Involvement of individual hippocampal signaling protein levels in spatial memory formation is strain-dependent

Although a series of signaling cascades involved in spatial memory have been identified, their link to spatial memory and strain-dependent expression has not been reported so far. Hippocampal levels of the abovementioned signaling proteins were determined in laboratory inbred strain C57BL/6J, the wild-derived inbred strain PWD/PhJ and the wild caught mouse Apodemus sylvaticus (AS) by immunoblotting. The resulting hippocampal protein levels were correlated with results from MWM. Hippocampal signaling protein (hSP) levels were tested also in yoked controls. Within-strain comparison between trained and yoked controls revealed significant differences between levels of Phospho-CaMKII (alpha), Phospho-CREB, Egr-1, c-Src, Phospho-ERK5, Phospho-MEK5 and NOS1 in all of the three strains tested. In addition, the three strains revealed different involvement of individual hSP levels clearly indicating that individual mouse strains were linked to individual hSPs in spatial memory. Phospho-ERK5 levels were not detectable in hippocampi of yoked controls of each strain. We learn from this study that a series of hSPs are associated with spatial memory and that different hSPs are linked to spatial memory in different strains that show different outcome in the MWM. Even correlational patterns in the individual hSPs differed between mouse strains. This is of importance for the interpretation of previous studies on the abovementioned signaling cascades as well as for the design of future studies on these hippocampal proteins. It is intriguing that individual mouse strains, laboratory or wild caught, may use different signaling pathways for spatial memory in the Morris water maze.     

Hippocampal lipids linked to spatial memory in the C57bl/6j mouse

Although the role of individual brain lipids for learning and memory has been reported, no systematic approach associating brain lipids with spatial memory has been carried out. It was therefore the aim of the study to determine brain lipids in hippocampus of mice forming and yoked controls that did not form spatial memory using the probe trial as the endpoint.10 animals were trained in the Morris water maze (MWM) and 10 mice were serving as yoked controls i.e. no platform was used during the whole experiment.Hippocampal tissue lipids were extracted and data were acquired with Fourier transformation ion cyclotron resonance mass spectrometry (LTQ-FT) coupled to HPLC.Glycerophosphatidylethanolamines (18:0/22:6, 18:0/20:4 and 18:1/18:1), plasmalogens (16:0-10/22:6 and 18:0-10/22:6) and ceramides (18:0) showed higher levels in the trained group, while glycerolysophosphatidylcholines (16:0, 18:1, 18:0, 20:4), sphingomyelins (16:0, 24:1), ether linked glycerophosphatidylcholines (16:0-10/18:0), glycerophosphatidylcholines (16:0/18:1, 16:0/18:0, 18:0/18:1, 38:7, 18:1/20:1, 20:4/20:4, 22:1/18:1, 22:0/18:1, 20:4/22:6, 22:6/22:6), glucosylceramide (24:1) and plasmalogen (18:0-10/20:1) revealed lower levels in the trained group.Decreased levels of certain species of lysophosphatidylcholine, sphingomyelin, plasmenylphosphatidylcholine, phosphatidylcholine, glycosylceramide and plasmalogen at the probe trial for spatial memory may indicate catabolism in terms of consumption during this process.Increased hippocampal levels of long chain highly unsaturated phosphatidylethanolamines, plasmalogens and ceramides may reflect increased synthesis or decreased degradation at the endpoint of memory testing, probably representing interactions in the brain lipid pathways. The study shows pathways involved in spatial memory, may propose the use of individual brain lipids as probable cognitive enhancers and forms the basis for further studies on the role of brain lipids per se.     

