(R,R)-1,12-Dimethylspermine can mitigate abnormal spermidine accumulation in Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS) is an X-linked intellectual disability syndrome caused by a loss-of-function mutation in the spermine synthase (SMS) gene. Primarily affecting males, the main manifestations of SRS include osteoporosis, hypotonic stature, seizures, cognitive impairment, and developmental delay. Because there is no cure for SRS, treatment plans focus on alleviating symptoms rather than targeting the underlying causes. Biochemically, the cells of individuals with SRS accumulate excess spermidine, whereas spermine levels are reduced. We recently demonstrated that SRS patient-derived lymphoblastoid cells are capable of transporting exogenous spermine and its analogs into the cell and, in response, decreasing excess spermidine pools to normal levels. However, dietary supplementation of spermine does not appear to benefit SRS patients or mouse models. Here, we investigated the potential use of a metabolically stable spermine mimetic, (R,R)-1,12-dimethylspermine (Me2SPM), to reduce the intracellular spermidine pools of SRS patient-derived cells. Me2SPM can functionally substitute for the native polyamines in supporting cell growth while stimulating polyamine homeostatic control mechanisms. We found that both lymphoblasts and fibroblasts from SRS patients can accumulate Me2SPM, resulting in significantly decreased spermidine levels with no adverse effects on growth. Me2SPM administration to mice revealed that Me2SPM significantly decreases spermidine levels in multiple tissues. Importantly, Me2SPM was detectable in brain tissue, the organ most affected in SRS, and was associated with changes in polyamine metabolic enzymes. These findings indicate that the (R,R)-diastereomer of 1,12-Me2SPM represents a promising lead compound in developing a treatment aimed at targeting the molecular mechanisms underlying SRS pathology.

Spermine protects aluminium chloride and iron-induced neurotoxicity in rat model of Alzheimer's disease via attenuation of tau phosphorylation, Amyloid-ß (1-42) and NF-κB pathway.

Alzheimer's disease (AD) is the most prevalent type of dementia, characterized by a gradual decline in cognitive and memory functions of the aged peoples. Long-term exposure to heavy metals (aluminium and iron) cause neurotoxicity by amyloid plaques accumulation, tau phosphorylation, increased oxidative stress, neuroinflammation, and cholinergic neurons degeneration, contributes to the development of AD-like symptoms. The present research work is designed to investigate the neuroprotective effect of spermine in aluminium chloride (AlCl3), and iron (Fe) induced AD-like symptoms in rats. Rats were administered of AlCl3 (100 mg/kg p.o.) alone and in combination with iron (120 µg/g, p.o.) for 28 days. Spermine (5 and 10 mg/kg) through intraperitoneal (i.p.) route was given for 14 days. The recognition and spatial memory impairment were tasted using Morris water maze (MWM), actophotometer, and Novel Object Recognition test (NORT). All the rats were sacrificed on day 29, brains were isolated, and tissue homogenate was used for neuroinflammatory, biochemical, neurotransmitters, metals concentration, and nuclear factor-kappa B (NF-κB) analysis. In the present study, AlCl3 and iron administration elevated oxidative stress, cytokines release, dysbalanced neurotransmitters concentration, and biochemical changes. Rats treated with spermine dose-dependently improved the recognition and spatial memory, attenuated proinflammatory cytokine release, and restored neurotransmitters concentration and antioxidant enzymes. Spermine also mitigated the increased beta-amyloid (Aß42), with downregulation of tau phosphorylation. Furthermore, spermine augmented the hippocampal levels of B cell leukaemia/lymphoma-2 (Bcl-2), diminished nuclear factor-kappa B (NF-κB) and caspase-3 (casp-3) expression. Moreover, spermine exhibited the neuroprotective effect through anti-inflammatory, antioxidant, neurotransmitters restoration, anti-apoptotic Aß42 concentration.

The protective role of spermine against male reproductive aberrations induced by exposure to electromagnetic field - An experimental investigation in the rat.

