Production of recombinant proteins by microbes and higher organisms

Large proteins are usually expressed in a eukaryotic system while smaller ones are expressed in prokaryotic systems. For proteins that require glycosylation, mammalian cells, fungi or the baculovirus system is chosen. The least expensive, easiest and quickest expression of proteins can be carried out in Escherichia coli. However, this bacterium cannot express very large proteins. Also, for S–S rich proteins, and proteins that require post-translational modifications, E. coli is not the system of choice. The two most utilized yeasts are Saccharomyces cerevisiae and Pichia pastoris. Yeasts can produce high yields of proteins at low cost, proteins larger than 50 kD can be produced, signal sequences can be removed, and glycosylation can be carried out. The baculoviral system can carry out more complex post-translational modifications of proteins. The most popular system for producing recombinant mammalian glycosylated proteins is that of mammalian cells. Genetically modified animals secrete recombinant proteins in their milk, blood or urine. Similarly, transgenic plants such as Arabidopsis thaliana and others can generate many recombinant proteins.     

Biomimetic synthesis and characterisation of protein capped silver nanoparticles

A controlled and up-scalable route for the biosynthesis of silver nanopartilces (NPs) mediated by fungal proteins of Coriolus versicolor has been undertaken for the first time. The fungus when challenged with silver nitrate solution accumulated silver NPs on its surface in 72h which could be reduced to 1h by tailoring the reaction conditions. Under alkaline conditions, the reaction was much faster and could easily proceed at room temperature even without stirring. The resulting Ag NPs displayed controllable structural and optical properties depending on the experimental parameters such as pH and reaction temperatures. The average size, morphology, and structure of particles were determined by AFM, TEM, XRD and UV/Visible absorption spectrophotometry. Fourier transform infrared study disclosed that the amino groups were bound to the particles, which was accountable for the stability of NPs. It further confirmed the presence of protein as the stabilizing and capping agent surrounding the silver NPs. Experiments were conducted both with, media in which fungus was initially harvested and that of pristine fungal mycelium alone. Under normal conditions, in the case of media extracellular synthesis took place whereby other than the fungal proteins, glucose was also responsible for the reduction. In the case of fungal mycelium, the intracellular formation of Ag NPs, could be tailored to give both intracellular and extracellular Ag NPs under alkaline conditions whereby the surface S-H groups of the fungus played a major role.     

Evaluation of the toxicity of different phytoextracts of Ocimum basilicum against Anopheles stephensi and Culex quinquefasciatus

The larvicidal effect of the crude carbon tetrachloride, methanol and petroleum ether leaf extracts of a widely grown medicinal plant, Ocimum basilicum, against Anopheles stephensi and Culex quinquefasciatus was evaluated. Petroleum ether extract was found to be the most effective against the larvae of both mosquitoes, with LC₅₀ values of 8.29, 4.57; 87.68, 47.25 ppm and LC₉₀ values of 10.06, 6.06; 129.32, 65.58 ppm against A. stephensi and C. quinquefasciatus being observed after 24 and 48 h of treatment, respectively. The efficacy of petroleum ether was followed by that of the carbon tetrachloride and methanol extracts, which had LC₅₀ values of 268.61, 143.85; 446.61, 384.84 ppm and LC₉₀ values of 641.23, 507.80; 923.60, 887.00 ppm against A. stephensi after 24 and 48 h, respectively, and LC₅₀ values of 24.14, 17.02; 63.48, 53.77 ppm and LC₉₀ values of 295.38, 204.23; 689.71, 388.87 ppm against C. quinquefasciatus after 24 and 48 h of treatment, respectively. These extracts are highly toxic against mosquito larvae from a range of species; therefore, they may be useful for the management of mosquito larvae to control vector borne diseases.     

Changes in species composition,diversity and biomass of herbaceous plant traits due to N amendment in a dry tropical environment of India

