Cellular Polyamine Research Articles

Occurrence of homospermidine and thermospermine as a cellular polyamine in unicellular chlorophyte and multicellular charophyte green algae

Occurrence of homospermidine and thermospermine as a cellular polyamine in unicellular chlorophyte and multicellular charophyte green algae

Polyamines and Gut Mucosal Homeostasis

The epithelium of gastrointestinal (GI) mucosa has the most rapid turnover rate of any tissue in the body and its integrity is preserved through the dynamic balance between cell migration, proliferation, growth arrest and apoptosis. To maintain tissue homeostasis of the GI mucosa, the rates of epithelial cell division and apoptosis must be highly regulated by various extracellular and intracellular factors including cellular polyamines. Natural polyamines spermidine, spermine and their precursor putrescine, are organic cations in eukaryotic cells and are implicated in the control of multiple signaling pathways and distinct cellular functions. Normal intestinal epithelial growth depends on the available supply of polyamines to the dividing cells in the crypts, and polyamines also regulate intestinal epithelial cell (IEC) apoptosis. Although the specific molecular processes controlled by polyamines remains to be fully defined, increasing evidence indicates that polyamines regulate intestinal epithelial integrity by modulating the expression of various growth-related genes. In this review, we will extrapolate the current state of scientific knowledge regarding the roles of polyamines in gut mucosal homeostasis and highlight progress in cellular and molecular mechanisms of polyamines and their potential clinical applications.

Temporal regulation of somatic embryogenesis in guava (Psidium guajava L.)

SummarySomatic embryogenesis was induced in immature zygotic embryos of guava (Psidium guajava L.) at 10-weeks post anthesis using 2 – 38 d pulses and continuous treatments with different concentrations (0.001 – 3.0 mg l–1) of 2,4-dichlorophenoxyacetic acid (2,4-D). Subsequent development of somatic embryo was observed by sub-culturing onto plant growth regulator (PGR)-free, full-strength Murashige and Skoog (MS) agar medium containing 3% (w/v) sucrose. The maximum responses were shifted gradually from 0.01 mg l–1 2,4-D (continuous or 38 d treatment), to 0.05 mg l–1 2,4-D (28 – 18 d treatment), to 0.1 mg l–1 2,4-D (14 – 12 d treatment) and, finally, to 0.5 mg l–1 2,4-D (10 – 8 d treatment).The highest frequency of embryogenesis (68.8%) and embryogenic intensity (69.2 embryos per explant per culture), as well as moderate frequencies of convertible elongated and short torpedo-stage somatic embryos (15% and 42.9%, respectively), with the highest efficiency (27.5) achieved in the 8 d treatment compared to continuous treatment of zygotic embryo explants with 0.5 mg l–1 2,4-D in MS agar medium containing 3% (w/v) sucrose at both the induction and development phases. Cellular pools of total, free, conjugated, and bound forms of various polyamines (e.g., putrescine, spermidine, and spermine) showed slow increases in the presence of 2,4-D during the 8 d induction phase. This was followed by rapid increases immediately after sub-culturing explant tissue onto the PGR-free MS development medium. The concentrations of various polyamines declined gradually over time on MS development medium. The production, metabolism, and use of different forms of various polyamines at various times (d) after culture initiation correlated with their temporal regulation during the induction and development phases of somatic embryogenesis. Exogenous application of different polyamines during the induction phase resulted in slightly increased frequencies, intensities, and efficiencies of embryogenesis, but with no observable change in the frequencies of the various categories of somatic embryos. The present study indicated that the process of somatic embryogenesis in guava may be less sensitive to PGRs and was induced even in the continuous presence of 2,4-D. Furthermore, somatic embryogenesis in guava was temporally regulated as a function of the exogenous concentration of 2,4-D. In addition, the temporal regulation of somatic embryogenesis was also associated with high levels of production, metabolism, and use of cellular polyamines during the different phases. Hence, improvements in the process of somatic embryogenesis in guava could be regulated by timed applications of 2,4-D alone, or in combination with polyamines, thereby modulating endogenous levels of the latter.

