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Cadaverine is an endogenous metabolite produced by the gut microbiome with various activity in physiological and pathological conditions. However, whether cadaverine regulates pain or itch remains unclear. In this study, we first found that cadaverine may bind to histamine 4 receptor (H4R) with higher docking energy score using molecular docking simulations, suggesting cadaverine may act as an endogenous ligand for H4R. We subsequently found intradermal injection of cadaverine into the nape or cheek of mice induces a dose-dependent scratching response in mice, which was suppressed by a selective H4R antagonist JNJ-7777120, transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine and PLC inhibitor U73122, but not H1R antagonist or TRPA1 antagonist or TRPV4 antagonist. Consistently, cadaverine-induced itch was abolished in Trpv1 -/- but not Trpa1 -/- mice. Pharmacological analysis indicated that mast cells and opioid receptors were also involved in cadaverine-induced itch in mice. scRNA-Seq data analysis showed that H4R and TRPV1 are mainly co-expressed on NP2, NP3 and PEP1 DRG neurons. Calcium imaging analysis showed that cadaverine perfusion enhanced calcium influx in the dissociated dorsal root ganglion (DRG) neurons, which was suppressed by JNJ-7777120 and capsazepine, as well as in the DRG neurons from Trpv1 -/- mice. Patch-clamp recordings found that cadaverine perfusion significantly increased the excitability of small diameter DRG neurons, and JNJ-7777120 abolished this effect, indicating involvement of H4R. Together, these results provide evidences that cadaverine is a novel endogenous pruritogens, which activates H4R/TRPV1 signaling pathways in the primary sensory neurons., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cadaverine (Cad), which has an independent synthesis pathway compared to other polyamine (PA) types, contributes to the health of plants by regulating plant growth and development, abiotic stress tolerance and antioxidant defense mechanisms. In this work, experiments were carried out to understand the effects of exogenous Cad (10 µM) application under drought stress (%22 PEG 6000) and without stress on cell cycle, total protein content, endogenous PA levels, and biochemical enzyme activities in barley (Hordeum vulgare cv. Burakbey) considering the potential of Cad to stimulate the drought-related tolerance system. Cad application in a stress-free environment showed an effect almost like low-impact drought stress, causing changes in all parameters examined compared to samples grown in distilled water environment (Control). The results clearly show that Cad applied against the negative effects of drought stress on all parameters creates a drought resistance mechanism of the plant. Accordingly, Cad applied together with drought stress increased the density of cells in the cell cycle (G1-S and S-G2 phases) and the amount of endogenous (spermidine 10% and spermine 40%) PAs. In addition, while superoxide dismutase (SOD) (5%), (CAT) (55%) and ascorbate peroxidase (APX) (18%) enzyme levels increased, a stress response mechanism occurred due to the decrease in total protein content (20%) and malondialdehyde (MDA) (80%). As a result, exogenous application of 10 µM Cad showed that it reduced the negative effects of drought stress on endogenous PA amounts, cell division and biochemical activities in barley., (© 2023. Springer Nature Limited.)

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine ( R-I-Cad ) into N , N -dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates.

Polyamines are essential for biofilm formation in Escherichia coli , but it is still unclear which polyamines are primarily responsible for this phenomenon. To address this issue, we constructed a series of E. coli K-12 strains with mutations in genes required for the synthesis and metabolism of polyamines. Disruption of the spermidine synthase gene ( speE ) caused a severe defect in biofilm formation. This defect was rescued by the addition of spermidine to the medium but not by putrescine or cadaverine. A multidrug/spermidine efflux pump membrane subunit (MdtJ)-deficient strain was anticipated to accumulate more spermidine and result in enhanced biofilm formation compared to the MdtJ + strain. However, the mdtJ mutation did not affect intracellular spermidine or biofilm concentrations. E. coli has the spermidine acetyltransferase (SpeG) and glutathionylspermidine synthetase/amidase (Gss) to metabolize intracellular spermidine. Under biofilm-forming conditions, not Gss but SpeG plays a major role in decreasing the too-high intracellular spermidine concentrations. Additionally, PotFGHI can function as a compensatory importer of spermidine when PotABCD is absent under biofilm-forming conditions. Last, we report here that, in addition to intracellular spermidine, the periplasmic binding protein (PotD) of the spermidine preferential ABC transporter is essential for stimulating biofilm formation. IMPORTANCE Previous reports have speculated on the effect of polyamines on bacterial biofilm formation. However, the regulation of biofilm formation by polyamines in Escherichia coli has not yet been assessed. The identification of polyamines that stimulate biofilm formation is important for developing novel therapies for biofilm-forming pathogens. This study sheds light on biofilm regulation in E. coli Our findings provide conclusive evidence that only spermidine can stimulate biofilm formation in E. coli cells, not putrescine or cadaverine. Last, Δ potD inhibits biofilm formation even though the spermidine is synthesized inside the cells from putrescine. Since PotD is significant for biofilm formation and there is no ortholog of the PotABCD transporter in humans, PotD could be a target for the development of biofilm inhibitors., (Copyright © 2021 American Society for Microbiology.)

The liver is the primary target organ for perfluorooctane sulphonate (PFOS), a recently discovered persistent organic pollutant. However, the mechanisms mediating hepatotoxicity remain unclear. Herein, we explored the relationship between reactive oxygen species (ROS) and autophagy and apoptosis induced by PFOS in L-02 cells, which are incubated with different concentrations of PFOS (0, 50, 100, 150, 200, or 250  μ mol/L) for 24 or 48 hrs at 37°C. The results indicated that PFOS exposure decreased cell activities, enhanced ROS levels in a concentration-dependent manner, decreased mitochondrial membrane potential (MMP), and induced autophagy and apoptosis. Compared with the control, 200  μ mol/L PFOS increased ROS levels; enhanced the expression of Bax, cleaved-caspase-3, and LC3-II; induced autophagy; decreased MMP; and lowered Bcl-2, p62, and Bcl-2/Bax ratio. The antioxidant N-acetyl cysteine (NAC) protected MMP against PFOS-induced changes and diminished apoptosis and autophagy. Compared with 200  μ mol/L PFOS treatment, NAC pretreatment reversed the increase in ROS, Bax, and cleaved-caspase-3 protein caused by PFOS, lowered the apoptosis rate increased by PFOS, and increased the levels of MMP and Bcl-2/Bax ratio decreased by PFOS. The autophagy inhibitor 3-methyladenine and chloroquine decreased apoptosis and cleaved-caspase-3 protein level and increased the Bcl-2/Bax ratio. In summary, our results suggest that ROS-triggered autophagy is involved in PFOS-induced apoptosis in L-02 cells., Competing Interests: The authors have no conflict of interests to declare regarding the publication of this work., (Copyright © 2021 Huai-cai Zeng et al.)

The significant increase of periodontitis, chronic kidney disease (CKD), Alzheimer's disease and cancer can be attributed to an ageing population. Each disease produces a range of biomarkers that can be indicative of disease onset and progression. Biomarkers are defined as cellular (intra/extracellular components and whole cells), biochemical (metabolites, ions and toxins) or molecular (nucleic acids, proteins and lipids) alterations which are measurable in biological media such as human tissues, cells or fluids. An interesting group of biomarkers that merit further investigation are the polyamines. Polyamines are a group of molecules consisting of cadaverine, putrescine, spermine and spermidine and have been implicated in the development of a range of systemic diseases, in part due to their production in periodontitis. Cadaverine and putrescine within the periodontal environment have demonstrated cell signalling interfering abilities, by way of leukocyte migration disruption. The polyamines spermine and spermidine in tumour cells have been shown to inhibit cellular apoptosis, effectively prolonging tumorigenesis and continuation of cancer within the host. Polyamine degradation products such as acrolein have been shown to exacerbate renal damage in CKD patients. Thus, the use of such molecules has merit to be utilized in the early indication of such diseases in patients.

