scholarly journals Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport

2021 ◽  
Vol 8 (12) ◽  
pp. 289
Author(s):  
Joseph M. Autry ◽  
Bengt Svensson ◽  
Samuel F. Carlson ◽  
Zhenhui Chen ◽  
Razvan L. Cornea ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Transport Rate ◽  
Inhibitory Peptide ◽  
Gluteal Muscle ◽  
Exertional Rhabdomyolysis ◽  
Sds Page ◽  
High Incidence ◽  
Fast Twitch

We have analyzed the enzymatic activity of the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) from the horse gluteal muscle. Horses are bred for peak athletic performance yet exhibit a high incidence of exertional rhabdomyolysis, with elevated levels of cytosolic Ca2+ proposed as a correlative linkage. We recently reported an improved protocol for isolating SR vesicles from horse muscle; these horse SR vesicles contain an abundant level of SERCA and only trace-levels of sarcolipin (SLN), the inhibitory peptide subunit of SERCA in mammalian fast-twitch skeletal muscle. Here, we report that the in vitro Ca2+ transport rate of horse SR vesicles is 2.3 ± 0.7-fold greater than rabbit SR vesicles, which express close to equimolar levels of SERCA and SLN. This suggests that horse myofibers exhibit an enhanced SR Ca2+ transport rate and increased luminal Ca2+ stores in vivo. Using the densitometry of Coomassie-stained SDS-PAGE gels, we determined that horse SR vesicles express an abundant level of the luminal SR Ca2+ storage protein calsequestrin (CASQ), with a CASQ-to-SERCA ratio about double that in rabbit SR vesicles. Thus, we propose that SR Ca2+ cycling in horse myofibers is enhanced by a reduced SLN inhibition of SERCA and by an abundant expression of CASQ. Together, these results suggest that horse muscle contractility and susceptibility to exertional rhabdomyolysis are promoted by enhanced SR Ca2+ uptake and luminal Ca2+ storage.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

scholarly journals Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+Signaling

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Nahed El-Najjar ◽  
Rashmi P. Kulkarni ◽  
Nancy Nader ◽  
Rawad Hodeify ◽  
Khaled Machaca
Keyword(s):  
Insulin Resistance ◽  
Smooth Muscle ◽  
Sarcoplasmic Reticulum ◽  
Vascular Smooth Muscle ◽  
Complex Disease ◽  
Prolonged Exposure ◽  
In Vitro System ◽  
A7r5 Cells

Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+signaling, including most prominently an inhibition of the passive ER Ca2+leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+signaling machinery are different.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Glycation of Host Proteins Increases Pathogenic Potential of Porphyromonas gingivalis

2021 ◽  
Vol 22 (21) ◽  
pp. 12084
Author(s):  
Michał Śmiga ◽  
John W. Smalley ◽  
Paulina Ślęzak ◽  
Jason L. Brown ◽  
Klaudia Siemińska ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Disease Process ◽  
Human Keratinocytes ◽  
Bacterial Biofilm ◽  
Pathogenic Potential ◽  
Glycated Proteins ◽  
Sds Page

The non-enzymatic addition of glucose (glycation) to circulatory and tissue proteins is a ubiquitous pathophysiological consequence of hyperglycemia in diabetes. Given the high incidence of periodontitis and diabetes and the emerging link between these conditions, it is of crucial importance to define the basic virulence mechanisms employed by periodontopathogens such as Porphyromonas gingivalis in mediating the disease process. The aim of this study was to determine whether glycated proteins are more easily utilized by P. gingivalis to stimulate growth and promote the pathogenic potential of this bacterium. We analyzed the properties of three commonly encountered proteins in the periodontal environment that are known to become glycated and that may serve as either protein substrates or easily accessible heme sources. In vitro glycated proteins were characterized using colorimetric assays, mass spectrometry, far- and near-UV circular dichroism and UV–visible spectroscopic analyses and SDS-PAGE. The interaction of glycated hemoglobin, serum albumin and type one collagen with P. gingivalis cells or HmuY protein was examined using spectroscopic methods, SDS-PAGE and co-culturing P. gingivalis with human keratinocytes. We found that glycation increases the ability of P. gingivalis to acquire heme from hemoglobin, mostly due to heme sequestration by the HmuY hemophore-like protein. We also found an increase in biofilm formation on glycated collagen-coated abiotic surfaces. We conclude that glycation might promote the virulence of P. gingivalis by making heme more available from hemoglobin and facilitating bacterial biofilm formation, thus increasing P. gingivalis pathogenic potential in vivo.

