Demonstration by heterologous expression that the Leishmania SCA1 gene encodes an arabinopyranosyltransferase

In part of the life cycle within their sand fly vector, Leishmania major parasites first attach to the fly's midgut through their main surface adhesin lipophosphoglycan (LPG) and later resynthesize a structurally distinct LPG that results in detachment and eventual transmission. One of these structural modifications requires the addition of alpha1,2-D-arabinopyranose caps to beta1,3-galactose side chains in the phosphoglycan repeat unit domain of LPG. We had previously identified two side chain arabinose genes (SCA1/2) that were involved in the alpha1,2-D-Arap capping. SCA1/2 exhibit canonical glycosyltransferase motifs, and overexpression of either gene leads to elevated microsomal alpha1,2-D-ArapT activity, resulting in arabinopyranosylation of beta1,3-Gal side chains in LPG (hereafter called side chain D-arabinopyranosyltransferase [sc-D-ArapT]). Heterologous expression in a null arabinose background was used to determine whether the SCA1 gene encodes the actual sc-D-ArapT. SCA1 expression constructs introduced into both mammalian COS-7 cells and the baculovirus-sf9 cell system exhibited considerable expression of the protein. However, functional sc-D-ArapT activity was observed only in the latter. In in vitro assays incubated with guanidine 59-diphosphate (GDP)-D-[3H]Arap as the sugar donor and utilizing exogenous LPG as an acceptor, significant sc-D-ArapT activity was observed when microsomes from the baculovirus-sf9 cells were incubated in presence of the LPG acceptor. No activity was observed in the absence of LPG. These results demonstrate that SCA1 encodes a sc-D-ArapT and provide the first example of heterologous expression of a D-ArapT gene.     

Leishmania infantum: Lipophosphoglycan intraspecific variation and interaction with vertebrate and invertebrate hosts

Interspecies variations in lipophosphoglycan (LPG) have been the focus of intense study over the years due its role in specificity during sand fly-Leishmania interaction. This cell surface glycoconjugate is highly polymorphic among species with variations in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO(4) backbone of repeat units. However, the degree of intraspecies polymorphism in LPG of Leishmania infantum (syn. Leishmania chagasi) is not known. In this study, intraspecific variation in the repeat units of LPG was evaluated in 16 strains of L. infantum from Brazil, France, Algeria and Tunisia. The structural polymorphism in the L. infantum LPG repeat units was relatively slight and consisted of three types: type I does not have side chains; type II has one β-glucose residue that branches off the disaccharide-phosphate repeat units and type III has up to three glucose residues (oligo-glucosylated). The significance of these modifications was investigated during in vivo interaction of L. infantum with Lutzomyia longipalpis, and in vitro interaction of the parasites and respective LPGs with murine macrophages. There were no consequential differences in the parasite densities in sand fly midguts infected with Leishmania strains exhibiting type I, II and III LPGs. However, higher nitric oxide production was observed in macrophages exposed to glucosylated type II LPG.     

Exposure to 50 Hz electromagnetic field raises the levels of the anti-apoptotic protein BAG3 in melanoma cells

The expression of the anti‐apoptotic protein BAG3 is induced in several cell types by exposure to high temperature, oxidants, and other stressful agents. We investigated whether exposure to 50 Hz electromagnetic fields raised BAG3 levels in the human melanoma cell line M14, in vitro and in orthotopic xenografts. Exposure of cultured cells or xenografts for 6 h or 4 weeks, respectively, produced a significant (P < 0.01) increase in BAG3 protein amounts. Interestingly, at the same times, we could not detect any significant variation in the levels of HSP70/72 protein or cell apoptosis. These results confirm the stressful effect of exposure to ELF in human cells, by identifying BAG3 protein as a marker of ELF‐induced stress. Furthermore, they suggest that BAG3 induction by ELF may contribute to melanoma cell survival and/or resistance to therapy. J. Cell. Physiol. 226: 2901–2907, 2011. © 2011 Wiley‐Liss, Inc.     

