scholarly journals Functional analysis of the biochemical activity of mammalian phosphatidylinositol 5 phosphate 4-kinase enzymes

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Swarna Mathre ◽  
K. Balasankara Reddy ◽  
Visvanathan Ramya ◽  
Harini Krishnan ◽  
Avishek Ghosh ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Cell Size ◽  
Kinase Activity ◽  
Gland Cell ◽  
Specific Activity ◽  
Salivary Gland Cell ◽  
Drosophila Genome ◽  
Biochemical Activity

Abstract Phosphatidylinositol 5 phosphate 4-kinase (PIP4K) are enzymes that catalyse the phosphorylation of phosphatidylinositol 5-phosphate (PI5P) to generate PI(4,5)P2. Mammalian genomes contain three genes, PIP4K2Α, 2B and 2C and murine knockouts for these suggested important physiological roles in vivo. The proteins encoded by PIP4K2A, 2B and 2C show widely varying specific activities in vitro; PIP4K2A is highly active and PIP4K2C 2000-times less active, and the relationship between this biochemical activity and in vivo function is unknown. By contrast, the Drosophila genome encodes a single PIP4K (dPIP4K) that shows high specific activity in vitro and loss of this enzyme results in reduced salivary gland cell size in vivo. We find that the kinase activity of dPIP4K is essential for normal salivary gland cell size in vivo. Despite their highly divergent specific activity, we find that all three mammalian PIP4K isoforms are able to enhance salivary gland cell size in the Drosophila PIP4K null mutant implying a lack of correlation between in vitro activity measurements and in vivo function. Further, the kinase activity of PIP4K2C, reported to be almost inactive in vitro, is required for in vivo function. Our findings suggest the existence of unidentified factors that regulate PIP4K enzyme activity in vivo.

scholarly journals Non-Lethal Concentration of MeHg Causes Marked Responses in the DNA Repair, Integrity, and Replication Pathways in the Exposed Human Salivary Gland Cell Line

2021 ◽  
Vol 12 ◽  
Author(s):  
Lygia Sega Nogueira ◽  
Carolina P. Vasconcelos ◽  
Jessica Rodrigues Plaça ◽  
Geovanni Pereira Mitre ◽  
Leonardo Oliveira Bittencourt ◽  
...  
Keyword(s):  
Dna Repair ◽  
Salivary Gland ◽  
Cell Line ◽  
Gland Cell ◽  
Salivary Gland Cell ◽  
Human Salivary Gland ◽  
Gland Cells ◽  
Salivary Gland Cells

In Brazilian northern Amazon, communities are potentially exposed and vulnerable to methylmercury (MeHg) toxicity through the vast ingestion of fish. In vivo and in vitro studies demonstrated that the salivary glands as a susceptible organ to this potent environmental pollutant, reporting alterations on physiological, biochemical, and proteomic parameters. However, the alterations caused by MeHg on the gene expression of the exposed human salivary gland cells are still unknown. Therefore, the goal was to perform the transcriptome profile of the human salivary gland cell line after exposure to MeHg, using the microarray technique and posterior bioinformatics analysis. The cell exposure was performed using 2.5 µM MeHg. A previously published study demonstrated that this concentration belongs to a range of concentrations that caused biochemical and metabolic alterations in this linage. As a result, the MeHg exposure did not cause lethality in the human salivary gland cells line but was able to alter the expression of 155 genes. Downregulated genes (15) are entirety relating to the cell metabolism impairment, and according to KEGG analysis, they belong to the glycosphingolipid (GSL) biosynthesis pathway. On the other hand, most of the 140 upregulated genes were related to cell-cycle progression, DNA repair, and replication pathway, or cellular defenses through the GSH basal metabolism. These genomic changes revealed the effort to the cell to maintain physiological and genomic stability to avoid cell death, being in accordance with the nonlethality in the toxicity test. Last, the results support in-depth studies on nonlethal MeHg concentrations for biomarkers identification that interpret transcriptomics data in toxicological tests serving as an early alert of physiological changes in vitro biological models.

scholarly journals Perlecan Domain IV Peptide Stimulates Salivary Gland Cell Assembly In Vitro

