Effect of diisobutyl adipate on the expression of biomarker genes that respond to endocrine disruption and on gonadal sexual differentiation in Japanese medaka (Oryzias latipes).

Phthalate and non-phthalate plasticizers are used in polymer materials, such as plastic and rubber. It has recently been found that diisobutyl adipate (DIBA), which is considered an environmentally safe non-phthalate plasticizer, potentially acts as a thyroid disruptor in fish. Here, we investigated the sexual hormone effects of DIBA based on the expression levels of genes that respond to endocrine disruption and sexual hormone activity in the livers and gonads, and on gonadal sexual differentiation in Japanese medaka. Compared with the control group, the mRNA expression of chgH, vtg1, vtg2, and esr1 was significantly suppressed in the livers of DIBA exposed XX individuals. Furthermore, the mRNA expression of gsdf was significantly upregulated and downregulated in the gonads of XX and XY individuals, respectively. The mRNA expressions of esr1 and esr2b were significantly suppressed by DIBA exposure in the gonads of both XX and XY individuals. These observations suggest that DIBA has potential androgenic activity in Japanese medaka. However, normal testes and ovaries were observed in respective XY and XX medaka after DIBA exposure; therefore, these results suggest that DIBA may have weak androgenic activity.

Novel functions for 2-phenylbenzimidazole-5-sulphonic acid: Inhibition of ovarian cancer cell responses and tumour angiogenesis.

In this study, we investigated the effects and molecular mechanisms of 2-phenylbenzimidazole-5-sulphonic acid (PBSA), an ultraviolet B protecting agent used in sunscreen lotions and moisturizers, on ovarian cancer cell responses and tumour angiogenesis. PBSA treatment markedly blocked mitogen-induced invasion through down-regulation of matrix metalloproteinase (MMP) expression and activity in ovarian cancer SKOV-3 cells. In addition, PBSA inhibited mitogen-induced cell proliferation by suppression of cyclin-dependent kinases (Cdks), but not cyclins, leading to pRb hypophosphorylation and G1 phase cell cycle arrest. These anti-cancer activities of PBSA in ovarian cancer cell invasion and proliferation were mediated by the inhibition of mitogen-activated protein kinase kinase 3/6-p38 mitogen-activated protein kinase (MKK3/6-p38MAPK ) activity and subsequent down-regulation of MMP-2, MMP-9, Cdk4, Cdk2 and integrin ß1, as evidenced by treatment with p38MAPK inhibitor SB203580. Furthermore, PBSA suppressed the expression and secretion of vascular endothelial growth factor in SKOV-3 cells, leading to inhibition of capillary-like tubular structures in vitro and angiogenic sprouting ex vivo. Taken together, our results demonstrate the pharmacological effects and molecular targets of PBSA on modulating ovarian cancer cell responses and tumour angiogenesis, and suggest further evaluation and development of PBSA as a promising chemotherapeutic agent for the treatment of ovarian cancer.

Mitochondrial oxodicarboxylate carrier deficiency is associated with mitochondrial DNA depletion and spinal muscular atrophy-like disease.

PURPOSE: To understand the role of the mitochondrial oxodicarboxylate carrier (SLC25A21) in the development of spinal muscular atrophy-like disease. METHODS: We identified a novel pathogenic variant in a patient by whole-exome sequencing. The pathogenicity of the mutation was studied by transport assays, computer modeling, followed by targeted metabolic testing and in vitro studies in human fibroblasts and neurons. RESULTS: The patient carries a homozygous pathogenic variant c.695A>G; p.(Lys232Arg) in the SLC25A21 gene, encoding the mitochondrial oxodicarboxylate carrier, and developed spinal muscular atrophy and mitochondrial myopathy. Transport assays show that the mutation renders SLC25A21 dysfunctional and 2-oxoadipate cannot be imported into the mitochondrial matrix. Computer models of central metabolism predicted that impaired transport of oxodicarboxylate disrupts the pathways of lysine and tryptophan degradation, and causes accumulation of 2-oxoadipate, pipecolic acid, and quinolinic acid, which was confirmed in the patient's urine by targeted metabolomics. Exposure to 2-oxoadipate and quinolinic acid decreased the level of mitochondrial complexes in neuronal cells (SH-SY5Y) and induced apoptosis. CONCLUSION: Mitochondrial oxodicarboxylate carrier deficiency leads to mitochondrial dysfunction and the accumulation of oxoadipate and quinolinic acid, which in turn cause toxicity in spinal motor neurons leading to spinal muscular atrophy-like disease.

