scholarly journals CCL2 affects fat metabolism in liver regeneration by regulating ADRP expression

2020 ◽  
Vol 76 (11) ◽  
pp. 652-655
Author(s):  
XUEKUN XING ◽  
HUI WAN ◽  
LAN ZHAO
Keyword(s):  
Liver Regeneration ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Fat Metabolism ◽  
Serum Levels ◽  
Shrna Expression ◽  
Expression Levels ◽  
Liver Index ◽  
Adipose Differentiation ◽  
Green Fluorescent

This study aimed to elucidate the effect of chemokine (c-c motif) ligand 2 (CCL2) on fat metabolism in liver regeneration. CCL2 shRNA expression plasmids were constructed and transfected into rats using hydraulic transgenic technology. Transfection efficiency was measured using a fluorescence microscope. We also measured serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to test liver functioning. The weights of regenerative livers were recorded and the liver index was calculated. We used immunohistochemistry to determine the expression of PCNA, and used western blot to measure expression levels of adipose differentiation related protein (ADRP). After transfection of pGenesil-1.0-ccl2 into the liver, expression levels of green fluorescent protein were 35% at 6 h, and the liver index as well as levels of ALT, AST, PCNA, and ADRP were all lower than those in the group that underwent partial hepatectomy. We conclude that CCL2 may affect fat metabolism in liver regeneration by inhibiting the expression of ADRP.

scholarly journals Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

2021 ◽  
Vol 22 (4) ◽  
pp. 2127
Author(s):  
Jakub Suchodolski ◽  
Anna Krasowska
Keyword(s):  
Candida Albicans ◽  
Multidrug Resistance ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
De Novo ◽  
Pathogenic Fungus ◽  
Serum Levels ◽  
Efflux Transporters ◽  
Fluconazole Resistance ◽  
Green Fluorescent

Candida albicans is a pathogenic fungus that is increasingly developing multidrug resistance (MDR), including resistance to azole drugs such as fluconazole (FLC). This is partially a result of the increased synthesis of membrane efflux transporters Cdr1p, Cdr2p, and Mdr1p. Although all these proteins can export FLC, only Cdr1p is expressed constitutively. In this study, the effect of elevated fructose, as a carbon source, on the MDR was evaluated. It was shown that fructose, elevated in the serum of diabetics, promotes FLC resistance. Using C. albicans strains with green fluorescent protein (GFP) tagged MDR transporters, it was determined that the FLC-resistance phenotype occurs as a result of Mdr1p activation and via the increased induction of higher Cdr1p levels. It was observed that fructose-grown C. albicans cells displayed a high efflux activity of both transporters as opposed to glucose-grown cells, which synthesize Cdr1p but not Mdr1p. Additionally, it was concluded that elevated fructose serum levels induce the de novo production of Mdr1p after 60 min. In combination with glucose, however, fructose induces Mdr1p production as soon as after 30 min. It is proposed that fructose may be one of the biochemical factors responsible for Mdr1p production in C. albicans cells.

scholarly journals New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters

2016 ◽  
Vol 82 (8) ◽  
pp. 2240-2246 ◽  
Author(s):  
Alex I. Kanno ◽  
Cibelly Goulart ◽  
Henrique K. Rofatto ◽  
Sergio C. Oliveira ◽  
Luciana C. C. Leite ◽  
...  
Keyword(s):  
Mycobacterium Bovis ◽  
Fluorescent Protein ◽  
Vaccine Development ◽  
Expression Vectors ◽  
Content Type ◽  
Expression Levels ◽  
Wide Range ◽  
Mycobacteriophage L5 ◽  
Green Fluorescent ◽  
Selection Of

ABSTRACTThe expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.

A quantitative method for normalization of transfection efficiency using enhanced green fluorescent protein

2005 ◽  
Vol 342 (2) ◽  
pp. 341-344 ◽  
Author(s):  
Dineshkumar H. Dandekar ◽  
Manish Kumar ◽  
Jayashree S. Ladha ◽  
Krishna N. Ganesh ◽  
Debashis Mitra
Keyword(s):  
Green Fluorescent Protein ◽  
Quantitative Method ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Green Fluorescent

scholarly journals Nucleofection Efficiency of Sheep Primary Fibroblasts Established from Refrigerated Skin

2018 ◽  
Vol 10 (4) ◽  
pp. 12
Author(s):  
Mahipal Singh ◽  
Xiaoling Ma
Keyword(s):  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Genetically Engineered ◽  
Dermal Fibroblasts ◽  
Biologically Active ◽  
Cell Type ◽  
Gfp Expression ◽  
Primary Fibroblasts ◽  
Species Specific ◽  
Green Fluorescent

Dermal fibroblasts are useful for production of genetically engineered biologically active factors for development of cellular therapies and tissue engineering products for regenerative medicine. However, their transfection efficiencies using traditional non-viral methods are low and vary based on cell-type and species-specific differences. Using nucleofection technology, here we show that the transfection efficiency of primary fibroblasts established after 0-, 35-, and 65-days of postmortem storage of sheep skin tissues in a refrigerator was 59.49 % ± 9.66 %, 59.33 % ± 11.59 %, and 43.48 % ± 8.09 % respectively, as determined by analysis of green fluorescent protein (GFP) expression. 

