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Identification of a Protein Kinase A Regulatory Subunit from Leishmania having Importance in Metacyclogenesis Through Induction of Autophagy
No full textcAMP-mediated responses act as modulators of environmental
sensing and cellular differentiation of many
kinetoplastidae parasites including Leishmania.
Although cAMP synthesizing (adenylate cyclase) and
degrading (phosphodiesterase) enzymes have been
cloned and characterized from Leishmania, no cAMPbinding
effector molecule has yet been identified from
this parasite. In this study, a regulatory subunit of
cAMP-dependent protein kinase (Ldpkar1), homologous
to mammalian class I cAMP-dependent protein
kinase regulatory subunit, has been identified from
L. donovani. Further characterization suggested possible
interaction of LdPKAR1 with PKA catalytic
subunits and inhibition of PKAactivity. This PKAregulatory
subunit is expressed in all life cycle stages and
its expression attained maximum level in stationary
phase promastigotes, which are biochemically similar
to the infective metacyclic promastigotes. Starvation
condition, the trigger for metacyclogenesis in the
parasite, elevates LdPKAR1 expression and under
starvation condition promastigotes overexpressing
Ldpkar1 attained metacyclic features earlier than
normal cells. Furthermore, Ldpkar1 overexpression
accelerates autophagy, a starvation-induced cytological
event necessary for metacyclogenesis
and amastigote formation. Conditional silencing of
Ldpkar1 delays the induction of autophagy in the parasite.
The study, for the first time, reports the identification
of a functional cAMP-binding effector molecule from Leishmania that may modulate important cytological
events affecting metacyclogenesi
The Presence of the Iron-Sulfur Motif Is Important for the Conformational Stability of the Antiviral Protein, Viperin
No full textViperin, an antiviral protein, has been shown to contain a CX3CX2C motif, which is conserved in the radical S-adenosylmethionine
(SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to
have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination
of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a
low-resolution predicted structure. The results show that Viperin is an a -b protein containing iron-sulfur cluster at the
center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary
structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants
individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by
alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the
mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational
stability of the mutant proteins may have direct relevance to the absence of their antiviral activity
TLR4 and NKT Cell Synergy in Immunotherapy against Visceral Leishmaniasis
No full textNKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most
cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune
responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to
dendritic cells mediated via Toll like Receptors (TLR) can be modulated by activated invariant Natural Killer T (iNKT) cells. The
terminal b-(1–4)-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-c and
TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and
iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the
protective efficacy of the b-(1–4)-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL) antigen of L. donovani
parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory
signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that
contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected
animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in
visceral leishmaniasis immunotherapeutic
Cigarette Smoke induces p-Benzoquinone–Albumin Adduct in Blood Serum:Implications on Structure and Ligand Binding Properties
No full textEarlier we had reported that irrespective of the source cigarette smoke (CS) contains substantial amounts
of p-benzosemiquinone, which is readily converted to p-benzoquinone (p-BQ) by disproportionation and
oxidation by transition metal containing proteins. Here we show that after CS-exposure, p-BQ-protein
adducts are formed in the lungs as well as serum albumin of guinea pigs. We also show that serum of
human smokers contains p-BQ-albumin adduct. It is known that human serum albumin (HSA) plays a
very important role in binding and transport of a variety of ligands, including fatty acids and drugs. We
show in vitro that p-BQ forms covalent adducts with free amino groups of all twenty amino acids as well as
�-amino groups of lysine residues of HSA in a concentration dependent manner. When HSA is incubated
with p-BQ in the molar ratio of 1:1, the number of p-BQ incorporated is 1. At the molar ratio of 1:60,
the number of p-BQ incorporated is 40. The formation of HSA–p-BQ adduct has been demonstrated by