Sorting of an exocrine secretory protein to the regulated secretory pathway in endocrine cells

The effect of exogenous polyamines on electrolyte leakage, chilling index, polygalacturonase activity (PG), ethylene production, and firmness in zucchini squash fruits stored for 12 days at 2 degrees C or 10 degrees C, 85-90% RH was evaluated. Fruits were infiltrated with putrescine (PUT) spermidine (SPD) and spermine (SPM) at 0.1, 0.25, 0.5, 2.0, and 4.0 mM. All polyamines exerted a protective effect on cell and organelle membranes. The most effective was SPD, which reduced electrolyte leakage between 62% and 82%, compared to control fruits stored at 2 degrees C. At 10 degrees C they did not exhibit chilling injury (CI) symptoms, while at 2 degrees C SPM (0.5 mM) and SPD (0.5 mM) diminished them 92% and 100%, respectively; which extended storage life for 8-10 days at 2 degrees C. High concentrations of polyamines (>2.0 mM) caused the appearance of CI symptoms. PG activity diminished proportionally to the concentration of polyamine except for the concentration at 4.0 mM. No significant changes were observed in ethylene production.     

Inhibitory and promotive effects of polyamines on transglutaminase-induced protein polymerization

Transglutaminase (TGase) has been reported to be involved in the regulation of cell growth. We examined the effects of polyamines on TGase activity. The polymerization of casein was inhibited by putrescine (PUT) and spermidine (SPD). On the other hand, polymerization of N,N-dimethylcasein was increased by spermine (SPM) and SPD. These results suggested polyamines played two distinct roles as inhibitor and promoter for TGase-catalyzed protein polymerization.     

Strain-independent global effect of hippocampal proteins in mice trained in the Morris water maze

A series of individual proteins have been linked to performance in the Morris water maze (MWM) but no global effects have been reported. It was therefore the aim of the study to show which proteins were strain-independent, global factors for training in the MWM. Strains C57BL/6J, apodemus sylvaticus and PWD/PhJ were used. MWM and gels from trained animals were from a previous own study and corresponding yoked groups were generated. Hippocampal proteins were extracted and run on two-dimensional gel electrophoresis. Spots with different expressional levels between trained and yoked groups were punched and identified by mass spectrometry (nano-LC-ESI-MS/MS, ion trap). Two-way ANOVA with two factors (strain and training) was carried out and a Bonferroni test was used to compare groups. 12 proteins from several pathways and cascades showed different levels in trained mice versus corresponding yoked animals in all strains tested. Four out of these proteins were verified by immunoblotting: beta-synuclein, profilin 2, nucleoside diphosphate kinase A (NME1) and isocitrate dehydrogenase 3. Four proteins verified by immunoblotting could be shown to be involved in training in the MWM as a global effect, independent of the strain tested.     

Immunomodulatory action of spermine and spermidine on NR8383 macrophage line in various culture conditions

We examined the effect of spermine (SPM) and spermidine (SPD) on tumor necrosis factor (TNF)alpha and monocyte chemoattractant protein-1 (MCP-1) secretion from macrophages in various culture conditions, including several protocols of polyamines addition and media supplemented with 0, 1 or 15% fetal bovine serum. TNFalpha secretion was inhibited by SPM or SPD added 18h before stimulation in a concentration-dependent manner. Their effect was directly related to the presence of FBS. When SPM or SPD was added simultaneously to the stimulus, the TNFalpha secretion inhibition was higher than that obtained after pre-treatment. In this case, the effect was inversely proportional to the presence of FBS. The addition of polyamines also inhibited the secretion of MCP-1 in NR8383 cells. We conclude that SPM and SPD inhibited the secretion of inflammatory cytokines TNFalpha and MCP-1 in different ways, depending on culture conditions. In any case, SPM was more effective than SPD.     

Role of polyamines in the stimulation of synthesis and secretion of plasminogen activator from bovine aortic endothelial cells

The effects of the polyamines putrescine (PUT), spermidine (SPD), and spermidine (SPM) on the secretion of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) were evaluated using cultured bovine aortic endothelial cells. All three polyamines enhanced PA secretion in a time- and dose-dependent manner, with a potency rank order of SPM greater than SPD greater than PUT. The PA stimulation required both RNA and protein synthesis, as evidenced by inhibition of polyamine-induced PA secretion by actinomycin D and cycloheximide. The inhibitors of polyamine biosynthesis methylglyoxal bis-(guanylhydrazone) (MGBG) and dl-(difluoromethyl) ornithine (DFMO) alone did not affect basal or polyamine-induced PA secretion, with the exception that MGBG reduced the effect of PUT. Polyamine-treated cells enhanced secretions of both tissue-type and urokinase-type PA. The results of the present study suggest that polyamines may play a role in the regulation of PA synthesis and secretion and that this function can be modified under pathophysiological conditions affecting cellular and tissue levels of polyamines.     

