Induction and differential expression of certain novel proteins in Anabaena L31 under UV-B radiation stress.

For examining how UV-B radiation alters the proteome of the N2-fixing cyanobacterium, Anabaena L31, we extracted proteins from cultures irradiated with UV-B + white light and controls (white light irradiated) and analyzed the proteins using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Twenty one proteins, including two hypothetical proteins (HPs) were identified and placed in eight functional categories. However several of the proteins were housekeeping proteins involved in key metabolic processes such as carbon, amino acid biosynthesis and energy metabolism, certain proteins seem to have a role in stress (antioxidative enzymes), translation, cellular processes and reductases. Two novel HPs (all3797 and all4050) were characterized in detail. These two were over-expressed after UV-B irradiation and characterized as FAS 1 (all3797) and PRC barrel-like (all4050) proteins. Bioinformatics analysis revealed that the genes of both the HPs have promoter regions as well as transcription binding sites in their upstream region (UTR). Promoters present in all3797 genes suggest their crucial role against UV-B and certain other abiotic stresses. To our knowledge these novel proteins have not been previously reported in any Anabaena strains subjected to UV-B stress. Although we have focused our study on a limited number of proteins, results obtained shed light on the highly complicated but poorly studied aspect of UV-B radiation-mediated changes in the proteome and expression of proteins in cyanobacteria.

Green synthesis of silver nanoparticles using cell extracts of Anabaena doliolum and screening of its antibacterial and antitumor activity.

In the present work, we describe a simple, cheap, and unexplored method for "green" synthesis of silver nanoparticles using cell extracts of the cyanobacterium Anabaena doliolum. An attempt was also made to test the antimicrobial and antitumor activities of the synthesized nanoparticles. Analytical techniques, namely UV-vis spectroscopy, X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and TEMselected area electron diffraction, were used to elucidate the formation and characterization of silver-cyanobacterial nanoparticles (Ag-CNPs). Results showed that the original color of the cell extract changed from reddish blue to dark brown after addition of silver nitrate solution (1 mM) within 1 h, suggesting the synthesis of Ag-CNPs. That the formation Ag-CNPs indeed occurred was also evident from the spectroscopic analysis of the reaction mixture, wherein a prominent peak at 420 nm was noted. TEM images revealed well-dispersed, spherical Ag- CNPs with a particle size in the range of 10-50 nm. The X-ray diffraction spectrum suggested a crystalline nature of the Ag-CNPs. FTIR analysis indicated the utilization of a hydroxyl (-OH) group in the formation of Ag-CNPs. Ag-CNPs exhibited strong antibacterial activity against three multidrug-resistant bacteria. Additionally, Ag-CNPs strongly affected the survival of Dalton's lymphoma and human carcinoma colo205 cells at a very low concentration. The Ag-CNPs-induced loss of survival of both cell types may be due to the induction of reactive oxygen species generation and DNA fragmentation, resulting in apoptosis. Properties exhibited by the Ag-CNP suggest that it may be used as a potential antibacterial and antitumor agent.

Synthesis of ZnO nanoparticles using the cell extract of the cyanobacterium, Anabaena strain L31 and its conjugation with UV-B absorbing compound shinorine.

In the present work, we describe a cheap, unexplored and simple procedure for the synthesis of zinc oxide nanoparticles (ZnONPs) using the cell extract of the cyanobacterium, Anabaena strain L31. An attempt was also made to conjugate synthesized ZnONPs with a UV-absorbing water soluble compound shinorine. UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infra-red (FTIR) spectroscopy, transmission electron microscopy (TEM) and TEM-selected area electron diffraction (SAED) analyses were made to elucidate the formation and characterization of ZnONPs and ZnONPs-shinorine conjugate. The synthesized ZnONPs were characterized by a sharp peak at 370 nm in UV-vis spectrum. TEM images showed the formation of spherical shaped nanoparticles with an average size of 80 nm. Results of selective area electron diffraction (SAED) pattern showed a set of rings which suggested uniform shape with hexagonal structure of ZnONPs. XRD spectra confirmed the crystalline structure of particles. Conjugation of ZnONPs with shinorine was successfully achieved at pH 7.0 and 10mM concentration of shinorine. The conjugate showed a zeta potential value of -3.75 mV as compared to +30.25 mV of ZnONPs. The change in zeta potential value of ZnONPs-shinorine conjugate was attributed to the changes in the surface functionalities after conjugation. The generation of in vivo reactive oxygen species (ROS) by Anabaena strain L31 with treatment of ZnONPs-shinorine conjugate showed approximately 75% less ROS generation as compared to ZnONPs. Properties exhibited by the ZnONPs-shinorine conjugate suggest that it may be used as a potential agent in developing environmental-friendly sunscreen filters of biological origin.

