Attenuation of Transient Focal Cerebral Ischemic Injury in Transgenic Mice Expressing a Mutant ICE Inhibitory Protein

1997 ◽  
Vol 17 (4) ◽  
pp. 370-375 ◽  
Author(s):  
Hideaki Hara ◽  
Klaus Fink ◽  
Matthias Endres ◽  
Robert M. Friedlander ◽  
Valeria Gagliardini ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Transgenic Mice ◽  
Artery Occlusion ◽  
Dominant Negative ◽  
Neurological Deficits ◽  
Wild Type ◽  
Brain Swelling ◽  
Inhibitory Protein ◽  
Endogenous Expression ◽  
Dominant Negative Mutation

We used transgenic mice expressing a dominant negative mutation of interleukin-1β converting enzyme (ICE) (C285G) in a model of transient focal ischemia in order to investigate the role of ICE in ischemic brain damage. Transgenic mutant ICE mice (n = 11) and wild-type littermates (n = 9) were subjected to 3 h of middle cerebral artery occlusion followed by 24 h of reperfusion. Cerebral infarcts and brain swelling were reduced by 44% and 46%, respectively. Neurological deficits were also significantly reduced. Regional CBF, blood pressure, core temperature, and heart rate did not differ between groups when measured for up to 1 h after reperfusion. Increases in immunoreactive IL-1β levels, observed in ischemic wild-type brain at 30 min after reperfusion, were 77% lower in the mutant strain, indicating that proIL-1β cleavage is inhibited in the mutants. DNA fragmentation was reduced in the mutants 6 and 24 h after reperfusion. Hence, endogenous expression of an ICE inhibitor confers resistance to cerebral ischemia and brain swelling. Our results indicate that down-regulation of ICE expression might provide a useful therapeutic target in cerebral ischemia.

scholarly journals Knockout of Silent Information Regulator 2 (SIRT2) Preserves Neurological Function after Experimental Stroke in Mice

2015 ◽  
Vol 35 (12) ◽  
pp. 2080-2088 ◽  
Author(s):  
Lea Krey ◽  
Fred Lühder ◽  
Kathrin Kusch ◽  
Bozena Czech-Zechmeister ◽  
Birte Könnecke ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Cerebral Ischemia ◽  
Focal Cerebral Ischemia ◽  
Brain Regions ◽  
Artery Occlusion ◽  
Neurological Deficits ◽  
Experimental Stroke ◽  
Wild Type ◽  
Significant Difference ◽  
Stroke Models

Sirtuin-2 (Sirt2) is a member of the NAD+-dependent protein deacetylase family. Various members of the sirtuin class have been found to be involved in processes related to longevity, regulation of inflammation, and neuroprotection. Induction of Sirt2 mRNA was found in the whole hemisphere after experimental stroke in a recent screening approach. Moreover, Sirt2 protein is highly expressed in myelin-rich brain regions after stroke. To examine the effects of Sirt2 on ischemic stroke, we induced transient focal cerebral ischemia in adult male Sirt2-knockout and wild-type mice. Two stroke models with different occlusion times were applied: a severe ischemia (45 minutes of middle cerebral artery occlusion (MCAO)) and a mild one (15 minutes of MCAO), which was used to focus on subcortical infarcts. Neurological deficit was determined at 48 hours after 45 minutes of MCAO, and up to 7 days after induction of 15 minutes of cerebral ischemia. In contrast to recent data on Sirt1, Sirt2−/− mice showed less neurological deficits in both models of experimental stroke, with the strongest manifestation after 48 hours of reperfusion. However, we did not observe a significant difference of stroke volumes or inflammatory cell count between Sirt2-deficient and wild-type mice. Thus we postulate that Sirt2 mediates myelin-dependent neuronal dysfunction during the early phase after ischemic stroke.

scholarly journals The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 651
Author(s):  
Hsiao-Cheng Tsai ◽  
Che-Hong Chen ◽  
Daria Mochly-Rosen ◽  
Yi-Chen Ethan Li ◽  
Min-Huey Chen
Keyword(s):  
Bone Loss ◽  
Bacterial Infection ◽  
Bone Regeneration ◽  
Alcohol Drinking ◽  
Bacterial Species ◽  
Dominant Negative ◽  
Enzyme Inactivation ◽  
Wild Type ◽  
Micro Computed Tomography ◽  
Dominant Negative Mutation

