Norspermidine is not a self-produced trigger for biofilm disassembly.

Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 µM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 µM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 µM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.

Polyethylenimine linked with chitosan improves astaxanthin production in Haematococcus pluvialis.

Astaxanthin is receiving increasing interest as an antioxidant and high value-added secondary metabolite. Haematococcus pluvialis is the main source for astaxanthin production, and many studies are being conducted to increase the production of astaxanthin. In this study, we linked polyethylenimine (PEI) with chitosan to maintain astaxanthin-inducing ability while securing the recyclability of the inducer. Astaxanthin accumulation in H. pluvialis was induced to 86.4 pg cell-1 with the PEI-chitosan fiber (PCF) treatment prepared by cross-linking of 10 µM PEI and low molecular weight (MW) chitosan via epichlorohydrin. PEI concentration affected the astaxanthin accumulation, whereas the MW of chitosan did not. In addition, the PCF treatment in H. pluvialis increased the reactive oxygen species (ROS) content in cells, thereby upregulating the transcription of enzymes involved in astaxanthin biosynthesis. PCF can be reused multiple times with the maintenance of over 90% of the astaxanthin production efficiency. This study offers a reusable PCF stimulation strategy for enhancing natural astaxanthin content, and PCF treatment will easily increase the production scale or reduce production costs by using recyclability that is not available in current methods. KEY POINTS: • Polyethylenimine-chitosan fiber (PCF) was applied to Haematococcus pluvialis • PCF promotes astaxanthin accumulation by enhancing oxidative stress in H. pluvialis • PCF can be reused multiple times with maintaining over 90% production efficiency.

Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin.

The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

Amination of cotton fiber using polyethyleneimine and its application as an adsorbent to directly remove a harmful cyanobacterial species, Microcystis aeruginosa, from an aqueous medium.

In the present study, we applied an adsorption-based strategy for the removal of a harmful cyanobacterial species, Microcystis aeruginosa, using cotton fiber. Considering the negatively charged surface properties of M. aeruginosa cells in aqueous phases, aminated cotton fibers were prepared through polyethyleneimine (PEI) modification on the pristine cotton fibers. The aminated surface properties of PEI-modified cotton fiber (PEI-cotton) were confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and potentiometric titration analyses. The pristine cotton fiber could not remove the M. aeruginosa cells, but the PEI-cotton could efficiently remove 98.7% of M. aeruginosa cells from the aqueous medium. In addition, removed cells could be observed on the sorbent surface by field emission scanning electron microscopy (FE-SEM) analysis. PEI-cotton fabricated in 3% PEI solution could remove M. aeruginosa cells (97.9%) more efficiently compared to that fabricated in 1% (82.1%) and 2% (86.2%) of PEI solutions. From the toxicity assessment of the PEI-cotton using Daphnia magna, negligible toxicity of PEI-cotton was confirmed. Our results indicate that the application of PEI-cotton fibers for the removal of M. aeruginosa cells could be suggested as a feasible, effective, and eco-friendly method of harmful algal bloom (HAB) control in water resources.

Polyamine-independent growth and biofilm formation, and functional spermidine/spermine N-acetyltransferases in Staphylococcus aureus and Enterococcus faecalis.

Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes, respectively, and are devoid of any polyamine when grown in polyamine-free media. In contrast to bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni and Agrobacterium tumefaciens, which absolutely require polyamines for growth, S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore, S. aureus and E. faecalis form biofilms normally without polyamines, and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines, including norspermidine, eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N-acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine, spermine and norspermidine, that spermine is the more preferred substrate, and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy, polyamine-independent growth and biofilm formation, and presence of functional polyamine N-acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology.

Application of a polyethylenimine-modified polyacrylonitrile-biomass waste composite fiber sorbent for the removal of a harmful cyanobacterial species from an aqueous solution.