2009 ◽  
Vol 15 (11) ◽  
pp. 3309-3320 ◽  
Author(s):  
Swati Pradhan ◽  
Chu Zhang ◽  
Xinqiao Jia ◽  
Daniel D. Carson ◽  
Robert Witt ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Gland Cell ◽  
Salivary Gland Cell ◽  
Cell Assembly

scholarly journals AnIn VitroCulture System for Long-Term Expansion of Epithelial and Mesenchymal Salivary Gland Cells: Role of TGF-β1 in Salivary Gland Epithelial and Mesenchymal Differentiation

2013 ◽  
Vol 2013 ◽  
pp. 1-20 ◽  
Author(s):  
Kajohnkiart Janebodin ◽  
Worakanya Buranaphatthana ◽  
Nicholas Ieronimakis ◽  
Aislinn L. Hays ◽  
Morayma Reyes
Keyword(s):  
Salivary Gland ◽  
Culture System ◽  
Gland Cell ◽  
Mesenchymal Cells ◽  
Salivary Gland Cell ◽  
Salivary Gland Cells ◽  
Gland Development

Despite a pivotal role in salivary gland development, homeostasis, and disease, the role of salivary gland mesenchyme is not well understood. In this study, we used theCol1a1-GFPmouse model to characterize the salivary gland mesenchymein vitroandin vivo. TheCol1a1-GFPtransgene was exclusively expressed in the salivary gland mesenchyme.Ex vivoculture of mixed salivary gland cells in DMEM plus serum medium allowed long-term expansion of salivary gland epithelial and mesenchymal cells. The role of TGF-β1 in salivary gland development and disease is complex. Therefore, we used thisin vitroculture system to study the effects of TGF-β1 on salivary gland cell differentiation. TGF-β1 induced the expression of collagen, and inhibited the formation of acini-like structures in close proximity to mesenchymal cells, which adapted a fibroblastic phenotype. In contrast, TGF-βR1 inhibition increased acini genes and fibroblast growth factors (Fgf-7andFgf-10), decreased collagen and induced formation of larger, mature acini-like structures. Thus, inhibition of TGF-βsignaling may be beneficial for salivary gland differentiation; however, due to differential effects of TGF-β1 in salivary gland epithelial versus mesenchymal cells, selective inhibition is desirable. In conclusion, this mixed salivary gland cell culture system can be used to study epithelial-mesenchymal interactions and the effects of differentiating inducers and inhibitors.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

scholarly journals A novel PET tracer 18F-deoxy-thiamine: synthesis, metabolic kinetics, and evaluation on cerebral thiamine metabolism status

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Changpeng Wang ◽  
Siwei Zhang ◽  
Yuefei Zou ◽  
Hongzhao Ma ◽  
Donglang Jiang ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
High Accumulation ◽  
Metabolic Kinetics ◽  
Thiamine Metabolism ◽  
Pet Tracer ◽  
The Status ◽  
The Brain

Abstract Background Some neuropsychological diseases are associated with abnormal thiamine metabolism, including Korsakoff–Wernicke syndrome and Alzheimer’s disease. However, in vivo detection of the status of brain thiamine metabolism is still unavailable and needs to be developed. Methods A novel PET tracer of 18F-deoxy-thiamine was synthesized using an automated module via a two-step route. The main quality control parameters, such as specific activity and radiochemical purity, were evaluated by high-performance liquid chromatography (HPLC). Radiochemical concentration was determined by radioactivity calibrator. Metabolic kinetics and the level of 18F-deoxy-thiamine in brains of mice and marmosets were studied by micro-positron emission tomography/computed tomography (PET/CT). In vivo stability, renal excretion rate, and biodistribution of 18F-deoxy-thiamine in the mice were assayed using HPLC and γ-counter, respectively. Also, the correlation between the retention of cerebral 18F-deoxy-thiamine in 60 min after injection as represented by the area under the curve (AUC) and blood thiamine levels was investigated. Results The 18F-deoxy-thiamine was stable both in vitro and in vivo. The uptake and clearance of 18F-deoxy-thiamine were quick in the mice. It reached the max standard uptake value (SUVmax) of 4.61 ± 0.53 in the liver within 1 min, 18.67 ± 7.04 in the kidney within half a minute. The SUV dropped to 0.72 ± 0.05 and 0.77 ± 0.35 after 60 min of injection in the liver and kidney, respectively. After injection, kidney, liver, and pancreas exhibited high accumulation level of 18F-deoxy-thiamine, while brain, muscle, fat, and gonad showed low accumulation concentration, consistent with previous reports on thiamine distribution in mice. Within 90 min after injection, the level of 18F-deoxy-thiamine in the brain of C57BL/6 mice with thiamine deficiency (TD) was 1.9 times higher than that in control mice, and was 3.1 times higher in ICR mice with TD than that in control mice. The AUC of the tracer in the brain of marmosets within 60 min was 29.33 ± 5.15 and negatively correlated with blood thiamine diphosphate levels (r = − 0.985, p = 0.015). Conclusion The 18F-deoxy-thiamine meets the requirements for ideal PET tracer for in vivo detecting the status of cerebral thiamine metabolism.

