Molecular cloning of a thermo-alkaliphilic lipase from Bacillus subtilis DR8806: Expression and biochemical characterization

A thermo-alkaliphilic lipase from Bacillus subtilis DR8806 was functionally expressed as an N-terminal 6xHis-tagged recombinant enzyme in Escherichia coli BL21 using pET-28a(+) expression vector. Sequence analysis revealed an open reading frame of 639 bp encoding a 212-amino acid protein containing the well-conserved Ala-His-Ser-Met-Gly motif. One-step purification of the His-tagged recombinant lipase was achieved using Ni-NTA affinity chromatography with a specific activity of 1364 U/mg. The purified enzyme with an apparent molecular mass of 26.8 kDa demonstrated the maximum activity at 70 °C and pH 8.0 for hydrolysis of p-nitrophenylbutyrate as substrate. The enzyme activity was strongly inhibited by divalent ions of heavy metals such as Hg²⁺ and Cu²⁺, while retained over 90% of the original activity in the presence of several reagents including DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)), SDS (sodium dodecyl sulfate), urea, DMF (dimethylformamide), DTT (dithiothreitol), glycerol and Triton X-100. While being considerably stable in organic solvents, imidazolium-based ionic liquids (ILs) had stimulatory effects on the activity of purified lipase. Remarkable stabilization of enzyme at alkaline pH and in ionic liquids as well as its thermostability/thermoactivity are among the most fundamental characteristics which offer great potential for various biotechnological applications including detergent formulation, bioremediation processes and biotransformation in non-aqueous media.     

Molecular genetic diversity within Myrmeleontidae family

Antlions are insects which feed on ants, insect which dig a pit and lies in wait for ants and other insects. Twelve species of Myrmeleontidae family as antlions and many specimens were identified in different locations in Fars province in Iran. To unveil the genetic similarity between these species, their DNA was extracted by modified CTAB method and with the use of seventeen 10-nucleotides primers of random amplified polymorphic DNA (RAPD); the genetic analysis of them was investigated. After PCR, agarose 1.5?% was used for electrophoresis. The obtained electrophoresis bands had base pairs range between 150 and 1,000?bp. The maximum of polymorphic bands belonged to OPH5, N13, and the minimum of polymorphic bands belonged to OPA7 primers. Different genetic similarity indices were found between eight species of antlions. Possibility of use of RAPD marker together with morphological studies for classification and identification of antlions is discussed.     

Alleviation of chromium toxicity in rice seedlings by applying exogenous glutathione

The effect of exogenous reduced glutathione (GSH) on alleviation of hexavalent chromium (Cr⁶⁺) toxicity to rice seedlings and its physiological mechanisms were comprehensively investigated in a series of experiments. Our results showed that growth and nutrient uptake of rice seedlings were dramatically reduced under 100 μM Cr⁶⁺ stress, and the reduction was significantly alleviated by exogenous GSH. Cr⁶⁺ stress also reduced cell viability in root tips and damaged ultrastructure of both chloroplasts and root cells, while the addition of GSH alleviates those negative effects. Cr-induced toxicity and GSH-caused Cr alleviation differed significantly between Cr-tolerant Line 117 (L117) and Cr-sensitive Line 41 (L41). Under Cr⁶⁺ stress, cystine content was increased and GSH content was decreased in rice plants, exogenous GSH, however, mitigated the Cr-toxicity by reversing the Cr-induced changes of the two compounds. The types of Cr-induced secretion of organic acids varied between the genotypes, where reduction in the contents of acetic and lactic acids and tartaric and malic acids were observed in L117 and L41, respectively. The addition of GSH alleviated the reduction of secretion of these organic acids. Exogenous GSH also altered the forms of Cr ions in the rhizosphere and the fraction of distribution at subcellular level in both shoots and roots. It may be concluded that the alleviation of Cr⁶⁺ toxicity by exogenous GSH is directly attributed to its regulation on forms of Cr ions in rhizosphere and their distribution at subcellular levels.     

Proteomic analysis of sensitive and multi drug resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> strains

Multidrug-resistant tuberculosis (MDR-TB) is caused by bacteria that are resistant to the most effective anti TB drugs (Isoniazid and Rifampicin) with or without resistance to other drugs. Novel intervention strategies to eliminate this disease based on finding proteins can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profile of MDR-TB with sensitive isolates. Two-dimensional gel electrophoresis (2DE) along with mass spectrometry is a powerful and effective tool to identification and characterization of Mycobacterium tuberculosis. Two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was used for diagnosis and comparison of proteins. We identified 14 protein spots in MDR-TB isolates that 2DE analysis showed these spots absent in M. tuberculosis sensitive isolates (Rv1876, Rv0379, Rv0147, Rv2031c, Rv3597c, Rv1886c, MT0493, Rv0440, Rv3614c, Rv1626, Rv0443, Rv0475, Rv3057 and unknown protein. The results showed 22 protein spots which were up regulated (or expressed) by the MDR-TB isolates, (Rv1240, Rv3028c, Rv2971, Rv2114c, Rv3311, Rv3699, Rv1023, Rv1308, Rv3774, Rv0831c, Rv2890c, Rv1392, Rv0719, Rv0054, Rv3418c, Rv0462, Rv2215, Rv2986c, Rv3248c and Rv1908c)). Two up regulated protein spots were identified in sensitive isolate (Rv1133c and Rv0685). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug resistant and sensitive of M. tuberculosis.     

