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Synthesis of Substituted Derivatives of Plant Alkaloid Berberine And Studies on Their dna And Rna Binding Aspects
No full textBerberine is one of the most widely distributed alkaloids in the plant kingdom. Berberine and related alkaloids have remarkable biological activity ranging from antileishmanial, antimalarial, antifungal to anticancer activity. More recently the antidiabetic activity and novel cholesterol lowering activity have also been revealed. DNA and RNA are believed to be the primary cellular target of this alkaloid in manifesting its biological activity particularly the anticancer activity. More efficient derivatives of this alkaloid with enhanced DNA and RNA binding efficacy are essential for effective biological applications. There are few reports that 9-position is an important determinant in the DNA and topoisomerase I binding of berberine. But no detailed studies exist on the interaction of 9-substituted analogs with DNA and RNA. Hence in this thesis a series of new 9-O-substituted berberine analogs have been synthesized and their DNA and poly(A) binding studies investigated using various biophysical technique.In the second chapter these analogs were evaluated for their binding to double stranded calf thymus DNA by a wide variety of techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry. The results revealed that these analogs showed more than six times higher binding affinity to DNA compared to berberine. The viscosity and ferrocyanide quenching experiments confirmed that the analogs are stronger intercalative binders to DNA, useful for further potential biological applications. Stronger binding of the analogs was also inferred from circular dichroism studies and thermal melting experiments. Thermodynamics of the binding from isothermal titration calorimetry experiments revealed an entropy driven binding for these analogs compared to the enthalpy driven binding of berberine. A comparative study also revealed that the spacer length is also of significant importance in modulating the DNA binding affinities.
In the third and fourth chapters the novel analogs of berberine with aryl/arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore were evaluated for their binding to single (ss) and double stranded(ds) poly(A) by a wide variety of biophysical techniques. The results suggested that these analogs exhibited several fold higher binding affinity to ss poly(A) relative to berberine. Optical melting and the unique dilution experiments revealed that berberine analogs induced self-structure formation in ss poly(A) and the nature of the substituent was important for the ease of formation of self-structure. Energetics of the binding suggested an entropy driven binding for the analogs in sharp contrast to the enthalpy driven binding of berberine. The study also revealed that the spacer length at the 9-position had an important role in the self-structure formation.In the fifth chapter absorption, fluorescence, circular dichroism and microcalorimetry tchniques were employed to study the interaction of five 9-O-(ω-amino) alkyl ether analogs of berberine with double-stranded calf thymus DNA. The C-9 derivatization resulted in dramatic enhancements in the fluorescence emission of these compounds. Interaction of these analogs, which have an additional recognition motif with DNA, were evaluated through different spectroscopic and calorimetric titration experiments. The analogs remarkably enhanced the DNA binding affinity and the same was directly dependent on the alkyl chain length. The analog with six –CH2 groups enhanced the DNA binding affinity by about thirty three times compared to berberine. The binding became more entropically driven with increasing chain length.
In the sixth and seventh chapters the interaction of berberine and these five 9-O-(ω-amino) alkyl ether analogs with single and double stranded polyadenylic acid has been studied to understand the role of an additional anchoring module in the formation and/or amplification of self-structure in poly(A). The binding was characterized by absorption and fluorescence titration, Job plot and isothermal titration calorimetry. Self-structure formation was confirmed by circular dichroic melting, optical melting, and dilution experiments. Energetics of the interaction revealed that as the alkyl chain length increased, the binding was more entropy dominated. The results showed that berberine and all the analogs induced self-structure formation in poly(A). The length of the alkyl chain had a significant influence on the ease of formation of self-structure. New insights in terms of structural and thermodynamic aspects into self-structure formation in poly(A) by berberine analogs are revealed from these studies. Finally, the significant conclusions from these studies have been summarized at the end of the thesis
Studies on the Anti-Cancer Activity of Corchorusin-d, a Saikosaponin Like Compound Isolated from Corchorus Acutangulus, a Wild Herb
No full textLeukemia is a heterogeneous group of neoplasm arising from the malignant transformation of hematopoietic cells. Development of multi-drug resistance is one of the
major problems in leukemia. On the other hand melanoma is a less common but deadly type of cancer among all types of skin cancer. Therefore, exploration of new molecules
that could revolutionize the management of the disease is urgently needed. Drug discovery from medicinal plants has played an important role in the treatment of cancer
and indeed clinical applications of plant secondary metabolites and their derivatives towards combating cancer have been common over the last half centuries. Considering
the huge natural resources of India, there is scope for development of potential therapeutic agents from plants. Keeping in mind the thrust of present scenario and based
