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1.
Cell Signal ; 74: 109712, 2020 10.
Article in English | MEDLINE | ID: mdl-32659264

ABSTRACT

The ubiquitin (Ub)-conjugating enzyme variants (Uev) Uev1A and Mms2 interact with Ubc13 to form heterodimeric complexes with different biological functions. Uev1A-Ubc13 is involved in NF-κB activation while Mms2-Ubc13 is required for the DNA-damage response. The structural comparison of the core domains of these two Uevs reveals no obvious difference, suggesting that the amino terminal extension of Uev1A plays a critical role in the functional determination. Indeed, truncated Uev1A lacking the N-terminal extension behaves like Mms2, while a chimeric protein containing the N-terminal Uev1A fused to Mms2 functionally resembles Uev1A. Interestingly, the N-terminal extension of Uev1A also dictates whether to assemble di- or poly-Ub chains in an in vitro reaction. Both thermodynamic measurements and enzymatic assays revealed that the Uev1A N-terminal extension weakens the Uev-Ubc13 interaction; however, other means capable of causing a reduced Uev1A-Ubc13 affinity and poly-Ub chain assembly do not necessarily promote NF-κB activation, indicating that the poly-Ub chain formation is not the only component contributed by the N-terminal extension of Uev1A. The physiological relevance of the Uev1A N-terminal truncation is presented and discussed.


Subject(s)
NF-kappa B/metabolism , Transcription Factors/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Binding Sites , Cell Line, Tumor , Gene Expression Regulation , Humans , Protein Binding
2.
Biotechnol Prog ; 36(4): e2976, 2020 07.
Article in English | MEDLINE | ID: mdl-32012477

ABSTRACT

Studying age-related neuropathologies in vitro requires a three-dimensional (3D) culture system presenting mature phenotypes. In this study, we aimed to determine whether aged reaggregate cultures physiologically represent mature brain tissue. Results support that embryo-derived rat central nervous system (CNS) reaggregate cultures develop into mature-like tissues, comparable to in vivo maturation, including the following characteristics: (a) progressive reduction in cell proliferation (reduced anti-Ki-67 immunoreactivity), (b) progressive restriction of long neurite growth potential (as explant cultures), and (c) increased and sustained synaptic enzyme (acetylcholine esterase, AChE) activity. The acquisition of mature-like reaggregate cultures has allowed us to pursue the hypothesis that the physiological integrity of 3D CNS cultures may be monitored by synaptic enzyme activity. To assess this hypothesis, mature-like reaggregates were exposed to H2 O2 , glutamate, or amyloid ß(1-42); each resulted in diminished AChE activity. H2 O2 exposure resulted in nuclear fragmentation. Glutamate and amyloid ß(1-42) exposure resulted in acetylcholine content reduction. Simultaneous reduction of AChE activity and acetylcholine content verified diminished cholinergic integrity. This scheme exploiting synapse enzyme activity of mature-like 3D CNS tissue is therefore applicable to age-related neuropathology research including in vitro screening of conditions potentially affecting synapse integrity, including the promotion of dementia.


Subject(s)
Brain/cytology , Cell Culture Techniques , Central Nervous System/cytology , Cholinergic Neurons/metabolism , Acetylcholinesterase/genetics , Acetylcholinesterase/metabolism , Animals , Brain/metabolism , Cell Aggregation/genetics , Central Nervous System/metabolism , Cholinergic Neurons/cytology , Glutamic Acid/genetics , Rats
3.
Biotechnol Prog ; 35(2): e2745, 2019 03.
Article in English | MEDLINE | ID: mdl-30421867

ABSTRACT

The only cure available for Type 1 diabetes involves the transplantation of islets of Langerhans isolated from donor organs. However, success rates are relatively low. Disconnection from vasculature upon isolation and insufficient rate of revascularization upon transplantation are thought to be a major cause, as islet survival and function depend on extensive vascularization. Research has thus turned toward the development of pretransplantation culture techniques to enhance revascularization of islets, so far with limited success. With the aim to develop a technique to enhance islet revascularization, this work proposes a method to isolate and culture pancreas-derived blood vessels. Using a mild multistep digestion method, pancreatic blood vessels were retrieved from whole murine pancreata and cultured in collagen Type 1. After 8 days, 50% of tissue explants had formed anastomosed microvessels which extended up to 300 µm from the explant tissue and expressed endothelial cell marker CD31 but not ductal marker CK19. Cocultures with islets of Langerhans revealed survival of both tissues and insulin expression by islets up to 8 days post-embedding. Microvessels were frequently found to encapsulate islets, however no islet penetration could be detected. This study reports for the first time the isolation and culture of pancreatic blood vessels. The methods and results presented in this work provide a novel explant culture model for angiogenesis and tissue engineering research with relevance to islet biology. It opens the door for in vivo validation of the potential of these pancreatic blood vessel explants to improve islet transplantation therapies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2745, 2019.


