Lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates for use in biomedicine. ... more Lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates for use in biomedicine. The interaction of nanomaterials with biological systems determines whether they are suitable for use in living cells. In-depth knowledge of the nano-bio interactions is therefore a prerequisite for the development of biomedical applications. The current study evaluates fundamental aspects of the NPcell interface for square bipyramidal UCNPs containing a LiYF 4 :Yb 3+ , Tm 3+ core and two different silica surface coatings. Given their importance for mammalian physiology, fibroblast and renal proximal tubule epithelial cells were selected as cellular model systems. We have assessed the toxicity of the UCNPs and measured their impact on the homeostasis of living non-malignant cells. Rigorous analyses were conducted to identify possible toxic and sub-lethal effects of the UCNPs. To this end, we examined biomarkers that reveal if UCNPs induce cell killing or stress. Quantitative measurements demonstrate that short-term exposure to the UCNPs had no profound effects on cell viability, cell size or morphology. Indicators of oxidative, endoplasmic reticulum, or nucleolar stress, and the production of molecular chaperones varied with the surface modification of the UCNPs and the cell type analyzed. These differences emphasize the importance of evaluating cells of diverse origin that are relevant to the intended use of the nanomaterials. Taken together, we established that short-term, our square bipyramidal UCNPs are not toxic to non-malignant fibroblast and proximal renal epithelial cells. Compared with established inducers of cellular stress, these UCNPs have minor effects on cellular homeostasis. Our results build the foundation to explore square bipyramidal UCNPs for future in vivo applications.
Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize l... more Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize lines. A 3.5-kb mRNA with an abundance of 1-3 x i07 of the poly(A) RNA, however, is found exclusively in those lines that carry an active Ac. Plants with two Ac elements contain slightly more 3.5-kb Ac transcript than those with only one Ac. Overlapping cDNA clones spanning most of the message have been isolated and sequenced. The 5'-end of the transcript was determined by Northern hybridization and Si mapping. It starts at several sites over a distance of nearly 100 bases, contains an AUGfree leader 600-700 nucleotides long, has a long open reading frame encoding 807 amino acids and an untranslated 3'-sequence of 239 nucleotides. Four introns with a combined length of 654 bases are removed from the primary transcript. Radiosequencing of in vitro translation products shows that translation of the long open reading frame begins at the frst AUG, even though it is located in an unfavourable sequence context. The transcript is found in all organs investigated, provided an active Ac is present in the stock.
The smooth muscle cells of chicken gizzard harbor the ectoenzyme 5'-nucleotidase. The purified en... more The smooth muscle cells of chicken gizzard harbor the ectoenzyme 5'-nucleotidase. The purified enzyme was reconstituted into 3H-labeled proteoliposomes which were used as a model to study the association of a membrane protein with fibronectin. We demonstrated that the binding process between proteoliposomes and fibronectin has the qualities of a receptor-ligand interaction, i.e., is saturable and specific. In contrast to the association of fibronectin with integrins, the interaction with 5'-nucleotidase does not require divalent metal ions. Synthetic peptides containing the RGD-sequence or a monoclonal antibody interfering with binding of other receptors to the cell-binding domain of fibronectin did not abolish the interaction with 5'-nucleotidase. This indicates that the RGDS-sequence does not represent the major contact site for the AMPase and that the 5'-nucleotidase belongs to a separate class of fibronectin receptors with distinct properties as compared to the integrins.
