scholarly journals Endothelial cell-specific reduction of heparan sulfate suppresses glioma growth in mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Takamasa Kinoshita ◽  
Hiroyuki Tomita ◽  
Hideshi Okada ◽  
Ayumi Niwa ◽  
Fuminori Hyodo ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Heparan Sulfate ◽  
Vascular Endothelium ◽  
Wild Type ◽  
Brain Capillaries ◽  
Gene Encoding ◽  
Glioma Growth ◽  
Underlying Mechanisms ◽  
The Brain

Abstract Purpose Heparan sulfate (HS) is one of the factors that has been suggested to be associated with angiogenesis and invasion of glioblastoma (GBM), an aggressive and fast-growing brain tumor. However, it remains unclear how HS of endothelial cells is involved in angiogenesis in glioblastoma and its prognosis. Thus, we investigated the effect of endothelial cell HS on GBM development. Methods We generated endothelial cell-specific knockout of Ext1, a gene encoding a glycosyltransferase and essential for HS synthesis, and murine GL261 glioblastoma cells were orthotopically transplanted. Two weeks after transplantation, we examined the tumor progression and underlying mechanisms. Results The endothelial cell-specific Ext1 knockout (Ext1CKO) mice exhibited reduced HS expression specifically in the vascular endothelium of the brain capillaries compared with the control wild-type (WT) mice. GBM growth was significantly suppressed in Ext1CKO mice compared with that in WT mice. After GBM transplantation, the survival rate was significantly higher in Ext1CKO mice than in WT mice. We investigated how the effect of fibroblast growth factor 2 (FGF2), which is known as an angiogenesis-promoting factor, differs between Ext1CKO and WT mice by using an in vivo Matrigel assay and demonstrated that endothelial cell-specific HS reduction attenuated the effect of FGF2 on angiogenesis. Conclusions HS reduction in the vascular endothelium of the brain suppressed GBM growth and neovascularization in mice.

scholarly journals DJ-1 regulates the integrity and function of ER-mitochondria association through interaction with IP3R3-Grp75-VDAC1

2019 ◽  
Vol 116 (50) ◽  
pp. 25322-25328 ◽  
Author(s):  
Yi Liu ◽  
Xiaopin Ma ◽  
Hisashi Fujioka ◽  
Jun Liu ◽  
Shengdi Chen ◽  
...  
Keyword(s):  
Autosomal Recessive ◽  
Cellular Mechanism ◽  
Loss Of Function ◽  
Wild Type ◽  
Calcium Efflux ◽  
And Function ◽  
The Brain ◽  
Early Onset Parkinson’S Disease

Loss-of-function mutations in DJ-1 are associated with autosomal recessive early onset Parkinson’s disease (PD), yet the underlying pathogenic mechanism remains elusive. Here we demonstrate that DJ-1 localized to the mitochondria-associated membrane (MAM) both in vitro and in vivo. In fact, DJ-1 physically interacts with and is an essential component of the IP3R3-Grp75-VDAC1 complexes at MAM. Loss of DJ-1 disrupted the IP3R3-Grp75-VDAC1 complex and led to reduced endoplasmic reticulum (ER)-mitochondria association and disturbed function of MAM and mitochondria in vitro. These deficits could be rescued by wild-type DJ-1 but not by the familial PD-associated L166P mutant which had demonstrated reduced interaction with IP3R3-Grp75. Furthermore, DJ-1 ablation disturbed calcium efflux-induced IP3R3 degradation after carbachol treatment and caused IP3R3 accumulation at the MAM in vitro. Importantly, similar deficits in IP3R3-Grp75-VDAC1 complexes and MAM were found in the brain of DJ-1 knockout mice in vivo. The DJ-1 level was reduced in the substantia nigra of sporadic PD patients, which was associated with reduced IP3R3-DJ-1 interaction and ER-mitochondria association. Together, these findings offer insights into the cellular mechanism in the involvement of DJ-1 in the regulation of the integrity and calcium cross-talk between ER and mitochondria and suggests that impaired ER-mitochondria association could contribute to the pathogenesis of PD.

scholarly journals Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

2000 ◽  
Vol 74 (7) ◽  
pp. 3353-3365 ◽  
Author(s):  
Chi-Long Lin ◽  
Che-Sheng Chung ◽  
Hans G. Heine ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

scholarly journals SUN-260 Dual Role of Carboxypeptidase E in Prohormone Processing and a Novel Neurotrophic Factor Mediating Neuroprotection and Cognitive Functions in Hippocampal CA3 Neurons in Mice

