Role Of polyP Research Articles

Mammalian Mitochondrial Inorganic Polyphosphate (polyP) and Cell Signaling: Crosstalk Between PolyP and the Activity of AMPK.

Inorganic polyphosphate (polyP) is an evolutionary ancient polymer composed by orthophosphate units linked by phosphoanhydride bonds. In mammalian cells, polyP shows a high localization in mammalian mitochondria, and its regulatory role in various aspects of bioenergetics has already been demonstrated, via molecular mechanism(s) yet to be fully elucidated. In recent years, a role for polyP in signal transduction, from brain physiology to bloodstream, has also emerged. The intriguing possibility is that the effects of polyP on signal transduction could be mechanistically linked to those exerted on bioenergetics. Here, using a combination of cellular and animal models, we demonstrate for the first time the intimate crosstalk between the levels of polyP and the activation status of the AMPK signaling pathway, via a mechanism involving free phosphate homeostasis. AMPK is a key player in mammalian cell signaling, and a crucial regulator of cellular and mitochondrial homeostasis. Our results show that the depletion of mitochondrial polyP in mammalian cells downregulates the activity of AMPK. Moreover, increased levels of polyP activate AMPK. Accordingly, the genetic downregulation of AMPKα1 impairs polyP levels in both SH-SY5Y cells and in the brains of female mice. Our findings shed new light on the regulation of AMPK and position polyP as a potent regulator of mammalian cell physiology beyond mere bioenergetics, paving the road for using its metabolism as an innovative pharmacological target in pathologies characterized by dysregulated bioenergetics.

Characterization of polyphosphate dynamics in the widespread freshwater diatom Achnanthidium minutissimum under varying phosphorus supplies.

Polyphosphates (polyP) are ubiquitous biomolecules that play a multitude of physiological roles in many cells. We have studied the presence and role of polyP in a unicellular alga, the freshwater diatom Achnanthidium minutissimum. This diatom stores up to 2.0 pg·cell-1 of polyP, with chain lengths ranging from 130 to 500 inorganic phosphate units (Pi). We applied energy dispersive X-ray spectroscopy, Raman/fluorescence microscopy, and biochemical assays to localize and characterize the intracellular polyP granules that were present in large apical vacuoles. We investigated the fate of polyP in axenic A. minutissimum cells grown under phosphorus (P), replete (P(+)), or P deplete (P(-)) cultivation conditions and observed that in the absence of exogenous P, A. minutissimum rapidly utilizes their internal polyP reserves, maintaining their intrinsic growth rates for up to 8 days. PolyP-depleted A. minutissimum cells rapidly took up exogenous P a few hours after Pi resupply and generated polyP three times faster than cells that were not initially subjected to P limitation. Accordingly, we propose that A. minutissimum deploys a succession of acclimation strategies regarding polyP dynamics where the production or consumption of polyP plays a central role in the homeostasis of the diatom.

Liquid-Liquid Phase Separation (LLPS)-Driven Fibrilization of Amyloid-β Protein.

Amyloid-β [Aβ(1-40)] aggregation into a fibrillar network is one of the major hallmarks of Alzheimer's disease (AD). Recently, a few studies reported that polyphosphate (polyP), an anionic biopolymer that participates in various cellular physiological processes in humans, induces fibrilization in many amyloidogenic proteins [ 2020 Alzheimer's Disease Facts and Figures; John Wiley and Sons Inc., 2020; Tanzi, R. E.; Bertram, L. Cell 2005, 120, 545-555; Selkoe, D. J. Proc. Natl. Acad. Sci. U.S.A. 1995, 275, 630-631; and Rambaran, R. N.; Serpell, L. C. Prion 2008, 2, 112-117]. However, the role of polyP in Aβ(1-40) fibrilization and the underlying mechanism are unclear. In this study, we report experimental investigations on the role of polyP in the fibrilization kinetics of Aβ(1-40). It is found that polyP exhibits a dual effect depending upon the pH value. At pH = 7 (neutral), polyP inhibits amyloid fibrilization in a dose-dependent manner similar to negatively charged nanoparticles. On the contrary, at pH = 3 (acidic), polyP accelerates amyloid fibrilization kinetics via liquid-liquid phase separation (LLPS), wherein the protein-rich droplets contain mature fibrils. In the parameter space spanned by concentrations of Aβ(1-40) and polyP, a phase diagram is constructed to demark the domain where LLPS is observed at pH = 3. Characterization of the protein aggregates, secondary structure content in the aggregates, and cell viability studies in the presence of aggregates are discussed at both pH values. This study reveals that anionic biopolymers can modulate amyloid fibrilization kinetics, linked to neurodegenerative diseases, depending upon their local concentrations and pH.

