Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry

Lead (Pb) is a heavy metal with a proven neurotoxic effect. Exposure is particularly dangerous to the developing brain in the pre- and neonatal periods. One postulated mechanism of its neurotoxicity is induction of inflammation. This study analyzed the effect of exposure of rat pups to Pb during periods of brain development on the concentrations of selected cytokines and prostanoids in the forebrain cortex, hippocampus and cerebellum. Methods: Administration of 0.1% lead acetate (PbAc) in drinking water ad libitum, from the first day of gestation to postnatal day 21, resulted in blood Pb in rat pups reaching levels below the threshold considered safe for humans by the Centers for Disease Control and Prevention (10 µg/dL). Enzyme-linked immunosorbent assay (ELISA) method was used to determine the levels of interleukins IL-1β, IL-6, transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2) and thromboxane B2 (TXB2). Western blot and quantitative real-time PCR were used to determine the expression levels of cyclooxygenases COX-1 and COX-2. Finally, Western blot was used to determine the level of nuclear factor kappa B (NF-κB). Results: In all studied brain structures (forebrain cortex, hippocampus and cerebellum), the administration of Pb caused a significant increase in all studied cytokines and prostanoids (IL-1β, IL-6, TGF-β, PGE2 and TXB2). The protein and mRNA expression of COX-1 and COX-2 increased in all studied brain structures, as did NF-κB expression. Conclusions: Chronic pre- and neonatal exposure to Pb induces neuroinflammation in the forebrain cortex, hippocampus and cerebellum of rat pups.

Download Full-text

Anti-inflammation conferred by stimulation of CD200R1 via Dok1 pathway in rat microglia after germinal matrix hemorrhage

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x17725211 ◽

2017 ◽

Vol 39 (1) ◽

pp. 97-107 ◽

Cited By ~ 10

Author(s):

Zhanhui Feng ◽

Lan Ye ◽

Damon Klebe ◽

Yan Ding ◽

Zhen-Ni Guo ◽

...

Keyword(s):

Western Blot ◽

Tnf Alpha ◽

Protective Effects ◽

Germinal Matrix ◽

Germinal Matrix Hemorrhage ◽

Beneficial Effects ◽

Cd200 Receptor ◽

Rat Pups ◽

Enhanced Expression ◽

Time Dependent Effect

CD200 has been reported to be neuroprotective in neurodegenerative diseases. However, the potential protective effects of CD200 in germinal matrix hemorrhage (GMH) have not been investigated. We examined the anti-inflammatory mechanisms of CD200 after GMH. A total of 167 seven-day-old rat pups were used. The time-dependent effect of GMH on the levels of CD200 and CD200 Receptor 1 (CD200R1) was evaluated by western blot. CD200R1 was localized by immunohistochemistry. The short-term (24 h) and long-term (28 days) outcomes were evaluated after CD200 fusion protein (CD200Fc) treatment by neurobehavioral assessment. CD200 small interfering RNA (siRNA) and downstream of tyrosine kinase 1 (Dok1) siRNA were injected intracerebroventricularly. Western blot was employed to study the mechanisms of CD200 and CD200R1. GMH induced significant developmental delay and caused impairment in both cognitive and motor functions in rat pups. CD200Fc ameliorated GMH-induced damage. CD200Fc increased expression of Dok1 and decreased IL-1beta and TNF-alpha levels. CD200R1 siRNA and Dok1 siRNA abolished the beneficial effects of CD200Fc, as demonstrated by enhanced expression levels of IL-1beta and TNF-alpha. CD200Fc inhibited GMH-induced inflammation and this effect may be mediated by CD200R1/Dok1 pathway. Thus, CD200Fc may serve as a potential treatment to ameliorate brain injury for GMH patients.

Download Full-text

PGE2 Modulates GABAA Receptors via an EP1 Receptor-Mediated Signaling Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000430143 ◽

2015 ◽

Vol 36 (5) ◽

pp. 1699-1711 ◽

Cited By ~ 2

Author(s):

Guang Yang ◽

Wen-Hao Dong ◽

Chang-Long Hu ◽

Yan-Ai Mei

Keyword(s):

