Ketohexokinase: Expression and Localization of the Principal Fructose-metabolizing Enzyme

Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact.

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Large and small splice variants of collagen XII: differential expression and ligand binding.

The Journal of Cell Biology ◽

10.1083/jcb.130.4.1005 ◽

1995 ◽

Vol 130 (4) ◽

pp. 1005-1014 ◽

Cited By ~ 77

Author(s):

M Koch ◽

B Bohrmann ◽

M Matthison ◽

C Hagios ◽

B Trueb ◽

...

Keyword(s):

Ligand Binding ◽

Splice Variants ◽

Expression Patterns ◽

Collagen I ◽

Heparin Binding ◽

Fibronectin Type Iii ◽

Alternatively Spliced ◽

Collagenous Domain ◽

Fibronectin Type

Collagen XII has a short collagenous tail and a very large, three-armed NC3 domains consisting primarily of fibronectin type III repeats. Differential splicing within this domain gives rise to a large (320 kD) and a small (220 kD) subunit; the large but not the small can carry glycosaminoglycan. To investigate whether collagen XII variants have distinct expression patterns and functions, we generated antibody and cDNA probes specific for the alternatively spliced domain. We report here that the large variant has a more restricted expression in embryonic tissue than the small. For example, whereas the small variant is widespread in the dermis, the large is limited to the base of feather buds. Distinct proportions of mRNA for the two variants were detected depending on the tissue. Monoclonal antibodies allowed us to separate collagen XII variants, and to show that homo- and heterotrimers exist. Collagen XII variants differ in ligand binding. Small subunits interact weakly with heparin via their COOH-terminal domain. Large subunits have additional, stronger heparin-binding site(s) in their NH2-terminal extra domain. In vivo, both large and small collagen XII are associated with interstitial collagen. Here we show biochemically and ultrastructurally that collagen XII can be incorporated into collagen I fibrils when it is present during, but not after, fibril formation. Removal of the collagenous domain of collagen XII reduces its coprecipitation with collagen I. Our results indicate that collagen XII is specifically associated with fibrillar collagen, and that the large variant has binding sites for extracellular ligands not present in the small variant.

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Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2003662117 ◽

2020 ◽

Vol 117 (38) ◽

pp. 23527-23538 ◽

Cited By ~ 1

Author(s):

Lisa Redlingshöfer ◽

Faye McLeod ◽

Yu Chen ◽

Marine D. Camus ◽

Jemima J. Burden ◽

...

Keyword(s):

Synaptic Vesicle ◽

Light Chain ◽

Splice Variants ◽

Membrane Deformation ◽

Functional Balance ◽

Electrophysiological Recordings ◽

Alternatively Spliced ◽

Vesicle Pool

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genesCLTAandCLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.

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Cellular Localization of Enzymatically Active Thrombin in Intact Human Tissues by Hirudin Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653870 ◽

1995 ◽

Vol 73 (05) ◽

pp. 793-797 ◽

Cited By ~ 60

Author(s):

Leo R Zacharski ◽

Vincent A Memoli ◽

William D Morain ◽

Jean-Marc Schlaeppi ◽

Sandra M Rousseau

Keyword(s):

Tumor Cell ◽

Cell Carcinoma ◽

Cellular Localization ◽

Thrombin Generation ◽

Normal Lung ◽

Cancer Tissue ◽

Immunohistochemical Techniques ◽

Tumor Types ◽

Negative Controls

SummaryCellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inihibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen in colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Honeysuckle Aqueous Extracts Induced let-7a Suppress EV71 Replication and Pathogenesis In Vitro and In Vivo and Is Predicted to Inhibit SARS-CoV-2

Viruses ◽

10.3390/v13020308 ◽

2021 ◽

Vol 13 (2) ◽

pp. 308

Author(s):

Ying-Ray Lee ◽

Chia-Ming Chang ◽

Yuan-Chieh Yeh ◽

Chi-Ying F. Huang ◽

Feng-Mao Lin ◽

...

Keyword(s):

Protein Expression ◽

Western Blotting ◽

Expression Profiles ◽

Plaque Assay ◽

Virus Titer ◽

Enterovirus 71 ◽

Luciferase Reporter ◽

Pathogen Invasion

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

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A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

International Journal of Molecular Sciences ◽

10.3390/ijms22042141 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2141

Author(s):

Srinu Tumpara ◽

Elena Korenbaum ◽

Mark Kühnel ◽

Danny Jonigk ◽

Beata Olejnicka ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Western Blotting ◽

Complex Mixture ◽

Immunoglobulin M ◽

Confocal Laser ◽

Enzyme Linked Immunosorbent Assay ◽

Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

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Extracellular signal-regulated kinase inhibition prevents venous adaptive remodeling via regulation of Eph-B4

Vascular ◽

10.1177/1708538121999854 ◽

2021 ◽

pp. 170853812199985

Author(s):

Yuanyuan Guo ◽

Fan Zhu ◽

Xiong Zhang ◽

Guangmin Wu ◽

Pinting Fu ◽

...

