifidobacteria, and Streptococci) contained in VSL#3, the Lactobacillus species were the strongest potentiator of mucin secretion in vitro. A preliminary characterization of the putative mucin secretagogue suggested that it was a heat-resistant soluble compound, which is not sensitive to protease and DNase treatment. These findings contribute to a better understanding of the complex and beneficial interaction between colonic epithelial cells and intestinal bacteria.

scholarly journals Small-Molecule Inhibitors of the Rce1p CaaX Protease

2007 ◽  
Vol 12 (7) ◽  
pp. 983-993 ◽  
Author(s):  
Surya P. Manandhar ◽  
Emily R. Hildebrandt ◽  
Walter K. Schmidt
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Proteolytic Processing ◽  
In Vivo Studies ◽  
In Vitro Assays ◽  
Selective Agents ◽  
Wild Type Yeast ◽  
Micromolar Range

The Rce1p protease is required for the maturation of the Ras GTPase and certain other isoprenylated proteins and is considered a chemotherapeutic target. To identify new small-molecule inhibitors of Rce1p, the authors screened the National Cancer Institute Diversity Set compound library using in vitro assays to monitor the proteolytic processing of peptides derived from Ras and the yeast a-factor mating pheromone. Of 46 inhibitors initially identified with a Ras-based assay, only 9 were effective in the pheromone-based assay. The IC50 values of these 9 compounds were in the low micromolar range for both yeast (6-35 µM) and human Rce1p (0.4-46 µM). Four compounds were somewhat Rce1p selective in that they partially inhibited the Ste24p protease and did not inhibit Ste14p isoprenylcysteine carboxyl methyltransferase, 2 enzymes also involved in the maturation of isoprenylated proteins. The remaining 5 compounds inhibited all 3 enzymes. The 2 most Rce1p-selective agents were ineffective trypsin inhibitors, further supporting the specificity of these agents for Rce1p. The 5 least specific compounds formed colloidal aggregates, a proposed common feature of promiscuous inhibitors. Interestingly, the most specific Rce1p inhibitor also formed a colloidal aggregate. In vivo studies revealed that treatment of wild-type yeast with 1 compound induced a Ras2p delocalization phenotype that mimics observed effects in rce1 ste24 null yeast. The 9 compounds identified in this study represent new tools for understanding the enzymology of postisoprenylation-modifying enzymes and provide new insight for the future development of Rce1p inhibitors. ( Journal of Biomolecular Screening 2007:983-993)

scholarly journals Testing the Efficacy and Toxicity of Adenylyl Cyclase Inhibitors against Enteric Pathogens Using In Vitro and In Vivo Models of Infection

2010 ◽  
Vol 78 (4) ◽  
pp. 1740-1749 ◽  
Author(s):  
Scott T. Moen ◽  
Carla A. Blumentritt ◽  
Terry M. Slater ◽  
Shilpa D. Patel ◽  
Christopher B. Tutt ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Adenylyl Cyclase ◽  
Cultured Cells ◽  
Potent Inhibitor ◽  
Suppressive Effect ◽  
Intestinal Epithelial ◽  
In Vivo Models ◽  
Adp Ribosyltransferase

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) produces the ADP-ribosyltransferase toxin known as heat-labile enterotoxin (LT). In addition to the toxic effect of LT resulting in increases of cyclic AMP (cAMP) and disturbance of cellular metabolic processes, this toxin promotes bacterial adherence to intestinal epithelial cells (A. M. Johnson, R. S. Kaushik, D. H. Francis, J. M. Fleckenstein, and P. R. Hardwidge, J. Bacteriol. 191:178-186, 2009). Therefore, we hypothesized that the identification of a compound that inhibits the activity of the toxin would have a suppressive effect on the ETEC colonization capabilities. Using in vivo and in vitro approaches, we present evidence demonstrating that a fluorenone-based compound, DC5, which inhibits the accumulation of cAMP in intoxicated cultured cells, significantly decreases the colonization abilities of adenylyl cyclase toxin-producing bacteria, such as ETEC. These findings established that DC5 is a potent inhibitor both of toxin-induced cAMP accumulation and of ETEC adherence to epithelial cells. Thus, DC5 may be a promising compound for treatment of diarrhea caused by ETEC and other adenylyl cyclase toxin-producing bacteria.

scholarly journals Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit.

1993 ◽  
Vol 121 (1) ◽  
pp. 171-178 ◽  
Author(s):  
F Balzac ◽  
A M Belkin ◽  
V E Koteliansky ◽  
Y V Balabanov ◽  
F Altruda ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Subcellular Localization ◽  
Functional Properties ◽  
Cultured Cells ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Variant Form ◽  
Integrin Subunit ◽  
Stable Transfectants

We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties.

scholarly journals Mapping in vivo associations of cytoplasmic proteins with integrin beta 1 cytoplasmic domain mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 151-160 ◽  
Author(s):  
J M Lewis ◽  
M A Schwartz
Keyword(s):  
Intracellular Signaling ◽  
Focal Adhesions ◽  
Cytoplasmic Domain ◽  
Cytoskeletal Proteins ◽  
In Vitro Assays ◽  
Integrin Subunit ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
Beta 1 Integrin

