Enhanced method for High Spatial Resolution surface imaging and analysis of fungal spores using Scanning Electron Microscopy.

Efficient, fast and new micro-analytical methods for characterization of ultrastructures of fungal spores with electron microscopy are very much required and essential. SEM analysis of biological materials, especially fungi, requires optimal preparation of the specimen and often requires the usage of dried samples which demands a challenging sample preparation. In the present investigation, we described a fast and improved method for the preparation of fungal specimen for scanning electron microscopy (SEM). The fungus, Curvularia lunata was grown on the surface of sterile Whatman No.1 filter paper which was overlaid on Potato Dextrose Agar (PDA) medium, gold coated immediately after removal from the growth medium and subjected to imaging. Generally, SEM imaging is done with samples that were fixed with chemical fixatives, dehydrated and gold coated specimens, but here we describe an easy and more efficient sample preparation for SEM which enabled enhanced image quality and precision visualization of fungal cells, especially the spores. The developed method has enabled the analysis of even the robust samples like fungal spores that to eliminating special temperature requirement. The ultimate goal was to develop an improved protocol/method applied to analysis of fungal spores with greater coverage about fungal specimen preparation. This method permits the use of rapid sample preparation and will allow us to imaging of individual spore or conidia structures in the context of fungal cell architecture which clarifies our understanding in fungal taxonomy/biology.

Impact of geography on adaptation of Phyllanthus amarus seeds.

The present study was aimed to evaluate adaptive mechanism in terms of seed characters of Phyllanthus amarus collected from ten different locations of Tamil Nadu, India. The adaptive variations among the collected populations were assessed based on the sink and float percentages of the seeds in water, the percentage of seed germination, total protein, carbohydrates and their seedling's growth ability such as shoot and root lengths. From this, we observed that the population had a significantly higher germination percentage of sinking seeds that were attributed to its relatively higher carbohydrate and protein contents than the floating seeds. A comparison of the seed population by cluster analysis and principal coordinate analysis showed that the Chennai population constituted a single clade that was very distinct from the other nine populations, which were further grouped into two sub-clusters. They exhibited a trend consistent with their geographical proximity. Standardised Mantel's t tests had revealed that the adaptive diversity of the P. amarus population was significantly affected by the geographic distance (r = 0.78, t = 2.68, P > 0.001), altitude (r = 0.35, t = 21.53, P > 0.05), minimum temperature (r = 0.43, t = 1.49, P > 0.01) and maximum temperature (r = 0.49, t = 1.67, P > 0.001). Seed's characteristics and geographical conditions were correlated along with 19 bioclimatic variables. In dry season, the seedling's rooting ability showed positive correlation, while its protein content exhibited a negative correlation. It is clearly evident from this study that the geographical variables significantly influence the adaptive ability of the P. amarus.

Antibacterial cysteine protease from Cissus quadrangularis L.

An antibacterial Cp was extracted from the stem of Cissus quadrangularis and purified with a 5.39 fold increase in specific activity and 8.67% recovery. The molecular weight of the purified enzyme was estimated to be 39kDa by SDS-PAGE. The purified enzyme appeared as a single band on Native-PAGE. The optimum pH and temperature for protease activity were around 6.0 and 50°C respectively. The Cp showed pH stability from 3 to 10 and retained more than 90% of its relative protease activity. The addition of metal ions such as Mg2+ and Ca2+ also exhibited relative protease activity. Cp showed a potent antibacterial activity against pathogenic bacteria. About 4.74Uml-1 of Cp from C. quadrangularis was tested for antibacterial activity against Bacillus cereus and Bacillus megaterium which subsequently showed zone of inhibition of 21 and 20mm respectively. Cp from C. quadrangularis degraded the peptidoglycan layer of bacteria by Cp was confirmed by transmission electron microscopic analysis.

Antibacterial serine protease from Wrightia tinctoria: Purification and characterization.

A serine protease was purified from the leaves of Wrightia tinctoria by sequential flow through method comprising screening, optimization, ammonium sulfate precipitation, gel filtration and ion exchange column chromatography. The yield and purification fold obtained were 11.58% and 9.56 respectively. A single band of serine protease was visualized on SDS-PAGE and 2-D gel electrophoretic analyses were revealed with the molecular mass of 38.5 kDa. Serine protease had an optimum pH of 8.0 and was stable at 45°C with high relative protease activity. The addition of metal ions such as Mg2+ and Mn2+ exhibits a high relative activity. Serine protease had a potent antibacterial activity against both Gram-positive and Gram-negative bacteria. A 10 µg/ml of serine protease was tested against S. aureus, M. luteus, P. aeruginosa and K. pneumoniae which had 21, 20, 18 and 17 mm of zone of inhibition respectively. Serine protease from W. tinctoria degrades the peptidoglycan layer of bacteria which was visualized by transmission electron microscopic analysis.

Qualitative and quantitative determination of R-phycoerythrin from Halymenia floresia (Clemente) C. Agardh by polyacrylamide gel using electrophoretic elution technique.

R-Phycoerythrin is one of the phycobiliproteins widely found in seaweeds. In this study, we have shown to extract and purify R-Phycoerythrin from the south east cost Indian red seaweed Halymenia floresia. R-Phycoerythrin was extracted in 50mM phosphate buffer (pH 7.0). The preparative native PAGE purification was employed alternative to the chromatography and therefore can be scaled up efficiently. Both the yield and the purity of R-Phycoerythrin are very effective. The purified R-Phycoerythrin showed a single band on the examination by native PAGE electrophrosis. SDS-PAGE analysis showed five bands at 16kDa, 21kDa, 30kDa, 39kDa and 47kDa which corresponds to the α, ß and γ', γ and αß subunits. This preparative method for R-Phycoerythrin purification can offer a reference for R-Phycoerythrin purification from other marine red macro algae.