The emerging landscape of eukaryotic polyphosphatases.

Polyphosphate (polyP) is a conserved polymer of inorganic phosphate residues that can reach thousands of moieties in length. PolyP has been implicated in cellular functions ranging from energy and phosphate homeostasis to cell signalling in eukaryotes from yeast to humans. Despite the interest in the role of polyP as a signalling molecule, the spatiotemporal regulation of polyP itself remains poorly understood. This knowledge gap limits our ability to understand how polyP impacts the physiology of normal and diseased cells and how this might be exploited in a therapeutic context. Polyphosphatases, enzymes that degrade polyP to generate shorter chains and free inorganic phosphate are ideally positioned to mediate polyP dynamics. However, little is known about how the activities of these enzymes are linked to specific cellular functions and how they might be regulated. Here, we provide an in-depth overview of polyphosphatase enzymes in budding yeast, which has served as a workhorse for polyP research, and in mammalian cells where the enzymes that make and degrade polyP have remained elusive. We identify critical open questions in both systems and propose strategies to guide future work.

Depletion of mitochondrial polyP levels increase the phosphorylation of AMPK protein and compromises oxygen consumption in SH-SY5Y cells.

Inorganic polyphosphate (polyP) is a highly negatively charged polymer that is well-conserved through evolution and present in every studied organism. PolyP is composed of orthophosphates linked by high-energy phosphoanhydride bonds. While its metabolism has been characterized in microorganisms, it is poorly explored in mammals. Previous studies showed that polyP can be found in diverse mammalian subcellular compartments but that it is especially abundant in mitochondria, where it could function as a key regulator of mitochondrial bioenergetics, via an unknown mechanism. We have demonstrated that decreased levels of polyP in mammalian mitochondria induce the dysfunction of the organelle, which results in compromised bioenergetics. AMP-activated protein kinase (AMPK) is a key energy gauge, and it regulates diverse metabolic processes. Therefore, this study aimed to investigate whether polyP levels can participate in the activation of AMPK and how this pathway can impact mitochondrial physiology and represent an important and novel approach that can contribute to improving our knowledge of mammalian bioenergetics and mitochondrial physiology. We used undifferentiated and differentiated SH-SY5Y cells, under wild-type conditions and after the enzymatical depletion of mitochondrial polyP (MitoPPX). Our data show that depletion of mitochondrial polyP impacts mitochondrial bioenergetics by affecting ATP-linked respiration. It also increases the levels of activated AMPK in differentiated SH-SY5Y cells. Our findings suggest an important novel role of polyP as a key regulator of cellular bioenergetics probably via the regulation of the AMPK pathway. AMPK activity and mitochondrial dysfunction are commonly implicated in the pathogenesis of many neurodegenerative diseases. Therefore, the manipulation of the metabolism of polyP in neuronal cells might represent a plausible target for developing therapeutic approaches against neurodegenerative diseases.

Polyphosphate Dynamics in Cable Bacteria.