Molecular control of gene expression by Brucella BaaR, an IclR-type transcriptional repressor.

The general stress response sigma factor σE1 directly and indirectly regulates the transcription of dozens of genes that influence stress survival and host infection in the zoonotic pathogen Brucella abortus Characterizing the functions of σE1-regulated genes therefore would contribute to our understanding of B. abortus physiology and infection biology. σE1 indirectly activates transcription of the IclR family regulator Bab2_0215, but the function of this regulator remains undefined. Here, we present a structural and functional characterization of Bab2_0215, which we have named B rucella adipic acid-activated regulator (BaaR). We found that BaaR adopts a classic IclR-family fold and directly represses the transcription of two operons with predicted roles in carboxylic acid oxidation. BaaR binds two sites on chromosome II between baaR and a divergently transcribed hydratase/dehydrogenase (acaD2), and it represses transcription of both genes. We identified three carboxylic acids (adipic acid, tetradecanedioic acid, and ϵ-aminocaproic acid) and a lactone (ϵ-caprolactone) that enhance transcription from the baaR and acaD2 promoters. However, neither the activating acids nor caprolactone enhanced transcription by binding directly to BaaR. Induction of baaR transcription by adipic acid required the gene bab2_0213, which encodes a major facilitator superfamily transporter, suggesting that Bab2_0213 transports adipic acid across the inner membrane. We conclude that a suite of structurally related organic molecules activate transcription of genes repressed by BaaR. Our study provides molecular-level understanding of a gene expression program in B. abortus that is downstream of σE1.

Structural basis for high-affinity adipate binding to AdpC (RPA4515), an orphan periplasmic-binding protein from the tripartite tricarboxylate transporter (TTT) family in Rhodopseudomonas palustris.

The tripartite tricarboxylate transporter (TTT) family is a poorly characterised group of prokaryotic secondary solute transport systems, which employ a periplasmic substrate-binding protein (SBP) for initial ligand recognition. The substrates of only a small number of TTT systems are known and very few SBP structures have been solved, so the mechanisms of SBP-ligand interactions in this family are not well understood. The SBP RPA4515 (AdpC) from Rhodopseudomonas palustris was found by differential scanning fluorescence and isothermal titration calorimetry to bind aliphatic dicarboxylates of a chain length of six to nine carbons, with KD values in the µm range. The highest affinity was found for the C6-dicarboxylate adipate (1,6-hexanedioate). Crystal structures of AdpC, either adipate or 2-oxoadipate bound, revealed a lack of positively charged amino acids in the binding pocket and showed that water molecules are involved in bridging hydrogen bonds to the substrate, a conserved feature in the TTT SBP family that is distinct from other types of SBP. In AdpC, both of the ligand carboxylate groups and a linear chain conformation are needed for coordination in the binding pocket. RT-PCR showed that adpC expression is upregulated by low environmental adipate concentrations, suggesting adipate is a physiologically relevant substrate but as adpC is not genetically linked to any TTT membrane transport genes, the role of AdpC may be in signalling rather than transport. Our data expand the known ligands for TTT systems and identify a novel high-affinity binding protein for adipate, an important industrial chemical intermediate and food additive. DATABASES: Protein structure co-ordinates are available in the PDB under the accession numbers 5OEI and 5OKU.

α-Ketoadipic Acid and α-Aminoadipic Acid Cause Disturbance of Glutamatergic Neurotransmission and Induction of Oxidative Stress In Vitro in Brain of Adolescent Rats.