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.

scholarly journals Cellular Werner Phenotypes in Mice Expressing a Putative Dominant-Negative Human WRN Gene

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 357-362
Author(s):  
Lan Wang ◽  
Charles E Ogburn ◽  
Carol B Ware ◽  
Warren C Ladiges ◽  
Hagop Youssoufian ◽  
...  
Keyword(s):  
Mouse Model ◽  
Transgenic Mice ◽  
Werner Syndrome ◽  
Dominant Negative ◽  
Dominant Negative Mutation ◽  
Fibroblast Cultures ◽  
Age Related

Abstract Mutations at the Werner helicase locus (WRN) are responsible for the Werner syndrome (WS). WS patients prematurely develop an aged appearance and various age-related disorders. We have generated transgenic mice expressing human WRN with a putative dominant-negative mutation (K577M-WRN). Primary tail fibroblast cultures from K577M-WRN mice showed three characteristics of WS cells: hypersensitivity to 4-nitroquinoline-1-oxide (4NQO), reduced replicative potential, and reduced expression of the endogenous WRN protein. These data suggest that K577M-WRN mice may provide a novel mouse model for the WS.

scholarly journals Suppressor Mutations Bypass the Requirement of fluG for Asexual Sporulation and Sterigmatocystin Production in Aspergillus nidulans

Genetics ◽  
2003 ◽  
Vol 165 (3) ◽  
pp. 1083-1093
Author(s):  
Jeong-Ah Seo ◽  
Yajun Guan ◽  
Jae-Hyuk Yu
Keyword(s):  
Aspergillus Nidulans ◽  
Molecular Mechanisms ◽  
Dominant Negative ◽  
Wild Type ◽  
Dominant Mutation ◽  
Site Mutation ◽  
Asexual Sporulation ◽  
Dominant Negative Mutation ◽  
Suppressor Mutations ◽  
Early Developmental

Abstract Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.

Impairment of TGF-β signaling in T cells increases susceptibility to experimental autoimmune hepatitis in mice

2003 ◽  
Vol 284 (3) ◽  
pp. G525-G535 ◽  
Author(s):  
Christoph Schramm ◽  
Martina Protschka ◽  
Heinz H. Köhler ◽  
Jürgen Podlech ◽  
Matthias J. Reddehase ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Autoimmune Hepatitis ◽  
Mononuclear Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Histological Score ◽  
Tgf Β1 ◽  
Increased Susceptibility ◽  
Experimental Autoimmune

In autoimmune hepatitis, strong TGF-β1 expression is found in the inflamed liver. TGF-β overexpression may be part of a regulatory immune response attempting to suppress autoreactive T cells. To test this hypothesis, we determined whether impairment of TGF-β signaling in T cells leads to increased susceptibility to experimental autoimmune hepatitis (EAH). Transgenic mice of strain FVB/N were generated expressing a dominant-negative TGF-β type II receptor in T cells under the control of the human CD2 promoter/locus control region. On induction of EAH, transgenic mice showed markedly increased portal and periportal leukocytic infiltrations with hepatocellular necroses compared with wild-type mice (median histological score = 1.8 ± 0.26 vs. 0.75 ± 0.09 in wild-type mice; P < 0.01). Increased IFN-γ production (118 vs. 45 ng/ml) and less IL-4 production (341 vs. 1,256 pg/ml) by mononuclear cells isolated from transgenic livers was seen. Impairment of TGF-β signaling in T cells therefore leads to increased susceptibility to EAH in mice. This suggests an important role for TGF-β in immune homeostasis in the liver and may teleologically explain TGF-β upregulation in response to T cell-mediated liver injury.

scholarly journals Agmatine Attenuates Brain Edema through Reducing the Expression of Aquaporin-1 after Cerebral Ischemia

2009 ◽  
Vol 30 (5) ◽  
pp. 943-949 ◽  
Author(s):  
Jae Hwan Kim ◽  
Yong Woo Lee ◽  
Kyung Ah Park ◽  
Won Taek Lee ◽  
Jong Eun Lee
Keyword(s):  
Cerebral Ischemia ◽  
Water Content ◽  
Brain Edema ◽  
Arginine Decarboxylase ◽  
Artery Occlusion ◽  
Brain Swelling ◽  
Ischemic Brain ◽  
Cerebral Artery Occlusion ◽  
Swelling Volume ◽  
Brain Water

