scholarly journals Human IgA as a heterovalent ligand: switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte.

1986 ◽  
Vol 102 (3) ◽  
pp. 920-931 ◽  
Author(s):  
J M Schiff ◽  
M M Fisher ◽  
A L Jones ◽  
B J Underdown
Keyword(s):  
Secretory Component ◽  
Binding Activity ◽  
Asialoglycoprotein Receptor ◽  
Galactose Oxidase ◽  
Rat Hepatocyte ◽  
Binding Properties ◽  
Affinity Adsorption ◽  
Switching Event ◽  
Transit Times

Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly share a common intracellular compartment.

scholarly journals A novel cycle involving fatty acyl-coenzyme A regulates asialoglycoprotein receptor activity in permeable hepatocytes.

1994 ◽  
Vol 5 (2) ◽  
pp. 227-235 ◽  
Author(s):  
P H Weigel ◽  
J D Medh ◽  
J A Oka
Keyword(s):  
Rat Hepatocytes ◽  
Fatty Acyl ◽  
Binding Activity ◽  
Asialoglycoprotein Receptor ◽  
Receptor Activity ◽  
Independent Effect ◽  
Palmitoyl Carnitine ◽  
Acyl Coenzyme A ◽  
Chain Lengths

Asialoglycoprotein receptors (ASGP-Rs) in permeable rat hepatocytes can be inactivated in the absence of ligand. This cytosol-independent effect is relatively slow (t1/2 approximately 12 min) and is temperature and ATP dependent. Here we show that in the absence of cytosol, the addition of palmitoyl-CoA (Pal-CoA) rapidly (t1/2 < 0.4 min) and quantitatively reactivates the inactivated receptors. Receptor reactivation was half-maximal at approximately 10-12 microM free Pal-CoA at 37 degrees C. Although substantially higher total concentrations were used, much of the added Pal-CoA was cell associated and not free. The effects of Pal-CoA were eliminated by bovine serum albumin at concentrations sufficient to bind all free monomeric fatty acyl-CoA, suggesting that micellar effects are not responsible for the ability to reactivate ASGP-Rs. Also, palmitoyl-carnitine did not substitute for Pal-CoA. The initial ASGP-R inactivation is not affected by treating cells with N-ethylmaleimide or by a KCl wash but is inhibited by sodium orthovanadate or high Ca2+ levels. Myristoyl-CoA (C14) was also able to reactivate inactive ASGP-Rs about as well as Pal-CoA. Fatty acyl-CoAs with chain lengths of C12 (lauroyl) or C18 (steroyl) were < 50% as active. The ligand binding activity of these receptors can subsequently be modulated within minutes by the further addition of ATP or Pal-CoA to achieve additional rounds of ASGP-R inactivation or reactivation, respectively. These in vitro data demonstrate the occurrence of a novel asialoglycoprotein receptor inactivation-reactivation cycle that could regulate receptor activity during endocytosis and receptor recycling.

scholarly journals Luteolin and GroESL Modulate In Vitro Activity of NodD

2002 ◽  
Vol 184 (2) ◽  
pp. 525-530 ◽  
Author(s):  
Kuo-Chen Yeh ◽  
Melicent C. Peck ◽  
Sharon R. Long
Keyword(s):  
Dna Binding ◽  
Sinorhizobium Meliloti ◽  
Binding Activity ◽  
Binding Properties ◽  
E Coli ◽  
Early Stages ◽  
Dna Binding Activity ◽  
Dna Binding Properties ◽  
Lysr Family

ABSTRACT In the early stages of symbiosis between the soil bacterium Sinorhizobium meliloti and its leguminous host plant, alfalfa, bacterial nodulation (nod) genes are controlled by NodD1, NodD2, and NodD3, members of the LysR family of transcriptional regulators, in response to flavonoid and other inducers released by alfalfa. To gain an understanding of the biochemical aspects of this action, epitope-tagged recombinant NodD1 and NodD3 were overexpressed in Escherichia coli. The DNA binding properties of the purified recombinant NodD proteins were indistinguishable from those of NodD isolated from S. meliloti. In addition, the E. coli GroEL chaperonin copurified with the recombinant NodD proteins. In this study, we showed that NodD proteins are in vitro substrates of the GroESL chaperonin system and that their DNA binding activity is modulated by GroESL. This confirmed the earlier genetic implication that the GroESL chaperonin system is essential for the function of these regulators. Increased DNA binding activity by NodD1 in the presence of luteolin confirmed that NodD1 is involved in recognizing the plant signal during the early stages of symbiosis.

