RGD conjugation to polyethyleneimine does not improve DNA delivery to bone marrow stromal cells

Bone marrow stromal cells (BMSC) modified with therapeutic genes are being actively pursued for gene therapy protocols. To develop safe and effective nonviral methods for BMSC modification, the cationic polymer polyethyleneimine (PEI) has been utilized to condense plasmid DNA for intracellular delivery. This study was conducted to explore the feasibility of increasing the PEI's effectiveness by coupling integrin-binding arginine-glycine-aspartic acid (RGD) peptides to the polymer. BMSC from rats were isolated and expanded in culture for gene transfer studies. In contrast to our expectations, RGD-conjugated PEI did not exhibit an enhanced binding to BMSC. This was the case where the peptides were conjugated to PEI by short, disulfide linkages or long poly(ethylene glycol) linkages. Using a reporter gene for the enhanced green fluorescent protein, the transfection efficiency of RGD-conjugated PEI was also lower than the delivery by the native PEI, which exhibited equivalent transfection efficiency to that of an adenovirus. We conclude that native PEI was sufficient for the transformation of BMSC and that coupling of the integrin-binding RGD-peptides did not improve the effectiveness of this polymer for BMSC transfection.     

C2-Symmetric azobenzene-amino acid conjugates and their inhibition of Subtilisin Kexin Isozyme-1

C₂-Symmetric azobenzene-amino acid/peptide hybrids containing stable E-azo moiety have been synthesized. Upon irradiation with long wavelength UV, these compounds isomerized to the Z-form, whose thermal reisomerization to the E isomer slowed down considerably. These compounds exhibited in vitro moderate to strong inhibition of mammalian cellular protease Subtilisin Kexin Isozyme-1, also called Site 1 Protease, which plays vital roles in cholesterol synthesis, lipid metabolism, bone formation, and viral infections.     

Design, synthesis and inhibition activity of novel cyclic peptides against protein tyrosine phosphatase A from Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent for tuberculosis has employed several signalling molecules to sense the host cellular environment and act accordingly. For example, protein tyrosine phosphatase A (MPtpA) of M. tuberculosis, a signalling protein belonging to the tyrosine phosphatase superfamily, is involved in phagocytosis and is active in virulent mycobacterial form. Starting from a β-lactam framework a new class of structure based cyclic peptide (CP) inhibitors was designed. The synthesis involves a crucial intramolecular transamidation via a ring opening reaction. All the compounds show moderate to good inhibitory activities against MPtpA in micromolar concentrations. The results of inhibition kinetics suggest mixed mode of inhibition. The binding constant determined from circular dichroism (CD) and fluorescence quenching studies shows strong binding of the hydrophilic side chain of CPs with the enzyme active site residues. All these are well supported by docking studies.     

Infection of dendritic cells by a gamma2-herpesvirus induces functional modulation

The murine gamma-herpesvirus-68 (gammaHV68) establishes viral latency in dendritic cells (DCs). In the present study, we examined the specific consequences of DC infection by gammaHV68, both in vivo and in vitro. Ex vivo analysis of infected mice showed that the virus colonizes respiratory DCs very early after infection and that all subsets of splenic DCs analyzed are viral targets. We have developed and characterized an in vitro model of gammaHV68 infection of DCs. Using this model, we demonstrated that viral infection neither induces full DC maturation nor interferes with exogenous activation, which is assessed by cell surface phenotypic changes. However, whereas gammaHV68 infection alone failed to elicit cytokine secretion, IL-10 secretion of exogenously activated DCs was enhanced. Furthermore, gammaHV68-infected DCs efficiently stimulated virus-specific T cell hybridomas but failed to induce alloreactive stimulation of normal T cells. These data indicate that viral infection doesn't interfere with Ag processing and presentation but does interfere with the ability of DCs to activate T cells. The inhibition of T cell activation was partially reversed by blocking IL-10. Analysis of infected mice shows elevated levels of IL-10 expression in DCs and that lack of endogenous IL-10 is associated with decreased gammaHV68 long-term latency. Taken together, these observations indicate that gamma2-herpesvirus infection of DCs is a mechanism of viral immune evasion, partially mediated by IL-10.     

Curcumin stimulates angiogenesis through VEGF and expression of HLA‐G in first‐trimester human placental trophoblasts

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real‐time polymerase chain reaction (qRT‐PCR), promoter DNA methylation, qRT‐PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG‐3 (trophoblast), and HMEC‐1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor‐2 and fatty acid‐binding protein‐4 (FABP4) in these cells. Curcumin‐stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA‐G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down‐regulated mRNA expression of HSD11β2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA‐G by curcumin, hitherto unknown, is a novel finding since HLA‐G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.     