Cable bacteria are multicellular sulfide oxidizing bacteria that display a unique metabolism based on long-distance electron transport. Cells in deeper sediment layers perform the sulfide oxidizing half-reaction whereas cells in the surface layers of the sediment perform the oxygen-reducing half-reaction. These half-reactions are coupled via electron transport through a conductive fiber network that runs along the shared cell envelope. Remarkably, only the sulfide oxidizing half-reaction is coupled to biosynthesis and growth whereas the oxygen reducing half-reaction serves to rapidly remove electrons from the conductive fiber network and is not coupled to energy generation and growth. Cells residing in the oxic zone are believed to (temporarily) rely on storage compounds of which polyphosphate (poly-P) is prominently present in cable bacteria. Here we investigate the role of poly-P in the metabolism of cable bacteria within the different redox environments. To this end, we combined nanoscale secondary ion mass spectrometry with dual-stable isotope probing (13C-DIC and 18O-H2O) to visualize the relationship between growth in the cytoplasm (13C-enrichment) and poly-P activity (18O-enrichment). We found that poly-P was synthesized in almost all cells, as indicated by 18O enrichment of poly-P granules. Hence, poly-P must have an important function in the metabolism of cable bacteria. Within the oxic zone of the sediment, where little growth is observed, 18O enrichment in poly-P granules was significantly lower than in the suboxic zone. Thus, both growth and poly-P metabolism appear to be correlated to the redox environment. However, the poly-P metabolism is not coupled to growth in cable bacteria, as many filaments from the suboxic zone showed poly-P activity but did not grow. We hypothesize that within the oxic zone, poly-P is used to protect the cells against oxidative stress and/or as a resource to support motility, while within the suboxic zone, poly-P is involved in the metabolic regulation before cells enter a non-growing stage.

Inorganic Polyphosphate, Mitochondria, and Neurodegeneration.

With an aging population, the presence of aging-associated pathologies is expected to increase within the next decades. Regrettably, we still do not have any valid pharmacological or non-pharmacological tools to prevent, revert, or cure these pathologies. The absence of therapeutical approaches against aging-associated pathologies can be at least partially explained by the relatively lack of knowledge that we still have regarding the molecular mechanisms underlying them, as well as by the complexity of their etiopathology. In fact, a complex number of changes in the physiological function of the cell has been described in all these aging-associated pathologies, including neurodegenerative disorders. Based on multiple scientific manuscripts produced by us and others, it seems clear that mitochondria are dysfunctional in many of these aging-associated pathologies. For example, mitochondrial dysfunction is an early event in the etiopathology of all the main neurodegenerative disorders, and it could be a trigger of many of the other deleterious changes which are present at the cellular level in these pathologies. While mitochondria are complex organelles and their regulation is still not yet entirely understood, inorganic polyphosphate (polyP) could play a crucial role in the regulation of some mitochondrial processes, which are dysfunctional in neurodegeneration. PolyP is a well-preserved biopolymer; it has been identified in every organism that has been studied. It is constituted by a series of orthophosphates connected by highly energetic phosphoanhydride bonds, comparable to those found in ATP. The literature suggests that the role of polyP in maintaining mitochondrial physiology might be related, at least partially, to its effects as a key regulator of cellular bioenergetics. However, further research needs to be conducted to fully elucidate the molecular mechanisms underlying the effects of polyP in the regulation of mitochondrial physiology in aging-associated pathologies, including neurodegenerative disorders. With a significant lack of therapeutic options for the prevention and/or treatment of neurodegeneration, the search for new pharmacological tools against these conditions has been continuous in past decades, even though very few therapeutic approaches have shown potential in treating these pathologies. Therefore, increasing our knowledge about the molecular mechanisms underlying the effects of polyP in mitochondrial physiology as well as its metabolism could place this polymer as a promising and innovative pharmacological target not only in neurodegeneration, but also in a wide range of aging-associated pathologies and conditions where mitochondrial dysfunction has been described as a crucial component of its etiopathology, such as diabetes, musculoskeletal disorders, and cardiovascular disorders.

Inorganic polyphosphate as an energy source in tumorigenesis.

Cancer cells have high demands for energy to maintain their exceedingly proliferative growth. However, the mechanism of energy expenditure in cancer is not well understood. We hypothesize that cancer cells might utilize energy-rich inorganic polyphosphate (polyP), as energetic reserve. PolyP is comprised of orthophosphates linked by phosphoanhydride bonds, as in ATP. Here, we show that polyP is highly abundant in several types of cancer cells, including brain tumor-initiating cells (BTICs), i.e., stem-like cells derived from a mouse brain tumor model that we have previously described. The polymer is avidly consumed during starvation of the BTICs. Depletion of ATP by inhibiting glycolysis and mitochondrial ATP-synthase (OXPHOS) further decreases the levels of polyP and alters morphology of the cells. Moreover, enzymatic hydrolysis of the polymer impairs the viability of cancer cells and significantly deprives ATP stores. These results suggest that polyP might be utilized as a source of phosphate energy in cancer. While the role of polyP as an energy source is established for bacteria, this finding is the first demonstration that polyP may play a similar role in the metabolism of cancer cells.