Subject(s)
Islets of Langerhans/cytology , Pancreas/cytology , Animals , Cell Survival , Cells, Cultured , Coculture Techniques , Female , Fluorescent Dyes/chemistry , Islets of Langerhans/diagnostic imaging , Mice , Optical Imaging , Pancreas/diagnostic imaging
4.
Assay Drug Dev Technol ; 16(8): 462-471, 2018 12.
Article in English | MEDLINE | ID: mdl-30106594

ABSTRACT

The lack of understanding on how to treat pancreas-related diseases and develop new therapeutics is partly due to the unavailability of appropriate models. In vitro models fail to provide a physiological environment. Testing new drug targets in these models can give rise to bias and misleading results. Therefore, we developed an in vivo model for drug testing on full pancreatic digests, which maintains the interactions between endo- and exocrine tissues and allows retrieving the samples for further analyses. The use of full pancreatic digest eliminates the need to isolate islets, reducing time and cost. In this model, four different conditions can be implanted subcutaneously within the same animal. Each condition consists of full pancreatic tissue digests embedded in alginate beads. All alginate beads in one animal contained full pancreatic digest of the same donor and, after 5-day implantation, were retrieved for analysis focusing on survival, function, and/or organization. Proof-of-principle of the platform was evidenced by showing the effect of hyaluronic acid and vascular endothelial growth factor on the overall function of the full pancreatic digest and on endothelial cells in the pancreatic digest, respectively. Retrieval from identical animals allows direct comparison between conditions. Metabolism (MTT) quantification, dithizone staining, and glucose-stimulated insulin secretion assessment allow to discriminate, using a minimal number of animals, between treatments and validate the system. Because of its simplicity, the model is highly adaptable to specific needs of the user.


Subject(s)
Alginates/metabolism , Drug Carriers/metabolism , Models, Animal , Pancreas/metabolism , Subcutaneous Absorption/physiology , Tissue Distribution/physiology , Alginates/administration & dosage , Animals , Drug Carriers/administration & dosage , Drug Evaluation, Preclinical/methods , Female , Pancreas/drug effects , Rats , Rats, Sprague-Dawley , Subcutaneous Absorption/drug effects , Tissue Distribution/drug effects
5.
Biotechnol Prog ; 34(4): 1059-1068, 2018 07.
Article in English | MEDLINE | ID: mdl-29603910

ABSTRACT

Type II diabetes progresses with inadequate insulin secretion and prolonged elevated circulating glucose levels. Also, pancreatic islets isolated for transplantation or tissue engineering can be exposed to glucose over extended timeframe. We hypothesized that isolated pancreatic islets can secrete insulin over a prolonged period of time when incubated in glucose solution and that not all islets release insulin in unison. Insulin secretion kinetics was examined and modeled from single mouse islets in response to chronic glucose exposure (2.8-20 mM). Results with single islets were compared to those from pools of islets. Kinetic analysis of 58 single islets over 72 h in response to elevated glucose revealed distinct insulin secretion profiles: slow-, fast-, and constant-rate secretors, with slow-secretors being most prominent (ca., 50%). Variations in the temporal response to glucose therefore exist. During short-term (<4 h) exposure to elevated glucose few islets are responding with sustained insulin release. The model allowed studying the influence of islet size, revealing no clear effect. At high-glucose concentrations, when secretion is normalized to islet volume, the tendency is that smaller islets secrete more insulin. At high-glucose concentrations, insulin secretion from single islets is representative of islet populations, while under low-glucose conditions pooled islets did not behave as single ones. The characterization of insulin secretion over prolonged periods complements studies on insulin secretion performed over short timeframe. Further investigation of these differences in secretion profiles may resolve open-ended questions on pre-diabetic conditions and transplanted islets performance. This study deliberates the importance of size of islets in insulin secretion. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1059-1068, 2018.