We recently reported that necroptosis contributed to compression-induced nucleus pulposus (NP) cells death. In the current study, we investigated the regulative effect of necroptosis inhibitor Necrostatin-1 on NP cells apoptosis and autophagy. Necrostatin-1, autophagy inhibitor 3-Methyladenine and apoptosis inhibitor Z-VAD-FMK were employed, and NP cells were exposed to 1.0 MPa compression for 0, 24 and 36 h. Necroptosis-associated molecules were measured by Western blot and RT-PCR. Autophagy and apoptosis levels were evaluated by Western blot and quantified by flow cytometry after monodansylcadaverine and Annexin V-FITC/propidium iodide staining, respectively. The cell viability and cell death were also examined. Furthermore, we measured mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (MPTP) and indices of oxidative stress to assess mitochondrial dysfunction. The results established that Necrostatin-1 blocked NP cells autophagy, and 3-Methyladenine had little influence on NP cells necroptosis. The Necrostatin-1+3-Methyladenine treatment exerted almost the same role as Necrostatin-1 in reducing NP cells death. Necrostatin-1 restrained NP cells apoptosis, while Z-VAD-FMK enhanced NP cells necroptosis. The Necrostatin-1+Z-VAD-FMK treatment provided more prominent role in blocking NP cells death compared with Necrostatin-1, consistent with increased MMP, reduced opening of MPTP and oxidative stress. In summary, the synergistic utilization of Necrostatin-1 and Z-VAD-FMK is a very worthwhile solution in preventing compression-mediated NP cells death, which might be largely attributed to restored mitochondrial function., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Songfeng Chen et al.)

A nutritional intervention, exclusive enteral nutrition (EEN) can induce remission in patients with pediatric Crohn's disease (CD). We characterized changes in the fecal microbiota and metabolome to identify the mechanism of EEN. Feces of 43 children were collected prior, during and after EEN. Microbiota and metabolites were analyzed by 16S rRNA gene amplicon sequencing and NMR. Selected metabolites were evaluated in relevant model systems. Microbiota and metabolome of patients with CD and controls were different at all time points. Amino acids, primary bile salts, trimethylamine and cadaverine were elevated in patients with CD. Microbiota and metabolome differed between responders and non-responders prior to EEN. EEN decreased microbiota diversity and reduced amino acids, trimethylamine and cadaverine towards control levels. Patients with CD had reduced microbial metabolism of bile acids that partially normalized during EEN. Trimethylamine and cadaverine inhibited intestinal cell growth. TMA and cadaverine inhibited LPS-stimulated TNF-alpha and IL-6 secretion by primary human monocytes. A diet rich in free amino acids worsened inflammation in the DSS model of intestinal inflammation. Trimethylamine, cadaverine, bile salts and amino acids could play a role in the mechanism by which EEN induces remission. Prior to EEN, microbiota and metabolome are different between responders and non-responders.

Polyamines are small amines that accumulate during stress and contribute to disease resistance through as yet unknown signaling pathways. Using a comprehensive RNA-sequencing analysis, we show that early transcriptional responses triggered by each of the most abundant polyamines (putrescine, spermidine, spermine, thermospermine and cadaverine) exhibit specific quantitative differences, suggesting that polyamines (rather than downstream metabolites) elicit defense responses. Signaling by putrescine, which accumulates in response to bacteria that trigger effector triggered immunity (ETI) and systemic acquired resistance (SAR), is largely dependent on the accumulation of hydrogen peroxide, and is partly dependent on salicylic acid (SA), the expression of ENHANCED DISEASE SUSCEPTIBILITY (EDS1) and NONEXPRESSOR of PR GENES1 (NPR1). Putrescine elicits local SA accumulation as well as local and systemic transcriptional reprogramming that overlaps with SAR. Loss-of-function mutations in arginine decarboxylase 2 (ADC2), which is required for putrescine synthesis and copper amine oxidase (CuAO), which is involved in putrescine oxidation, compromise basal defenses, as well as putrescine and pathogen-triggered systemic resistance. These findings confirm that putrescine elicits ROS-dependent SA pathways in the activation of plant defenses., (© 2020 John Wiley & Sons Ltd.)

A tight junction (TJ) makes a physical barrier in the epidermal cells of skin. Ultraviolet (UV) light may disrupt the TJ barrier, but the mechanism has not been well clarified. Weak UVB (5 mJ/cm 2 ) caused mislocalization of claudin-1 (CLDN1), a component of the TJ strand, and disruption of TJ barrier in human keratinocyte-derived HaCaT cells. The UVB-induced mislocalization of CLDN1 was inhibited by monodansylcadaverine (MDC), a clathrin-dependent endocytosis inhibitor, suggesting that UVB enhances the internalization of CLDN1. Transepidermal electrical resistance and paracellular flux of lucifer yellow, a fluorescent hydrophilic marker, were rescued by MDC. UVB changed neither the total nor phosphorylation levels of CLDN1, but it increased both mono-ubiquitination and tyrosine nitration levels of CLDN1. Fluorescence measurements revealed that UVB increased intracellular free Ca 2+ , nitric oxide (NO), and peroxynitrite contents, which were inhibited by Opsin2 (OPN2) siRNA, suggesting that OPN2 functions as a UVB sensor. The effects of UVB were inhibited by an antagonist of transient receptor potential type vanilloid 1 (TRPV1) and Ca 2+ chelator. Both NO donor and peroxynitrite donor induced the mislocalization of CLDN1 and disruption of TJ barrier, which were rescued by a NO synthase (NOS) inhibitor and a peroxynitrite scavenger. Weak UVB irradiation induced the disruption of TJ barrier mediated by mislocalization of CLDN1 in HaCaT cells. The OPN2/TRPV1/NOS signaling pathway may be a novel target for preventing destruction of the TJ barrier by UVB irradiation.

Plant responses to salinity have been extensively studied over the last decades. Despite the vast accumulated knowledge, the ways Arabidopsis lateral roots (LR) cope with lethal salinity has not been fully resolved. Here we compared the primary root (PR) and the LR responses during events leading to lethal salinity (NaCl 200 mM) in Arabidopsis. We found that the PR and young LR responded differently to lethal salinity: While the PR died, emerging and young LR's remained strikingly viable. Moreover, "age acquired salt tolerance" (AAST) was observed in the PR. During the 2 days after germination (DAG) the PR was highly sensitive, but at 8 DAG there was a significant increase in the PR cell survival. Nevertheless, the young LR exhibited an opposite pattern and completely lost its salinity tolerance, as it elongated beyond 400 µm. Examination of several cell death signatures investigated in the young LR showed no signs of an active programmed cell death (PCD) during lethal salinity. However, Autophagic PCD (A-PCD) but not apoptosis-like PCD (AL-PCD) was found to be activated in the PR during the high salinity conditions. We further found that salinity induced NADPH oxidase activated ROS, which were more highly distributed in the young LR compared to the PR, is required for the improved viability of the LR during lethal salinity conditions. Our data demonstrated a position-dependent resistance of Arabidopsis young LR to high salinity. This response can lead to identification of novel salt stress coping mechanisms needed by agriculture during the soil salinization challenge.