Cellular polyamines of alkaliphilic, halophilic and thermophilic methanogens, and acidophilic and alkaliphilic extreme halophiles, and acidophilic thermophiles belonging to the domain Archaea

Cellular polyamines of alkaliphilic, halophilic and thermophilic methanogens, and acidophilic and alkaliphilic extreme halophiles, and acidophilic thermophiles belonging to the domain Archaea

Targeted expression of ornithine decarboxylase antizyme prevents upper aerodigestive tract carcinogenesis in p53-deficient mice

Upper aerodigestive tract (UADT) cancers of the oral cavity and esophagus are a significant global health burden, and there is an urgent need to develop relevant animal models to identify chemopreventive and therapeutic strategies to combat these diseases. Antizyme (AZ) is a multifunctional negative regulator of cellular polyamine levels, and here, we evaluate the susceptibility of keratin 5 (K5)-AZ transgenic mice to tumor models that combine chemical carcinogenesis with dietary and genetic risk factors known to influence human susceptibility to UADT cancer and promote UADT carcinogenesis in mice. First, p53(+/-) and K5-AZ/p53(+/-) (AZ/p53(+/-)) mice were placed on a zinc-deficient (ZD) or zinc-sufficient (ZS) diet and chronically exposed to 4-nitroquinoline 1-oxide. Tongue tumor incidence, multiplicity and size were substantially reduced in both ZD and ZS AZ/p53(+/-) mice compared with p53(+/-). AZ expression also reduced progression to carcinoma in situ or invasive carcinoma and decreased expression of the squamous cell carcinoma biomarkers K14, cyclooxygenase-2 and metallothionein. Next, AZ-expressing p53(+/-) and p53 null mice were placed on the ZD diet and treated with a single dose of N-nitrosomethylbenzylamine. Regardless of p53 status, forestomach (FST) tumor incidence, multiplicity and size were greatly reduced with AZ expression, which was also associated with a significant decrease in FST epithelial thickness along with reduced proliferation marker K6 and increased differentiation marker loricrin. These studies demonstrate the powerful tumor suppressive effects of targeted AZ expression in two distinct and unique mouse models and validate the polyamine metabolic pathway as a target for chemoprevention of UADT cancers.

Polyamines and carcinogenesis

SUMMARY The naturally occurring polyamines, spermine, spermidine and the diamine putrescine are widespread in nature. They have been implicated in growth and differentiation processes. In 1967, we reported that cancer cells are rich in polyamines. Subsequently, it has been shown that polyamines are released from cancer cells and may be detected in body fluids such as urine, blood and cerebrospinal fluids. It has also been demonstrated that the increase in cellular polyamine levels is an early and an obligatory event in the process of malignant transformation. This increase in cellular polyamine concentration is due to the activation of ornithine decarboxylase (ODC), which catalyses the rate limiting step in polyamine synthesis by converting ornithine to putrescine. Assays of urinary and blood polyamines have been used to detect cancer and to determine the success of therapy. A sensitive, rapid, chemiluminescence-based method for the determination of diamines and polyamines was developed and 2.000 urine samples were tested. An interesting "gene therapy" system for injecting amine oxidases into normal and transformed cells was developed as follows: serum amine oxidase and porcine kidney damine oxidase were trapped within reconstituted Sendai virus envelopes. Chick or rat fibroblasts, transformed by Rous sarcoma virus, were more susceptible to the injected enzymes, compared to the normal culture, when macromolecular synthesis was tested. An in vitro chemosensitivity assay for the testing of the sensitivity of cancer cells from individual patients ("tailored treatment") was also developed. All these studies stress the importance of polyamines in carcinogenesis.

