A molecular analysis of desiccation tolerance mechanisms in the anhydrobiotic nematode Panagrolaimus superbus using expressed sequenced tags

<p>Abstract</p> <p>Background</p> <p>Some organisms can survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. <it>Panagrolaimus superbus </it>is a free-living anhydrobiotic nematode that can survive rapid environmental desiccation. The mechanisms that <it>P. superbus </it>uses to combat the potentially lethal effects of cellular dehydration may include the constitutive and inducible expression of protective molecules, along with behavioural and/or morphological adaptations that slow the rate of cellular water loss. In addition, inducible repair and revival programmes may also be required for successful rehydration and recovery from anhydrobiosis.</p> <p>Results</p> <p>To identify constitutively expressed candidate anhydrobiotic genes we obtained 9,216 ESTs from an unstressed mixed stage population of <it>P. superbus</it>. We derived 4,009 unigenes from these ESTs. These unigene annotations and sequences can be accessed at <url>http://www.nematodes.org/nembase4/species_info.php?species=PSC</url>. We manually annotated a set of 187 constitutively expressed candidate anhydrobiotic genes from <it>P. superbus</it>. Notable among those is a putative lineage expansion of the <it>lea </it>(late embryogenesis abundant) gene family. The most abundantly expressed sequence was a member of the nematode specific <it>sxp/ral-2 </it>family that is highly expressed in parasitic nematodes and secreted onto the surface of the nematodes' cuticles. There were 2,059 novel unigenes (51.7% of the total), 149 of which are predicted to encode intrinsically disordered proteins lacking a fixed tertiary structure. One unigene may encode an exo-β-1,3-glucanase (GHF5 family), most similar to a sequence from <it>Phytophthora infestans</it>. GHF5 enzymes have been reported from several species of plant parasitic nematodes, with horizontal gene transfer (HGT) from bacteria proposed to explain their evolutionary origin. This <it>P. superbus </it>sequence represents another possible HGT event within the Nematoda. The expression of five of the 19 putative stress response genes tested was upregulated in response to desiccation. These were the antioxidants <it>glutathione peroxidase, dj-1 </it>and <it>1-Cys peroxiredoxin</it>, an <it>shsp </it>sequence and an <it>lea </it>gene.</p> <p>Conclusions</p> <p><it>P. superbus </it>appears to utilise a strategy of combined constitutive and inducible gene expression in preparation for entry into anhydrobiosis. The apparent lineage expansion of <it>lea </it>genes, together with their constitutive and inducible expression, suggests that LEA3 proteins are important components of the anhydrobiotic protection repertoire of <it>P. superbus</it>.</p

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF RELATED SUBSTANCES IN FAMPRIDINE DRUG SUBSTANCE AND TABLET DOSAGE FORMS

  Objective: The objective of this method is to develop a stability-indicating reversed phase high performance liquid chromatographic method for the quantification of related substances in the drug substance and tablet dosage form of Fampridine.Methods: Inertsil ODS 3V, (150 mm × 4.6 mm, 5 μm particle size) column was used for the separation of analytes. Mobile phase A was prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate (0.05 mol) and 1 g of 1-octane sulfonic acid into a 1000 ml of water, pH was adjusted to 4.0±0.05 with diluted orthophosphoric acid. Mobile phase B was prepared by mixing the above phosphate buffer (pH 4.0) and acetonitrile in 20:80 (% v/v). Gradient mode was used with the flow rate of 1.0 ml/minutes, and the peaks were monitored at 260 nm.Results: Linearity results showed that the correlation coefficient (r2) is &gt;0.995 for individual active drug substances as well as their related substances in the range of limit of quantification to 150% of the specification concentration (0.5% with respect to sample concentration of 0.4 mg/ml). Accuracy of the method was established with their recovery values in the range of 98.5-104.5% with the % RSD not more than 1.7%. The method was proved by highly precise (% RSD of intra-day and inter-day study was not more than 4.3%) and more robust.Conclusion: Present method is able to separate two related compounds with each other and with the main drug substance with the resolution more than 2.0. The test standard solution and test solution were found to be stable in diluent up to 24 hrs. The mass balance of forced degradation of formulations is close to 99% made this method as a stability indicating method