Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid

Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells.     

Positive hyaluronan/PEI/DNA complexes as a target-specific intracellular delivery to malignant breast cancer

In the present study, The PEI/DNA (PD) complexes was first prepared with positive surface charge under a suitable N/P ratio of 10. The redundant positive charge was partially and excessively shielded by a polysaccharide, hyaluronic acid (HA), in aqueous solution. The HA/PEI/DNA ternary complexes were characterized by assessing the zeta potential and size, then transferred to MDA-MB-435, MDA-MB-231, and MCF-7 cell lines with different amounts of HA-specific CD44 receptors on the surface. Consequently, The transfection efficiency of all the prepared complexes show a little increased to MCF-7 (low CD44 level) while a large increased to MDA-MB-231 and MDA-MB-435 cells (high CD44 level) with adding HA. Also, when HA:PEI charge ratio was 7.5%, the ternary complexes show the highest transfection efficiency. The prepared ternary complexes exhibited increased 2–13-fold fluorescence intensity and lower cell toxicity compared to the PD (N/P, 10). These results indicated that the positive HA/PEI/DNA ternary complexes (HA:PEI charge ratio, 7.5%) can target malignant breast cancer cells with high CD44 level and might be a promising candidate vector for gene therapy.     

Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid

Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells.     

Ternary Complexes with Core-Shell Bilayer for Double Level Targeted Gene Delivery: In Vitro and In Vivo Evaluation

Purpose

Hyaluronic acid (HA)/polyethyleneimine-dexamethasone (PEI-Dex)/DNA ternary complexes with “core-shell” bilayer were developed for double level targeted gene delivery. A PEI₁₈₀₀-Dex, as a core, was applied to compact DNA into a nano-sized structure and facilitate the nuclear translocation of DNA after endocytosis into tumor cells, and a polyanion HA, as the outer corona, was employed to improve targeted tumor delivery and reduce cytotoxicity.

Methods

PEI-Dex was synthesized and characterized by ¹H NMR. The characterizations of ternary complexes were investigated. Their biological properties, including transfection efficiency, cytotoxicity, cellular uptake and in vivo efficacy were evaluated systemically.

Results

Ternary complexes with the size of about 160 nm exhibited the lowest cytotoxicity and the highest transfection efficiency in B16F10 cells among investigated complexes. The sub-cellular localization study confirmed that ternary complexes could facilitate more efficient cell uptake and nuclear transport of DNA than binary complexes. Moreover, Cy7-labeled ternary complexes obviously accumulated in the tumor after i.v. administration, indicating that ternary complexes could assist the DNA targeting to the tumor. In in vivo studies, HA/PEI₁₈₀₀-Dex/DNA ternary complexes showed confirmed anti-inflammation activity, and could significantly suppress tumor growth of tumor-bearing nude mice.

Conclusions

HA/PEI-Dex/DNA ternary complexes might be a promising targeted gene delivery system.     

Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis

A new PLA₂ (Cdcum6) from crotoxin complex of Colombian Crotalus durissus cumanensis rattlesnake was purified using molecular exclusion chromatography and RP-HPLC. The molecular mass of Cdcum6 was determined by SDS-PAGE ∼14 KDa and confirmed by MALDI-TOF (14321.98 Da). The enzyme showed K_m 6.0 mM, V_max 3.44 nmol/min, optimum pH was 8.0 and temperature was between 30 and 45 °C, and it had a strict requirement of Ca²⁺ for its activity. The N-terminal sequence of PLA₂ was SLVQF EKMIK EVAGK NGVPWY. Comparison of amino acid sequence data with other PLA₂ from South American Crotalus durissus rattlesnakes showed that Cdcum6 shares the highest sequence identity with Cdr13 an isoform PLA₂ from Crotalus durissus ruruima, nevertheless, Cdcum6 showed high content of basic and hydrophobic amino acids. In mice, Cdcum6 presented higher LD₅₀ than crotoxin complex from C. d. cumanensis. Additionally, Cdcum6 induced a conspicuous local myotoxic effect and moderate footpad edema; in vitro, it was antigoagulant in doses as low as 0.5 μg/ml, and it was not cytotoxic on myoblast but Cdcum6 was able to lyse myotubes.     