Tissue accumulation of α-ketoadipic (KAA) and α-aminoadipic (AAA) acids is the biochemical hallmark of α-ketoadipic aciduria. This inborn error of metabolism is currently considered a biochemical phenotype with uncertain clinical significance. Considering that KAA and AAA are structurally similar to α-ketoglutarate and glutamate, respectively, we investigated the in vitro effects of these compounds on glutamatergic neurotransmission in the brain of adolescent rats. Bioenergetics and redox homeostasis were also investigated because they represent fundamental systems for brain development and functioning. We first observed that AAA significantly decreased glutamate uptake, whereas glutamate dehydrogenase activity was markedly inhibited by KAA in a competitive fashion. In addition, AAA and more markedly KAA induced generation of reactive oxygen and nitrogen species (increase of 2',7'-dichloroflurescein (DCFH) oxidation and nitrite/nitrate levels), lipid peroxidation (increase of malondialdehyde concentrations), and protein oxidation (increase of carbonyl formation and decrease of sulfhydryl content), besides decreasing the antioxidant defenses (reduced glutathione (GSH)) and aconitase activity. Furthermore, KAA-induced lipid peroxidation and GSH decrease were prevented by the antioxidants α-tocopherol, melatonin, and resveratrol, suggesting the involvement of reactive species in these effects. Noteworthy, the classical inhibitor of NMDA glutamate receptors MK-801 was not able to prevent KAA-induced and AAA-induced oxidative stress, determined by DCFH oxidation and GSH levels, making unlikely a secondary induction of oxidative stress through overstimulation of glutamate receptors. In contrast, KAA and AAA did not significantly change brain bioenergetic parameters. We speculate that disturbance of glutamatergic neurotransmission and redox homeostasis by KAA and AAA may play a role in those cases of α-ketoadipic aciduria that display neurological symptoms.

Adipic acid tolerance screening for potential adipic acid production hosts.

BACKGROUND: Biobased processes for the production of adipic acid are of great interest to replace the current environmentally detrimental petrochemical production route. No efficient natural producer of adipic acid has yet been identified, but several approaches for pathway engineering have been established. Research has demonstrated that the microbial production of adipic acid is possible, but the yields and titres achieved so far are inadequate for commercialisation. A plausible explanation may be intolerance to adipic acid. Therefore, in this study, selected microorganisms, including yeasts, filamentous fungi and bacteria, typically used in microbial cell factories were considered to evaluate their tolerance to adipic acid. RESULTS: Screening of yeasts and bacteria for tolerance to adipic acid was performed in microtitre plates, and in agar plates for A. niger in the presence of adipic acid over a broad range of concentration (0-684 mM). As the different dissociation state(s) of adipic acid may influence cells differently, cultivations were performed with at least two pH values. Yeasts and A. niger were found to tolerate substantially higher concentrations of adipic acid than bacteria, and were less affected by the undissociated form of adipic acid than bacteria. The yeast exhibiting the highest tolerance to adipic acid was Candida viswanathii, showing a reduction in maximum specific growth rate of no more than 10-15% at the highest concentration of adipic acid tested and the tolerance was not dependent on the dissociation state of the adipic acid. CONCLUSIONS: Tolerance to adipic acid was found to be substantially higher among yeasts and A. niger than bacteria. The explanation of the differences in adipic acid tolerance between the microorganisms investigated are likely related to fundamental differences in their physiology and metabolism. Among the yeasts investigated, C. viswanathii showed the highest tolerance and could be a potential host for a future microbial cell factory for adipic acid.

Spinal astrocytic activation contributes to both induction and maintenance of pituitary adenylate cyclase-activating polypeptide type 1 receptor-induced long-lasting mechanical allodynia in mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP-PACAP receptors signaling system in the modulation of spinal nociceptive transmission. We have previously reported that a single intrathecal injection of PACAP or a PACAP specific (PAC1) receptor selective agonist, maxadilan, in mice induced dose-dependent aversive behaviors, which lasted more than 30 min, and suggested that the maintenance of the nociceptive behaviors was associated with the spinal astrocytic activation. RESULTS: We found that a single intrathecal administration of PACAP or maxadilan also produced long-lasting hind paw mechanical allodynia, which persisted at least 84 days without affecting thermal nociceptive threshold. In contrast, intrathecal application of vasoactive intestinal polypeptide did not change mechanical threshold, and substance P, calcitonin gene-related peptide, or N-methyl-D-aspartate induced only transient mechanical allodynia, which disappeared within 21 days. Western blot and immunohistochemical analyses with an astrocytic marker, glial fibrillary acidic protein, revealed that the spinal PAC1 receptor stimulation caused sustained astrocytic activation, which also lasted more than 84 days. Intrathecal co-administration of L-α-aminoadipate, an astroglial toxin, with PACAP or maxadilan almost completely prevented the induction of the mechanical allodynia. Furthermore, intrathecal treatment of L-α-aminoadipate at 84 days after the PAC1 stimulation transiently reversed the mechanical allodynia accompanied by the reduction of glial fibrillary acidic protein expression level. CONCLUSION: Our data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia.