Aging increases the susceptibility to a diverse set of diseases and disorders, including neurodeg... more Aging increases the susceptibility to a diverse set of diseases and disorders, including neurodegeneration, cancer, diabetes, and arthritis. Natural compounds are currently being explored as alternative or complementary agents to treat or prevent aging-related malfunctions. Curcumin, a phytochemical isolated from the spice turmeric, has garnered great interest in recent years. With anti-oxidant, anti-inflammatory, anti-microbial, and other physiological activities, curcumin has great potential for health applications. However, the benefits of curcumin are restricted by its low bioavailability and stability in biological systems. Curcumin nanoformulations, or nano-curcumin, may overcome these limitations. This review discusses different forms of nano-curcumin that have been evaluated in vitro and in vivo to treat or prevent aging-associated health impairments. We describe current barriers for the routine use of curcumin nanoformulations in the clinic. Our review highlights outstanding questions and future work that is needed to ensure nano-curcumin is efficient and safe to lessen the burden of aging-related health problems.
Biochimica Et Biophysica Acta - Reviews On Cancer, May 1, 2022
Nanomaterials are at the forefront of health research and development. Among different nanomateri... more Nanomaterials are at the forefront of health research and development. Among different nanomaterials, nanoparticles are especially promising for cancer theranostics. However, despite great potential, the clinical translation of nano-based applications continues to face obstacles. A major hurdle to the localized eradication of tumors is the efficient targeting of nanomaterials to the desired tissues and cells. In particular, nanoparticle properties and the route of administration impact the efficacy of precision nanomedicine. This review focuses on nanoparticles that have been produced for the detection and treatment of cancer. Common and tissue-specific barriers that limit the accumulation of nanoparticles in malignant tumors are discussed. The in-depth discussion focuses on the physicochemical properties of nanoparticles and the surface modifications that achieve efficient accumulation at tumor sites. Furthermore, limitations of current strategies and open questions are presented. The review concludes with an outlook on future directions and the trajectories that will drive the field forward to advance nano-oncology in the clinic.
We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which c... more We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Highermolecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms. MATERIALS AND METHODS Materials Phospholipase C from Bacillus cereus (type III), phospholipase A2 from Naja naja venom, phospholipase C from Clostridium welchii, phospholipase D from cabbage, aprotinin, antipain, chymostatin, leupeptin, pepstatin, phenylmethanesulphonyl fluoride, iodoacetamide, concanavalin A and methyl CX-D-mannopyranoside were obtained from Sigma. Triton X-1 14, Triton X-100, sodium deoxycholate and 1,10-phenanthroline were purchased from Serva. PI-specific phospholipase C from Bacillus thuringiensis (PIPLC) was kindly provided by
5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrol... more 5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membranebound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.
Introduction: Human mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, an... more Introduction: Human mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, and tolerogenic effects. Long-term in vitro expansion of MSCs to generate clinical grade products results in the accumulation of senescent-functionally impaired MSCs. Markers to assess the 'senescent load' of MSC products are needed. Methods: Early and late passage human adipose tissue (AT) MSCs from pediatric and adult donors were characterized using established senescent markers [i.e., MSC size, granularity, and autofluorescence by flow cytometry; β-galactosidase staining (SA-β-gal); CDKN2A and CDKN1A by qRT-PCR]. In gene set enrichment analysis, DPP4 (also known as adenosine deaminase complexing protein 2 or CD26) was found as a prominent dysregulated transcript that was increased in late passage MSC(AT). This was confirmed in a larger number of MSC samples by PCR, flow cytometry, Western blotting, and immunofluorescence. In vitro immunopotency assays compared the function of CD26 high and CD26 low MSC(AT). The effect of senolytics on the CD26 high subpopulation was evaluated in senescent MSC(AT). Results: Late passage MSC(AT) had a senescence transcriptome signature. DPP4 was the most differentially enriched gene in senescent MSCs. Late passage senescent MSC(AT) had higher CD26 surface levels and total protein abundance. Moreover, CD26 surface levels were higher in early passage MSC(AT) from adults compared to pediatric donors. CD26 abundance correlated with established senescence markers. CD26 high MSC(AT) had reduced immunopotency compared to CD26 low MSC(AT). Senolytic treatment induced MSC apoptosis, which decreased the frequencies of CD26 high MSC(AT). Conclusions: DPP4 gene expression and DPP4/CD26 protein abundance are markers of replicative senescence in MSC(AT). Samples enriched in CD26 high MSC(AT) have reduced immunopotency and CD26 high MSCs are reduced with senolytics.