Cyanobacterial harmful algal blooms (Cyano-HABs) in water resources involving algal species such as Microcystis aeruginosa have become a serious environmental issue due to their severely negative effects. In the present study, an adsorption-based strategy was employed to control M. aeruginosa, with industrial waste-derived Escherichia coli biomass valorized to produce polyethylenimine-modified polyacrylonitrile-E. coli biomass composite fiber (PEI-PANBF). PEI-PANBF removed approximately 80% of M. aeruginosa cells from an aqueous solution without causing any cell damage. Interestingly, the thickness of PEI-PANBF had a strong influence on the efficiency of M. aeruginosa cell removal. In addition, PEI-PANBF simultaneously removed M. aeruginosa cells and their toxic secondary metabolite, microcystin-LR, from aqueous media. Thus, our proposed fiber represents a feasible utilization method of industrial waste biomass as a biosorbent for the control of Cyano-HABs.

Mitochondrial localization of telomeric protein TIN2 links telomere regulation to metabolic control.

Both mitochondria, which are metabolic powerhouses, and telomeres, which help maintain genomic stability, have been implicated in cancer and aging. However, the signaling events that connect these two cellular structures remain poorly understood. Here, we report that the canonical telomeric protein TIN2 is also a regulator of metabolism. TIN2 is recruited to telomeres and associates with multiple telomere regulators including TPP1. TPP1 interacts with TIN2 N terminus, which contains overlapping mitochondrial and telomeric targeting sequences, and controls TIN2 localization. We have found that TIN2 is posttranslationally processed in mitochondria and regulates mitochondrial oxidative phosphorylation. Reducing TIN2 expression by RNAi knockdown inhibited glycolysis and reactive oxygen species (ROS) production and enhanced ATP levels and oxygen consumption in cancer cells. These results suggest a link between telomeric proteins and metabolic control, providing an additional mechanism by which telomeric proteins regulate cancer and aging.

Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a posttranscriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

A polyamine-independent role for S-adenosylmethionine decarboxylase.

The only known function of S-adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that 'eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.

Homospermidine biosynthesis in the cyanobacterium Anabaena requires a deoxyhypusine synthase homologue and is essential for normal diazotrophic growth.

Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym-Homospermidine is the dominant polyamine in the filamentous, N2 -fixing, heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst-specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena.

Spermidine promotes Bacillus subtilis biofilm formation by activating expression of the matrix regulator slrR.

Ubiquitous polyamine spermidine is not required for normal planktonic growth of Bacillus subtilis but is essential for robust biofilm formation. However, the structural features of spermidine required for B. subtilis biofilm formation are unknown and so are the molecular mechanisms of spermidine-stimulated biofilm development. We report here that in a spermidine-deficient B. subtilis mutant, the structural analogue norspermidine, but not homospermidine, restored biofilm formation. Intracellular biosynthesis of another spermidine analogue, aminopropylcadaverine, from exogenously supplied homoagmatine also restored biofilm formation. The differential ability of C-methylated spermidine analogues to functionally replace spermidine in biofilm formation indicated that the aminopropyl moiety of spermidine is more sensitive to C-methylation, which it is essential for biofilm formation, but that the length and symmetry of the molecule is not critical. Transcriptomic analysis of a spermidine-depleted B. subtilis speD mutant uncovered a nitrogen-, methionine-, and S-adenosylmethionine-sufficiency response, resulting in repression of gene expression related to purine catabolism, methionine and S-adenosylmethionine biosynthesis and methionine salvage, and signs of altered membrane status. Consistent with the spermidine requirement in biofilm formation, single-cell analysis of this mutant indicated reduced expression of the operons for production of the exopolysaccharide and TasA protein biofilm matrix components and SinR antagonist slrR Deletion of sinR or ectopic expression of slrR in the spermidine-deficient ΔspeD background restored biofilm formation, indicating that spermidine is required for expression of the biofilm regulator slrR Our results indicate that spermidine functions in biofilm development by activating transcription of the biofilm matrix exopolysaccharide and TasA operons through the regulator slrR.

Adsorptive removal of harmful algal species Microcystis aeruginosa directly from aqueous solution using polyethylenimine coated polysulfone-biomass composite fiber.