Keyword(s):

Western Blotting ◽

Vein Graft ◽

Artery Bypass ◽

Bypass Graft ◽

Graft Loss ◽

Elastic Fibers ◽

Sprague Dawley ◽

Vein Grafts ◽

Graft Stenosis

Objectives Vein graft adaptation (VGA) is a process that vein as a vascular graft conduits in arterial reconstructive surgery; VGA can lead to postoperative vein graft stenosis (VGS) and complications after coronary artery bypass graft and other peripheral artery bypass surgeries. VGA is characterized by vein graft loss the venous features without exhibiting arterial features; furthermore, the activation of ERK inhibited the maintenance of venous properties of the vein graft. We hypothesized that ERK inhibition can affect vein VGS through regulating the expression of EphB4. Methods Rat vein transplantation model was established using wild-type and EphB4+/− Sprague-Dawley rats. Hematoxylin-eosin, Masson, Verhoeff, actin staining, and immunohistochemistry were applied to observe the structure of the vein grafts. Vascular smooth muscle cells (VSMCs) were isolated from the vein and vein grafts. Western blotting was used to determine the expression of p-ERK1/2 and EphB4, and immunofluorescence was applied to detect the expression and location of EphB4. Cell wound scratch assay and CCK8 assay were used to determine the migration and proliferation of VSMCs. Real-time polymerase chain reaction was used to determine the mRNA expression of EphB4. Results Western blotting in vein sample and vein graft sample detected p-ERK1/2 and ERK1/2 expression in both EphB4+/+ and EphB4+/− rats. The expression of p-ERK was increased in vein graft compared to vein. Immunofluorescence in VSMCs form EphB4+/+ and EphB4+/− rats detected EphB4 expression in both cells, and the expression of EphB4 was increased in VSMCs form EphB4+/+ rats. SCH772984 reduces the proliferation and migration of VSMCs. Inhibition of ERK suppressed the increase of vein graft wall thickness, and the expression of collagen fibers, elastic fibers, and α-actin was decreased. Vein graft from EphB4+/− rats reduces the expression of EphB4, and SCH772984 suppressed the decrease of EphB4 in vivo. Vein graft from EphB4+/− rats increased the expression of EphB4, and SCH772984 suppressed the increase of EphB4 in vivo. Conclusions The inhibition of ERK1/2 suppressed the process of VGS by decreasing the proliferation of VSMCs. The ERK-inhibitor SCH772984 suppressed the level of VGS by extending the time of EphB4 expression during the process of VGA, thus maintaining the venousization of vein graft. The mechanism may be that the inhibitor SCH772984 suppresses the level of VGS by extending the time of EphB4 expression during the process of VGA. Therefore, our research provides a new target of VGS treatment by inhibiting the expression of ERK1/2 through the process of VGA.

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GMP-compliant fully automated radiosynthesis of [18F]FEPPA for PET/MRI imaging of regional brain TSPO expression

EJNMMI Research ◽

10.1186/s13550-021-00768-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chi-Wei Chang ◽

Chuang-Hsin Chiu ◽

Ming-Hsien Lin ◽

Hung-Ming Wu ◽

Tsung-Hsun Yu ◽

...

Keyword(s):