Chikungunya virus (CHIKV) is a re-emerging arbovirus known to cause chronic myalgia and arthralgia with high morbidity. CHIKV is now considered endemic in many countries across Asia and Africa. In this study, the susceptibility of various human, mammalian and mosquito cell lines to CHIKV infection was evaluated. CHIKV infection was found to be cell-type dependent and virus strain-specific. Furthermore, SJCRH30 (human rhabdomyosarcoma cell line) was showed to be highly permissive to CHIKV infection, with maximum production of infectious virions observed at 12 h.p.i. Pre-infection treatment of SJCRH30 with various inhibitors of endocytosis, including monodansylcadaverine (receptor-mediated endocytic inhibitor), dynasore (clathrin-mediated endocytic inhibitor), as well as filipin (caveolin-mediated endocytosis inhibitor), resulted in minimal inhibition of CHIKV infection. In contrast, dose-dependent inhibition of CHIKV infection was observed with the treatment of macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Furthermore, siRNA-mediated knockdown of sortin nexin 9 (SNX9) a protein involved in macropinosome formation, also resulted in a significant dose-dependent reduction in viral titre. By performing a virus entry assay, CHIKV particles were also observed to colocalize with FITC-dextran, a macropinosome marker. This study shows for the first time, that the infectious entry of CHIKV into human muscle cells is mediated by macropinocytosis. Together, the data from this study may pave the way for the development of specific inhibitors that target the entry process of CHIKV into cells., Competing Interests: The authors have declared that no competing interests exist.

Despite evidence linking the human microbiome to health and disease, how the microbiota affects human physiology remains largely unknown. Microbiota-encoded metabolites are expected to play an integral role in human health. Therefore, assigning function to these metabolites is critical to understanding these complex interactions and developing microbiota-inspired therapies. Here, we use large-scale functional screening of molecules produced by individual members of a simplified human microbiota to identify bacterial metabolites that agonize G-protein-coupled receptors (GPCRs). Multiple metabolites, including phenylpropanoic acid, cadaverine, 9-10-methylenehexadecanoic acid, and 12-methyltetradecanoic acid, were found to interact with GPCRs associated with diverse functions within the nervous and immune systems, among others. Collectively, these metabolite-receptor pairs indicate that diverse aspects of human health are potentially modulated by structurally simple metabolites arising from primary bacterial metabolism., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent studies showed that changes to the gut microbiome alters the microbiome-derived metabolome, potentially promoting carcinogenesis in organs that are distal to the gut. In this study, we assessed the relationship between breast cancer and cadaverine biosynthesis. Cadaverine treatment of Balb/c female mice (500 nmol/kg p.o. q.d.) grafted with 4T1 breast cancer cells ameliorated the disease (lower mass and infiltration of the primary tumor, fewer metastases, and lower grade tumors). Cadaverine treatment of breast cancer cell lines corresponding to its serum reference range (100-800 nM) reverted endothelial-to-mesenchymal transition, inhibited cellular movement and invasion, moreover, rendered cells less stem cell-like through reducing mitochondrial oxidation. Trace amino acid receptors (TAARs), namely, TAAR1, TAAR8 and TAAR9 were instrumental in provoking the cadaverine-evoked effects. Early stage breast cancer patients, versus control women, had reduced abundance of the CadA and LdcC genes in fecal DNA, both responsible for bacterial cadaverine production. Moreover, we found low protein expression of E. coli LdcC in the feces of stage 1 breast cancer patients. In addition, higher expression of lysine decarboxylase resulted in a prolonged survival among early-stage breast cancer patients. Taken together, cadaverine production seems to be a regulator of early breast cancer.

A whole-cell (cadaverine-producing strain, Escherichia coli AST3) immobilization method was developed for improving catalytic activity and cadaverine tolerance during cadaverine production. Cell-immobilized beads were prepared by polyvinyl alcohol (PVA) and sodium alginate (SA) based on their advantages in biocatalyst activity recovery and mechanical strength. The following optimal immobilization conditions were established using response surface methodology: 3.62% SA, 4.71% PVA, 4.21% CaCl 2 , calcification, 12 h, and freezing for 16 h at - 80 °C, with a cell concentration of 0.3% (g dry cell weight (DCW) per 100 mL) of immobilized beads. After a 2-h bioconversion, the immobilized beads maintained 85% of their original biocatalyst activity, which was 1.8-fold higher than that of free cells. Furthermore, the effects of cell protectants on immobilized biocatalyst activity were examined by fed-batch bioconversion experiments. The results showed that the addition of polyvinylpyrrolidone (PVP) into the immobilized matrix effectively protected biocatalyst activity, with 95% of the relative activity remaining after the 2-h bioconversion. The performance of PVA-SA-PVP-immobilized E. coli AST3 showed continuous production of cadaverine, with an average cadaverine yield of 29 ± 1 g gDCW -1  h -1 after 12 h, suggesting that this method is capable of industrial scale cadaverine production.

Aims: Accumulating evidence supports that cerebrospinal fluid (CSF) in the subarachnoid space (SAS) could reenter the brain parenchyma via the glymphatic influx. The present study was designed to characterize the detailed pathway of subarachnoid CSF influx by using a novel CSF tracer., Main Methods: Fluorescently conjugated cadaverine (A488-ca), for the first time, was employed to investigate CSF movement in the brain. Following intracisternal infusion of CSF tracers, mice brain was sliced and prepared for fluorescence imaging. Some brain sections were immunostained in order to observe tracer distribution and cellular uptake., Key Findings: A488-ca moved into the brain parenchyma rapidly, and the influx was time and region dependent. A488-ca entered the mice brain more readily and spread more widely than another commonly used CSF tracer-fluorescently conjugated ovalbumin (OA-45). Furthermore, A488-ca could enter the brain parenchyma either along the paravascular space or across the pial surface. Suppression of glymphatic transport by administration with acetazolamide strikingly reduced the influx of A488-ca. More importantly, relative to OA-45 largely remained in the extracellular space, A488-ca exhibited obvious cellular uptake by astrocytes surrounding the blood vessels and neurons in the cerebral cortex., Significance: Subarachnoid CSF could flow into the brain parenchyma via the glymphatic influx, in which the transcellular pathway was faithfully traced by intracisternal infusion with fluorescently conjugated cadaverine. These observations extend our comprehension on the glymphatic influx pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objectives: Indium compounds are used in manufacturing displays of mobile phones and televisions. However, these materials cause interstitial pneumonia in exposed workers. Animal experiments demonstrated that indium compounds caused lung cancer. Chronic inflammation is considered to play a role in lung carcinogenesis and fibrosis induced by particulate matters. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation and may participate in carcinogenesis. To clarify the mechanism of carcinogenesis, we examined 8-nitroG formation in indium-exposed cultured cells., Methods: We treated RAW 264.7 mouse macrophages with indium oxide (In 2 O 3 ) nanoparticles (primary diameter: 30-50 nm), and performed fluorescent immunocytochemistry to detect 8-nitroG. The extent of 8-nitroG formation was evaluated by quantitative image analysis. We measured the amount of nitric oxide (NO) in the culture supernatant of In 2 O 3 -treated cells by the Griess method. We also examined the effects of inhibitors of inducible NO synthase (iNOS) and endocytosis on In 2 O 3 -induced 8-nitroG formation., Results: In 2 O 3 significantly increased the intensity of 8-nitroG formation in RAW 264.7 cells in a dose-dependent manner. In 2 O 3 -induced 8-nitroG formation was observed at 2 h and further increased at 4 h, and the amount of NO released from In 2 O 3 -exposed cells was significantly increased at 2-4 h compared with the control. 8-NitroG formation was suppressed by 1400W (an iNOS inhibitor), methyl-β-cyclodextrin and monodansylcadaverine (inhibitors of caveolae- and clathrin-mediated endocytosis, respectively)., Conclusions: These results suggest that endocytosis and NO generation participate in indium-induced 8-nitroG formation. NO released from indium-exposed inflammatory cells may induce DNA damage in adjacent lung epithelial cells and contribute to carcinogenesis.

