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Vaccination with Leishmania Hemoglobin Receptor–Encoding DNA Protects Against Visceral Leishmaniasis
No full textLeishmaniasis is a severe infectious disease. Drugs used for leishmaniasis are very toxic, and no vaccine is available.We found that the hemoglobin receptor (HbR) of Leishmania was conserved across various strains of Leishmania,and anti-HbR antibody could be detected in kala-azar patients’ sera. Our results showed that immunization with HbR-DNA induces complete protection against virulent Leishmania donovani infection in both BALB/c mice and
hamsters. Moreover, HbR-DNA immunization stimulated the production of protective cytokines like interferon-g
(IFN-g), interleukin-12 (IL-12), and tumor necrosis factor–a (TNF-a) with concomitant down-regulation of diseasepromoting cytokines like IL-10 and IL-4. HbR-DNA vaccination also induced a protective response by generating
multifunctional CD4+ and CD8+ T cells. All HbR-DNA–vaccinated hamsters showed sterile protection and survived
during an experimental period of 8 months. These findings demonstrate the potential of HbR as a vaccine candidate
against visceral leishmaniasi
Validation of a Simple Resazurin-Based Promastigote Assay for the Routine Monitoring of Miltefosine Susceptibility in Clinical Isolates of Leishmania Donovani
No full textSimple, cost-effective approach for routine surveillance
of parasite susceptibility to antileishmanial drug
miltefosine (MIL) is highly desirable for controlling emergence
of drug resistance in visceral leishmaniasis (VL). We
validated a simple resazurin-based fluorimetric assay using
promastigotes to track natural MIL tolerance in Leishmania
donovani parasites from VL cases (n017) against standard
amastigote assay, in two different labs in India. The interstage
MIL susceptibility correlated strongly (r00.70, p0
0.0018) using J774.A.1 macrophage cell line-based amastigote
assay and fluorescence-based resazurin assay for promastigotes.
Investigation of inter-stage MIL susceptibility
for the same set of clinical isolates in another lab also
showed a strong correlation (r00.72, p00.0012) using
mouse peritoneal macrophages for amastigote assay and
resazurin-based alamar blue assay for promastigotes. Additionally,
parasites from post-kala-azar dermal leishmaniasis
(PKDL) lesions (n07, r00.78, p00.046) and MIL-induced
parasites (r00.92, p00.0001; n03) also exhibited a strongly
correlated inter-stage miltefosine susceptibility. Thus, our
results support the utility of resazurin assay as a simplified
biological tool for MIL susceptibility monitoring in clinical
isolates from MIL-treated VL/PKDL patient
Role and Regulation of the E3 Ubiquitin Ligase Chip in Glioma
No full textCells use incredibly diverse mechanisms to regulate protein stability and degradation, which
in turn is dependent on the proper functioning of two multi-subunit molecular machineries.
The molecular chaperones help nascent polypeptides fold correctly while 26S proteasome degrades
faulty polypeptides and proteins in response to specific signals. This balance between
folding and degradation is highly regulated and is essential to maintaining normal physiology
and development. Multiple genetic and epigenetic alterations in tumour cells upsets this
balance. Many of the chaperone clients are oncogenes and cancer cells use the chaperone machinery
to protect an array of mutated and over-expressed oncoproteins from misfolding and
degradation. Therefore, the chaperones particularly HSP90 is recognized as a crucial facilitator
of “oncogene addiction” and cancer cell survival. Similarly, and surprisingly, cancer cells
are also extremely sensitive to inhibition of the 26S proteasome. It is now well established
that both chaperones and proteasomes are crucial for survival and propagation of tumour
cells.
Carboxy-terminus of Hsc70 interacting protein or CHIP is a chaperone associated E3 ligase
that ubiquitinates its substrates thus channeling them towards proteasomal degradation.
