scholarly journals Probing tissue transglutaminase mediated vascular smooth muscle cell aging using a novel transamidation-deficient Tgm2-C277S mouse model

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Huilei Wang ◽  
James Chen ◽  
Sandeep Jandu ◽  
Sean Melucci ◽  
William Savage ◽  
...  
Keyword(s):  
Tissue Transglutaminase ◽  
Significant Loss ◽  
In Vivo Studies ◽  
Cell Aging ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Independent Functions ◽  
Vascular Stiffening

AbstractTissue transglutaminase (TG2), a multifunctional protein of the transglutaminase family, has putative transamidation-independent functions in aging-associated vascular stiffening and dysfunction. Developing preclinical models will be critical to fully understand the physiologic relevance of TG2’s transamidation-independent activity and to identify the specific function of TG2 for therapeutic targeting. Therefore, in this study, we harnessed CRISPR-Cas9 gene editing technology to introduce a mutation at cysteine 277 in the active site of the mouse Tgm2 gene. Heterozygous and homozygous Tgm2-C277S mice were phenotypically normal and were born at the expected Mendelian frequency. TG2 protein was ubiquitously expressed in the Tgm2-C277S mice at levels similar to those of wild-type (WT) mice. In the Tgm2-C277S mice, TG2 transglutaminase function was successfully obliterated, but the transamidation-independent functions ascribed to GTP, fibronectin, and integrin binding were preserved. In vitro, a remodeling stimulus led to the significant loss of vascular compliance in WT mice, but not in the Tgm2-C277S or TG2−/− mice. Vascular stiffness increased with age in WT mice, as measured by pulse-wave velocity and tensile testing. Tgm2-C277S mice were protected from age-associated vascular stiffening, and TG2 knockout yielded further protection. Together, these studies show that TG2 contributes significantly to overall vascular modulus and vasoreactivity independent of its transamidation function, but that transamidation activity is a significant cause of vascular matrix stiffening during aging. Finally, the Tgm2-C277S mice can be used for in vivo studies to explore the transamidation-independent roles of TG2 in physiology and pathophysiology.

Factor VIIa induces extracellular vesicles from the endothelium: A potential mechanism for its hemostatic effect

Blood ◽  
2021 ◽  
Author(s):  
Kaushik Das ◽  
Shiva Keshava ◽  
Shabbir A Ansari ◽  
Vijay Kumar Reddy Kondreddy ◽  
Charles Esmon ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Extracellular Vesicles ◽  
Factor Viia ◽  
Mutant Mice ◽  
In Vivo Studies ◽  
Cell Protein ◽  
Wild Type ◽  
Hemostatic Effect

Recombinant FVIIa (rFVIIa) is used as a hemostatic agent to treat bleeding disorders in hemophilia patients with inhibitors and other groups of patients. Our recent studies showed that FVIIa binds endothelial cell protein C receptor (EPCR) and induces protease-activated receptor 1 (PAR1)-mediated biased signaling. The importance of FVIIa-EPCR-PAR1-mediated signaling in hemostasis is unknown. In the present study, we show that FVIIa induces the release of extracellular vesicles (EVs) from endothelial cells both in vitro and in vivo. Silencing of EPCR or PAR1 in endothelial cells blocked the FVIIa-induced generation of EVs. Consistent with these data, FVIIa treatment enhanced the release of EVs from murine brain endothelial cells isolated from wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice. In vivo studies revealed that administration of FVIIa to wild-type, EPCR overexpressors, and PAR1-R46Q mutant mice, but not EPCR-deficient or PAR1-R41Q mutant mice, increase the number of circulating EVs. EVs released in response to FVIIa treatment exhibit enhanced procoagulant activity. Infusion of FVIIa-generated EVs and not control EVs to platelet-depleted mice increased thrombin generation at the site of injury and reduced blood loss. Administration of FVIIa-generated EVs or generation of EVs endogenously by administering FVIIa augmented the hemostatic effect of FVIIa. Overall, our data reveal that FVIIa treatment, through FVIIa-EPCR-PAR1 signaling, releases EVs from the endothelium into the circulation, and these EVs contribute to the hemostatic effect of FVIIa.

scholarly journals Structural Requirements of Tom40 for Assembly into Preexisting TOM Complexes of Mitochondria

2001 ◽  
Vol 12 (5) ◽  
pp. 1189-1198 ◽  
Author(s):  
Doron Rapaport ◽  
Rebecca D. Taylor ◽  
Michael Käser ◽  
Thomas Langer ◽  
Walter Neupert ◽  
...  
Keyword(s):  
Intermediate Stage ◽  
In Vivo Studies ◽  
Mitochondrial Outer Membrane ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Blue Native