Male Pheromones Influence the Mating Behavior of Echinothrips americanus.

Two dibasic esters, the dimethyl ester of hexanedioic acid (dimethyl adipate, DBE-6) and the dimethyl ester of pentanedioic acid (dimethyl glutarate, DBE-5) were found in head-thorax extracts of male Echinothrips americanus. DBE-5 induced abdomen wagging and raising in males and females, which is typically exhibited when encountering a male. DBE-6 was avoided by males and was detected on mated, but not on virgin, females. Both substances applied to virgin females lead to females being ignored by males. The role of both substances is discussed with regard to the male mating system.

Adjuvant Effect of an Alternative Plasticizer, Diisopropyl Adipate, on a Contact Hypersensitivity Mouse Model: Link with Sensory Ion Channel TRPA1 Activation.

Due to health concerns about phthalate esters, the use of alternative plasticizers is being considered. Phthalate esters enhance skin sensitization to fluorescein isothiocyanate (FITC) in mouse models. We have demonstrated that phthalate esters stimulate transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. We also found a correlation between TRPA1 activation and the enhancing effect on FITC-induced contact hypersensitivity (CHS) when testing various types of phthalate esters. Here we investigated the effects of an alternative plasticizer, diisopropyl adipate (DIA). Activation of TRPA1 by DIA was demonstrated by calcium mobilization using Chinese hamster ovary cells expressing TRPA1 in vitro. The effect of DIA was inhibited by a TRPA1-specific antagonist, HC-030031. The presence of DIA or dibutyl phthalate (DBP; positive control) during skin sensitization of BALB/c mice to FITC augmented the CHS response, as revealed by the level of ear-swelling. The enhancing effect of DIA was inhibited by in vivo pretreatment with HC-030031. FITC-presenting CD11c(+) dendritic cell (DC)-trafficking to draining lymph nodes was facilitated both by DIA and by DBP. DBP and DIA were similarly active in the enhancement of interferon-γ production by draining lymph nodes, but the effect on interleukin-4 production was weaker with DIA. Overall, DIA activated TRPA1 and enhanced FITC-induced CHS, as DBP did. The adjuvant effects of adipate esters may need to be considered because they are used as ingredients in cosmetics and drug formulations topically applied to the skin.

Involvement of trigeminal astrocyte activation in masseter hyperalgesia under stress.

It is commonly accepted that psychological stress contributes to the development of temporomandibular joint disorders, in which chronic orofacial pain is the main symptom. However, the central mechanism underlying the development of these disorders has remained unclear. The current study was performed to determine the involvement of the glia in the trigeminal spinal subnucleus caudalis in stress-induced increases in masseter muscle hyperalgesia in rats. After being subjected to chronic restraint stress, the animals showed decreased body weight gain, behavioral changes and marked masseter allodynia. We also found that astrocytes, but not microglia, in the trigeminal subnucleus caudalis (Vc) were dramatically activated. A further analysis was undertaken to investigate the contribution of the glia; we intrathecally injected l-α-aminoadipate (astrocyte-specific inhibitor) and/or minocycline (microglia-specific inhibitor) into the stressed rats. Our results showed that l-α-aminoadipate (LAA), but not minocycline, could significantly attenuate the mechanical masseter allodynia and behavioral changes induced by restraint stress. In addition, the expression of interleukin-1ß (IL-1ß) and phosphorylated N-methyl-d-aspartic acid receptor 1 (p-NR1) in the Vc was significantly increased after chronic restraint stress, whereas LAA dramatically inhibited the overexpression of IL-1ß and p-NR1. Taken together, these results suggest that activated astrocytes in the Vc may be one of the most important factors in the pathophysiology of masseter hyperalgesia induced by restraint stress and the following overexpression of IL-1ß and excessive NMDAR phosphorylation may ultimately contribute to masseter hyperalgesia. Thus, inhibiting spinal astrocytic activation may represent a novel therapeutic strategy for the treatment of orofacial pain induced by stress.