Micelle‐enhanced spectrofluorimetric method for determination of sitagliptin and identification of potential alkaline degradation products using LC‐MS

A novel, quick, simple and highly sensitive spectrofluorimetric method was developed and validated for the determination of sitagliptin (SG) in its pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behavior of sitagliptin in an SDS micellar system. In an aqueous solution of phosphate buffer pH 4.0, the fluorescence intensity of SG in the presence of SDS was greatly enhanced, by 200%, i.e. twofold enhancement. The fluorescence intensity of SG was measured at 300 nm after excitation at 270 nm. The method showed good linearity in the range 0.03–10.0 µg/mL with a good correlation coefficient (r = 0.9998). The limits of detection and quantitation values were 5.31 and 16.1 ng/mL, respectively. The proposed method was successfully applied to the analysis of SG in its single and co‐formulated commercial tablets; the results were in good agreement with those obtained using a reference method. Application of the proposed method was extended to stability studies of SG after exposure to different forced degradation conditions according to the ICH guidelines, such as acidic, alkaline, thermal, photo‐ and oxidative stress. The chemical structure of certain potential degradation products (DPs) were investigated using LC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

Integrated stratigraphy and astronomical calibration of the Serravallian/Tortonian boundary section at Monte Gibliscemi (Sicily, Italy)

Results are presented of an integrated stratigraphic (calcareous plankton biostratigraphy, cyclostratigraphy and magnetostratigraphy) study of the Serravallian/Tortonian (S/T) boundary section of Monte Gibliscemi (Sicily, Italy). Astronomical calibration of the sedimentary cycles provides absolute ages for calcareous plankton bio-events in the interval between 9.8 and 12.1 Ma. The first occurrence (FO) of Neogloboquadrina acostaensis, usually taken to delimit the S/T boundary, is dated astronomically at 11.781 Ma, pre-dating the migratory arrival of the species at low latitudes in the Atlantic by almost 2 million years. In contrast to delayed low-latitude arrival of N. acostaensis, Paragloborotalia mayeri shows a delayed low-latitude extinction of slightly more than 0.7 million years with respect to the Mediterranean (last occurrence (LO) at 10.49 Ma at Ceara Rise; LO at 11.205 Ma in the Mediterranean). The Discoaster hamatus FO, dated at 10.150 Ma, is clearly delayed with respect to the open ocean. The ages of D. kugleri first and last common occurrence (FCO and LCO), Catinaster coalitus FO, Coccolithus miopelagicus last (regular) occurrence (L(R)O) and the D. hamatus/neohamatus cross-over, however, are in good to excellent agreement with astronomically tuned ages for the same events at Ceara Rise (tropical Atlantic), suggesting that both independently established timescales are consistent with one another. The lack of a reliable magnetostratigraphy hampers a direct comparison with the geomagnetic polarity timescale of Cande and Kent (1995; CK95), but ages of calcareous nannofossil events suggests that CK95 is significantly younger over the studied time interval. Approximate astronomical ages for the polarity reversals were obtained by exporting astronomical ages of selected nannofossil events from Ceara Rise (and the Mediterranean) to eastern equatorial Pacific ODP Leg 138 Site 845, which has a reliable magnetostratigraphy.Our data from the Rio Mazzapiedi–Castellania section reveal that the base of the Tortonian stratotype corresponds almost exactly with the first regular occurrence (FRO) of N. acostaensis s.s. as defined in the present study, dated at 10.554 Ma. An extrapolated age of 11.8 Ma calculated for the top of the Serravallian stratotype indicates that there is a gap between the top of the Serravallian and the base of the Tortonian stratotype, potentially rendering all bio-events in the interval between 11.8 and 10.554 Ma suitable for delimiting the S/T boundary. Despite the tectonic deformation and the lack of a magnetostratigraphy, Gibliscemi remains a candidate to define the S/T boundary by means of the Tortonian global boundary stratotype section and point (GSSP).