In recent times, the treatment of harmful algal blooms (HABs) became an important environmental issue to preserve and remediate water resources globally. In the present study, the adsorptive removal of harmful algal species Microcystis aeruginosa directly from an aqueous medium was attempted. Waste biomass (Escherichia coli) was immobilized using polysulfone and coated using the cationic polymer polyethylenimine (PEI) to generate PEI-coated polysulfone-biomass composite fiber (PEI-PSBF). The density of M. aeruginosa in an aqueous medium (BG11) was significantly decreased by treatment with PEI-PSBF. additionally, analysis using FE-SEM, confirmed that the removal of M. aeruginosa algal cells by PEI-PSBF was caused by the adsorption mechanism. According to the profiles of phosphorus for the algal cell growth in M. aeruginosa cultivating samples, we found that the adsorbed M. aeruginosa onto the PEI-PSBF lost their biological activity compared to the non-treated M. aeruginosa cells.

Desalted Salicornia europaea powder and its active constituent, trans-ferulic acid, exert anti-obesity effects by suppressing adipogenic-related factors.

CONTEXT: Salicornia europaea (Amaranthaceae) (SE) has been shown to reduce obesity, but it remains a problem as a food supplement because of its high salt content (25-35% NaCl). OBJECTIVES: This study investigated the anti-obesity effects and mechanism of action of desalted SE powder (DSP). MATERIALS AND METHODS: Sprague-Dawley rats (n = 50) were divided into a normal control group (NC), a high-fat diet (HFD)-induced obesity control group (HFD), and HFD groups co-administered DSP (250 and 500 mg/kg) or Garcinia cambogia (Clusiaceae) extract (GE, 200 mg/kg, standard control) orally each day for 12 weeks. RESULTS: The body weight was significantly reduced by co-administration of DSP (596.51 ± 19.84 kg, 4.60% and 562.08 ± 9.74 kg, 10.10%, respectively) and GE (576.00 ± 11.29 kg, 7.88%) relative to the HFD group (625.25 ± 14.02 kg) and was accompanied by reduced abdominal fat mass, and serum lipid levels, with no effects on feed intake. To find the underlying mechanism of the anti-obesity effects, trans-ferulic acid (TFA) was identified as the main ingredient and investigated with regard to whether it attenuated adipogenesity in 3T3L-1 cells. DSP-derived TFA suppressed adipocyte differentiation and accumulation of intracellular lipids. TFA also down-regulated the adipogenesis-related gene expression of sterol regulatory element-binding protein 1, peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein-α and fatty acid synthase. CONCLUSIONS: These findings suggest that DSP may be considered for use as a food supplement intent of controlling obesity through its antiobesity and antiadipogenic properties.

Analysis of Cation-Dependent DNA (G3T1)4 Shape Change Using Fluorescence Correlation Spectroscopy.

In G-rich DNA, it is well known that the form changes from single-strand DNA to G-quadruplex due to cations. In this study, we analyze the diffusion coefficient and fluorescence intensity obtained by fluorescence correlation spectroscopy for short G-rich DNA of the (G3T1)4 sequence labeled as 5-Carboxytetramethylrhodamine (TAMRA) with variation of the K+ ion concentration. At a K+ ion concentration of more than 200 mM, the single-strand DNA was changed to the G-quadruplex. The size of the G-quadruplex decreased to 86% than the size of the single strand DNA at K+ ion concentration of 0 M. The size of the G-quadruplex and the fluorescence intensity of TAMRA attached to the DNA were constant with an increase in the K+ ion concentration between 200 and 800 mM. This means that the size of the DNA and the fluorescence intensity of the TAMRA are not affected by the K+ ion concentration at the G-quadruplex structure because the binding structure of DNA and TAMRA dye leads to stability at a concentration of less than 100 mM K+. Based on our short G-rich DNA results, longer G-rich DNA is analyzed for the diffusion coefficient of the DNA and the fluorescence intensity variation of fluorescence dye attached to the DNA.

Persistence Length and Cooperativity Estimation of Single Stranded DNA using FCS Combined with HYDRO Program.