Integrins promote formation of focal adhesions and trigger intracellular signaling pathways through cytoplasmic proteins such as talin, alpha-actinin, and focal adhesion kinase (FAK). The beta 1 integrin subunit has been shown to bind talin and alpha-actinin in in vitro assays, and these proteins may link integrin to the actin cytoskeleton either directly or through linkages to other proteins such as vinculin. However, it is unknown which of these associations are necessary in vivo for formation of focal contacts, or which regions of beta 1 integrin bind to specific cytoskeletal proteins in vivo. We have developed an in vivo assay to address these questions. Microbeads were coated with anti-chicken beta 1 antibodies to selectively cluster chicken beta 1 integrins expressed in cultured mouse fibroblasts. The ability of cytoplasmic domain mutant beta 1 integrins to induce co-localization of proteins was assessed by immunofluorescence and compared with that of wild-type integrin. As expected, mutant beta 1 lacking the entire cytoplasmic domain had a reduced ability to induce co-localization of talin, alpha-actinin, F-actin, vinculin, and FAK. The ability of beta 1 integrin to co-localize talin and FAK was found to require a sequence near the C-terminus of beta 1. The region of beta 1 required to co-localize alpha-actinin was found to reside in a different sequence, several amino acids further from the C-terminus of beta 1. Deletion of 13 residues from the C-terminus blocked co-localization of talin, FAK, and actin, but not alpha-actinin. Association of alpha-actinin with clustered integrin is therefore not sufficient to induce the co-localization of F-actin.

scholarly journals In silico Elucidation of Dihydroquinine Mechanism of Action against Toxoplasma gondii

2022 ◽  
Author(s):  
Joseph A. Ayariga ◽  
Aarin M. Huffman ◽  
Audrey Napier ◽  
BK Robertson ◽  
Daniel Abugri
Keyword(s):  
Mechanism Of Action ◽  
In Silico ◽  
In Silico Analysis ◽  
Trna Synthetase ◽  
In Vitro Assays ◽  
In Vivo Models ◽  
Translational Machinery ◽  
Calcium Dependent

Dihydroquinine (DHQ), is a quinine-based compound with anti-malarial properties. However, little is known about its mechanism of action against T. gondii inhibition, which shares similar biology with Plasmodium spp. In order to explore DHQ activity as an inhibitor of T. gondii using in vitro assays, we first used an in silico approach to decipher its mechanisms of action based on previous knowledge about its disruption of nucleic acid and protein synthesis. An in silico study was performed on T. gondii parasite replication, transcriptional and translational machinery to decipher the binding potentials of DHQ to some top selected enzymes. We report for the first time, using an in silico analysis that showed that DHQ binds strongly to DNA gyrase, Calcium Dependent Protein Kinase 1 (CDPK 1), and prolyl tRNA synthetase and thus could affect DNA replication, transcriptional and translational activities in T. gondii. Also, we found DHQ to effectively bind to mitochondria detoxifying enzymes (i.e., superoxide dismutase (SOD), peroxidoxin, and Catalase (CAT)). In conclusion, DHQ could be a lead compound for the treatment of toxoplasmosis when successfully evaluated using in vitro and in vivo models to confirm its effectiveness and safety.

scholarly journals Minireview: Putting Physiology Back into Estrogens' Mechanism of Action

Endocrinology ◽  
2011 ◽  
Vol 152 (12) ◽  
pp. 4481-4488 ◽  
Author(s):  
Robert D. Koos
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Hypoxia Inducible Factor ◽  
Estrogen Receptor Α ◽  
Proliferative Effect ◽  
High Oxygen Level ◽  
Endothelial Growth Factor ◽  
Endometrial Epithelial Cells

After decades of research, the mechanism by which estrogens stimulate the proliferation of epithelial cells in the endometrium and mammary gland, and in the carcinomas that arise in those tissues, is still not understood. Cells do not proliferate in response to 17β-estradiol (E2) alone, and although it is widely recognized that growth factors play a role in E2's proliferative effect, exactly how they are involved is unclear. It has long been known that the proliferation of endometrial epithelial cells is preceded by dramatic increases in blood flow and microvascular permeability, filling the subepithelial stroma with plasma and the proteins it contains, such as IGF-I, which is known to synergize with E2 in the induction of cell proliferation. The hyperpermeability is caused by vascular endothelial growth factor (VEGF), which is rapidly induced by E2, via the transcription factors hypoxia-inducible factor 1 and estrogen receptor α, in luminal epithelial cells in vivo. As we recently showed, VEGF is also strongly induced in endometrial cancer cells in vitro when excessive degradation of hypoxia-inducible factor 1α, caused by the abnormally high oxygen level to which cultured cells are exposed, is prevented. Putting these facts together, we now propose a new model of E2-induced proliferation in which VEGF-induced vascular hyperpermeability plays an essential role. E2 first induces the expression by endometrial epithelial cells of VEGF, which then acts in a paracrine manner to induce interendothelial cell gaps in subepithelial blood vessels, through which plasma and the proteins therein enter the adjacent stroma. Plasma carries even more E2, which circulates bound to proteins, and IGF-l, which together drive epithelial cells completely through the cell cycle.

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