Modulation of polyamine levels in ginseng hairy root cultures subjected to salt stress

Modulation of differential gene expression and change of polyamine content by salt stress are analyzed for the first time in a well-known medicinal plant, Panax ginseng C.A. Meyer. Three ginseng genes (PgSPD, PgSAMDC, and PgADC) involved in polyamine biosynthesis showed differential up-regulation patterns after 1 and 7 days of salt treatments. The modulation of gene expression resulted in the elevation of total polyamine content with relatively high levels of spermidine and spermine, while putrescine level diminished depending on the salt concentration. Conversely, salt stress led to a significant increase in diamine oxidase and subsequent decline in polyamine oxidase. The proline content caused by salinity follows a similar pattern as the total polyamine content and exogenous spermidine also resulted in the alleviation of proline content under salinity. Further, polyamine biosynthesis inhibitors, such as cyclohexylamine and methylglyoxal bis-(guanylhydrazone) mediated down-regulation of PgSPD and PgSAMDC, and affected cellular polyamine levels. Thus, polyamines may enhance the ginseng plant tolerance in response to the salt stress by increasing the levels of endogenous polyamines.

Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1.

A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H(2)O(2). The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenously-applied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H(2)O(2). Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn.

Polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca²+ signaling by differentially modulating STIM1 and STIM2.

Early epithelial restitution occurs as a consequence of intestinal epithelial cell (IEC) migration after wounding, and its defective regulation is implicated in various critical pathological conditions. Polyamines stimulate intestinal epithelial restitution, but their exact mechanism remains unclear. Canonical transient receptor potential-1 (TRPC1)-mediated Ca(2+) signaling is crucial for stimulation of IEC migration after wounding, and induced translocation of stromal interaction molecule 1 (STIM1) to the plasma membrane activates TRPC1-mediated Ca(2+) influx and thus enhanced restitution. Here, we show that polyamines regulate intestinal epithelial restitution through TRPC1-mediated Ca(2+) signaling by altering the ratio of STIM1 to STIM2. Increasing cellular polyamines by ectopic overexpression of the ornithine decarboxylase (ODC) gene stimulated STIM1 but inhibited STIM2 expression, whereas depletion of cellular polyamines by inhibiting ODC activity decreased STIM1 but increased STIM2 levels. Induced STIM1/TRPC1 association by increasing polyamines enhanced Ca(2+) influx and stimulated epithelial restitution, while decreased formation of the STIM1/TRPC1 complex by polyamine depletion decreased Ca(2+) influx and repressed cell migration. Induced STIM1/STIM2 heteromers by polyamine depletion or STIM2 overexpression suppressed STIM1 membrane translocation and inhibited Ca(2+) influx and epithelial restitution. These results indicate that polyamines differentially modulate cellular STIM1 and STIM2 levels in IECs, in turn controlling TRPC1-mediated Ca(2+) signaling and influencing cell migration after wounding.

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.

1113 Caveolin 1 Stimulates Intestinal Epithelial Restitution by Activating CA2+ Signaling and is Regulated by Cellular Polyamines

Background & Aims: Micro-RNAs (miRNAs) are short, non-coding RNAs that regulate gene expression post transcriptionally. Several studies have demonstrated distinct miRNA profiles in a wide range of human disorders including inflammatory bowel disease and colon cancer. Surprisingly little is known about miRNA responses in immune cells that are challenged with relevant proinflammatory stimuli, i.e. cytokines or pathogen-associated microbial patterns. With this study we thus aim to generate a prototypic picture of inflammatory miRNA networks in monocytes as a relevant immune cell population and to understand functional consequences of selected regulated miRNAs on inflammatory signalling pathways. Methods: Levels of 330 miRNAs upon stimulation with a panel of pro-inflammatory components such as microbial pattern molecules (flagellin, diacylated lipopeptide lipopolysaccharide, muramyl dipeptide), infection with Listeria monocytogenes and TNF-alpha as pro-inflammatory control in primary human monocytes were qunatified using real time PCR. Selected miRNAs were verified in parallel in LPMNCs and biopsies from IBD patients. Transfection of premiRs and antago-miRs in THP1 monocytic cells were employed in order to understand functional consequences of miRNA regulation. Results: We identified distinct miRNA response clusters pointing to unique and shared mechanisms for different proinflammatory stimuli even in a single cell type. Additionally, we identified potential target genes of three selected miRNAs miR-129-5p, miR-146a and miR-378 which were part of PAMP-specific response clusters by transfecting THP1 monocytes with the corresponding preor antimiRNAs . The miRNAs induced distinct transcriptomal signatures, e.g. overexpression of miRNA129-5p, which was selectively upregulated by the NOD2-elicitor MDP, led to an upregulation of DEFB1, IRAK1, FBXW7 and IKK-gamma (Nemo). Interestingly, regulation of several "overlap" miRNAs shared between stimuli could also be detected in samples from IBD patients. Conclusions: Our findings on co-regulated clusters of miRNAs support the hypothesis that miRNAs act in functional groups. This study indicates that miRNAs play an important role in fine-tuning inflammatory mechanisms. Investigation in the field of miRNA responses in intestinal inflammation will help to understand their effects on gene expression and may close the regulatory gap between mRNA and protein expression in inflammatory diseases.