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Lan Xiao ◽  
Vinay Sharma ◽  
Leila Toulabi ◽  
Xuyu Yang ◽  
Cheol Lee ◽  
...  
Keyword(s):  
Neurotrophic Factor ◽  
Neuronal Degeneration ◽  
Tyrosine Kinase Receptor ◽  
Wild Type ◽  
Prohormone Processing ◽  
Hippocampal Ca3 ◽  
Ca3 Neurons ◽  
The Brain

Abstract Stress causes release of glucocorticoids from the adrenals which then circulate to the brain. High concentrations glucocorticoid from chronic severe stress results in pathophysiology in the brain, including neuronal degeneration, cell death and cognitive dysfunction, leading to diseases such as Alzheimer Disease and Major Depressive Disorders. Neurotrophic/growth factors such as BDNF, NGF and NT3 have been linked to these pathological conditions. Carboxypeptidase E (CPE), a proneuropeptide/prohormone processing enzyme, also named neurotrophic factor-α1(NFα1) is highly expressed in the stress-vulnerable hippocampal CA3 neurons, and was shown to have neuroprotective activity from in vitro studies. Here we investigated if CPE-NFα1 functions in vivo, independent of its enzymatic activity, and the mechanism underlying its action. We generated knock-in mice expressing a non-enzymatic form of CPE, CPE-E342Q, but not wild-type CPE. The CPE-E342Q mice showed significantly decreased neuropeptide content and exhibited obesity, diabetes and infertility due to lack of prohormone processing activity, similar to CPE-KO mice. However, they showed no hippocampal CA3 degeneration, exhibited neurogenesis in the dentate gyrus, and displayed normal spatial learning and memory, similar to CPE wild-type mice, after weaning stress; unlike CPE-KO mice which showed hippocampal CA3 neuronal degeneration and cognitive deficits. Binding studies showed that radiolabeled CPE bound hippocampal cell membrane specifically, in a saturable manner. Binding of CPE and CPE-E342Q to hippocampal neurons activated Erk signaling and pre-treatment with either of these proteins protected neurons against H2O2- or glutamate-induced neurotoxcity by increasing BCL2 expression. In vitro and in vivo inhibitor studies demonstrated that this neuroprotective effect was independent of tyrosine kinase receptor signaling. Taken together, the data provide evidence that CPE-NFα1 is a unique neurotrophic factor which acts through a non-tyrosine kinase receptor to activate Erk-BCL2 signaling to protect hippocampal CA3 neurons against stress-induced neurodegeneration and maintaining normal cognitive functions in mice.

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

Endothelial cell-based cytokine gene delivery inhibits 9L glioma growth in vivo

1996 ◽  
Vol 731 (1-2) ◽  
pp. 161-170 ◽  
Author(s):  
Myeong Nam ◽  
Peter Johnston ◽  
Bachchu Lal ◽  
Ravinder Indurti ◽  
Mary Ann Wilson ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Gene Delivery ◽  
Cytokine Gene ◽  
Glioma Growth ◽  
9L Glioma

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance

2016 ◽  
Vol 116 (12) ◽  
pp. 1022-1031 ◽  
Author(s):  
Yuki Takagi ◽  
Moe Murata ◽  
Toshihiro Kozuka ◽  
Yukiko Nakata ◽  
Ryo Hasebe ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Residual Activity ◽  
Base Substitution ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Missense Mutations ◽  
Wild Type ◽  
Gene Encoding ◽  
Single Base

SummaryAntithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values of all variants were maintained at above 80%. The thrombin–AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM- mediated anticoagulation.

Identification of an octamer element required for in vivo expression of the TIE1 gene in endothelial cells

2001 ◽  
Vol 360 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Stephane C. BOUTET ◽  
Thomas QUERTERMOUS ◽  
Bahaa M. FADEL
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Molecular Mechanisms ◽  
Vascular Development ◽  
Common Mechanism ◽  
Tyrosine Kinase Receptor ◽  
Wild Type ◽  
Binding Studies ◽  
In Vivo Expression

TIE1, an endothelial-cell-specific tyrosine kinase receptor, is required for the survival and growth of microvascular endothelial cells during the capillary sprouting phase of vascular development. To investigate the molecular mechanisms that regulate the expression of TIE1 in the endothelium, we analysed transgenic mouse embryos carrying wild-type or mutant TIE1 promoter/LacZ constructs. Our data indicate that an upstream DNA octamer element (5′-ATGCAAAT-3′) is required for the in vivo expression of TIE1 in embryonic endothelial cells. Transgenic embryos carrying the wild-type TIE1 promoter (−466 to +78bp) fused to LacZ and spanning the octamer element demonstrate endothelial-cell-specific expression of the reporter transgene. Point mutations introduced within the octamer element result in a significant decrease of endothelial LacZ expression, suggesting that the octamer site functions as a positive regulator for TIE1 gene expression in endothelial cells. DNA–protein binding studies show that the octamer element exhibits an endothelial-cell-specific pattern of binding via interaction with endothelial-cell-restricted factor(s). Our findings suggest an important role for the octamer element in regulating the expression of the TIE1 receptor in the embryonic endothelium and suggest a common mechanism for the regulation of the angiogenic and cell-specific TIE1 and TIE2 genes during vascular development.