Dl Calcyclin (SlOOA6) is an "EFhand" calcium binding protein of the S-100 family which is present... more Dl Calcyclin (SlOOA6) is an "EFhand" calcium binding protein of the S-100 family which is present in the brain, lung, stomach and placenta. The search for calcyclin target has led us to the identification of a 30 kDa protein. This protein was named calcyclin binding protein (CacyBP) A clone of CacyBP was isolated !?om the mouse brain cDNA library and sequenced. The full sequence of this clone showed no homology to sequence in a GenBank that indicated the CacyBP is a novel protein. Recombinant CacyBP was expressed in E. coli and shown to bind calcyclin in a physiological range of calcium concentration. Polyclonal antibodies raised against CacyBP were used for the analysis of its tissue and cellular distribution in rat Western blots using these antibodies and northern blots using fill length cDNA as a probe showed the highest expression of CacyBP in the rat brain, liver, spleen and stomach. By immunohistochemistry and in situ hybridization we found that CacyBP is expressed in some neurons of the hippocampus, cerebellum and cortex of the rat brain. Subcellular fractionation and western blotting showed the highest level of CacyBP in the cytosol and moderate in the endoplasmic reticulum.The results show that this novel protein target of calcyclin is a predominantly neuronal protein and suggest its involvement in Ca2'-dependent signaling pathway in neurons This work was supported by grant 6P04A05415 from the State Committee to Scientific Research.
Poly (ADP-ribose) polymerase (PARP) inhibitors are a novel therapeutic approach for the selective... more Poly (ADP-ribose) polymerase (PARP) inhibitors are a novel therapeutic approach for the selective targeting of tumours with BRCA1/2 hereditary deficiencies. BRCA1/2 are two key proteins involved in homologous recombination (HR) repair. Although the therapeutic benefit of PARP inhibitors has now been proven in the clinic in patients with BRCA1/2 mutations, much effort has been deployed to expand the use of PARP inhibitors beyond tumours harbouring inherited deficiencies in HR- mediated DNA repair. Several combination modalities with agents that can induce HR deficiency and exploit the concept of “contextual synthetic lethality” have been reported for PARP inhibitors. Here we exploited the contextual synthetic lethality between PARP and the epidermal growth factor receptor (EGFR) to design and synthesize a novel dual EGFR targeted PARP inhibitor, termed ZSMR06. The results showed that: a) ZSMR06 is capable of inhibiting EGFR and PARP in whole cells in the nanomolar range (36-63 nM); b) it was selectively potent against BRCA2 mutant and EGFR expressing isogenic cells; c) as monotherapy, ZSMR06 was extremely potent with growth inhibitory activities superior to that of an equimolar combination of olaparib (a clinical PARP inhibitor) and gefitinib (a clinical EGFR inhibitor) in a large panel of tumour cell lines; d) ZSMR06 strongly potentiated the effect of temozolomide (TMZ) and induced significantly stronger (p<0.05-0.001) growth inhibition than olaparib + TMZ combination in cells expressing O6-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers a robust resistance to TMZ; e) ZSMR06 selectively potentiated TMZ on EGFR expressing cells; f) it was able to strongly sensitize bladder cancer cells to radiation (DMF50= 2.96-9.5). These results in toto showed that ZSMR06 due to its dual mechanism of action is capable of behaving as a strong potentiator of TMZ and radiation in resistant cells. Its potency in non-BRCA1/2 mutant tumour cells indicates that it may be the first prototype of hybrid molecules capable of expanding the use of PARP inhibition beyond BRCA1/2 mutations. Citation Format: Martin Rupp, Zhor Senhaji Mouhri, Ursula Stochaj, Bertrand Jean-Claude. Tandem targeting of poly (ADP-ribose) polymerase (PARP) and epidermal growth factor receptor (EGFR) as a novel strategy for enhancing radio- and chemosensitivity of refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2925.
