scholarly journals Identification and Characterization of the Endocytic Transmembrane Glycoprotein Endo180 as a Novel Collagen Receptor

2003 ◽  
Vol 14 (9) ◽  
pp. 3592-3604 ◽  
Author(s):  
Dirk Wienke ◽  
John R. MacFadyen ◽  
Clare M. Isacke
Keyword(s):  
Physiological Role ◽  
Collagen Matrix ◽  
Mannose Receptor ◽  
In Vitro Assays ◽  
Type Ii ◽  
Urokinase Plasminogen Activator Receptor ◽  
Coated Pits ◽  
Rich Domain ◽  
Fibronectin Type

Endo180, a member of the mannose receptor family, is constitutively recycled between clathrin-coated pits on the cell surface and intracellular endosomes. Its large extracellular domain contains an N-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. The second of these lectin-like domains has been shown to mediate Ca2+-dependent mannose binding. In addition, cross-linking studies have identified Endo180 as a urokinase plasminogen activator receptor–associated protein and this interaction can be blocked by collagen V. Here we demonstrate directly using in vitro assays, cell-based studies and tissue immunohistochemistry that Endo180 binds both to native and denatured collagens and provide evidence that this is mediated by the fibronectin type II domain. In cell culture systems, expression of Endo180 results in the rapid uptake of soluble collagens for delivery to lysosomal degradative compartments. Together with the observed restricted expression of Endo180 in both embryonic and adult tissue, we propose that Endo180 plays a physiological role in mediating collagen matrix remodelling during tissue development and homeostasis and that the observed receptor upregulation in pathological conditions may contribute to disease progression.

scholarly journals Collagen binding by the mannose receptor mediated through the fibronectin type II domain

2006 ◽  
Vol 395 (3) ◽  
pp. 579-586 ◽  
Author(s):  
Catherine E. Napper ◽  
Kurt Drickamer ◽  
Maureen E. Taylor
Keyword(s):  
Mannose Receptor ◽  
Type I ◽  
Type Ii ◽  
Carbohydrate Recognition ◽  
Collagen Binding ◽  
Type Iii ◽  
Rich Domain ◽  
Type Iv ◽  
Extracellular Region ◽  
Fibronectin Type

The macrophage mannose receptor is the prototype for a family of receptors each having an extracellular region consisting of an N-terminal cysteine-rich domain related to the R-type carbohydrate-recognition domain of ricin, a fibronectin type II domain and eight to ten domains related to C-type carbohydrate-recognition domains. The mannose receptor acts as a molecular scavenger, clearing harmful glycoconjugates or micro-organisms through recognition of their defining carbohydrate structures. Cell-adhesion assays, as well as collagen-binding assays, have now been used to show that the mannose receptor can also bind collagen and that the fibronectin type II domain mediates this activity. Neither of the two types of sugar-binding domain in the receptor is involved in collagen binding. Fibroblasts expressing the mannose receptor adhere to type I, type III and type IV collagens, but not to type V collagen, and the adherence is inhibited by isolated mannose receptor fibronectin type II domain. The fibronectin type II domain shows the same specificity for collagen as the whole receptor, binding to type I, type III and type IV collagens. This is the first activity assigned to the fibronectin type II domain of the mannose receptor. The results suggest additional roles for this multifunctional receptor in mediating collagen clearance or cell–matrix adhesion.

scholarly journals Ubiquitously Expressed Dynamin-II Has a Higher Intrinsic GTPase Activity and a Greater Propensity for Self-assembly Than Neuronal Dynamin-I

1997 ◽  
Vol 8 (12) ◽  
pp. 2553-2562 ◽  
Author(s):  
Dale E. Warnock ◽  
Takeshi Baba ◽  
Sandra L. Schmid
Keyword(s):  
Self Assembly ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Gtpase Activity ◽  
Coated Pits ◽  
Gtp Hydrolysis ◽  
Rich Domain ◽  
Dynamin I ◽  
Intrinsic Gtpase Activity

