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.     

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.     

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.     

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.     

Polyamine metabolism in McCoy cells: III. Comparative studies of the metabolic fate of exogenous putrescine in human fibroblasts and McCoy cultures

The fate of exogenously added 14C-putrescine following incubation for 24 hours with McCoy and human skin fibroblast cultures was examined. The nature of the polyamine derivatives found were quite different indicative of a difference in the cellular metabolism of polyamines. Exogenously added putrescine (PUT) was metabolized by both McCoy and human skin fibroblast cultures to form spermidine (SPD), spermine (SPM), gamma-aminobutyric acid (GABA) and some unidentified compounds. Within the experimental period of observation, human cultured fibroblasts metabolized PUT more efficiently than McCoy cells and converted more than 50% of it into SPD, SPM, GABA and unknown compounds. Monoacetyl putrescine (MAP) was formed by human skin fibroblasts. It was mainly identified in the culture medium. No MAP was detectable either intracellularly or extracellularly in McCoy cultures. The percentage of 14C-radioactivity found as PUT in the culture medium was greater in McCoy cells (86.0%) than in human fibroblasts (53.9%). The reverse was true for the percentage distribution of 14C-radioactivity as PUT inside the cells. No low Mr conjugates of SPD or SPM were found in the medium or intracellularly with either culture type. Some low Mr putrescine conjugates were found in the culture media; these were identified by the liberation of PUT upon acid hydrolysis.     

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.     

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.     

Spermidine,a sensor for antizyme 1 expression regulates intracellular polyamine homeostasis

Although intracellular polyamine levels are highly regulated, it is unclear whether intracellular putrescine (PUT), spermidine (SPD), or spermine (SPM) levels act as a sensor to regulate their synthesis or uptake. Polyamines have been shown to induce AZ1 expression through a unique +1 frameshifting mechanism. However, under physiological conditions which particular polyamine induces AZ1, and thereby ODC activity, is unknown due to their inter-conversion. In this study we demonstrate that SPD regulates AZ1 expression under physiological conditions in IEC-6 cells. PUT and SPD showed potent induction of AZ1 within 4 h in serum-starved confluent cells grown in DMEM (control) medium. Unlike control cells, PUT failed to induce AZ1 in cells grown in DFMO containing medium; however, SPD caused a robust AZ1 induction in these cells. SPM showed very little effect on AZ1 expression in both the control and polyamine-depleted cells. Only SPD induced AZ1 when S-adenosylmethionine decarboxylase (SAMDC) and/or ODC were inhibited. Surprisingly, addition of DENSpm along with DFMO restored AZ1 induction by putrescine in polyamine-depleted cells suggesting that the increased SSAT activity in response to DENSpm converted SPM to SPD, leading to the expression of AZ1. This study shows that intracellular SPD levels controls AZ1 synthesis.     

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%).     

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.     

Electric organ polyamines and their effects on the acetylcholine receptor

1. The electric organ of Torpedo nobiliana contained putrescine (PUT), spermidine (SPD), spermine (SPM), and cadaverine (CAD). Traces of acetylated SPD and SPM were occasionaly seen. 2. Upon fractionation of the tissue by differential centrifugation, the polyamines (PA) were found predominantly in the soluble fraction. The postsynaptic membrane fraction, containing a high concentration of acetylcholine receptor (AChR), was proportionally enriched in SPM. The molar ratio of SPM to AChR was approximately two in these membranes. 3. The effect of exogeneous PA on AChR function was studied by two methods: carbamoylcholine (CCh)-dependent 86Rb+ influx into receptor-rich membrane vesicles and [alpha-125I]bungarotoxin (Bgt) binding to the AChR. 4. SPM inhibited both ion influx and the rate of Bgt binding at concentrations above 1 mM, and therefore it appears to act as a competitive antagonist of the AChR. 5. At submicromolar concentrations, and only after preincubation with the receptor-rich membrane, SPM and PUT increased the ion influx by about 20% over control values. 6. Preincubation with 100 nM SPM did not affect the equilibrium binding of iodinated toxin or the rate of toxin binding, and therefore SPM was not uncovering new receptors. 7. By measuring the initial rate of toxin binding after different periods of preincubation with 1 microM CCh, the rate of the slow phase of receptor desensitization was determined. This rate was not changed by 100 nM SPM. 8. Although these results suggest that at low concentrations SPM is a positive modulator of the AChR, the precise mechanism of action is not determined yet.     

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.     

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.     

Polyamine- and insulin-like growth factor-I-mediated proliferation of porcine uterine endometrial cells: a potential role for spermidine/spermine N(1)-acetyltransferase during peri-implantation

Insulin-like growth factor-I (IGF-I) and the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) are progesterone-regulated genes with maximal expression at peri-implantation in the porcine uterine endometrium. However, while IGF-I stimulates cell proliferation, SSAT, by acetylating the naturally occurring polyamines (PA) spermine (SPM) and spermidine (SPD), typically functions as a cell growth inhibitor. The present study examined the functional relationships of IGF-I, SSAT, and PA in the control of endometrial cell proliferation. Northern blot analysis indicated that SSAT mRNA levels change with distinct pregnancy stages, in contrast to those for the PA biosynthetic enzyme ornithine decarboxylase (ODC). Primary cultures of luminal and glandular epithelial (LE, GE) and stromal (ST) cells isolated from Day 12 pregnant pig endometrium had IGF-I mRNA levels for ST > LE > GE cells. The mRNA levels for SSAT and ODC were transiently diminished by IGF-I treatment, but only in GE cells. By contrast, SPM and SPD increased SSAT mRNA levels in GE and ST cells, but increased ODC mRNA levels only in GE cells. IGF-I, putrescine (PUT), and SPM individually increased cellular DNA synthesis as measured by tritiated thymidine incorporation in GE and ST cells, while SPD had an effect only in ST cells. IGF-I enhanced the proliferative effect of each PA in GE cells, but only of SPD in ST cells. The mitogen-activated protein kinase inhibitor, PD98059, inhibited the induction by SPM of GE cell DNA synthesis but not that of IGF-I. Wortmannin, a phosphatidylinositol-3-kinase inhibitor had no effect on either IGF-I or SPM induction of GE cell DNA synthesis. The relative concentrations of SPM, SPD, and PUT in uterine luminal fluids differed, with the levels for each PA higher at pregnancy Day 12 than at 11.5. These results suggest that IGF-I and PA act through distinct signaling pathways to mediate cell-type-specific growth of early pregnancy pig uterine endometrium. Further, SSAT, through its control of intracellular PA levels, likely plays a modulatory role in the establishment of an optimal uterine environment for successful embryo attachment.     