Nanomedicine: Nanotechnology, Biology and Medicine, Nov 1, 2019
Gold nanoparticles have excellent potential for theranostic applications, but their impact on liv... more Gold nanoparticles have excellent potential for theranostic applications, but their impact on living cells is only partially understood. Many gold nanoparticles enter cells through endosomes/lysosomes which are linked to different cell organelles and compartments. Our study focuses on the unfolded protein response (UPR) in the endoplasmic reticulum (ER), cytoplasmic RNA-granules and proteostasis, because they are established indicators of cell stress and key regulators of cellular homeostasis. Using HeLa and renal proximal tubule cells as model systems, we show that gold nanourchins reduce cell proliferation, cause ER stress and impair proteostasis. Specifically, gold nanourchins activate the PERK-branch of the UPR, promote RNA oxidation, enhance P-body formation, and accumulate the oxidative stress marker Nrf2 and NFκB in nuclei. Taken together, our study demonstrates that gold nanourchins compromise ER, redox, protein, and RNA homeostasis. These insights provide new information on the cellular responses and molecular changes that gold nanourchins elicit in mammalian cells.
Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across... more Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across the cytoplasmic membrane. To define regions important for membrane insertion as well as for biological function, we constructed plasmids encoding different portions of the lactose carrier. Among several lacY deletions, two were obtained that encoded mutant proteins with complementary amino acid sequences. The truncated polypeptide Y71/1 (amino acid residues 1 to 71) comprises the first two alpha-helices predicted for the intact protein, and polypeptide delta Y4-69 carries an internal deletion of this region. Regulated coexpression of these lacY-DNA segments governed by separate but identical lacOP control regions resulted in functional complementation with the following characteristics. (i) Simultaneous synthesis of both incomplete proteins restored transport activity in transport-negative cells, measured as accumulation of [14C]lactose. (ii) Under complementing conditions, but not in t...
5′-Nucleotidase is a glycoprotein concentrated in the plasma membranes of many eucaryotic cells. ... more 5′-Nucleotidase is a glycoprotein concentrated in the plasma membranes of many eucaryotic cells. It is an ectoenzyme, i.e. its catalytic center faces the cell exterior. By its enzymatic activity AMP is hydrolysed into adenosine and Pi. 5′-Nucleotidase purified from chicken gizzard exhibits a molecular mass of 79 kDa after SDS-PAGE and behaves like an integral membrane protein, i.e. it is only soluble in the presence of detergents and can be reconstituted into artificial phospholipid vesicles (Dieckhoff et al. 1985, 1987)
The AAA+ ATPase valosin containing protein (VCP) is essential for cell and organ homeostasis, esp... more The AAA+ ATPase valosin containing protein (VCP) is essential for cell and organ homeostasis, especially in cells of the nervous system. As part of a large network, VCP collaborates with many cofactors to ensure proteostasis under normal, stress, and disease conditions. A large number of mutations have revealed the importance of VCP for human health. In particular, VCP facilitates the dismantling of protein aggregates and the removal of dysfunctional organelles. These are critical events to prevent malfunction of the brain and other parts of the nervous system. In line with this idea, VCP mutants are linked to the onset and progression of neurodegeneration and other diseases. The intricate molecular mechanisms that connect VCP mutations to distinct brain pathologies continue to be uncovered. Emerging evidence supports the model that VCP controls cellular functions on multiple levels and in a cell type specific fashion. Accordingly, VCP mutants derail cellular homeostasis through sev...
Lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates for use in biomedicine. ... more Lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates for use in biomedicine. The interaction of nanomaterials with biological systems determines whether they are suitable for use in living cells. In-depth knowledge of the nano-bio interactions is therefore a prerequisite for the development of biomedical applications. The current study evaluates fundamental aspects of the NPcell interface for square bipyramidal UCNPs containing a LiYF 4 :Yb 3+ , Tm 3+ core and two different silica surface coatings. Given their importance for mammalian physiology, fibroblast and renal proximal tubule epithelial cells were selected as cellular model systems. We have assessed the toxicity of the UCNPs and measured their impact on the homeostasis of living non-malignant cells. Rigorous analyses were conducted to identify possible toxic and sub-lethal effects of the UCNPs. To this end, we examined biomarkers that reveal if UCNPs induce cell killing or stress. Quantitative measurements demonstrate that short-term exposure to the UCNPs had no profound effects on cell viability, cell size or morphology. Indicators of oxidative, endoplasmic reticulum, or nucleolar stress, and the production of molecular chaperones varied with the surface modification of the UCNPs and the cell type analyzed. These differences emphasize the importance of evaluating cells of diverse origin that are relevant to the intended use of the nanomaterials. Taken together, we established that short-term, our square bipyramidal UCNPs are not toxic to non-malignant fibroblast and proximal renal epithelial cells. Compared with established inducers of cellular stress, these UCNPs have minor effects on cellular homeostasis. Our results build the foundation to explore square bipyramidal UCNPs for future in vivo applications.
Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize l... more Transcripts of various sizes hybridize to the transposable element Ac of Zea mays in most maize lines. A 3.5-kb mRNA with an abundance of 1-3 x i07 of the poly(A) RNA, however, is found exclusively in those lines that carry an active Ac. Plants with two Ac elements contain slightly more 3.5-kb Ac transcript than those with only one Ac. Overlapping cDNA clones spanning most of the message have been isolated and sequenced. The 5'-end of the transcript was determined by Northern hybridization and Si mapping. It starts at several sites over a distance of nearly 100 bases, contains an AUGfree leader 600-700 nucleotides long, has a long open reading frame encoding 807 amino acids and an untranslated 3'-sequence of 239 nucleotides. Four introns with a combined length of 654 bases are removed from the primary transcript. Radiosequencing of in vitro translation products shows that translation of the long open reading frame begins at the frst AUG, even though it is located in an unfavourable sequence context. The transcript is found in all organs investigated, provided an active Ac is present in the stock.
The smooth muscle cells of chicken gizzard harbor the ectoenzyme 5'-nucleotidase. The purified en... more The smooth muscle cells of chicken gizzard harbor the ectoenzyme 5'-nucleotidase. The purified enzyme was reconstituted into 3H-labeled proteoliposomes which were used as a model to study the association of a membrane protein with fibronectin. We demonstrated that the binding process between proteoliposomes and fibronectin has the qualities of a receptor-ligand interaction, i.e., is saturable and specific. In contrast to the association of fibronectin with integrins, the interaction with 5'-nucleotidase does not require divalent metal ions. Synthetic peptides containing the RGD-sequence or a monoclonal antibody interfering with binding of other receptors to the cell-binding domain of fibronectin did not abolish the interaction with 5'-nucleotidase. This indicates that the RGDS-sequence does not represent the major contact site for the AMPase and that the 5'-nucleotidase belongs to a separate class of fibronectin receptors with distinct properties as compared to the integrins.
Aging increases the susceptibility to a diverse set of diseases and disorders, including neurodeg... more Aging increases the susceptibility to a diverse set of diseases and disorders, including neurodegeneration, cancer, diabetes, and arthritis. Natural compounds are currently being explored as alternative or complementary agents to treat or prevent aging-related malfunctions. Curcumin, a phytochemical isolated from the spice turmeric, has garnered great interest in recent years. With anti-oxidant, anti-inflammatory, anti-microbial, and other physiological activities, curcumin has great potential for health applications. However, the benefits of curcumin are restricted by its low bioavailability and stability in biological systems. Curcumin nanoformulations, or nano-curcumin, may overcome these limitations. This review discusses different forms of nano-curcumin that have been evaluated in vitro and in vivo to treat or prevent aging-associated health impairments. We describe current barriers for the routine use of curcumin nanoformulations in the clinic. Our review highlights outstanding questions and future work that is needed to ensure nano-curcumin is efficient and safe to lessen the burden of aging-related health problems.
