Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum).Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris.A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and 1 H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active 3 SN-1 in P. pastoris.4
To the Editor, Pollen-food allergy syndromes (PFAS) have been clinically reported and are based on structurally similar sensitizing allergens in pollen and plant food. Besides PR-10 or LTP allergens, the recently described Gibberellin-regulated proteins (GRP) are also allergens found in pollen and plant derived food, therefore are allergens involved in PFAS. 1,2 They are found in Cupressaceae pollen and some fruits such as peach/apricot, citrus, pomegranate, and cherry. 3 The aim of the study was to compare GRP sensitization in two cohorts of Japanese children allergic to Japanese cedar (Cryptomeria japonica, Cryj) pollen: group 1 allergic to pollen-only (n = 21), group 2 allergic to pollen and fruit (n = 22) suffering from systemic reaction (Table 1). Clinical data of recruited allergic patients were recorded at the department of pediatrics, Sagamihara National Hospital. Allergomics analysis was performed using direct or competitive IgE immunoblots after separation of the Japanese cedar pollen extracts in electrophoresis in 1 dimension (1-DE) or 2-DE followed by identification of allergens by mass spectrometry. Recombinant pollen and fruit GRPs were produced.Associated fruit IgE-dependent allergy in group 2 mainly involved peach, citrus, and apple but also cherry, Japanese apricot, strawberry, and grape. In a cohort of fruit-only allergic children,
The methanolic crude extract of the bark and seed of Entada phaseoloides (L.) Merr. and its different organic soluble partitionates were screened for antioxidant, cytotoxic, membrane stabilizing and antimicrobial activities. The crude extract and carbon tetrachloride and aqueous soluble fractions of both bark and seed showed higher level of total phenolic content (TPC, 245.59, 240.22, 240.03 & 117.0 mg of GAE/gm of dried extract). In the DPPH assay, the crude extract of bark and its chloroform and aqueous soluble fractions demonstrated strong antioxidant property with the IC50 of 3.24, 1.55 and 3.6 µg/ml, respectively whereas all the fractions of seed extract revealed mild antioxidant activity. The petroleum ether soluble fraction of both seed and bark exhibited significant cytotoxicity (LC50 = 1.54 & 5.4 µg/ml) which confers the presence of bioactive metabolites in this plant. On the other hand, the crude extract of seed and petroleum ether soluble fraction of bark inhibited the hemolysis of RBC of rat's blood by 78.89 and 57.43%, respectively as compared to 84.44% exerted by acetyl salicylic acid (0.10 mg/ml). In antimicrobial screening, the carbon tetrachloride soluble fraction of bark showed significant antimicrobial activity against Staphylococcus aureus (zone of inhibition = 17.0 mm) with MIC and MBC values of 7.81 µg/ml and 125 µg/ml, respectively.
Aims:In the present study, the crude methanol extract of tuber of Polianthes tuberosa Linn along with its all Kupchan fractions were investigated for antioxidant, cytotoxic, antimicrobial, membrane stabilizing and thrombolytic activities.
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