Characterization of the arginine deiminase ofStreptococcus equisubsp.zooepidemicus

2006 ◽  
Vol 52 (9) ◽  
pp. 868-876 ◽  
Author(s):  
Kyongsu Hong
Keyword(s):  
Apoptotic Cell ◽  
Immunoblot Analysis ◽  
Arginine Deiminase ◽  
Open Reading Frames ◽  
Dependent Manner ◽  
Acid Glycoprotein ◽  
Protective Antigens ◽  
Streptococcus Equi ◽  
Arca Gene

Streptococcus equi subsp. zooepidemicus is an important cause of infectious diseases in horses and rarely humans. Little is known about the virulence factors or protective antigens of S. equi subsp. zooepidemicus. In the present study, I designed original primers based on an alignment of the gene sagp(arcA) from Streptococcus pyogenes encoding streptococcal acid glycoprotein – arginine deiminase (SAGP/AD) to amplify the S. equi subsp. zooepidemicus counterpart sequence by polymerase chain reaction, and I analyzed the sagp(arcA) gene of the organism. Using chromosomal walking steps, I identified a contiguous eight-gene locus involved in SAGP/AD production. Their open reading frames were found to share significant homologies and to correspond closely in molecular mass to previously sequenced arc genes of S. pyogenes, thus they were designated ahrC.2 (arginine repressor), arcR (CRP/FNR transcription regulator), sagp(arcA) (streptococcal acid glycoprotein – arginine deiminase), putative acetyltransferase gene, arcB (ornithine carbamyl transferase), arcD (arginine–ornithine antiporter), arcT (Xaa-His peptidase), and arcC (carbamate kinase). The SAGP homologue of S. equi subsp. zooepidemicus (SzSAGP), encoded by arcA gene of the bacteria (arcA(SZ)), was successfully expressed in Escherichia coli and purified to homogeneity. When in vitro growth inhibitory activity of the recombinant SzSAGP was tested against MOLT-3 cells, it inhibited the growth of the cells during the 3 days of culture in a dose-dependent manner, accompanied by the induction of apoptotic cell death. The recombinant protein also possessed AD activity. By immunoblot analysis using both anti-SzSAGP-SfbI(H8) and anti-SfbI(H8) sera, I was able to demonstrate that the SzSAGP protein is expressed on the streptococcal surface.Key words: SAGP, arginine deiminase, Streptococcus equi subsp. zooepidemicus.

scholarly journals Binding of Dipyridamole of Human Platelets and to α-Acid Glycoprotein and its Significance for the Inhibition of Adenosine Uptake

1977 ◽  
Author(s):  
K. Subbarao ◽  
B. Rucinski ◽  
A. Summers ◽  
S. Niewiarowski
Keyword(s):  
Binding Sites ◽  
Ex Vivo ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Acid Glycoprotein ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport

The interactions of dipyridamole with α1-acid glycoprotein of plasma and with human platelets are related to inhibition of adenosine uptake by platelets. One mole of dipyridamole binds to one mole of α1-acid glycoprotein with a dissociation constant (Kd) of 1.3 μM. It was found that platelets contain both high and low affinity binding sites for the drug. The binding of dipyridamole to the high affinity sites follows a Michaelis Menten binding pattern with a Kd of 0.04 μM. Approximately 2x104 dipyridamole molecules are bound at the high affinity sites of each platelet. The lower affinity sites bind the drug with a Kd of 4 μM. In the presence of α1acid glycoprotein the binding of dipyridamole to platelets is inhibited. Correspondingly, the dipyridamole inhibition of adenosine uptake by platelets is reduced 1000-fold by α1acid glycoprotein. Binding of dipyridamole to human platelets is essential for its inhibition of adenosine uptake by platelets. Dipyridamole reduced the [14C]-ATP to [14C]-ADP ratio in the platelets. Purified α1acid glycoprotein reversed these effects of dipyridamole on adenosine metabolism of platelets in a concentration dependent manner. A correlationwas observed between the level of circulating dipyridamole in plasma and the inhibition of [14C]-adenosine uptake by platelets of PRP samples of 12 human volunteers given different amounts of dipyridamole. The in vitro and ex vivo effects of dipyridamole on the [14C]-adenosine uptake by platelets were found to be identical. Our data suggest the presence of dipyridamole binding sites in platelets that regulate adenosine transport across the cell surface.

scholarly journals The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development.

