RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for poly... more RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for beta globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives' yields for various size real-time PCR amplicons. Longer amplicons (above approximately 225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.
Intraneuronal accumulation of amyloid-β(1–42) (Aβ1–42) is one of the earliest signs of Alzheimer’... more Intraneuronal accumulation of amyloid-β(1–42) (Aβ1–42) is one of the earliest signs of Alzheimer’s disease (AD). Cell surface heparan sulfate proteoglycans (HSPGs) have profound influence on the cellular uptake of Aβ1–42 by mediating its attachment and subsequent internalization into the cells. Colocalization of amyloid plaques with members of the syndecan family of HSPGs, along with the increased expression of syndecan-3 and -4 have already been reported in postmortem AD brains. Considering the growing evidence on the involvement of syndecans in the pathogenesis of AD, we analyzed the contribution of syndecans to cellular uptake and fibrillation of Aβ1–42. Among syndecans, the neuron specific syndecan-3 isoform increased cellular uptake of Aβ1–42 the most. Kinetics of Aβ1–42 uptake also proved to be fairly different among SDC family members: syndecan-3 increased Aβ1–42 uptake from the earliest time points, while other syndecans facilitated Aβ1–42 internalization at a slower pace. I...
Although syndecan-1 (SDC1) is known to be dysregulated in various cancer types, its implication i... more Although syndecan-1 (SDC1) is known to be dysregulated in various cancer types, its implication in tumorigenesis is poorly understood. Its effect may be detrimental or protective depending on the type of cancer. Our previous data suggest that SDC1 is protective against hepatocarcinogenesis. To further verify this notion, human SDC1 transgenic (hSDC1+/+) mice were generated that expressed hSDC1 specifically in the liver under the control of the albumin promoter. Hepatocarcinogenesis was induced by a single dose of diethylnitrosamine (DEN) at an age of 15 days after birth, which resulted in tumors without cirrhosis in wild-type and hSDC1+/+ mice. At the experimental endpoint, livers were examined macroscopically and histologically, as well as by immunohistochemistry, Western blot, receptor tyrosine kinase array, phosphoprotein array, and proteomic analysis. Liver-specific overexpression of hSDC1 resulted in an approximately six month delay in tumor formation via the promotion of SDC1 ...
International Journal of Peptide Research and Therapeutics
It is well established that cationic peptides can enter cells following attachment to polyanionic... more It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane tra...
Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated v... more Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9’s cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of sy...
Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enz... more Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enzyme 2 (ACE2) is still viewed as the main cell entry receptor mediating SARS-CoV-2 internalization. It has been reported that wild-type mice, like other rodent species of the Muridae family, cannot be infected with SARS-CoV-2 due to differences in their ACE2 receptors. On the other hand, the consensus heparin-binding motif of SARS-CoV-2’s spike protein, PRRAR, enables the attachment to rodent heparan sulfate proteoglycans (HSPGs), including syndecans, a transmembrane HSPG family with a well-established role in clathrin- and caveolin-independent endocytosis. As mammalian syndecans possess a relatively conserved structure, we analyzed the cellular uptake of inactivated SARS-CoV-2 particles in in vitro and in vivo mice models. Cellular studies revealed efficient uptake into murine cell lines with established syndecan-4 expression. After intravenous administration, inactivated SARS-CoV-2 was ...
Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more t... more Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more transmissible than the original virus, the SARS-CoV-2 B.1.617 lineage, named the Delta variant, swept through the world in 2021. The mutations in the Delta’s spike protein shift the protein towards a net positive electrostatic potential. To understand the key molecular drivers of the Delta infection, we investigate the cellular uptake of the Delta spike protein and Delta spike-bearing SARS-CoV-2 pseudoviruses. Specific in vitro modification of ACE2 and syndecan expression enabled us to demonstrate that syndecan-4, the syndecan isoform abundant in the lung, enhances the transmission of the Delta variant by attaching its mutated spike glycoprotein and facilitating its cellular entry. Compared to the wild-type spike, the Delta one shows a higher affinity towards heparan sulfate proteoglycans than towards ACE2. In addition to attachment to the polyanionic heparan sulfate chains, the Delta spi...
Csak a novenyek rendelkeznek sejtciklus-specifikusan, a G2/M fazisban expresszalodo ciklin fuggő ... more Csak a novenyek rendelkeznek sejtciklus-specifikusan, a G2/M fazisban expresszalodo ciklin fuggő kinazokkal (CDKB-k). Klonoztuk es funkcionalisan jellemeztuk a lucerna CDKB2;1 kinaz előtti kb. 360 bp hosszu upstream regiot. Bizonyitottuk, hogy ez a szakasz kepes a riporter genek expressziojat az osztodo merisztematikus sejtekre korlatozni transzgen lucerna novenyekben, es G2/M fazis specifikus expressziot biztositani szinkronizalt, lucerna transzgen novenyekből generalt sejtszuszpenzioban. Immunohisztokemiai festessel igazoltuk, hogy transzgen noveny gyokereben a riporter genek az endogen kinaznak megfelelő mintazatban voltak jelen. Megallapitottuk, hogy az eddig szigoruan sejtciklus-specifikus szabalyozasunak hitt CDKB2;1 kinaz a sebzes hatasara is expresszalodik. Az etilen, amely egyik lehetseges sebzes-valasz mediator, szinten bekapcsolta a promotert, de sem a jazminsav, sem a szalicilsav nem hatott a promoter szabalyzasara, amelyek meg lehetseges szignalatvivői lehetnek a sebzes...
