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Cells Oct 2022Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures,...
Cell detachment techniques using animal-derived enzymes are necessary for the production of biopharmaceuticals that are made with the help of adherent cell cultures, although the majority of protein therapeutics (>USD 100 billion of income per year) are made under suspension cultures that do not require animal-derived proteins for manufacture. In this study, we establish the optimal Vero cell detachment process, and analyze physiological changes during cell detachment at the cellular and molecular levels. Using flow cytometry, we find that animal-based enzymes are more likely to induce apoptosis than animal-origin-free enzymes. We analyze the levels of RNAs, proteins, and metabolites in cells treated with two detachment strategies, and identify 1237 differentially expressed genes, 2883 differential proteins, and 210 differential metabolites. Transcriptomic analysis shows that animal-origin-free enzymes have a less significant effect on gene expression levels. Combined with proteomic analysis, animal-based enzymes affect the oxidative phosphorylation process and reduce the mRNA and protein levels of Cytochrome C Oxidase Assembly Protein 17 (COX17), which is a Cytochrome C Oxidase Copper Chaperone involved in the mitochondrial respiratory chain. Metabolomics analysis indicates that the levels of spermine and spermidine, which are involved in the glutathione metabolism pathway and apoptosis inhibition, are significantly reduced. Therefore, COX17, spermine, and spermidine may be biomarkers for evaluating the cell subculture process. In conclusion, we have deeply characterized the cell subculture process through multi-omics, which may provide important guidance for research and process evaluation to optimize cell detachment processes.
Topics: Chlorocebus aethiops; Animals; Vero Cells; Electron Transport Complex IV; Spermidine; Spermine; Proteomics
PubMed: 36359792
DOI: 10.3390/cells11213396 -
Biosensors Aug 2022The biogenic aliphatic polyamines (spermine, spermidine, and putrescine) are responsible for numerous cell functions, including cell proliferation, the stabilization of... (Review)
Review
The biogenic aliphatic polyamines (spermine, spermidine, and putrescine) are responsible for numerous cell functions, including cell proliferation, the stabilization of nucleic acid conformations, cell division, homeostasis, gene expression, and protein synthesis in living organisms. The change of polyamine concentrations in the urine or blood is usually related to the presence of malignant tumors and is regarded as a biomarker for the early diagnosis of cancer. Therefore, the detection of polyamine levels in physiological fluids can provide valuable information in terms of cancer diagnosis and in monitoring therapeutic effects. In this review, we summarize the recent advances in fluorescent methods for polyamine detection (supramolecular fluorescent sensing systems, fluorescent probes based on the chromophore reaction, fluorescent small molecules, and fluorescent nanoparticles). In addition, tumor polyamine-suppressing strategies (such as polyamine conjugate, polyamine analogs, combinations that target multiple components, spermine-responsive supramolecular chemotherapy, a combination of polyamine consumption and photodynamic therapy, etc.) are highlighted. We hope that this review promotes the development of more efficient polyamine detection methods and provides a comprehensive understanding of polyamine-based tumor suppressor strategies.
Topics: Humans; Neoplasms; Polyamines; Putrescine; Spermidine; Spermine
PubMed: 36005029
DOI: 10.3390/bios12080633 -
Plant Physiology Oct 1990The diamine putrescine, the triamine spermidine, and the tetramine spermine are ubiquitous in plant cells, while other polyamines are of more limited occurrence. Their... (Review)
Review
The diamine putrescine, the triamine spermidine, and the tetramine spermine are ubiquitous in plant cells, while other polyamines are of more limited occurrence. Their chemistry and pathways of biosynthesis and metabolism are well characterized. They occur in the free form as cations, but are often conjugated to small molecules like phenolic acids and also to various macromolecules. Their titer varies from approximately micromolar to more than millimolar, and depends greatly on environmental conditions, especially stress. In cereals, the activity of one of the major polyamine biosynthetic enzymes, arginine decarboxylase, is rapidly and dramatically increased by almost every studied external stress, leading to 50-fold or greater increases in putrescine titer within a few hours. The physiological significance of this increase is not yet clear, although most recent work suggests an adaptive, protective role. Polyamines produced through the action of ornithine decarboxylase, by contrast, seem essential for DNA replication and cell division. The application of exogenous polyamines produces effects on patterns of senescence and morphogenesis, suggesting but not proving a regulatory role for polyamines in these processes. The evidence for such a regulatory role is growing.