Morphological Distinction Unravels Mechanisms Of Platelet Biogenesis From Bone Marrow Megakaryocytes

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2428-2428
Author(s):  
Satoshi Nishimura ◽  
Koji Eto ◽  
Ryozo Nagai
Keyword(s):  
Bone Marrow ◽  
Fluorescent Protein ◽  
Conflicts Of Interest ◽  
Human Subjects ◽  
Blood Platelets ◽  
Serum Levels ◽  
Cytokine Balance ◽  
Green Fluorescent

Abstract Blood platelets are generated in the bone marrow (BM) from their precursors, megakaryocytes (MK). Although we know that MKs produce platelets throughout life, precisely how platelets are produced in vivo remains uncertain, largely because of the rarity of MKs in the BM and the lack an adequate visualization technique. In the present study, we were able to visualize MK dynamics leading to platelet release in living animals at high resolution. To clearly understand the nature of thrombopoiesis in BM MKs, we optimized an in vivo imaging technique based on two-photon microscopy that enabled us to visualize living BM in CAG- enhanced green fluorescent protein (eGFP) mice. By visualizing living bone marrow in vivo, we observed that two modes (fragmentation and proplatelet formation) can be ongoing simultaneously in the same mouse. We observed that these two modes detectable by different morphological behavior can be ongoing simultaneously in the same BM of mouse, and are regulated by specific cytokines. Short proplatelets from megakaryocytes predominated at steady state, and more elongated proplatelets were accelerated by thrombopoietin (TPO) with responding to chronic platelet needs including recovery form BM transplantations. In contrast, acute platelet needs by blood loss, 5-FU administration or pritoneal acute inflammation increased cytoplasmic fragmentation following rapid ‘rupture’. Observed two modes are both dependent on tubulin reorganization on platelet biogenesis. In addition, platelet increase at acute phase is independent of proliferation by MK progenitors and this factor might exert apoptosis machinery on already reserved mature type of MKs. This humoral factor was identified by combination of in vitro screening systems and in vivo MK visualization analysis. Factor serum levels were reduced independently of the thrombopoietin level in human subjects with low platelet counts. It thus appears the cytokine balance dynamically regulates the mode of thrombopoiesis and the cellular programming of MKs. Thus, these novel factor may be a novel therapeutic target in thrombocytopenic situations, especially when associated with acute loss of platelets or when platelet transfusion is limited or unsuccessful. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Isolation, Characterization, and Localization of a Capsule-Associated Gene, CAP10, of Cryptococcus neoformans

1999 ◽  
Vol 181 (18) ◽  
pp. 5636-5643 ◽  
Author(s):  
Y. C. Chang ◽  
K. J. Kwon-Chung
Keyword(s):  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Wild Strain ◽  
Extracellular Polysaccharide ◽  
Capsule Formation ◽  
Expression Levels ◽  
Model Studies ◽  
Isolation And Characterization ◽  
Green Fluorescent

ABSTRACT Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene, CAP10, which is required for capsule formation. Complementation of the acapsularcap10 mutant produced an encapsulated strain and the deletion of CAP10 from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When CAP10 was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that CAP10 was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of CAP10 are modulated by the transcriptional factorSTE12α. Deletion of STE12α downregulated the expression levels of CAP10 while overexpression ofSTE12α upregulated the expression levels ofCAP10. Animal model studies indicate that deletion of theCAP10 gene results in the loss of virulence, and complementation of the acapsular phenotype of cap10restores virulence. Thus, CAP10 is required for capsule formation and virulence.

scholarly journals Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

2018 ◽  
Vol 2 (4) ◽  
pp. 671-678
Author(s):  
Pedro Esponda
Keyword(s):  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Foreign Gene ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

scholarly journals Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

2000 ◽  
Vol 84 (09) ◽  
pp. 460-467 ◽  
Author(s):  
M. L. M. Lamfers ◽  
M. J. Wijnberg ◽  
J. M. Grimbergen ◽  
L. G. M. Huisman ◽  
M. C. Aalders ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Adenoviral Gene Transfer ◽  
Amino Terminal ◽  
Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

scholarly journals Novel Histone-Based DNA Carrier Targeting Cancer-Associated Fibroblasts

Polymers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1695
Author(s):  
Alexey Kuzmich ◽  
Olga Rakitina ◽  
Dmitry Didych ◽  
Victor Potapov ◽  
Marina Zinovyeva ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Growth Factor Receptor ◽  
Surface Protein ◽  
Firefly Luciferase ◽  
Luciferase Assay ◽  
Cancer Associated Fibroblasts ◽  
Histone H2a ◽  
Green Fluorescent

Nuclear proteins, like histone H2A, are promising non-viral carriers for gene delivery since they are biocompatible, biodegradable, bear intrinsic nuclear localization signal, and are easy to modify. The addition of surface-protein-binding ligand to histone H2A may increase its DNA delivery efficiency. Tumor microenvironment (TME) is a promising target for gene therapy since its surface protein repertoire is more stable than that of cancer cells. Cancer-associated fibroblasts (CAFs) are important components of TME, and one of their surface markers is beta-type platelet-derived growth factor receptor (PDGFRβ). In this study, we fused histone H2A with PDGFRβ-binding peptide, YG2, to create a novel non-viral fibroblast-targeting DNA carrier, H2A-YG2. The transfection efficiency of histone complexes with pDNA encoding a bicistronic reporter (enhanced green fluorescent protein, EGFP, and firefly luciferase) in PDGFRβ-positive and PDGFRβ-negative cells was estimated by luciferase assay and flow cytometry. The luciferase activity, percentage of transfected cells, and overall EGFP fluorescence were increased due to histone modification with YG2 only in PDGFRβ-positive cells. We also estimated the internalization efficiency of DNA-carrier complexes using tetramethyl-rhodamine-labeled pDNA. The ligand fusion increased DNA internalization only in the PDGFRβ-positive cells. In conclusion, we demonstrated that the H2A-YG2 carrier targeted gene delivery to PDGFRβ-positive tumor stromal cells.

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