absorption spectroscopy, MALDI-MS and MALDI-TOF–TOF-MS analyses. Upon complexation with p-BQ,
the secondary structure and conformation of HSA are altered, as evidenced by steady state and timeresolved
fluorescence, circular dichroism, 8-anilino-1-napthalenesulfonic acid binding and differential
scanning calorimetry. Alteration of the structure and conformation of HSA results in impairment of its
ligand binding properties with respect to myristic acid, quercitin and paracetamol. This might be one of
the reasons why transport and distribution of lipids and drugs are impaired in smoker
Role of K+ Binding Residues in Stabilization of Heme Spin state of Leishmania Major Peroxidase
No full textThe endogenous cation in peroxidases may contribute to the type of heme coordination. Here a series of ferric
and ferrous derivatives of wild-type Leishmania major peroxidase (LmP) and of engineered K+ site mutants of
LmP, lacking potassium cation binding site, has been examined by electronic absorption spectroscopy at
25 °C. Using UV–visible spectrophotometry, we show that the removal of K+ binding site causes substantial
changes in spin states of both the ferric and ferrous forms. The spectral changes are interpreted to be, most
likely, due to the formation of a bis-histidine coordination structure in both the ferric and ferrous oxidation
states at neutral pH 7.0. Stopped flow spectrophotometric techniques revealed that characteristics of Compound
I were not observed in the K+ site double mutants in the presence of H2O2. Similarly electron donor
oxidation rate was two orders less for the K+ site double mutants compared to the wild type. These data
show that K+ functions in preserving the protein structure in the heme surroundings as well as the spin
state of the heme iron, in favor of the enzymatically active form of Lm
Synthesis of Some Novel Polynuclear Heteroaromatics Based on Quinolines and Isoquinolines
No full textThe thesis embodies the results of studies on “Synthesis of some novel polynuclear heteroaromatics mainly based on quinoline and isoquinoline”. Quinoline derivatives
are a class of synthetic antibiotics with broad spectrum antibacterial as well as antimalarial activities. The fused quinoline derivatives are also known to be effective in
the treatment of autoimmune conditions (such as rhenumatoid arthritis) with their anti inflammatory effects. Among the numerous structurally diverse derivatives many quinolines show significant biological activity such as quinine, morphine, and multifloramine. Hence, it is not surprising that this structural motif is also an important component in many of today’s pharmaceuticals. Nevertheless, the diversity
of quinoline as well as their biological and pharmaceutical relevance is still motivating academic and industrial researchers to look for new and improved syntheses for quinoline derivatives. Clearly, a number of practical methods have been developed for the synthesis of quinolines in the past century. More recently, especially transition metal catalysis has become a powerful tool for synthetic
methodology but chemical and biological research has now presented a great challenge to synthesize and optimise highly efficient and cost-effective synthetic
routes to some unique and novel biologically active substances
Studies of Protein Conformation and Dynamics Using Fluorescence Correlation Spectroscopy (FCS) and Other Biophysical Methods
No full textThe mechanism by which the unfolded chain of a protein folds into its functional three
dimensional structure has been a matter of intense debate. Some of the major challenges in
protein folding are as follow: a) how do the initial contacts in the unfolded state modulate the
late stage of protein folding? b) How to design experimentation to study the inherent
heterogeneity of the protein folding pathway? This is important because the conventional bulk
spectroscopic measurements have been found inadequate to monitor the intricacies of protein
folding pathways. c) How to study the mechanism of aggregation and design inhibitor molecules
to block protein aggregation? This is because protein aggregation has been shown to have direct
relevance in a number of neurodegenerative diseases.
In this thesis, we have used fluorescence correlation spectroscopy (FCS) to address some of the
above questions. FCS measures fluorescence fluctuations in a small observation volume while
the system is kept under thermodynamic equilibrium. These fluctuations can originate either
from the molecular diffusion inside the observation volume or through any chemical kinetics or
conformational events. FCS is a unique experimental technique which can measure both, the
conformation of the protein molecule and its fluctuation dynamics in the μsec time scale at single
molecule resolution.