Paraquat toxicity is reduced by polyamines in rice leaves

The protective effect of polyamines against paraquat (PQ) toxicity of rice (Oryza sativa) leaves was investigated. PQ treatment resulted in a higher putrescine (PUT) and lower spermidine (SPD) and spermine (SPM) levels in rice leaves. Pretreatment with SPD and SPM, which resulted in a 10- and 20-fold increase in endogenous level of SPD and SPM, respectively, reduced PQ toxicity (30%). Limited reduction of PQ toxicity by exogenous SPD and SPM is most likely due to the fact that they are not readily transported in rice leaf cells and localized to those areas along the cut edges of detached rice leaves [4]. PUT pretreatment did not increase endogenous SPD and SPM levels and had no effect on reducing PQ toxicity. It was found that 1,10-phenanthroline, an iron chelator, treatment reduced the toxicity of PQ (35%) and increased the levels of SPD (27%). The results indicate that reduction of PQ toxicity by SPD and SPM is due to increased activities of catalase (18%) and peroxidase (40%).     

Covalent modifications of antitetanus F(ab')2 fragments with natural and synthetic polyamines and their effects on the antibody endocytosis in cultured HL60 cells

For antibody therapeutics to succeed when intracellular target molecules are involved, a strategy must be applied to increase the delivery of antibodies into cells to reach their targets. Antibody cationization by chemical conjugation of a polyamine could be one such strategy. Both natural polyamines with increasing net charge valencies (putrescine, PUT; spermidine, SPD; and spermine, SPM) and a synthetic polyamine (hexamethylenediamine, HMD) can be used to cationize antibodies, but no comparison of the respective effects of these polyamines on intracellular delivery of antibodies has been performed yet. This study describes the covalent modification of antitetanus F(ab') 2 with these four polyamines using different reaction conditions, and compares the effects of these modifications on antibody interaction with cultured HL60 cells. The cationized antibodies retained > or =80% of the binding activity of the unmodified F(ab') 2 with regard to tetanus toxin, as measured by an antigen-binding capture enzyme immunoassay. This same method was used to quantify the amount of cell-associated F(ab') 2 following incubation with HL60 cells. Cationization was shown to enhance cell interaction of the F(ab') 2 : the higher the number of coupled polyamine molecules, the greater the amount of antibody associated with the cells. Moreover, coupling the F(ab') 2 to the SPD and SPM polyamines had greater effect on cell interaction than coupling the F(ab') 2 to the PUT and HMD diamines. Internalization of the cationized antibodies by the HL60 cells was demonstrated by confocal microscopy. This technique also showed that SPD and SPM were more effective than PUT and HMD in terms of intracellular delivery of the F(ab') 2 . It follows from all these results that electrostatic interaction involving charge density plays a predominant role in the endocytic transport mechanism of the F(ab') 2 modified with these polyamines. However, coupling the F(ab') 2 to SPM and SPD yielded the same maximum effects in terms of cell interaction, although coupling SPM was expected to increase the antibody net charge valency more than coupling SPD. This finding suggests that the effective global charge for the cell interaction and uptake of polyamine-modified antibodies does not simply correspond to the addition of the ionizable amine functions on the coupled polyamines, and that other factors may come into play.     