Abstract 1522: Elevated polyamine levels generate an immunosuppressive oncogenic microenvironment

Abstract Increased suprabasal epidermal activity of ornithine decarboxylase (ODC), a key regulatory enzyme in polyamine biosynthesis, stimulates cellular proliferation and vascularization in the underlying dermis, and promotes skin tumorigenesis after treatment with a single, subthreshold dose of a carcinogen in ODCER transgenic mice. Interestingly, skin tumor formation is also induced in ODCER transgenic mice by wounding and does not require prior carcinogen treatment. This wound-induced tumor formation is dependent on the polyamine-induction of sustained recruitment of inflammatory cells to the dermal wound site. Moreover, when weakly transformed (Ras activation) primary keratinocytes (Ker/Ras) were injected subcutaneously in syngeneic ODCER transgenic mice, tumor growth occurred while it did not in normal littermates. These data suggest the generation of an oncogenic stromal microenvironment in mice with elevated epidermal polyamine levels. Polyamine-Based Therapy (PaBT: combination of the well-characterized ODC inhibitor, difluoromethylornithine (DFMO), and an inhibitor of cellular polyamine transport, AMXT 1501) significantly inhibited tumor growth in wildtype FVB mice that were orthotopically injected with either syngeneic Ker/Ras cells or neu02 mammary tumor cells. Flow cytometry analyses of tumor inflammatory infiltrates revealed that PaBT significantly increased the percentage of CD3+ cell population (includes T cells and NK cells) and decreased the percentage of both F4/80+ (macrophage) and Gr-1+ (neutrophil and monocyte) subpopulations of CD45+ cells compared to tumors in control, non-treated mice. When PaBT treatment was stopped and tumors were resected, we re-challenged two weeks later with a second injection of neu02 tumor cells. Mice that had been previously treated with PaBT were protected from secondary tumor growth (demonstrating no or very little tumor growth) compared to mice that never received PaBT. In order to examine whether elevated polyamine biosynthesis suppresses a T-cell mediated adaptive immune response, we examined the effect of ODC overexpression in keratinocytes on a classic contact hypersensitivity (CHS) response test using ODCER transgenic mice. These mice showed reduced ear swelling in comparison to normal littermates together with reduced neutrophil infiltration and decreased migration of FITC-loaded dendritic cells to draining lymph nodes following hapten elicitation of CHS. Collectively, these data suggest that the immunosuppression mediated by increased epidermal ODC activity may contribute to an environment conducive to tumor growth, and, conversely, that depletion of tumor polyamines will block tumorigenesis in part by normalizing the stromal microenvironment and restoring tumor immune surveillance. (Supported by NIH grant CA070739 and funding from Aminex Therapeutics Inc) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1522. doi:1538-7445.AM2012-1522

Over-expression of Arginine Decarboxylase Gene in Tapetal Tissue Results in Male Sterility in Tomato Plants