The exponentially increasing use of electromagnetic field (EMF)-emitting devices imposes substantial health burden on modern societies with particular concerns of male infertility. Limited studies have addressed the modulation of this risk by protective agents. We investigated the hazardous effects of rat exposure to EMF (900 MHz, 2 h/day for 8 weeks) on male fertility and evaluated the possible protective effect of the polyamine, spermine, against EMF-induced alterations. Exposure to EMF significantly decreased sperm count, viability and motility, and increased sperm deformities. EMF-exposed rats exhibited significant reductions in serum inhibin B and testosterone along with elevated activin A, follicle-stimulating hormone, luteinizing hormone and estradiol concentrations. Testicular steroidogenic acute regulatory protein (StAR), c-kit mRNA expression and testicular activities of the key androgenic enzymes 3ß- and 17ß-hydroxysteroid dehydrogenases were significantly attenuated following exposure to EMF. Exposure led to testicular lipid peroxidation, decreased catalase and glutathione peroxidase activities and triggered nuclear factor-kappa B p65, inducible nitric oxide synthase, cyclooxygenase-2 and caspase-3 overexpression. EMF-exposed rats showed testicular DNA damage as indicated by elevated comet parameters. Spermine administration (2.5 mg/Kg/day intraperitoneally for 8 weeks) prevented EMF-induced alterations in the sperm and hormone profiles, StAR and c-kit expression and androgenic enzyme activities. Spermine hampered EMF-induced oxidative, inflammatory, apoptotic and DNA perturbations. Histological and histomorphometric analysis of the testes supported all biochemical findings. In conclusion, rat exposure to EMF disrupts sperm and hormone profiles with underlying impairment of steroidogenesis and spermatogenesis. Spermine confers protection against EMF-associated testicular and reproductive aberrations, at least in part, via antioxidant, anti-inflammatory and anti-apoptotic mechanisms.

Roles of spermine in modulating the antioxidant status and Nrf2 signalling molecules expression in the thymus and spleen of suckling piglets-new insight.

This study aimed to investigate the effects of spermine and extended spermine administration on the antioxidant status and expression of NF-E2-related factor 2 (Nrf2) signalling molecules in the thymus and spleen in suckling piglets. One half of eighty 12-day-old suckling piglets obtained sufficient nutrient intake supplemented with spermine (0.4 mmol/kg body weight), and another half received restricted nutrient intake supplemented with physiological saline in equal doses once a day for 7 hr or 3, 6 or 9 days in pairs. Spermine supplementation and its extended duration significantly decreased malondialdehyde (MDA) and protein carbonyl (PC) contents (p < .05), but markedly improved antisuperoxide anion (ASA), antihydroxyl radical (AHR), catalase (CAT), total superoxide dismutase (T-SOD), total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activities (p < .05) and glutathione (GSH) content (p < .05) in the thymus and spleen. Additionally, real-time PCR analysis showed that spermine administration and extended spermine intake reduced Kelch-like ECH-associated protein 1 (Keap1) gene transcription and enhanced SOD1, GPx1, CAT, glutathione reductase (GR) and Nrf2 mRNA levels of the thymus and spleen (p < .05), and also improved GST gene expression in the thymus (p < .05). Notably, the spermine-supplemented time for the optimal effects of suckling piglet was determined to be 6 days. Collectively, the current study suggested that spermine supplementation and extended spermine administration could protect the health of the thymus and spleen from early weaning by enhancing the antioxidant status and regulating the expression of antioxidant-related signalling molecules.

Impact of the Pd2Spermine Chelate on Osteosarcoma Metabolism: An NMR Metabolomics Study.

A metabolomics study of Pd2Spermine(Spm) on osteosarcoma MG-63 and osteoblastic HOb cells is presented to assess the impact of the potential palladium drug on cell metabolism compared with cisplatin (cDDP). Despite its higher cytotoxicity, Pd2Spm induced lower (and reversible) metabolic impact on MG-63 cells and the absence of apoptosis; conversely, it induced significant deviations in osteoblastic amino acid metabolism. However, when in combination with doxorubicin and methotrexate, Pd2Spm induced strong metabolic deviations on lipids, choline compounds, amino acids, nucleotides, and compounds related to antioxidative mechanisms (e.g., glutathione, inositol, hypoxanthine), similarly to the cDDP cocktail. Synergetic effects included triggering of lipid biosynthesis by Pd2Spm in the presence of doxorubicin (and reinforced by methotrexate) and changes in the glycosylation substrate uridine diphosphate acetylgalactosamine and methionine and serine metabolisms. This work provides promising results related to the impact of Pd2Spm on osteosarcoma cellular metabolism, particularly in drug combination protocols. Lipid metabolism, glycosylation, and amino acid metabolisms emerge as relevant features for targeted studies to further understand a potential anticancer mechanism of combined Pd2Spm.