Tom40 is the major subunit of the translocase of the outer mitochondrial membrane (the TOM complex). To study the assembly pathway of Tom40, we have followed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 protein. Upon import into isolated mitochondria, Tom40 precursor proteins lacking the first 20 or the first 40 amino acid residues were assembled as the wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested at an intermediate stage of assembly. We constructed mutant versions of Tom40 affecting this region and transformed the genes into a sheltered heterokaryon containing a tom40 null nucleus. Homokaryotic strains expressing the mutant Tom40 proteins had growth rate defects and were deficient in their ability to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobility and a tendency to lose Tom40 subunits from the complex. Thus, both in vitro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Finally, we found that TOM complexes in the mitochondrial outer membrane were capable of exchanging subunits in vitro. A model is proposed for the integration of Tom40 subunits into the TOM complex.

Niacin stimulates adiponectin secretion through the GPR109A receptor

2009 ◽  
Vol 296 (3) ◽  
pp. E549-E558 ◽  
Author(s):  
Eric P. Plaisance ◽  
Martina Lukasova ◽  
Stefan Offermanns ◽  
Youyan Zhang ◽  
Guoqing Cao ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Surface Expression ◽  
In Vivo Studies ◽  
Serum Adiponectin ◽  
Cell Surface Expression ◽  
Wild Type ◽  
The Metabolic Syndrome ◽  
In The Wild

Niacin (nicotinic acid) has recently been shown to increase serum adiponectin concentrations in men with the metabolic syndrome. However, little is known about the mechanism(s) by which niacin regulates the intracellular trafficking and secretion of adiponectin. Since niacin appears to exert its effects on lipolysis through receptor (GPR109A)-dependent and -independent pathways, the purpose of this investigation was to examine the role of the recently identified GPR109A receptor in adiponectin secretion. Initial in vivo studies in rats demonstrated that niacin (30 mg/kg po) acutely increases serum adiponectin concentrations, whereas it decreases NEFAs. Further in vitro studies demonstrated an increase in adiponectin secretion and a decrease in lipolysis in primary adipocytes following treatment with niacin or β-hydroxybutyrate (an endogenous ligand of the GPR109A receptor), but these effects were blocked when adipocytes were pretreated with pertussis toxin. Niacin had no effect on adiponectin secretion or lipolysis in 3T3-L1 adipocytes, which have limited cell surface expression of the GPR109A receptor. To further substantiate these in vitro findings, wild-type and GPR109A receptor knockout mice were administered a single dose of niacin or placebo, and serum was obtained for the determination of adiponectin and NEFA concentrations. Serum adiponectin concentrations increased and serum NEFAs decreased in the wild-type mice within 10 min following niacin administration. However, niacin administration had no effect on adiponectin and NEFA concentrations in the GPR109A receptor knockout mice. These results demonstrate that the GPR109A receptor plays an important role in the dual regulation of adiponectin secretion and lipolysis.

scholarly journals Involvement of the RNA Polymerase α-Subunit C-Terminal Domain in LuxR-Dependent Activation of the Vibrio fischeri Luminescence Genes

1999 ◽  
Vol 181 (15) ◽  
pp. 4704-4707 ◽  
Author(s):  
Ann M. Stevens ◽  
Nobuyuki Fujita ◽  
Akira Ishihama ◽  
E. P. Greenberg
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Vibrio Fischeri ◽  
In Vivo Studies ◽  
Wild Type ◽  
Α Subunit ◽  
Subunit C ◽  
Terminal Domain

ABSTRACT LuxR is a ς70 RNA polymerase (RNAP)-dependent transcriptional activator that controls expression of the Vibrio fischeri lux operon in response to an acylhomoserine lactone-cell density signal. We have investigated whether the α-subunit C-terminal domain (αCTD) of RNAP is required for LuxR activity. A purified signal-independent, LuxR C-terminal domain-containing polypeptide (LuxRΔN) was used to study the activation of transcription from theluxI promoter in vitro. Initiation of luxoperon transcription was observed in the presence of LuxRΔN and wild-type RNAP but not in the presence of LuxRΔN and RNAPs with truncated αCTDs. We also studied the in vivo role of the RNAP αCTD in activation of lux transcription in Escherichia coli. This enabled a comparison of results obtained with full-length LuxR to those obtained with LuxRΔN. These in vivo studies indicated that both LuxR and LuxRΔN require the RNAP αCTD for activity. The results of DNase I protection studies showed that LuxRΔN-RNAP complexes can bind and protect the luxIpromoter, but with less efficacy when the αCTD is truncated in comparison to the wild type. Thus, both in vitro and in vivo experiments demonstrated that LuxR-dependent transcriptional activation of the lux operon involves the RNAP αCTD and suggest that αCTD-LuxR interactions may play a role in recruitment of RNAP to theluxI promoter.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

scholarly journals The SH3 Domain Contributes to BCR/ABL-Dependent Leukemogenesis In Vivo: Role in Adhesion, Invasion, and Homing

