Bioassay-guided identification of an anti-inflammatory prenylated acylphloroglucinol from Melicope ptelefolia and molecular insights into its interaction with 5-lipoxygenase

A bioassay-guided investigation of Melicope ptelefolia Champ ex Benth (Rutaceae) resulted in the identification of an acyphloroglucinol, 2,4,6-trihydroxy-3-geranylacetophenone or tHGA, as the active principle inhibiting soybean 15-LOX. The anti-inflammatory action was also demonstrated on human leukocytes, where the compound showed prominent inhibitory activity against human PBML 5-LOX, with an IC(50) value of 0.42 μM, very close to the effect produced by the commonly used standard, NDGA. The compound concentration-dependently inhibited 5-LOX product synthesis, specifically inhibiting cysteinyl leukotriene LTC(4) with an IC(50) value of 1.80 μM, and showed no cell toxicity effects. The anti-inflammatory action does not seem to proceed via redox or metal chelating mechanism since the compound tested negative for these bioactivities. Further tests on cyclooxygenases indicated that the compound acts via a dual LOX/COX inhibitory mechanism, with greater selectivity for 5-LOX and COX-2 (IC(50) value of 0.40 μM). The molecular features that govern the 5-LOX inhibitory activity was thus explored using in silico docking experiments. The residues Ile 553 and Hie 252 were the most important residues in the interaction, each contributing significant energy values of -13.45 (electrostatic) and -5.40 kcal/mol (electrostatic and Van der Waals), respectively. The hydroxyl group of the phloroglucinol core of the compound forms a 2.56? hydrogen bond with the side chain of the carboxylate group of Ile 553. Both Ile 553 and Hie 252 are crucial amino acid residues which chelate with the metal ion in the active site. Distorting the geometry of these ligands could be the reason for the inhibition activity shown by tHGA. The molecular simulation studies supported the bioassay results and served as a good model for understanding the way tHGA binds in the active site of human 5-LOX enzyme.     

New class of acetylcholinesterase inhibitors from the stem bark of Knema laurina and their structural insights

Bioassay-guided extraction of the stem bark of Knema laurina showed the acetylcholinesterase (AChE) inhibitory activity of DCM and hexane fractions. Further repeated column chromatography of hexane and DCM fractions resulted in the isolation and purification of five alkenyl phenol and salicylic acid derivatives. New compounds, (+)-2-hydroxy-6-(10′-hydroxypentadec-8′(E)-enyl)benzoic acid (1) and 3-pentadec-10′(Z)-enylphenol (2), along with known 3-heptadec-10′(Z)-enylphenol (3), 2-hydroxy-6-(pentadec-10′(Z)-enyl)benzoic acid (4), and 2-hydroxy-6-(10′(Z)-heptadecenyl)benzoic acid (5) were isolated from the stem bark of this plant. Compounds (1-5) were tested for their acetylcholinesterase inhibitory activity. The structures of these compounds were elucidated by the 1D and 2D NMR spectroscopy, mass spectrometry and chemical derivatizations. Compound 5 showed strong acetylcholinesterase inhibitory activity with IC₅₀ of 0.573 ± 0.0260 μM. Docking studies of compound 5 indicated that the phenolic compound with an elongated side chain could possibly penetrate deep into the active site of the enzyme and arrange itself through π-π interaction, H-bonding, and hydrophobic contacts with some critical residues along the complex geometry of the active gorge.     

Microbiological quality and related factors of sheep milk produced in farms of NE Greece

Sheep herds are a significant capital of dairy industry in Greece, but as dairy farming becomes more complex and intense the need to provide assistance to dairy producers on milk quality is critical. In this study, the quality of raw milk produced in sheep farms of NE Greece was surveyed and the role of various factors was explored. In total, 21 dairy ewe’s farms from the regions of Xanthi and Evros, in the north-eastern Greece were monitored. Milk samples were sampled after the morning milking every 15 days throughout the dairy period (March–June). For the study, a questionnaire was also filled by personal interview with the owners in order to collect information about herd characteristics, health status, handling practices etc. From each farm, air was also sampled for microbiological analysis. Milk samples were examined for chemical components: fat content, protein, lactose, non-fat dry matter (NFDM) and somatic cells count (SCC). Microbiological examination involved the estimation of Total Bacterial Counts (TBC), coliform count (CC), Staphylococcus aureus, Streptococcus sp., and preliminary incubation count (PIC). The possible correlation among different bacterial species and their interaction with SCC and chemical components of milk was also considered. It was examined whether farm management practices could influence the hygiene and the quality of milk. Our results show that as an average TBCs were 5.48 log cfu/ml, SCC: 6.05 log cells/ml, CC: 4.49 log cfu/ml, S. aureus: 3.94 log cfu/ml, Streptococcus sp.: 4.95 log cfu/ml and PIC: 5.7 log cfu/ml. The mean fat, protein, lactose and NFDM were 6.17%, 5.28%, 4.73% and 10.95% respectively. The study revealed significant positive correlation between TBC and PIC (0.825), while SCC was marginally positive correlated with protein and NFDM. No statistically significant correlations observed among SCC with any of the bacterial species. Herd size and farm management practices had considerable influence on SCC and bacterial species.     