Pharmacological inhibition of protein tyrosine phosphatase 1B protects against atherosclerotic plaque formation in the LDLR-/- mouse model of atherosclerosis.

Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with type 1 or type 2 diabetes, due to accelerated atherosclerosis. Recent evidence suggests a strong link between atherosclerosis and insulin resistance, due to impaired insulin receptor (IR) signalling. Here, we demonstrate that inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of the IR prevents and reverses atherosclerotic plaque formation in an LDLR-/- mouse model of atherosclerosis. Acute (single dose) or chronic PTP1B inhibitor (trodusquemine) treatment of LDLR-/- mice decreased weight gain and adiposity, improved glucose homeostasis and attenuated atherosclerotic plaque formation. This was accompanied by a reduction in both, circulating total cholesterol and triglycerides, a decrease in aortic monocyte chemoattractant protein-1 (MCP-1) expression levels and hyperphosphorylation of aortic Akt/PKB and AMPKα. Our findings are the first to demonstrate that PTP1B inhibitors could be used in prevention and reversal of atherosclerosis development and reduction in CVD risk.

Involvement of Carrier-Mediated Transport at the Blood-Cerebrospinal Fluid Barrier in Spermine Clearance from Rat Brain.

Spermine is the end-product in the polyamine biosynthetic pathway, and its excess accumulation induces neuroexcitatory responses and neurotoxicity. The purpose of this study was to elucidate the involvement of transport systems at the brain barriers in the clearance of spermine. In vivo rat spermine elimination from brain parenchyma across the blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier (BCSFB) was assessed by intracerebral and intracerebroventricular administration techniques, respectively. To characterize spermine transport at the BCSFB, a transport study using rat choroid plexus was performed. After the intracerebral microinjection of [3H]spermine, no time-dependent decrease in [3H]spermine in the ipsilateral cerebrum was observed, suggesting the low contribution of the BBB to spermine clearance from the brain. In contrast, the [3H]spermine concentration in the CSF after intracerebroventricular administration was time-dependently decreased with an elimination rate constant of 0.352 min-1, and the elimination clearance of [3H]spermine was 6.6-fold greater than that of [14C]D-mannitol, reflecting bulk flow of the CSF. This [3H]spermine elimination was attenuated by co-administration of unlabeled excess spermine, indicating carrier-mediated elimination of spermine from the CSF. [3H]Spermine transport into the choroid plexus was strongly inhibited by unlabeled spermine, other polyamines (spermidine and putrescine), and organic cation transporter substrates such as corticosterone and 1-methyl-4-phenylpyridinium. However, other substrates/inhibitors for organic cation transporters (decynium-22 and tetraethylammonium) had little effect. Consequently, our study indicates that transporting molecules at the BCSFB, distinct from typical organic cation transporters, are involved in spermine clearance from the CSF.

Spermine inhibits Endoplasmic Reticulum Stress-induced Apoptosis: a New Strategy to Prevent Cardiomyocyte Apoptosis.

BACKGROUND/AIMS: Endoplasmic reticulum stress (ERS) plays an important role in the progression of acute myocardial infarction (AMI), in part by mediating apoptosis. Polyamines, including putrescine, spermidine, and spermine, are polycations with anti-oxidative, anti-aging, and cell growth-promoting activities. This study aimed to determine the mechanisms by which spermine protects against ERS-induced apoptosis in rats following AMI. METHODS AND RESULTS: AMI was established by ligation of the left anterior descending coronary artery (LAD) in rats, and exogenous spermine was administered by intraperitoneal injection (2.5 mg/ml daily for 7 days pre-AMI). Spermine treatment limited infarct size, attenuated cardiac troponin I and creatinine kinase-MB release, improved cardiac function, and decreased ERS and apoptosis related protein expression. Isolated cardiomyocytes subjected to hypoxia showed significant increase in reactive oxygen species (ROS) and the expression of apoptosis and ERS related proteins; these effects occurred through PERK and eIF2α phosphorylation. The addition of spermine attenuated cardiomyocyte apoptosis, suppressed the production of ROS, and inhibited ERS related pathways. CONCLUSIONS: Spermine was an effective pre-treatment strategy to attenuate cardiac ERS injury in rats, and the cardioprotective mechanism occurring through inhibition of ROS production and down regulation of the PERK-eIF2α pathway. These findings provide a novel target for the prevention of apoptosis in the setting of AMI.