Influence on the physicochemical properties of fish collagen gels using self-assembly and simultaneous cross-linking with the N-hydroxysuccinimide adipic acid derivative.

Collagen gels from Southern catfish (Silurus meridionalis Chen) skins were prepared via the self-assembly of collagen molecules and simultaneous cross-linking with the N-hydroxysuccinimide adipic acid derivative (NHS-AA). The doses of NHS-AA were converted to [NHS-AA]/[NH2] ratios (0.025-1.6, calculated by the [active ester group] of NHS-AA and [ε-NH2] of lysine and hydroxylysine residues of collagen). When the ratio < 0.05, collagen gels were formed by collagen molecule self-assembly, resulting in the opalescent appearance of collagen gels and the characteristic D-periodicity of partial collagen fibrils, the collagen gel ([NHS-AA]/[NH2] = 0.05) displayed a small increase in denaturation temperature (Td, 42.8 °C), remaining weight (12.59%), specific water content (SWC 233.7) and elastic modulus (G' 128.4 Pa) compared with uncross-linked collagen gel (39.1 °C, 9.12%, 222.4 and 85.4 Pa, respectively). As the ratio > 0.05, disappearance of D-periodicity and a gradual change in appearance from opalescent to transparent suggested that the inhibition of NHS-AA in the self-assembly of collagen molecules was more obvious. As a result, the collagen gel ([NHS-AA]/[NH2] = 0.2) had the lowest Td (35.8 °C), remaining weight (7.96%), SWC (130.9) and G' (31.9 Pa). When the ratio was 1.6, the collagen molecule self-assembly was markedly suppressed and the formation of collagen gel was predominantly via the covalent cross-linking bonds which led to the transparent appearance, and the maximum values of Td (47.0 °C), remaining weight (45.92%) and G' (420.7 Pa) of collagen gel. These results indicated that collagen gels with different properties can be prepared using different NHS-AA doses.

Diverse influences of androgen-disrupting chemicals on immune responses mounted by macrophages.

Androgen-disrupting chemicals (ADCs) can alter male sexual development. Although the effects of ADCs on hormone disruption have been studied, their influence on the immune response is not fully understood. To investigate the effects of ADCs on innate immunity, we tested eight candidate ADCs for their influence on macrophages by measuring nitric oxide (NO) production and cell viability. Our results showed that treatment with a mixture of lipopolysaccharide and hexachlorobenzene increased NO production in RAW 264.7 cells, a murine macrophage cell line. In contrast, compared to exposure to a negative control, exposure to di-2-ethylhexyl adipate (DEHA), benzylbutyl phthalate (BBP), testosterone (TTT), or permethrin decreased NO production. DEHA, BBP, and TTT inhibited NO production in an inducible nitric oxide synthase-dependent manner. Treatment with bisphenol A (BPA), nonylphenol (NNP), or tributyltin chloride (TBTC) reduced NO production and induced cell death. While BPA induced RAW 264.7 cell death through apoptosis, NNP and TBTC caused cell death through necrosis. These results offer insights into the influences of ADCs on the innate immune system.

Degradation of polyester polyurethane by a newly isolated soil bacterium, Bacillus subtilis strain MZA-75.

A polyurethane (PU) degrading bacterial strain MZA-75 was isolated from soil through enrichment technique. The bacterium was identified through 16S rRNA gene sequencing, the phylogenetic analysis indicated the strain MZA-75 belonged to genus Bacillus having maximum similarity with Bacillus subtilis strain JBE0016. The degradation of PU films by strain MZA-75 in mineral salt medium (MSM) was analyzed by scanning electron microscopy (SEM), fourier transform infra-red spectroscopy (FT-IR) and gel permeation chromatography (GPC). SEM revealed the appearance of widespread cracks on the surface. FTIR spectrum showed decrease in ester functional group. Increase in polydispersity index was observed in GPC, which indicates chain scission as a result of microbial treatment. CO2 evolution and cell growth increased when PU was used as carbon source in MSM in Sturm test. Increase in both cell associated and extracellular esterases was observed in the presence of PU indicated by p-Nitrophenyl acetate (pNPA) hydrolysis assay. Analysis of cell free supernatant by gas chromatography-mass spectrometry (GC-MS) revealed that 1,4-butanediol and adipic acid monomers were produced. Bacillus subtilis strain MZA-75 can degrade the soft segment of polyester polyurethane, unfortunately no information about the fate of hard segment could be obtained. Growth of strain MZA-75 in the presence of these metabolites indicated mineralization of ester hydrolysis products into CO2 and H2O.