In vitro binding activity between HCK SH3 domain and HIV-1 Nef protein

2011 ◽  
Vol 31 (3) ◽  
pp. 262-265
Author(s):  
Xiao-lin QIN ◽  
Chao-qi LIU ◽  
Dong-ming REN ◽  
Yong-qin ZHOU
Keyword(s):  
Sh3 Domain ◽  
Binding Activity ◽  
Hiv 1

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

184 Two types of anti-TIGIT antibodies with distinct binding epitope and functional activities

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A198-A198
Author(s):  
Tingting Zhong ◽  
Xinghua Pang ◽  
Zhaoliang Huang ◽  
Na Chen ◽  
Xiaoping Jin ◽  
...  
Keyword(s):  
T Cells ◽  
Mixed Culture ◽  
Cell Activity ◽  
Cross Reactivity ◽  
Binding Activity ◽  
Jurkat Cells ◽  
List Type ◽  
Dependent Manner ◽  
Vitro Assay

BackgroundTIGIT is an inhibitory receptor mainly expressed on natural killer (NK) cells, CD8+ T cells, CD4+ T cells and Treg cells. TIGIT competes with CD226 for binding with CD155. In cancers, CD155 has been reported to up-regulate on tumor cells, and TIGIT was found to increase on TILs.1 Activation of TIGIT/CD155 pathway would mediate immunosuppression in tumor; while blockade of TIGIT promotes anti-tumor immune response.MethodsAK126 and AK113 are two humanized anti-human TIGIT monoclonal antibodies developed by Akesobio. Binding activity of AK126 and AK113 to human TIGIT, and competitive binding activity with CD155 and CD112, were performed by using ELISA, Fortebio, and FACS assays. Cross-reactivity with cynomolgus monkey TIGIT and epitope binning were also tested by ELISA assay. In-vitro assay to investigate the activity to promote IL-2 secretion was performed in mixed-culture of Jurkat-TIGIT cells and THP-1 cells.ResultsAK126 and AK113 could specifically bind to human TIGIT with comparative affinity and effectively blocked the binding of human CD155 and CD112 to human TIGIT. X-ray crystal structure of TIGIT and PVR revealed the C’-C’’ loop and FG loop regions of TIGIT are the main PVR interaction regions.2 The only amino acid residue differences in these regions between human and monkey TIGIT are 70C and 73D. AK126 binds to both human and monkey TIGIT, AK113 binds only to monkey TIGIT. This suggests that these residues are required for AK113 binding to human TIGIT, but not required for AK126. Interestingly, results from cell-based assays indicated that AK126 and AK113 showed significantly different activity to induce IL-2 secretion in mixed-culture of Jurkat-TIGIT cells and THP-1 cells (figure 1A and B), in which AK126 had a comparable capacity of activity to 22G2, a leading TIGIT mAb developed by another company, to induce IL-2 secretion, while, AK113 showed a significantly higher capacity than 22G2 and AK126.Abstract 184 Figure 1Anti-TIGIT Antibodies Rescues IL-2 Production in Vitro T-Cell Activity Assay in a dose dependent manner. Jurkat-TIGIT cells (Jurkat cells engineered to over-express human TIGIT) were co-cultured with THP-1 cells, and stimulated with plate-bound anti-CD3 mAb in the presence of TIGIT ligand CD155 (A) or CD112 (B) with anti-TIGIT antibodies. After incubated for 48h at 37° C and 5.0% CO2, IL-2 levels were assessed in culture supernatants by ELISA. Data shown as mean with SEM for n = 2.ConclusionsWe discovered two distinct types of TIGIT antibodies with differences in both epitope binding and functional activity. The mechanism of action and clinical significance of these antibodies require further investigation.ReferencesSolomon BL, Garrido-Laguna I. TIGIT: a novel immunotherapy target moving from bench to bedside. Cancer Immunol Immunother 2018;67:1659–1667.Stengel KF, Harden-Bowles K, Yu X, et al. Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering. Proc Natl Acad Sci USA 2012;109:5399–5404.

scholarly journals An In Vivo Predictive Dissolution Methodology (iPD Methodology) with a BCS Class IIb Drug Can Predict the In Vivo Bioequivalence Results: Etoricoxib Products

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 507
Author(s):  
Isabel Gonzalez-Alvarez ◽  
Marival Bermejo ◽  
Yasuhiro Tsume ◽  
Alejandro Ruiz-Picazo ◽  
Marta Gonzalez-Alvarez ◽  
...  
Keyword(s):  
Plasma Concentrations ◽  
In Vitro Dissolution ◽  
Case Examples ◽  
Dissolution Studies ◽  
Weak Bases ◽  
Transit Times ◽  
Class Iib ◽  
The Impact

The purpose of this study was to predict in vivo performance of three oral products of Etoricoxib (Arcoxia® as reference and two generic formulations in development) by conducting in vivo predictive dissolution with GIS (Gastro Intestinal Simulator) and computational analysis. Those predictions were compared with the results from previous bioequivalence (BE) human studies. Product dissolution studies were performed using a computer-controlled multicompartmental dissolution device (GIS) equipped with three dissolution chambers, representing stomach, duodenum, and jejunum, with integrated transit times and secretion rates. The measured dissolved amounts were modelled in each compartment with a set of differential equations representing transit, dissolution, and precipitation processes. The observed drug concentration by in vitro dissolution studies were directly convoluted with permeability and disposition parameters from literature to generate the predicted plasma concentrations. The GIS was able to detect the dissolution differences among reference and generic formulations in the gastric chamber where the drug solubility is high (pH 2) while the USP 2 standard dissolution test at pH 2 did not show any difference. Therefore, the current study confirms the importance of multicompartmental dissolution testing for weak bases as observed for other case examples but also the impact of excipients on duodenal and jejunal in vivo behavior.

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