Subject(s)
Insulin Secretion/physiology , Insulin/metabolism , Islets of Langerhans/metabolism , Animals , Female , Kinetics , Mice , Organ Culture Techniques
6.
Colloids Surf B Biointerfaces ; 159: 166-173, 2017 Nov 01.
Article in English | MEDLINE | ID: mdl-28802201

ABSTRACT

This study describes a method to screen biomimetic surfaces based on intracellular insulin content of either fully or partly dissociated primary endocrine islet tissue. It is challenging to maintain endocrine pancreatic islets and more so, dissociated ones. Physiological activity of isolated islet cells in vitro declines due to loss of cell-to-cell and cell-to-extracellular matrix interactions. An in vitro model was developed to evaluate specific extracellular binding components potentially affecting islet biology, with the intention to identify in vivo-like peptides promoting survival and function. Synthetic peptides were bound to low-fouling carboxy-methyl-dextran surfaces, effectively presenting defined surfaces while minimizing non-specific interactions. These biomimetic surfaces were screened based on intracellular insulin content of applied mouse primary islet tissue by analysis with an anti-insulin cell-ELISA. Three active biomimetic surfaces were identified, two laminin- (IKLLI and PDSGR) and one cadherin (HAVDI)-derived, which supported adhesion and survival of insulin-containing cultures for 5days, respectively suggesting a benefit from both cell-extracellular matrix and cell-cell interactions. Cells from dissociated islets show progression over 10days on the HAVDI-biomimetic for the insulin immunoreactivity and cell density. The three surfaces did not act additively or synergistically. A favorable reaction to glucose-stimulated insulin secretion on the cadherin-biomimetic indicated the cultures were physiologically functional. This supportive role of biomimetic peptides represents initial progress in defining minimal extracellular binding requirements influencing islet cell physiology. This will influence further optimization of growth surfaces and promote the basic understanding of islet biology. Low-fouling biomimetics are predicted to be applicable to additional diverse culture systems.


Subject(s)
Biomimetics , Animals , Glucose/metabolism , Insulin/chemistry , Islets of Langerhans/cytology , Mice , Peptides/chemistry
7.
Nucleic Acids Res ; 41(4): 2328-39, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23303771

ABSTRACT

It has been long speculated that mammalian Rev3 plays an important, yet unknown role(s) during mammalian development, as deletion of Rev3 causes embryonic lethality in mice, whereas no other translesion DNA synthesis polymerases studied to date are required for mouse embryo development. Here, we report that both subunits of Polζ (Rev3 and Rev7) show an unexpected increase in expression during G(2)/M phase, but they localize independently in mitotic cells. Experimental depletion of Rev3 results in a significant increase in anaphase bridges, chromosomal breaks/gaps and common fragile site (CFS) expression, whereas Rev7 depletion primarily causes lagging chromosome defect with no sign of CFS expression. The genomic instability induced by Rev3 depletion seems to be related to replication stress, as it is further enhanced on aphidicolin treatment and results in increased metaphase-specific Fanconi anemia complementation group D type 2 (FANCD2) foci formation, as well as FANCD2-positive anaphase bridges. Indeed, a long-term depletion of Rev3 in cultured human cells results in massive genomic instability and severe cell cycle arrest. The aforementioned observations collectively support a notion that Rev3 is required for the efficient replication of CFSs during G(2)/M phase, and that the resulting fragile site instability in Rev3 knockout mice may trigger cell death during embryonic development.


Subject(s)
Chromosome Fragile Sites , DNA-Binding Proteins/physiology , DNA-Directed DNA Polymerase/physiology , Cell Division , Cell Proliferation , Cells, Cultured , Chromosome Breakage , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Fanconi Anemia Complementation Group D2 Protein/analysis , G2 Phase , Genomic Instability , Histones/analysis , Humans , Mad2 Proteins , Mitosis/genetics , Nucleic Acid Synthesis Inhibitors , Proteins/antagonists & inhibitors , Proteins/metabolism
8.
Mol Biol Cell ; 22(13): 2373-83, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21551069

ABSTRACT

In response to DNA damage such as from UV irradiation, mammalian Y-family translesion synthesis (TLS) polymerases Polη and Rev1 colocalize with proliferating cell nuclear antigen at nuclear foci, presumably representing stalled replication sites. However, it is unclear whether the localization of one polymerase is dependent on another. Furthermore, there is no report on the in vivo characterization of the Rev3 catalytic subunit of the B-family TLS polymerase Polζ. Here we describe the detection of endogenous human Polη, Rev1, and Rev3 by immunocytochemistry using existing or newly created antibodies, as well as various means of inhibiting their expression, which allows us to examine the dynamics of endogenous TLS polymerases in response to UV irradiation. It is found that Rev1 and Polη are independently recruited to the nuclear foci, whereas the Rev3 nuclear focus formation requires Rev1 but not Polη. In contrast, neither Rev1 nor Polη recruitment requires Rev3. To further support these conclusions, we find that simultaneous suppression of Polη and Rev3 results in an additive cellular sensitivity to UV irradiation. These observations suggest a cooperative and sequential assembly of TLS polymerases in response to DNA damage. They also support and extend the current polymerase switch model.