Inorganic polyphosphate potentiates lipopolysaccharide-induced macrophage inflammatory response

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate units that are linked by phosphoanhydride bonds and is involved in various pathophysiological processes. However, the role of polyP in immune cell dysfunction is not well-understood. In this study, using several biochemical and cell biology approaches, including cytokine assays, immunofluorescence microscopy, receptor-binding assays with quartz crystal microbalance, and dynamic light scanning, we investigated the effect of polyP on in vitro lipopolysaccharide (LPS)-induced macrophage inflammatory response. PolyP up-regulated LPS-induced production of the inflammatory cytokines, such as tumor necrosis factor α, interleukin-1β, and interleukin-6, in macrophages, and the effect was polyP dose- and chain length-dependent. However, orthophosphate did not exhibit this effect. PolyP enhanced the LPS-induced intracellular macrophage inflammatory signals. Affinity analysis revealed that polyP interacts with LPS, inducing formation of small micelles, and the polyP-LPS complex enhanced the binding affinity of LPS to Toll-like receptor 4 (TLR4) on macrophages. These results suggest that inorganic polyP plays a critical role in promoting inflammatory response by enhancing the interaction between LPS and TLR4 in macrophages.

Accumulation of Nucleolar Inorganic Polyphosphate Is a Cellular Response to Cisplatin-Induced Apoptosis.

The chemotherapeutic drug cisplatin, which targets DNA, serves as one of the main staples in cancer treatment. Yet, the therapeutic application of cisplatin is limited by two major challenges: the occurrence of reversible and irreversible side effects due to non-specific toxicity, and the intrinsic or developing resistance of tumor cells toward cisplatin. Here we demonstrate that cancer cells respond to cisplatin treatment with the nucleolar accumulation of inorganic polyphosphate (polyP), a universally conserved high-energy compound. PolyP accumulation positively correlates with the levels of activated caspase-3, suggesting a novel role of polyP in cisplatin-mediated apoptosis. In support of this finding, we discovered that administration of exogenous polyP increases cisplatin-induced toxicity in select cancer cell lines, raising the exciting possibility that enhancing endogenous polyP levels might be a novel mechanism to sensitize cancer cells to cisplatin treatment.

Polyphosphate in Lactobacillus and Its Link to Stress Tolerance and Probiotic Properties.

The synthesis of the inorganic polymer polyphosphate (poly-P) in bacteria has been linked to stress survival and to the capacity of some strains to sequester heavy metals. In addition, synthesis of poly-P by certain strains of probiotic lactobacilli has been evidenced as a probiotic mechanism due to the homeostatic properties of this compound at the intestinal epithelium. We analyzed the link between poly-P synthesis, stress response, and mercury toxicity/accumulation by comparing wild-type strains of Lactobacillus and their corresponding mutants devoid of poly-P synthesis capacity (defective in the poly-P kinase, ppk, gene). Results showed that resistance to salt (NaCl) and acidic (pH 4) stresses upon ppk mutation was affected in Lactobacillus casei, while no effect was observed in two different Lactobacillus plantarum strains. Inorganic [Hg(II)] and organic (CH3Hg) mercury toxicity was generally increased upon ppk mutation, but no influence was seen on the capacity to retain both mercurial forms by the bacteria. Notwithstanding, the culture supernatants of ppk-defective L. plantarum strains possessed a diminished capacity to induce HSP27 expression, a marker for cell protection, in cultured Caco-2 cells compared to wild-type strains. In summary, our results illustrate that the role of poly-P in stress tolerance can vary between strains and they reinforce the idea of probiotic-derived poly-P as a molecule that modulates host-signaling pathways. They also question the relevance of this polymer to the capacity to retain mercury of probiotics.

Polyphosphate is involved in cell cycle progression and genomic stability in Saccharomyces cerevisiae.

