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SynthesisofNewSomatostatinReceptorBindingRadio-labelledPeptides: Physicochemical and Biological Evaluation and Scintigraphic Studies
No full textPeptides are important regulators of growth and cellular functions not only in normal tissue but also in tumors. Peptides are becoming of increasing interest in nuclear oncology for targeted tumor diagnosis and therapy. 111In-diethylenetriaminepentaacetic acid (111In-DTPA) labelled Octreotide (Octreoscan; Mallinckrodt / Inc.) was the first routinely used tracer in nuclear medicine for the localization and staging of somatostatin receptor positive tumors. Stable somatostatin derivatives such as Octreotide / Lantrotide are the most frequently used radiopharmaceuticals acting through specific binding to somatostatin receptors; however, they do not bind with high affinity to all five receptor subtypes. 111In-DTPA-Octreotide based imaging served as a sensitive and specific technique for somatostatin receptor scintigraphy that can detect in-vivo somatostatin receptors in various tumors mainly of endocrine origin. 111In-DTPA-Octreotide is commercially available for imaging; however the use of 111In as a radioisotope entails high cost, limited availability, suboptimal image quality and a high radiation burden to the patient. These disadvantages were overcome by the use of 99mTc labelled somatostatin analogs. Thus newly developed somatostatin analogs labelled with 99mTc were intensively studied in the proposed research work.New somatostatin analogs (octreotide derivatives) like HYNIC-Tyr3-Octreotide (TOC), HYNIC-His3-Octreotate and HYNIC-Met3-Octreotate (TATE) were synthesized by standard Fmoc Solid Phase Peptide Synthesis (SPPS) on O-t-butylthreoninol-2-chloro trityl resin for TOC derivatives and H-Thr(tBu)-2-Cl trityl resin for TATE derivatives on a semi automated peptide synthesizer according to our laboratory protocol. Coupling of prochelator to peptide was performed in the presence of excess of HYNIC-BOC in solid phase. BOC protected HYNIC was also synthesized and characterized in our laboratory. Then the peptide chains were cyclized by formation of cys-cys disulfide bond through Iodine oxidation method. These peptides were deblocked and deprotected from resin and lateral protecting groups by using trifluoro acetic acid cocktail. Then purification was achieved by semi-preparative HPLC. The structures of the peptides were then elucidated by 1D NMR (1H NMR, 13C NMR, DEPT 90, 135), 2D NMR (HSQC, HMBC, COSY), MALDI mass and FT-IR spectroscopy. The peptides were again quantitated by amino acid analysis of a 24 h acid hydrolysate using Waters Accu-Tag chemistry together with a Waters HPLC system. Conformational analysis of the peptides was performed by circular dichorism spectroscopy in pure water and trifluroethanol-water (1:1)
Wnt/�-Catenin Pathway Is Regulated by PITX2 Homeodomain Protein and Thus Contributes to the Proliferation of Human Ovarian Adenocarcinoma Cell, SKOV-3
No full textWnt pathway and homeodomain proteins are associated with cancer, but their interaction in ovarian cancer
cells has not been studied.PITX2 itself and through inducing Wnt ligands activates the canonical Wnt pathway and cell proliferation. Downregulation
of Frizzled receptors limits further Wnt activation.PITX2 enhances proliferation of SKOV-3 cells by inducing canonical Wnt signaling. This study will help understand the mechanism of proliferation in ovarian cancer cells
Comprehensive SNP Scan of DNA Repair and DNA Damage Response Genes Reveal Multiple Susceptibility Loci Conferring Risk to Tobacco Associated Leukoplakia and Oral Cancer
No full textPolymorphic variants of DNA repair and damage response genes play major role in carcinogenesis. These variants are
suspected as predisposition factors to Oral Squamous Cell Carcinoma (OSCC). For identification of susceptible variants
affecting OSCC development in Indian population, the ‘‘maximally informative’’ method of SNP selection from HapMap data
to non-HapMap populations was applied. Three hundred twenty-five SNPs from 11 key genes involved in double strand
break repair, mismatch repair and DNA damage response pathways were genotyped on a total of 373 OSCC, 253
leukoplakia and 535 unrelated control individuals. The significantly associated SNPs were validated in an additional cohort
of 144 OSCC patients and 160 controls. The rs12515548 of MSH3 showed significant association with OSCC both in the
discovery and validation phases (discovery P-value: 1.43E-05, replication P-value: 4.84E-03). Two SNPs (rs12360870 of
MRE11A, P-value: 2.37E-07 and rs7003908 of PRKDC, P-value: 7.99E-05) were found to be significantly associated only with
leukoplakia. Stratification of subjects based on amount of tobacco consumption identified SNPs that were associated with
either high or low tobacco exposed group. The study reveals a synergism between associated SNPs and lifestyle factors in
predisposition to OSCC and leukoplakia
Oxidative inhibition of Hsp90 disrupts the super-chaperone complex and attenuates pancreatic adenocarcinoma in vitro and in vivo