Spermine reduces reactive oxygen species levels and decreases cryocapacitation in canine sperm cryopreservation.

The objective of this study was to determine the ability of spermine to act as an antioxidant in scavenging reactive oxygen species (ROS), maintaining sperm function and decreasing cryocapacitation after cryopreservation. Although motility did not increase with spermine treatment, values for membrane integrity were significantly increased (P < 0.05). Higher percentages of linearity and straightness with a lower amplitude of lateral head displacement (ALH) indicated that spermine inhibits hyperactivation. Concentrations of intracellular and extracellular ROS were decreased in the treatment group (P < 0.05). Higher expression of an anti-apoptotic gene (Bcl-2) and lower expression of a pro-apoptotic gene (Bax), together with decreased expression of the mitochondrial ROS modulator ROMO1, DNA repair due to oxidative damage (OGG1), spermine synthase (SMS), NADPH oxidase associated with motility (NOX5) and spermine amino oxidase (SMOX), all showed that 5.0 mM spermine treatment was beneficial to spermatozoa. Furthermore, the proportion of live spermatozoa with intact acrosomes after thawing in the treatment group was higher than in the control. After incubation in canine capacitating medium, numbers of live capacitated spermatozoa with reacted acrosomes were higher than in the control. Our results indicate that 5.0 mM spermine is an optimal concentration for maintaining sperm function, reducing ROS production, preventing apoptosis and adverse effects of cryocapacitation during canine sperm cryopreservation.

Oral administration of polyamines ameliorates liver ischemia/reperfusion injury and promotes liver regeneration in rats.

Polyamines are essential for cell growth and differentiation. They play important roles in protection from liver damage and promotion of liver regeneration. However, little is known about the effect of oral exogenous polyamine administration on liver damage and regeneration. This study investigated the impact of polyamines (spermidine and spermine) on ischemia/reperfusion injury (IRI) and liver regeneration. We used a rat model in which a 70% hepatectomy after 40 minutes of ischemia was performed to mimic the clinical condition of living donor partial liver transplantation (LT). Male Lewis rats were separated into 2 groups: a polyamine group given polyamines before and after operation as treatment and a vehicle group given distilled water as placebo. The levels of serum aspartate aminotransferase and alanine aminotransferase at 6, 24, and 48 hours after reperfusion were significantly lower in the polyamine group compared with those in the vehicle group. Polyamine treatment reduced the expression of several proinflammatory cytokines and chemokines at 6 hours after reperfusion. Histological analysis showed significantly less necrosis and apoptosis in the polyamine group at 6 hours after reperfusion. Sinusoidal endothelial cells were also well preserved in the polyamine group. In addition, the regeneration of the remnant liver at 24, 48, and 168 hours after reperfusion was significantly accelerated, and the Ki-67 labeling index and the expressions of proliferating cell nuclear antigen and phosphorylated retinoblastoma protein at 24 hours after reperfusion were significantly higher in the polyamine group compared with those in the vehicle group. In conclusion, perioperative oral polyamine administration attenuates liver IRI and promotes liver regeneration. It might be a new therapeutic option to improve the outcomes of partial LT. Liver Transplantation 22 1231-1244 2016 AASLD.

Poly-ion Complex of Chondroitin Sulfate and Spermine and Its Effect on Oral Chondroitin Sulfate Bioavailability.

Chondroitin sulfate (CS) has been accepted as an ingredient in health foods for the treatment of symptoms related to arthritis and cartilage repair. However, CS is poorly absorbed through the gastrointestinal tract because of its high negative electric charges and molecular weight (MW). In this study, poly-ion complex (PIC) formation was found in aqueous solutions through electrostatic interaction between CS and polyamines-organic molecules having two or more primary amino groups ubiquitously distributed in natural products at high concentrations. Characteristic properties of various PICs generated by mixing CS and natural polyamines, including unusual polyamines, were studied based on the turbidity for PIC formation, the dynamic light scattering for the size of PIC particles, and ζ-potential measurements for the surface charges of PIC particles. The efficiency of PIC formation between CS and spermine increased in a CS MW-dependent manner, with 15 kDa CS being critical for the formation of PIC (particle size: 3.41 µm) having nearly neutral surface charge (ζ-potential: -0.80 mV). Comparatively, mixing tetrakis(3-aminopropyl)ammonium and 15 kDa of CS afforded significant levels of PIC (particle size: 0.42±0.16 µm) despite a strongly negative surface charge (-34.67±1.15 mV). Interestingly, the oral absorption efficiency of CS was greatly improved only when PIC possessing neutral surface charges was administered to mice. High formation efficiency and electrically neutral surface charge of PIC particles are important factors for oral CS bioavailability.