To begin to understand mechanistic differences in endocytosis in neurons and nonneuronal cells, we have compared the biochemical properties of the ubiquitously expressed dynamin-II isoform with those of neuron-specific dynamin-I. Like dynamin-I, dynamin-II is specifically localized to and highly concentrated in coated pits on the plasma membrane and can assemble in vitro into rings and helical arrays. As expected, the two closely related isoforms share a similar mechanism for GTP hydrolysis: both are stimulated in vitro by self-assembly and by interaction with microtubules or the SH3 domain-containing protein, grb2. Deletion of the C-terminal proline/arginine-rich domain from either isoform abrogates self-assembly and assembly-dependent increases in GTP hydrolysis. However, dynamin-II exhibits a ∼threefold higher rate of intrinsic GTP hydrolysis and higher affinity for GTP than dynamin-I. Strikingly, the stimulated GTPase activity of dynamin-II can be >40-fold higher than dynamin-I, due principally to its greater propensity for self-assembly and the increased resistance of assembled dynamin-II to GTP-triggered disassembly. These results are consistent with the hypothesis that self-assembly is a major regulator of dynamin GTPase activity and that the intrinsic rate of GTP hydrolysis reflects a dynamic, GTP-dependent equilibrium of assembly and disassembly.

Insulin secretion by rat lachrymal glands: effects of systemic and local variables

2005 ◽  
Vol 289 (5) ◽  
pp. E768-E775 ◽  
Author(s):  
Daniel Andrade Cunha ◽  
Everardo M. Carneiro ◽  
Mônica de C. Alves ◽  
Angélica Gobbi Jorge ◽  
Sylvia Morais de Sousa ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Time Course ◽  
Physiological Role ◽  
Tear Film ◽  
Insulin Level ◽  
Glucose Consumption ◽  
In Vitro Assays ◽  
Corneal Tissue ◽  
Metabolic Role

To understand the secretory mechanisms and physiological role of insulin in the tear film, the present study examined 1) the time course of insulin secretion in the tear film under glucose intravenous stimulation, 2) the glucose- and carbachol-induced insulin secretion from isolated lachrymal gland (LG), 3) the effect of insulin on glucose consumption by the cornea, and 4) the expression of insulin, pancreatic duodenal homeobox-1 (PDX-1), and glucose transport proteins (GLUTs) in LG tissue. The insulin level in the tear film of 8-wk-old male Wistar rats increased from 0.6 ± 0.45 to 3.7 ± 1.3 ng/ml in the initial minutes after glucose stimulation. In vitro assays demonstrated that higher glucose concentrations from 2.8 to 16.7 mM, 200 μM carbachol, or 40 mM KCl significantly increased insulin secretion from lacrimal glands compared with controls, but did not detect C-peptide as measured by RIA. Glucose consumption by corneal tissue, evaluated by radiolabeled d-[U-14C]glucose uptake, was 24.07 ± 0.61 and was enhanced to 31.63 ± 3.15 nmol·cornea−1·2 h−1 in the presence of 6 nM insulin ( P = 0.033) and to 37.5 ± 3.7 nmol·cornea−1·2 h−1 in the presence of 11.2 mM glucose ( P = 0.015). Insulin and PDX-1 mRNA was detected in LG. Insulin was located in the apical areas of acinar cells by immunoperoxidase and the expression of GLUT-1, but not PDX-1, was confirmed by Western blot. These findings suggest that insulin secretion in the tear film is influenced by local stimuli such as nutrient and neural inputs and that this hormone plays a metabolic role in ocular surface tissues. These data also indicate that under normal conditions the insulin secreted by LG is stored, but it is not clear that is locally produced in the LG.

scholarly journals HANP1/H1T2, a Novel Histone H1-Like Protein Involved in Nuclear Formation and Sperm Fertility

2005 ◽  
Vol 25 (16) ◽  
pp. 7107-7119 ◽  
Author(s):  
Hiromitsu Tanaka ◽  
Naoko Iguchi ◽  
Ayako Isotani ◽  
Kouichi Kitamura ◽  
Yoshiro Toyama ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Nuclear Protein ◽  
Physiological Role ◽  
Histone H1 ◽  
Vitro Fertilization ◽  
Rich Domain ◽  
And Function ◽  
Specific Nuclear Protein

