Low concentration of silver nanoparticles not only enhances the activity of horseradish peroxidase but alter the structure also

Chemical synthesis of Ag-NPs was carried out using reduction method. The reduction mechanistic approach of silver ions was found to be a basic clue for the formation of the Ag-NPs. The nanoparticles were characterized by UV-vis, FT-IR and TEM analysis. We had designed some experiments in support of our hypothesis, "low concentrations of novel nanoparticles (silver and gold) increases the activity of plant peroxidases and alter their structure also", we had used Ag-NPs and HRP as models. The immobilization/interaction experiment had demonstrated the specific concentration range of the Ag-NPs and within this range, an increase in HRP activity was reported. At 0.08 mM concentration of Ag-NPs, 50% increase in the activity yield was found. The U.V-vis spectra had demonstrated the increase in the absorbance of HRP within the reported concentration range (0.06-0.12 mM). Above and below this concentration range there was a decrease in the activity of HRP. The results that we had found from the fluorescence spectra were also in favor of our hypothesis. There was a maximum increase in ellipticity and α-helix contents in the presence of 0.08 mM concentration of Ag-NPs, demonstrated by circular dichroism (CD) spectra. Finally, incubation of a plant peroxidase, HRP with Ag-NPs, within the reported concentration range not only enhances the activity but also alter the structure.     

Trivanillic polyphenols with anticancer cytostatic effects through the targeting of multiple kinases and intracellular Ca2+ release

Cancer cells exhibit de-regulation of multiple cellular signalling pathways and treatments of various types of cancers with polyphenols are promising. We recently reported the synthesis of a series of 33 novel divanillic and trivanillic polyphenols that displayed anticancer activity, at least in vitro, through inhibiting various kinases. This study revealed that minor chemical modifications of a trivanillate scaffold could convert cytotoxic compounds into cytostatic ones. Compound 13c, a tri-chloro derivative of trivanillic ester, displayed marked inhibitory activities against FGF-, VEGF-, EGF- and Src-related kinases, all of which are implicated not only in angiogenesis but also in the biological aggressiveness of various cancer types. The pan-anti-kinase activity of 13c occurs at less than one-tenth of its mean IC(50) in vitro growth inhibitory concentrations towards a panel of 12 cancer cell lines. Of the 26 kinases for which 13c inhibited their activity by >75%, eight (Yes, Fyn, FGF-R1, EGFR, Btk, Mink, Ret and Itk) are implicated in control of the actin cytoskeleton organization to varying degrees. Compound 13c accordingly impaired the typical organization of the actin cytoskeleton in human U373 glioblastoma cells. The pan-anti-kinase activity and actin cytoskeleton organization impairment provoked by 13c concomitantly occurs with calcium homeostasis impairment but without provoking MDR phenotype activation. All of these anticancer properties enabled 13c to confer therapeutic benefits in vivo in a mouse melanoma pseudometastatic lung model. These data argue in favour of further chemically modifying trivanillates to produce novel and potent anticancer drugs.     

Characterization of adenosine receptor in its native environment: insights from molecular dynamics simulations of palmitoylated/glycosylated, membrane-integrated human A2B adenosine receptor

Selective A(2B) receptor antagonists and agonists may play a role in important pathologies such as gastrointestinal, neurological (i.e., Alzheimer disease and dementia) and hypersensitive disorders (i.e., asthma), diabetes, atherosclerosis, restenosis and cancer. Hence, it is regarded as a good target for the development of clinically useful agents. In this study, the effects of lipid bilayer, N-acetylglucosamine and S-palmitoyl on the dynamic behavior of A(2B)AR model is explored. Homology modeling, molecular docking and molecular dynamics simulations were performed to explore structural features of A(2B)AR in the presence of lipid bilayer. Twenty ns MD simulation was performed on the constructed model inserted in a hydrated lipid bilayer to examine stability of the best model. OSIP339391 as the most potent antagonist was docked in the active site of the model. Another MD simulation was performed on the ligand-protein complex to explore effects of the bilayer on this complex. A similar procedure was performed for the modified protein with N-acetylglucosamine and S-palmitoyl moieties in its structure. Phe173 and Glu174 located in EL2 were determined to be involved in ligand-receptor interactions through π-π stacking and hydrogen bonding. Asn254 was crucial to form hydrogen-bonding. The reliability of the model was assessed through docking using both commercial and synthetic antagonists and an r(2) of 0.70 was achieved. Our results show that molecular dynamics simulations of palmitoylated/glycosylated, membrane-integrated human A(2B)AR in its native environment is a possible approach and this model can be used for designing potent and selective A(2B)AR antagonists.     