Root growth architecture is a major determinant of agricultural productivity and plant fitness in natural ecosystems. Here we describe the methods used in a Quantitative Trait Loci (QTL) study that allowed the identification of ORGANIC CATION TRANSPORTER 1 (OCT1) as a determinant of root growth response to cadaverine treatment in Arabidopsis thaliana. This protocol screens natural accessions to characterize the variation in root growth response to the naturally occurring polyamine cadaverine, then uses recombination mapping to identify loci that are responsible for the variation existing between two accessions with contrasting phenotypes.

The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF1 and IGF2. The IGF1R is involved in both physiological and pathological activities and is usually overexpressed in most types of cancer. In addition to its classical mechanism of action, recent evidence has shown a nuclear presence of IGF1R, associated with novel genomic/transcriptional types of activities. The present study was aimed at evaluating the hypothesis that nuclear IGF1R localization is not restricted to cancer cells and might constitute a novel physiologically relevant regulatory mechanism. Our data shows that nuclear translocation takes place in a wide array of cells, including normal diploid fibroblasts. In addition, we provide evidence for a synergistic effect of a nuclear translocation blocker along with selective IGF1R inhibitors in terms of decreasing cell proliferation. Given the important role of the IGF1R in mitogenesis, the present results may be of translational relevance in cancer research. In conclusion, results are consistent with the concept that nuclear IGF1R fulfills important physiological and pathological roles.

The present study investigated the effect of cinnamon essential oil on the quality of vacuum-packaged common carp (Cyprinus carpio) fillets stored at 4±1°C in terms of sensory scores, physicochemical characteristics (total volatile basic nitrogen (TVB-N), biogenic amines, and color), and presence of spoilage microbiota. A total of 290,753 bacterial sequences and 162 different genera belonging to 14 phyla were observed by a high-throughput sequencing technique targeting the V3-V4 region of 16S rDNA, which showed a more comprehensive estimate of microbial diversity in carp samples compared with microbial enumeration. Before storage, Macrococcus and Aeromonas were the prevalent populations in the control samples, but cinnamon essential oil decreased the relative abundance of Macrococcus in the treated samples. Variability in the predominant microbiota in different samples during chilled storage was observed. Aeromonas followed by Lactococcus were the major contaminants in the spoiled control samples. Microbial enumeration also observed relatively higher counts of Aeromonas than other spoilage microorganisms. Compared with the control samples, cinnamon essential oil inhibited the growth of Aeromonas and Lactococcus were the predominant components in the treated samples on day 10; plate counts also revealed a relatively high level of lactic acid bacteria during refrigerated storage. However, there were no significant differences (P>0.05) in the composition of dominant microbiota between these two treatments at the end of the shelf-life. Furthermore, cinnamon essential oil treatment was more effective in inhibiting the increase of TVB-N and the accumulation of biogenic amines (especially for putrescine and cadaverine levels). Based primarily on sensory analysis, the use of cinnamon essential oil extended the shelf-life of vacuum-packaged common carp fillets by about 2days., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tumor necrosis factor-alpha (TNF-α) exists in two forms: secretory TNF-α (sTNF-α) and transmembrane TNF-α (tmTNF-α). Although both forms of TNF-α induce tumor cell apoptosis, tmTNF-α is able to kill tumor cells that are resistant to sTNF-α-mediated cytotoxicity, indicating their differences in signal transduction. Here, we demonstrate that internalization of TNFR1 is crucial for sTNF-α- but not for tmTNF-α-induced apoptosis. sTNF-α induces binding of tumor necrosis factor receptor type 1-associated death domain protein (TRADD) to the death domain (DD) of TNFR1 and subsequent activation of nuclear factor kappa B (NF-κB), and the formation of death-inducing signaling complexes (DISCs) in the cytoplasm after internalization. In contrast, tmTNF-α induces DISC formation on the membrane in a DD-independent manner. It leads to the binding of signal transducer and activator of transcription 1 (STAT1) to a region spanning amino acids 319-337 of TNFR1 and induces phosphorylation of serine at 727 of STAT1. The phosphorylation of STAT1 promotes its binding to TRADD, and thus recruits Fas-associated protein with DD (FADD) and caspase 8 to form DISC complexes. This STAT1-dependent signaling results in apoptosis but not NF-κB activation. STAT1-deficiency in U3A cells counteracts tmTNF-α-induced DISC formation and apoptosis. Conversely, reconstitution of STAT1 expression restores tmTNF-α-induced apoptotic signaling in the cell line. Consistently, tmTNF-α suppresses the growth of STAT1-containing HT1080 tumors, but not of STAT1-deficient U3A tumors in vivo. Our data reveal an unappreciated molecular mechanism of tmTNF-α-induced apoptosis and may provide a new clue for cancer therapy.

The death-associated odor cadaverine, generated by bacteria-mediated decarboxylation of lysine, has been described as the principal activator of a particular olfactory receptor in zebrafish, TAAR13c. Low concentrations of cadaverine activated mainly TAAR13c-expressing olfactory sensory neurons, suggesting TAAR13c as an important element of the neuronal processing pathway linking cadaverine stimulation to a strongly aversive innate behavioral response. Here, we characterized the initial steps of this neuronal pathway. First we identified TAAR13c-expressing cells as ciliated neurons, equivalent to the situation for mammalian taar genes, which shows a high degree of conservation despite the large evolutionary distance between teleost fishes and mammals. Next we identified the target area of cadaverine-responsive OSNs in the olfactory bulb. We report that cadaverine dose-dependently activates a group of dorsolateral glomeruli, at the lowest concentration down to a single invariant glomerulus, situated at the medial border of the dorsolateral cluster. This is the first demonstration of a single stereotyped target glomerulus in the fish olfactory system for a non-pheromone odor. A mix of different amines activates many glomeruli within the same dorsolateral cluster, suggesting this area to function as a general amine response region.