Nanocomposite scaffold seeded with mesenchymal stem cells for bone repair

The mechanical property of bone tissue scaffolds is one of the most important aspects in bone tissue engineering that has remained problematic. In our previous study, we fabricated a three‐dimensional scaffold from nano‐hydroxyapatite/gelatin (nHA/Gel) and investigated its efficiency in promoting bone regeneration both in vitro and in vivo. In the present study, the effect of adding silicon carbide (SiC) on the mechanical and biological behaviors of the nHA/Gel/SiC and bone regeneration in vivo were determined. nHA and SiC were synthesized and characterized by the X‐ray diffraction pattern and transmission electron microscope image. Layer solvent casting, freeze drying, and lamination techniques were applied to prepare these scaffolds. Then, the biocompatibility and cell adhesion behavior of the synthesized nHA/Gel/SiC scaffolds were investigated. For in vivo studies, rats were categorized into three groups: blank defect, blank scaffold, and rat bone marrow mesenchymal stem cells (rBM‐MSCs)/scaffold. After 1, 4, and 12 weeks post‐injury, the rats were sacrificed and the calvaria were harvested. Sections with a thickness of 5 µm thickness were prepared and stained with hematoxylin–eosin and Masson's Trichrome, and immunohistochemistry was performed. Our results showed that SiC effectively increased the mechanical properties of the nHA/Gel/SiC scaffold. No significant differences were observed in biocompatibility, cell adhesion, and cytotoxicity of the nHA/Gel/SiC in comparison with the nHA/Gel nanocomposite. Based on histological and immunohistochemical studies, both osteogenesis and collagenization were significantly higher in the rBM‐MSCs/scaffold group, quantitatively and qualitatively. The present study strongly suggests the potential of SiC as an alternative strategy to improve the mechanical and biological properties of bone tissue engineering scaffolds, and shows that the pre‐seeded nHA/Gel/SiC scaffold with rBM‐MSCs improves osteogenesis in the engineered bone implant.     

Enhancing crop growth, nutrients availability, economics and beneficial rhizosphere microflora through organic and biofertilizers

Field experiment was conducted on fodder maize to explore the potential of integrated use of chemical, organic and biofertilizers for improving maize growth, beneficial microflora in the rhizosphere and the economic returns. The treatments were designed to make comparison of NPK fertilizer with different combinations of half dose of NP with organic and biofertilizers viz. biological potassium fertilizer (BPF), Biopower, effective microorganisms (EM) and green force compost (GFC). Data reflected maximum crop growth in terms of plant height, leaf area and fresh biomass with the treatment of full NPK; and it was followed by BPF+full NP. The highest uptake of NPK nutrients by crop was recorded as: N under half NP+Biopower; P in BPF+full NP; and K from full NPK. The rhizosphere microflora enumeration revealed that Biopower+EM applied along with half dose of GFC soil conditioner (SC) or NP fertilizer gave the highest count of N-fixing bacteria (Azotobacter, Azospirillum, Azoarcus andZoogloea). Regarding the P-solubilizing bacteria,Bacillus was having maximum population with Biopower+BPF+half NP, andPseudomonas under Biopower+EM+half NP treatment. It was concluded that integration of half dose of NP fertilizer with Biopower+BPF / EM can give similar crop yield as with full rate of NP fertilizer; and through reduced use of fertilizers the production cost is minimized and the net return maximized. However, the integration of half dose of NP fertilizer with biofertilizers and compost did not give maize fodder growth and yield comparable to that from full dose of NPK fertilizers.     

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex,a sequence-selective N3-adenine methylating agent

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild type < deltamag1< deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GC > AT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity.     

Cadmium Toxicity in Spermatogenesis and Protective Effects of <Emphasis Type="SmallCaps">l</Emphasis>-Carnitine in Adult Male Rats

In this study, the effects of cadmium toxicity and the protective effects of l-carnitine on spermatogenesis in Sprague–Dawley rat were evaluated. Animals were subdivided into five groups. Cadmium chloride (1-mg/kg body weight) was injected intraperitoneally during 16 days at intervals of 48 h between subsequent treatments. l-Carnitine (500 mg/kg b.w., IP) was pretreated in both of control and cadmium-injected rats. Animals were killed on day 17 after the first treatment. The left cauda epididymis was removed and immediately immersed into Hank’s balanced salt solution for evaluation of sperm count and viability. Following contamination with cadmium, a decrease in the number and viability of cauda epididymis sperm, the number of cell proliferation, and Johnsen Scores in the seminiferous tubules was observed. Consequently, l-carnitine treatment caused an increase in the number and viability of cauda epididymis sperm, the number of cell proliferation, and Johnsen Scores in the cadmium-induced group.