Polyphosphate (polyP) is a linear chain of up to hundreds of inorganic phosphate residues that is necessary for many physiological functions in all living organisms. In some bacteria, polyP supplies material to molecules such as DNA, thus playing an important role in biosynthetic processes in prokaryotes. In the present study, we set out to gain further insight into the role of polyP in eukaryotic cells. We observed that polyP amounts are cyclically regulated in Saccharomyces cerevisiae, and those mutants that cannot synthesise (vtc4Δ) or hydrolyse polyP (ppn1Δ, ppx1Δ) present impaired cell cycle progression. Further analysis revealed that polyP mutants show delayed nucleotide production and increased genomic instability. Based on these findings, we concluded that polyP not only maintains intracellular phosphate concentrations in response to fluctuations in extracellular phosphate levels, but also muffles internal cyclic phosphate fluctuations, such as those produced by the sudden demand of phosphate to synthetize deoxynucleotides just before and during DNA duplication. We propose that the presence of polyP in eukaryotic cells is required for the timely and accurate duplication of DNA.

Inorganic polyphosphate in cardiac myocytes: from bioenergetics to the permeability transition pore and cell survival.

Inorganic polyphosphate (polyP) is a linear polymer of Pi residues linked together by high-energy phosphoanhydride bonds as in ATP. PolyP is present in all living organisms ranging from bacteria to human and possibly even predating life of this planet. The length of polyP chain can vary from just a few phosphates to several thousand phosphate units long, depending on the organism and the tissue in which it is synthesized. PolyP was extensively studied in prokaryotes and unicellular eukaryotes by Kulaev's group in the Russian Academy of Sciences and by the Nobel Prize Laureate Arthur Kornberg at Stanford University. Recently, we reported that mitochondria of cardiac ventricular myocytes contain significant amounts (280±60pmol/mg of protein) of polyP with an average length of 25 Pi and that polyP is involved in Ca(2+)-dependent activation of the mitochondrial permeability transition pore (mPTP). Enzymatic polyP depletion prevented Ca(2+)-induced mPTP opening during ischaemia; however, it did not affect reactive oxygen species (ROS)-mediated mPTP opening during reperfusion and even enhanced cell death in cardiac myocytes. We found that ROS generation was actually enhanced in polyP-depleted cells demonstrating that polyP protects cardiac myocytes against enhanced ROS formation. Furthermore, polyP concentration was dynamically changed during activation of the mitochondrial respiratory chain and stress conditions such as ischaemia/reperfusion (I/R) and heart failure (HF) indicating that polyP is required for the normal heart metabolism. This review discusses the current literature on the roles of polyP in cardiovascular health and disease.

Minimally invasive surgical options for congenital and acquired uterine factors associated with recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is defined as two or more failed clinical pregnancies before 20 weeks' gestation and may be caused by genetic, endocrinologic, anatomic and immunologic abnormalities. Anatomic uterine anomalies include congenital malformations (bicornuate, didelphic, septate and unicornuate uteri) and acquired defects (fibroids, adenomas, adhesions and polyps). Women with septate and bicornuate uteri, intrauterine adhesions, and some adenomas and fibroids are at increased risk of RPL. Data support surgical treatment of all of these lesions except bicornuate uteri. The role of polyps in RPL is unclear. Minimally invasive options for surgical correction of intrauterine lesions include hysteroscopy, laparoscopy with and without robotic assistance and minilaparotomy.

Polyphosphate kinase of Lysinibacillus sphaericus and its effects on accumulation of polyphosphate and bacterial growth