ABSTRACT We cloned a testis-specific cDNA from mice that encodes a histone H1-like, haploid germ cell-specific nuclear protein designated HANP1/H1T2. The HANP1/H1T2 protein was specifically localized to the nuclei of murine spermatids during differentiation steps 5 to 13 but not to the nuclei of mature sperm. HANP1/H1T2 contains an arginine-serine-rich domain and an ATP/GTP binding site, and it binds to DNA, ATP, and protamine. To investigate the physiological role of HANP1/H1T2, we generated Hanp1/H1T2-disrupted mutant mice. Homozygous Hanp1/H1T2 mutant males were infertile, but females were fertile. Although a substantial number of sperm were recovered from the epididymides, their shape and function were abnormal. During sperm morphogenesis, the formation of nuclei was disturbed and protamine-1 and -2 were only weakly detectable in the nuclei. The chromatin packaging was aberrant, as demonstrated by electron microscopy and biochemical analysis. The mutant sperm exhibited deficient motility and were not competent to fertilize eggs under in vitro fertilization conditions; however, they were capable of fertilizing eggs via intracytoplasmic sperm injection that resulted in the birth of healthy progeny. Thus, we found that HANP1/H1T2 is essential for nuclear formation in functional spermatozoa and is specifically involved in the replacement of histones with protamines during spermiogenesis. At the time of submission of the manuscript, we found an independent publication by Martianov et al. (I. Martianov, S. Brancorsini, R. Catena, A. Gansmuller, N. Kotaja, M. Parvinen, P. Sassone-Corsi, and I. Davidson, Proc. Natl. Acad. Sci. USA 102:2808-2813, 2005) that reported similar results.

scholarly journals AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion of Dictyostelium Contractile Vacuoles

2009 ◽  
Vol 20 (20) ◽  
pp. 4278-4288 ◽  
Author(s):  
Yujia Wen ◽  
Irene Stavrou ◽  
Kirill Bersuker ◽  
Rebecca J. Brady ◽  
Arturo De Lozanne ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Contractile Vacuole ◽  
Snare Proteins ◽  
In Vitro Assays ◽  
Accessory Proteins ◽  
Coated Pits ◽  
Contractile Vacuoles ◽  
Null Mutants

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane–coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.

scholarly journals Comparison of D0870, a new triazole antifungal agent, to fluconazole for inhibition of Candida albicans cytochrome P-450 by using in vitro assays.

1996 ◽  
Vol 40 (6) ◽  
pp. 1382-1386 ◽  
Author(s):  
K Venkateswarlu ◽  
D W Denning ◽  
N J Manning ◽  
S L Kelly
Keyword(s):  
Candida Albicans ◽  
Spectrophotometric Assay ◽  
In Vitro Assays ◽  
Type Ii ◽  
Intracellular Accumulation ◽  
Cell Extracts ◽  
Biochemical Comparison ◽  
Cytochrome P 450 ◽  
Differential Affinity

D0870 was 12 to 15 times more active than fluconazole in experiments to determine the MIC for growth arrest for two isolates of Candida albicans. A biochemical comparison of in vitro sterol biosynthesis in cell extracts showed only a twofold superiority of D0870 over fluconazole. A large differentiation (10-fold) in 50% saturating concentrations obtained by examining the binding of the azoles to microsomal P-450 was observed in a type II binding spectrophotometric assay, possibly reflecting the differential affinity for more than one P-450 enzyme. Additional mechanisms besides affinity for the target enzyme sterol 14 alpha-demethylase, such as differential intracellular accumulation of drug, may contribute to the differences in antifungal activity.

scholarly journals Binding and uptake of pulmonary surfactant protein (SP-A) by pulmonary type II epithelial cells.

1989 ◽  
Vol 37 (4) ◽  
pp. 429-440 ◽  
Author(s):  
R M Ryan ◽  
R E Morris ◽  
W R Rice ◽  
G Ciraolo ◽  
J A Whitsett
Keyword(s):  
Epithelial Cells ◽  
Pulmonary Surfactant ◽  
Surfactant Protein ◽  
Uptake System ◽  
Type Ii ◽  
Electron Microscopic ◽  
Coated Pits ◽  
Type Ii Cell ◽  
Bridging Technique

A glycoprotein of Mr 26-36,000 (SP-A) is an abundant phospholipid-associated protein in pulmonary surfactant. SP-A enhances phospholipid reuptake and inhibits secretion by Type II epithelial cells in vitro. We have used two electron microscopic cytochemical methods to demonstrate selective binding and uptake of SP-A by rat pulmonary Type II epithelial cells. Using an immunogold bridging technique, we showed that SP-A binding was selective for Type II cell surfaces. Binding was dose dependent and saturable, reaching maximal binding at approximately 10 ng/ml. On warming to 23 degrees C, SP-A binding sites were clustered in coated pits on the cell surface. To characterize the internalization and intracellular routing of SP-A, we used the biotinyl ligand-avidin-gold technique. Biotinyl SP-A was bound by rat Type II epithelial cells as described above. On warming, biotinyl SP-A was seen in association with coated vesicles and was subsequently located in endosomes and multivesicular bodies. Biotinyl SP-A-gold complexes were seen in close approximation to lamellar bodies 10-60 min after warming. Binding of biotinyl SP-A was inhibited by competition with unlabeled SP-A. These results support the concept that Type II epithelial cells bind and internalize SP-A by receptor-mediated endocytosis. This newly described uptake system may play a role in the recycling of surfactant components or mediate the actions of SP-A on surfactant phospholipid secretion.