Changes in polyamines, c-myc and c-fos gene expression in osteoblast-like cells exposed to pulsed electromagnetic fields

Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.     

Involvement of polyamines in the adventitious rooting of micropropagated shoots of the apple rootstock MM106

Apple rootstock MM106 shoots, raised in vitro, rooted at 96.7% after culture on a medium supplemented with an auxin for 5 d in darkness followed by culture on a second medium without growth regulators for 25 d in light. In control conditions (in absence of auxin in the first medium), these shoots did not root. Putrescine (PUT), spermidine (SPD), cyclohexylamine (CHA), and aminoguanidine (AG) enhanced rooting when applied during the first d of culture in the absence of IBA; on the contrary, α-difluoromethylornithine (DFMO) added to the first medium with IBA inhibited rooting. The endogenous levels of indole 3-acetic acid (IAA) and indole 3-acetylaspartic acid (IAAsp) increased up to a maximum concentration at days 2 and 3, respectively, in initial rooting conditions. PUT, when added with IBA, did not affect the typical IAA and IAAsp increase; when applied alone, it provoked an increase of their levels. Similar results were recorded with CHA. SPD, AG, and DFMO did not induce an increase of IAA and IAAsp in nonrooting conditions. The levels of endogenous PUT increased to a maximum at day 2 in rooting conditions; it was slightly affected by exogenous PUT and CHA application but reduced by SPD, AG, and DFMO. In rooting conditions, if the first medium was supplemented with SPD or AG, a small increase in peroxidase activity was observed, similar to that obtained with PUT treatment. The present work indicates an involvement of polyamines in the control of rooting and an interaction with auxins during the physiological phase of rooting. The consequence of this relationship was a different rooting expression, according especially to the content of these regulators in the culture medium.     

Comparative effects of insulin and insulin-like growth factor-1 on follicle-stimulating hormone-induced responses in porcine granulosa cells

The effect of insulin and insulin-like growth factor-1 (IGF-1) on progesterone secretion by porcine granulosa cells and their modulatory effect on follicle-stimulating hormone (FSH)-induced responses were examined. For comparative purposes, growth hormone (GH), previously shown to stimulate IGF-1 secretion, was also included. Granulosa cells from ovarian follicles (3 to 5 mm) were cultured in multiwell plates for the first 48 hours, either in the presence or absence of 1% fetal bovine serum (FBS). Following plating, all cultures were maintained in serum-free media. The addition of only insulin, but not IGF-1 or GH, enhanced progesterone secretion under both culture conditions. When low-density lipoprotein was provided as steroid substrate, a stimulatory effect of insulin on progesterone accumulation was observed with a minimum dose of 10 ng/ml. Granulosa cells cultured in serum-free media from the time of plating secreted less progesterone and were less responsive to FSH compared with cultures plated with 1% FBS. Only insulin, but not IGF-1, enhanced FSH responses to threefold in cells cultured with 1% FBS. However, when cells were cultured in serum-free media from the time of plating, both insulin and IGF-1, but not GH, potentiated the responses to FSH, but insulin was more potent than IGF-1. Insulin-like growth-factor-1 binding studies with granulosa cells indicate the presence of specific high-affinity binding sites (Kd 3.96 nM). A dose of 100 ng/ml of insulin had negligible cross-reactivity with IGF-1 receptors.     

Epigallocatechin-3-O-gallate inhibits TNFalpha-induced monocyte chemotactic protein-1 production from vascular endothelial cells

Monocyte chemotactic protein-1 (MCP-1) plays a pivotal role in the recruitment of monocytes and thus in the development of inflammatory cardiovascular diseases. Epigallocatechin-3-O-gallate (EGCG), the major catechin derived from green tea, has multiple beneficial effects to reduce cardiovascular disease but the effects of EGCG on vascular endothelial MCP-1 production is not known. In this study, we investigated the mechanisms by which EGCG may inhibit tumor necrosis factor-alpha (TNFalpha)-induced MCP-1 production in bovine coronary artery endothelial cells. TNFalpha increased MCP-1 production in both a concentration and time-dependent manner. Inhibitors of phosphatidylinositol-3-OH kinase (PI-3 kinase), LY294002 and wortmannin, decreased TNFalpha-induced MCP-1 production. EGCG prevented TNFalpha-mediated MCP-1 production and reduced phosphorylation of Akt (Ser473). In addition, EGCG attenuated TNFalpha mediated down-regulation of TNFalpha receptor 1 (TNFR1), but not TNFR2. In conclusion, EGCG inhibited TNFalpha-induced MCP-1 production. Moreover, EGCG inhibited Akt phosphorylation as well as TNF activation of TNFR1, which subsequently resulted in reduced MCP-1 production. These data provide a novel mechanism where the green tea flavonoid, EGCG, could provide direct vascular benefits in inflammatory cardiovascular diseases.     

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.