scholarly journals A Dictyostelium myosin II lacking a proximal 58-kDa portion of the tail is functional in vitro and in vivo.

1992 ◽  
Vol 3 (12) ◽  
pp. 1455-1462 ◽  
Author(s):  
E W Kubalek ◽  
T Q Uyeda ◽  
J A Spudich
Keyword(s):  
Molecular Genetic ◽  
Myosin Ii ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Fruiting Bodies ◽  
Wild Type ◽  
In Vitro Motility Assay ◽  
Dictyostelium Myosin

We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 microns/s at 30 degrees C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.

scholarly journals Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
Author(s):  
Gary M. Leong ◽  
Sofia Moverare ◽  
Jesena Brce ◽  
Nathan Doyle ◽  
Klara Sjögren ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Hepatoma Cells ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Wild Type ◽  
Hepatic Expression ◽  
Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

scholarly journals Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

2021 ◽  
Author(s):  
Fatima Amanat ◽  
Shirin Strohmeier ◽  
Wen-Hsin Lee ◽  
Sandhya Bangaru ◽  
Andrew B Ward ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Severe Acute Respiratory Syndrome ◽  
Mouse Model ◽  
Neutralizing Antibodies ◽  
Dependent Manner ◽  
Receptor Binding Domain ◽  
Wild Type ◽  
Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Bin Ren ◽  
Arpita Mukhopadhyay* ◽  
Anthony A Lanahan ◽  
Zhen W Zhuang ◽  
Karen L Moodie ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Branching Morphogenesis ◽  
Erk Signaling ◽  
Dependent Manner ◽  
Wild Type ◽  
Erk Activation ◽  
Arterial Development ◽  
Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

Protective Effects on Mitochondria and Anti-Aging Activity of Polysaccharides from Cultivated Fruiting Bodies of Cordyceps militaris

2010 ◽  
Vol 38 (06) ◽  
pp. 1093-1106 ◽  
Author(s):  
Xing-Tai Li ◽  
Hong-Cheng Li ◽  
Chun-Bin Li ◽  
De-Qiang Dou ◽  
Ming-Bo Gao
Keyword(s):  
Hydroxyl Radicals ◽  
Cordyceps Militaris ◽  
Mitochondrial Swelling ◽  
Dependent Manner ◽  
Protective Effects ◽  
Fruiting Bodies ◽  
Concentration Dependent Manner ◽  
Mitochondrial Injury

Cordyceps militaris (L.) Link is an entomopathogenic fungus parasitic to Lepidoptera larvae, and is widely used as a folk tonic or invigorant for longevity in China. Although C. militaris has been used in traditional Chinese medicine for millennia, there is still a lack convincing evidence for its anti-aging activities. This study was performed to investigate the effects of polysaccharides from cultivated fruiting bodies of C. militaris (CMP) on mitochondrial injury, antioxidation and anti-aging activity. Fruiting bodies of C. militaris were cultivated artificially under optimized conditions. The spectrophotometric method was used to measure thiobarbituric acid reactive substances (TBARS), mitochondrial swelling, and activities of scavenging superoxide anions in vitro. D-galactose (100 mg/kg/day) was injected subcutaneously into back of the neck of mice for 7 weeks to induce an aging model. The effects of CMP on the activities of catalase (CAT), surperoxide dismutase (SOD), glutathione peroxidase (GPx) and anti-hydroxyl radicals were assayed in vivo using commercial monitoring kits. The results showed that CMP could inhibit mitochondrial injury and swelling induced by Fe2+ -L-Cysteine in a concentration- dependent manner and it also had a significant superoxide anion scavenging effect. Moreover, the activities of CAT, SOD, GPx and anti-hydroxyl radicals in mice liver were increased significantly by CMP. These results indicate that CMP protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting mitochondrial swelling, and increasing the activities of antioxidases. Therefore, CMP may have pharmaceutical values for mitochondrial protection and anti-aging. CMP was the major bioactive component in C. militaris.

scholarly journals Substitutions in the Periplasmic Domain of Low-Abundance Chemoreceptor Trg That Induce or Reduce Transmembrane Signaling: Kinase Activation and Context Effects

2001 ◽  
Vol 183 (2) ◽  
pp. 671-679 ◽  
Author(s):  
Bryan D. Beel ◽  
Gerald L. Hazelbauer
Keyword(s):  
Context Effects ◽  
High Abundance ◽  
In Vitro Assays ◽  
Wild Type ◽  
Kinase Activation ◽  
Interaction Sites ◽  
Natural Enzyme ◽  
Signaling Receptors

