Fanconi anemia proteins FANCD2 and FANCI exhibit different DNA damage responses during S-phase

Fanconi anemia (FA) pathway members, FANCD2 and FANCI, contribute to the repair of replication-stalling DNA lesions. FA pathway activation relies on phosphorylation of FANCI by the ataxia telangiectasia and Rad3-related (ATR) kinase, followed by monoubiquitination of FANCD2 and FANCI by the FA core complex. FANCD2 and FANCI are thought to form a functional heterodimer during DNA repair, but it is unclear how dimer formation is regulated or what the functions of the FANCD2-FANCI complex versus the monomeric proteins are. We show that the FANCD2-FANCI complex forms independently of ATR and FA core complex, and represents the inactive form of both proteins. DNA damage-induced FA pathway activation triggers dissociation of FANCD2 from FANCI. Dissociation coincides with FANCD2 monoubiquitination, which significantly precedes monoubiquitination of FANCI; moreover, monoubiquitination responses of FANCD2 and FANCI exhibit distinct DNA substrate specificities. A phosphodead FANCI mutant fails to dissociate from FANCD2, whereas phosphomimetic FANCI cannot interact with FANCD2, indicating that FANCI phosphorylation is the molecular trigger for FANCD2-FANCI dissociation. Following dissociation, FANCD2 binds replicating chromatin prior to-and independently of-FANCI. Moreover, the concentration of chromatin-bound FANCD2 exceeds that of FANCI throughout replication. Our results suggest that FANCD2 and FANCI function separately at consecutive steps during DNA repair in S-phase.     

Reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatin-mediated acute kidney injury

Peroxisome proliferator-activated receptor-α (PPARα) activation attenuates cisplatin (CP)-mediated acute kidney injury by increasing fatty acid oxidation, but mechanisms leading to reduced renal triglyceride (TG) accumulation could also contribute. Here, we investigated the effects of PPARα and CP on expression and enzyme activity of kidney lipoprotein lipase (LPL) as well as on expression of angiopoietin protein-like 4 (Angptl4), glycosylphosphatidylinositol-anchored-HDL-binding protein (GPIHBP1), and lipase maturation factor 1 (Lmf1), which are recognized as important proteins that modulate LPL activity. CP caused a 40% reduction in epididymal white adipose tissue (WAT) mass, with a reduction of LPL expression and activity. CP also reduced kidney LPL expression and activity. Angptl4 mRNA levels were increased by ninefold in liver and kidney tissue and by twofold in adipose tissue of CP-treated mice. Western blots of two-dimensional gel electrophoresis identified increased expression of a neutral pI Angptl4 protein in kidney tissue of CP-treated mice. Immunolocalization studies showed reduced staining of LPL and increased staining of Angptl4 primarily in proximal tubules of CP-treated mice. CP also increased TG accumulation in kidney tissue, which was ameliorated by PPARα ligand. In summary, a PPARα ligand ameliorates CP-mediated nephrotoxicity by increasing LPL activity via increased expression of GPHBP1 and Lmf1 and by reducing expression of Angptl4 protein in the proximal tubule.     

Ferric haem forms of Mycobacterium tuberculosis catalase-peroxidase probed by EPR spectroscopy: Their stability and interplay with pH

Low temperature EPR spectroscopy was used to characterise Mycobacterium tuberculosis catalase-peroxidase in its resting ferric haem state. Several high spin ferric haem forms and no low spin forms were found in the enzyme samples frozen in methanol on dry ice. The EPR spectra depended not only on the pH but also on the buffer type. As a general trend, the higher the pH, the greater the ‘rhombic’ fraction of the high spin ferric haem that was observed. The rhombic form was characterised by well separated two lines in the g = 6 region whereas in the ‘axial’ form the two lines overlap. This pH dependence of the equilibrium of axial and rhombic ferric haem forms is also seen in rapidly freeze-quenched samples. Different high spin ferric haem forms were monitored during a 3 week storage of the enzyme at 4 °C. For some forms, extremal dependences, i.e. those progressing via maxima or minima over storage time, were found. This indicates that the mechanism of the time-dependent transition from one high spin ferric haem form to another must be more complex than a simple single site oxidation.     

