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Salmonella has evolved several strategies to counteract intracellular microbicidal agents like reactive oxygen and nitrogen species. However, it is not yet clear how Salmonella escapes lysosomal degradation. Some studies have demonstrated that Salmonella can inhibit phagolysosomal fusion, whereas other reports have shown that the Salmonella-containing vacuole (SCV) fuses/interacts with lysosomes. Here, we have addressed this issue from a different perspective by investigating if the infected host cell has a sufficient quantity of lysosomes to target Salmonella. Our results suggest that SCVs divide along with Salmonella, resulting in a single bacterium per SCV. As a consequence, the SCV load per cell increases with the division of Salmonella inside the host cell. This demands more investment from the host cell to counteract Salmonella. Interestingly, we observed that Salmonella infection decreases the number of acidic lysosomes inside the host cell both in vitro and in vivo. These events potentially result in a condition in which an infected cell is left with insufficient acidic lysosomes to target the increasing number of SCVs, which favors the survival and proliferation of Salmonella inside the host cell.

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host ␦-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit M r of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20 -30% upon addition of Mg 2؉ . The enzyme is inhibited by Zn 2؉ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg 2؉ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.Studies in this laboratory had demonstrated that the malaria parasites (Plasmodium falciparum and Plasmodium berghei) are capable of heme biosynthesis de novo, despite acquiring large amounts of heme from the host red cell hemoglobin in the intraerythrocytic stage (1, 2). Inhibition of the de novo heme biosynthetic pathway leads to death of the parasite and the pathway is therefore a drug target (1-3). Studies on the enzymes of the heme-biosynthetic pathway have revealed that the first enzyme, ␦-aminolevulinate synthase, is coded for by the parasite genome and is localized in the parasite mitochondrion (4, 5). Studies from this laboratory have shown that the second enzyme of the pathway, ␦-aminolevulinate dehydratase (ALAD), 1 in the parasite is of host origin and evidence was provided to show that the enzyme is translocated (imported) from the host red cell into the parasite and is functional (2, 3).At the same time genome sequence data available for P. falciparum indicates that the parasite genome codes for ALAD (PfALAD) and other enzymes of the heme-biosynthetic pathway. Sato et al. (6) have shown by phylogenetic amino acid sequence analysis derived from truncated...

Background: The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients. Objective: To evaluate the protective role of BDNF in a model of sporadic ALS patients. Methods: The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively. Results: ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca²⁺ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed. Conclusion: Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients.

The precise role of autophagy in P. falciparum remains largely unknown. Although a limited number of autophagy genes have been identified in this apicomplexan, only PfAtg8 has been characterized to a certain extent. On the basis of the expression levels of PfAtg8 and the putative PfAtg5, we report that the basal autophagy in this parasite is quite robust and mediates not only the intraerythrocytic development but also fresh invasion of red blood cells (RBCs) in the subsequent cycles. We demonstrate that the basal autophagy responds to both inducers and inhibitors of autophagy. In addition, the parasite survival upon starvation is temporally governed by the autophagy status. Brief periods of starvation, which induces autophagy, help survival while prolonged starvation decreases autophagy leading to stalled parasite growth and reduced invasion. Thus, starvation-induced autophagy is context dependent. Importantly, we report characterization of another autophagy marker in this parasite, the putative PfAtg5 (Pf3D7_1430400). PfAtg5 is expressed in all the intraerythrocytic stages and partially colocalizes with ER, mitochondria, apicoplast and PfAtg8. It is also present on the double membrane bound vesicles. Altogether, these studies pave way for the detailed dissection of P. falciparum autophagy machinery and insights into molecular and functional characterization of its players for developing new therapeutics as antimalarials.