Targeting efflux pumps—In vitro investigations with acridone derivatives and identification of a lead molecule for MDR modulation

To search multi drug resistance modulators, acridones carrying hydroxyl amine substituent at N-10 and COOH/Cl at C-4 were investigated for their interactions with the three components of efflux pump viz. P-gp, ATP, and Mg²⁺. Experimental and theoretical results indicated that the compounds with COOH group at C-4 interact with P-gp and Mg²⁺ while other set of compounds with Cl at C-4 interact with ATP and Mg²⁺. Spot assay and R6G influx/efflux assay of compound 3 using Candida albicans showed decrease in the fungal growth and efflux of R6G, respectively, in presence of compound 3 suggesting the suitability of this compound for MDR modulation.     

Identification and Partial Characterization of a Dynamin-Like Protein,EhDLP1, from the Protist Parasite Entamoeba histolytica

The dynamin superfamily of proteins includes a large repertoire of evolutionarily conserved GTPases that interact with different subcellular organelle membranes in eukaryotes. Dynamins are thought to participate in a number of cellular processes involving membrane remodeling and scission. Dynamin-like proteins (DLPs) form a subfamily of this vast class and play important roles in cellular processes, such as mitochondrial fission, cytokinesis, and endocytosis. In the present study, a gene encoding a dynamin-like protein (EhDLP1) from the protist parasite Entamoeba histolytica was identified and the protein was partially characterized using a combination of in silico, biochemical, and imaging methods. The protein was capable of GTP binding and hydrolysis, lipid binding, and oligomerization. Immunofluorescence studies showed the protein to be associated with the nuclear membrane. A mutant of EhDLP1 lacking GTP binding and hydrolyzing activities did not associate with the nuclear membrane. The results suggest a nucleus-associated function for EhDLP1.Dynamins are a vast family of GTPases implicated in myriad processes, some of which lead to alteration of membrane structure (22). Classical dynamins, such as mammalian dynamins 1 to 3 (5) and the shibire protein from Drosophila melanogaster (29), are required mainly for scission of vesicles, acting as mechanoenzymes or molecular switches (12). In addition, several dynamin-like proteins (DLPs) have been identified in different organisms ranging from yeast to mammals. DLPs play a key role in the division of organelles such as chloroplasts, mitochondria, and peroxisomes (15, 22). For example, Candida albicans Vps1 has been shown to be associated with virulence-related phenotypes like filamentation and biofilm formation (2). DLPs have also been identified in protists. Downregulation or ablation of the gene products in protists by RNA interference or other methods has helped to decipher the multiple functions carried out by these proteins. These include mitochondrial division and endocytosis in Trypanosoma brucei (6, 20), cytokinesis in Dictyostelium discoideum (31), phagocytosis in Paramecium species (30), endocytic transport in Giardia lamblia (11), and biogenesis of secretory vesicles in Toxoplasma gondii (4). Apart from cellular membranes, some DLPs may also associate with nuclear membranes. Recently, a study on Tetrahymena thermophila reported the requirement of Drp6 for macronuclear development (23). The human DLP MxB has been shown previously to localize to the cytoplasmic face of the nuclear envelope and is involved in regulation of nuclear import (14). Dynamins and DLPs share a minimal domain architecture which includes an N-terminal GTPase domain, a middle domain, and a GTPase effector domain (GED). The GED is involved in enzyme oligomerization and the regulation of the GTPase activity. The GTPase domain contains a well-conserved GTP binding motif required for guanine-nucleotide binding and hydrolysis (22). DLPs lack a pleckstrin homology (PH) domain and a proline-rich domain (PRD), normally associated with protein-lipid and protein-protein interaction.The endocytic, secretory, and adhesion pathways of the parasite Entamoeba histolytica play crucial roles in nutrient uptake, host cell destruction, and the endocytosis of gut resident bacteria, erythrocytes, and cell debris (21). The trophozoites of E. histolytica are known to have robust endocytic capabilities, turning over approximately a third of their cellular volume every hour (1, 19). The presence of a classical receptor-mediated pathway has not yet been clearly demonstrated, though some of the molecules involved in this pathway, such as clathrin, have been identified in E. histolytica (28). Typical eukaryotic cytoplasmic organelles have not been observed in this organism. However, the functional equivalents of a Golgi network and an endoplasmic reticulum are reported to be present (3, 26). Entamoeba also contains a genomeless variant of mitochondria, termed mitosomes (17). The division or biogenesis of these organelles during cell division is not understood. Nuclear division in E. histolytica occurs without nuclear membrane dissolution and reassembly. Since dynamins and DLPs are known to be involved in endocytosis and organelle division, it is likely that these proteins may be performing similar functions in this organism. Although the E. histolytica genome encodes putative dynamins and DLPs, none of these have been characterized. In order to understand the roles of these molecules in amebic biology, we have initiated studies to characterize these proteins from E. histolytica. Here, we report the basic characterization of E. histolytica dynamin-like protein 1 (EhDLP1).     