Comparative analysis of the antioxidant and DNA protection capacities of Anadenanthera colubrina, Libidibia ferrea and Pityrocarpa moniliformis fruits

This study aimed to explore the antioxidant and DNA protection abilities of hydroalcoholic extracts from fruits of Anadenanthera colubrina (ACHE), Libidibia ferrea (LFHE) and Pityrocarpa moniliformis (PMHE). These extracts were tested by five antioxidant methods (phosphomolibdenium and reducing power assays; superoxide, hydrogen peroxide and nitric oxide scavenging) and DNA protection capacity. Total phenolic content was measured by Folin-Ciocalteu method. ACHE exhibited the highest phenolic content (578 mg/g GAE), followed by LFHE (460 mg/g GAE) and PMHE (448 mg/g GAE). In phosphomolibdenium assay, ACHE showed 24.81% of activity in relation to ascorbic acid, whereas LFHE and PMHE had 21.08% and 18.05%, respectively. These plants showed high ability to inhibit reactive species tested with IC50 values ranged from 10.66 to 14.37 μg/mL for superoxide radical; 26.05 to 45.43 μg/mL for hydrogen peroxide; 178.42 to 182.98 μg/mL for reducing power; and 199.2 to 283 μg/mL for nitric oxide. Furthermore, these extracts had capacity to break the DNA damage induced by hydroxyl radicals. The antioxidant activity of these plants is related with their higher phenolic content and show that they may be used as source of bioactive compounds, relevant to the maintenance of oxidative stability of the food matrix, cosmetics and/or pharmaceutical preparations.     

Effect of multifold charge groups and imidazole-4-carboxaldehyde on physicochemical characteristics and transfection of cationic polyphosphazenes/DNA complexes

To understand the dual influence of multifold charge groups and conjugation of imidazole moiety on the physicochemical characteristics and the transfection activity of polymer complexes, a series of cationic polyphosphazenes based on poly(2-(2-aminoethyoxy)ethoxy) phosphazene (PAEP) with different components of multifold charge groups was synthesized by means of introducing imidazole-4-carboxaldehyde (IC) into PAEP through the formation of Schiff base. Though the polymers with primary amino groups (1°) alone or with abundant primary amino groups could bind DNA more efficiently than the ones with mainly or totally secondary (2°) and tertiary (3°) amino groups, all of the polymers could condense DNA into small particles within 100 nm at the N/P ratio of 24. The cell viability of complexes and the pH buffering capacity of polymers increased with substitution degree of IC increasing. Among all the PAEP-based polymers, the highest transfection activity was found for poly(2-(2-aminoethyoxy)ethoxy/IC)phosphazene (PAEIC) 18 complexes containing 1°, 2° and 3° amines at a ratio of 3.5:1:1 with 18% substitution degree of IC, which indicated that either the coexistence of 1°, 2° and 3° amines or the conjugation of imidazole moiety played an important role in transfection activity. These results suggested that the most efficient gene carrier could be these polymers with 1°, 2° and 3° amines at an appropriate ratio, together with the presence of imidazole moiety in a small fraction.     

Genetic variants in SMARC genes are associated with DNA damage levels in Chinese population

The switching defective/sucrose nonfermenting (SWI/SNF) related, matrix associated, actin dependent regulators of chromatin (SMARC) are components of human SWI/SNF like chromatin remodeling protein complexes, which are essential in the process of DNA damage repair. In this study, we hypothesized that genetic variants in SMARC genes may modify the capacity of DNA repair to damage. To test this hypothesis, we genotyped a total of 20 polymorphisms in five key SMARC genes (SMARCA5, SMARCC2, SMARCD1, SMARCD2, SMARCD3) to evaluate their associations with DNA damage levels in 307 subjects. The DNA damage levels were measured with comet assay. The multiple linear regression was used to assess the relationship between each polymorphism and DNA damage levels in additive model. We found that the genotypes of rs6857360 (β = 0.23, 95% CI = 0.06–0.40, P = 0.008) in SMARCA5, rs6919 (β = 0.20, 95% CI = 0.05–0.34, P = 0.008) and rs2727280 (β = 0.18, 95% CI = 0.04–0.33, P = 0.013) in SMARCD2, and rs17173769 (β = −0.27, 95% CI = −0.52 to −0.01, P = 0.045) in SMARCD3 were significantly associated with DNA damage levels. After combining these four polymorphisms, we found that the more unfavorable alleles the subjects carried, the heavier DNA damage they suffered, suggesting a locus-dosage effect between combined genotypes and DNA damage levels (P for trend = 0.006). These findings suggest that genetic variants in SMARC genes may contribute the individual variations of DNA damage levels in Chinese population. Further larger and functional studies are warranted to confirm our findings.     