Novel molecular anti-colorectalcancer conjugate:chlorambucil-adipic acid dihydrizide-glutamine.

Cancer is one of the most fatal diseases in the world and it has been years that finding new drugs and chemotherapeutic techniques with lowest side effects become one of the most important challenging matters needs really hard efforts. Chlorambucil (CBL), an ancient direct-acting alkylating anticancer agent, is commonly used for initial treatment of some kinds of cancers but the use of CBL is often limited because of the unpleasant side effects due to its lack of specificity for targeting cancer cells. In this research we tried to increase the specificity of CBL by producing a novel conjugate by using glutamine amino acid (Glut). Based on previous studies, poly amines and nitrogen compounds noticeably are used by cancer cells increasingly; therefore we decided to increase the efficiency and specificity of CBL by designing and producing a novel anti cancer conjugate using glutamine amino acid as an uptake enhancer, CBL, and Adipic acid Dihydrazide (ADH) as a spacer and linker. The biological tests were carried out on HT29 colorectal cancer cell line to evaluate its anticancer properties. Biological tests like MTT assay, finding IC50, evaluating the induced mechanism of the death of our novel CBL-Glutamine conjugate on HT29 cells, testing abnormal toxicity of this conjugate on mice in comparison with CBL drug were careid out. We found that not only CBL-Glutamine conjugate preserved its anti cancer property with regard to CBL drug, but also it represent lower abnormal toxicity in mice. Apoptosis was detected as its mechanism of the death. Our present study provides a promising strategy for targeting cancer cells using amino acids nano-conjugate drugs. The future perspectives have also been highlighted in continuing similar and relative researches.

Effect of dietary adipic acid and corn dried distillers grains with solubles on laying hen performance and nitrogen loss from stored excreta with or without sodium bisulfate.

Effects of dietary adipic acid (0 vs. 1%) and corn dried distillers grains with solubles (DDGS; 0 vs. 20%) were evaluated on hen performance and egg characteristics from 26 to 34 wk of age. Four isocaloric and isonitrogenous diets were randomly assigned to blocks of 6 consecutive cages (36 cages per diet; 2 hens per cage). On wk 2 and 7 of the experiment, excreta were collected by cage block, mixed, and equally split into 2 containers. Sodium bisulfate (SBS) was spread (8.8 kg/100 m(2)) on the top surface of half of the containers. All containers were stored uncovered for 14 d at room temperature. Excreta pH, DM, and N content were measured on d 0, 7, and 14 of storage. Feed intake (112 g/d per hen), egg production (96.1%), and egg specific gravity (1.079 g/g) were not affected by diet. On excreta collection day, a synergy (P = 0.014) between dietary adipic acid and DDGS was detected, as the lowest excreta pH was obtained with the diet including both adipic acid and DDGS. On d 7 of storage, excreta pH was still reduced by dietary adipic acid (P = 0.046) and DDGS (P < 0.001), but a week later, only dietary DDGS decreased excreta pH (8.91 vs. 9.21; P < 0.001). Whereas dietary adipic acid had no influence on excreta N loss, excreta from hens fed 20% DDGS lost 19.7% more N (P = 0.039) during storage than hens not eating DDGS. Surface amendment of excreta with SBS increased excreta DM content, with the effect being even more marked on d 14 of storage (increase of 6.7 percentage units; P < 0.001), consistently decreased excreta pH during storage (P < 0.001) and reduced N loss by 26.1% for the 14 d of storage period.

Synthesis of biocompatible poly(ɛ-caprolactone)- block-poly(propylene adipate) copolymers appropriate for drug nanoencapsulation in the form of core-shell nanoparticles.