1994 ◽  
Vol 14 (8) ◽  
pp. 5360-5370 ◽  
Author(s):  
M E Kraus ◽  
J T Lis
Keyword(s):  
Alternative Splice ◽  
Splice Site ◽  
Developmental Stages ◽  
Immunoblot Analysis ◽  
Splicing Factor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Alternative Splice Site

B52 is a Drosophila melanogaster protein that plays a role in general and alternative splicing in vitro. It is homologous to the human splicing factor ASF/SF2 which is essential for an early step(s) in spliceosome assembly in vitro and also regulates 5' and 3' alternative splice site choice in a concentration-dependent manner. In vitro, B52 can function as both a general splicing factor and a regulator of 5' alternative splice site choice. Its activity in vivo, however, is largely uncharacterized. In this study, we have further characterized B52 in vivo. Using Western blot (immunoblot) analysis and whole-mount immunofluorescence, we demonstrate that B52 is widely expressed throughout development, although some developmental stages and tissues appear to have higher B52 levels than others do. In particular, B52 accumulates in ovaries, where it is packaged into the developing egg and is localized to nuclei by the late blastoderm stage of embryonic development. We also overexpressed this protein in transgenic flies in a variety of developmental and tissue-specific patterns to examine the effects of altering the concentration of this splicing factor in vivo. We show that, in many cell types, changing the concentration of B52 adversely affects the development of the organism. We discuss the significance of these observations with regard to previous in vitro results.

A prominent environmental endocrine disruptor, 4-nonylphenol, promotes endometriosis development via plasmacytoid dendritic cells

2020 ◽  
Vol 26 (8) ◽  
pp. 601-614
Author(s):  
Pooja Sharma ◽  
Hsin-Han Tseng ◽  
Jo-Yu Lynn Lee ◽  
Eing-Mei Tsai ◽  
Jau-Ling Suen
Keyword(s):  
Dendritic Cells ◽  
Endocrine Disruptor ◽  
Apoptotic Cell ◽  
Plasmacytoid Dendritic Cell ◽  
Local Treatment ◽  
Dependent Manner ◽  
Endometriotic Lesion ◽  
Lesion Development ◽  
Lesion Growth

Abstract Endometriosis is an estrogen-dependent chronic inflammatory disease and is associated etiologically with environmental endocrine disruptor (EED) exposure. 4-nonylphenol (NP), a widely found EED, has weak estrogenic activity and modulates plasmacytoid dendritic cell (pDC) function in vitro and in vivo. We aimed to elucidate the immunomodulatory effect of NP on the development of endometriosis, particularly focusing on pDCs. This study established a surgically induced endometriosis murine model (C57BL/6) under conditions of NP treatment that are relevant to the level and route of human exposure. Multi-parametric flow cytometry was used for analysis of infiltrated immune cell subsets in lesions. The results showed that NP exposure significantly promoted endometriotic lesion growth, survival and angiogenesis development of lesions as well as pDC accumulation in the lesions in mice. Adoptive transfer of NP-conditioned pDCs into mice significantly enhanced lesion development and local pDC infiltration, whereas NP-conditioned conventional dendritic cells did not affect lesion growth. In vitro functional analysis showed that NP-conditioned pDCs in lesions expressed high levels of CD36, a scavenger receptor and NP-conditioned splenic pDCs secreted an enhanced level of IL-10 in response to apoptotic cell recognition in a CD36-dependent manner. Furthermore, we observed that local treatment with blocking antibodies against IL-10 and CD36 on the day of surgery significantly inhibited lesion development. NP exposure also altered the estrous cycle in mice. The results suggest that chronic and low-dose exposure to NP enhances endometriotic lesion growth by altering pDC homeostasis and function. This study has important implications for understanding the environment-innate immunity interaction in human endometriosis.

scholarly journals Role of the BBA64 Locus of Borrelia burgdorferi in Early Stages of Infectivity in a Murine Model of Lyme Disease