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 406-418 ◽  
Author(s):  
Tomasz Skorski ◽  
Malgorzata Nieborowska-Skorska ◽  
Pawel Wlodarski ◽  
Mariusz Wasik ◽  
Rossana Trotta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Sh3 Domain ◽  
In Vivo Studies ◽  
Collagen Type Iv ◽  
Wild Type ◽  
Type Iv

To determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (▵SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. ▵SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of ▵SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing ▵SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of ▵SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of α2 integrin by ▵SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/ membrane fraction, ▵SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.

scholarly journals Evaluation of 2′-Deoxy-2′-Flouro-5-Methyl-1-β-d-Arabinofuranosyluracil as a Potential Gene Imaging Agent for HSV-tk Expression In Vivo

2002 ◽  
Vol 1 (2) ◽  
pp. 153535002002021
Author(s):  
Mian M. Alauddin ◽  
Atranik Shahinian ◽  
Erlinda M. Gordon ◽  
Peter S. Conti
Keyword(s):  
Emission Tomography ◽  
In Vivo Studies ◽  
In Vitro Experiments ◽  
Tumor Uptake ◽  
Wild Type ◽  
Positron Emission ◽  
Additional Tumor ◽  
Ht 29

2′-Deoxy-2′-flouro-5-methyl-1-β-d-arabinofuranosyluracil (FMAU) has been evaluated in HT-29 cells as a potential positron emission tomography (PET) radiotracer for imaging HSV-tk gene expression in vivo. In vitro experiments demonstrate that the accumulation of [14C]-FMAU in HSV-tk-expressing cells is 2.4-fold ( p < .02), 4.0-fold ( p < .001), and 5.3-fold ( p < .001) higher than the wild-type cells at 1, 3, and 5 hr, respectively. In vivo studies revealed that the tumor uptake in HSV-tk-expressing cells was 2.3-fold ( p < .001), 3.0-fold ( p < .001), and 5.5-fold ( p < .001) higher than the control cells at 1, 2, and 5 hr, respectively. FMAU was found to be more sensitive compared to our earlier studies using 9-[(3-18F-fluoro-1-hydroxy-2-propoxy)methyl]-guanine ([18F]-FHPG) and 9-(4-[18F]-fluoro-3-hydroxy-methylbutyl)guanine ([18F]-FHBG) in the same cell lines, although, the specificity was less than FHBG. These results suggest that while FMAU labeled with PET isotopes may be useful for imaging HSV-tk-expressing tumors in vivo, multitracer studies across additional tumor models are necessary in order to identify an optimal PET radiotracer.

In vivo studies of the γ subunit of retinal cGMP-phophodiesterase with a substitution of tyrosine-84

2001 ◽  
Vol 353 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Stephen H. TSANG ◽  
Clyde K. YAMASHITA ◽  
Kentaro DOI ◽  
Daniel J. SALCHOW ◽  
Nicole BOUVIER ◽  
...  
Keyword(s):  
Regulatory Control ◽  
In Vivo Studies ◽  
Wild Type ◽  
Targeted Disruption ◽  
Catalytic Core ◽  
Γ Subunit ◽  
Biochemical Assays ◽  
Fold Reduction

The inhibitory rod cGMP phosphodiesterase γ subunit (PDEγ) is a major component of the photoresponse and is required to support rod integrity. Pdegtm1/Pdegtm1 mice (which lack PDEγ owing to a targeted disruption of the Pdeg gene) suffer from a very rapid and severe photoreceptor degeneration. The Y84G (Tyr84 → Gly) allele of PDEγ has previously been shown in experiments carried out in vitro to reduce the regulatory control of the PDE catalytic core (PDEαβ) exerted by the wild-type γ subunit. To determine the effects of this mutation on in vivo function, the murine opsin promoter was used to direct expression to the photoreceptors of +/Pdegtm1 mice of a mutant Y84G and a wild-type PDEγ control transgene. The transgenic mice were crossed with Pdegtm1/Pdegtm1 mice to generate animals able to synthesize only the transgenic PDEγ. Our results showed that wild-type PDEγ and Y84G transgenes could complement the Pdegtm1/Pdegtm1 mutant for photoreceptor survival. The mutation caused a significant biochemical defect in PDE activation by transducin. However, the Y84G mutation did not fully eliminate the control of PDEγ on the PDE catalytic core in vivo; the expression of the mutant subunit was associated with only a 10-fold reduction in the amplitude of the a-wave and a 1.5-fold decrease in the b-wave of the corneal electroretinogram. Unexpectedly, the mutation caused a much ‘milder’ phenotype in vivo than was predicted from the biochemical assays in vitro.