Effects of Age and Parity on Mammary Gland Lesions and Progenitor Cells in the FVB/N-RC Mice

The FVB/N mouse strain is extensively used in the development of animal models for breast cancer research. Recently it has been reported that the aging FVB/N mice develop spontaneous mammary lesions and tumors accompanied with abnormalities in the pituitary glands. These observations have a great impact on the mouse models of human breast cancer. We have developed a population of inbred FVB/N mice (designated FVB/N-RC) that have been genetically isolated for 20 years. To study the effects of age and parity on abnormalities of the mammary glands of FVB/N-RC mice, twenty-five nulliparous and multiparous (3-4 pregnancies) females were euthanized at 16-22 months of age. Examination of the mammary glands did not reveal macroscopic evidence of mammary gland tumors in either aged-nulliparous or multiparous FVB/N-RC mice (0/25). However, histological analysis of the mammary glands showed rare focal nodules of squamous changes in 2 of the aged multiparous mice. Mammary gland hyperplasia was detected in 8% and 71% of the aged-nulliparous and aged-multiparous mice, respectively. Epithelial contents and serum levels of triiodothyronine were significantly higher in the experimental groups than the 14-wk-old control mice. Immuno-histochemical staining of the pituitary gland pars distalis showed no difference in prolactin staining between the control and the aged mice. Tissue transplant and dilution studies showed no effect of age and/or parity on the ability of putative progenitor cells present among the injected mammary cells to repopulate a cleared fat pad and develop a full mammary gland outgrowth. This FVB/N-RC mouse substrain is suitable to develop mouse models for breast cancer.     

Impact of AT2 Receptor Deficiency on Postnatal Cardiovascular Development

Background

The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear.

Methodology/Principal Findings

Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of α-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca²⁺ transient in response to isoprenaline when stimulated concomitantly with angiotensin II.

Conclusion

The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.     

Expansion of human cytomegalovirus (HCMV) immediate-early 1-specific CD8+ T cells and control of HCMV replication after allogeneic stem cell transplantation

Recovery of human cytomegalovirus (HCMV)-specific T immunity is critical for protection against HCMV disease in the early phase after allogeneic stem cell transplantation (SCT). Using an enzyme-linked immunospot assay with overlapping 15-mer peptides spanning pp65 and immediate-early 1 HCMV proteins, we investigated which HCMV-specific CD8⁺ gamma interferon-positive (IFN-γ⁺) T-cell responses against pp65 and IE-1 were associated with control of HCMV replication in 48 recipients of unmanipulated HLA-matched allografts at 3 months (M3) and 6 months (M6) after SCT and in 23 donors. At M3 after SCT, the magnitude of the pp65-specific IFN-γ-producing CD8⁺ T-cell response was greater in recipients than in donors, regardless of HCMV status. In contrast, expansion of IE-1-specific CD8⁺ T cells at M3 was associated with protection against HCMV, and no patient with this expansion had HCMV replication at M3. At M6, the number of HCMV-specific CD8⁺ T cells against both pp65 and IE-1 had expanded in all recipients, regardless of their previous levels of HCMV replication. The recipients' HCMV-specific CD8⁺ T cells already detectable in related donors were predominantly targeting pp65. In contrast, in 40% of the cases, the HCMV-specific CD8⁺ T cells in recipients involved new CD8⁺ T-cell specificities undetectable in their related donors and preferentially targeting IE-1. Taken together, these results showed that the delay in reconstituting IE-1-specific CD8⁺ T cells is correlated with the lack of protection against HCMV in the first 3 months after SCT. They also show that IE-1 is a major antigenic determinant of the early restoration of protective immunity to HCMV after SCT.     

Infectious bronchitis viruses with a novel genomic organization

A number of novel infectious bronchitis viruses (IBVs) were previously identified in commercial poultry in Australia, where they caused significant economic losses. Since there has been only limited characterization of these viruses, we investigated the genomic and phenotypic differences between these novel IBVs and other, classical IBVs. The 3' 7.5 kb of the genomes of 17 Australian IBV strains were sequenced, and growth properties of 6 of the strains were compared. Comparison of sequences of the genes coding for structural and nonstructural proteins revealed the existence of two IBV genotypes: classical and novel. The genomic organization of the classical IBVs was typical of those of other group III coronaviruses: 5'-Pol-S-3a-3b-E-M-5a-5b-N-untranslated region (UTR)-3'. However, the novel IBV genotype lacked either all or most of the genes coding for nonstructural proteins at the 3' end of the genome and had a unique open reading frame, X1. The gene order was either 5'-Pol-S-X1-E-M-N-UTR-3' or 5'-Pol-S-X1-E-M-5b-N-UTR-3'. Phenotypically, novel and classical IBVs also differed; novel IBVs grew at a slower rate and reached lower titers in vitro and in vivo and were markedly less immunogenic in chicks. Although the novel IBVs induced histopathological lesions in the tracheas of infected chicks that were comparable to those induced by classical strains, they did not induce lesions in the kidneys. This study has demonstrated for the first time the existence of a naturally occurring IBV genotype devoid of some of the genes coding for nonstructural proteins and has also indicated that all of the accessory genes are dispensable for the growth of IBV and that such viruses are able to cause clinical disease and economic loss. The phylogenic differences between these novel IBVs and other avian coronaviruses suggest a reservoir host distinct from domestic poultry.