Many areas have been heavily contaminated by heavy metals from industry and are not suitable for food production. The consumption of contaminated foods represents a health risk in humans, although some heavy metals are essential at low concentrations. Increasing the concentrations of essential elements in foods is one goal to improve nutrition. The aim of this study was to increase the accumulation of heavy metals in plant foods by the external application of putrescine. The levels of cadmium, zinc and iron were measured in different vegetables grown in hydroponic medium supplemented with heavy metals and compared with those grown in a reference medium. The estimated daily intake, based on the average daily consumption for various vegetable types, and the influence of polyamines on metal uptake were calculated., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Changes in the two main intracellular degradation systems, the Ubiquitin-Proteasome System and the Autophagy-Lysosome pathway (ALP) are widely discussed as a hallmark of the aging process. To follow the age-related behavior of both degradation systems we examined their impact on ferritin, known to be degradable by both. Ferritin H was analyzed in young and senescent human fibroblasts, revealing a higher steady-state level in the senescent cells. By blocking both proteolytic systems, we confirmed that particularly the ALP plays a crucial role in ferritin H turnover. However, an unexpected increase in lysosomal activity in the senescent cells, suggests a dysregulation in the autophagy pathway. To further investigate the impaired ferritin H turnover, confocal microscopic colocalization studies of ferritin H with lysosomal-associated membrane protein 2a (Lamp2a) and monodansylcadaverine (MDC) were performed and clearly revealed the degradation of ferritin by macroautophagy. By induction of autophagy via inhibition of mTOR using rapamycin an increase of ferritin H turnover was obtained in senescent cells, demonstrating a mTOR dependent reduction of autophagy in senescent human fibroblasts., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Transglutaminase 2 (TGM2), a member of the transglutaminase family of enzymes, is a multifunctional protein involved in numerous events spanning from cell differentiation, to signal transduction, apoptosis, and wound healing. It is expressed in a variety of cells, macrophages included. Macrophage TGM2 promotes the clearance of apoptotic cells (efferocytosis) and emerging evidence suggests that defective efferocytosis contributes to the consequences of inflammation-associated diseases, including atherosclerotic lesion progression and its sequelae. Of interest, active TGM2 identified in human atherosclerotic lesions plays critical roles in plaque stability through effects on matrix cross-linking and TGFβ activity. This study explores the mechanisms by which TGM2 controls efferocytosis in human macrophages., Methods and Results: Herein we show that TGM2 increases progressively during monocyte differentiation towards macrophages and controls their efferocytic potential as well as morphology and viability. Two experimental approaches that took advantage of the inhibition of TGM2 activity and protein silencing give proof that TGM2 reduction significantly impairs macrophage efferocytosis. Among the mechanisms involved we highlighted a role of the receptors CD14 and SR-AI whose levels were markedly reduced by TGM2 inhibition. Conversely, CD36 receptor and αvβ3 integrin levels were not influenced. Of note, lipid accumulation and IL-10 secretion were reduced in macrophages displaying defective efferocytosis., Conclusion: Overall, our data define a crucial role of TGM2 activity during macrophage differentiation via mechanisms involving CD14 and SR-AI receptors and show that TGM2 inhibition triggers a pro-inflammatory phenotype., (Copyright © 2016 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon.

Key Message: Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

The olfactory-type signaling machinery has been known to be involved not only in odorant detection but also in other tissues with unsuspected sensory roles. As a barrier, the choroid plexus (CP) is an active participant in the monitoring of the cerebrospinal fluid (CSF), promptly responding to alterations in its composition. We hypothesized that olfactory signaling could be active in CP, contributing to the surveillance of the CSF composition. We determined the mRNA and protein expression of the major components of the olfactory transduction pathway in the rat CP, including odorant receptors, the olfactory G-protein (Gαolf), adenylate cyclase 3 and cyclic nucleotide-gated channel 2. The functionality of the transduction pathway and the intracellular mechanisms involved were analyzed by DC field potential recording electrophysiological analysis, in an ex vivo CP-brain setup, using polyamines as stimuli and blockers of the downstream signaling pathways. Concentration-dependent responses were obtained for the polyamines studied (cadaverine, putrescine, spermine and spermidine), all known to be present in the CSF. Transfection of a CP epithelial cell line with siRNA against Gαolf effectively knocked down protein expression and reduced the CP cells' response to spermine. Thus, the key components of the olfactory chemosensory apparatus are present and are functional in murine CP, and polyamines seem to trigger both the cAMP and the phospholipase C-inositol 1,4,5-trisphosphate pathways. Olfactory-like chemosensory signaling may be an essential component of the CP chemical surveillance apparatus to detect alterations in the CSF composition, and to elicit responses to modulate and maintain brain homeostasis., (© 2016 Federation of European Biochemical Societies.)

Trace amine-associated receptors (TAARs) are G Protein-Coupled Receptors that function as vertebrate olfactory receptors. Like odorant receptors, TAARs constitute an ever-evolving sensory subsystem, with individual TAARs recognizing particular chemicals and some evoking stereotyped behaviors. Several TAARs mediate aversion or attraction towards volatile amines that include the mouse odor trimethylamine, the predator odor 2-phenylethylamine, and the death-associated odor cadaverine. TAAR-expressing sensory neurons achieve monoallelic receptor expression, use canonical olfactory signaling molecules, and target a dedicated olfactory bulb region. In mouse, TAAR4 and TAAR5 are encoded by adjacent genes and localize to adjacent glomeruli, yet mediate opposing behaviors. Future studies are needed to understand how TAAR-expressing sensory neurons engage higher-order neural circuits to encode odor valence., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that cross-links proteins with monoamines such as serotonin (5-hydroxytryptamine, 5-HT) via a transglutamidation reaction, and is associated with pathophysiologic vascular responses. 5-HT is a mitogen for pulmonary artery smooth muscle cells (PASMCs) that has been linked to pulmonary vascular remodeling underlying pulmonary hypertension development. We previously reported that 5-HT-induced PASMC proliferation is inhibited by the TG2 inhibitor monodansylcadaverine (MDC); however, the mechanisms are poorly understood. In the present study we hypothesized that TG2 contributes to 5-HT-induced signaling pathways of PASMCs. Pre-treatment of bovine distal PASMCs with varying concentrations of the inhibitor MDC led to differential inhibition of 5-HT-stimulated AKT and ROCK activation, while p-P38 was unaffected. Concentration response studies showed significant inhibition of AKT activation at 50 μM MDC, along with inhibition of the AKT downstream targets mTOR, p-S6 kinase and p-S6. Furthermore, TG2 depletion by siRNA led to reduced 5-HT-induced AKT activation. Immunoprecipitation studies showed that 5-HT treatment led to increased levels of serotonylated AKT and increased TG2-AKT complex formations which were inhibited by MDC. Overexpression of TG2 point mutant cDNAs in PASMCs showed that the TG2 C277V transamidation mutant blunted 5-HT-induced AKT activation and 5-HT-induced PASMC mitogenesis. Finally, 5-HT-induced AKT activation was blunted in SERT genetic knock-out rat cells, but not in their wild-type counterpart. The SERT inhibitor imipramine similarly blocked AKT activation. These results indicate that TG2 contributes to 5-HT-induced distal PASMC proliferation via promotion of AKT signaling, likely via its serotonylation. Taken together, these results provide new insight into how TG2 may participate in vascular smooth muscle remodeling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Expression of genes encoding polyamine transporters from plasmids and polyamine supplements increased furfural tolerance (growth and ethanol production) in ethanologenic Escherichia coli LY180 (in AM1 mineral salts medium containing xylose). This represents a new approach to increase furfural tolerance and may be useful for other organisms. Microarray comparisons of two furfural-resistant mutants (EMFR9 and EMFR35) provided initial evidence for the importance of polyamine transporters. Each mutant contained a single polyamine transporter gene that was upregulated over 100-fold (microarrays) compared to that in the parent LY180, as well as a mutation that silenced the expression of yqhD. Based on these genetic changes, furfural tolerance was substantially reconstructed in the parent, LY180. Deletion of potE in EMFR9 lowered furfural tolerance to that of the parent. Deletion of potE and puuP in LY180 also decreased furfural tolerance, indicating functional importance of the native genes. Of the 8 polyamine transporters (18 genes) cloned and tested, half were beneficial for furfural tolerance (PotE, PuuP, PlaP, and PotABCD). Supplementing AM1 mineral salts medium with individual polyamines (agmatine, putrescine, and cadaverine) also increased furfural tolerance but to a smaller extent. In pH-controlled fermentations, polyamine transporter plasmids were shown to promote the metabolism of furfural and substantially reduce the time required to complete xylose fermentation. This increase in furfural tolerance is proposed to result from polyamine binding to negatively charged cellular constituents such as nucleic acids and phospholipids, providing protection from damage by furfural., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A bacterium isolated from Lake Taihu was identified as Pseudomonas sp. A3CT, which performed different effects on Microcystis spp. Growth of Microcystis flos-aquae and Microcystis aeruginosa was assessed in co-culture with A3CT to determine the stimulatory or inhibitory effects on these toxic, bloom-forming Microcystis strains. Results demonstrated that the impacts of A3CT were species specific. A3CT promoted the growth of M. aeruginosa but inhibited growth of M. flos-aquae. To investigate the cause of this phenomenon, the chemical composition of A3CT exudates and the impact of exposure to A3CT exudates on the two Microcystis species were determined. Results suggested that the observed differential growth responses of the two microalgae to A3CT exposure might be related to two components in A3CT exudates NH4 (+) and cadaverine. Growth stimulation of M. aeruginosa by A3CT was significantly related to NH4 (+) concentration. Cadaverine possibly acted as a growth inhibitor of M. flos-aquae. The different effects of cadaverine on growth of the two Microcystis strains suggested that A3CT might play a role in intrageneric succession patterns observed during Microcystis blooms in Lake Taihu.