Biochimica Et Biophysica Acta - Reviews On Cancer, May 1, 2022
Nanomaterials are at the forefront of health research and development. Among different nanomateri... more Nanomaterials are at the forefront of health research and development. Among different nanomaterials, nanoparticles are especially promising for cancer theranostics. However, despite great potential, the clinical translation of nano-based applications continues to face obstacles. A major hurdle to the localized eradication of tumors is the efficient targeting of nanomaterials to the desired tissues and cells. In particular, nanoparticle properties and the route of administration impact the efficacy of precision nanomedicine. This review focuses on nanoparticles that have been produced for the detection and treatment of cancer. Common and tissue-specific barriers that limit the accumulation of nanoparticles in malignant tumors are discussed. The in-depth discussion focuses on the physicochemical properties of nanoparticles and the surface modifications that achieve efficient accumulation at tumor sites. Furthermore, limitations of current strategies and open questions are presented. The review concludes with an outlook on future directions and the trajectories that will drive the field forward to advance nano-oncology in the clinic.
We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which c... more We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Highermolecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms. MATERIALS AND METHODS Materials Phospholipase C from Bacillus cereus (type III), phospholipase A2 from Naja naja venom, phospholipase C from Clostridium welchii, phospholipase D from cabbage, aprotinin, antipain, chymostatin, leupeptin, pepstatin, phenylmethanesulphonyl fluoride, iodoacetamide, concanavalin A and methyl CX-D-mannopyranoside were obtained from Sigma. Triton X-1 14, Triton X-100, sodium deoxycholate and 1,10-phenanthroline were purchased from Serva. PI-specific phospholipase C from Bacillus thuringiensis (PIPLC) was kindly provided by
5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrol... more 5'-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membranebound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5'-p-fluorosulphonylbenzoyladenosine (5'-FSBA). 5'-FSBA irreversibly inactivates 5'-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5'-nucleotidase, abolished the inactivation by 5'-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5'-FSBA, which was employed for the radiolabelling of chicken gizzard 5'nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5'-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5'-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5'-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5'nucleotidase by 5'-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.
Introduction: Human mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, an... more Introduction: Human mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, and tolerogenic effects. Long-term in vitro expansion of MSCs to generate clinical grade products results in the accumulation of senescent-functionally impaired MSCs. Markers to assess the 'senescent load' of MSC products are needed. Methods: Early and late passage human adipose tissue (AT) MSCs from pediatric and adult donors were characterized using established senescent markers [i.e., MSC size, granularity, and autofluorescence by flow cytometry; β-galactosidase staining (SA-β-gal); CDKN2A and CDKN1A by qRT-PCR]. In gene set enrichment analysis, DPP4 (also known as adenosine deaminase complexing protein 2 or CD26) was found as a prominent dysregulated transcript that was increased in late passage MSC(AT). This was confirmed in a larger number of MSC samples by PCR, flow cytometry, Western blotting, and immunofluorescence. In vitro immunopotency assays compared the function of CD26 high and CD26 low MSC(AT). The effect of senolytics on the CD26 high subpopulation was evaluated in senescent MSC(AT). Results: Late passage MSC(AT) had a senescence transcriptome signature. DPP4 was the most differentially enriched gene in senescent MSCs. Late passage senescent MSC(AT) had higher CD26 surface levels and total protein abundance. Moreover, CD26 surface levels were higher in early passage MSC(AT) from adults compared to pediatric donors. CD26 abundance correlated with established senescence markers. CD26 high MSC(AT) had reduced immunopotency compared to CD26 low MSC(AT). Senolytic treatment induced MSC apoptosis, which decreased the frequencies of CD26 high MSC(AT). Conclusions: DPP4 gene expression and DPP4/CD26 protein abundance are markers of replicative senescence in MSC(AT). Samples enriched in CD26 high MSC(AT) have reduced immunopotency and CD26 high MSCs are reduced with senolytics.
