Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates

Background  

The application of molecular tools to the analysis of tuberculosis has revealed examples of clonal complexity, such as exogenous reinfection, coinfection, microevolution or compartmentalization. The detection of clonal heterogeneity by standard genotyping approaches is laborious and often requires expertise. This restricts the rapid availability of Mycobacterium tuberculosis (MTB) genotypes for clinical or therapeutic decision-making. A new PCR-based technique, MIRU-VNTR, has made it possible to genotype MTB in a time frame close to real-time fingerprinting. Our purpose was to evaluate the capacity of this technique to provide clinicians with a rapid discrimination between reactivation and exogenous reinfection and whether MIRU-VNTR makes it possible to obtain data directly from stored MTB isolates from recurrent episodes.     

Steroidal dihydrocarbothioic acid amido pyrazoles: synthesis,characterization, cytotoxicity and genotoxicity studies

A new series of steroidal dihydrocarbothioic acid amido pyrazole analogues were synthesized, and after characterization, evaluation for cytotoxicity, comet assay and western blotting was carried out. The synthesis of these analogues is convenient and involves two steps, i.e. aldol condensation as first step followed by nucleophilic addition of thiosemicarbazide across α, β-unsaturated carbonyl as a later step. Quantitative yields of more than 80 % are obtained in both the steps. After characterization by IR, ¹H NMR, ¹³C NMR, MS and analytical data, all the compounds of both series were tested for cytotoxic activity against a panel of different human cancer cell lines by MTT assay, during which compound 3e, 3f, 4e, 4f and 4h are very potent especially against HepG2 and MCF-7 cancer cell lines. Cell cycle analysis depicted the cell death in S-phase while as annexin V-FITC/PI analysis showed that compounds effectively induce apoptosis. Apoptotic degradation of DNA of MCF-7 cells in the presence of different steroidal derivatives was analysed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay). In western blotting analysis, the relative expressions of relevant apoptotic markers depicted an apoptosis by steroidal dihydropyrazole in MCF-7 cancer cells.     

Luminal adenosine stimulates chloride secretion through A1 receptor in mouse jejunum

Adenosine is known to stimulate chloride secretion by mouse jejunum. Whereas the receptor on the basolateral side is believed to be A2B, the receptor involved in the luminal effect of adenosine has not been identified. We found that jejuna expressed mRNA for all adenosine receptor subtypes. In this study, we investigated the stimulation of chloride secretion by adenosine in jejuna derived from mice lacking the adenosine receptors of A1 (A1R) and A2A (A(2A)R) or control littermates. The jejunal epithelium was mounted in a Ussing chamber, and a new method on the basis of impedance analysis was used to calculate the short-circuit current (I(sc)) values. Chloride secretion was assessed by the I(sc) after inhibition of the sodium-glucose cotransporter by adding phloridzin to the apical bathing solution. The effect of apical adenosine on chloride secretion was lost in jejuna from mice lacking the A1R. There was no difference in the response to basolaterally applied adenosine or to apical forskolin. Furthermore, in jejuna from control mice, the effect of apical adenosine was also abolished in the presence of 8-cyclopentyl-1,3-dipropylxanthine, a specific A1R antagonist. Responses to adenosine were identical in jejuna from control and A(2A)R knockout mice. This study demonstrates that A1R (and not A(2A)R) mediates the enhancement of chloride secretion induced by luminal adenosine in mice jejunum.     

Synthesis of tricyclic analogs of stephaoxocanidine and their evaluation as acetylcholinesterase inhibitors

The synthesis of simplified analogs of the novel isoquinoline alkaloid stephaoxocanidine, carrying the oxazaphenalene ABC-ring system of the natural product, and their activity as inhibitors of the enzyme acetylcholinesterase, are reported. 5,6-Dimethoxy-7H -8-oxa-1-aza-phenalen-9-one (5) was as active as a Narcissus extract enriched in galantamine.     

Spatial and temporal transmission risk of Dirofilaria immitis in Argentina

The aim of this work was to assess spatial and seasonal Dirofilaria immitis transmission risk throughout Argentina with models based on the temperature threshold below which filarial development will not proceed in the mosquito (i.e. 14 °C), the occurrence and the number of potential vector mosquito species, and the Heartworm Development Units derived from the degree-days concept. The four models showed a similar increasing southwest-northeast tendency and correlated significantly with canine prevalences used as external validation data. About one-third of Argentina would be suitable for heartworm transmission and the highest risk areas include the north-eastern provinces. According to our models, heartworm transmission is markedly seasonal with peaks in January and February; no region would support transmission throughout the year. To improve the present models, it is necessary to know which mosquito species are competent rather than potential vectors in the country. We believe the present study provides the first risk assessment maps for D. immitis transmission in the Southern Hemisphere and provides a useful guide for heartworm prevention during the transmission periods in different regions of Argentina.     

Calreticulin-melatonin. An unexpected relationship.

Increasing evidence suggests that melatonin can exert some effect at nuclear level. Previous experiments using binding techniques clearly showed the existence of specific melatonin binding sites in cell nucleus of rat liver. To further identify these sites, nuclear extracts from rat hepatocytes were treated with different percentages of ammonium sulfate and purified by affinity chromatography. Subsequent ligand blot analysis shows the presence of two polypeptides of approximately 60 and approximately 74 kDa that bind specifically to melatonin. N-Terminal sequence analysis showed that the 60 kDa protein shares a high homology with rat calreticulin, whereas the 74 kDa protein shows no homology with any known protein. The binding of melatonin to calreticulin was further characterized incubating 2-[125I]melatonin with recombinant calreticulin. Binding kinetics show a Kd = 1.08 +/- 0.2 nm and Bmax = 290 +/- 34 fmol.mg protein-1, compatible with other binding sites of melatonin in the cell. The presence of calreticulin was further identified by Western blot analysis, and the lack of endoplasmic reticulum contamination in our material was assessed by Western blot and immunostaining with anti-calnexin Ig. The results suggest that calreticulin may represent a new class of high-affinity melatonin binding sites involved in some functions of the indoleamine including genomic regulation.     

Proteomic approaches to identifying carbonylated proteins in brain tissue

Oxidative stress plays a critical role in the pathogenesis of a number of diseases. The carbonyl end products of protein oxidation are among the most commonly measured markers of oxidation in biological samples. Protein carbonyl functional groups may be derivatized with 2,4-dinitrophenylhydrazine (DNPH) to render a stable 2,4-dinitrophenylhydrazone-protein (DNP-protein) and the carbonyl contents of individual proteins then determined by two-dimensional electrophoresis followed by immunoblotting using specific anti-DNP antibodies. Unfortunately, derivatization of proteins with DNPH could affect their mass spectrometry (MS) identification. This problem can be overcome using nontreated samples for protein identification. Nevertheless, derivatization could also affect their mobility, which might be solved by performing the derivatization step after the initial electrophoresis. Here, we compare two-dimensional redox proteome maps of mouse cerebellum acquired by performing the DNPH derivatization step before or after electrophoresis and detect differences in protein patterns. When the same approach is used for protein detection and identification, both methods were found to be useful to identify carbonylated proteins. However, whereas pre-DNPH derivatized proteins were successfully analyzed, high background staining complicated the analysis when the DNPH reaction was performed after transblotting. Comparative data on protein identification using both methods are provided.