Brain edema is frequently shown after cerebral ischemia. It is an expansion of brain volume because of increasing water content in brain. It causes to increase mortality after stroke. Agmatine, formed by the decarboxylation of L-arginine by arginine decarboxylase, has been shown to be neuroprotective in trauma and ischemia models. The purpose of this study was to investigate the effect of agmatine for brain edema in ischemic brain damage and to evaluate the expression of aquaporins (AQPs). Results showed that agmatine significantly reduced brain swelling volume 22 h after 2 h middle cerebral artery occlusion in mice. Water content in brain tissue was clearly decreased 24 h after ischemic injury by agmatine treatment. Blood–brain barrier (BBB) disruption was diminished with agmatine than without. The expressions of AQPs-1 and -9 were well correlated with brain edema as water channels, were significantly decreased by agmatine treatment. It can thus be suggested that agmatine could attenuate brain edema by limitting BBB disruption and blocking the accumulation of brain water content through lessening the expression of AQP-1 after cerebral ischemia.

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

2002 ◽  
Vol 283 (2) ◽  
pp. E338-E345 ◽  
Author(s):  
Masatoshi Tsuru ◽  
Hideki Katagiri ◽  
Tomoichiro Asano ◽  
Tetsuya Yamada ◽  
Shigeo Ohno ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Dominant Negative ◽  
Transport Activity ◽  
Wild Type ◽  
Atypical Pkc ◽  
Endogenous Expression ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Ζ

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-α, novel PKC-δ, and atypical PKC isoforms of PKC-λ and PKC-ζ, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-α and PKC-λ/ζ, but not of PKC-δ, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-α and exogenous PKC-δ but not atypical PKC-λ/ζ. Insulin also activated the overexpressed PKC-δ but not PKC-α. Expression of the wild-type PKC-α or PKC-δ resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-α expression, which inhibited the PMA activation of PKC-α, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-δ but not of PKC-α. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-λ/ζ was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

scholarly journals Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain.

1992 ◽  
Vol 118 (1) ◽  
pp. 203-212 ◽  
Author(s):  
M Metsäranta ◽  
S Garofalo ◽  
G Decker ◽  
M Rintala ◽  
B de Crombrugghe ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Endochondral Ossification ◽  
Electron Microscopic Examination ◽  
Type Ii Collagen ◽  
Dominant Negative ◽  
Collagen Molecule ◽  
Type Ii ◽  
Electron Microscopic ◽  
Dominant Negative Mutation ◽  
Collagen Chain

We have generated transgenic mice by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7. This mutation was expected to disturb the assembly and processing of the homotrimeric type II collagen molecule in cartilage. Expression of transgene mRNA at levels equivalent or higher than the endogenous mRNA in the offspring of two founder animals resulted in a severe chondrodysplastic phenotype with short limbs, hypoplastic thorax, abnormal craniofacial development, and other skeletal deformities. The affected pups died at birth due to respiratory distress. Light microscopy of epiphyseal growth plates of transgenic pups demonstrated a marked reduction in cartilaginous extracellular matrix and disruption of the normal organization of the growth plate. The zone of proliferating chondrocytes was greatly reduced whereas the zone of hypertrophic chondrocytes was markedly increased extending deep into the diaphysis suggestive of a defect in endochondral ossification. Electron microscopic examination revealed chondrocytes with extended RER, a very severe reduction in the amount of cartilage collagen fibrils, and abnormalities in their structure. We postulate that the deletion in the alpha 1(II) collagen acts as a dominant negative mutation disrupting the assembly and secretion of type II collagen molecules. The consequences of the mutation include interference with normal endochondral ossification. These mice constitute a valuable model to study the mechanisms underlying human chondrodysplasias and normal bone formation.

scholarly journals EphrinB2 Activation Enhances Vascular Repair Mechanisms and Reduces Brain Swelling After Mild Cerebral Ischemia

2017 ◽  
Vol 37 (5) ◽  
pp. 867-878 ◽  
Author(s):  
Adnan Ghori ◽  
Florian B. Freimann ◽  
Melina Nieminen-Kelhä ◽  
Irina Kremenetskaia ◽  
Karen Gertz ◽  
...  
Keyword(s):  
Cerebral Ischemia ◽  
Blood Brain Barrier ◽  
Major Complication ◽  
Artery Occlusion ◽  
Brain Barrier ◽  
Tyrosine Kinase Receptor ◽  
Vascular Repair ◽  
Brain Swelling ◽  
Blood Brain ◽  
Repair Mechanisms

