Tetraoxanes as inhibitors of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii growth and anti-cancer molecules

New cyclohexylidene 1,2,4,5-tetraoxanes with polar guanidine and urea based groups were synthesized and evaluated for their antimalarial activity against chloroquine resistant and susceptible Plasmodium falciparum strains. The derivatives showed moderate, nM range antimalarial activities and low cytotoxicity. The N-phenylurea derivative 24 exhibited the best resistance indices (RIW2 = 0.44, RITM91C235 = 0.80) and was not toxic against human normal peripheral blood mononuclear cells (IC50 gt 200 mu M). Seven derivatives were tested in vitro against four human cancer cell lines and they demonstrated high selectivity toward leukaemia K562 cells. One compound, derivative 21 with a primary amino group, was the first tetraoxane tested in vivo against Toxoplasma gondii as another apicomplexan parasite. Subcutaneous administration at a dose of 10 mg kg(-1) day(-1) for 8 days allowed the survival of 20 % of infected mice, thus demonstrating the high potential of tetraoxanes for the treatment of apicomplexan parasites

Utilization of miscanthus waste biomass for xylanase production by soil bacterium Sinorhizobium meliloti

Miscanthus × giganteus, as a lignocellulosic material, consists of about 30% xylan and represents a good basis for research as a possible raw material in the production of xylanases. Among the soil bacteria, rhizobium is rarely investigated as an enzyme producer. For the first time, Sinorhizobium meliloti strain 207 was used to obtain xylanase during submerged and solid fermentation using miscanthus biomass as a substrate. During submerged fermentation (28°C), the maximum xylanase activity was achieved after 48 h with 10% inoculum and H2SO4 substrate modification. The maximum xylanase activity of 1.215 U/mL was obtained during solid-state fermentation (28°C) by using also H2SO4 modified miscanthus biomass which was moistened with distillate water. The crude enzyme, produced by strain 207, could be further used in eco-friendly processes of lignocellulose material bioconversion to useful products.Саопштење са међународног скупа штампано у целини (M33

Decreased levels of BAG3 in a family with a rare variant and in idiopathic dilated cardiomyopathy.

The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease; however, in a third of all patients the cause remains undefined and patients are diagnosed as having idiopathic dilated cardiomyopathy (IDC). Recent studies suggest that many patients with IDC have a family history of HF and rare genetic variants in over 35 genes have been shown to be causative of disease. We employed whole-exome sequencing to identify the causative variant in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10-nucleotide deletion in the BCL2-associated athanogene 3 (BAG3) gene (Ch10:del 121436332_12143641: del. 1266_1275 [NM 004281]) that segregated with all affected individuals. The deletion predicted a shift in the reading frame with the resultant deletion of 135 amino acids from the C-terminal end of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins there was a considerable amount of genetic heterogeneity in the affected family members. Interestingly, we also found that the levels of BAG3 protein were significantly reduced in the hearts from unrelated patients with end-stage HF undergoing cardiac transplantation when compared with non-failing controls. Diminished levels of BAG3 protein may be associated with both familial and non-familial forms of dilated cardiomyopathy

Bag3-Induced Autophagy Is Associated with Degradation of JCV Oncoprotein, T-Ag

JC virus, JCV, is a human neurotropic polyomavirus whose replication in glial cells causes the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). In addition, JCV possesses oncogenic activity and expression of its transforming protein, large T-antigen (T-Ag), in several experimental animals induces tumors of neural origin. Further, the presence of JCV DNA and T-Ag have been repeatedly observed in several human malignant tissues including primitive neuroectodermal tumors and glioblastomas. Earlier studies have demonstrated that Bag3, a member of the Bcl-2-associated athanogene (Bag) family of proteins, which is implicated in autophagy and apoptosis, is downregulated upon JCV infection of glial cells and that JCV T-Ag is responsible for suppressing the activity of the BAG3 promoter. Here, we investigated the possible impact of Bag3 on T-Ag expression in JCV-infected human primary glial cells as well as in cells derived from T-Ag-induced medulloblastoma in transgenic animals. Results from these studies revealed that overexpression of Bag3 drastically decreases the level of T-Ag expression by inducing the autophagic degradation of the viral protein. Interestingly, this event leads to the inhibition of JCV infection of glial cells, suggesting that the reduced levels of T-antigen seen upon the overexpression of Bag3 has a biological impact on the viral lytic cycle. Results from protein-protein interaction studies showed that T-Ag and Bag3 physically interact with each other through the zinc-finger of T-Ag and the proline rich domains of Bag3, and this interaction is important for the autophagic degradation of T-Ag. Our observations open a new avenue of research for better understanding of virus-host interaction by investigating the interplay between T-Ag and Bag3, and their impact on the development of JCV-associated diseases

Effect of Bag3 on T-Ag expression in JCV transformed cells.

<p>A. BSB8 cells were transfected with empty vector or with an expression plasmid encoding Bag3 in a dose dependent manner. Western blot analyses were performed using whole cell protein extracts prepared at 48 hour post-transfections using specific antibodies recognizing T-Ag, Myc-tag, and Bag3 proteins. Western blot analysis of the same extracts using anti-Grb2 antibody was used as loading control. B. Relative intensity of the bands corresponding to T-Ag from panel A determined by densitometry is shown as a bar graph. C. Western blot analyses of protein extracts from U-87MG cells after transfection with plasmids encoding T-Ag and Bag3 using specific antibodies for T-Ag, Myc-tag, and Bag3 proteins. Western blot analyzsis of same extracts with anti-Grb2 antibody was used as loading control. D. Relative intensity of the bands from experiments illustrated in Panel C are shown as a bar graph. E. BSB8 cells were transfected with a Bag3 siRNA or a control non-targeted siRNA and expression of T-Ag and Bag3 was examined by Western blot analysis. Relative intensity of the bands corresponding to T-Ag and Bag3 were densitometrically determined and normalized to those from Grb2, and illustrated by a bar graph.</p

Effect of proteasomal inhibitors on Bag3-mediated downregulation of Large T antigen expression.

<p>A. Effect of Bag3 on transcription of JCV promoters. Primary culture of human fetal glial cells was transfected with CAT reporter genes under the control of JCV early or late gene promoters along with a plasmid expressing Bag3 (pCDNA6-Bag3), or a control empty vector (pCDNA6). Relative promoter activity was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045000#s4" target="_blank">Materials and Methods</a>. B. Protein extracts were prepared from BSB8 cells transfected with plasmid vector (control) or expression plasmid encoding Bag3. Large T-Ag was immunoprecipitated with a monoclonal antibody, separated on SDS-PAGE gel, and processed for Western blotting using anti-Ubiquitin antibody. Normal mouse serum (NMS) was used as negative control for immunoprecipitations. C. BSB8 cells were transfected with an expression plasmid encoding Bag3, and treated with MG-115 or DMSO (control). Whole cell extracts were prepared at 48 hour post-transfection, and Western blot analysis was performed for the detection of Large T, and Bag3 proteins. Western blot analysis of the same extracts with anti-Grb2 antibody was used as loading control. D. Relative band intensity of T-Ag bands from Panel B is shown as a bar graph.</p

Bag3 inhibits replication of JCV in primary culture of human fetal glial cells.

<p>A. Western blot analyses of whole cell protein extracts prepared from JCV infected cells using specific antibodies against T-Ag, VP1, Agnoprotein, Bag3, and the housekeeping protein, Grb2. In lane 1, extracts from uninfected cells were loaded as a control. B. Q-PCR analyses of the viral particles in the JCV infected-cells culture medium as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045000#s4" target="_blank">Materials and Methods</a>.</p