ABSTRACT We extended characterization of mutational substitutions in the ligand-binding region of Trg, a low-abundance chemoreceptor ofEscherichia coli. Previous investigations using patterns of adaptational methylation in vivo led to the suggestion that one class of substitutions made the receptor insensitive, reducing ligand-induced signaling, and another mimicked ligand occupancy, inducing signaling in the absence of ligand. We tested these deductions with in vitro assays of kinase activation and found that insensitive receptors activated the kinase as effectively as wild-type receptors and that induced-signaling receptors exhibited the low level of kinase activation characteristic of occupied receptors. Differential activation by the two mutant classes was not dependent on high-abundance receptors. Cellular context can affect the function of low-abundance receptors. Assays of chemotactic response and adaptational modification in vivo showed that increasing cellular dosage of mutant forms of Trg to a high-abundance level did not significantly alter phenotypes, nor did the presence of high-abundance receptors significantly correct phenotypic defects of reduced-signaling receptors. In contrast, defects of induced-signaling receptors were suppressed by the presence of high-abundance receptors. Grafting the interaction site for the adaptational-modification enzymes to the carboxyl terminus of induced-signaling receptors resulted in a similar suppression of phenotypic defects of induced-signaling receptors, implying that high-abundance receptors could suppress defects in induced-signaling receptors by providing their natural enzyme interaction sites intrans in clusters of suppressing and suppressed receptors. As in the case of cluster-related functional assistance provided by high-abundance receptors for wild-type low-abundance receptors, suppression by high-abundance receptors of phenotypic defects in induced-signaling forms of Trg involved assistance in adaptation, not signaling.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Inhibition of Cellular Proteasome Activities Enhances Hepadnavirus Replication in an HBX-Dependent Manner

2004 ◽  
Vol 78 (9) ◽  
pp. 4566-4572 ◽  
Author(s):  
Zhensheng Zhang ◽  
Ulrike Protzer ◽  
Zongyi Hu ◽  
James Jacob ◽  
T. Jake Liang
Keyword(s):  
Recombinant Adenovirus ◽  
Hepatitis Virus ◽  
Proteasome Inhibitors ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Dependent Manner ◽  
Type Virus ◽  
Wild Type

ABSTRACT The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.

scholarly journals Cnu, a Novel oriC-Binding Protein of Escherichia coli

2005 ◽  
Vol 187 (20) ◽  
pp. 6998-7008 ◽  
Author(s):  
Myung Suk Kim ◽  
Sung-Hun Bae ◽  
Sang Hoon Yun ◽  
Hee Jung Lee ◽  
Sang Chun Ji ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Replication Initiation ◽  
In Vitro Assays ◽  
Wild Type ◽  
Genetic Method ◽  
Dna Replication Initiation ◽  
Pair Sequence

ABSTRACT We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

scholarly journals De novo disease-associated mutations in KIF1A dominant negatively inhibit axonal transport of synaptic vesicle precursors

2021 ◽  
Author(s):  
Yuzu Anazawa ◽  
Tomoki Kita ◽  
Kumiko Hayashi ◽  
Shinsuke Niwa
Keyword(s):  
Synaptic Vesicle ◽  
Axonal Transport ◽  
De Novo ◽  
Molecular Motor ◽  
Model Systems ◽  
In Vitro Assays ◽  
In Vivo Model ◽  
Wild Type

KIF1A is a kinesin superfamily molecular motor that transports synaptic vesicle precursors in axons. Mutations in Kif1a lead to a group of neuronal diseases called KIF1A-associated neuronal disorder (KAND). KIF1A forms a homodimer and KAND mutations are mostly de novo and autosomal dominant; however, it is not known whether the function of wild-type KIF1A is inhibited by disease-associated KIF1A. No reliable in vivo model systems to analyze the molecular and cellular biology of KAND have been developed; therefore, here, we established Caenorhabditis elegans models for KAND using CRISPR/cas9 technology and analyzed defects in axonal transport. In the C. elegans models, heterozygotes and homozygotes exhibited reduced axonal transport phenotypes. In addition, we developed in vitro assays to analyze the motility of single heterodimers composed of wild-type KIF1A and disease-associated KIF1A. Disease-associated KIF1A significantly inhibited the motility of wild-type KIF1A when heterodimers were formed. These data indicate the molecular mechanism underlying the dominant nature of de novo KAND mutations.

scholarly journals A stealth adhesion factor contributes toVibrio vulnificuspathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation

2019 ◽  
Author(s):  
Tra–My Duong–Nu ◽  
Kwangjoon Jeong ◽  
Soo Young Kim ◽  
Wenzhi Tan ◽  
Sao Puth ◽  
...  
Keyword(s):  
Host Cell ◽  
Complement Activation ◽  
Vibrio Vulnificus ◽  
Alternative Pathway ◽  
Molecular Genetic ◽  
Wild Type ◽  
Triple Mutant ◽  
Mutant Cells

AbstractThe tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis.Vibrio vulnificus, an opportunistic pathogen, harbors three distincttadloci. Among them, onlytad1locus was highly upregulated inin vivogrowing bacteria compared toin vitroculture condition. To understand the pathogenic roles of the threetadloci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only theΔtad123triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on theΔtad123mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. TheΔtad123mutant cells showed attenuated host cell adhesion, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. TheΔtad123mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Taken together, all threetadloci cooperate to confer successful invasion ofV. vulnificusinto deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.Author SummaryTo understand the roles of the three Tad operons in the pathogenesis ofV. vulnificusinfection, we constructed mutant strain with single, double and triple Tad loci deletions. Employing a variety of mouse infection models coupled with molecular genetic analyses, we demonstrate here that all three Tad operons are required forV. vulnificuspathogenicity as the cryptic pili contribute to host cell and tissue invasion, survival in the blood, and resistance to complement activation.

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