2007 ◽  
Vol 76 (1) ◽  
pp. 391-402 ◽  
Author(s):  
Mahulena Maruskova ◽  
M. Dolores Esteve-Gassent ◽  
Valerie L. Sexton ◽  
J. Seshu
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Immunoblot Analysis ◽  
Open Reading Frames ◽  
Mammalian Host ◽  
Environmental Signals ◽  
Significant Difference ◽  
Linear Plasmid 54

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.

scholarly journals Surfactant-associated protein A is important for maintaining surfactant large-aggregate forms during surface-area cycling

1996 ◽  
Vol 313 (3) ◽  
pp. 835-840 ◽  
Author(s):  
Ruud A. W. VELDHUIZEN ◽  
Li-Juan YAO ◽  
Stephen A. HEARN ◽  
Fred POSSMAYER ◽  
James F. LEWIS
Keyword(s):  
Surface Area ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Dependent Manner ◽  
Large Aggregate ◽  
Surfactant Aggregates ◽  
Alveolar Surfactant ◽  
Surface Active

Alveolar surfactant can be separated into two major subfractions, the large surfactant aggregates (LAs) and the small surfactant aggregates (SAs). The surface-active LAs are the metabolic precursors of the inactive SAs. This conversion of LAs into SAs can be studied in vitro using a technique called surface-area cycling. We have utilized this technique to examine the effect of trypsin on aggregate conversion. Our results show that trypsin increases the conversion of LAs into SAs in a concentration- and time-dependent manner. Immunoblot analysis revealed that surfactant-associated Protein A (SP-A) was the main target of trypsin. To examine further the role of SP-A in aggregate conversion, we tested the effect of Ca2+ and mannan on this process. The absence of Ca2+ (1 mM EDTA) and the presence of mannan both increased the formation of SAs. Electron microscopy revealed that highly organized multilamellar and tubular myelin structures were present in samples that converted slowly to SAs. We concluded that SP-A is important for maintaining LA forms during surface-area cycling by stabilizing tubular myelin and multilamellar structures.

scholarly journals JAK Inhibitors Suppress Colon Cancer Cachexia-Associated Anorexia and Adipose Wasting in Mice

2020 ◽  
Author(s):  
Gurpreet Arora ◽  
Arun Gupta ◽  
Tong Guo ◽  
Aakash Gandhi ◽  
Aaron Laine ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Colon Adenocarcinoma ◽  
Serum Levels ◽  
Immunoblot Analysis ◽  
Dependent Manner ◽  
Jak Inhibitors ◽  
Rt Pcr ◽  
Stat3 Phosphorylation

ABSTRACTBackgroundCachexia (CX), a syndrome of muscle atrophy, adipose loss, and anorexia, is associated with reduced survival in cancer patients. The colon adenocarcinoma C26c20 cell line secretes the cytokine leukemia inhibitor factor (LIF) which induces CX. We characterized how LIF promotes CX-associated weight loss and anorexia in mice through JAK-dependent changes in adipose and hypothalamic tissues.MethodsCX was induced in vivo with C26c20 colon adenocarcinoma cells or recombinant LIF administration in the absence or presence of JAK inhibitors. Blood, adipose, and hypothalamic tissues were collected and processed for cyto/adipokine ELISAs, immunoblot analysis, and quantitative RT-PCR. CX was induced in vitro by stimulating differentiated adipocytes with recombinant LIF or IL-6 in the absence or presence of lipase or JAK inhibitors. These activated adipocytes were processed for lipolysis, immunoblot analysis, and RT-PCR.ResultsTumor-secreted LIF induced changes in adipose tissue expression and serum levels of IL-6 and leptin in a JAK-dependent manner influencing CX-associated adipose wasting and anorexia. We identified two JAK inhibitors that block cytokine-mediated adipocyte lipolysis and IL-6 induction using an in vitro CX lipolysis assay. JAK inhibitors administered to in vivo colon cancer CX mouse models led to 1) a decrease in STAT3 phosphorylation in hypothalamic and adipose tissues, 2) a reverse in the CX serum cyto/adipokine signature, 3) a delay in colon cancer CX-associated anorexia and adipose loss, and 4) an improvement in overall survival.ConclusionsJAK inhibitors suppress cytokine-associated adipose loss and anorexia in multiple in vitro and in vivo models of cancer CX.