CHIP is thought to be the central player of the cellular protein quality control system and
several of its reported targets are known to play critical roles in multiple aspects of normal
cell function. Given the number of key nodal oncoproteins that are chaperone clients, manipulation
of the protein CHIP may serve the purpose of collapsing, or significantly weakening, a
cancer cell’s safety net. Therefore in an effort to understand how CHIP relates to the process
of tumourigenesis this thesis asks two broad questions. First, what are the protein targets of
CHIP (i.e., the role) and second, whether and how the intracellular distribution of CHIP is
maintained (i.e., the regulation). Our objective in this work is to understand these aspects of
CHIP in order to gain insights into its personality. In addition to providing important functional
understanding of this critical biological molecule, these insights will help in devising
novel strategies for cancer treatment
Characterization of NAD(P)H Cytochrome b5 Oxidoreductase in Leishmania Major
No full textNAD(P)H cytochrome b5 oxidoreductase (Ncb5or) belongs to the cytochrome b5 family of flavohemoprotein that is widely expressed in eukaryotic organisms. Although, Ncb5or is an endoplasmic reticulum-bound protein that has been implicated in lipid metabolism and diabetes, so far no Ncb5or gene has been reported in the unicellular parasitic protozoa Leishmania species. We have cloned, expressed and characterized Ncb5or gene from Leishmania major. Steady state catalysis and spectral studies showed that NADH can quickly reduce the ferric state of enzyme to ferrous state and able to donate electron(s) to external acceptors. To elucidate its exact physiological role in Leishmania, we attempted to create LmNcb5or knock out (KO) mutants by targeted gene replacement technique. Free fatty acid profile in knock out cells revealed marked deficiency in linoleate and linolenate when compared to wild type (WT) or overexpressing (OE) cells. We have shown that Ncb5or deletion results in dramatic increments in O2 consumption, ROS generation, ATP production, and apoptosis in Leishmania. Wild type characteristics are restored in KO cells on pre-treatment with BSA-bound linoleate. Real time PCR studies demonstrate a higher Δ12 desaturase, superoxide dismutase and GAPDH mRNA with a concomitant fall in Δ9 desaturase mRNA expression in LmNcb5or null cell line. We report LmNcb5or to be the redox partner of Δ12 fatty acid desaturase in linoleate biosynthesis of Leishmania and increased oxidative stress and apoptosis are the major consequences of LmNcb5or deficiency in Leishmani
In-Silico Structural and Functional Characterization of a V. cholerae O395 Hypothetical Protein Containing a PDZ1 and an Uncommon Protease Domain
No full textVibrio cholerae, the causative agent of epidemic cholera, has been a constant source of concern for decades. It has
constantly evolved itself in order to survive the changing environment. Acquisition of new genetic elements through
genomic islands has played a major role in its evolutionary process. In this present study a hypothetical protein was
identified which was present in one of the predicted genomic island regions of the large chromosome of V. cholerae O395 showing a strong homology with a conserved phage encoded protein. In-silico physicochemical analysis revealed that the hypothetical protein was a periplasmic protein. Homology modeling study indicated that the hypothetical protein was an unconventional and atypical serine protease belonging to HtrA protein family. The predicted 3D-model of the hypothetical protein revealed a catalytic centre serine utilizing a single catalytic residue for proteolysis. The predicted catalytic triad may
help to deduce the active site for the recruitment of the substrate for proteolysis. The active site arrangements of this predicted serine protease homologue with atypical catalytic triad is expected to allow these proteases to work in different environments of the host
Vibrio cholerae Classical Biotype Is Converted to the Viable Non-Culturable State when Cultured with the El Tor Biotype
No full textA unique event in bacterial epidemiology was the emergence of the El Tor biotype of Vibrio cholerae O1 and the subsequent rapid displacement of the existing classical biotype as the predominant cause of epidemic cholera. We demonstrate that when the El Tor and classical biotypes were cocultured in standard laboratory medium a precipitous decline in colony forming units (CFU) of the classical biotype occurred in a contact dependent manner. Several lines of evidence including DNA release, microscopy and flow cytometric analysis indicated that the drastic reduction in CFU of the classical biotype in
cocultures was not accompanied by lysis, although when the classical biotype was grown individually in monocultures, lysisof the cells occurred concomitant with decrease in CFU starting from late stationary phase. Furthermore, uptake of a membrane potential sensitive dye and protection of genomic DNA from extracellular DNase strongly suggested that the classical biotype cells in cocultures retained viability in spite of loss of culturability. These results suggest that coculturing the classical biotype with the El Tor biotype protects the former from lysis allowing the cells to remain viable in spite of the
loss of culturability. The stationary phase sigma factor RpoS may have a role in the loss of culturability of the classical biotype in cocultures. Although competitive exclusion of closely related strains has been reported for several bacterial species, conversion of the target bacterial population to the viable non-culturable state has not been demonstrated previously and may have important implications in the evolution of bacterial strain
Antimony-resistant but not antimony-sensitive Leishmania donovani up-regulates host IL-10 to overexpress multidrug-resistant protein 1
No full textThe molecular mechanism of antimony-resistant Leishmania donovani (SbRLD)–driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (Mϕs) has been investigated. This study showed that both promastigote and amastigote forms of SbRLD, but not the antimony-sensitive form of LD,express a unique glycan with N-acetylgalactosamine as a terminal
sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in SbRLD (KDSbRLD), compromises the ability to induce the above effects. Infection of Mϕs with KD SbRLD enhanced the sensitivity towardantimonials compared with infection with SbRLD, and infection of BALB/c mice with KD SbRLD caused significantly less organ parasite burden compared with infection induced by SbRLD. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by SbRLD to activate ERK and nuclear translocation of NF-κB involving p50/c-Rel leading to IL-10 induction, whereas
MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway.Interestingly both recombinant IL-10 and SbRLD up-regulate MDR1 in Mϕ with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but Mϕs derived from IL-10−/− mice are unable to show MDR1 up-regulation on infection with SbRLD. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10–driven MDR1 up-regulation is c-Fos/c-Jun and
not NF-κB, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs
Role of WISP3 (Wnt Induced Secreted Protein 3) in Cartilage Maintenance
No full textWISP3 (Wnt induced secreted protein 3) is a multi domain protein which is expressed mostly in cells of mesenchymal origin and in breast epithelial cells. WISP3 shares a similar modular structure with existing CCN family proteins Cyr 61, CTGF and NOV along with WISP1 and WISP2. Mutations in the WISP3 gene are associated with progressive pseudorheumatoid dysplasia(PPRD), an autosomal recessive skeletal disorder manifested in children at 3-8 years of age.Progressive cartilage loss in between articular bone joints and vertebra is one of the important
prognostic factors in patients with PPRD. There is experimental evidence suggesting that WISP3 promotes the expression of extra cellular cartilage specific matrix proteins such as collagen II and aggrecan. Contemporary literature also suggests that WISP3 may be involved in regulating the accumulation of reactive oxygen species, which could otherwise damage cartilage integrity.