Poly(propylene adipate)-block-poly(ɛ-caprolactone) copolymers were synthesized using a combination of polycondensation and ring-opening polymerization of ɛ-caprolactone in the presence of poly(propylene adipate). Gel permeation chromatography was used for molecular weight determination, whereas hydrogen-1 nuclear magnetic resonance and carbon-13 nuclear magnetic resonance spectroscopy were employed for copolymer characterization and composition evaluation. The copolymers were found to be block while their composition was similar to the feeding ratio. They formed semicrystalline structures, while only poly(ɛ-caprolactone) formed crystals, as shown by wide angle X-ray diffraction. Differential scanning calorimetry data suggest that the melting point and heat of fusion of copolymers decreased by increasing the poly(propylene adipate) amount. The synthesized polymers exhibited low cytotoxicity and were used to encapsulate desferrioxamine, an iron-chelating drug. The desferrioxamine nanoparticles were self-assembled into core shell structures, had mean particle size <250 nm, and the drug remained in crystalline form. Further studies revealed that the dissolution rate was mainly related to the melting temperature, as well as to the degree of crystallinity of copolymers.

Effects of ethylenediamine--a putative GABA-releasing agent--on rat hippocampal slices and neocortical activity in vivo.

The simple diamine diaminoethane (ethylenediamine, EDA) has been shown to activate GABA receptors in the central and peripheral nervous systems, partly by a direct action and partly by releasing endogenous GABA. These effects have been shown to be produced by the complexation of EDA with bicarbonate to form a carbamate. The present work has compared EDA, GABA and ß-alanine responses in rat CA1 neurons using extracellular and intracellular recordings, as well as neocortical evoked potentials in vivo. Superfusion of GABA onto hippocampal slices produced depolarisation and a decrease of field epsps, both effects fading rapidly, but showing sensitivity to blockade by bicuculline. EDA produced an initial hyperpolarisation and increase of extracellular field epsp size with no fade and only partial sensitivity to bicuculline, with subsequent depolarisation, while ß-alanine produces a much larger underlying hyperpolarisation and increase in fepsps, followed by depolarisation and inhibition of fepsps. The responses to ß-alanine, but not GABA or EDA, were blocked by strychnine. In vivo experiments, recording somatosensory evoked potentials, confirmed that EDA produced an initial increase followed by depression, and that this effect was not fully blocked by bicuculline. Overall the results indicate that EDA has actions in addition to the activation of GABA receptors. These actions are not attributable to activation of ß-alanine-sensitive glycine receptors, but may involve the activation of sites sensitive to adipic acid, which is structurally equivalent to the dicarbamate of EDA. The results emphasise the complex pharmacology of simple amines in bicarbonate-containing solutions.

Injectable oxidized hyaluronic acid/adipic acid dihydrazide hydrogel for nucleus pulposus regeneration.

Injectable hydrogel allows irregular surgical defects to be completely filled, lessens the risk of implant migration, and minimizes surgical defects due to the solution-gel state transformation. Here, we first propose a method for preparing oxidized hyaluronic acid/adipic acid dihydrazide (oxi-HA/ADH) injectable hydrogel by chemical cross-linking under physiological conditions. Fourier transform infrared spectrometry and trinitrobenzene sulfonate assay were used to confirm the oxidation of hyaluronic acid. Rheological properties were measured to evaluate the working ability of the hydrogel for further clinical application. The oxi-HA/ADH in situ forming hydrogel can transform from liquid form into a gel-like matrix within 3-8 min, depending on the operational temperature. Furthermore, hydrogel degradation and cell assessment is also a concern for clinical application. Injectable oxi-HA/ADH8 hydrogel can maintain its gel-like state for at least 5 weeks with a degradation percentage of 40%. Importantly, oxi-HA/ADH8 hydrogel can assist in nucleus pulposus cell synthesis of type II collagen and aggrecan mRNA gene expression according to the results of real-time PCR analysis, and shows good biocompatibility based on cell viability and cytotoxicity assays. Based on the results of the current study, oxi-HA/ADH hydrogel may possess several advantages for future application in nucleus pulposus regeneration.