Lysinibacillus sphaericus produces parasporal crystal proteins (Bin toxins) that are specifically toxic to Culex and Anopheles mosquito larvae. It has long persistence in mosquito breeding sites due to the recycle of spores in mosquito cadaver and certain environments. This bacterium is unable to metabolize sugar (except N-acetylglucosamine) but can use proteinaceous materials. Previous studies have shown that inorganic polyphosphate (polyP) is essential for cell growth and responses to nutritional stringencies and environmental stresses and branched-chain amino acids (BCAAs) are useful intracellular signals for bacterial adaption to nutritional environments. However, relatively little is known about the role of polyP and the regulation link between polyP and BCAAs in nutritional limitation adaptation in a bacterium incapable of polysaccharide utilization. Here, a primary enzyme involved in polyP biosynthesis, polyphosphate kinase (PPK), was identified from L. sphaericus, and the polyP accumulation in different nutritional media was investigated. Furthermore, a ppk null mutant and the ppk-complemented strain were constructed and the effect of deletion on cell growth, polyP accumulation and expression of BCAAs biosynthetic genes (ilvBHC) was surveyed. The result showed that deletion of ppk resulted in a deficiency in polyP accumulation rather than cell growth in Luria-Bertani (LB) medium. Further studies demonstrated that the cell growth was evidently retarded and the ilvBHC expression was significantly reduced when ppk mutant was transferred from LB to limited-amino acid (LA) medium and that these phenotypes were significantly restored in the ppk-complemented strain. These results suggested that PPK plays a role in polyP accumulation and expression of genes involved in BCAAs biosynthesis, providing a hint for further understanding of the role of PPK in nutritional limitation in L. sphaericus.

Role of Polyphosphate in Cancer Cell Proliferation

Inorganic polyphosphates (polyPs) are linear polymers of orthophosphate residues linked by phosphoanhydride bonds similar to ATP. PolyPs are ubiquitously distributed in nature from bacteria to mammalian cells where they play multiple roles. Here we investigated intracellular localization of polyP in cancer cell lines and tested the idea about the involvement of polyP in cell growth.To study PolyP localization and cellular levels we used DAPI-based method of polyP staining. Experiments were performed using the following cell lines: H1299 (lung cancer), U251 (glioma) and HEK293 as a control cell line. PolyP was also visualized by immunocytochemistry with X-press tagged PolyP-binding domain of exopolyphosphatase (PPX) and subsequent use of specific antibodies. The role of PolyP in cancer cell proliferation was studied using MTS proliferation assay. In these experiments endogenous PolyP was either blocked by spermine, a polyamine with high PolyP-binding affinity, or depleted by transient expression of PPX, an enzyme that specifically hydrolyzes PolyP into orthophosphate.PolyP levels were increased in glioma and lung cancer cells compare to HEK 293 cells. Proliferation of lung carcinoma (H1299) cells was significantly inhibited when PolyP was blocked with spermine compare to control cells. However, proliferation of glioma cells (U251) was not affected by spermine, even at high concentrations of this polyamine (60 μM), suggesting a cancer type dependent effect or, involvement of divers mechanisms of actions of PolyP in different cell types. These results suggest that PolyP plays an important role in the regulation of proliferation of cancer cells. Further investigation of PolyP contribution to cancer development and progression may provide tools to detect, prevent and/or treat cancer.

Growth characteristics ofProtoheliolites norvegicus(Tabulata; Upper Ordovician; Estonia)

Protoheliolites is an early heliolitine coral characterized by closely spaced corallites separated in places by sparse coenenchyme. Growth characteristics in the type species, P. norvegicus, are revealed by detailed analysis based on serial peels and thin sections of coralla from the uppermost Katian of north-western Estonia. Colonies of this species had a strong ability to recover from damage and partial mortality, resulting in various forms of rejuvenation, regeneration, fusion and reorganization of corallites; in some cases, this involved relatively large areas of undifferentiated soft parts. The shells of commensal cornulitids became enclosed in host coralla during colony growth. Coralla of P. norvegicus exhibit distinctive growth cycles due to responses to seasonal changes. The production of new corallites by coenenchymal increase usually occurred in low-density bands, in which corallites generally display round to subrounded transverse outlines. In high-density bands, the corallites became crenulated, their wall thickness increased, septal development was more pronounced, and the amount of coenenchyme increased. In addition to these cyclomorphic changes, there were significant astogenetic changes during growth. Compared with the early stage of colony development, distinctive characteristics in the late astogenetic stage include a decrease in the growth rate of the colony, better coordination among corallites, maximum development of corallite crenulations and septa in high-density bands, more numerous coenenchymal tubules and a greater proportion of corallum area occupied by coenenchyme. In general, the role of polyps in determining morphological characteristics of individual corallites, such as tabularium area, corallite crenulations and wall thickness, was subordinate to the astogeny of the colony. Growth characteristics including colony-wide coordination of polyp behaviour and subjugation of individuals to restore the colony following damage suggest a strong astogenetic control and high level of colony integration. Protoheliolites probably arose from a heliolitine genus rather than from a nonheliolitine group as some authors have proposed.