WISP3 has a putative IGF binding protein domain, but the precise mechanism by which WISP3 modulates IGF1 functions with respect to cartilage homeostasis is not known. My experimental results suggest that WISP3 interacts with IGF1 and inhibits IGF1 secretion. In addition, WISP3
reduces IGF1 induced collagen X expression and alkaline phosphatase activity most likely through
modulating signaling events that promote RUNX2 transcription factor activation. Furthermore,
WISP3 curbs the IGF1 induced accumulation of ROS, which in turn potentiates WISP3 expression. My findings thus indicate that WISP3 is a potent regulator of chondrocyte
hypertrophy. It is quite possible that PPRD patients suffer from cartilage loss at least partly on account of uncontrolled chondrocyte hypertrophy in the absence of an appropriately functional WISP3 protein and thus strengthen the existing data featuring WISP3 being important for the
maintenance of chondrocyte / cartilage differentiation
An Unusual Diastereoselective Pictet–Spengler Reaction: Synthesis of Novel Tetrahydro-β-Carboline Glycosides
No full textAn unusual kinetic approach to the Pictet–Spengler reaction
was investigated, in which L- or D-tryptophan methyl ester
reacted with aldehydes of 1,2-O-cyclohexylidene-3-allyloxy-α-Dxylofuranose, yielding exclusively the cis or trans diastereomer of tetrahydro-β-carboline glycoside, respectively, with complete stereocontrol
Experimental Evaluation of Anticancer and Antileishmanial Activity of Bungarus Fasciatus and Bungarus Caeruleus Snake Venom
No full textNatural products are used from ancient time as potential therapeutic agents against various pathophysiological conditions throughout the world. Worldwide search is
continuously going on for better treatment and management of the diseases at different stages, which include several natural products or their purified compounds as drugs on
experimental basis. Among the natural products; animals and animal products played significant beneficial role against several diseases (Costa-Neto and Marques, 2000). In
Indian, Chinese, Cuban traditional medicine, Homeopathy, Ayurveda use of animal products (venom, horn, bone, dung, etc) has been mentioned (Gomes et al., 2011). Among the animal products venom of different species had been reported to be used in several pathophysiological conditions. Venom-toxins are of interest in drug design for
their capability to mimic interesting pharmacological properties (Harvey et al., 1998).Venom is one of the deadly poisons of several species have immense biomedical
importance. Snake venom, which is secreted from specialized venom gland, is the mixture of proteins, enzymes, non-protein substances, metal, combination of bioactive
proteins and polypeptides and acts like toxin (Doley and Kini, 2009). These toxins not only help snakes for prey capture and defense but they also possess bioactive molecules/properties. This natural resource has been used as powerful probes for elucidating complex biological processes of vital importance (Pal et al., 2002). Snake venom may serve as starting material for drug development to combat several pathophysiological conditions. Snake venom as medicine was used by human civilization for centuries.Snake venom toxins have widely diverse characteristics. Sushruta (7th century, B.C.), an
Indian physician used snake venom to prolong life. Charak used snake venom for “Udara Roga”. Ayurvedic system of medicine used the venom after “Shodhana” i.e.
detoxification. Chinese physicians used snake venom routinely to treat stroke. In homeopathy medicines vipers, crotalus, cobra venoms are used. Two antibacterial
components from Pseudechis australis snake venom had been isolated which were more potent than standard drug tetracycline used in Aeromonas infections (Stiles et al., 1991). Aggrastat isolated from Echis carinatus had been used as antiplatelet drug (Hartman et al., 1992). Disintegrins from American copperhead snake venom(Agkistrodon contortrix) was used against gliomas as it prevents cells from sticking together and prevent interaction with surrounding tissue (Trikha et al., 1994). In deep vein thrombosis myocardial infarction, pulmonary embolus like condition, defibrinating agents like ancrod from Malayan pitviper, batroxobin from Bothrops atrox and crotalase from Crotalus adamanteus had been used (Markland, 1998). Naja kaouthia (Indian monocellate cobra) venom showed anti-arthritic activity in experimental rat model