Comparison of ibuprofen release from minitablets and capsules containing ibuprofen: β-Cyclodextrin complex

Mixtures containing ibuprofen (IB) complexed with β-cyclodextrin (βCD) obtained by two complexation methods [suspension/solution (with water removed by air stream, spray- and freeze-drying) and kneading technique] were processed into pharmaceutical dosage forms (minitablets and capsules). Powders (IB, βCD and IBβCD) were characterized for moisture content, densities (true and bulk), angle of repose and Carr’s index, X-ray and NMR. From physical mixtures and IBβCD complexes without other excipients were prepared 2.5-mm-diameter minitablets and capsules. Minitablets were characterized for the energy of compaction, tensile strength, friability, density and IB release (at pH 1.0 and 7.2), whereby capsules were characterized for IB release. The results from the release of IB were analyzed using different parameters, namely, the similarity factor (f₂), the dissolution efficiency (DE) and the amounts released at a certain time (30, 60 and 180 min) and compared statistically (α = 0.05). The release of IB from the minitablets showed no dependency on the amount of water used in the formation of the complexes. Differences were due to the compaction force used or the presence of a shell for the capsules. The differences observed were mostly due to the characteristics of the particles (dependent on the method considered on the formation of the complexes) and neither to the dosage form nor to the complex of the IB.     

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and l-amino acid oxidase

Some proteins present in snake venom possess enzymatic activities, such as phospholipase A₂ and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA₂ (BmarPLA₂) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA₂ was isolated from the venom, and N-terminal amino acid sequencing of sPLA₂ showed high amino acid identity with other lysine K49 sPLA₂s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC = 50 μg/mL and MLC = 200 μg/mL. However, the BmarTV and BmarPLA₂ did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC₅₀ = 2.55 μg/mL and 2.86 μg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC₅₀ of 86.56 μg/mL for L. amazonensis and 79.02 μg/mL for L. chagasi. BmarPLA₂ did not promote any inhibition of the growth of these parasites. The BmarLAAO and BmarTV presented low toxicity at the concentrations studied. In conclusion, whole venom as well as the l-amino acid oxidase from Bothrops marajoensis was able to inhibit the growth of several microorganisms, including S. aureus, Candida albicans, Pseudomonas aeruginosa, and Leishmania sp.     

The hOGG1 Ser326Cys polymorphism and Huntington's disease

Increasing evidence supports a role for oxidative DNA damage and impaired DNA repair mechanisms in the pathogenesis of age related neurodegenerative diseases. Within this context there is a current interest in the understanding of the role played by polymorphisms of DNA repair genes in the inter-individual risk to develop neurodegenerative pathologies, as well as in the onset and the progression of disease symptoms. Particularly, somatic CAG repeat expansion of the gene encoding for huntingtin has been observed in tissues of patients affected by Huntington's disease (HD), including blood and brain. Recent evidence suggests that somatic CAG repeat expansion in HD cells might contribute to disease age at onset and is mediated by the DNA repair OGG1 enzyme, during the removal of 8-oxoguanine (8-oxoG) from the DNA. There is also evidence that the expression of hMTH1, which removes 8-oxoG from the nucleotide pool, protects mice from HD-like symptoms, and progenitor striatal cells from the toxic effects of the mutant huntingtin. The hOGG1 Ser326Cys polymorphism results in reduced OGG1 activity and increased 8-oxoG formation. In the present study, performed on blood DNA from 91 HD subjects, we observed that bearers of the mutant Cys326 allele (Ser326Cys + Cys326Cys) tend to have an increased number of CAG repeats of the expanded HD allele (P = 0.049); moreover bearers of at least one copy of the mutant Cys326 allele, mainly heterozygous subjects, showed a significant (P = 0.041) earlier disease onset than Ser326Ser wild-type individuals, suggesting a possible role of the hOGG1 Ser326Cys polymorphism in HD phenotype.     

Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H2O2-induced DNA damage in cultured human leukocytes

Essential oil (EO) of Alpinia zerumbet leaves, at non-toxic concentrations (50–300 μg/mL), did not induce genotoxicity in human leukocytes. However, at the highest concentration (500 μg/mL) tested caused a reduction in cell proliferation and viability, and an increase in DNA damage. Moreover, in vivo experiments showed that EO (400 mg/kg) did not exert mutagenicity on peripheral blood cells and bone marrow in mice. In DPPH test, EO showed scavenging effects against DPPH radicals, and other free radicals (determination of intracellular GSH and lipid peroxidation assays). Furthermore, EO was able to reduce the intracellular levels of ROS, and prevented leukocytes DNA against oxidative damage. The ability of EO to reduce H₂O₂ toxicity was observed only when cells were treated with EO during and after exposure to H₂O₂. With the co- and post-treatment procedures, EO decreased the frequency of apoptotic and micronucleated leukocytes as well DNA strand breaks. However, a synergistic effect was observed in cultures exposed to 500 μg/mL EO. In conclusion, EO at concentrations up to 300 μg/mL or doses up to 400 mg/kg are not mutagenic in leukocytes and in mice, but do have antioxidative and protective effects against the cytotoxicity and clastogenesis induced by H₂O₂.     