Biochemical estimation of Moringa oleifera leaf extract for synthesis of silver nanoparticle mediated drug delivery system

Breast tissue remodeling occurs throughout a women’s life from pre-menstrual to post menopausal time, hence there is a high risk of DNA damage. Estrogen metabolite generates free radical that causes DNA damage leading to cancer cell formation. Moringa oleifera leaf extract contain rich amount of Polyphenolic and Flavonoids compound which trap the free radical and thereby prevent cancer cell progression. Moreover anti-gonadotrophic hormonal activity of the leaf extract slows down the proliferation rate of T47D cell line. Hence our aim of study mainly focused to develop an anticancer drug delivery system using M. oleifera leaf extract. Silver nanoparticle (AgNp) is formed by green synthesis method using M. oleifera leaf extract. Reducing sugar, Flavonoids, Polyphenolic compound, and NADPH dependent dehydrogenase present in M. oleifera leaf extract are responsible for reduction of the silver ion (Ag+) followed by formation of metallic silver (Ag0). The formation of spherical and rectangular shaped AgNp with average size of 50 nm is reflected with sharp SPR band near 420 nm and from SEM, TEM analysis. The nanoparticle mediated polyvinyl alcohol composite film (nano-rod) is prepared for delivery of the purified anti-neoplastic phyto-compound from leaf extract. The large surface area of AgNp permits its coordination with purified compound molecule by metal ligand bonding. Therefore it provide duel effect, it acts as carrier molecule for phyto-compound and also creates cytotoxicity effect on cancer cells. Single dose treatment on HeLa cell indicates that the composite film is more effective in killing the cancer cell than the purified anti-neoplastic phyto-compound.     

Crystal Structure of 1-(2′,3′,5′-Tri -O-Acetyl-β-D-ribofuranosyl)-3-acetoxy-2-pyridone: An Antitumour Agent

Abstract

1–C3′, 3′, 5′–Tri—O—acetyl— β—D—ribof uranosyll)—3—acetoxy —2—pyridone,crystallised in space group P2 with z=2 and cell parameters a=12. 446(2), b=10. 415(2), c=7. 600(2) A, β=03. 3O(4). The structure was solved by direct methods and refined by full—matrix least—squares to a final R value of 0·251 for 1847 observed reflections. The sugar—pucker is found to be ³ECC3′ endo) with P = 17.7° and x_CN=170. 2(2)° in the range. The C4′-C5′ conformation is gauche minus. Because of the absence of H—bond donor atoms. the crystal structure is stabilised by a network of C-H—-O close contacts. No base stacking is observed.     

Cloning, characterization and expression of a chloroplastic fructose-1,6-bisphosphatase from Porteresia coarctata conferring salt-tolerance in transgenic tobacco

A gene coding for the chloroplastic fructose 1,6-bisphosphatase (PcCFR) in Porteresia coarctata Tateoka (Roxb.), a halophytic wild rice, has been isolated along with its rice (Oryza sativa; var. indica) homologue (OsCFR), cloned and sequenced. Comparison between the nucleotide and deduced amino acid sequences of these two revealed a difference in five amino acid residues, namely Glu¹⁴, Thr²⁴, Ala⁴⁸, Ala¹⁶³ and Arg²⁹⁶ in OsCFR which have been found to be replaced by Ser¹⁴, Ile²⁴, Ser⁴⁸, Ser¹⁶³ and Lys²⁹⁶ in PcCFR respectively. The purified recombinant PcCFR is found to retain its enzymatic activity in presence of up to 500 mM NaCl in vitro as opposed to OsCFR, which is inactivated even at lower salt concentration. The six in vitro point mutant proteins of PcCFR showed varied degree of sensitivity towards high salt, with the maximum OsCFR-like effect in the triple mutant S¹⁴A-S⁴⁸A-S¹⁶³A suggesting a possible concerted role of all three serine residues in the in vitro salt tolerance property of PcCFR protein. Transgenic tobacco plants with chloroplast targeted PcCFR and OsCFR gene(s) have been developed under constitutive expression of CaMV 35S promoter and NOS terminator. The PcCFR transgenics showed better plant growth during exposure to salt stress in comparison to either the OsCFR or the empty vector transformed plants. The PcCFR transgenics also revealed enhanced photosynthetic efficiency coupled with protection to both photodamage of PSII and chlorophyll degradation through better reactive oxygen species scavenging at higher concentration of NaCl during late salt-stress growth.     

A chemo-enzymatic route to differentially protected aryl-naphthalenes

Aryl-naphthalene diacetates prepared from bispropargyl sulfones, ethers and amines via Garratt–Braverman Cyclization have been selectively hydrolysed by AK lipase to the monoacetates 12a–c in high yields. The regioisomeric mono acetates 13a–c have been prepared by acetylation of the corresponding diols using the same enzyme. In both cases, the more exposed acetoxymethyl or hydroxy methyl attached to the naphthalene ring binds to the active site of the enzyme and underwent hydrolysis/acetylation. The method provides easy access to differentially protected aryl-naphthalenes which should allow further modifications.