scholarly journals Gonadotropin Dependent Neuregulin1 Signaling Regulates Luteal Cell Survival

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A766-A767
Author(s):  
Jennifer Jones ◽  
Saswati Banerjee ◽  
Winston E Thompson ◽  
Indrajit Chowdhury
Keyword(s):  
Signaling Pathway ◽  
First Trimester ◽  
Immunoblot Analysis ◽  
National Institutes Of Health ◽  
Luteal Cell ◽  
Dependent Manner ◽  
Experimental Conditions ◽  
Pregnant Rat ◽  
Factor Α

Abstract The formation of a functional corpus luteum (CL) is an absolute requirement for reproductive success and is induced by the mid-cycle surge of luteinizing hormone (LH). The CL is a transient ovarian endocrine structure that maintains pregnancy in primate during the first trimester and in rodents during the entire pregnancy by producing steroid hormone progesterone (P4). CL growth and differentiation are tightly regulated by both survival and cell death signals, including endocrine (LH), intra-ovarian regulators, and cell-cell interactions. Neuregulin-1 (NRG1) is a member of the epidermal growth factor-like factor family that mediates it’s effect through the erythroblastoma (ErbB) family. However, the detailed mechanisms associated with the interplay of NRG1 and its receptors in CL function is not known. Therefore, we examined the role and action of NRG1 and its receptors in the gonadotropin signaling pathway that impacts CL functions. Immunocolocalization of NRG1 and ErbB2/3 in pregnant rat CL on day 14 and 21 suggest that both NRG1 and ErbB2/3 are differentially expressed in CL. Moreover, both NRG1 and ErbB2/3 are highly expressed in rat CL on day 14 compared to day 21. Furthermore, in vitro studies revealed that rat luteal cells (LCs) treated with exogenous tumor necrosis factor-α (TNFα, an inflammatory cytokine) promoted apoptosis in LCs in a dose and time-dependent manner. However, the effects of TNFα was attenuated in presence of exogenous NRG1. Under these experimental conditions, immunoblot analysis indicated that exogenous TNFα treatment in the presence of NRG1 inhibits apoptosis through increased levels of the anti-apoptotic proteins Bcl2 and Bclxl, and activation of ErbB2-ErbB3-PI3K-Akt signaling pathway. Collectively, these studies provide new insights on the NRG1-mediated anti-apoptotic mechanism in LCs through ErbB3-ErbB2-PI3K-Akt→Bcl/Bcl-xL pathway and may have important clinical implications. Acknowledgements: This study was supported in part by National Institutes of Health Grants 1 SC1 GM130544-01A1, 1SC3GM113751 and G12RR03034. This research was conducted in a facility constructed with support from the Research Facilities Improvement Grant C06RR018386 from the National Institutes of Health National Center for Research Resources.

scholarly journals Pro-apoptotic caspase deficiency reveals a cell-extrinsic mechanism of NK cell regulation

2021 ◽  
Author(s):  
Tayla M. Olsen ◽  
Wei Hong Tan ◽  
Arne C. Knudsen ◽  
Anthony Rongvaux
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Nk Cell ◽  
Apoptotic Cell ◽  
Type I ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
A Cell

AbstractRegulated cell death is essential for the maintenance of cellular and tissue homeostasis. In the hematopoietic system, genetic defects in apoptotic cell death generally produce the accumulation of immune cells, inflammation and autoimmunity. In contrast, we found that genetic deletion of caspases of the mitochondrial apoptosis pathway reduces natural killer (NK) cell numbers and makes NK cells functionally defective in vivo and in vitro. Caspase deficiency results in constitutive activation of a type I interferon (IFN) response, due to leakage of mitochondrial DNA and activation of the cGAS/STING pathway. The NK cell defect in caspase-deficient mice is independent of the type I IFN response, but the phenotype is partially rescued by cGAS or STING deficiency. Finally, caspase deficiency alters NK cells in a cell-extrinsic manner. Type I IFNs and NK cells are two essential effectors of antiviral immunity, and our results demonstrate that they are both regulated in a caspase-dependent manner. Beyond caspase-deficient animals, our observations may have implications in infections that trigger mitochondrial stress and caspase-dependent cell death.