Novel 16S rRNA based PCR method targeting Deinococcus spp. and its application to assess the diversity of deinococcal populations in environmental samples

The members of the genus Deinococcus are extensively studied because of their exemplary radiation resistance. Both ionizing and non-ionizing rays are routinely employed to select upon the radiation resistant deinococcal population and isolate them from the majority of radiation sensitive population. There are no studies on the development of molecular tools for the rapid detection and identification of deinococci from a mixed population without causing the bias of radiation enrichment. Here we present a Deinococcus specific two-step hemi-nested PCR for the rapid detection of deinococci from environmental samples. The method is sensitive and specific to detect deinococci without radiation exposure of the sample. The new protocol was successfully employed to detect deinococci from several soil samples from different geographical regions of India. The PCR method could be adapted to a three-step protocol to study the diversity of the environmental deinococcal population by denaturing gradient gel electrophoresis (DGGE). Sequence analysis of the DGGE bands revealed that the samples harbor diverse populations of deinococci, many of which were not recovered by culturing and may represent novel clades. We demonstrate that the genus specific primers are also suitable for the rapid identification of the bacterial isolates that are obtained from a typical radiation enrichment isolation technique. Therefore the primers and the protocols described in this study can be used to study deinococcal diversity from environmental samples and can be employed for the rapid detection of deinococci in samples or identifying pure culture isolates as Deinococcus species.     

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).     

Purification and partial characterization of bacteriocin produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> ssp. <Emphasis Type="Italic">lactis</Emphasis> LL171

Lactic acid bacteria (LAB) are possessing ability to synthesize antimicrobial compounds (like bacteriocin) during their growth. In this regard, novel bacteriocin compound secreting capability of LAB isolated from Tulum Cheese in Turkey was demonstrated. The synthesized bacteriocin was purified by ammonium sulphate precipitation, dialysis and gel filtration. The molecular weight (≈3.4 kDa) of obtained bacteriocin was confirmed by SDS-PAGE, which revealed single peptide band. Molecular identification of LAB strain isolated from Tulum Cheese was conducted using 16S rDNA gene sequencing as Lactococcus lactis ssp. lactis LL171. The amino acid sequences (KKIDTRTGKTMEKTEKKIELSLKNMKTAT) of the bacteriocin from Lactococcus lactis ssp. lactis LL171 was found unique and novel than reported bacteriocins. Further, the bacteriocin was possessed the thermostable property and active at wide range of pH values from 1 to 11. Thus, bacteriocin reported in this study has the potential applications property as food preservative agent.     

Abiotic elicitation of gymnemic acid in the suspension cultures of Gymnema sylvestre

Elicitation is one of the few strategies that find commercial application in the enhancement of secondary metabolite production from plants as well as cell culture systems. Due to their immense medicinal value, production of saponins in suspension cultures has been attempted by many researchers. Gymnema sylvestre is a rich source of gymnemic acids (saponins) that find application in the treatment of diabetes. The present study is an attempt to evaluate the effect of various metal salts (cadmium chloride, mercuric chloride, silver nitrate, cupric chloride, cobaltous chloride and calcium chloride) in eliciting the response from G. sylvestre suspension cultures. The maximum gymnemic acid production in the suspensions was achieved on day 12 of culture, though the maximum biomass was obtained on day 16. Among the different salts, CdCl₂ gave maximum response (59.97 mg/gDCW) at 2 mM concentration after a 24 h time period, while, AgNO₃ gave the least response (18.35 mg/gDCW) on incubation of 48 h at 1 mM concentration, in terms of gymnemic acid accumulation. The accumulation of gymnemic acid was found to be dependent on treatment time and concentration of the elicitor. The enhanced gymnemic acid production shown by the suspensions in response to the metal salts indicates their role in evoking the plant defense mechanisms. These elicitation studies help in providing a platform for improved commercial supply of bioactive gymnemic acids.