Blockage of mitochondrial calcium uniporter prevents iron accumulation in a model of experimental subarachnoid hemorrhage.

Previous studies have shown that iron accumulation is involved in the pathogenesis of brain injury following subarachnoid hemorrhage (SAH) and chelation of iron reduced mortality and oxidative DNA damage. We previously reported that blockage of mitochondrial calcium uniporter (MCU) provided benefit in the early brain injury after experimental SAH. This study was undertaken to identify whether blockage of MCU could ameliorate iron accumulation-associated brain injury following SAH. Therefore, we used two reagents ruthenium red (RR) and spermine (Sper) to inhibit MCU. Sprague-Dawley (SD) rats were randomly divided into four groups including sham, SAH, SAH+RR, and SAH+Sper. Biochemical analysis and histological assays were performed. The results confirmed the iron accumulation in temporal lobe after SAH. Interestingly, blockage of MCU dramatically reduced the iron accumulation in this area. The mechanism was revealed that inhibition of MCU reversed the down-regulation of iron regulatory protein (IRP) 1/2 and increase of ferritin. Iron-sulfur cluster dependent-aconitase activity was partially conserved when MCU was blocked. In consistence with this and previous report, ROS levels were notably reduced and ATP supply was rescued; levels of cleaved caspase-3 dropped; and integrity of neurons in temporal lobe was protected. Taken together, our results indicated that blockage of MCU could alleviate iron accumulation and the associated injury following SAH. These findings suggest that the alteration of calcium and iron homeostasis be coupled and MCU be considered to be a therapeutic target for patients suffering from SAH.

An iGlu receptor antagonist and its simultaneous use with an anticancer drug for cancer therapy.

Glutamate receptor antagonists have been known to play a crucial role in the treatment of many neuronal diseases. Recently, these antagonists have also shown therapeutic effects in the treatment of cancer. In this study, an ionotropic glutamate (iGlu) receptor antagonist, 4-hydroxyphenylacetyl spermine (L1), was used concurrently with a common anticancer drug, doxorubicin (Dox), for simultaneous cancer therapy. Mesoporous silica nanoparticles (MSNPs) were employed as the delivery vehicle for both L1 and Dox by conjugating the iGlu receptor antagonist on the surface and encapsulating Dox within the mesopores. Dox was then trapped within the mesopores by functionalizing a redox-cleavable capping group on the MSNP surface, and it could be released upon exposure to the reductive glutathione. In vitro studies on B16F10 and NIH3T3 cell lines revealed that the iGlu receptor antagonist L1 exhibited therapeutic as well as targeting effects. In addition, the simultaneous use of therapeutic L1 and Dox proved to be synergistic in the treatment of cancer. The present work demonstrated the feasibility of employing a delivery system to deliver both neuroprotective drug and anticancer drug for efficient anticancer treatment.

The LIM domain only 4 protein is a metabolic responsive inhibitor of protein tyrosine phosphatase 1B that controls hypothalamic leptin signaling.

Protein tyrosine phosphatase 1B (PTP1B) counteracts leptin signaling and is a therapeutic target for obesity and diabetes. Here we found that LIM domain only 4 (LMO4) inhibits PTP1B activity by increasing the oxidized inactive form of PTP1B. Mice with neuronal ablation of LMO4 have elevated PTP1B activity and impaired hypothalamic leptin signaling, and a PTP1B inhibitor normalized PTP1B activity and restored leptin control of circulating insulin levels. LMO4 is palmitoylated at its C-terminal cysteine, and deletion of this residue prevented palmitoylation and retention of LMO4 at the endoplasmic reticulum and abolished its inhibitory effect on PTP1B. Importantly, LMO4 palmitoylation is sensitive to metabolic stress; mice challenged with a brief high-fat diet or acute intracerebroventricular infusion of saturated fatty acid had less palmitoylated LMO4, less oxidized PTP1B, and increased PTP1B activity in the hypothalamus. Thus, unleashed PTP1B activity attributable to loss of LMO4 palmitoylation may account for rapid loss of central leptin signaling after acute exposure to saturated fat.