The effects of salt stress condition on the growth, morphology, photosynthetic performance, and p... more The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR...
Scientific evidence suggests that α-synuclein and tau have prion-like properties and that prion-l... more Scientific evidence suggests that α-synuclein and tau have prion-like properties and that prion-like spreading and seeding of misfolded protein aggregates constitutes a central mechanism for neurodegeneration. Heparan sulfate proteoglycans (HSPGs) in the plasma membrane support this process by attaching misfolded protein fibrils. Despite of intense studies, contribution of specific HSPGs to seeding and spreading of α-synuclein and tau has not been explored yet. Here we report that members of the syndecan family of HSPGs mediate cellular uptake of α-synuclein and tau fibrils via a lipid-raft dependent and clathrin-independent endocytic route. Among syndecans, the neuron predominant syndecan-3 exhibits the highest affinity for both α-synuclein and tau. Syndecan-mediated internalization of α-synuclein and tau depends heavily on conformation as uptake via syndecans start to dominate once fibrils are formed. Overexpression of syndecans, on the other hand, reduces cellular uptake of monom...
RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for poly... more RNA recovered from paraffin-embedded tissue has been reported to be a suitable substrate for polymerase chain reaction. During tissue fixation and paraffin embedding, RNA undergoes degradation, but with certain restrictions, it can be used for gene expression studies. At the same time, formalin-fixed, paraffin embedded histopathology archives contain an unestimable collection, which could be analyzed to investigate changes in mRNA expression in pathologic processes. To decide for future tissue conservation of pathology samples, it would be reasonable to satisfy both histologic and molecular biologic needs. The effect of three different fixation methods, RNAlater (SIGMA R 0901, St Louis, MO), acetone, and formalin, were compared by histology, immunohistochemistry, and real-time PCR. To assess tissue structure preservation and antigenicity, hematoxylin-eosin staining and immunohistochemistry were performed; to assess RNA quality, RNA was extracted and the transcription of different amplicon sizes (121, 225, 406 bp for GAPDH; 166, 310, 536 bp for beta globin) were examined on human endometrium samples. The most adequate tissue preservation was found in case of formalin fixation, while there were no significant differences in the three fixatives' yields for various size real-time PCR amplicons. Longer amplicons (above approximately 225 bp) have limited use for gene expression studies, while shorter amplicons could give more reliable results.
Intraneuronal accumulation of amyloid-β(1–42) (Aβ1–42) is one of the earliest signs of Alzheimer’... more Intraneuronal accumulation of amyloid-β(1–42) (Aβ1–42) is one of the earliest signs of Alzheimer’s disease (AD). Cell surface heparan sulfate proteoglycans (HSPGs) have profound influence on the cellular uptake of Aβ1–42 by mediating its attachment and subsequent internalization into the cells. Colocalization of amyloid plaques with members of the syndecan family of HSPGs, along with the increased expression of syndecan-3 and -4 have already been reported in postmortem AD brains. Considering the growing evidence on the involvement of syndecans in the pathogenesis of AD, we analyzed the contribution of syndecans to cellular uptake and fibrillation of Aβ1–42. Among syndecans, the neuron specific syndecan-3 isoform increased cellular uptake of Aβ1–42 the most. Kinetics of Aβ1–42 uptake also proved to be fairly different among SDC family members: syndecan-3 increased Aβ1–42 uptake from the earliest time points, while other syndecans facilitated Aβ1–42 internalization at a slower pace. I...
Although syndecan-1 (SDC1) is known to be dysregulated in various cancer types, its implication i... more Although syndecan-1 (SDC1) is known to be dysregulated in various cancer types, its implication in tumorigenesis is poorly understood. Its effect may be detrimental or protective depending on the type of cancer. Our previous data suggest that SDC1 is protective against hepatocarcinogenesis. To further verify this notion, human SDC1 transgenic (hSDC1+/+) mice were generated that expressed hSDC1 specifically in the liver under the control of the albumin promoter. Hepatocarcinogenesis was induced by a single dose of diethylnitrosamine (DEN) at an age of 15 days after birth, which resulted in tumors without cirrhosis in wild-type and hSDC1+/+ mice. At the experimental endpoint, livers were examined macroscopically and histologically, as well as by immunohistochemistry, Western blot, receptor tyrosine kinase array, phosphoprotein array, and proteomic analysis. Liver-specific overexpression of hSDC1 resulted in an approximately six month delay in tumor formation via the promotion of SDC1 ...
International Journal of Peptide Research and Therapeutics
It is well established that cationic peptides can enter cells following attachment to polyanionic... more It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane tra...
Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated v... more Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9’s cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of sy...
Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enz... more Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enzyme 2 (ACE2) is still viewed as the main cell entry receptor mediating SARS-CoV-2 internalization. It has been reported that wild-type mice, like other rodent species of the Muridae family, cannot be infected with SARS-CoV-2 due to differences in their ACE2 receptors. On the other hand, the consensus heparin-binding motif of SARS-CoV-2’s spike protein, PRRAR, enables the attachment to rodent heparan sulfate proteoglycans (HSPGs), including syndecans, a transmembrane HSPG family with a well-established role in clathrin- and caveolin-independent endocytosis. As mammalian syndecans possess a relatively conserved structure, we analyzed the cellular uptake of inactivated SARS-CoV-2 particles in in vitro and in vivo mice models. Cellular studies revealed efficient uptake into murine cell lines with established syndecan-4 expression. After intravenous administration, inactivated SARS-CoV-2 was ...
Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more t... more Emerging SARS-CoV-2 variants pose threats to vaccination campaigns against COVID-19. Being more transmissible than the original virus, the SARS-CoV-2 B.1.617 lineage, named the Delta variant, swept through the world in 2021. The mutations in the Delta’s spike protein shift the protein towards a net positive electrostatic potential. To understand the key molecular drivers of the Delta infection, we investigate the cellular uptake of the Delta spike protein and Delta spike-bearing SARS-CoV-2 pseudoviruses. Specific in vitro modification of ACE2 and syndecan expression enabled us to demonstrate that syndecan-4, the syndecan isoform abundant in the lung, enhances the transmission of the Delta variant by attaching its mutated spike glycoprotein and facilitating its cellular entry. Compared to the wild-type spike, the Delta one shows a higher affinity towards heparan sulfate proteoglycans than towards ACE2. In addition to attachment to the polyanionic heparan sulfate chains, the Delta spi...
Csak a novenyek rendelkeznek sejtciklus-specifikusan, a G2/M fazisban expresszalodo ciklin fuggő ... more Csak a novenyek rendelkeznek sejtciklus-specifikusan, a G2/M fazisban expresszalodo ciklin fuggő kinazokkal (CDKB-k). Klonoztuk es funkcionalisan jellemeztuk a lucerna CDKB2;1 kinaz előtti kb. 360 bp hosszu upstream regiot. Bizonyitottuk, hogy ez a szakasz kepes a riporter genek expressziojat az osztodo merisztematikus sejtekre korlatozni transzgen lucerna novenyekben, es G2/M fazis specifikus expressziot biztositani szinkronizalt, lucerna transzgen novenyekből generalt sejtszuszpenzioban. Immunohisztokemiai festessel igazoltuk, hogy transzgen noveny gyokereben a riporter genek az endogen kinaznak megfelelő mintazatban voltak jelen. Megallapitottuk, hogy az eddig szigoruan sejtciklus-specifikus szabalyozasunak hitt CDKB2;1 kinaz a sebzes hatasara is expresszalodik. Az etilen, amely egyik lehetseges sebzes-valasz mediator, szinten bekapcsolta a promotert, de sem a jazminsav, sem a szalicilsav nem hatott a promoter szabalyzasara, amelyek meg lehetseges szignalatvivői lehetnek a sebzes...
The effects of salt stress condition on the growth, morphology, photosynthetic performance, and p... more The effects of salt stress condition on the growth, morphology, photosynthetic performance, and paramylon content were examined in the mixotrophic, unicellular, flagellate Euglena gracilis. We found that salt stress negatively influenced cell growth, accompanied by a decrease in chlorophyll (Chl) content. Circular dichroism (CD) spectroscopy revealed the changes in the macro-organization of pigment-protein complexes due to salt treatment, while the small-angle neutron scattering (SANS) investigations suggested a reduction in the thylakoid stacking, an effect confirmed by the transmission electron microscopy (TEM). At the same time, the analysis of the thylakoid membrane complexes using native-polyacrylamide gel electrophoresis (PAGE) revealed no significant change in the composition of supercomplexes of the photosynthetic apparatus. Salt stress did not substantially affect the photosynthetic activity, as reflected by the fact that Chl fluorescence yield, electron transport rate (ETR...
Scientific evidence suggests that α-synuclein and tau have prion-like properties and that prion-l... more Scientific evidence suggests that α-synuclein and tau have prion-like properties and that prion-like spreading and seeding of misfolded protein aggregates constitutes a central mechanism for neurodegeneration. Heparan sulfate proteoglycans (HSPGs) in the plasma membrane support this process by attaching misfolded protein fibrils. Despite of intense studies, contribution of specific HSPGs to seeding and spreading of α-synuclein and tau has not been explored yet. Here we report that members of the syndecan family of HSPGs mediate cellular uptake of α-synuclein and tau fibrils via a lipid-raft dependent and clathrin-independent endocytic route. Among syndecans, the neuron predominant syndecan-3 exhibits the highest affinity for both α-synuclein and tau. Syndecan-mediated internalization of α-synuclein and tau depends heavily on conformation as uptake via syndecans start to dominate once fibrils are formed. Overexpression of syndecans, on the other hand, reduces cellular uptake of monom...
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