Subject(s)
DNA Damage , DNA, Neoplasm/radiation effects , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Nuclear Proteins/metabolism , Nucleotidyltransferases/metabolism , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , DNA Replication/genetics , DNA Replication/physiology , DNA-Directed DNA Polymerase/genetics , HCT116 Cells , Humans , Nuclear Proteins/genetics , Nucleotidyltransferases/genetics , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Ultraviolet Rays
9.
Cell Res ; 18(1): 162-73, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18157158

ABSTRACT

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance (DDT) has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen (PCNA) by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis (TLS) with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.


Subject(s)
Base Pair Mismatch/physiology , DNA Damage/physiology , DNA Replication/physiology , Mutagenesis/genetics , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/physiology , Animals , Binding Sites , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/physiology , Eukaryotic Cells/metabolism , Genomic Instability/physiology , Humans , Models, Biological , Models, Molecular , Neoplasms/genetics , Prokaryotic Cells/metabolism , Protein Binding , Protein Processing, Post-Translational , SUMO-1 Protein/metabolism , Saccharomyces cerevisiae , Ubiquitination
10.
Apoptosis ; 11(12): 2147-57, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17041755

ABSTRACT

We have previously shown that UEV1 is up-regulated in all tumor cell lines examined and when SV40-transformed human embryonic kidney cells undergo immortalization; however, it is unclear whether and how UEV1 plays a critical role in this process. UEV1A encodes a ubiquitin conjugating enzyme variant, which is required for Ubc13 (ubiquitin conjugating enzyme) catalyzed poly-ubiquitination of target proteins through Lys63-linked chains. One of the target proteins is NEMO/IKKgamma (nuclear factor-kappaB essential modulator/inhibitor of kappaB protein kinase), a regulatory subunit of IkappaB kinase in the NF-kappaB signaling pathway. In this report, we show that constitutive high-level expression of UEV1A alone in cultured human cells was sufficient to cause a significant increase in NF-kappaB activity as well as the expression of its target anti-apoptotic protein, Bcl-2 (B-cell leukemia/lymphoma 2). Overexpression of UEV1A also conferred prolonged cell survival under serum-deprived conditions, and protected cells against apoptosis induced by diverse stressing agents. All of the effects of Uev1A were reversible upon suppression of UEV1 expression by RNA interference. Our observations presented in this report provide evidence that Uev1A is a critical regulatory component in the NF-kappaB signaling pathway in response to environmental stresses and identify UEV1A as a potential proto-oncogene.


Subject(s)
Apoptosis , NF-kappa B/metabolism , Transcription Factors/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Apoptosis/drug effects , Cell Survival/drug effects , Culture Media, Serum-Free , Gene Expression/drug effects , HeLa Cells , Humans , Models, Biological , Proto-Oncogene Mas , Proto-Oncogene Proteins c-bcl-2/metabolism , Tumor Necrosis Factor-alpha/pharmacology
11.
J Cell Biol ; 170(5): 745-55, 2005 Aug 29.
Article in English | MEDLINE | ID: mdl-16129784

ABSTRACT

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.


Subject(s)
Ligases/metabolism , Polyubiquitin/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Amino Acid Sequence , Animals , Carrier Proteins/metabolism , Cell Line , Cell Nucleus/metabolism , DNA Damage , DNA Repair , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , I-kappa B Kinase , Ligases/genetics , Lipopolysaccharides/metabolism , Lysine/metabolism , Macromolecular Substances , Mice , Molecular Sequence Data , NF-kappa B/metabolism , Rad51 Recombinase , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , TNF Receptor-Associated Factor 2/metabolism , TNF Receptor-Associated Factor 6/metabolism , Transcription Factors/genetics , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Protein Ligases
12.
Cell Mol Neurobiol ; 23(2): 205-10, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12735632

ABSTRACT

1. We hypothesized that non-neuronal cells could be eliminated from primary dorsal root ganglion (DRG) cultures by including a DNA topoisomerase inhibitor (camptothecin) during culture. 2. Exposure to 20 microM camptothecin for 48 h, beginning at 3 days in vitro, reliably eliminates proliferating non-neuronal cells. 3. Following camptothecin treatment, neurons survived and continued to extend neurites for several weeks without obvious defects in morphology or viability. 4. Transient camptothecin exposure is therefore an efficient and fast-acting method to purify DRG neurons in culture.


Subject(s)
Cell Culture Techniques/methods , Ganglia, Spinal/cytology , Animals , Animals, Newborn , Cell Division/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Ganglia, Spinal/physiology , Mice , Schwann Cells/cytology , Schwann Cells/physiology
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