scholarly journals Catestatin prevents endothelial inflammation and promotes thrombus resolution in acute pulmonary embolism in mice

2019 ◽  
Vol 39 (11) ◽  
Author(s):  
Hua Chen ◽  
Dongxia Liu ◽  
Lan Ge ◽  
Tao Wang ◽  
Zhenzhen Ma ◽  
...  
Keyword(s):  
Pulmonary Embolism ◽  
Acute Pulmonary Embolism ◽  
Thrombus Formation ◽  
Vascular Dysfunction ◽  
In Vivo Model ◽  
Protective Effects ◽  
Inhibitory Peptide ◽  
Endothelial Inflammation

AbstractCatestatin (CTS), a catecholamine-release inhibitory peptide, exerts pleiotropic cardiac protective effects. Pulmonary embolism caused by deep vein thrombosis involving vascular dysfunction. The present study aims to investigate the effects of CTS on thrombus formation that may inhibit the development of pulmonary embolism and its potential pathway. Acute pulmonary embolism (APE) model was developed as an in vivo model. The effects of CTS on mice with APE were examined. Human pulmonary artery endothelial cells (HPAECs) were pretreated with CTS before thrombin stimulation, and endothelial inflammation and underlying mechanisms were evaluated in vitro. That plasma CTS level was decreased in APE mice, while the number of platelets was significantly increased. The decreased circulating CTS level negatively associated with the number of platelets. CTS administration increased the survival rate of APE mice and protected against microvascular thrombosis in lung. APE-induced the increase in platelets number and plasma von Willebrand factor (VWF) were inhibited by CTS. Platelets from CTS-treated APE mice showed impaired agonist-induced platelets aggregation and spreading. CTS also ameliorated APE-induced the systemic inflammatory response. In in vivo study, thrombin-induced the increase in inflammation, TLR-4 expression and p38 phosphorylation were abrogated by CTS in HPAECs. Furthermore, TLR-4 overexpression inhibited the effect of CTS on VWF release and inflammation in HPAECs. Collectively, CTS increases thrombus resolution by attenuating endothelial inflammation at partially via inhibiting TLR-4-p38 pathway. The present study may provide a novel approach for anti-thrombosis.

Evidence of an Intrinsic Defect of Lymphocyte Reactivity in AKR Mice.

1975 ◽  
Vol 61 (4) ◽  
pp. 339-349 ◽  
Author(s):  
Dino Collavo ◽  
Giovanni Biasi ◽  
Alfonso Colombatti ◽  
Mario Varotto ◽  
Roberto Fabbris
Keyword(s):  
Immunocompetent Cells ◽  
Intrinsic Defect ◽  
Lymphocyte Response ◽  
High Incidence ◽  
Splenic Cells ◽  
Pha Stimulation ◽  
Akr Mice ◽  
Lymphocyte Reactivity

Like their AKR/J parent, (CBAT6T6 × AKR/J)F1 mice are carriers of endogenous G-MuLV and present a high incidence of spontaneous lymphoma. However, the F1. hybrids do not present the immunological deficits seen in pre-leukemic AKR/J mice since they respond normally to in vitro PHA stimulation and to in vivo LPS immunization. These observations suggest that there is probably no direct relationship between the presence of MuLV and immunological impairment. Studies have been carried out to ascertain whether the altered immunological reactivity seen in AKR/J mice is related to factors intrinsic to the immunocompetent cells or to environmental inadequacy. Thus, (CBAT6T6 × AKR/J)F1 mice were thymectomized, irradiated, reconstituted with syngeneic bone marrow and simultaneous transplant of CBAT6T6 and AKR/J thymus, and their lymphocyte response to PHA was assayed. In addition, antibody production following LPS immunization was studied by transferring AKR/J splenic cells to irradiated CBAT6T6 or (CBAT6T6 × AKR/J)F1 mice, and vice-versa. The results of these investigations indicate that an intrinsic lymphocyte hyporeactivity is present in AKR/J mice and environmental factors do not modify the reactivity of the transferred immunocompetent cells.