The receptor for advanced glycation end products (RAGE) is involved in diabetes or angiogenesis in tumors. Under pathological conditions, RAGE is overexpressed and upon ligand binding and internalization stimulates signaling pathways that promote cell proliferation. In this work, amino dendritic polymers PEI 25 kDa and alkylated derivatives of PAMAM-G2 were engineered by the nonenzymatic Maillard glycation reaction to generate novel AGE-containing gene delivery vectors targeting the RAGE. The glycated dendritic polymers were easily prepared and retained the capability to bind and protect DNA from endonucleases. Furthermore, while glycation decreased the transfection efficiency of the dendriplexes in CHO-k1 cells which do not express RAGE, glycated dendriplexes acted as efficient transfection reagents in CHO-k1 cells which stably express recombinant RAGE. In addition, preincubation with BSA-AGEs, a natural ligand of the RAGE, or dansyl cadaverine, an inhibitor of the RAGE internalization, blocked transfection, confirming their specificity toward RAGE. The results were confirmed in NRK and RAW264.7 cell lines, which naturally express the receptor. The glycated compounds retain their transfection efficiency in the presence of serum and promote in vivo transfection in a mouse model. Accordingly, RAGE is a suitable molecular target for the development of site-directed engineered glycated nonviral gene vectors.

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

Tau immunotherapy is effective in transgenic mice, but the mechanisms of Tau clearance are not well known. To this end, Tau antibody uptake was analyzed in brain slice cultures and primary neurons. Internalization was rapid (<1 h), saturable, and substantial compared with control mouse IgG. Furthermore, temperature reduction to 4 °C, an excess of unlabeled mouse IgG, or an excess of Tau antibodies reduced uptake in slices by 63, 41, and 62%, respectively (p = 0.002, 0.04, and 0.005). Uptake strongly correlated with total and insoluble Tau levels (r(2) = 0.77 and 0.87 and p = 0.002 and 0.0002), suggesting that Tau aggregates influence antibody internalization and/or retention within neurons. Inhibiting phagocytosis did not reduce uptake in slices or neuronal cultures, indicating limited microglial involvement. In contrast, clathrin-specific inhibitors reduced uptake in neurons (≤ 78%, p < 0.0001) and slices (≤ 35%, p = 0.03), demonstrating receptor-mediated endocytosis as the primary uptake pathway. Fluid phase endocytosis accounted for the remainder of antibody uptake in primary neurons, based on co-staining with internalized dextran. The receptor-mediated uptake is to a large extent via low affinity FcγII/III receptors and can be blocked in slices (43%, p = 0.04) and neurons (53%, p = 0.008) with an antibody against these receptors. Importantly, antibody internalization appears to be necessary for Tau reduction in primary neurons. Overall, these findings clarify that Tau antibody uptake is primarily receptor-mediated, that these antibodies are mainly found in neurons with Tau aggregates, and that their intracellular interaction leads to clearance of Tau pathology, all of which have major implications for therapeutic development of this approach.

Carrion smell is strongly repugnant to humans and triggers distinct innate behaviors in many other species. This smell is mainly carried by two small aliphatic diamines, putrescine and cadaverine, which are generated by bacterial decarboxylation of the basic amino acids ornithine and lysine. Depending on the species, these diamines may also serve as feeding attractants, oviposition attractants, or social cues. Behavioral responses to diamines have not been investigated in zebrafish, a powerful model system for studying vertebrate olfaction. Furthermore, olfactory receptors that detect cadaverine and putrescine have not been identified in any species so far. Here, we show robust olfactory-mediated avoidance behavior of zebrafish to cadaverine and related diamines, and concomitant activation of sparse olfactory sensory neurons by these diamines. The large majority of neurons activated by low concentrations of cadaverine expresses a particular olfactory receptor, trace amine-associated receptor 13c (TAAR13c). Structure-activity analysis indicates TAAR13c to be a general diamine sensor, with pronounced selectivity for odd chains of medium length. This receptor can also be activated by decaying fish extracts, a physiologically relevant source of diamines. The identification of a sensitive zebrafish olfactory receptor for these diamines provides a molecular basis for studying neural circuits connecting sensation, perception, and innate behavior.

Androgens regulate body development and differentiation through a variety of genotropic mechanisms, mostly in reproductive organs. In recent years a different scenario for sex hormone actions has emerged: the intestinal muscle. Thus, although estrogens relax intestinal muscle, androgens are powerful inducers of mechanical potentiation. This effect of androgens was intriguing because it is observed at physiological concentrations, is mediated by nongenomic mechanisms, and involves a phenomenon of calcium sensitization of contractile machinery by stimulating phosphorylation of 20 kDa myosin light chain by Rho-associated kinase. Here we have deciphered the molecular mechanisms underlying calcium sensitization and mechanical potentiation by androgens in male intestinal muscle as well as its tight relationship to polyamine metabolism. Thus, androgens stimulate polyamine synthesis, and the inhibition of polyamine synthesis abolishes androgen-induced calcium sensitization and 20 kDa myosin light chain phosphorylation. We demonstrate that the first molecular step in the induction of calcium sensitization is a nonconventional activation of the adaptor protein RhoA, triggered by a transglutaminase-catalyzed polyamination of RhoA, which is then targeted to the membrane to activate Rho-associated kinase. Altogether, these results demonstrate that the physiological levels of androgens, through the modulation of polyamine metabolism and posttanslational modification of RhoA, activate a new signal transduction pathway in the intestinal smooth muscle to induce calcium sensitization. Furthermore, apart from being one of the few physiologically relevant nongenomic effects of androgens, these results might underlie the well-known gender differences in intestinal transits, thus expanding the nature's inventory of sex hormones effects.

The cadaverine (Cad), an organic diamine was examined for its response on growth in salinity and metal stressed B. juncea cv RH-30 vis-à-vis compared the response of ammonium nitrate. The Cad (1 mM) application ameliorated the effect caused by salinity and metal stress on seed germination and plant growth. The plant growth recovery (dry biomass accumulation) was dependent on stress and diamine type. The higher growth recovery potential of Cad under both stresses was due to elevation in photosynthetic pigments, nitrate reductase activity and organic nitrogen as well as soluble protein, It is inferred that growth in stressed seedlings was mediated by Cad through lowering endogenous Cd/Pb and Na+ / K+ level in leaf and shoot tissues.