Translational diffusion properties of single-stranded DNA (ssDNA) and G-quadruplexes were studied to determine the persistence length and cooperativity of G-quadruplex formation using FCS combined with HYDRO in which wormlike chain (WLC)-based Monte Carlo simulation are implemented. The presence of a guanine instead of a thymine shortened the contour length of nucleic acids and increased the vulnerability to ion screening. For cooperativity estimation, the telomeric sequence HT72 was assumed to undergo 27 intermediate states, which can be classified as ssDNA, single-G-quadruplex, double-G-quadruplex, and three consecutive G-quadruplexes. Each state type was modeled using a series of beads and appropriate bond lengths, which were obtained from the WLC model. Using the HYDRO program, we calculated diffusion times for each species, and these were used to calculate simulated HT72 diffusion times for mixtures of species in arbitrary KCl concentrations. By comparison between simulated and experimental diffusion properties, we obtained a positive cooperativity of C = 200 from FCS combined with HYDRO.

Spermidine Inversely Influences Surface Interactions and Planktonic Growth in Agrobacterium tumefaciens.

UNLABELLED: In bacteria, the functions of polyamines, small linear polycations, are poorly defined, but these metabolites can influence biofilm formation in several systems. Transposon insertions in an ornithine decarboxylase (odc) gene in Agrobacterium tumefaciens, predicted to direct synthesis of the polyamine putrescine from ornithine, resulted in elevated cellulose. Null mutants for odc grew somewhat slowly in a polyamine-free medium but exhibited increased biofilm formation that was dependent on cellulose production. Spermidine is an essential metabolite in A. tumefaciens and is synthesized from putrescine in A. tumefaciens via the stepwise actions of carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase (CASDC). Exogenous addition of either putrescine or spermidine to the odc mutant returned biofilm formation to wild-type levels. Low levels of exogenous spermidine restored growth to CASDH and CASDC mutants, facilitating weak biofilm formation, but this was dampened with increasing concentrations. Norspermidine rescued growth for the odc, CASDH, and CASDC mutants but did not significantly affect their biofilm phenotypes, whereas in the wild type, it stimulated biofilm formation and depressed spermidine levels. The odc mutant produced elevated levels of cyclic diguanylate monophosphate (c-di-GMP), exogenous polyamines modulated these levels, and expression of a c-di-GMP phosphodiesterase reversed the enhanced biofilm formation. Prior work revealed accumulation of the precursors putrescine and carboxyspermidine in the CASDH and CASDC mutants, respectively, but unexpectedly, both mutants accumulated homospermidine; here, we show that this requires a homospermidine synthase (hss) homologue. IMPORTANCE: Polyamines are small, positively charged metabolites that are nearly ubiquitous in cellular life. They are often essential in eukaryotes and more variably in bacteria. Polyamines have been reported to influence the surface-attached biofilm formation of several bacteria. In Agrobacterium tumefaciens, mutants with diminished levels of the polyamine spermidine are stimulated for biofilm formation, and exogenous provision of spermidine decreases biofilm formation. Spermidine is also essential for A. tumefaciens growth, but the related polyamine norspermidine exogenously rescues growth and does not diminish biofilm formation, revealing that the growth requirement and biofilm control are separable. Polyamine control of biofilm formation appears to function via effects on the cellular second messenger cyclic diguanylate monophosphate, regulating the transition from a free-living to a surface-attached lifestyle.

Different polyamine pathways from bacteria have replaced eukaryotic spermidine biosynthesis in ciliates Tetrahymena thermophila and Paramecium tetaurelia.

The polyamine spermidine is absolutely required for growth and cell proliferation in eukaryotes, due to its role in post-translational modification of essential translation elongation factor eIF5A, mediated by deoxyhypusine synthase. We have found that free-living ciliates Tetrahymena and Paramecium lost the eukaryotic genes encoding spermidine biosynthesis: S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SpdSyn). In Tetrahymena, they were replaced by a gene encoding a fusion protein of bacterial AdoMetDC and SpdSyn, present as three copies. In Paramecium, a bacterial homospermidine synthase replaced the eukaryotic genes. Individual AdoMetDC-SpdSyn fusion protein paralogues from Tetrahymena exhibit undetectable AdoMetDC activity; however, when two paralogous fusion proteins are mixed, AdoMetDC activity is restored and spermidine is synthesized. Structural modelling indicates a functional active site is reconstituted by sharing critical residues from two defective protomers across the heteromer interface. Paramecium was found to accumulate homospermidine, suggesting it replaces spermidine for growth. To test this concept, a budding yeast spermidine auxotrophic strain was found to grow almost normally with homospermidine instead of spermidine. Biosynthesis of spermidine analogue aminopropylcadaverine, but not exogenously provided norspermidine, correlated with some growth. Finally, we found that diverse single-celled eukaryotic parasites and multicellular metazoan Schistosoma worms have lost the spermidine biosynthetic pathway but retain deoxyhypusine synthase.