We have shown that FCS can be used to study the effect of a number of protein stabilizers on the
conformation and μsec dynamics of fluorescently labelled cytochrome c. Measuring ureainduced
unfolding of cytochrome c in the absence and presence of arginine, we show that
arginine inhibits the formation of a partially folded aggregation prone intermediate. Other
stabilizers, e.g. sucrose, NaCl, and proline, do not show this behaviour. Our result implicate that
the effect of arginine on protein self-association may have come from its interaction with side
chains, while a traditional osmolyte like sucrose influences only the protein backbone. To
validate this hypothesis, we have used surfactant self association as a “super simplified” model
of protein aggregation. Using two common surfactants, namely SDS and DTAB, we have
established the dual nature of arginine. In a separate study, we have shown that initial
hydrophobic collapse and the formation of secondary structure of a protein take place differently
depending on the solution conditions
Modeling the Closed and Open State Conformations of the GABAA Ion Channel - Plausible Structural Insights for Channel Gating
No full textRecent disclosure of high resolution crystal
structures of Gloeobacter violaceus (GLIC) in open state and
Erwinia chrysanthemii (ELIC) in closed state provides newer
avenues to advance our knowledge and understanding of the
physiologically and pharmacologically important ionotropic
GABAA ion channel. The present modeling study envisions
understanding the complex molecular transitions involved in
ionic conductance, which were not evident in earlier disclosed
homology models. In particular, emphasis was put on
understanding the structural basis of gating, gating transition
from the closed to the open state on an atomic scale. Homology modeling of two different physiological states of GABAA was
carried out using their respective templates. The ability of induced fit docking in breaking the critical inter residue salt bridge
(Glu155β2 and Arg207β2) upon endogenous GABA docking reflects the perceived side chain rearrangements that occur at the
orthosteric site and consolidate the quality of the model. Biophysical calculations like electrostatic mapping, pore radius
calculation, ion solvation profile, and normal-mode analysis (NMA) were undertaken to address pertinent questions like the
following: How the change in state of the ion channel alters the electrostatic environment across the lumen; How accessible is
the Cl− ion in the open state and closed state; What structural changes regulate channel gating. A “Twist to Turn” global motion
evinced at the quaternary level accompanied by tilting and rotation of the M2 helices along the membrane normal rationalizes the
structural transition involved in gating. This perceived global motion hints toward a conserved gating mechanism among pLGIC.
To paraphrase, this modeling study proves to be a reliable framework for understanding the structure function relationship of the
hitherto unresolved GABAA ion channel. The modeled structures presented herein not only reveal the structurally distinct
conformational states of the GABAA ion channel but also explain the biophysical difference between the respective state
Use of the Cryptogein Gene to Stimulate the Accumulation of Bacopa Saponins in Transgenic Bacopa Monnieri Plants
No full textGenetic transformation of the Indian medicinal
plant, Bacopa monnieri, using a gene encoding cryptogein,
a proteinaceous elicitor, via Ri and Ti plasmids, were
established and induced bioproduction of bacopa saponins
in crypt-transgenic plants were obtained. Transformed
roots obtained with A. rhizogenes strain LBA 9402 crypt on
selection medium containing kanamycin (100 mg l-1) dedifferentiated
forming callus and redifferentiated to roots
which, spontaneously showed shoot bud induction. Ri
crypt-transformed plants thus obtained showed integration
and expression of rol genes as well as crypt gene. Ti crypttransformed
B. monnieri plants were established following
transformation with disarmed A. tumefaciens strain harboring
crypt. Transgenic plants showed significant
enhancement in growth and bacopa saponin content.
Bacopasaponin D (1.4–1.69 %) was maximally enhanced
in transgenic plants containing crypt. In comparison to
Ri-transformed plants, Ri crypt-transformed plants showed
significantly (p B 0.05) enhanced accumulation of bacoside
A3, bacopasaponin D, bacopaside II, bacopaside III
and bacopaside V. Produced transgenic lines can be used
for further research on elicitation in crypt-transgenic plants
as well as for large scale production of saponins.
Key message The cryptogein gene, which encodes a proteinaceous
elicitor is associated with increase in secondary
metabolite accumulation—either alone or in addition to the
increases associated with transformation by A. rhizogene
Is germline transmission of MAD2 gene deletion associated with human fetal loss?
No full textThe spindle assembly checkpoint (SAC) monitors proper attachment of spindles to the kinetochore during mitotic and
meiotic cell divisions and thus prevents aneuploidy. Chromosomal aneuploidy has been found to be associated with pregnancy loss and birth defects. Mad2 is one of the critical molecules of SAC. Deregulated Mad2 expression has been found to be associated with defective SAC-mediated abnormal meiotic progression in cell studies using animal models. Whether mutation in MAD2L1 is associated with the loss of Mad2 expression in aborted human fetuses is unknown. In this study, a correlation between aneuploidy and MAD2 defect was examined in primary fibroblast cultures obtained from abortuses.We report three trisomic abortuses with undetectable Mad2 expression. Further, quantitative
real-time PCR revealed copy number deletion of MAD2 gene in these fetuses. Analysis of parental DNA samples available from two families revealed copy number loss of the same gene, suggesting Mendelian inheritance of MAD2 deletion. This germline transmission of exonic deletion of MAD2 is possibly associated with its loss of expression resulting in abnormal SAC function, subsequent aneuploidy and pregnancy loss