Polyamines decrease the permeability of the outer membrane of Escherichia coli to fluoroquinolones and β-lactams. In this study, we tested the effect of four polyamines (spermidine, spermine, cadaverine and putrescine) on fluoroquinolone uptake in Mycobacterium bovis BCG. Our results show that polyamines are also capable of reducing the permeability of the mycobacterial outer membrane to fluoroquinolones. Spermidine was most effective and demonstrated reversible dose- and pH-dependent inhibition of ciprofloxacin accumulation. The extent of this inhibition was demonstrated across the fluoroquinolone compound class to varying degrees. Furthermore, we have shown that the addition of spermidine increases the survival of M. bovis BCG after a 5-day exposure to ciprofloxacin by up to 25 times. The treatment of actively-replicating Mycobacterium tuberculosis with spermidine reduced ciprofloxacin accumulation by half while non-replicating nutrient-starved M. tuberculosis cultures lacked similar sensitivity to polyamines. Gene expression studies showed that several outer membrane proteins are significantly down-regulated during the shift to non-replication. Collectively, these characteristics of fluoroquinolone uptake in M. bovis BCG are consistent with facilitated transport by porin-like proteins and suggest that a reduction in intracellular uptake contributes to the phenotypic drug resistance demonstrated by M. tuberculosis in the non-replicating state.

At acidic pH and in the presence of lysine, the pH sensor CadC activates transcription of the cadBA operon encoding the lysine/cadaverine antiporter CadB and the lysine decarboxylase CadA. In effect, these proteins contribute to acid stress adaptation in Escherichia coli. cadBA expression is feedback inhibited by cadaverine, and a cadaverine binding site is predicted within the central cavity of the periplasmic domain of CadC on the basis of its crystallographic analysis. Our present study demonstrates that this site only partially accounts for the cadaverine response in vivo. Instead, evidence for a second, pivotal binding site was collected, which overlaps with the pH-responsive patch of amino acids located at the dimer interface of the periplasmic domain. The temporal response of the E. coli Cad module upon acid shock was measured and modeled for two CadC variants with mutated cadaverine binding sites. These studies supported a cascade-like binding and deactivation model for the CadC dimer: binding of cadaverine within the pair of central cavities triggers a conformational transition that exposes two further binding sites at the dimer interface, and the occupation of those stabilizes the inactive conformation. Altogether, these data represent a striking example for the deactivation of a pH sensor., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Different cell types have been reported to internalize lactoferrin (Lf) by specific or nonspecific receptors. Our studies focused on the endocytic pathway of human Lf in macrophage-like THP-1 cells. Lactoferrin was found to be internalized by THP-1 cells differentiated with phorbol myristate acetate. Incubation of cells with chlorpromazine and dansylcadaverine, inhibitors of clathrin-dependent endocytosis, led to a 50% inhibition of Lf internalization compared with untreated cells. Bafilomycin A1 and NH(4)Cl treatment also resulted in 40%-60% inhibition, respectively, suggesting that the internalization of Lf may partly be mediated by acidic endosome-like organelles. Endocytic uptake of Lf was also cholesterol-dependent, as shown by methyl-β-cyclodextrin or nystatin treatment of the cells prior to internalization. Partial colocalization of Lf and EEA-1, a marker specific for early endosomes, could be observed. Colocalization of Lf with a specific endoplasmic reticulum marker was also detected. Our results suggest that Lf is internalized mainly by the clathrin-dependent pathway in THP-1 cells and targets the ER. The physiological consequences of this intracellular trafficking will be the subject of future investigations.

Recognition of lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) activates two district proinflammatory signaling pathway and initiates LPS internalization. To investigate roles of LPS internalization, a traditionally regarded metabolic pathway for LPS, in regulation of these two pathways, three internalization inhibitors, monodansylcadaverine (MDC, a clathrin inhibitor), dynasore (DS, a dynamin inhibitor) and chloroquine (CQ, an endosome acidifying maturation inhibitor) were applied to induce internalization dysfunction in macrophages. Results showed MDC and DS affected LPS internalization but did not interfere with their colocalization. Additionally, they decreased cytokines and chemokines release and inhibited signaling molecules activation mediated by TRAM-TRIF-dependent pathway as determined by protein array. In contrast, CQ did not inhibit LPS internalization but affected the colocalization. It also suppressed macrophage activation mediated by both MyD88-dependent and TRAM-TRIF-dependent pathways. The above data indicated that LPS internalization was clathrin/dynamin dependent and it was essential for activation of TRAM-TRIF-dependent signaling pathway., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A new bisamide N₁-acetyl-N₇-phenylacetyl cadaverine (1) and a series of diketopiperazines including a new diketopiperazine cyclo(2-hydroxy-Pro-R-Leu) (2), together with a new natural product cyclo(4-hydroxy-S-Pro-S-Trp) (3) and two known leucine-based diketopiperazines cyclo(4-hydroxy-R-Pro-S-Leu) (4) and cyclo (S-Pro-R-Leu) (5), were isolated from ethyl acetate extract of a fermentation broth of a marine-derived Streptomyces sp. Their structures were elucidated by the interpretation of spectroscopic analysis. The antitumor activities of compounds 1-3 against HL-60 cell lines were tested by MTT assay.

Epidermal growth factor (EGF) decreases the mRNA and protein levels of claudin-2 (CLDN2) in Madin-Darby canine kidney (MDCK) II cells. Here we examined whether EGF affects the stability and intracellular distribution of CLDN2 protein. EGF decreased surface and total levels of CLDN2, which was inhibited by U0126, a MEK inhibitor. CLDN2 was co-localized at the tight junctions (TJs) with ZO-1, a scaffolding protein. The fluorescence signal for CLDN2 disappeared on treatment with EGF, which was inhibited by U0126. EGF accelerated the decrease in CLDN2 in the presence of cycloheximide, a translation inhibitor, indicating that EGF reduces the stability of the protein. Chloroquine, a lysosomal protease inhibitor, blocked the EGF-induced decrease in CLDN2 protein and caused the co-localization of CLDN2 with Lamp-1, a marker of lysosome. Monodancylcadaverine, an inhibitor of endocytosis, and clathrin siRNA blocked the EGF-induced decrease in CLDN2 and the translocation of CLDN2 from the TJs to the lysosome. EGF increased the association of CLDN2 with clathrin and adaptin α which was inhibited by U0126. These results suggest that EGF accelerates clathrin-dependent endocytosis and lysosomal degradation of CLDN2 protein mediated by the activation of a MEK/ERK pathway., (Copyright © 2011 Wiley-Liss, Inc.)

Matrine has a wide range of pharmacological effects including antitumor activity in vitro and in vivo. Autophagy is closely associated with tumors and plays an important role in human tumor suppression, so inducing autophagy is a potential therapeutic strategy in adjuvant chemotherapy. The aim of this study was to investigate whether or not autophagy is involved in antitumor effects of matrine on human gastric cancer SGC-7901 cells, and to further elucidate the underlying molecular mechanisms. Sulphorhodamine B (SRB) assay was used to examine matrine's cytotoxicity against SGC-7901 gastric cancer cells. The effects of matrine on the cell cycle and apoptosis were measured by flow cytometry, and cellular morphology was observed under an inverted phase contrast microscope and transmission electron microscope. Monodansylcadaverine (MDC) staining was used to detect autophagy. The expression levels of Bax and Beclin 1 in SGC-7901 cells were monitored by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed that matrine significantly inhibited the proliferation of SGC-7901 gastric cancer cells and induced G1-phase cell cycle arrest. Furthermore, both autophagy and apoptosis were activated during the matrine-induced death of SGC-7901 cells. Beclin 1 is involved in matrine-induced autophagy and the pro-apoptotic mechanisms of matrine may be associated with its up-regulation of Bax expression. These findings indicate that matrine is a potent antitumor agent for treating gastric cancer. The ability of matrine to induce autophagy underlines its potential utility as a new gastric cancer treatment modality.