No full textPancreatic cancer is almost always fatal, in part because of its delayed diagnosis, poor prognosis, rapid progression and chemoresistance. Oncogenic proteins are stabilized by the Hsp90, making it a potential therapeutic target. We investigated the oxidative stress-mediated dysfunction of Hsp90 and the hindrance of its chaperonic activity by a carbazole alkaloid,mahanine, as a strategic therapeutic in pancreatic cancer. Mahanine exhibited antiproliferative activity against several pancreatic cancer cell lines through apoptosis. It induced early accumulation of reactive oxygen species (ROS) leading to thiol oxidation, aggregation and dysfunction of Hsp90 in MIAPaCa-2. N-acetyl-L-cysteine prevented mahanine-induced ROS
accumulation, aggregation of Hsp90, degradation of client proteins and cell death. Mahanine disrupted Hsp90-Cdc37 complex in MIAPaCa-2 as a consequence of ROS generation. Client proteins were restored by MG132, suggesting a possible role of ubiquitinylated protein degradation pathway. Surface plasmon resonance study demonstrated that the rate of interaction of mahanine with recombinant Hsp90 is in the range of seconds. Molecular dynamics simulation showed its weak interactions
with Hsp90. However, no disruption of the Hsp90-Cdc37 complex was observed at an early time point, thus ruling out that mahanine directly disrupts the complex. It did not impede the ATP binding pocket of Hsp90. Mahanine also reduced in vitro migration and tube formation in cancer cells. Further, it inhibited orthotopic pancreatic tumor growth in nude mice. Taken together, these results provide evidence for mahanine-induced ROS-mediated destabilization of Hsp90 chaperone activity resulting in Hsp90-Cdc37 disruption leading to apoptosis, suggesting its potential as a specific target in pancreatic cancer
Successful Therapy of Visceral Leishmaniasis With Curdlan Involves T-Helper 17 Cytokines
No full textThe aim of this study was to evaluate and characterize the therapeutic potential of curdlan, a naturally occurring
β-glucan immunomodulator, against visceral leishmaniasis, a fatal parasitic disease. Curdlan eliminated
the liver and spleen parasite burden in a 45-day BALB/c mouse model of visceral leishmaniasis at a dosage of
10 mg/kg/day as determined by Giemsa-stained organ impression smears. Curdlan was associated with production
of the disease-resolving T-helper (Th) 1 and Th17-inducing cytokines interleukin (IL)-6, IL-1β, and
IL-23, as well as with production of Th17 cytokines IL-17 and IL-22, as determined by enzyme-linked immunosorbent
assay (ELISA) and real time polymerase chain reaction (RT-PCR). Reversal of curdlan-mediated
protection by anti-IL-17 and anti-IL-23 monoclonal antibodies showed the importance of Th17 cytokines.
Significantly decreased production of both IL-17 and IL-22 by mice that received anti-IL-23 antibody
suggested the essential role of IL-23 in Th17 differentiation. Although administration of recombinant IL-17
or IL-23 caused significant suppression of the organ parasite burden, with marked generation of interferon γ
and nitric oxide (NO), effects were much faster for IL-17. These results documented that although both IL-23
and IL-17 play major roles in the antileishmanial effect of curdlan, the effect of IL-23 may occur indirectly,
through the induction of IL-17 productio
Biophysical Studies on the Interaction of Aristololactam- -D-glucosideandDaunomycinwithRibonucleicAcids
No full textThe sentence “This structure has novel features which are of considerable biological interest” may be one of science’s most famous understatement appeared in April 1953 in the scientific paper in ‘Nature’ where James Watson and Francis Crick presented the unique structure of the Deoxyribonucleic acid-helix (Watson and Crick; 1953). A new understanding of heredity and hereditary diseases was possible once it was determined that DNA consists of two chains twisted around each other, or double helix, of alternating phosphate and sugar groups, and that the two chains are held together by hydrogen bonds between pairs of organic bases— adenine (A) with thymine (T), and guanine (G) with cytosine (C). Modern biotechnology also has its basis in the structural knowledge of DNA. The discovery of the structure of the molecule that carries genetic information from one generation to the other solved the mystery of fundamental living cell. In 1961, Francois Jacob and Jacques Monod demonstrated that the products of certain genes regulated the expression of other genes by acting upon specific sites at the edge of those genes. They also hypothesized the existence of an intermediary between DNA and its protein products, which they called messenger RNA. Between 1961 and 1965, the relationship between the information contained in DNA and the structure of proteins was determined: there is a code, the genetic code, which creates a correspondence between the succession of nucleotides in the DNA sequence and a series of amino acids in proteins. The paradigm that “DNA is transcribed into RNA, which is translated into a protein that exerts a phenotype” has instilled a new notion to important biological discoveries. All cells from bacteria to human express their genetic information from DNA to RNA to protein, a principle so fundamental that is termed as ‘the central dogma of molecular biology