Dl Calcyclin (SlOOA6) is an "EFhand" calcium binding protein of the S-100 family which is present... more Dl Calcyclin (SlOOA6) is an "EFhand" calcium binding protein of the S-100 family which is present in the brain, lung, stomach and placenta. The search for calcyclin target has led us to the identification of a 30 kDa protein. This protein was named calcyclin binding protein (CacyBP) A clone of CacyBP was isolated !?om the mouse brain cDNA library and sequenced. The full sequence of this clone showed no homology to sequence in a GenBank that indicated the CacyBP is a novel protein. Recombinant CacyBP was expressed in E. coli and shown to bind calcyclin in a physiological range of calcium concentration. Polyclonal antibodies raised against CacyBP were used for the analysis of its tissue and cellular distribution in rat Western blots using these antibodies and northern blots using fill length cDNA as a probe showed the highest expression of CacyBP in the rat brain, liver, spleen and stomach. By immunohistochemistry and in situ hybridization we found that CacyBP is expressed in some neurons of the hippocampus, cerebellum and cortex of the rat brain. Subcellular fractionation and western blotting showed the highest level of CacyBP in the cytosol and moderate in the endoplasmic reticulum.The results show that this novel protein target of calcyclin is a predominantly neuronal protein and suggest its involvement in Ca2'-dependent signaling pathway in neurons This work was supported by grant 6P04A05415 from the State Committee to Scientific Research.
Poly (ADP-ribose) polymerase (PARP) inhibitors are a novel therapeutic approach for the selective... more Poly (ADP-ribose) polymerase (PARP) inhibitors are a novel therapeutic approach for the selective targeting of tumours with BRCA1/2 hereditary deficiencies. BRCA1/2 are two key proteins involved in homologous recombination (HR) repair. Although the therapeutic benefit of PARP inhibitors has now been proven in the clinic in patients with BRCA1/2 mutations, much effort has been deployed to expand the use of PARP inhibitors beyond tumours harbouring inherited deficiencies in HR- mediated DNA repair. Several combination modalities with agents that can induce HR deficiency and exploit the concept of “contextual synthetic lethality” have been reported for PARP inhibitors. Here we exploited the contextual synthetic lethality between PARP and the epidermal growth factor receptor (EGFR) to design and synthesize a novel dual EGFR targeted PARP inhibitor, termed ZSMR06. The results showed that: a) ZSMR06 is capable of inhibiting EGFR and PARP in whole cells in the nanomolar range (36-63 nM); b) it was selectively potent against BRCA2 mutant and EGFR expressing isogenic cells; c) as monotherapy, ZSMR06 was extremely potent with growth inhibitory activities superior to that of an equimolar combination of olaparib (a clinical PARP inhibitor) and gefitinib (a clinical EGFR inhibitor) in a large panel of tumour cell lines; d) ZSMR06 strongly potentiated the effect of temozolomide (TMZ) and induced significantly stronger (p<0.05-0.001) growth inhibition than olaparib + TMZ combination in cells expressing O6-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers a robust resistance to TMZ; e) ZSMR06 selectively potentiated TMZ on EGFR expressing cells; f) it was able to strongly sensitize bladder cancer cells to radiation (DMF50= 2.96-9.5). These results in toto showed that ZSMR06 due to its dual mechanism of action is capable of behaving as a strong potentiator of TMZ and radiation in resistant cells. Its potency in non-BRCA1/2 mutant tumour cells indicates that it may be the first prototype of hybrid molecules capable of expanding the use of PARP inhibition beyond BRCA1/2 mutations. Citation Format: Martin Rupp, Zhor Senhaji Mouhri, Ursula Stochaj, Bertrand Jean-Claude. Tandem targeting of poly (ADP-ribose) polymerase (PARP) and epidermal growth factor receptor (EGFR) as a novel strategy for enhancing radio- and chemosensitivity of refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2925.