scholarly journals Plant Physiology, 2021 Functional redox links between lumen thiol oxidoreductase1 and serine/threonine-protein kinase STN7

2021 ◽  
Author(s):  
Jianghao Wu ◽  
Liwei Rong ◽  
Weijun Lin ◽  
Lingxi Kong ◽  
Dengjie Wei ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Disulfide Bonds ◽  
Kinase Activity ◽  
Light Harvesting ◽  
Photosynthetic Electron Transport ◽  
State Transitions ◽  
Light Harvesting Complex ◽  
Light Harvesting Complex Ii

Abstract In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1.

scholarly journals Design of PSMA ligands with modifications at the inhibitor part: an approach to reduce the salivary gland uptake of radiolabeled PSMA inhibitors?

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Veronika Barbara Felber ◽  
Manuel Amando Valentin ◽  
Hans-Jürgen Wester
Keyword(s):  
Salivary Gland ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
In Vivo Studies ◽  
Tumor Xenograft ◽  
Tumor Uptake ◽  
Lncap Cells ◽  
High Affinity

Abstract Aim To investigate whether modifications of prostate-specific membrane antigen (PSMA)-targeted radiolabeled urea-based inhibitors could reduce salivary gland uptake and thus improve tumor-to-salivary gland ratios, several analogs of a high affinity PSMA ligand were synthesized and evaluated in in vitro and in vivo studies. Methods Binding motifs were synthesized ‘on-resin’ or, when not practicable, in solution. Peptide chain elongations were performed according to optimized standard protocols via solid-phase peptide synthesis. In vitro experiments were performed using PSMA+ LNCaP cells. In vivo studies as well as μSPECT/CT scans were conducted with male LNCaP tumor xenograft-bearing CB17-SCID mice. Results PSMA ligands with A) modifications within the central Zn2+-binding unit, B) proinhibitor motifs and C) substituents & bioisosteres of the P1′-γ-carboxylic acid were synthesized and evaluated. Modifications within the central Zn2+-binding unit of PSMA-10 (Glu-urea-Glu) provided three compounds. Thereof, only natLu-carbamate I (natLu-3) exhibited high affinity (IC50 = 7.1 ± 0.7 nM), but low tumor uptake (5.31 ± 0.94% ID/g, 1 h p.i. and 1.20 ± 0.55% ID/g, 24 h p.i.). All proinhibitor motif-based ligands (three in total) exhibited low binding affinities (> 1 μM), no notable internalization and very low tumor uptake (< 0.50% ID/g). In addition, four compounds with P1′-ɣ-carboxylate substituents were developed and evaluated. Thereof, only tetrazole derivative natLu-11 revealed high affinity (IC50 = 16.4 ± 3.8 nM), but also this inhibitor showed low tumor uptake (3.40 ± 0.63% ID/g, 1 h p.i. and 0.68 ± 0.16% ID/g, 24 h p.i.). Salivary gland uptake in mice remained at an equally low level for all compounds (between 0.02 ± 0.00% ID/g and 0.09 ± 0.03% ID/g), wherefore apparent tumor-to-submandibular gland and tumor-to-parotid gland ratios for the modified peptides were distinctly lower (factor 8–45) than for [177Lu]Lu-PSMA-10 at 24 h p.i. Conclusions The investigated compounds could not compete with the in vivo characteristics of the EuE-based PSMA inhibitor [177Lu]Lu-PSMA-10. Although two derivatives (3 and 11) were found to exhibit high affinities towards LNCaP cells, tumor uptake at 24 h p.i. was considerably low, while uptake in salivary glands remained unaffected. Optimization of the established animal model should be envisaged to enable a clear identification of PSMA-targeting radioligands with improved tumor-to-salivary gland ratios in future studies.

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