Topics: Aging; Carboxy-Lyases; Morphogenesis; Ornithine Decarboxylase; Plant Development; Plant Physiological Phenomena; Plants; Polyamines; Putrescine; Spermidine; Spermine
PubMed: 11537482
DOI: 10.1104/pp.94.2.406 -
Chemical & Pharmaceutical Bulletin 2016Polyamines, such as spermine (1), thermospermine (2) and norspermine (3), are widely distributed in nature, and have multiple biological activities. In addition, many of...
Polyamines, such as spermine (1), thermospermine (2) and norspermine (3), are widely distributed in nature, and have multiple biological activities. In addition, many of their conjugates have potential for pharmacological use. Here, we present a solid-phase synthesis using our nitrobenzenesulfonyl (Ns) strategy, which can provide 1, 2 and 3 on a gram scale. This approach should be suitable for facile construction of a diverse library of polyamines.
Topics: Molecular Structure; Spermine
PubMed: 27581645
DOI: 10.1248/cpb.c16-00355 -
Allergy Oct 2018Airway epithelial injury is a crucial component of acute and severe asthma pathogenesis and a promising target for treatment of refractory asthma. However, the...
BACKGROUND
Airway epithelial injury is a crucial component of acute and severe asthma pathogenesis and a promising target for treatment of refractory asthma. However, the underlying mechanism of epithelial injury remains poorly explored. Although high levels of polyamines, mainly spermine, have been found in asthma and comorbidity, their role in airway epithelial injury and the cause of their altered levels in asthma have not been explored.
METHODS
We measured key polyamine metabolic enzymes in lung samples from normal and asthmatic subjects and in mice with OVA-induced allergic airway inflammation (AAI). Polyamine metabolism was modulated using pharmacologic/genetic modulators. Epithelial stress and apoptosis were measured by TSLP levels and TUNEL assay, respectively.
RESULTS
We found loss of the polyamine catabolic enzymes spermidine/spermine-N (1)-acetyltransferase-1 (SAT1) and spermine oxidase (SMOX) predominantly in bronchial epithelial cells (BECs) of human asthmatic lung samples and mice with AAI. In naïve mice, SAT1 or SMOX knockdown led to airway hyper-responsiveness, remodeling, and BEC apoptosis. Conversely, in mice with AAI, overexpression of either SAT1 or SMOX alleviated asthmatic features and reduced TSLP levels and BEC apoptosis. Similarly, while pharmacological induction of SAT1 and SMOX using the polyamine analogue bis(ethyl)norspermine (BENSPM) alleviated asthmatic features with reduced TSLP levels and BEC apoptosis, pharmacological inhibition of these enzymes using BERENIL or MDL72527, respectively, worsened them. Spermine accumulation in lungs correlated with BEC apoptosis, and spermine treatment caused apoptosis of human BEAS-2B cells in vitro.
CONCLUSIONS
Spermine induces BEC injury. Induction of polyamine catabolism may represent a novel therapeutic approach for asthma via reversing BEC stress.
Topics: Animals; Apoptosis; Asthma; Epithelial Cells; Epithelium; Humans; Lung; Mice; Polyamines; Respiratory System; Spermine
PubMed: 29729200
DOI: 10.1111/all.13472 -
Amino Acids Feb 2010N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus...