Analysis of the Fluorescence Correlation Function of Quantum Rods with Different Lengths.

We built a polarization fluorescence correlation spectroscopy system to analyze the variation of the correlation function in rotational diffusion based on the length of rod-like fluorescent particles. Because the rotational diffusion of particles in liquid depends on the relative polarization states of the laser source and particle fluorescence, we compared the amplitudes of the rotational diffusion using the autocorrelation function in different polarization states. For experiments that depend on the length of the fluorescent particles, we prepared three kinds of quantum rod samples with a width of 6.5 ± 0.5 nm and lengths of 17 ± 3, 40 ± 3, and 46 ± 3 nm. Through the experiment, we obtained the hydrodynamic radii of each particle using the rotational diffusion coefficient: 10.7 ± 0.8, 13.4 ± 0.7, and 14.1 ± 0.4 nm with the length of the particles. All the obtained values for radii are 3 nm larger than the calculated equivalent radii of spheres with the same volume as the rod samples. Through a fraction analysis by polarization state, we confirmed that the ratio of rotational fraction for polarization increases with the aspect ratio of the actual particle.

Investigation of the nanoviscosity effect of a G-quadruplex and single-strand DNA using fluorescence correlation spectroscopy.

Guanine (G)-quadruplexes are of interest because of their presence in the telomere sequence and the oncogene promoter region. Their diffusion and change of structure, especially in high viscosity solutions, are important for understanding their dynamics. G-quadruplexes may have less effective viscosity (nanoviscosity) when they are smaller than the solvent molecules. In this paper, we report the difference in the diffusion dynamics of the G-rich DNA sequences of single-strand DNA (ssDNA) and the G-quadruplex in aqueous, sucrose, and polyethylene glycol (PEG) solutions. From experiments with aqueous and sucrose solutions, we confirm that a simple diffusion model according to the viscosity is appropriate. In the PEG experiments, the nanoviscosity effect is observed according to PEG's molecular weight. In the PEG 200 solution, both the ssDNA and the G-quadruplex possess macroviscosity. In the PEG 10,000 solution, the G-quadruplex possesses nanoviscosity and the ssDNA possesses macroviscosity, whereas, in the PEG 35,000 solution, both ssDNA and the G-quadruplex possess nanoviscosity. The experimental results are consistent with the theoretical predictions.

Product feedback regulation implicated in translational control of the Trypanosoma brucei S-adenosylmethionine decarboxylase regulatory subunit prozyme.

Human African sleeping sickness (HAT) is caused by the parasitic protozoan Trypanosoma brucei. Polyamine biosynthesis is an important drug target in the treatment of HAT. Previously we showed that trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme for biosynthesis of the polyamine spermidine, is activated by heterodimer formation with an inactive paralogue termed prozyme. Furthermore, prozyme protein levels were regulated in response to reduced AdoMetDC activity. Herein we show that T. brucei encodes three prozyme transcripts. The 3'UTRs of these transcripts were mapped and chloramphenicol acetyltransferase (CAT) reporter constructs were used to identify a 1.2 kb region that contained a 3'UTR prozyme regulatory element sufficient to upregulate CAT protein levels (but not RNA) upon AdoMetDC inhibition, supporting the hypothesis that prozyme expression is regulated translationally. To gain insight into trans-acting factors, genetic rescue of AdoMetDC RNAi knock-down lines with human AdoMetDC was performed leading to rescue of the cell growth block, and restoration of prozyme protein to wild-type levels. Metabolite analysis showed that prozyme protein levels were inversely proportional to intracellular levels of decarboxylated AdoMet (dcAdoMet). These data suggest that prozyme translation may be regulated by dcAdoMet, a metabolite not previously identified to play a regulatory role.