INFLUENCE OF POLYAMINES ON THE SPORULATION OF GRATELOUPIA (HALYMENIACEAE,RHODOPHYTA)1

Polyamines (PAs) such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are ubiquitous aliphatic amines involved in widely varying physiological behavior, but particularly they are actively involved in cell growth, division, and differentiation during reproductive events in plants. The contents of PUT, SPD, and SPM in infertile and fertile thalli of the red macroalga Grateloupia sp. were compared, and the results revealed a significant decrease in quantity from infertile to fertile status. At the enzymatic level, l ‐ornithine decarboxylase (ODC) was mainly detected, and l ‐arginine decarboxylase activity was not diminished by the inhibition of ODC. The maximum enzymatic activities, within the range of activities observed, correlated with the lower levels of polyamines in fertile thalli. In culture, SPM promoted the maturation of cystocarps to the eventual liberation of spores from aseptic fertile explants. PAs accumulated in cultivated explants as compared with noncultivated, but exogenous SPM addition further increased the endogenous SPM. The addition of berenyl, cordycepin, cyclohexylamine, dicyclohexylamine, and aurintricarboxylic acid blocked the synthesis in culture at the level of PUT, and partially at SPD and SPM synthesis, but the addition of SPM restored the levels of SPD and SPM as SPM accumulated, and they appeared to interconvert each other. The results obtained suggest that the culture in presence of SPM restored a deficient SPM situation in fertile explants, thus promoting sporulation.     

Hippocampal lipids linked to spatial memory in the C57BL/6J mouse

Although the role of individual brain lipids for learning and memory has been reported, no systematic approach associating brain lipids with spatial memory has been carried out. It was therefore the aim of the study to determine brain lipids in hippocampus of mice forming and yoked controls that did not form spatial memory using the probe trial as the endpoint. 10 animals were trained in the Morris water maze (MWM) and 10 mice were serving as yoked controls i.e. no platform was used during the whole experiment. Hippocampal tissue lipids were extracted and data were acquired with Fourier transformation ion cyclotron resonance mass spectrometry (LTQ-FT) coupled to HPLC. Glycerophosphatidylethanolamines (18:0/22:6, 18:0/20:4 and 18:1/18:1), plasmalogens (16:0-10/22:6 and 18:0-10/22:6) and ceramides (18:0) showed higher levels in the trained group, while glycerolysophosphatidylcholines (16:0, 18:1, 18:0, 20:4), sphingomyelins (16:0, 24:1), ether linked glycerophosphatidylcholines (16:0-10/18:0), glycerophosphatidylcholines (16:0/18:1, 16:0/18:0, 18:0/18:1, 38:7, 18:1/20:1, 20:4/20:4, 22:1/18:1, 22:0/18:1, 20:4/22:6, 22:6/22:6), glucosylceramide (24:1) and plasmalogen (18:0-10/20:1) revealed lower levels in the trained group. Decreased levels of certain species of lysophosphatidylcholine, sphingomyelin, plasmenylphosphatidylcholine, phosphatidylcholine, glycosylceramide and plasmalogen at the probe trial for spatial memory may indicate catabolism in terms of consumption during this process. Increased hippocampal levels of long chain highly unsaturated phosphatidylethanolamines, plasmalogens and ceramides may reflect increased synthesis or decreased degradation at the endpoint of memory testing, probably representing interactions in the brain lipid pathways. The study shows pathways involved in spatial memory, may propose the use of individual brain lipids as probable cognitive enhancers and forms the basis for further studies on the role of brain lipids per se.     

Polyamine Changes and Chilling Injury in Cold-stored Loquat Fruits

Free polyamine levels (spermine (SPM), spermidine (SPD), and putrescine (PUT)) were determined using thin-layer chromatography and fluorometric method in loquat (Eriobotrya japonica Lindl. cv. Dahongpao) fruits stored at 1℃ and 12℃ and in postharvest SPM treated fruits stored at 1℃ respectively to investigate the relationship between changes in polyamines and chilling injury. In the loquat fruits stored at 1℃, SPM level decreased gradually in the first two weeks, then increased sharply and reached a peak value after three weeks, thereafter it decreased rapidly. SPD level decreased steadily during the first three weeks and increased significantly afterwards. PUT level evolved in a similar way as the SPM level did except that it increased slowly in the first two weeks. The fruit showed symptom of chilling injury manifested as flesh leatheriness after three weeks. However, no significant increase and decrease of these three polyamines was detected during storage at the nonchilling temperature (12℃). The SPM-treated fruits maintained high levels of SPM and SPD and remained low level of PUT during storage at 1℃, and no symptom of chilling injury was observed. These results suggested that the increase in SPM level in response to chilling exposure might serve as a defense mechanism against chilling injury while the accumulation of PUT could be a cause of the stress-induced injury and the increase in SPD level could be a consequence of this kind of stress.     