N-alkylated polyamine analogues have potential as anticancer and antiparasitic drugs. However, their metabolism in the host has remained incompletely defined thus potentially limiting their utility. Here, we have studied the degradation of three different spermine analogues N,N'-bis-(3-ethylaminopropyl)butane-1,4-diamine (DESPM), N-(3-benzyl-aminopropyl)-N'-(3-ethylaminopropyl)butane-1,4-diamine (BnEtSPM) and N,N'-bis-(3-benzylaminopropyl)butane-1,4-diamine (DBSPM) and related mono-alkylated derivatives as substrates of recombinant human polyamine oxidase (APAO) and spermine oxidase (SMO). APAO and SMO metabolized DESPM to EtSPD [K(m(APAO)) = 10 microM, k(cat(APAO)) = 1.1 s(-1) and K(m(SMO)) = 28 microM, k(cat(SMO)) = 0.8 s(-1), respectively], metabolized BnEtSPM to EtSPD [K(m(APAO)) = 0.9 microM, k(cat(APAO)) = 1.1 s(-1) and K(m(SMO)) = 51 microM, k(cat(SMO)) = 0.4 s(-1), respectively], and metabolized DBSPM to BnSPD [K(m(APAO)) = 5.4 microM, k(cat(APAO)) = 2.0 s(-1) and K(m(SMO)) = 33 microM, k(cat(SMO)) = 0.3 s(-1), respectively]. Interestingly, mono-alkylated spermine derivatives were metabolized by APAO and SMO to SPD [EtSPM K(m(APAO)) = 16 microM, k(cat(APAO)) = 1.5 s(-1); K(m(SMO)) = 25 microM, k(cat(SMO)) = 8.2 s(-1); BnSPM K(m(APAO) )= 6.0 microM, k(cat(APAO)) = 2.8 s(-1); K(m(SMO)) = 19 muM, k(cat(SMO)) = 0.8 s(-1), respectively]. Surprisingly, EtSPD [K(m(APAO)) = 37 microM, k(cat(APAO)) = 0.1 s(-1); K(m(SMO)) = 48 microM, k(cat(SMO)) = 0.05 s(-1)] and BnSPD [K(m(APAO)) = 2.5 microM, k(cat(APAO)) = 3.5 s(-1); K(m(SMO)) = 60 microM, k(cat(SMO)) = 0.54 s(-1)] were metabolized to SPD by both the oxidases. Furthermore, we studied the degradation of DESPM, BnEtSPM or DBSPM in the DU145 prostate carcinoma cell line. The same major metabolites EtSPD and/or BnSPD were detected both in the culture medium and intracellularly after 48 h of culture. Moreover, EtSPM and BnSPM were detected from cell samples. Present data shows that inducible SMO parallel with APAO could play an important role in polyamine based drug action, i.e. degradation of parent drug and its metabolites, having significant impact on efficiency of these drugs, and hence for the development of novel N-alkylated polyamine analogues.
Topics: Cell Line, Tumor; Enzyme Stability; Humans; Kinetics; Oxidoreductases Acting on CH-NH Group Donors; Polyamines; Spermine; Substrate Specificity; Polyamine Oxidase
PubMed: 20012116
DOI: 10.1007/s00726-009-0429-2 -
The Biochemical Journal Jun 2015The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney...
Depletion of the polyamines spermidine and spermine by overexpression of spermidine/spermine N¹-acetyltransferase 1 (SAT1) leads to mitochondria-mediated apoptosis in mammalian cells.
The polyamines putrescine, spermidine and spermine are intimately involved in the regulation of cellular growth and viability. Transduction of human embryonic kidney (HEK) 293T cells with an adenovirus encoding a key polyamine catabolic enzyme, spermidine N¹-acetyltransferase 1 (SSAT1)/SAT1 (AdSAT1), leads to a rapid depletion of spermidine and spermine, arrest in cell growth and a decline in cell viability. Annexin V/propidium iodide FACS analyses, terminal uridine nucleotide end-labelling (TUNEL) and caspase 3 assays showed a clear indication of apoptosis in AdSAT1-transduced cells (at 24-72 h), but not in cells transduced with GFP-encoding adenovirus (AdGFP). Apoptosis in the polyamine-depleted cells occurs by the mitochondrial intrinsic pathway, as evidenced by loss of mitochondrial membrane potential, increase in pro-apoptotic Bax, decrease in anti-apoptotic Bcl-xl, Bcl2 and Mcl-1 and release of cytochrome c from mitochondria, upon transduction with AdSAT1. Moreover, TEM images of AdSAT1-transduced cells revealed morphological changes commonly associated with apoptosis, including cell shrinkage, nuclear fragmentation, mitochondrial alteration, vacuolization and membrane blebbing. The apoptosis appears to result largely from depletion of the polyamines spermidine and spermine, as the polyamine analogues α-methylspermidine (α-MeSpd) and N¹,N¹²-dimethylspermine (Me₂Spm) that are not substrates for SAT1 could partially restore growth and prevent apoptosis of AdSAT1-transduced cells. Inhibition of polyamine oxidases did not restore the growth of AdSAT1-transduced cells or block apoptosis, suggesting that the growth arrest and apoptosis were not induced by oxidative stress resulting from accelerated polyamine catabolism. Taken together, these data provide strong evidence that the depletion of the polyamines spermidine and spermine leads to mitochondria-mediated apoptosis.