GOLVEN secretory peptides regulate auxin carrier turnover during plant gravitropic responses

Growth and development are coordinated by an array of intercellular communications. Known plant signaling molecules include phytohormones and hormone peptides. Although both classes can be implicated in the same developmental processes, little is known about the interplay between phytohormone action and peptide signaling within the cellular microenvironment. We show that genes coding for small secretory peptides, designated GOLVEN (GLV), modulate the distribution of the phytohormone auxin. The deregulation of the GLV function impairs the formation of auxin gradients and alters the reorientation of shoots and roots after a gravity stimulus. Specifically, the GLV signal modulates the trafficking dynamics of the auxin efflux carrier PIN-FORMED2 involved in root tropic responses and meristem organization. Our work links the local action of secretory peptides with phytohormone transport.     

In Situ Normoxia Enhances Survival and Proliferation Rate of Human Adipose Tissue-Derived Stromal Cells without Increasing the Risk of Tumourigenesis

Adipose tissue-derived stromal cells (ASCs) natively reside in a relatively low-oxygen tension (i.e., hypoxic) microenvironment in human body. Low oxygen tension (i.e., in situ normoxia), has been known to enhance the growth and survival rate of ASCs, which, however, may lead to the risk of tumourigenesis. Here, we investigated the tumourigenic potential of ASCs under their physiological condition to ensure their safe use in regenerative therapy. Human ASCs isolated from subcutaneous fat were cultured in atmospheric O₂ concentration (21% O₂) or in situ normoxia (2% O₂). We found that ASCs retained their surface markers, tri-lineage differentiation potential, and self-renewal properties under in situ normoxia without altering their morphology. In situ normoxia displayed a higher proliferation and viability of ASCs with less DNA damage as compared to atmospheric O₂ concentration. Moreover, low oxygen tension significantly up-regulated VEGF and bFGF mRNA expression and protein secretion while reducing the expression level of tumour suppressor genes p16, p21, p53, and pRb. However, there were no significant differences in ASCs telomere length and their relative telomerase activity when cultured at different oxygen concentrations. Collectively, even with high proliferation and survival rate, ASCs have a low tendency of developing tumour under in situ normoxia. These results suggest 2% O₂ as an ideal culture condition for expanding ASCs efficiently while maintaining their characteristics.     

Aflatoxin M1 in breast milk of nursing Sudanese mothers

The presence of aflatoxin M₁ (AFM₁) in the breast milk of nursing Sudanese mothers was investigated using AOAC official method 980.21 as the extraction method and HPLC with fluorescence detector for separation and detection. Following informed consent, 94 breast milk samples of mothers were collected, and 51 samples were found to be positive for AFM₁, with an average concentration of 0.401?±?0.525?ng?g^?1 and a maximum level of 2.561?ng?g^?1. The volunteers completed a questionnaire concerning their dietary preferences. The data collected suggest that peanut butter, vegetable oils and rice are the main sources responsible for the AFM₁ burden in breast milk. The toxin levels are alarmingly high, and indicate that Sudanese infants are exposed to high levels of AFM₁. A wide range of harmful effects, and consequently health problems, can be expected due AFM₁ toxicity.     

DPPH free radical scavenger components from the fruits of Alpinia rafflesiana Wall. ex. Bak. (Zingiberaceae)

The methanol extract of the dried ripe fruits of Alpinia rafflesiana was investigated for its DPPH free radical scavenger constituents. 2',3',4',6'-Tetrahydroxychalcone (7), which has never been isolated from natural sources was found to be most active as a DPPH free radical scavenger with the IC50 value of 55 microM. Other known compounds isolated from this species include 5,6-dehydrokawain (1), flavokawin B (2). 1,7-diphenyl-5-hydroxy-6-hepten-3-one (3), (-)-pinocembrin (4), cardamonin (5) and (-)-pinostrobin (6). The DPPH free radical scavenger compounds were detected using TLC autographic analysis. The percentage inhibition of DPPH free radical scavenging activity was measured on isolates (5-7) using colorimetric analysis.     

Prevalence of ESBL and MBL encoding genes in Acinetobacter baumannii strains isolated from patients of intensive care units (ICU)

The aim of this study was to investigate the prevalence of ESBL and MBL encoding genes among A. baumannii isolates. In this cross sectional study, 100 A. baumannii strains were isolated from ICU wards of 3 educational hospitals of Hamadan City, Iran in 2011. Phenotypic identification of the production of ESBLs and MBLs has been carried out by using E-test and DDST methods, respectively. PCR technique was used for amplification of the ESBL and MBL encoding genes, namely: CTX-M, SHV, TEM, OXA-51, VIM-Family, IMP-Family, SPM-1, SIM-1, and GIM-1. Eighty seven (87%), 95 (95%), 98 (98%) and 95 (95%) out of 100 A. baumannii isolates were resistant to imipenem, meropenem, ceftazidime and cefotaxime, respectively. Also, 99% and 7% of the isolates were MBLs and ESBLs produced phenotypically. Thirty (30%), 20 (20%) and 58 (58%) out of 100 A. baumannii isolates have been confirmed to harbor the bla_VIM-family, TEM and SHV genes, respectively. Our results show no significant relationship between the detected gens with production of MBLs and ESBLs in spite of high prevalence of MBL encoding and drug resistant A. baumannii. Probably some other genes rather than what we studied are involved in phenotypic production of MBLs and ESBLs and subsequent drug resistance in Hamadan area, Iran.