on established reports, the present study was carried out to explore constituents Corchorus acutangulus Lam. (C. aestuans) for anticancer activity. Except one isolated
study by a Chinese group, not much work has been done to find out the biological activity of the plant extract and isolates thereof
Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis
No full textVisceral leishmaniasis (VL) or kala-azar, a disseminated infection of the lymphoreticular system of the body, is marked by severe defect in immune system of the host. Successful cure of VL depends on the immune status of the host in combination with the effects of the antileishmanial drugs. The rationale approach towards eradication of this disease would be to potentiate the immune functioning of the host in addition to parasite killing. This review deals with different aspects of adaptive and innate immune
responses and explores their role in protection or pathogenesis of VL. IL-10 has emerged as the principal cytokine responsible
for disease pathogenesis, although evidences regarding its source during active VL remain inconclusive. On the other hand, IFNγ,
under the influence of IL-12, is mostly correlated with healing of the disease. Chemokines are important in mounting cell-mediated
immune response as they can prevent parasite invasion in association with cytokines. Different types of T cells like CD4, CD8
and NK T cells also contribute to the immunology of this disease. In spite of conflicting reports, the role of regulatory T cells in
VL pathogenesis is important. Recently discovered Th17 subset and its different members have been reported to perform diverse
functions in the course of VL and leishmaniasis as a whole. Innate immune responses, depending on the cell types, are essential
in early parasite detection and subsequent development of an efficient NK cell response. Immunotherapy targeting IL-10 could
be looked upon as an interesting option for the treatment of V
A Study on Cybrid Model of Parkinson’s Disease
No full textParkinson’s disease (PD) is a neurodegenerative disease characterized by specific loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the
brain. Of the many known causes or risk factors till date mitochondrial dysfunction is considered to play one of the major roles in the pathogenesis of the disease. Existence
of mitochondrial dysfunction is reported in the blood and brain of parkinsonian patients. Reports suggest complex I inhibition in the postmortem brain samples of idiopathic PD patients. Though there are various cell and animal models to approach and study the mitochondrial genetics and proteomics the correct representation of the mitochondrial dysfunction occurring in humans cannot be mirrored in such condition. Human cell line study is one way to represent the disease pathology occurring and for investigating the causes and also for designing novel drugs for the treatment of the disease
Insight Into the Embryo-Uterus Molecular Cross Talk in the Process of Implantation: A Study Using Uterine Specific Stat-3 Knockout Mice Model.
No full textFrom the research and health perspectives, there is an imperative need for a better understanding of reproductive mechanisms and related health issues. Regulation of
fertility is a major factor in population control and, indirectly, in the eradication of poverty. Conversely studies in reproduction can also provide solutions to infertility. These domains along with the successful completion of gestation to a healthy birth are
of direct relevance to human maternal and fetal health. Also recent studies have shown that the completion of normal gestation is an important key to long term health
far into adulthood, and therefore of benefit to society
Mechanism of Amphotericin B Resistance in Clinical Isolates of Leishmania donovani
No full textThe clinical value of amphotericin B, the mainstay therapy for visceral leishmaniasis in sodium antimony gluconatenonresponsive
zones of Bihar, India, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding
of the underlying mechanisms is the need of the hour. We have selected an amphotericin B-resistant clinical isolate
which demonstrated 8-fold-higher 50% lethal doses (LD50) than an amphotericin B-sensitive strain to explore the mechanism of
amphotericin B resistance. Fluorimetric analysis demonstrated lower anisotropy in the motion of the diphenylhexatriene fluorescent
probe in the resistant strain, which indicated a higher fluidity of the membrane for the resistant strain than for the sensitive
strain. The expression patterns of the two transcripts of S-adenosyl-L-methionine:C-24-�-sterol methyltransferase and the
absence of ergosterol, replaced by cholesta-5,7,24-trien-3�-ol in the membrane of the resistant parasite, indicate a decreased
amphotericin B affinity, which is evidenced by decreased amphotericin B uptake. The expression level of MDR1 is found to be
higher in the resistant strain, suggesting a higher rate of efflux of amphotericin B. The resistant parasite also possesses an upregulated
tryparedoxin cascade and a more-reduced intracellular thiol level, which helps in better scavenging of reactive oxygen
species produced by amphotericin B. The resistance to amphotericin B was partially reverted by the thiol metabolic pathway and
ABC transporter inhibitors. Thus, it can be concluded that altered membrane composition, ATP-binding cassette transporters,
and an upregulated thiol metabolic pathway have a role in conferring amphotericin B resistance in clinical isolates of Leishmania donovani
Thionine Interaction to DNA: Comparative Spectroscopic Studies on Double Stranded Versus Single Stranded DNA
No full textInteraction of thionine with double stranded and
single stranded calf thymus DNA has been studied by
absorbance, fluorescence, competition dialysis, circular
dichroism and isothermal titration calorimetry. Binding to
the native double stranded DNA conformation induced
strong quenching in fluorescence spectrum of thionine.
