Identification of novel proteins regulating lipid droplet biogenesis in filamentous fungi.

Lipid droplets (LDs) are storage organelles for neutral lipids which are critical for lipid homeostasis. Current knowledge of fungal LD biogenesis is largely limited to budding yeast, while LD regulation in multinucleated filamentous fungi which exhibit considerable metabolic activity remains unexplored. In this study, 19 LD-associated proteins were identified in the multinucleated species Aspergillus oryzae using a colocalization screening of a previously established enhanced green fluorescent protein (EGFP) fusion library. Functional screening identified 12 lipid droplet-regulating (LDR) proteins whose loss of function resulted in irregular LD biogenesis, particularly in terms of LD number and size. Bioinformatics analysis, targeted mutagenesis, and microscopy revealed four LDR proteins that localize to LD via the putative amphipathic helices (AHs). Further analysis revealed that LdrA with an Opi1 domain is essential for cytoplasmic and nuclear LD biogenesis involving a novel AH. Phylogenetic analysis demonstrated that the patterns of gene evolution were predominantly based on gene duplication. Our study identified a set of novel proteins involved in the regulation of LD biogenesis, providing unique molecular and evolutionary insights into fungal lipid storage.

Characterization and evaluation of antibacterial and antiproliferative activities of crude protein extracts isolated from the seed of Ricinus communis in Bangladesh.

BACKGROUND: Ricinus communis (Euphorbiaceae) has previously been reported to possess analgesic, antihistamine, antioxidant and anti-inflammatory activities. This study was designed for isolation, characterization and evaluation of antibacterial and anti-proliferative activities of R. communis seed protein. METHODS: The concentration and molecular weight of R. communis seed protein were estimated by SDS-PAGE and spectrophotometric analysis, respectively. Lectin activity was evaluated by hemagglutination assay on mice blood. In vitro susceptibility of four human pathogenic bacteria including Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes and Staphylococcus aureus was detected using disk diffusion assay, and minimum inhibitory concentration (MIC) value was determined using micro-dilution method. A total of twenty four Swiss albino mice containing Ehrlich's ascites carcinoma (EAC) cells were treated with the crude protein of R. communis at 50 and 100 µg/ml/d/mouse for 6 days. Growth inhibitory activity of R. communis seed protein on EAC cells was determined by haemocytometer counting using trypan blue dye and DAPI (4΄,6-diamidino-2-phenylindole) staining was used to assess apoptotic cells. RESULTS: The protein concentration of six R. communis (castor) varieties ranged between 21-35 mg/ml and molecular weight between 14-200 kDa. Castor protein agglutinated mice blood at 3.125 µg/wall. The seed protein shows considerable antimicrobial activity against E. coli, P. aeruginosa and S. aureus, exhibiting MIC values of 250, 125 and 62.5 µg/ml, respectively. Administration of seed protein led to 54 % growth inhibition of EAC cells at 100 µg/ml. DAPI staining indicates marked features of apoptosis including condensation of cytoplasm, nuclear fragmentation and aggregation of apoptotic bodies etc. CONCLUSION: Our study suggests that the lectin rich R. communis seed protein has strong antibacterial and anticancer activities.

Assessment of antioxidant, anticancer and antimicrobial activity of two vegetable species of Amaranthus in Bangladesh.

BACKGROUND: Amaranthus (Amaranthaceae) has previously been reported to possess different bioactive phytochemicals including phenols, tannins and flavonoids. The current study was designed to evaluate the antioxidant, anti-proliferative and antimicrobial activity of stem and seed extracts of Amaranthus lividus (AL) and Amaranthus hybridus (AH), respectively. METHODS: Antioxidant activity of methanol extract was assessed by DPPH radical scavenging assay. Determination of lectin activity of Amaranthus extract was carried out using hemagglutination assay on mouse blood. A total of thirty six Swiss albino mice containing Ehrlich's ascites carcinoma (EAC) cells were treated with AL and AH extract at 25, 50 and 100 µg/ml/day/mouse for six days. Growth inhibitory activity was determined by haemocytometer counting of EAC cells using trypan blue dye and DAPI (4΄,6-diamidino-2-phenylindole) staining was used to assess apoptotic cells. Gene amplification study was conducted to observe the expression pattern of p53, Bax, Bcl-2 and caspase-3 mRNA using PCR (polymer chain reaction) technique. In vitro susceptibility of five pathogenic bacteria including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella typhi and Staphylococcus aureus was detected using disk diffusion assay. RESULTS: The radical scavenging assay indicated that AH and AL possesses potent antioxidant potential, exhibiting IC50 value of 28 ± 1.5 and 93 ± 3.23 µg/ml, respectively. Hemagglutination assay revealed that AH and AL agglutinated mice blood at 1.565 and 3.125 µg/wall, respectively. Administration of AH and AL extract led to 45 and 43 % growth inhibition of EAC cells, respectively at 100 µg/ml with marked features of apoptosis including cell shrinkage, condensation of cytoplasm and aggregation of apoptotic bodies etc. Up-regulation of p53, Bax and caspase-3 and down-regulation of Bcl-2 mRNA in Amaranthus treated mice indicated mitochondria mediated apoptosis of EAC cells in comparison with control. None of the bacterial species showed susceptibility to the extract of both the Amaranthus species. CONCLUSION: Our current findings suggest that both of the Amaranthus species have strong antioxidant, lectin and anti-proliferative activity on EAC cells. The current anticancer potential was observed due mainly to the mitochondria mediated apoptosis of EAC cells.

Gene structures of trocarin D and coagulation factor X, two functionally diverse prothrombin activators from Australian rough scaled snake.

Activation of prothrombin to thrombin is the key reaction in blood coagulation cascade. We have recently shown that Australian rough scaled snake, Tropidechis carinatus, possesses two parallel prothrombin activator systems. Trocarin D, a venom prothrombin activator produced in the venom gland, plays an offensive role as a toxin, whereas factor X is produced in the liver and plays a role in the hemostatic mechanism. These two proteins are structurally similar and have identical domain architecture. Because of the differences in their physiological roles, and tissue-specific expression, we determined the gene structure of these closely related proteins. Both the genes have eight exons similar to all mammalian factor X genes. All the exon-intron boundaries of these two genes are at the same position and the splice junctions are almost identical. Partial sequencing of the introns shows that they share a very high degree of sequence identity indicating that the gene duplication is a recent event. Further studies on the characterization of these two genes particularly the promoter regions are in progress.