Raphe AMPA receptors and nicotinic acetylcholine receptors mediate ketamine-induced serotonin release in the rat prefrontal cortex.

Several lines of evidence indicate that ketamine has a rapid antidepressant-like effect in rodents and humans, but underlying mechanisms are unclear. In the present study, we investigated the effect of ketamine on serotonin (5-HT) release in the rat prefrontal cortex by in vivo microdialysis. A subcutaneous administration of ketamine (5 and 25 mg/kg) significantly increased the prefrontal 5-HT level in a dose-dependent manner, which was attenuated by local injection of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonists into the dorsal raphe nucleus (DRN). Direct stimulation of AMPARs in the DRN significantly increased prefrontal 5-HT level, while intra-DRN injection of ketamine (36.5 nmol) had no effect. Furthermore, intra-DRN injection of an α 4 ß 2-nicotinic acetylcholine receptor (nAChR) antagonist, dihydro-ß-erythroidine (10 nmol), significantly attenuated the subcutaneous ketamine-induced increase in prefrontal 5-HT levels. These results suggest that AMPARs and α 4 ß 2-nAChRs in the DRN play a key role in the ketamine-induced 5-HT release in the prefrontal cortex.

Resembling breast milk: influence of polyamine-supplemented formula on neonatal BALB/cOlaHsd mouse microbiota.

Infant microbiota is influenced by numerous factors, such as delivery mode, environment, prematurity and diet (breast milk or formula). In addition to its nutritional value, breast milk contains bioactive substances that drive microbial colonisation and support immune system development, which are usually not present in infant formulas. Among these substances, polyamines have been described to be essential for intestinal and immune functions in newborns. However, their effect on the establishment of microbiota remains unclear. Therefore, the aim of the present study was to ascertain whether an infant formula supplemented with polyamines has an impact on microbial colonisation by modifying it to resemble that in breast-fed neonatal BALB/c mice. In a 4 d intervention, a total of sixty pups (14 d old) were randomly assigned to the following groups: (1) breast-fed group; (2) non-enriched infant formula-fed group; (3) three different groups fed an infant formula enriched with increasing concentrations of polyamines (mixture of putrescine, spermidine and spermine), following the proportions found in human milk. Microbial composition in the contents of the oral cavity, stomach and small and large intestines was analysed by quantitative PCR targeted at fourteen bacterial genera and species. Significantly different (P< 0·05) microbial colonisation patterns were observed in the entire gastrointestinal tract of the breast-fed and formula-fed mice. In addition, our findings demonstrate that supplementation of polyamines regulates the amounts of total bacteria, Akkermansia muciniphila, Lactobacillus, Bifidobacterium, Bacteroides-Prevotella and Clostridium groups to levels found in the breast-fed group. Such an effect requires further investigation in human infants, as supplementation of an infant formula with polyamines might contribute to healthy gastrointestinal tract development.

Safety profile of dextran-spermine gene delivery vector in mouse lungs.

A nano-sized polymer, dextran-spermine (D-SPM), was shown to have the capacity to deliver gene to the lung of mouse via intranasal route. In this study, assessments on the safety profile of D-SPM were performed to complement the gene expression results. African green monkey kidney fibroblast (COS-7) and human adenocarcinoma breast (MCF-7) cells transfected with D-SPM/pDNA showed massive reduction in the number of viable cells. As for in vivo study, elevated level of neutrophils was observed, despite the minimal level of pro-inflammatory cytokines (TNF-alpha, IL-12, IFN-gamma) detected in the bronchoalveolar lavage fluid (BALF) of mice treated with the D-SPM/pDNA complexes. Histology profile examinations of the lungs showed mild inflammatory responses, with inflamed areas overlap with healthy areas. Although reduction of mice weight was seen at day 1 post administration, the mice did not show any sign of abnormal behavior or physical appearance. Biodistribution study was performed to determine the ability of the D-SPM/pDNA complexes to infiltrate to other non-intended organs. The result showed that the D-SPM/pDNA complexes were only localized at the lung and no gene expression was detected in other organs or blood. In short, these results indicate that the D-SPM/pDNA exhibited mild toxicity in the mouse lungs.

Increased ornithine-derived polyamines cause airway hyperresponsiveness in a mouse model of asthma.