Homeostasis of cellular Polyamines (PAs) is very important for proper growth and development of living organisms. Development of male sex organ and gametophyte in plants is a complex process involving many known and unknown pathways. PAs are actively involved in the development of anther and pollen. In the present study, oat arginine decarboxylase (ADC) gene has been over-expressed in tapetum of tomato using tapetum-specific TA29 promoter to perturb the PA homeostasis in order to see its implication on male fertility. Tapetum-specific ADC overexpression resulted in PA accumulation, mainly in free and bound fractions in the anther of transgenics. Transgenic lines displayed abnormal pollen and tapetum development. Pollen grains were aborted and distorted in morphology and tapetum showed premature degeneration in ADC transgenics. These transgenics failed to set seeds, but cross pollination with normal pollen from wild type plants restored the seed setting indicating that s ADC over-expression did not affect female fertility. ADC over-expression also did not affect the growth and morphology of transgenics. The present study suggests that the excess of PAs in anther is lethal for pollen and tapetum development and overexpression of ADC gene can be an effective approach for the engineering of male sterility in plants.

Autoimmune Diseases and Polyamines

Genetics and environmental factors have important roles in autoimmune diseases but neither has given us sufficient understanding of these mysterious diseases. Therefore, we are now looking closer at epigenetics, an interface between genetics and environmental factors. Epigenetics can be defined as reversible heritable changes to chromatin that can alter gene expression without altering the gene's DNA sequence. Methylation of DNA and histones are primary means of epigenetic control. By adding methyl groups to DNA and histones, it can limit accessibility of the underlying gene thereby altering the amount of gene expression. The methyl group is derived from an essential molecule in the cell, S-adenosylmethionine (SAM). However, a group of small molecules called polyamines also require SAM for their synthesis. Polyamines are essential for many cellular functions and polyamine activity is increased in many autoimmune diseases. Presented here is the "polyamine hypothesis" in which increased polyamine synthesis competes with cellular methylation (epigenetic control) for SAM. It is proposed that increased polyamine activity can cause disruption of cellular methylation, which can lead to abnormal expression of previously sequestered genes and disruption of other methylation-dependent cellular processes.

MiR-503 represses CUG-binding protein 1 translation by recruiting CUGBP1 mRNA to processing bodies

microRNAs (miRNAs) and RNA-binding proteins (RBPs) jointly regulate gene expression at the posttranscriptional level and are involved in many aspects of cellular functions. The RBP CUG-binding protein 1 (CUGBP1) destabilizes and represses the translation of several target mRNAs, but the exact mechanism that regulates CUGBP1 abundance remains elusive. In this paper, we show that miR-503, computationally predicted to associate with three sites of the CUGBP1 mRNA, represses CUGBP1 expression. Overexpression of an miR-503 precursor (pre-miR-503) reduced the de novo synthesis of CUGBP1 protein, whereas inhibiting miR-503 by using an antisense RNA (antagomir) enhanced CUGBP1 biosynthesis and elevated its abundance; neither intervention changed total CUGBP1 mRNA levels. Studies using heterologous reporter constructs revealed a greater repressive effect of miR-503 through the CUGBP1 coding region sites than through the single CUGBP1 3'-untranslated region target site. CUGBP1 mRNA levels in processing bodies (P-bodies) increased in cells transfected with pre-miR-503, while silencing P-body resident proteins Ago2, RCK, or LSm4 decreased miR-503-mediated repression of CUGBP1 expression. Decreasing the levels of cellular polyamines reduced endogenous miR-503 levels and promoted CUGBP1 expression, an effect that was prevented by ectopic miR-503 overexpression. Repression of CUGBP1 by miR-503 in turn altered the expression of CUGBP1 target mRNAs and thus increased the sensitivity of intestinal epithelial cells to apoptosis. These findings identify miR-503 as both a novel regulator of CUGBP1 expression and a modulator of intestinal epithelial homoeostasis.