Aim European and North American studies have suggested that nitrogen (N) depositions reduce plant diversity and increase primary productivity due to changes in plant traits. To predict the vegetation response to future global change, experimental validations from other regions are widely needed. We assessed the effects of N treatment by urea fertilization on the diversity and biomass of the herbaceous plant traits (HPTs) in a dry tropical environment of India.Methods Diversity and biomass of different HPTs were determined on the basis of data collected in year 2010, from 135, 1 m × 1 m plots distributed over 15 locations. The plots were treated with urea fertilizer in different doses (Control, 60kgNha^-1 yr^-1 and 120kg N ha^-1 yr^-1) since 1st January 2007. The plots were ordinated and data were subjected to appropriate statistical analyses.Important findings Correspondence analysis (CA) suggested uniqueness of species composition due to N amendment. Species number and biomass of the trait categories varied due to N fertilization and traits. All studied trait categories (except N-fixers) yielded maximum mean species number at moderate level of N fertilization. Different levels of N fertilization exhibited different species diversity–primary productivity (D-P) relationships. Further, study showed reduction in plant diversity due to increase in biomass at high rates of N addition.Conclusions Tall, erect, non N-fixers, annuals, grasses HPTs were favoured by N enrichment. N dose above 60kg enhanced the biomass of fast growing, erect, annuals, non N-fixers, nitrophilic HPTs. The changes in traits with N addition, especially the increase in annuals and grasses and decrease in typically N-rich N-fixers, have implications for sustainable cattle production.     

Elevated levels of urinary 17-ketosteroids in central Indian children residing near sewage treatment plant and solid waste disposal plant: A preliminary study

Urinary excretion of 17-ketosteroid (17-KS) was assessed in male pre-pubertal subjects aged (8–11 years; n = 90). Children living near sewage treatment plant and solid waste disposal plant (Group P) showed significantly higher levels of urinary 17-KS (Group P: 3.27 ± 1.63 µg/mL/CRE; p < 0.01) than children living in cleaner area (0.50 ± 0.53 µg/mL/CRE; Group C). Occurrence of urinary dibutyl phthalate in representative subjects of Group P (odds ratio: 9; p < 0.05; 95% of Confidence interval (CI) 1.93–72.99) was higher compared to Group C. Urinary concentrations of Cd (0.85 µg/g CRE ± 0.11), Mn (24.25 µg/g CRE ± 6.11) and Pb (12.39 µg/g CRE ± 2.86) in Group P were significantly (p < 0.01) higher than those found in Group C (Cd (0.28 µg/g CRE ± 0.03), Mn (13.33 µg/g CRE ± 3.20) and Pb (5.67 µg/g CRE ± 0.53)). Analyses of ambient air samples (PM₁₀) in polluted area revealed major occurrence of phthalates, whereas derivatives of trifluoromethyl, dione, etc. were identified in PM_2.5 fraction. Metal (Cd, Co, Mn and Pb) concentrations in ambient air (24 h, PM₁₀) were higher in polluted area compared to cleaner area. We conclude that elevated levels of urinary 17-KS in Group P could be attributed to higher exposure of these subjects to Endocrine disrupting chemicals (EDCs) compared to Group C.     

Cloning,Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.     

Role of serine/threonine phosphatase (SP-STP) in Streptococcus pyogenes physiology and virulence

Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.     

Unexpected applications of secondary metabolites

Secondary metabolites have been found to have interesting applications over and above their well-known medical uses, e.g., as antimicrobials, etc. These alternative applications include antitumor, cholesterol-lowering, immunosuppressant, antiprotozoal, antihelminth, antiviral and anti-ageing activities. Polyene antibiotics, such as amphotericin B, are of use as antiprion agents, antitumor drugs and against leishmaniasis. Other microbial natural products that show antibiotic activity are used against cancer e.g., doxorubicin, neomycin, β-lactams, bleomycin and rapamycin. Macrolide antibiotics, such as erythromycin, clarithromycin and azithromycin, improve pulmonary function in patients suffering from panbion cholitis. Pigments like prodigiosin and shikonin have antitumor activity, while violacein has anti-ulcer and antitumor activity and also acts as an antiprotozoal agent. Statins, in addition to lowering cholesterol and LDL levels, also decrease elevated C-reactive protein (CRP) levels independent of their cholesterol effects. Immunosppressants have many alternative effects: (i) Cyclosporin is proving useful in treatment of inflammatory disease such as asthma and muscular dystrophy. (ii) Rapamycin is extremely useful in preventing restenosis of stents grafted in balloon angioplasty. (iii) Tacrolimus and ascomycin help in treating inflammatory skin disease such as allergic contact dermatitis and psoriasis. Artemisinin, an antimalarial agent, is also showing antitumor activity. Other natural products, including those from plants (betulinic acid and shikonin), animals (bryostatins) and microbes (squalestatin and sophorolipids) have a multiplicity of potentially useful actions. Unexpected functions of known secondary metabolites are continuously being unraveled, and are fulfilling some of the needs of present day medicine and show great promise for the future.