Antizyme (AZ) regulates intestinal cell growth independent of polyamines

Since antizyme (AZ) is known to inhibit cell proliferation and to increase apoptosis, the question arises as to whether these effects occur independently of polyamines. Intestinal epithelial cells (IEC-6) were grown in control medium and medium containing 5 mM difluoromethylornithine (DFMO) to inhibit ODC, DFMO + 5 µM spermidine (SPD), DFMO + 5 µM spermine (SPM), or DFMO + 10 µM putrescine (PUT) for 4 days and various parameters of growth were measured along with AZ levels. Cell counts were significantly decreased and mean doubling times were significantly increased by DFMO. Putrescine restored growth in the presence of DFMO. However, both SPD and SPM when added with DFMO caused a much greater inhibition of growth than did DFMO alone, and both of these polyamines caused a dramatic increase in AZ. The addition of SPD or SPM to media containing DFMO + PUT significantly inhibited growth and caused a significant increase in AZ. IEC-6 cells transfected with AZ-siRNA grew more than twice as rapidly as either control cells or those incubated with DFMO, indicating that removal of AZ increases growth in cells in which polyamine synthesis is inhibited as well as in control cells. In a separate experiment, the addition of SPD increased AZ levels and inhibited growth of cells incubated with DFMO by 50 %. The addition of 10 mM asparagine (ASN) prevented the increase in AZ and restored growth to control levels. These results show that cell growth in the presence or absence of ODC activity and in the presence or absence of polyamines depends only on the levels of AZ. Therefore, the effects of AZ on cell growth are independent of polyamines.     

Effects of polyamines on the release of gonadotropin-releasing hormone and gonadotropins in developing female rats

Polyamines, putrescine (PUT), spermidine (SPD), spermine (SPM), and agmatine (AGM), are polycationic amines related to multiple cell functions found in high concentrations during the development of hypothalamus and pituitary. In previous works, we demonstrated that alpha-difluoromethylornithine (DFMO), an inhibitor of polyamines biosynthesis, induced a delay in puberty of female rats, accompanied by high, sustained follicle-stimulating hormone (FSH) levels during the infantile period. Also, DFMO treatment induced changes in polyamine concentration both in hypothalamus and pituitary of rats, mainly a decrease of PUT and SPD, an increase in SPM, and no change in AGM. In the present work, we investigated the direct effects of polyamines on the secretion of hypothalamic GnRH and pituitary gonadotropins in 6- and 15-day-old female rats. In 6-day-old animals, in vitro incubations with PUT, SPD, and AGM of hypothalami or anterior pituitaries were able to inhibit GnRH, FSH, and leutinizing hormone (LH) secretion, respectively. SPM showed a nonspecific transient inhibitory effect on FSH. When challenged with either high K(+) (hypothami) or GnRH (pituitaries), the tissues incubated in the presence of polyamines showed no differences when compared with their controls. No effects of polyamines in 15-day-old rats in either tissue were observed. Pituitary cell cultures of 6-day-old animals incubated with DFMO for 4 days showed a significant increase in FSH, but not in LH. We conclude that high PUT, SPD, and AGM levels during the first 10 days of life are important for the development of the hypothalamic-hypophyseal unit, probably related to an inhibitory effect on GnRH and gonadotropins. Therefore, polyamine participation, especially PUT and SPD, is of importance in the regulation of GnRH and gonadotropin secretion in the neonatal and infantile periods, critical stages in the establishment of sexual differentiation.     

Different pathways of uptake and degradation of sphingomyelin by lymphoblastoid cells and the potential participation of the neutral sphingomyelinase.