Nanomedicine: Nanotechnology, Biology and Medicine, Nov 1, 2019
Gold nanoparticles have excellent potential for theranostic applications, but their impact on liv... more Gold nanoparticles have excellent potential for theranostic applications, but their impact on living cells is only partially understood. Many gold nanoparticles enter cells through endosomes/lysosomes which are linked to different cell organelles and compartments. Our study focuses on the unfolded protein response (UPR) in the endoplasmic reticulum (ER), cytoplasmic RNA-granules and proteostasis, because they are established indicators of cell stress and key regulators of cellular homeostasis. Using HeLa and renal proximal tubule cells as model systems, we show that gold nanourchins reduce cell proliferation, cause ER stress and impair proteostasis. Specifically, gold nanourchins activate the PERK-branch of the UPR, promote RNA oxidation, enhance P-body formation, and accumulate the oxidative stress marker Nrf2 and NFκB in nuclei. Taken together, our study demonstrates that gold nanourchins compromise ER, redox, protein, and RNA homeostasis. These insights provide new information on the cellular responses and molecular changes that gold nanourchins elicit in mammalian cells.
Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across... more Escherichia coli lactose permease mediates the proton-driven translocation of galactosides across the cytoplasmic membrane. To define regions important for membrane insertion as well as for biological function, we constructed plasmids encoding different portions of the lactose carrier. Among several lacY deletions, two were obtained that encoded mutant proteins with complementary amino acid sequences. The truncated polypeptide Y71/1 (amino acid residues 1 to 71) comprises the first two alpha-helices predicted for the intact protein, and polypeptide delta Y4-69 carries an internal deletion of this region. Regulated coexpression of these lacY-DNA segments governed by separate but identical lacOP control regions resulted in functional complementation with the following characteristics. (i) Simultaneous synthesis of both incomplete proteins restored transport activity in transport-negative cells, measured as accumulation of [14C]lactose. (ii) Under complementing conditions, but not in t...
5′-Nucleotidase is a glycoprotein concentrated in the plasma membranes of many eucaryotic cells. ... more 5′-Nucleotidase is a glycoprotein concentrated in the plasma membranes of many eucaryotic cells. It is an ectoenzyme, i.e. its catalytic center faces the cell exterior. By its enzymatic activity AMP is hydrolysed into adenosine and Pi. 5′-Nucleotidase purified from chicken gizzard exhibits a molecular mass of 79 kDa after SDS-PAGE and behaves like an integral membrane protein, i.e. it is only soluble in the presence of detergents and can be reconstituted into artificial phospholipid vesicles (Dieckhoff et al. 1985, 1987)
The AAA+ ATPase valosin containing protein (VCP) is essential for cell and organ homeostasis, esp... more The AAA+ ATPase valosin containing protein (VCP) is essential for cell and organ homeostasis, especially in cells of the nervous system. As part of a large network, VCP collaborates with many cofactors to ensure proteostasis under normal, stress, and disease conditions. A large number of mutations have revealed the importance of VCP for human health. In particular, VCP facilitates the dismantling of protein aggregates and the removal of dysfunctional organelles. These are critical events to prevent malfunction of the brain and other parts of the nervous system. In line with this idea, VCP mutants are linked to the onset and progression of neurodegeneration and other diseases. The intricate molecular mechanisms that connect VCP mutations to distinct brain pathologies continue to be uncovered. Emerging evidence supports the model that VCP controls cellular functions on multiple levels and in a cell type specific fashion. Accordingly, VCP mutants derail cellular homeostasis through sev...
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