scholarly journals  Conglutinin is not specific to cattle

2011 ◽  
Vol 56 (No. 10) ◽  
pp. 510-519 ◽  
Author(s):  
M. Dec ◽  
A. Wernicki ◽  
A. Puchalski ◽  
R. Urban-Chmiel
Keyword(s):  
Western Blotting ◽  
Bovine Serum ◽  
Polyclonal Antibodies ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Ruminant Species ◽  
Reducing Conditions ◽  
Adult Bovine Serum ◽  
Molecular Masses ◽  
Cell Wall Extract

   Conglutinin is a high-molecular-weight mammalian lectin which binds in a calcium-dependent manner to cell-surface-bound complement fragment iC3b, yeast cell-wall extract and terminal non-reducing N-acetyl-D-glucosamine, mannose and fucose residues. This protein, originally detected in bovine serum, belongs to the family of collectins, which are effector molecules in innate immunity. Conglutinin appears to play an important role in defence mechanisms, showing antiviral and antibacterial activity. We have characterized the electrophoresis profile of bovine serum conglutinin and used Western blotting to compare profiles of this lectin derived from the sera of different breeds of cattle. The profile of non-reduced conglutinin is characterised by many bands with molecular masses ranging from 34 to 630 kDa. Reduced lectin takes the form of three main bands with molecular masses of 41, 47 and 96 kDa. We show that conglutinin is present not only in adult bovine serum, but also in foetal bovine serum, colostrum and milk. The sera of sheep, goats, gnu antelopes and deer, as well as some non-ruminant species such as llamas, horses, boars, pigs and humans, contain proteins which have similar antigenicity to that of bovine conglutinin. These reacted with monoclonal and polyclonal antibodies specific for bovine conglutinin under reducing and non-reducing conditions in Western blotting. The protein profiles of bison and swine lectin were observed to be particularly similar to bovine conglutinin.

Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVI

2003 ◽  
Vol 90 (10) ◽  
pp. 662-671 ◽  
Author(s):  
Wen-Hui Lee ◽  
Xiao-Yan Du ◽  
Qiu-Min Lu ◽  
Kenneth Clemetson ◽  
Yun Zhang
Keyword(s):  
Amino Acid ◽  
Platelet Aggregation ◽  
Molecular Mass ◽  
Mass Difference ◽  
Amino Acid Sequences ◽  
Dependent Manner ◽  
Reducing Conditions ◽  
Glycosylation Sites ◽  
Molecular Masses ◽  
Dose Dependent

SummaryStejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (α), 20 kDa (β1) and 22 kDa (β2), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that β1 and β2 subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between β1 and β2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the α and β, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against αIIbβ3 inhibited the aggregation response to stejnulxin, indicating that activation of αIIbβ3 and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbα or α2β1 as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.

scholarly journals Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ke Zhang ◽  
Zhuoying Li ◽  
Yunyang Lu ◽  
Linyi Xiang ◽  
Jiadong Sun ◽  
...  
Keyword(s):  
Western Blotting ◽  
Planar Cell Polarity ◽  
Type Ii Collagen ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Inflammatory Microenvironment ◽  
Functional Roles ◽  
Protein Levels ◽  
Oa Chondrocytes

Abstract Background The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes. Methods Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways. Results IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes. Conclusions For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

scholarly journals Influences of advanced glycosylation end products on the inner blood–retinal barrier in a co-culture cell model in vitro

2020 ◽  
Vol 15 (1) ◽  
pp. 619-628
Author(s):  
Chen Yuan ◽  
Ya Mo ◽  
Jie Yang ◽  
Mei Zhang ◽  
Xuejun Xie
Keyword(s):  
Electrical Resistance ◽  
Cell Model ◽  
Polyclonal Antibodies ◽  
Time Dependent ◽  
Dependent Manner ◽  
Blood Retinal Barrier ◽  
Advanced Glycosylation End Products ◽  
End Products ◽  
Advanced Glycosylation

