O6-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability

The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in O6-position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as protein tags for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of thermozymes, emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.

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Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

Analytical Methods ◽

10.1039/d1ay00020a ◽

2021 ◽

Author(s):

Lijuan Liu ◽

Shengting Zhang ◽

Xiaodan Zheng ◽

Hongmei Li ◽

Qi Chen ◽

...

Keyword(s):

Carbon Dots ◽

Fusobacterium Nucleatum ◽

Living Cells ◽

First Time ◽

Fluorescent Carbon Dots ◽

Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

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Diabetic Cataract—Pathogenesis, Epidemiology and Treatment

Journal of Ophthalmology ◽

10.1155/2010/608751 ◽

2010 ◽

Vol 2010 ◽

pp. 1-8 ◽

Cited By ~ 154

Author(s):

Andreas Pollreisz ◽

Ursula Schmidt-Erfurth

Keyword(s):

Experimental Studies ◽

Disease Process ◽

Basic Research ◽

Polyol Pathway ◽

Population Based ◽

Diabetic Patients ◽

Diabetic Cataract ◽

Cataract Development

Cataract in diabetic patients is a major cause of blindness in developed and developing countries. The pathogenesis of diabetic cataract development is still not fully understood. Recent basic research studies have emphasized the role of the polyol pathway in the initiation of the disease process. Population-based studies have greatly increased our knowledge concerning the association between diabetes and cataract formation and have defined risk factors for the development of cataract. Diabetic patients also have a higher risk of complications after phacoemulsification cataract surgery compared to nondiabetics. Aldose-reductase inhibitors and antioxidants have been proven beneficial in the prevention or treatment of this sightthreatening condition in in vitro and in vivo experimental studies. This paper provides an overview of the pathogenesis of diabetic cataract, clinical studies investigating the association between diabetes and cataract development, and current treatment of cataract in diabetics.

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SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

The Journal of General Physiology ◽

10.1085/jgp.16.2.233 ◽

1932 ◽

Vol 16 (2) ◽

pp. 233-242 ◽

Cited By ~ 27

Author(s):

B. G. Wilkes ◽

Elizabeth T. Palmer

Keyword(s):

Physiological Activity ◽

Outer Region ◽

Living Cells ◽

Yeast Cells ◽

Live Cells ◽

Intracellular Enzyme ◽

Relationship Of ◽

Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

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In Vitro and in Vivo Imaging of Nitroxyl with Copper Fluorescent Probe in Living Cells and Zebrafish

Molecules ◽

10.3390/molecules23102551 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2551 ◽

Cited By ~ 3

Author(s):

Sathyadevi Palanisamy ◽

Yu-Liang Wang ◽

Yu-Jen Chen ◽

Chiao-Yun Chen ◽

Fu-Te Tsai ◽

...

Keyword(s):

Critical Role ◽

Model Organism ◽

Living Cells ◽

Biological Species ◽

Zebrafish Model ◽

Physiological Processes ◽

Collective Data ◽

Angeli's Salt

Nitroxyl (HNO) plays a critical role in many physiological processes which includes vasorelaxation in heart failure, neuroregulation, and myocardial contractility. Powerful imaging tools are required to obtain information for understanding the mechanisms involved in these in vivo processes. In order to develop a rapid and high sensitive probe for HNO detection in living cells and the zebrafish model organism, 2-((2-(benzothiazole-2yl)benzylidene) amino)benzoic acid (AbTCA) as a ligand, and its corresponding copper(II) complex Cu(II)-AbTCA were synthesized. The reaction results of Cu(II)-AbTCA with Angeli’s salt showed that Cu(II)-AbTCA could detect HNO quantitatively in a range of 40–360 µM with a detection limit of 9.05 µM. Furthermore, Cu(II)-AbTCA is more selective towards HNO over other biological species including thiols, reactive nitrogen, and reactive oxygen species. Importantly, Cu(II)-AbTCA was successfully applied to detect HNO in living cells and zebrafish. The collective data reveals that Cu(II)-AbTCA could be used as a potential probe for HNO detection in living systems.

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Organoid based personalized medicine: from bench to bedside

Cell Regeneration ◽

10.1186/s13619-020-00059-z ◽

2020 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Yaqi Li ◽

Peiyuan Tang ◽

Sanjun Cai ◽

Junjie Peng ◽

Guoqiang Hua

Keyword(s):

Stem Cells ◽

Personalized Medicine ◽

Adult Stem Cells ◽

Three Dimensional ◽

Basic Research ◽

Normal Tissues ◽

Technology Applications ◽

Genetically Manipulated

AbstractThree-dimensional cultured organoids have become a powerful in vitro research tool that preserves genetic, phenotypic and behavioral trait of in vivo organs, which can be established from both pluripotent stem cells and adult stem cells. Organoids derived from adult stem cells can be established directly from diseased epithelium and matched normal tissues, and organoids can also be genetically manipulated by CRISPR-Cas9 technology. Applications of organoids in basic research involve the modeling of human development and diseases, including genetic, infectious and malignant diseases. Importantly, accumulating evidence suggests that biobanks of patient-derived organoids for many cancers and cystic fibrosis have great value for drug development and personalized medicine. In addition, organoids hold promise for regenerative medicine. In the present review, we discuss the applications of organoids in the basic and translational research.