Molecular mechanisms of ultraviolet radiation-induced DNA damage and repair.

DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280-315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

Evidences showing ultraviolet-B radiation-induced damage of DNA in cyanobacteria and its detection by PCR assay.

Impact of ultraviolet-B radiation in causing the damages to the DNA of the cyanobacterium, Anabaena strain BT2 has been investigated. Exposure of genomic DNA (in vitro) to UV-B radiation for 1 h did not cause any shift in the absorption peak (lambda(max)) but more than 30% increase in absorbance was noticed in comparison to untreated control DNA (no exposure to UV-B). This increase in absorbance in a way may be comparable to typical hypochromic effect but there was no decrease in absorbance following transfer of UV-B-treated DNA to fluorescent light or in the dark. That the damaging effect of UV-B radiation on native structure of DNA is indeed real was also evident from the PCR-based assay such as RAPD, rDNA amplification, and ARDRA. Template activity of UV-B-treated genomic DNA was drastically inhibited, there was no amplification in RAPD assay after prior exposure of DNA to UV-B for 60 min. Only one band of approximately 400 bp was observed even after 60 min of exposure which suggests that certain segment of DNA strand is resistant to UV-B effects. Similar to the effects on RAPD profile, amplification of rDNA was significantly inhibited following exposure of genomic DNA to UV-B. Our findings clearly demonstrate that UV-B does affect the DNA of cyanobacteria and the killings of these microbes might be due to the irreversible damages caused to DNA by this high energy radiation. It is felt that PCR assay may be conveniently used for screening the damages caused to DNA by UV-B radiation in cyanobacteria and other microorganisms.

Role of white light in reversing UV-B-mediated effects in the N2-fixing cyanobacterium Anabaena BT2.

The effects of various irradiances of artificial UV-B (280-315 nm) in the presence or absence of visible light (photosynthetically active radiation) on growth, survival, 14CO2 uptake and ribulose 1,5-bisphosphate carboxylase (RuBISCO) activity were studied in the N2-fixing cyanobacterium Anabaena BT2. We tested the hypothesis whether or not visible radiation offers any protection against UV-B-induced deleterious effects on growth and photosynthesis in Anabaena BT2. Attempts were also made to determine the irradiances of UV-B where inhibitory effects could be mitigated by simultaneous irradiation with visible light. Exposure of cultures to 0.2 W m(-2) or higher irradiance of UV-B caused inhibition of growth and survival and growth ceased above 1.0 W m(-2). 14CO uptake and RuBISCO activity were found to be more sensitive to UV-B and around 60% reduction in 14CO2 uptake and RuBISCO activity occurred after exposure of cultures to 0.4 W m(-2) for 1 h. However, growth, 14CO2 uptake and RuBISCO activity were nearly normal when UV-B (0.4 W m(-2)) and visible light (14.4 W m(-2)) were given simultaneously. Blue radiation (450 nm) was found to be the most effective in photoreactivation against UV-B, better than UV-A or any other light wavelength band. Our results demonstrate that the studied cyanobacterium possesses active photoreactivation mechanism(s) against UV-B-mediated damage which in turn probably allow survival under natural conditions in spite of being continuously exposed to the UV-B component present in the solar radiation. Continued growth of many algae and cyanobacteria in the presence of intense solar UV-B radiation under natural conditions seems to be due to the active role of photoreactivation.