AbstractAdvanced glycosylation end products (AGEs) are harmful factors that can damage the inner blood–retinal barrier (iBRB). Rat retinal microvascular endothelial cells (RMECs) were isolated and cultured, and identified by anti-CD31 and von Willebrand factor polyclonal antibodies. Similarly, rat retinal Müller glial cells (RMGCs) were identified by H&E staining and with antibodies of glial fibrillary acidic protein and glutamine synthetase. The transepithelial electrical resistance (TEER) value was measured with a Millicell electrical resistance system to observe the leakage of the barrier. Transwell cell plates for co-culturing RMECs with RMGCs were used to construct an iBRB model, which was then tested with the addition of AGEs at final concentrations of 50 and 100 mg/L for 24, 48, and 72 h. AGEs in the in vitro iBRB model constructed by RMEC and RMGC co-culture led to the imbalance of the vascular endothelial growth factor (VEGF) and pigment epithelial derivative factor (PEDF), and the permeability of the RMEC layer increased because the TEER decreased in a dose- and time-dependent manner. AGEs increased VEGF but lowered PEDF in a dose- and time-dependent manner. The intervention with AGEs led to the change of the transendothelial resistance of the RMEC layer likely caused by the increased ratio of VEGF/PEDF.

scholarly journals Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

2021 ◽  
Vol 22 (4) ◽  
pp. 1596
Author(s):  
Elsa Ronzier ◽  
Claire Corratgé-Faillie ◽  
Frédéric Sanchez ◽  
Christian Brière ◽  
Tou Cheu Xiong
Keyword(s):  
Arabidopsis Thaliana ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
In Planta ◽  
Knock Out ◽  
Calcium Dependent ◽  
Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

scholarly journals Pancreatic beta-cells expressing the Arg64 variant of the beta(3)-adrenergic receptor exhibit abnormal insulin secretory activity

2001 ◽  
Vol 27 (2) ◽  
pp. 133-144 ◽  
Author(s):  
R Perfetti ◽  
H Hui ◽  
K Chamie ◽  
S Binder ◽  
M Seibert ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Beta Cells ◽  
Western Blotting ◽  
Adrenergic Receptor ◽  
Pancreatic Beta Cells ◽  
Secretory Activity ◽  
Host Cells ◽  
Human Pancreas ◽  
Dependent Manner ◽  
Wild Type

The Arg64 beta(3)-adrenergic receptor (beta(3)AR) variant is associated with an earlier age of onset of diabetes and lower levels of insulin secretion in humans. The aims of this study were to investigate whether beta(3)AR is expressed by islet cells, if receptor binding affects insulin secretion and, finally, if the beta(3)AR Arg64 variant induces abnormal insulin secretory activity. Human pancreas extracts were subjected to RT-PCR, Western blotting and immunostaining analyses. DNA sequencing and Western blotting demonstrated that the beta(3)AR gene is transcribed and translated in the human pancreas; immunostaining showed that it is expressed by the islets of Langerhans. Cultured rat beta-cells responded to human beta(3)AR agonists in a dose- and time-dependent manner. Transfection of cultured rat beta-cells with the wild-type human beta(3)AR produced an increased baseline and ligand-dependent insulin secretion compared with parental cells. On the other hand, cells transfected with the Arg64 variant of the beta(3)AR secreted less insulin, both spontaneously and after exposure to human beta(3)AR agonists. Furthermore, while transfection with the wild-type beta(3)AR preserved the glucose-dependent secretion of insulin, expression of the variant receptor rendered the host cells significantly less responsive to glucose. In summary, cells express the beta(3)AR, and its activation contributes to the regulation of insulin secretion. These findings may help explain the low levels of insulin secretion in response to an i.v. glucose tolerance test observed in humans carrying the Arg64 polymorphism.

Monoclonal antibodies reveal the alteration of the rhodocetin structure upon α2β1 integrin binding

2011 ◽  
Vol 440 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Thilo Bracht ◽  
Flávia Figueiredo de Rezende ◽  
Jörg Stetefeld ◽  
Lydia M. Sorokin ◽  
Johannes A. Eble
Keyword(s):  
Monoclonal Antibodies ◽  
Dependent Manner ◽  
Hydrophilic Surfaces ◽  
Sequence Homologies ◽  
Molecular Masses ◽  
The Individual ◽  
Β Chain ◽  
Α2β1 Integrin ◽  
Characteristic Domain ◽  
Integrin Α2