Objective— Cerebral edema caused by the disruption of the blood–brain barrier is a major complication after stroke. Therefore, strategies to accelerate and enhance neurovascular recovery after stroke are of prime interest. Our main aim was to study the role of ephrinB2/EphB4 signaling in mediating the vascular repair and in blood–brain barrier restoration after mild cerebral ischemia occlusion/reperfusion. Approach and Results— Here, we show that the guidance molecule ephrinB2 plays a key role in neurovascular protection and blood–brain barrier restoration after stroke. In a focal stroke model, we characterize the stroke-induced damage to cerebral blood vessels and their subsequent endogenous repair on a cellular, molecular, and functional level. EphrinB2 and its tyrosine kinase receptor EphB4 are upregulated early after stroke by endothelial cells and perivascular support cells, in parallel to their reassembly during neurovascular recovery. Using both retroviral and pharmacological approaches, we show that the inhibition of ephrinB2/EphB4 signaling suppresses post-middle cerebral artery occlusion neurovascular repair mechanisms resulting in an aggravation of brain swelling. In contrast, the activation of ephrinB2 after brain ischemia leads to an increased pericyte recruitment and increased endothelial–pericyte interaction, resulting in an accelerated neurovascular repair after ischemia. Conclusions— We show that reducing swelling could result in improved outcome because of reduction in damaged brain tissue. We also identify a novel role for ephrinB2/EphB4 signaling in the maintenance of the neurovascular homeostasis and provide a novel therapeutic approach in reducing brain swelling after stroke.

scholarly journals Isolation of Glomerular Podocytes by Cationic Colloidal Silica-coated Ferromagnetic Nanoparticles

2016 ◽  
Vol 9 (1) ◽  
pp. 67-87
Author(s):  
Andreas Blutke
Keyword(s):  
Transgenic Mice ◽  
Transgenic Animals ◽  
Dominant Negative ◽  
Ferromagnetic Nanoparticles ◽  
Wild Type ◽  
Isolated Cells ◽  
Specific Expression ◽  
Specific Antibodies ◽  
Isolated Glomeruli ◽  
Fluorescent Markers

Background: Podocyte homeostasis plays a crucial role for the maintenance of physiological glomerular function and podocyte injury is regarded as a major determinant of development and progression of renal disease. Objective: Investigation of podocytes requires appropriate methods for their isolation. Previously reported methods use podocyte specific antibodies or transgenic mice with podocyte specific expression of fluorescent markers for isolation of podocytes by magnetic or fluorescence activated cell sorting. Method: Here, a novel, antibody-free method for isolation of podocyte protein and RNA from mouse glomeruli is described. Preparations of isolated glomeruli were added to a suspension of cationic silica-coated colloidal ferromagnetic nanoparticles. The nanoparticles bound to the negatively charged cell surfaces of podocytes residing on the outer surface of the isolated glomeruli. After enzymatic and mechanical dissociation of glomerular cells, nanoparticle-coated podocytes were isolated in a magnetic field. The method was tested in adult wild-type mice without renal lesions and in mice of two nephropathy models (Growth hormone (GH)-transgenic mice and transgenic mice expressing a dominant negative receptor for the glucose dependent insulinotropic polypeptide, GIPRdn) displaying albuminuria, glomerular hypertrophy and evidence for a reduced negative cell surface charge of podocytes. Results: The isolated cells displayed typical morphological and ultrastructural properties of podocytes. On average, 182,000 ± 37,000 cells were counted in the podocyte isolates harvested from ~10,000-12,000 glomeruli per mouse. On the average, the purity of podocyte isolates of these mice accounted for ~63 ± 18 % and the podocyte isolates displayed high mRNA and protein expression abundances of podocyte markers (nephrin and WT1), whereas the expression of endothelial (Cd31) and mesangial markers (Serpinb7) was significantly decreased in podocyte isolates, as compared to samples of isolated glomeruli. The numbers of cells isolated from GH- transgenic and GIPRdn-transgenic mice were not markedly different from that of wild-type mice. Conclusion: The described method represents an alternative for podocyte isolation, particularly in experiments where podocyte specific antibodies or transgenic animals with podocyte specific expression of fluorescent markers are not applicable.

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