Topics: Acetyltransferases; Antimetabolites; Apoptosis; Apoptosis Regulatory Proteins; Cell Proliferation; Cell Shape; Cell Size; HEK293 Cells; Humans; In Situ Nick-End Labeling; Kidney; Kinetics; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Mitochondria; Recombinant Proteins; Spermidine; Spermine
PubMed: 25849284
DOI: 10.1042/BJ20150168 -
Animal : An International Journal of... Jun 2018Previous studies showed that spermine could protect the organism from oxidative damage in vivo. However, in vivo information on the antioxidant-related underlying...
Previous studies showed that spermine could protect the organism from oxidative damage in vivo. However, in vivo information on the antioxidant-related underlying molecular mechanism of spermine is limited. In this experiment, we further evaluated the effects of spermine supplementation and extended spermine administration on the antioxidant status and antioxidant-related signaling molecules gene expression in the liver and longissimus dorsi of piglets. A total of 80 piglets were randomly distributed to two groups, that is, those with adequate nutrient intake administrated with spermine (0.4 mmol/kg BW) or those with restricted nutrient intake supplemented by saline. The piglets were fed in pairs for 7 h or 3, 6, or 9 days. The results are as follows: (1) spermine can promote the antioxidant capacity by increasing enzymatic antioxidant capacity, glutathione content and clearance of oxygen radicals; (2) spermine significantly increased the mRNA levels of enzymatic antioxidant substances, NF-E2-related nuclear factor 2, Kelch-like ECH-associated protein 1, and the mammalian target of rapamycin but decreased the mRNA levels of ribosomal p70 S6 kinase in the liver and longissimus dorsi of the piglets.
Topics: Animals; Animals, Newborn; Antioxidants; Dietary Supplements; Gene Expression; Liver; Spermine; Swine
PubMed: 29065949
DOI: 10.1017/S1751731117002737 -
Biomolecules Mar 2022Polyamines (PAs) are small, versatile molecules with two or more nitrogen-containing positively charged groups and provide widespread biological functions. Most of these... (Review)
Review
Polyamines (PAs) are small, versatile molecules with two or more nitrogen-containing positively charged groups and provide widespread biological functions. Most of these aspects are well known and covered by quite a number of excellent surveys. Here, the present review includes novel aspects and questions: (1) It summarizes the role of most natural and some important synthetic PAs. (2) It depicts PA uptake from nutrition and bacterial production in the intestinal system following loss of PAs via defecation. (3) It highlights the discrepancy between the high concentrations of PAs in the gut lumen and their low concentration in the blood plasma and cerebrospinal fluid, while concentrations in cellular cytoplasm are much higher. (4) The present review provides a novel and complete scheme for the biosynthesis of Pas, including glycine, glutamate, proline and others as PA precursors, and provides a hypothesis that the agmatine pathway may rescue putrescine production when ODC knockout seems to be lethal (solving the apparent contradiction in the literature). (5) It summarizes novel data on PA transport in brain glial cells explaining why these cells but not neurons preferentially accumulate PAs. (6) Finally, it provides a novel and complete scheme for PA interconversion, including hypusine, putreanine, and GABA (unique gliotransmitter) as end-products. Altogether, this review can serve as an updated contribution to understanding the PA mystery.
Topics: Central Nervous System; Neuroglia; Polyamines; Putrescine; Spermine
PubMed: 35454090
DOI: 10.3390/biom12040501 -
Langmuir : the ACS Journal of Surfaces... Mar 2020Coacervates are polymer-rich droplets that form through liquid-liquid phase separation in polymer solutions. Liquid-liquid phase separation and coacervation have...
Coacervates are polymer-rich droplets that form through liquid-liquid phase separation in polymer solutions. Liquid-liquid phase separation and coacervation have recently been shown to play an important role in the organization of biological systems. Such systems are highly dynamic and under continuous influence of enzymatic and chemical processes. However, it is still unclear how enzymatic and chemical reactions affect the coacervation process. Here, we present and characterize a system of enzymatically active coacervates containing spermine, RNA, free nucleotides, and the template independent RNA (de)polymerase PNPase. We find that these RNA coacervates display transient nonspherical shapes, and we systematically study how PNPase concentration, UDP concentration, and temperature affect coacervate morphology. Furthermore, we show that PNPase localizes predominantly into the coacervate phase and that its depolymerization activity in high-phosphate buffer causes coacervate degradation. Our observations of nonspherical coacervate shapes may have broader implications for the relationship between (bio)chemical activity and coacervate biology.
Topics: Polymers; RNA; Spermine; Temperature
PubMed: 31995710
DOI: 10.1021/acs.langmuir.9b02719