Linear Scatchard plots indicated the binding to be of one
type and the affinity values evaluated to be of the order of
105 M−1 with double stranded DNA. Fluorescence quenching
was much weaker with single stranded DNA and the
binding affinity was about one order lower. Ferrocyanide
quenching studies revealed that the fluorescence emission
of dye molecules bound to the double stranded DNA was
quenched much less compared to those bound to the single
stranded DNA. Furthermore, there was significant emission
polarization for the bound dye molecules and strong energy
transfer from the DNA base pairs to the dye molecules
indicating intercalative binding to ds DNA. Salt dependence
of the binding phenomenon revealed that electrostatic
forces played a significant role in the binding process. The
intercalation of the dye molecules to double stranded DNA
and simple stacking to single strands was proved from these
fluorescence techniques. Support to the fluorescence results
have been derived from absorption, circular dichroic and
dialysis results. Calorimetric studies suggested that the
binding to ds DNA conformation was both enthalpy and entropy favoured while that to ss DNA was predominantly
entropy favoured
Purification and Characterization of a Trehalase–Invertase Enzyme with Dual Activity from Candida Utilis
No full textTrehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by
two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active
against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate
specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose.
The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the
entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step
of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects
on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also
differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring
at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of
the purified protein to previously known neutral trehalases. While earlier workers mentioned independent
purification of neutral trehalase or invertase from different sources, the present study reports the
purification of a single protein showing dual activity
A Genetic Network That Balances Two Outcomes Utilizes Asymmetric Recognition of Operator Sites
No full textStability and induction of the lysogenic state of bacteriophage l are balanced by a complex regulatory network. A
key feature of this network is the mutually exclusive cooperative binding of a repressor dimer (CI) to one of two pairs of binding
sites, OR1-OR2 or OR2-OR3. The structural features that underpin the mutually exclusive binding mode are not well understood.
Recent studies have demonstrated that CI is an asymmetric dimer. The functional importance of the asymmetry is not fully clear.
Due to the asymmetric nature of the CI dimer as well as its binding sites, there are two possible bound orientations. By fluorescence
resonance energy transfer measurements we showed that CI prefers one bound orientation. We also demonstrated that
the relative configuration of the binding sites is important for CI dimer-dimer interactions and consequent cooperative binding.
We proposed that the operator configuration dictates the orientations of the bound CI molecules, which in turn dictates CI cooperative
interaction between the OR1-OR2 or OR2-OR3, but not both. Modeling suggests that the relative orientation of the C- and
N-terminal domains may play an important role in the mutually exclusive nature of the cooperative binding. This work correlates
unique structural features of a transcription regulatory protein with the functional properties of a gene regulatory networ
Redox Regulation in Malaria: Current Concepts and Pharmacotherapeutic Implications
No full textMalaria imposes a serious threat to human and becomes more prevalent due to the emergence of drug resistant parasite.
Understanding of the underlying mechanism of drug resistance and identification of novel drug targets are key effective processes for the
management of malaria. Malaria parasite is highly susceptible to oxidative stress but lives in a pro-oxidant rich environment containing
oxygen and iron, which produce a large amount of reactive oxygen species. Management of oxidative stress in malaria parasite is tightly
regulated through active redox and antioxidant defense systems. The elevation of oxidative stress as a result of inhibition of any
component of this defense system leads to redox imbalance and ultimately parasite death. Therefore, redox system plays an indispensable
role for the survival of parasite within the host. Identification of key molecules, which disrupt parasite redox balance by altering key
redox reactions and promote oxidative stress in parasites, would be an effective approach to develop novel antimalarial drugs. During the
last few decades, contributions by researchers around the globe provide even better understanding of redox biology of malaria parasite.
Here, in this review, we are highlighting the knowledge gathered so far regarding the essential redox-active processes and their
components in malaria parasite to overcome elevated oxidative insults. We have also given maximum efforts to enlist currently used
redox-active antimalarials, their mode of action and pharmacotherapeutic implications