scholarly journals Production of a novel heterodimeric two-chain insulin-Fc fusion protein

2020 ◽  
Vol 33 ◽  
Author(s):  
Christine Faust ◽  
Christian Ochs ◽  
Marcus Korn ◽  
Ulrich Werner ◽  
Jennifer Jung ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Physiological Role ◽  
Peptide Hormone ◽  
In Vitro Assays ◽  
Fusion Partner ◽  
Insulin Analogs ◽  
Fc Fusion Protein ◽  
A Chain

Abstract Insulin is a peptide hormone produced by the pancreas. The physiological role of insulin is the regulation of glucose metabolism. Under certain pathological conditions the insulin levels can be reduced leading to the metabolic disorder diabetes mellitus (DM). For type 1 DM and, dependent on the disease progression for type 2 DM, insulin substitution becomes indispensable. To relieve insulin substitution therapy for patients, novel insulin analogs with pharmacokinetic and pharmacodynamic profiles aiming for long-lasting or fast-acting insulins have been developed. The next step in the evolution of novel insulins should be insulin analogs with a time action profile beyond 1–2 days, preferable up to 1 week. Nowadays, insulin is produced in a recombinant manner. This approach facilitates the design and production of further insulin-analogs or insulin-fusion proteins. The usage of the Fc-domain from immunoglobulin as a fusion partner for therapeutic proteins and peptides is widely used to extend their plasma half-life. Insulin consists of two chains, the A- and B-chain, which are connected by two disulfide-bridges. To produce a novel kind of Fc-fusion protein we have fused the A-chain as well as the B-chain to Fc-fragments containing either ‘knob’ or ‘hole’ mutations. The ‘knob-into-hole’ technique is frequently used to force heterodimerization of the Fc-domain. Using this approach, we were able to produce different variants of two-chain-insulin-Fc-protein (tcI-Fc-protein) variants. The tcI-Fc-fusion variants retained activity as shown in in vitro assays. Finally, prolonged blood glucose lowering activity was demonstrated in normoglycemic rats. Overall, we describe here the production of novel insulin-Fc-fusion proteins with prolonged times of action.

Development of Nanophase β-Tricalcium Phosphate/Collagen Fiber Composites for Improving Cell Adhesion and Proliferation

2006 ◽  
Vol 309-311 ◽  
pp. 581-584 ◽  
Author(s):  
Bing Gang Guan ◽  
Wei Qi Yan ◽  
Di Sheng Yang ◽  
Chao Zou ◽  
Wen Jian Weng
Keyword(s):  
Cell Adhesion ◽  
Tricalcium Phosphate ◽  
Cell Attachment ◽  
Collagen Matrix ◽  
Sem Analysis ◽  
In Vitro Assays ◽  
Beta Tricalcium Phosphate ◽  
And Control ◽  
Cell Adhesion And Proliferation

A novel porous beta-tricalcium phosphate /collagen fibers (β-TCP/CF) composite, having a well-dispersed nano-sized β-TCP in collagen matrix, was developed by a wet-chemical method. The nano-composite was compared to conventional β-TCP on cytocompatibility by cell attachment, proliferation, alkaline phosphotatse (AKP) activity and scanning electron microscopy (SEM) analysis. These in vitro assays showed that the β-TCP/CF composite elicited cell adhesion and proliferation better then controls. Moreover experiments on osteoblast-like cells showed improved cell growth with the highly characterized nanophase structure. SEM micrographs also showed that the nano-sized composite exhibited much more viable cells in attachment on the surface compared with the controls. At 1, 3 and 5 days, AKP activity was not significant different for the tested and control samples, while at 7 day after culture, significantly increased AKP activity was observed for β-TCP/CF than for the control. The in vitro results obtained confirmed the remarkable improvement of cell adhesion and proliferation of the nano-sized β-TCP/CF composite, which may be a new promising candidate for tissue engineered bone substitute.

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