Mr Imaging ◽

Western Blotting ◽

Second Generation ◽

Mrna Levels ◽

Automated Radiosynthesis ◽

Lps Challenge ◽

Tnf Α ◽

The Brain ◽

Tspo Expression

Abstract Background Expression of translocator protein (TSPO) on the outer mitochondrial membrane of activated microglia is strongly associated with neuroinflammation. The second-generation PET ligand [18F]FEPPA specifically binds TSPO to enable in vivo visualization and quantification of neuroinflammation. We optimized a fully automated radiosynthesis method and evaluated the utility of [18F]FEPPA, the second-generation PET ligand specifically binds TSPO, in a mouse model of systemic LPS challenge to detect TSPO-associated signals of central and peripheral inflammation. In vivo dynamic PET/MR imaging was performed in LPS-induced and control mice after [18F]FEPPA administration. The relationship between the [18F]FEPPA signal and the dose of LPS was assessed. The cytokine levels (i.e., TNF-α, Il-1β, Il-6) in LPS-induced mice were measured by RT-PCR. Standard uptake value (SUV), total volume of distribution (VT) and area under the curve (AUC) were determined based on the metabolite-uncorrected plasma input function. Western blotting and immunostaining were used to measure TSPO expression in the brain. Results The fully automated [18F]FEPPA radiosynthesis produced an uncorrected radiochemical yield of 30 ± 2% within 80 min, with a radiochemical purity greater than 99% and specific activity of 148.9‒216.8 GBq/µmol. Significant differences were observed in the brain after [18F]FEPPA administration: SUV, VT and AUC were 1.61 ± 0.1, 1.25 ± 0.12 and 1.58 ± 0.09-fold higher in LPS-injected mice than controls. TNF-α, Il-1β and Il-6 mRNA levels were also elevated in the brains of LPS-injected mice. Western blotting revealed TSPO (p < 0.05) and Iba-1 (p < 0.01) were upregulated in the brain after LPS administration. In LPS-injected mice, TSPO immunoactivity colocalized with Iba-1 in the cerebrum and TSPO was significantly overexpressed in the hippocampus and cerebellum. The peripheral organs (heart, lung) of LPS-injected mice had higher [18F]FEPPA signal-to-noise ratios than control mice. Conclusions Based on the current data on ligand specificity and selectivity in central tissues using 7 T PET/MR imaging, we demonstrate that [18F]FEPPA accumulations significant increased in the specific brain regions of systemic LPS-induced neuroinflammation (5 mg/kg). Future investigations are needed to determine the sensitivity of [18F]FEPPA as a biomarker of neuroinflammation as well as the correlation between the PET signal intensity and the expression levels of TSPO.

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Generation of tetracycline-controllable CYP3A4-expressing Caco-2 cells by the piggyBac transposon system

Scientific Reports ◽

10.1038/s41598-021-91160-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Moe Ichikawa ◽

Hiroki Akamine ◽

Michika Murata ◽

Sumito Ito ◽

Kazuo Takayama ◽

...

Keyword(s):

Small Intestine ◽

Drug Absorption ◽

Cell Model ◽

Negative Effects ◽

Piggybac Transposon ◽

Drug Metabolizing Enzyme ◽

Metabolizing Enzyme ◽

Cyp3a4 Expression

AbstractCaco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.

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Adipocytes derived fibrinolytic components in peritoneum — a pilot study

Open Medicine ◽

10.2478/s11536-012-0042-8 ◽

2012 ◽

Vol 7 (5) ◽

pp. 604-609

Author(s):

Sylvie Opatrna ◽

Marie Korabečná ◽

Věra Křížková ◽

Zbynek Tonar ◽

Jitka Kočová ◽

...

Keyword(s):

Adipose Tissue ◽

Plasminogen Activator ◽

Cellular Localization ◽

Plasminogen Activator Inhibitor ◽

Immunohistochemical Localization ◽

Related Factors ◽

Mutual Correlation ◽

Transport Characteristic ◽

Diabetes Status ◽

Pai 1

AbstractThe proteins of the fibrinolytic system — urokinase plasminogen activator(uPA), tissue plasminogen activator (tPA)and plasminogen activator inhibitor type IPAI-I) — play important roles in fibrotization in various organs and including peritoneum. To study the cellular localization of PAI-1, tPA and uPA within the adipose tissue of the peritoneal membrane in patients at the onset of peritoneal dialysis(PD) we determined the initial expression of these proteins in relationship to multiple clinical variables. Methods: routinely performed parietal peritoneal biopsies in 12 patients undergoing peritoneal catheter implantation were examined. We used formalinfixed, paraffin-embedded specimens for immunohistochemical localization of these proteins along with the stereological pointcounting method for quantification of their expression within the peritoneal adipose tissue. Results: strong positive mutual correlation between the expression of PAI-1 and both uPA (SpearmanR=0.66) and tPA (R=0.59) as well as between the expression of uPA and tPA (R=0.77) was found without any relatioship to BMI, age, peritoneal transport characteristic or diabetes status. Conclusion: Adipose tissue within the peritoneum is capable of producing fibrinolysis regulators (independently on clinical parameters) thus possibly affecting the fibrotization and function of peritoneum as dialysis membrane. The effect of dialysis solution dosing, composition and other dialysis related factors should be clarified in future studies.

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