Up-regulation of arginase contributes to airways hyperresponsiveness (AHR) in asthma by reducing L-arginine bioavailability for the nitric oxide (NO) synthase isozymes. The product of arginase activity, L-ornithine, can be metabolized into polyamines by ornithine decarboxylase. We tested the hypothesis that increases in L-ornithine-derived polyamines contribute to AHR in mouse models of allergic airways inflammation. After measuring significantly increased polyamine levels in sputum samples from human subjects with asthma after allergen challenge, we used acute and subacute ovalbumin sensitization and challenge mouse models of allergic airways inflammation and naive mice to investigate the relationship of AHR to methacholine and polyamines in the lung. We found that spermine levels were elevated significantly in lungs from the acute model, which exhibits robust AHR, but not in the subacute murine model of asthma, which does not develop AHR. Intratracheal administration of spermine significantly augmented airways responsiveness to methacholine in both naive mice and mice with subacute airways inflammation, and reduced nitrite/nitrate levels in lung homogenates, suggesting that the AHR developed as a consequence of inhibition of constitutive NO production in the airways. Chronic inhibition of polyamine synthesis using an ornithine decarboxylase inhibitor significantly reduced polyamine levels, restored nitrite/nitrate levels to normal, and abrogated the AHR to methacholine in the acute model of allergic airways inflammation. We demonstrate that spermine increases airways responsiveness to methacholine, likely through inhibition of constitutive NO synthesis. Thus, inhibition of polyamine production may represent a new therapeutic target to treat airway obstruction in allergic asthma.

Modification of secondary head-forming activity of microinjected Ƨ-catenin mRNA by co-injected spermine and spermidine in Xenopus early embryos.

Polyamines are essential for cell growth and differentiation. In Xenopus early embryos, per embryo level of spermine is extremely low as compared with that of spermidine. To disclose the possible function of polyamines in Xenopus early embryos, we tested the effect of co-injection of spermine and spermidine on the functioning of exogenously microinjected in vitro-synthesized, Ƨ-catenin mRNA which is known to induce a secondary head after being microinjected into a ventral vegetal blastomere in 8-celled Xenopus embryos. Microinjection of Ƨ-catenin mRNA in fact induced a secondary axis with a secondary head, and co-injection of spermine or spermidine suppresses induction of the secondary head and secondary axis without drastic effects like induction of immediate cell death or execution of apoptosis at blastula stage. The inhibitory effects were dosage dependent, and at lower doses the inhibition was mainly on secondary head formation rather than on secondary axis formation. We performed similar experiments using GFP mRNA and confirmed that expression of GFP mRNA was also suppressed by co-injection of spermine. We mixed Ƨ-catenin mRNA with different amounts of spermine and performed electrophoresis on agarose gels, with a finding that the prior mixing greatly suppressed mRNA migration. These results suggest that an excess amount of spermine as well as spermidine exerts inhibitory effects on mRNA translation, and that the inhibition may be due to direct binding of polyamines to mRNA and a reduction of negative charges on mRNA molecules that might also induce the formation of cross link-like networks among mRNAs.

Hydrophobically modified dendrons: developing structure-activity relationships for DNA binding and gene transfection.

This paper develops a structure-activity relationship understanding of the way in which surfactant-like dendrons with hydrophilic spermine surface groups and a variety of lipophilic units at their focal points can self-assemble and subsequently bind to DNA with high affinity. The choice of functional group at the focal point of the dendron and the high tunability of the molecular structure have a very significant impact on DNA binding. Mesoscale modeling of the mode of dendron self-assembly provides a direct insight into how the mode of self-assembly exerts its effect on the DNA binding process. In particular, the hydrophobic unit controls the number of dendrons in the self-assembled micellar structures, and hence their diameters and surface charge density. The DNA binding affinity correlates with the surface charge density of the dendron aggregates. Furthermore, these structure-activity effects can also be extended to cellular gene delivery, as surface charge density plays a role in controlling the extent of endosomal escape. It is reported that higher generation dendrons, although binding DNA less strongly than the self-assembling lower generation dendrons, are more effective for transfection. The impact of the lipophilic group at the focal point is less significant for the DNA binding ability of these larger dendrons, which is predominantly controlled by the spermine surface groups, but it does modify the levels of gene transfection. Significant synergistic effects on gene delivery were observed when employing combinations of the dendrons and polyethyleneimine (PEI, 25 kDa), with transfection becoming possible at low loading levels where the two components would not transfect individually, giving practically useful levels of gene delivery.