Cell Surface ◽

Western Blot ◽

Gabaa Receptors ◽

Surface Expression ◽

Cell Surface Expression ◽

Granule Neurons ◽

Patch Clamp Technique ◽

Mammalian Tissues ◽

Ep1 Receptor ◽

Western Blot Data

Aims: PGE2 is one of the most abundant prostanoids in mammalian tissues, but its effect on neuronal receptors has not been well investigated. This study examines the effect of PGE2 on GABAA receptor currents in rat cerebellar granule neurons. Methods: GABAA currents were recorded using a patch-clamp technique. Cell surface and total protein of GABAA β1/2/3 subunits was carried out by Western blot analysis. Results: Upon incubation of neurons with PGE2 (1 µM) for 60 minutes, GABAA currents were significantly potentiated. This PGE2-driven effect could be blocked by PKC or CaMKII inhibitors as well as EP1 receptor antagonist, and mimicked by PMA or EP1 receptor agonist. Furthermore, Western blot data showed that PGE2 did not increase the total expression level of GABAA receptors, but significantly increased surface levels of GABAA β1/2/3 subunits after 1 h of treatment. Consistently, both PKC and CaMKII inhibitors were able to reduce PGE2-induced increases in cell surface expression of GABAA receptors. Conclusion: Activation of either the PKC or CaMKII pathways by EP1 receptors mediates the PGE2-induced increase in GABAA currents. This suggests that upregulation of postsynaptic GABAA receptors by PGE2 may have profound effects on cerebellar functioning under physiological and pathological conditions.

Download Full-text

Orogastric EGF enhances c-neu and EGF receptor phosphorylation in suckling rat jejunum in vivo

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1993.265.1.g63 ◽

1993 ◽

Vol 265 (1) ◽

pp. G63-G72 ◽

Cited By ~ 2

Author(s):

J. F. Thompson ◽

R. M. Lamprey ◽

P. C. Stokkers

Keyword(s):

Western Blot ◽

Kinase Activity ◽

Egf Receptor ◽

Basolateral Membrane ◽

Similar Response ◽

Rat Jejunum ◽

Rat Pups ◽

Epidermal Growth

Epidermal growth factor (EGF) in rat milk stimulates intestinal growth. We examined the role of EGF-stimulated tyrosine kinase activity in vivo in this process using an affinity-purified anti-phosphotyrosine antibody in Western blot. Jejunal sacs from fasted 8-day-old rat pups were incubated with intraluminal EGF and were then assayed for phosphotyrosyl proteins (p-Tyr) by Western blot. A 170-kDa p-Tyr was present in EGF-treated sacs but not in control. A 190-kDa p-Tyr was constitutively present in control but increased in abundance in EGF-treated sacs. Dose-response experiments demonstrated that the increase in p-Tyr was present at 100 ng/ml EGF, which is within the physiological range. The 170- and 190-kDa p-Tyr was confirmed by immunoprecipitation to be the EGF receptor and c-neu, respectively. A similar response was also observed in the jejunum after orogastric gavage feeding of EGF. By use of indirect immunofluorescence, the EGF receptor was localized primarily to the basolateral membrane of both the crypt and villus epithelium. c-neu was localized primarily in the villus enterocyte basolateral membrane. These data demonstrate that intraluminal EGF stimulates rapid tyrosine phosphorylation of the EGF receptor in vivo and that c-neu is a major substrate of the EGF receptor in suckling jejunum. The basolateral membrane localization of the EGF receptor and c-neu suggests that EGF is rapidly transported across the intestinal epithelium in suckling rat jejunum.

Download Full-text

Neuroglobin Plays a Protective Role in Arsenite-Induced Cytotoxicity by Inhibition of Cdc42 and Rac1GTPases in Rat Cerebellar Granule Neurons

Cellular Physiology and Biochemistry ◽

10.1159/000430323 ◽

2015 ◽

Vol 36 (4) ◽

pp. 1613-1627 ◽

Cited By ~ 5

Author(s):

Xiaona Liu ◽

Yanhui Gao ◽

Yuan An ◽

Xiaoyan Fu ◽

Yuanyuan Li ◽

...

Keyword(s):

Western Blot ◽

Rho Gtpases ◽

Primary Cultures ◽

Protective Role ◽

Cell Counting ◽

Cerebellar Granule Neurons ◽

Tunel Staining ◽

Granule Neurons ◽

Cytotoxic Effects ◽

Cerebellar Granule

Background and Aims: We have previously shown that neuroglobin (Ngb) expression can be regulated by sodium arsenite (NaAsO2) exposure in rat cerebellar granule neurons (CGNs). However, the precise molecular mechanisms of Ngb action are largely unknown. Ras homolog (Rho) guanosine triphosphatases (Rho GTPases) are involved in the regulation of a number of cellular processes, including cell cytotoxicity. It has been reported that Ngb can act as a guanine nucleotide dissociation inhibitior (GDI) role to inactivate Rho GTPases. Therefore, we investigated Rho GTPases activation induced by NaAsO2 exposure in rat CGNs and effects of Rho GTPases activation on the cells. We also investigated the role of Ngb in this process. Methods: Primary cultures of CGNs were prepared from 7-day-old Wistar rat pups. The cytotoxic effects of NaAsO2 on CGNs were evaluated using the Cell Counting Kit-8 assay and TUNEL staining. RNA interference technology was used to silence Ngb, and the subsequent effects were evaluated by quantitative RT-PCR and Western blot. Cdc42 and Rac1 activation were measured by pull-down assay and Western blot. Results: NaAsO2 induced cytotoxicity in rat CGNs, increased GTP-bound form of Cdc42 and Rac1 GTPases in the cells. Furthermore, inhibition of Cdc42 or Rac1 activity using the inhibitor ZCL278 or NSC23766 decreased apoptosis and increased cell viability in the cells exposed to NaAsO2. Using siRNA-mediated knockdown, we show that NaAsO2-induced cytotoxicity was exacerbated, activation of Cdc42 (GTP-Cdc42) and Rac1 (GTP-Rac1) was increased in Ngb RNA silencing cells. Conclusions: cytotoxic effects of NaAsO2 on rat CGNs is induced at least partly by Cdc42 and Rac1 activation, and Ngb can inhibit Cdc42 and Rac1 activation to play protective role in rat CGNs exposed to NaAsO2.