Diverse role of microbially bioconverted product of cabbage (Brassicaoleracea) by Pseudomonassyringe pv. T1 on inhibiting Candida species

This study was carried out to evaluate the anticandidal effects of bioconverted product, obtained from the microbial conversion of cabbage (Brassicaoleracea) by a bacterial strain Pseudomonassyringe pv. T1 (Ps-T1) against various isolates of Candida species. The diameters of zones of inhibition of bioconverted product of cabbage (10 μl, corresponding to 500 μg/disc) against Candidaalbicans KACC 30003 and 30062, Candidageochares KACC 30061, Candidasaitoana KACC 41238 and Candidaglabrata P00368 were found between 10 ± 1 and 16 ± 0.8 mm. The bioconverted product was tested for the minimum inhibitory and minimum fungicidal concentration values against the tested pathogens which were found in the range of 125-500 and 125-500 μg/ml, respectively. On the viable counts of the tested fungal pathogens, the bioconverted product showed a remarkable anticandidal effect. Also the study of using scanning electron microscopy on the morphology of C.albicans KACC 30062 revealed potential detrimental effect of bioconverted product at MIC concentration. The results of this study suggest that bioconverted product of cabbage by Ps-T1 holds potential therapeutic value and medicinal significance to control Candida species.     

In vitro antioxidant and in vivo anti-inflammatory potential of crude polysaccharide from Turbinaria ornata (Marine Brown Alga)

Water-soluble crude polysaccharide from a brown alga Turbinaria ornata (TCP) was screened for its antioxidant and anti-inflammatory potential. The major functional groups of polysaccharide were analyzed by Fourier Transmission-Infra Red (FT-IR). In vitro free radical quenching and total antioxidant activity of TCP was investigated by 1, 1-diphenyl-2-picryl hydrazyl (DPPH), nitric oxide (NO) scavenging, lipid peroxidation (LPO) inhibition and ABTS radical assay. Evaluation of anti-inflammatory activity of TCP was performed using carrageenan-induced paw edema in rats and vascular permeability test in mice. Phytochemical analysis of TCP showed the presence of carbohydrates, proteins and polyphenols further, the FT-IR analysis of TCP showed the presence of functional groups of sugar moiety, uronic acids and sulfate groups. TCP showed maximum LPO, NO and DPPH inhibition of 78.04%, 38.82% and 80.21% at a concentration of 1000, 125 and 500 μg/ml respectively. Oral administration of TCP (2.5, 5, 10, 20 mg/kg) reduced the paw edema considerably (p < 0.05) in a dose dependent manner compared to carrageenan induced rats. Similarly, oral administration of TCP (3, 10, 30 mg/kg) evoked a significant (p < 0.05) dose dependent inhibitory effect on vascular permeability in mice. Altogether, these results suggest that the crude polysaccharide of T.ornata could be considered as a potential antioxidant and anti-inflammatory agent.     

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708 bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 °C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aα-chain, apparently not degrading the Bβ and γ-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 °C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.     

Antioxidant activity and ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana and Rosa brunonii

Roses are one of the most important groups of ornamental plants and their fruits and flowers are used in a wide variety of food, nutritional products and different traditional medicines. The antioxidant activity of methanolic extracts from fresh flowers of three rose species (Rosa damascena, Rosa bourboniana and Rosa brunonii) was evaluated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical method. The ability to scavenge DPPH radical was measured by the discoloration of the solution. The methanolic extract from R. brunonii exhibited maximum free-radical-scavenging activity (64.5 ± 0.38%) followed by R. bourboniana (51.8 ± 0.46%) and R. damascena (43.6 ± 0.25%) at 100 μg/ml. Simultaneously, ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study phenolic composition in the methanolic extracts from the fresh flowers of rose species. The phenolic constituents were further investigated by direct infusion-ESI-QTOF-MS/MS in negative ion mode. Characteristic Electrospray ionization tandem mass spectrometry (ESI-MS/MS) spectra with other diagnostic fragment ions generated by retro Diels–Alder (RDA) fragmentation pathways were recorded for the flavonoids. Distinct similarities were observed in the relative distribution of polyphenolic compounds among the three species. The dominance of quercetin, kaempferol and their glycosides was observed in all the three species.     