The metabolism of sphingomyelin (SPM) was investigated in Epstein-Barr virus-transformed lymphoid cell lines from normal individuals and from patients with Niemann-Pick disease Type A (deficient in the acid, lysosomal sphingomyelinase) and familial hypercholesterolemia (lacking the low density lipoprotein receptor). Cells were incubated with the following radioactive or fluorescent SPMs: [choline-methyl-14C] SPM, [oleoyl-3H]SPM, pyrene-propenoyl-SPM (P3:1-SPM), pyrene-butanoyl-SPM (P4-SPM), pyrene-dodecanoyl-SPM (P12-SPM), and pyrene-sulfonylamino-undecanoyl-SPM (PSA11-SPM). Several pathways of uptake and subsequent metabolism of SPM in the lymphoblastoid cells were identified. [choline-methyl-14C]SPM and the P12-analog, administered to the cells in the presence of lipoproteins, were taken up through the apoB/E receptor-dependent pathway of endocytosis and degraded solely by the lysosomal sphingomyelinase. Under similar conditions, the other sphingomyelins, i.e. [oleoyl-3H]SPM, P3:1-SPM, P4-SPM, and PSA11-SPM, were taken up by a low density lipoprotein receptor-independent pathway and degraded mostly by a nonlysosomal sphingomyelinase which also catalyzed their hydrolysis in Niemann-Pick cells. In the absence of serum, all sphingomyelins were taken up by an apoB/E receptor-independent pathway and hydrolyzed by a nonlysosomal sphingomyelinase. Indeed, in vitro assays demonstrated the presence, in lymphoblastoid cells, of the neutral magnesium-activated sphingomyelinase, which was also fully active in the Niemann-Pick cells. In conclusion, our observations are consistent with: (i) the existence in lymphoblastoid cells of several pathways for the uptake and subsequent utilization of SPM; (ii) a major role of lipoproteins for the metabolic routing of the SPM; and (iii) the effect of the structure of the fatty acyl residue of SPM on its possible association with lipoproteins and/or cell membranes.     

Regulation of polyamine transport by polyamines and polyamine analogs

Regulation of polyamine transport in murine L1210 leukemia cells was characterized in order to better understand its relationship to specific intracellular polyamines and their analogs and to quantitate the sensitivity by which it is controlled. Up-regulation of polyamine uptake was evaluated following a 48-hr treatment with a combination of biosynthetic enzyme inhibitors to deplete intracellular polyamine pools. The latter declined gradually over 48 hr and was accompanied by a steady increase in spermidine (SPD) and spermine (SPM) transport as indicated by rises in V_max to levels ～4.5 times higher than control values. Restoration of individual polyamine pools during a 6-hr period following inhibitor treatment revealed that SPD and SPM uptake could not be selectively affected by specific pool changes. The effectiveness of individual polyamines in reversing inhibitor-induced stimulation of uptake was as follows: putrescine < SPD < SPM = the SPM analog, N¹, N¹²-bis(ethyl)spermine (BESPM). In contrast to stimulation of transport, down-regulation by exogenous polyamines or analogs occurred rapidly and in response to subtle increases in intracellular pools. Following a 1-hr exposure to 10 μM BESPM, V_max values for SPD and SPM fell by 70%, whereas the analog pool increased to only 400–500 pmol/10⁶ cells—about 15–20% of the total polyamine pool (～2.8 nmol/10⁶ cells). SPM produced nearly identical regulatory effects on transport kinetics. Both BESPM and SPM were even more effective at down-regulating transport that had been previously stimulated four to fivefold by polyamine depletion achieved with enzyme inhibitors. A dose response with BESPM at 48 hr revealed a biphasic effect on uptake whereby concentrations of analog < 3 μM produced an increase in SPD and SPM V_max values, whereas concentrations 3 μM and higher produced a marked suppression of these values. Cells treated with 3 μM BESPM for 2 hr and placed in analog-free medium recovered transport capability in only 3 hr. Thus, whereas stimulation of polyamine transport is a relatively insensitive and slowly responsive process that tends to parallel polyamine depletion, down-regulation of polyamine transport by exogenous polyamines and analogs and its reversal are rapidly responsive events that correlate with relatively small (i.e., 15–20%) changes in intracellular polyamine pools.     