scholarly journals Luteolin Exerts Cardioprotective Effects through Improving Sarcoplasmic Reticulum Ca2+-ATPase Activity in Rats during Ischemia/Reperfusion In Vivo

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Changsheng Nai ◽  
Haochen Xuan ◽  
Yingying Zhang ◽  
Mengxiao Shen ◽  
Tongda Xu ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Signaling Pathway ◽  
Myocardial Infarct ◽  
Ischemia Reperfusion ◽  
Akt Signaling ◽  
Myocardial Infarct Size ◽  
Akt Signaling Pathway ◽  
Cardioprotective Effects

The flavonoid luteolin exists in many types of fruits, vegetables, and medicinal herbs. Our previous studies have demonstrated that luteolin reduced ischemia/reperfusion (I/R) injury in vitro, which was related with sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. However, the effects of luteolin on SERCA2a activity during I/R in vivo remain unclear. To investigate whether luteolin exerts cardioprotective effects and to monitor changes in SERCA2a expression and activity levels in vivo during I/R, we created a myocardial I/R rat model by ligating the coronary artery. We demonstrated that luteolin could reduce the myocardial infarct size, lactate dehydrogenase release, and apoptosis during I/R injury in vivo. Furthermore, we found that luteolin inhibited the I/R-induced decrease in SERCA2a activity in vivo. However, neither I/R nor luteolin altered SERCA2a expression levels in myocardiocytes. Moreover, the PI3K/Akt signaling pathway played a vital role in this mechanism. In conclusion, the present study has confirmed for the first time that luteolin yields cardioprotective effects against I/R injury by inhibiting the I/R-induced decrease in SERCA2a activity partially via the PI3K/Akt signaling pathway in vivo, independent of SERCA2a protein level regulation. SERCA2a activity presents a novel biomarker to assess the progress of I/R injury in experimental research and clinical applications.

Immunochemical analysis of the human chorionic gonadotrophin-like material secreted by 'normal' and neoplastic urothelial cells

1989 ◽  
Vol 2 (2) ◽  
pp. 107-112 ◽  
Author(s):  
R. K. Iles ◽  
T. Chard
Keyword(s):  
Bladder Tumour ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Sds Page ◽  
Β Hcg ◽  
Bladder Carcinomas ◽  
Immunochemical Characteristics ◽  
Normal Urothelium

ABSTRACT Material with the immunochemical characteristics of human chorionic gonadotrophin (hCG) is produced by bladder tumour cells in vitro and in vivo. In order to characterize this material further, media were collected from 17 cell cultures (three choriocarcinomas, seven bladder carcinomas and seven 'normal' urothelium). The hCG-like material was compared with pregnancy hCG and purified α- and β-subunits by specific radioimmunoassays. Media were also submitted to affinity chromatography and the fractions further analysed by SDS-PAGE and Western blotting. It was shown that both the neoplastic and normal urothelium produced only free β-subunit-like material. This urothelial 'β-hCG' has the same molecular weight and electrophoretic mobility as that present in the intact hCG of pregnancy.

scholarly journals Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

Blood ◽  
1996 ◽  
Vol 87 (12) ◽  
pp. 5287-5296 ◽  
Author(s):  
YL Zu ◽  
Y Ai ◽  
A Gilchrist ◽  
ME Labadia ◽  
RI Sha'afi ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Enzymatic Activity ◽  
Map Kinase ◽  
Phorbol Ester ◽  
Map Kinases ◽  
Phosphorylation Site ◽  
Human Neutrophils ◽  
Inhibitory Peptide

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.

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