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Induction and Maturation of Hepatocyte-Like Cells In Vitro: Focus on Technological Advances and Challenges

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.765980 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ye Xie ◽

Jia Yao ◽

Weilin Jin ◽

Longfei Ren ◽

Xun Li

Keyword(s):

Genetic Manipulation ◽

Toxicity Testing ◽

Basic Research ◽

Drug Approval ◽

Cell Types ◽

Comprehensive Overview ◽

Technological Advances ◽

Degree Of Differentiation

Limited by the poor proliferation and restricted sources of adult hepatocytes, there is an urgent need to find substitutes for proliferation and cultivation of mature hepatocytes in vitro for use in disease treatment, drug approval, and toxicity testing. Hepatocyte-like cells (HLCs), which originate from undifferentiated stem cells or modified adult cells, are considered good candidates because of their advantages in terms of cell source and in vitro expansion ability. However, the majority of induced HLCs are in an immature state, and their degree of differentiation is heterogeneous, diminishing their usability in basic research and limiting their clinical application. Therefore, various methods have been developed to promote the maturation of HLCs, including chemical approaches, alteration of cell culture systems, and genetic manipulation, to meet the needs of in vivo transplantation and in vitro model establishment. This review proposes different cell types for the induction of HLCs, and provide a comprehensive overview of various techniques to promote the generation and maturation of HLCs in vitro.

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Real time imaging reveals a peroxisomal reticulum in living cells

Journal of Cell Science ◽

10.1242/jcs.113.20.3663 ◽

2000 ◽

Vol 113 (20) ◽

pp. 3663-3671 ◽

Cited By ~ 1

Author(s):

M. Schrader ◽

S.J. King ◽

T.A. Stroh ◽

T.A. Schroer

Keyword(s):

Real Time ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Real Time Imaging ◽

Peroxisomal Targeting ◽

Microtubule Motor ◽

Targeting Signal ◽

Peroxisomal Targeting Signal

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.

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Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.57-66.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 57-66

Author(s):

M Zuber ◽

E M Tan ◽

M Ryoji

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Living Cells ◽

Nuclear Antigen ◽

Plasmid Replication ◽

Dna Polymerase Delta ◽

Dna Polymerase Alpha ◽

Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

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Establishment of a novel human CIC-DUX4 sarcoma cell line, Kitra-SRS, with autocrine IGF-1R activation and metastatic potential to the lungs

Scientific Reports ◽

10.1038/s41598-019-52143-3 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Sho Nakai ◽

Shutaro Yamada ◽

Hidetatsu Outani ◽

Takaaki Nakai ◽

Naohiro Yasuda ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Fusion Gene ◽

Primary Tumour ◽

Chromosomal Rearrangements ◽

Basic Research ◽

Metastatic Potential ◽

Original Tumour

Abstract Approximately 60–70% of EWSR1-negative small blue round cell sarcomas harbour a rearrangement of CIC, most commonly CIC-DUX4. CIC-DUX4 sarcoma (CDS) is an aggressive and often fatal high-grade sarcoma appearing predominantly in children and young adults. Although cell lines and their xenograft models are essential tools for basic research and development of antitumour drugs, few cell lines currently exist for CDS. We successfully established a novel human CDS cell line designated Kitra-SRS and developed orthotopic tumour xenografts in nude mice. The CIC-DUX4 fusion gene in Kitra-SRS cells was generated by t(12;19) complex chromosomal rearrangements with an insertion of a chromosome segment including a DUX4 pseudogene component. Kitra-SRS xenografts were histologically similar to the original tumour and exhibited metastatic potential to the lungs. Kitra-SRS cells displayed autocrine activation of the insulin-like growth factor 1 (IGF-1)/IGF-1 receptor (IGF-1R) pathway. Accordingly, treatment with the IGF-1R inhibitor, linsitinib, attenuated Kitra-SRS cell growth and IGF-1-induced activation of IGF-1R/AKT signalling both in vitro and in vivo. Furthermore, upon screening 1134 FDA-approved drugs, the responses of Kitra-SRS cells to anticancer drugs appeared to reflect those of the primary tumour. Our model will be a useful modality for investigating the molecular pathology and therapy of CDS.

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