Genetic variations of CYP2B6 gene were associated with plasma BPDE-Alb adducts and DNA damage levels in coke oven workers

Polycyclic aromatic hydrocarbons (PAHs), the main components of coke oven emissions, can induce activation of cytochrome P450 (CYP) enzymes, which metabolize PAHs and result in DNA damage by forming adducts. This study was designed to know whether genetic variants of CYP genes are associated with plasma benzo[a]pyrene-7,8-diol-9,10-epoxide-albumin (BPDE-Alb) adducts and DNA damage in coke oven workers. In this study, 298 workers were divided into four groups according to the environmental PAHs exposure levels. The concentrations of plasma BPDE-Alb adducts were detected by reverse-phase high-performance liquid chromatography and the DNA damage levels were measured using comet assay. Twelve tag single nucleotide polymorphisms (tagSNPs) of 4 CYP genes were selected and genotyped by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In the top group, workers with CYP2B6 rs3760657GA genotype have lower BPDE-Alb adducts and DNA damage levels than those with rs3760657GG genotype (P < 0.05). In the control group, the DNA damage levels of subjects with CYP1A1 rs4646421AA or GA + AA genotypes were lower than those with GG genotype (P < 0.05). However, no such effects were shown for the other tagSNPs. These results suggested that genetic variations of CYP2B6 might be associated with low BPDE-Alb adducts and DNA damage levels in worker with high exposure to PAHs.     

Evaluation of the mutagenic effects of myosmine in human lymphocytes using the HPRT gene mutation assay

The minor tobacco alkaloid myosmine is implicated in DNA damage through pyridyloxobutylation similar to the tobacco-specific nitrosamines (TSNA). In contrast to TSNA, occurrence of myosmine is not restricted to tobacco. Myosmine is genotoxic to human cells in the comet assay. In this study, the mutagenic effect of myosmine was evaluated using the cloning hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. Four hour exposure of isolated peripheral blood lymphocytes from 14 subjects homozygous for the Leu84 wild-type of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene to 1 mM of myosmine increased mutant frequency from 0.73 ± 0.58 × 10⁻⁶ in control to 1.14 ± 0.89 × 10⁻⁶ lymphocytes (P < 0.05). These new data further confirm the mutagenic effects of myosmine.     

Simultaneous determination of amino acids in discrete brain areas in Suncus murinus by high performance liquid chromatography with electrochemical detection

An improved and simple reversed-phase high performance liquid chromatography method with electrochemical detection for the simultaneous determination of amino acids in brain tissue of Suncus murinus was developed. Homogenates from 5 different brain areas were derivatized with o-phthalaldehyde in the presence of sodium sulphite. Subsequent separation was achieved using linear gradient elution over 30 min. The derivatives were stable for up to 20 h at 4 °C. The method was accurate, reproducible, and showed good linearity. The recoveries were >88% for aspartate, glutamine, glutamate, glycine and γ-aminobutyric acid, with the limit of quantification varying from 5 to 30 pmol. The method was successfully applied for the measurement of amino acids under fed and fasted conditions.     

Genotypic identification of Penicillium expansum and the role of processing on patulin presence in juice

This work aimed at isolation and identification of patulin producing fungi and to follow the presence of patulin during apple juice processing. Among 34 Penicillium isolates, eight isolates (five from healthy appeared apples and 12 from rot spotted apples) were considered as patulin producers using thin-layer chromatography. These isolates were classically identified as a Penicillium expansum. PCR utilizing primers based on the polygalacturonase gene of P. expansum was applied for detecting this mold. The PCR amplified a 404-bp DNA product from all tested P. expansum isolates, but not in other common food spoilage Penicillium species. RAPD technique using P1 or M13 primers was applied to determine the similarity of the P. expansum isolates. RAPD results revealed that the tested strains showed high percentage of similarity and no correlation was observed between cluster analysis and the sources of isolation. Patulin could not be detected in healthy appeared apples and their extracted juice during different stages of juice process. In apple juice made from the healthy parts of apples decayed by P. expansum contained patulin which may present health hazard. The obtained results assured that patulin is known to be stable in apple juice even after pasteurization. In conclusion, the removal of the rotten part from the fruit is not sufficient to eliminate the mycotoxin patulin from apple juice. Although, the enzyme treatment (pectinase and amylase) and pasteurization (95 °C for 7 min) significantly (p < 0.05) reduced patulin level, its level is still higher than the level of <50 μg/kg considered by Codex alimentarius when the apple juice processed from the healthy parts of rot spotted fruits.