Evaluation and Biosynthesis of Enzymes from Tropical Medicinal Plants
No full textMedicinal plants grow naturally around us. Over centuries, cultures around the world have learned how to use plants to fight illness and maintain health. These readily available and culturally important traditional medicines form the basis of an accessible and affordable health-care regime for indigenous and rural populations. Written records of the use of plants as medicinal agents date back thousands of years. The oldest records come from Mesopotamia and date from about 2600 BC. Those records are not simply a case of one or two plant based ‘drugs’ finding their way into popular use, because the documents indicate that there were many drugs in use that contained plants (upto 1,000 in the case of Mesopotamia). Four thousand years ago, the medical knowledge of the Indian subcontinent was termed as ayurveda and from that time ayurveda remains an important system of medicine and drug therapy in India. Plant alkaloids are the primary active ingredients of ayurvedic drugs. Folk or ethno-medicinal uses represent leads, which may guide pharmaceutical researchers to discover modern therapeutic drugs. Skepticisms may still exist among some researchers regarding indigenous folk traditions that have been handed down from generation to generation. However, many medicinally important compounds for example, atropine, digitoxin, d-tubocurarine, ephedrine, morphine, reserpine, podophyllotoxin, bromelain and many other are of natural origin. Indeed, some have been discovered through following up leads derived from ethnographic research into folk use and information on integration of the indigenous people with their ecosystem
Biophysical Studies on the Interaction of Isoquinoline Alkaloids with Serum Albumins
No full textJoseph F. Foster first suggested that the model of albumin was a flexible linkage of semiindependent domains (Foster, 1960). The name albumin derives from the early German term albumen, generally indicating proteins. Albumen, on the other hand, derives from the Latin word albus (white) indicating the white part of the cooked egg surrounding the yolk. The protein component of the egg white makes part of a heterogeneous group of proteins, broadly indicated as albumins, including serum albumin, milk albumin and urinary proteins. More specifically, serum albumin is a member of a family of homologous proteins characterized by distinctive structural features with peculiar ligand binding properties. Members of this family are -fetoprotein (AFP), afamin (AFM; also named -albumin), and vitamin D binding protein (DBP) (Peters, 1996; Fasano et al., 2007). Serum albumin was recognized as the principal component of the blood as early as 1839. It has been a model protein for many years for physiological studies. It is the most abundant soluble protein in the circulatory system comprising of about 52-60% of the total plasma protein content
Identification and Molecular Characterization of YsaL (Ye3555): A Novel Negative Regulator of YsaN ATPase in Type Three Secretion System of Enteropathogenic Bacteria Yersinia enterocolitica
Get PDFType Three Secretion (T3S) ATPases are involved in delivery of virulent factors from bacteria to their hosts (through
injectisome) in an energy (ATP) dependent manner during pathogenesis. The activities of these ATPases are tightly
controlled by their specific regulators. In Yersinia enterocolitica, YsaN was predicted as a putative ATPase of the Ysa-Ysp Type Three Secretion System (T3SS) based on sequence similarity with other T3S ATPases. However detailed study and characterization of YsaN and its regulation remains largely obscure. Here, in this study, we have successfully cloned, overexpressed,purified and characterized the molecular properties of YsaN from Yersinia enterocolitica. YsaN acts as a Mg2+ dependent ATPase and exists in solution as higher order oligomer (dodecamer). The ATPase activity of oligomeric YsaN is
several fold higher than the monomeric form. Furthermore, by employing in silico studies we have identified the existence of a negative regulator of YsaN- a hypothetical protein YE3555 (termed ‘YsaL’). To verify the functionality of YsaL, we have evaluated the biochemical and biophysical properties of YsaL. Purified YsaL is dimeric in solution and strongly associates with YsaN to form a stable heterotrimeric YsaL-YsaN complex (stoichiometry- 2:1). The N terminal 6–20 residues of YsaN are invariably required for stable YsaL-YsaN complex formation. YsaL inhibited the ATPase activity of YsaN with a maximum inhibition at the molar ratio 2:1 (YsaL: YsaN). In short, our studies provide an insight into the presence of YsaN ATPase in Yersinia enterocolitica and its regulator YsaL. Our studies also correlate the functionality of one of the existing protein
interaction networks that possibly is indispensable for the energy dependent process of Ysa-Ysp T3SS in pathogenic Yersinia enterocolitic