Effect of steroid on hyperoxia-induced ICAM-1 expression in pulmonary endothelial cells

2000 ◽  
Vol 278 (2) ◽  
pp. L245-L252 ◽  
Author(s):  
Yukio Suzuki ◽  
Kazumi Nishio ◽  
Kei Takeshita ◽  
Osamu Takeuchi ◽  
Kenji Watanabe ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Oxygen Toxicity ◽  
Immunoblot Analysis ◽  
Nuclear Factor Κb ◽  
Intercellular Adhesion Molecule ◽  
Dependent Manner ◽  
Activator Protein 1 ◽  
Intercellular Adhesion Molecule 1 ◽  
Mobility Shift

Intercellular adhesion molecule-1 (ICAM-1) of the vascular endothelium plays a key role in the development of pulmonary oxygen toxicity. We studied the effect of steroid on hyperoxia-induced ICAM-1 expression using cultured endothelial cells in vitro. Human pulmonary artery endothelial cells (HPAECs) were cultured to confluence, and then the monolayers were exposed to either control (21% O2-5% CO2) or hyperoxic (90% O2-5% CO2) conditions with and without a synthetic glucocorticoid, methylprednisolone (MP). MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression in a dose-dependent manner. Neutrophil adhesion to hyperoxia-exposed endothelial cells was also inhibited by MP treatment. In addition, MP attenuated hyperoxia-induced H2O2 production in HPAECs as assessed by flow cytometry. An electrophoretic mobility shift assay demonstrated that hyperoxia activated nuclear factor-κB (NF-κB) but not activator protein-1 (AP-1) and that MP attenuated hyperoxia-induced NF-κB activation dose dependently. With Western immunoblot analysis, IκB-α expression was decreased by hyperoxia and increased by MP treatment. These results suggest that MP downregulates hyperoxia-induced ICAM-1 expression by inhibiting NF-κB activation via increased IκB-α expression.

scholarly journals SIRT3-SOD2-ROS pathway is involved in Linalool-induced glioma cell apoptotic death

2017 ◽  
Vol 64 (2) ◽  
Author(s):  
Yanhao Cheng ◽  
Chao Dai ◽  
Jian Zhang
Keyword(s):  
Cell Death ◽  
Protein Expression ◽  
Cell Viability ◽  
Glioma Cell ◽  
Apoptotic Cell ◽  
Immunoblot Analysis ◽  
Apoptotic Cell Death ◽  
Dependent Manner ◽  
Sod Activity ◽  
Mrna And Protein Expression

Glioma is the most prevalent type of adult primary brain tumor and chemotherapy of glioma was limited by drug-resistance. Linalool is an acyclic monoterpene alcohol possessing various pharmacological activities. The present study was conducted to evaluate the effect of Linalool on glioma cell growth. The effect of Linalool on U87-MG cells was investigated and the results showed that Linalool significantly reduced cell viability in U87-MG cells in a concentration and time-dependent manner. In addition, exposure of cells to Linalool resulted in concentration-dependent increase of TUNEL-stained cells, indicating the occurrence of apoptotic cell death. Linalool decreased mitochondrial oxygen consumption rate, increased the expression of Bax and Bcl-2, reduced the expression of Bcl-2 and Bcl-xl, and increased the activities of caspase 3 and caspase 9, leading to increase of apoptosis. Linalool resulted in a concentration-dependent decrease of SOD activity but had no significant effect on the mRNA and protein expression of SOD2. Moreover, Linalool resulted in a significant increase of acetylated SOD2. The mRNA and protein expression of SIRT3 was significantly inhibited by Linalool. Immunoblot analysis showed that there were protein/protein interaction of SOD2 and SIRT3 in control U87-MG cells. Linalool treatment significantly decreased the interaction of SOD2 and SIRT3. Overexpression of SIRT3 significantly inhibited Linalool-induced increase of mitochondrial ROS level, apoptotic cell death and decrease of cell viability. In summary, we found that Linalool exhibited inhibitory effect on glioma cells through regulation of SIRT3-SOD2-ROS signaling.

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