scholarly journals The SH3 Domain Contributes to BCR/ABL-Dependent Leukemogenesis In Vivo: Role in Adhesion, Invasion, and Homing

Blood ◽  
1998 ◽  
Vol 91 (2) ◽  
pp. 406-418 ◽  
Author(s):  
Tomasz Skorski ◽  
Malgorzata Nieborowska-Skorska ◽  
Pawel Wlodarski ◽  
Mariusz Wasik ◽  
Rossana Trotta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Sh3 Domain ◽  
In Vivo Studies ◽  
Collagen Type Iv ◽  
Wild Type ◽  
Type Iv

Abstract To determine the possible role of the BCR/ABL oncoprotein SH3 domain in BCR/ABL-dependent leukemogenesis, we studied the biologic properties of a BCR/ABL SH3 deletion mutant (▵SH3 BCR/ABL) constitutively expressed in murine hematopoietic cells. ▵SH3 BCR/ABL was able to activate known BCR/ABL-dependent downstream effector molecules such as RAS, PI-3kinase, MAPK, JNK, MYC, JUN, STATs, and BCL-2. Moreover, expression of ▵SH3 BCR/ABL protected 32Dcl3 murine myeloid precursor cells from apoptosis, induced their growth factor-independent proliferation, and resulted in transformation of primary bone marrow cells in vitro. Unexpectedly, leukemic growth from cells expressing ▵SH3 BCR/ABL was significantly retarded in SCID mice compared with that of cells expressing the wild-type protein. In vitro and in vivo studies to determine the adhesive and invasive properties of ▵SH3 BCR/ABL-expressing cells showed their decreased interaction to collagen IV- and laminin-coated plates and their reduced capacity to invade the stroma and to seed the bone marrow and spleen. The decreased interaction with collagen type IV and laminin was consistent with a reduced expression of α2 integrin by ▵SH3 BCR/ABL-transfected 32Dcl3 cells. Moreover, as compared with wild-type BCR/ABL, which localizes primarily in the cytoskeletal/ membrane fraction, ▵SH3 BCR/ABL was more evenly distributed between the cytoskeleton/membrane and the cytosol compartments. Together, the data indicate that the SH3 domain of BCR/ABL is dispensable for in vitro transformation of hematopoietic cells but is essential for full leukemogenic potential in vivo.

ATP-type DNA ligase requires other proteins for its activity in vitro and its operon components for radiation resistance in Deinococcus radiodurans in vivo

2010 ◽  
Vol 88 (5) ◽  
pp. 783-790 ◽  
Author(s):  
Swathi Kota ◽  
Vidya A. Kamble ◽  
Yogendra S. Rajpurohit ◽  
Hari S. Misra
Keyword(s):  
Dna Repair ◽  
Radiation Resistance ◽  
Deinococcus Radiodurans ◽  
Strand Break ◽  
Significant Loss ◽  
Dna Ligase ◽  
Protein Kinase Activity ◽  
Wild Type

A multiprotein DNA processing complex isolated from Deinococcus radiodurans contains the DNA repair protein PprA, an ATP-type DNA repair ligase (LigB) encoded by the drB0100 gene, and protein kinase activity. An ATP-dependent DNA end-joining activity was detected in the complex. To elucidate the function of the drB0100 gene, we generated the deletion mutant for the DR_B0100 ORF. The mutant exhibited a nearly 2-log cycle reduction in growth rate when exposed to a 10 000 Gray dose of γ-radiation, and a significant loss in mitomycin C and methylmethane sulphonate tolerance as compared with wild type. Functional complementation of these phenotypes required the wild-type copy of drB0100 along with other genes such as drb0099 and drb0098, organized downstream in the operon. The in vitro DNA ligase activity of LigB was stimulated severalfold by PprA in the presence of the recombinant DRB0098 protein. However, this activity did not improve when PprA was substituted with purified DRB0099 protein or when DRB0098 protein was substituted with the DRB0099 protein in the presence of PprA in solution. These results suggest that PprA and DRB0098 protein are required for LigB function. Furthermore, they also suggest that the LigB operon components contribute to radiation resistance and double-strand break (DSB) repair in D. radiodurans.

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