Mammalian oocytes are arrested at prophase I of meiosis until a preovulatory surge of LH stimulates them to resume meiosis. Prior to the LH surge, high levels of cAMP within the oocyte maintain meiotic arrest; this cAMP is generated in the oocyte through the activity of the constitutively active, G(s)-coupled receptor, G-protein-coupled receptor 3 (GPR3) or GPR12. Activated GPRs are typically targeted for desensitization through receptor-mediated endocytosis, but a continuously high level of cAMP is needed for meiotic arrest. The aim of this study was to examine whether receptor-mediated endocytosis occurs in the mouse oocyte and whether this could affect the maintenance of meiotic arrest. We found that constitutive endocytosis occurs in the mouse oocyte. Inhibitors of receptor-mediated endocytosis, monodansylcadaverine and dynasore, inhibited the formation of early endosomes and completely inhibited spontaneous meiotic resumption. A red fluorescent protein-tagged GPR3 localized in the plasma membrane and within early endosomes in the oocyte, demonstrating that GPR3 is endocytosed. However, overexpression of G-protein receptor kinase 2 and β-arrestin-2 had only a modest effect on stimulating meiotic resumption, suggesting that these proteins do not play a major role in GPR3 endocytosis. Inhibition of endocytosis elevated cAMP levels within oocytes, suggesting that there is an accumulation of GPR3 at the plasma membrane. These results show that endocytosis occurs in the oocyte, leading to a decrease in cAMP production, and suggest that there is a balance between cAMP production and degradation in the arrested oocyte that maintains cAMP levels at an appropriate level during the maintenance of meiotic arrest.

Although hypoxia can induce cell death, the cancer cells and endothelial cells within a solid tumor that remain active in the hypoxia microenvironment often possess an enhanced survival potential. Developing approaches aimed at increasing the sensitivity of endothelial cells to hypoxia-induced cell death represents a potentially important avenue for antiangiogenesis treatment. This study investigated approaches to increase the sensitivity of endothelial cells to hypoxia-induced apoptosis. Autophagy and apoptosis of endothelial cells induced by hypoxia were investigated by transmission electron microscopy, confocallaser microscopy, and western blotting. Moreover, cell invasion was observed by a transwell assay and F-actin quantitative analysis. In this study, it was found that hypoxia could induce both autophagy and apoptosis in hypoxia-inducible factor-1- and Beclin1-dependent endothelial cells. Hypoxia-induced autophagy was prohibited by phosphatidylinositol 3-kinase/Akt inhibitor but not mitogen-activated protein kinase inhibitor. Inhibition of autophagy promoted the rate of apoptosis. Further, the reversal of hypoxia-induced autophagy increased cell migration compared with the normoxia condition. This study concludes that hypoxia triggers a feedback mechanism that delays apoptosis of endothelial cells and that is driven by hypoxia-induced autophagy. Thus, approaches aimed at the disruption of this mechanism can be expected to enhance the susceptibility of endothelial cells to hypoxia-induced apoptosis.

Premature delivery remains a serious risk factor in pregnancy, with currently licensed tocolytics unable to offer significant improvement in neonatal outcome. Further understanding of the regulators of uterine contractility is required to enable the development of novel and more effective tocolytic therapies. The transglutaminase family is a class of calcium-dependent, transamidating enzymes, of which tissue transglutaminase 2 is a multifunctional enzyme with roles in cell survival, migration, adhesion, and contractility. The aim of the present study was to investigate the role of this enzyme in regulating the contractility of pregnant human myometrium. Tissue strips from biopsy samples obtained at elective cesarean section were either allowed to contract spontaneously or induced to contract with oxytocin, phenylephrine, or bradykinin. Activity integrals, used to measure contractile activity, were taken following cumulative additions of the reversible, polyamine transglutaminase inhibitors cystamine and mono-dansylcadaverine and the irreversible, site-specific transglutaminase inhibitors N-benzyloxycarbonyl-l-phenylalanyl-6-dimethylsulfonium-5-oxo-L-norleucine and 1,3-dimethyl-2[(oxopropyl)thio]imidazolium. The ability of cystamine and mono-dansylcadaverine to affect oxytocin-mediated calcium mobilization within primary cultured myometrial cells was also measured utilizing a calcium indicator. All inhibitors attenuated myometrial contractions in a concentration-dependent manner independent of the method of contraction stimulus. Similarly cultured myometrial cells preincubated with cystamine and mono-dansylcadaverine displayed an altered calcium response to oxytocin stimulation. Our findings demonstrate a potential role for tissue transglutaminase 2 in regulating uterine contractility in pregnant human myometrium that may be associated with the calcium signaling cascade required for contraction.

Application of exogenous putrescine (Put) increases nickel accumulation in rape shoots (Brassica napus), improving potential for phytoremediation of contaminated soils. Plants were grown within a growth chamber in water culture for five weeks, then 250-500 microM NiCl2 was added to rooting media. Within 5 days of treatment, damaging effects of nickel manifested as a reduction of root system size and a decrease of Cu and especially Fe content in young leaves. Spraying leaves of adult plants with Put markedly reduced toxic effects of Ni on root growth, enhanced leaf supply with Fe, and increased Ni content in young leaves by 2.5 times. Plant growth in medium with elevated levels of Ni stimulated accumulation of endogenous spermidine (Spd), spermine (Spm), and especially Put (by 4 times as compared with the control). Results suggest that Ni-induced accumulation of endogenous polyamines in rape leaves is caused by activation of long-distance metal transport within the plant and reduction of its toxicity due to Put chelating action.

Background and Purpose: The P2Y(1) receptor promotes chloride secretion in epithelial cells, a process critical for regulation of extracellular ion and fluid levels. Here we have examined the role of phosphorylation in agonist-induced internalization of P2Y(1) receptors., Experimental Approach: A high-affinity radiolabelled antagonist, MRS2500, was used to quantify cell surface-binding sites of P2Y(1) receptors in Madin-Darby canine kidney (MDCK) epithelial cells, following exposure to agonists. The regions in the carboxyl terminus involved in both agonist-induced internalization of the receptor and its phosphorylation were identified by mutational analysis., Key Results: Endogenous and stably expressed recombinant P2Y(1) receptors rapidly internalized with similar time courses in response to agonist in MDCK cells, ensuring that the levels of recombinant receptor achieved by retroviral infection did not adversely affect function of the internalization machinery. Four protein kinase C inhibitors of varying specificity did not affect internalization of recombinant receptors. Agonist-promoted internalization of a series of truncated P2Y(1) receptors identified a region between residues 349 and 359 in the carboxyl terminus as critical for regulation. Two amino acids within this region, Ser352 and Ser354, were shown to be both necessary and sufficient for agonist-promoted receptor phosphorylation and internalization., Conclusions and Implications: Our results firmly establish Ser352 and Ser354 in the carboxyl terminus of P2Y(1) receptors as critical residues for agonist-induced receptor internalization in MDCK cells. As the mechanism mediating this regulation requires phosphorylation of these key residues, the relevant receptor-regulated protein kinase can now be identified., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)