Identification, biochemical characterization, and in-vivo expression of the intracellular invertase BfrA from the pathogenic parasite Leishmania major

The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA–Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.

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A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

The Journal of Cell Biology ◽

10.1083/jcb.124.6.935 ◽

1994 ◽

Vol 124 (6) ◽

pp. 935-947 ◽

Cited By ~ 45

Author(s):

K Mensa-Wilmot ◽

JH LeBowitz ◽

KP Chang ◽

A al-Qahtani ◽

BS McGwire ◽

...

Keyword(s):

Phospholipase C ◽

Mammalian Cells ◽

Leishmania Major ◽

Protozoan Parasite ◽

T Lymphoma ◽

Major Surface ◽

Parasite Leishmania ◽

Negative Phenotype

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.

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The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146217 ◽

1993 ◽

Vol 51 ◽

pp. 74-75

Author(s):

Jason R. Swedlow ◽

Neil Osheroff ◽

Tim Karr ◽

John W. Sedat ◽

David A. Agard

Keyword(s):

Topoisomerase Ii ◽

Biochemical Characterization ◽

Developmental Cycle ◽

Dna Topoisomerase Ii ◽

Chromosomal Distribution ◽

Dna Topoisomerase ◽

Ideal System ◽

Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

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Faculty Opinions recommendation of Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725670185.793526669 ◽

2016 ◽

Author(s):

Martin Gruebele ◽

Max Platkov

Keyword(s):

In Vivo Expression ◽

Factor Governing

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Appropriate In Vivo Expression of a Muscle-Specific Promoter by Using Avian Retroviral Vectors for Gene Transfer

Journal of Virology ◽

10.1128/jvi.66.8.5175-5175.1992 ◽

1992 ◽

Vol 66 (8) ◽

pp. 5175-5175

Keyword(s):

Gene Transfer ◽

Retroviral Vectors ◽

In Vivo Expression

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Identification of a natural soluble neuropilin-1 that binds vascular endothelial growth factor: In vivo expression and antitumor activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.040337597 ◽

2000 ◽

Vol 97 (6) ◽

pp. 2573-2578 ◽

Cited By ~ 202

Author(s):

M. L. Gagnon ◽

D. R. Bielenberg ◽

Z.'e. Gechtman ◽

H.-Q. Miao ◽

S. Takashima ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Growth Factor ◽

Antitumor Activity ◽

Vascular Endothelial ◽

Neuropilin 1 ◽

In Vivo Expression ◽

Endothelial Growth Factor

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In Vivo Expression of Reprogramming Factor OCT4 Ameliorates Myelination Deficits and Induces Striatal Neuroprotection in Huntington’s Disease

Genes ◽

10.3390/genes12050712 ◽

2021 ◽

Vol 12 (5) ◽

pp. 712

Author(s):

Ji-Hea Yu ◽

Bae-Geun Nam ◽

Min-Gi Kim ◽

Soonil Pyo ◽

Jung-Hwa Seo ◽

...

Keyword(s):

Huntington's Disease ◽

Huntington’S Disease ◽

Cell Fate ◽

Gabaergic Neurons ◽

Oligodendrocyte Progenitor Cells ◽

Adeno Associated Virus ◽

In Vivo Expression ◽

Transcription Factor 4 ◽

Phosphate Buffered Saline

White matter atrophy has been shown to precede the massive loss of striatal GABAergic neurons in Huntington’s disease (HD). This study investigated the effects of in vivo expression of reprogramming factor octamer-binding transcription factor 4 (OCT4) on neural stem cell (NSC) niche activation in the subventricular zone (SVZ) and induction of cell fate specific to the microenvironment of HD. R6/2 mice randomly received adeno-associated virus 9 (AAV9)-OCT4, AAV9-Null, or phosphate-buffered saline into both lateral ventricles at 4 weeks of age. The AAV9-OCT4 group displayed significantly improved behavioral performance compared to the control groups. Following AAV9-OCT4 treatment, the number of newly generated NSCs and oligodendrocyte progenitor cells (OPCs) significantly increased in the SVZ, and the expression of OPC-related genes and glial cell-derived neurotrophic factor (GDNF) significantly increased. Further, amelioration of myelination deficits in the corpus callosum was observed through electron microscopy and magnetic resonance imaging, and striatal DARPP32+ GABAergic neurons significantly increased in the AAV9-OCT4 group. These results suggest that in situ expression of the reprogramming factor OCT4 in the SVZ induces OPC proliferation, thereby attenuating myelination deficits. Particularly, GDNF released by OPCs seems to induce striatal neuroprotection in HD, which explains the behavioral improvement in R6/2 mice overexpressing OCT4.

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Periplaneta americana Oligosaccharides Exert Anti-Inflammatory Activity through Immunoregulation and Modulation of Gut Microbiota in Acute Colitis Mice Model

Molecules ◽

10.3390/molecules26061718 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1718

Author(s):

Kaimin Lu ◽

Jing Zhou ◽

Jie Deng ◽

Yangjun Li ◽

Chuanfang Wu ◽

...

Keyword(s):

Side Effects ◽

Pathogenic Bacteria ◽

Periplaneta Americana ◽

Biochemical Characterization ◽

Antioxidant Activities ◽

Inflammatory Activity ◽

Mice Model ◽

Acute Colitis ◽

Anti Inflammatory

The incidence and prevalence of inflammatory bowel disorders (IBD) are increasing around the world due to bacterial infection, abnormal immune response, etc. The conventional medicines for IBD treatment possess serious side effects. Periplaneta americana (P. americana), a traditional Chinese medicine, has been used to treat arthritis, fever, aches, inflammation, and other diseases. This study aimed to evaluate the anti-inflammatory effects of oligosaccharides from P. Americana (OPA) and its possible mechanisms in vivo. OPA were purified and biochemical characterization was analyzed by HPGPC, HPLC, FT-IR, and GC–MS. Acute colitis mice model was established, the acute toxicity and anti-inflammatory activity were tested in vivo. The results showed OPA with molecular mass of 1.0 kDa were composed of 83% glucose, 6% galactose, 11% xylose, and the backbone was (1→4)-Glcp. OPA had potent antioxidant activities in vitro and significantly alleviated the clinical symptoms of colitis, relieved colon damage without toxic side effects in vivo. OPA exhibited anti-inflammatory activity by regulating Th1/Th2, reducing oxidative stress, preserving intestinal barrier integrity, and inhibiting TLR4/MAPK/NF-κB pathway. Moreover, OPA protected gut by increasing microbial diversity and beneficial bacteria, and reducing pathogenic bacteria in feces. OPA might be the candidate of complementary and alternative medicines of IBD with low-cost and high safety.

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