The Enzymatic Activities of the Escherichia coli Basic Aliphatic Amino Acid Decarboxylases Exhibit a pH Zone of Inhibition

The stringent response regulator ppGpp has recently been shown by our group to inhibit the Escherichia coli inducible lysine decarboxylase, LdcI. As a follow-up to this observation, we examined the mechanisms that regulate the activities of the other four E. coli enzymes paralogous to LdcI: the constitutive lysine decarboxylase LdcC, the inducible arginine decarboxylase AdiA, the inducible ornithine decarboxylase SpeF, and the constitutive ornithine decarboxylase SpeC. LdcC and SpeC are involved in cellular polyamine biosynthesis, while LdcI, AdiA, and SpeF are involved in the acid stress response. Multiple mechanisms of regulation were found for these enzymes. In addition to LdcI, LdcC and SpeC were found to be inhibited by ppGpp; AdiA activity was found to be regulated by changes in oligomerization, while SpeF and SpeC activities were regulated by GTP. These findings indicate the presence of multiple mechanisms regulating the activity of this important family of decarboxylases. When the enzyme inhibition profiles are analyzed in parallel, a "zone of inhibition" between pH 6 and pH 8 is observed. Hence, the data suggest that E. coli utilizes multiple mechanisms to ensure that these decarboxylases remain inactive around neutral pH possibly to reduce the consumption of amino acids at this pH.

Elstera litoralis gen. nov., sp. nov., isolated from stone biofilms of Lake Constance, Germany

An alphaproteobacterium, strain Dia-1(T), was isolated from algae-dominated biofilms on stones from the littoral zone of Lake Constance, Germany. This bacterium was isolated after initial enrichment in spent medium obtained after growth of a diatom culture. Numerous sugars and some organic acids and alcohols served as growth substrates. The bacterium grew slowly, was strictly aerobic but microaerophilic, and did not grow in cultures shaken under air. 16S rRNA gene sequence analysis indicated that strain Dia-1(T) was distantly related to representatives of the genera Azospirillum (90-91% sequence similarity), Skermanella (88-89%), Rhodocista (87-88%) and Dongia (88-89% sequence similarity). Based on this sequence comparison, on phenotypic characterization including substrate utilization patterns, and comparison of cellular fatty acids, quinones, polar lipids and polyamines, this isolate was found to be substantially different from the genera mentioned above. On the basis of these results, a novel genus and species is proposed for this strain. The name Elstera litoralis gen. nov., sp. nov. is suggested, with strain Dia-1(T) ( = DSM 19532(T) = LMG 24234(T)) as the type strain of the type species.

Antizyme Affects Cell Proliferation and Viability Solely through Regulating Cellular Polyamines

Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.

Lipopolysaccharide-induced anti-inflammatory acute phase response is enhanced in spermidine/spermine N 1-acetyltransferase (SSAT) overexpressing mice

Bacterial lipopolysaccharide (LPS) is an effective activator of the components of innate immunity. It has been shown that polyamines and their metabolic enzymes affect the LPS-induced immune response by modulating both pro- and anti-inflammatory actions. On the other hand, LPS causes changes in cellular polyamine metabolism. In this study, the LPS-induced inflammatory response in spermidine/spermine N(1)-acetyltransferase overexpressing transgenic mice (SSAT mice) was analyzed. In liver and kidneys, LPS enhanced the activity of the polyamine biosynthetic enzyme ornithine decarboxylase and increased the intracellular putrescine content in both SSAT overexpressing and wild-type mice. In survival studies, the enhanced polyamine catabolism and concomitantly altered cellular polyamine pools in SSAT mice did not affect the LPS-induced mortality of these animals. However, in the acute phase of LPS-induced inflammatory response, the serum levels of proinflammatory cytokines interleukin-1β and interferon-γ were significantly reduced and, on the contrary, anti-inflammatory cytokine interleukin-10 was significantly increased in the sera of SSAT mice compared with the wild-type animals. In addition, hepatic acute-phase proteins C-reactive protein, haptoglobin and α(1)-acid glycoprotein were expressed in higher amounts in SSAT mice than in the wild-type animals. In summary, the study suggests that SSAT overexpression obtained in SSAT mice enhances the anti-inflammatory actions in the acute phase of LPS-induced immune response.