Factors associated with colorectal cancer risk perception: the role of polyps and family history

It is unclear how objective risk factors influence the factors associated with colorectal cancer (CRC) risk perception. The goals of this study were to investigate factors associated with perceived risk of CRC and to explore how these relationships were modified by personal history of polyps or family history of CRC. The study involved a mailed questionnaire completed by 1646 men and women aged 50-75 years, which assessed perceived risk, demographic and health history variables and CRC worry. Participants were patients of primary care providers in a community medical group in central Massachusetts. The study sample seemed to have a generally accurate perception of CRC risk, which was appropriately increased in the presence of known risk factors. In multivariable analyses that controlled for all measured covariates, financial situation modified the association between perceived risk and a personal history of polyps, while age and insurance status modified the association between perceived risk and family history of CRC. CRC worry, self-reported health, personal history of other cancer and compliance with screening guidelines remained significant predictors of perceived risk. Potential interactions between objective risk factors and socioeconomic characteristics should be further explored in longitudinal studies.

Monitoring of Inorganic Polyphosphate Dynamics in Corynebacterium glutamicum Using a Novel Oxygen Sparger for Real Time 31P in vivo NMR

For the first time in intact bacterial cells, the dynamics of the build-up of soluble cytosolic inorganic polyphosphate (polyP) during aeration, and its breakdown during anaerobiosis have been observed with a time resolution of 50 s. Under conditions of 60-80% saturation with pure oxygen, the accumulation of high levels of intracellular polyP was detected when inorganic phosphate (Pi) and glucose or acetate were added to Corynebacterium glutamicum cell suspensions (3 ml, ∼40 mg dw/ml). The maximum levels of polyP reached were estimated to 600 mM P units in the cytosol or ∼3% phosphorus [w/w] in the cell dry weight. C. glutamicum polyP was apparently of high molecular weight (containing probably a few hundred units) as inferred from signal distribution, but a temporary average polyP chain length of about n = 40 could be estimated at the initial stages of polyP formation. After each addition of glucose or acetate, oxygen levels followed a steep decline to ∼20% and then an increase to the previous level. In contrast, polyP levels rose after the addition of substrate, and declined again, while the oxygen level recovered. When the oxygen supply was completely switched off, the polyP signal declined immediately, with concomitant re-appearance of phosphomonoester signals (sugar phosphates and related compounds). Both processes, the increase of polyP during aeration and supply with substrate and Pi, and the decrease during anaerobiosis, occurred within minutes. Only within these relatively brief windows of time between successive feedings with substrate or between aeration and anaerobiosis, high levels of polyP could be observed. Thus, our findings indicate that polyP occurs not only as the long known granular storage material in some Corynebacteria, such as C. diphtheriae or C. imitans, but that formation and breakdown of soluble polyP in C. glutamicum is a very dynamic process that may play a decisive role in C. glutamicum and in other strains of this genus. These investigations were made possible by combining nuclear magnetic resonance (NMR) techniques with novel methods of oxygen sparging and online substrate distribution. The sparger was custom made from titanium to fit into 10 mm o.d. NMR tubes. Both the size and the spacing of the holes in the sparger were calculated for optimum distribution of oxygen at 30 °C through 3 ml of C. glutamicum cell suspensions. The experiments were carried out using in vivo 31P NMR, and monitoring of oxygen was performed with a miniature oxygen optode in real time. Glucose or acetate and/or phosphate stock solutions could be added in situ. 31P NMR analyses of intracellular phosphorus metabolites were sampled with a time resolution of 50 s. The sparger unit, including optode and supply lines, could be easily switched from one sample to another after completion of an experiment. It is suggested to use these analytical tools to investigate other bacterial strains and even cell extracts, shedding further light on the novel roles of polyP in living cells [Schröder, H. C., Müller, W. E. G., (eds.). Inorganic polyphosphates - biochemistry, biology, biotechnology. Prog. Mol. Subcell. Biol. 23 (1999). Springer-Verlag, Berlin].