Autophagy in immune defense against Mycobacterium tuberculosis

Autophagy is a newly recognized innate and adaptive immunity defense against intracellular pathogens, in keeping with its role as a cytoplasmic maintenance pathway. Induction of autophagy by physiological, pharmacological or immunological means can eliminate intracellular Mycobacterium tuberculosis, providing one of the first examples of the immunological role of autophagy. Under normal circumstances, M. Tuberculosis survives in macrophages by inhibiting phagolysosome biogenesis. Induction of autophagy overcomes the mycobacterial phagosome maturation block, and delivers the tubercle bacilli to degradative compartments where they are eliminated.     

Mechanism of imipenem resistance in metallo-β-lactamases expressing pathogenic bacterial spp. and identification of potential inhibitors: An in silico approach

The World Health Organization reports that millions of people around the world are infected with antibiotic-resistant bacteria. Such resistance is more common in Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae strains because of the expression of the metallo-β-lactamases (MBLs) namely Imipenemase (IMP)-1, IMP-2, New Delhi metallo-β-lactamases-, Verona imipenemase (VIM)-4, VIM-5, and VIM-7. We did an in silico analysis to understand the resistance mechanism of imipenem at the structural level. Our modeling studies reveal that the VIM-4-imipenem complex has highest binding energy and forms a stable complex as indicated by a consensus score (C-score) value of 5.44. The intense interaction between the substrate and the β-lactamases leads to the increased hydrolysis of the substrate resulting in rapid hydrolysis of the antibiotic imipenem by VIM-4. Virtual screening of compounds from the ZINC database targeting VIM-4 was done, and we found compound ZINC44608383 as the high binding energy compound with the C-score value of 5.58. This compound could be exploited for inhibitor design and development. The current study helps us to understand the resistance mechanism of imipenem in MBL-expressing strains. Also, we have identified a probable inhibitor for VIM-4. We believe that our results will be useful for researchers in designing potent inhibitors for VIM-4.     

Putrescine Influences Growth and Production of Coumarins in Hairy Root Cultures of Witloof Chicory (Cichorium intybus L. cv. Lucknow Local)

The effect of putrescine (Put) on the growth and production of two coumarins, esculin and esculetin, in hairy roots of chicory (Cichorium intybus L. cv. Lucknow local) was examined. To study the role of Put on growth and production of coumarins, polyamine inhibitors, namely α-dl-difluromethylornithine and α-dl-difluromethylarginine were used at 1 mM concentration. Put treatment at 1.5 mM produced a 1.9-fold increase in the growth of hairy roots, as well as the production of esculin and esculetin. The treatments with polyamine (PA) inhibitors resulted in much lower growth and production of coumarins compared with both 1.5-mM Put treatment and the control. Both free and conjugated PAs were studied over the whole culture period, and conjugates of all three PAs, namely Put, spermidine, and spermine, were higher than free PAs throughout the culture period. The treatments with PA inhibitors showed lower levels of endogenous PAs compared with Put-treated samples. The treatment with 1.5 mM Put showed maximum accumulation of endogenous conjugated Put (2,098 ± 157 nmoles gm⁻¹ fresh weight). The production of esculin and esculetin was strictly correlated with growth in all treatments. Put at 1.5 mM resulted in greater length of primary root (18.3 ± 1.4 cm) as compared with the control (11 ± 0.9 cm) and larger numbers of secondary and tertiary roots. Received July 14, 1999; accepted October 5, 1999     

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.