The α2β1 antagonist rhodocetin from Calloselasma rhodostoma is a heterotetrameric CLRP (C-type lectin-related protein) consisting of four distinct chains, α, β, γ and δ. Via their characteristic domain-swapping loops, the individual chains form two subunits, αβ and γδ. To distinguish the four chains which share similar molecular masses and high sequence homologies, we generated 11 mAbs (monoclonal antibodies) with different epitope specificities. Four groups of distinct mAbs were generated: the first targeted the rhodocetin β chain, the second group bound to the αβ subunit mostly in a conformation-dependent manner, the third group recognized the γδ subunit only when separated from the αβ subunit, whereas a fourth group interacted with the γδ subunit both in the heterotetrameric molecule and complexed with the integrin α2 A-domain. Using the specific mAbs, we have shown that the rhodocetin heterotetramer dissociates into the αβ and γδ subunit upon binding to the integrin α2 A-domain at both the molecular and cellular levels. After dissociation, the γδ subunit firmly interacts with the α2β1 integrin, thereby blocking it, whereas the rhodocetin αβ subunit is released from the complex. The small molecular interface between the αβ and γδ subunits within rhodocetin is mostly mediated by charged residues, which causes the two dissociated subunits to have hydrophilic surfaces.

scholarly journals Characterization of Two Autographa californica Nucleopolyhedrovirus Proteins, Ac145 and Ac150, Which Affect Oral Infectivity in a Host-Dependent Manner

2004 ◽  
Vol 78 (12) ◽  
pp. 6439-6448 ◽  
Author(s):  
Renee Lapointe ◽  
Holly J. R. Popham ◽  
Ursula Straschil ◽  
David Goulding ◽  
David R. O'Reilly ◽  
...  
Keyword(s):  
Trichoplusia Ni ◽  
Polyclonal Antibodies ◽  
Heliothis Armigera ◽  
Autographa Californica ◽  
Dependent Manner ◽  
Significant Drop ◽  
Double Deletion ◽  
Autographa Californica Nucleopolyhedrovirus ◽  
Budded Virus ◽  
Nuclear Polyhedrosis

ABSTRACT The genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) contains two homologues, orf145 and orf150, of the Heliothis armigera Entomopoxvirus (HaEPV) 11,000-kDa gene. Polyclonal antibodies raised against the Ac145 or Ac150 protein were utilized to demonstrate that they are expressed from late to very late times of infection and are within the nuclei of infected Sf-21 cells. Transmission electron microscopy coupled with immunogold labeling of Ac145 found this protein within the nucleus in areas of nucleocapsid assembly and maturation, along with some association with the enveloped bundles of virions within the developing occlusion bodies (OBs). Ac150 was found to be mainly associated with enveloped bundles of virions within OBs and also with those not yet occluded. Both Ac145 and Ac150 were found to be present in budded virus as well as OBs. Both orf145 and orf150 were deleted from the AcMNPV genome, singly or together, and these deletion mutants were assessed for oral infectivity both in Trichoplusia ni and Heliothis virescens larvae. Deletion of Ac145 led to a small but significant drop in infectivity (sixfold) compared to wild-type (wt) AcMNPV for T. ni but not for H. virescens. Deletion of Ac150 alone had no effect on infectivity of the virus for either host. However, deletion of both Ac145 and Ac150 gave a recombinant virus with a drastic (39-fold) reduction in infectivity compared to wt virus for H. virescens. Intrahemocoelic injection of budded virus from the double-deletion virus into H. virescens larvae is as infectious to this host as wt budded virus, indicating that Ac145 and Ac150 play a role in primary oral infection of AcMNPV, the extent of which is host dependent.

scholarly journals Regulation of neuronal axon specification by glia-neuron gap junctions in C. elegans

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lingfeng Meng ◽  
Albert Zhang ◽  
Yishi Jin ◽  
Dong Yan
Keyword(s):  
Gap Junctions ◽  
Glial Cells ◽  
Neuronal Development ◽  
Dependent Manner ◽  
Critical Step ◽  
C Elegans ◽  
Calcium Dependent ◽  
Intracellular Pathways ◽  
Axon Specification

Axon specification is a critical step in neuronal development, and the function of glial cells in this process is not fully understood. Here, we show that C. elegans GLR glial cells regulate axon specification of their nearby GABAergic RME neurons through GLR-RME gap junctions. Disruption of GLR-RME gap junctions causes misaccumulation of axonal markers in non-axonal neurites of RME neurons and converts microtubules in those neurites to form an axon-like assembly. We further uncover that GLR-RME gap junctions regulate RME axon specification through activation of the CDK-5 pathway in a calcium-dependent manner, involving a calpain clp-4. Therefore, our study reveals the function of glia-neuron gap junctions in neuronal axon specification and shows that calcium originated from glial cells can regulate neuronal intracellular pathways through gap junctions.

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