Download Full-text

In Brief: Day of adoption affects behavior of rat pups and dams

PsycEXTRA Dataset ◽

10.1037/e384032004-011 ◽

2004 ◽

Author(s):

Keyword(s):

Rat Pups

Download Full-text

Vorkommen von Antikörpern gegen das bovine Immunschwächevirus bei Rindern in Bayern

Tierärztliche Praxis Ausgabe G Großtiere / Nutztiere ◽

10.1055/s-0038-1622979 ◽

2003 ◽

Vol 31 (05) ◽

pp. 248-253

Author(s):

Maren Bartels ◽

Katrin Hartmann ◽

L. Scobie ◽

O. Jarrett ◽

W. Klee

Keyword(s):

Western Blot

ZusammenfassungIm Rahmen einer epidemiologischen Untersuchung zur Infektion mit dem bovinen Immunschwächevirus (BIV) bei Rindern in Oberbayern erfolgten zwei Studien, in denen Serum mittels indirektem ELISA auf BIV-Antikörper untersucht wurde. Die ELISA-Ergebnisse der BIV-positiven Tiere der Studie I wurden mittels Western Blot bestätigt. In Studie I wurde Blut von 173 ungezielt ausgewählten Rinderpatienten der II. Medizinischen Tierklinik der Ludwig-Maximilians-Universität München untersucht. Von diesen waren acht Tiere BIV-infiziert. Das entspricht einer Prävalenz von 4,6%. Alle positiven Tiere waren über zwei Monate alt. In Studie II wurden 550 Kühe aus 11 oberbayerischen landwirtschaftlichen Betrieben untersucht. Hiervon waren 11 Tiere BIVAntikör-perpositiv. Dies entspricht einer Prävalenz von 2,0%. Die positiven Tiere stammten aus fünf Betrieben mit Boxenlaufstallhaltung. Kein Tier aus Betrieben mit Anbindehaltung war positiv. In Studie II lag das Durchschnittsalter der Kühe aus den Betrieben ohne BIV-infizierte Tiere signifikant höher als in den Betrieben mit BIV-infizierten Tieren. Die Prävalenz von BIV-Antikörpern war zwar unter den kranken Probanden aus Studie I signifikant höher als bei den klinisch unauffälligen Rindern der Studie II, die pathogene Bedeutung des BIV erscheint jedoch fraglich.

Download Full-text

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Download Full-text

Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648157 ◽

1991 ◽

Vol 65 (04) ◽

pp. 382-388 ◽

Cited By ~ 9

Author(s):

Dulce Veloso ◽

Robert W Colman

Keyword(s):

Complex Formation ◽

Western Blot ◽

Human Plasma ◽

Contact System ◽

Plasma Activation ◽

Normal Plasma ◽

Sds Page ◽

Activation Products ◽

Major Antigen ◽

Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

Download Full-text

Differences in the Interactions of Lupus Anticoagulant IgG with Human Prothrombin and Bovine Prothrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653838 ◽

1995 ◽

Vol 73 (04) ◽

pp. 668-674 ◽

Cited By ~ 34

Author(s):

L Vijaya Mohan Rao ◽

An D Hoang ◽

Samuel I Rapaport

Keyword(s):

Western Blot ◽

Lupus Anticoagulant ◽

Protein A ◽

Phospholipid Complex ◽

Experimental Conditions ◽

Bovine Plasma ◽

Activation System ◽

Rate Limiting ◽

Substantial Extent ◽

Prothrombin Activation

SummaryLupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin than the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell’s viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing >50% of inhibition. In contrast, only one LA IgG markedly inhibited (>50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin. In experiments with 125I-human prothrombin or 125I-bovine prothrombin in a solution containing Ca2+, the addition of PS/PC vesicles enhanced the binding of both human and bovine prothrombin to some LA IgG preparations. The enhanced binding was particularly evident for bovine prothrombin. Although seemingly related for some preparations, the ability of a LA IgG to bind to bovine prothrombin, either in the presence or absence of PS, and the ability of that LA IgG to inhibit the activation of bovine prothrombin was not consistently related for all preparations.

Download Full-text