Dephosphorylation of protamine 2 at serine 56 is crucial for murine sperm maturation in vivo

2019 ◽  
Vol 12 (574) ◽  
pp. eaao7232 ◽  
Author(s):  
Katsuhiko Itoh ◽  
Gen Kondoh ◽  
Hitoshi Miyachi ◽  
Manabu Sugai ◽  
Yoshiyuki Kaneko ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Sperm Maturation ◽  
Sperm Cells ◽  
Chromatin Binding ◽  
Wild Type ◽  
Underlying Mechanisms ◽  
Deficient Mice ◽  
Protamine 2

The posttranslational modification of histones is crucial in spermatogenesis, as in other tissues; however, during spermiogenesis, histones are replaced with protamines, which are critical for the tight packaging of the DNA in sperm cells. Protamines are also posttranslationally modified by phosphorylation and dephosphorylation, which prompted our investigation of the underlying mechanisms and biological consequences of their regulation. On the basis of a screen that implicated the heat shock protein Hspa4l in spermatogenesis, we generated mice deficient in Hspa4l (Hspa4l-null mice), which showed male infertility and the malformation of sperm heads. These phenotypes are similar to those of Ppp1cc-deficient mice, and we found that the amount of a testis- and sperm-specific isoform of the Ppp1cc phosphatase (Ppp1cc2) in the chromatin-binding fraction was substantially less in Hspa4l-null spermatozoa than that in those of wild-type mice. We further showed that Ppp1cc2 was a substrate of the chaperones Hsc70 and Hsp70 and that Hspa4l enhanced the release of Ppp1cc2 from these complexes, enabling the freed Ppp1cc2 to localize to chromatin. Pull-down and in vitro phosphatase assays suggested the dephosphorylation of protamine 2 at serine 56 (Prm2 Ser56) by Ppp1cc2. To confirm the biological importance of Prm2 Ser56 dephosphorylation, we mutated Ser56 to alanine in Prm2 (Prm2 S56A). Introduction of this mutation to Hspa4l-null mice (Hspa4l−/−; Prm2S56A/S56A) restored the malformation of sperm heads and the infertility of Hspa4l−/− mice. The dephosphorylation signal to eliminate phosphate was crucial, and these results unveiled the mechanism and biological relevance of the dephosphorylation of Prm2 for sperm maturation in vivo.

scholarly journals Rab27a Is an Essential Component of Melanosome Receptor for Myosin Va

2002 ◽  
Vol 13 (5) ◽  
pp. 1735-1749 ◽  
Author(s):  
Xufeng Wu ◽  
Fei Wang ◽  
Kang Rao ◽  
James R. Sellers ◽  
John A. Hammer
Keyword(s):  
Full Length ◽  
Tail Domain ◽  
Wild Type ◽  
Essential Component ◽  
Myosin Va ◽  
Alternatively Spliced ◽  
Additional Protein ◽  
The Brain ◽  
Alternatively Spliced Exons

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for myosin Va (Myo5a). Herein, we show that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null (dilute) phenotype in wild-type melanocytes is absolutely dependent on the presence of exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue their distribution indilute melanocytes requires exon F but not exon D. These results predict that an interaction between myosin Va and Rab27a should be exon F dependent. Consistent with this, Rab27a present in detergent lysates of melanocytes binds to beads coated with purified, full-length melanocyte myosin Va and melanocyte myosin Va lacking exon D, but not to beads coated with melanocyte myosin Va lacking exon F or brain myosin Va. Moreover, the preparation of melanocyte lysates in the presence of GDP rather than guanosine-5′-O-(3-thio)triphosphate reduces the amount of Rab27a bound to melanocyte myosin Va-coated beads by approximately fourfold. Finally, pure Rab27a does not bind to myosin Va-coated beads, suggesting that these two proteins interact indirectly. Together, these results argue that Rab27a is an essential component of a protein complex that serves as the melanosome receptor for myosin Va, suggest that this complex contains at least one additional protein capable of bridging the indirect interaction between Rab27a and myosin Va, and imply that the recruitment of myosin Va to the melanosome surface in vivo should be regulated by factors controlling the nucleotide state of Rab27a.

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