Polyamines, PI(4,5)P2, and actin polymerization

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.     

Infant formula supplemented with polyamines alters the intestinal microbiota in neonatal BALB/cOlaHsd mice

Polyamines play a critical role in the development of intestinal and immune systems during the infant breastfeeding period, but the effect of polyamines on the microbiota has not been reported. The aim of our study was to characterize the impact on the colonization pattern in neonatal BALB/cOlaHsd mice after supplementing an infant formula (IF) with a mixture of putrescine (PUT), spermidine (SPD) and spermine (SPM). A total of 48 pups (14 days old) were randomly assigned to 4-day intervention groups as follows: breast-fed (unweaned) pups (n=12); weaned pups (n=12) fed an infant formula (IF); weaned pups (n=12) fed an IF enriched with a low concentration of PUT, SPD and SPM (2.10, 22.05 and 38.00 μg/day, respectively); and weaned pups (n=12) fed with IF enriched with a high concentration of PUT, SPD and SPM (8.40, 88.20 and 152.00 μg/day, respectively) of polyamines in accordance with normal proportions found in human milk. Microbiota composition was analyzed by fluorescent in situ hybridization (FISH) with flow cytometry detection. Microbiota changes in formula-fed mice were significantly greater following supplementation with polyamines (P<.01). Bifidobacterium group bacteria, Akkermansia-like bacteria and Lactobacillus–Enterococcus group levels were higher in the groups fed infant formula supplemented with polyamines, resulting in even higher numbers of bacteria than in the breastfed pups. Our findings indicate that infant formulas enriched with polyamines may interact with gut microbiota, suggesting that further studies in human infants are required to assess the impact of polyamines on both growth and microbiota levels.     

Analyzing the effects of exogeneous polyamines and growth regulators on plating efficiency of sweet potato protoplasts using a central composite test design

The effects of exogenous polyamines and growth regulators on plating efficiency of greenhouse-grown sweet potato (Ipomoea batatas Lam.) petiole protoplasts after six days were analyzed using a central composite test design. The medium components screened were 1-naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), putrescine (PUT), spermidine (SPD), and spermine (SPM), each at five concentrations. Stepwise multiple regression analysis revealed significant interaction of NAA with BAP, PUT, and SPD as reflected in plating efficiencies. The interactions of NAA with BAP, and with SPD, were positive. The interaction of NAA and PUT appeared complex. A slight negative interaction was detected between PUT and SPM. These results indicated that plating efficiency of sweet potato protoplasts is highly sensitive to the concentrations of the medium components tested and it should be possible to further optimize the plating medium. Among the media formulations tested, the highest plating efficiency (10.8% after 6 days) was observed with NAA at 4.5 uM, BAP at 1.5 uM, PUT at 35.0 uM, SPD at 5.0 uM, and SPM at 2.5 uM.     

Conformation and ion channel properties of a five-helix Bundle protein.

The primary amphipathic peptide Ac-Met-Gly-Leu-Gly-Leu-Trp-Leu-Leu-Val-Leu10-Ala-Ala-Ala-Leu-Gln-Gly-Ala-Lys-Lys-Lys20-Arg-Lys-Val-NH-CH2-CH2-SH called SPM was able to induce formation of ion channels into planar lipid bilayers with main conductance values of 75 and 950 pS in 1 M KCl. The 75 pS value can be attributed to an aggregate composed of five monomers since the corresponding five-unit bundle (5-SPM) also presented a 70 pS channels under the same conditions. The upper 950 pS level would be generated by a hexameric aggregate. Ion channels induced by both SPM and its pentameric bundle are slightly cation selective but not voltage-dependent. The structural studies showed that the SPM and 5-SPM possess mainly an alpha-helical structure (approximately 40%) and are strongly embedded in the bilayer. This behaviour and the strong hydrophobic interactions occurring between helices in the bundle induce a strong stabilization of 5-SPM in the bilayer and would be responsible for the stepwise current fluctuations observed during the incorporation of 5-SPM into the membrane.