Effect of modification of 6-[(aminoalkyl)amino]-7H-benzo[e]-perimidin-7-ones on their cytotoxic activity toward sensitive and multidrug resistant tumor cell lines. Synthesis and biological evaluation

Benzoperimidines, a novel group of antitumor anthracenedione analogues, are of interest due to their ability to overcome multidrug resistance of tumor cells (Stefańska, B., Dzieduszycka, M., Bontemps-Gracz, M. M., Borowski, E., Martelli, S., Supino, R., Pratesi, G., De Cesare, MA., Zunino, F., Ku?nierczyk, H., Radzikowski, Cz. J. Med. Chem. 1999, 42, 3494). Although the structural factor essential for exhibiting this desirable property is the presence in the molecule of a fused heterocyclic ring, the cytotoxicity against resistant cells is highly influenced by the nature and location of the substituents. A series of novel synthetic derivatives, comprising monohydroxylated benzoperimidines and 2-aminobenzoperimidines, allowed the establishment of an in vitro structure-activity relationship for a panel of leukemia sensitive, as well as P-gp dependent multidrug resistance (MDR) and multidrug resistance associated protein dependent resistance (MRP) resistant cell lines. The membrane affinity for the compounds has also been determined.     

Productive infection of bovine papillomavirus type 2 in the placenta of pregnant cows affected with urinary bladder tumors

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.     

A case of multiple sclerosis improvement following removal of heavy metal intoxication

Multiple sclerosis (MS) is a chronic progressive disease of the central nervous system (CNS) provoking disability and neurological symptoms. The exact causes of SM are unknown, even if it is characterized by focal inflammatory lesions in CNS accompanied by autoimmune reaction against myelin. Indeed, many drugs able to modulate the immune response of patients have been used to treat MS. More recently, toxic metals have been proposed as possible causes of neurodegenerative diseases. The objective of this study is to investigate in vivo the impact of heavy metal intoxication in MS progression. We studied the case of a patient affected by MS, who has been unsuccessfully treated for some years with current therapies. We examined his levels of toxic heavy metals in the urine, following intravenous "challenge" with the chelating agent calcium disodium ethylene diamine tetraacetic acid (EDTA).The patient displayed elevated levels of aluminium, lead and mercury in the urine. Indeed, he was subjected to treatment with EDTA twice a month. Under treatment, the patient revealed in time improved symptoms suggestive of MS remission. The clinical data correlated with the reduction of heavy metal levels in the urine to normal range values. Our case report suggests that levels of toxic metals can be tested in patients affected by neurodegenerative diseases as MS.     

Structural determination of the O-specific chain of the lipopolysaccharide from the mushrooms pathogenic bacterium Pseudomonas tolaasii

The complete structure of the O-specific polysaccharide of the lipopolysaccharide isolated from the cultivated mushrooms pathogen Pseudomonas tolaasii is described. The structural determination, achieved by chemical and spectroscopical analyses, indicates a novel tetrasaccharide repeating unit built up of two units of 2-acetamido-2,6-di-deoxy-glucopyranose (Quinovosamine, QuipNAc) and two units of 2-acetamido-2-deoxy-gulopyranuronamide (GulpNAcAN), one of which is acetylated at C-3 position:     

Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex

Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.     

Mincle,an Innate Immune Receptor,Is Expressed in Urothelial Cancer Cells of Papillomavirus-Associated Urothelial Tumors of Cattle

Background

Mincle, macrophage-inducible C-type lectin, is a member of C-type lectin receptors. It plays an important role in anti-mycobacterial and anti-fungal immunity. Furthermore it senses dead cells through its primary ligand SAP130.

Materials and Findings

We examined ten urothelial tumors of the urinary bladder of cattle. Eight of them expressed E5 cDNA of bovine papillomaviruses type 2 (BPV-2) and type 13 (BPV-13) that belong to Deltapapillomavirus genus. Two of them were not examined for detection of E5 cDNA. Mincle expression appeared to occur in urothelial neoplastic cells only. No mincle expression was detected in urothelial cells from healthy cattle. Mincle expression was characterized by a membranous pattern in papillary urothelial cancers; isolated and/or clustered urothelial cells showing a strong cytoplasmic immunoreactivity were primarily seen in invasive urothelial cancers.

Conclusion

This is the first study about the expression of mincle in veterinary oncology and the first report which describes the expression of functional mincle receptor in neoplastic cells in medical literature. As it has been shown that urothelial cancer cells have the ability to function as antigen-presenting cells (APCs), it is conceivable that mincle expression is involved in the presentation of cancer cell antigens to cells of the immune system. Furthermore, since expression of mincle contributes to the control of Mycobacterium bovis BCG infection, this study has exciting clinical implications in comparative medicine keeping in mind that Bacillus Calmette-Guérin (BCG) immunotherapy is currently the most effective treatment of non-muscle invasive bladder cancer in man. Mincle expression in urothelial tumor cells warrants further study to better understand the role, if any, of this receptor in bladder cancer. Future studies will provide insights in the role of mincle receptor of urothelial cancer cells in antitumor immunotherapy.     

2,7-Dihydro-3H-pyridazino[5,4,3-kl]acridin-3-one derivatives, novel type of cytotoxic agents active on multidrug-resistant cell lines. Synthesis and biological evaluation

We have earlier postulated that the presence of a pyridazone ring fused with an anthracenedione moiety resulted in the analog's ability to overcome multidrug resistance of tumor cells [J. Med. Chem.1999, 42, 3494]. High cytotoxic activity of obtained anthrapyridazones [Bioorg. Med. Chem.2003, 11, 561] toward the resistant cell lines, prompted us to synthesize the similarly modified acridine compounds. A series of pyridazinoacridin-3-one derivatives (2b-h) were prepared from the reaction of 9-oxo-9,10-dihydroacridine-1-carboxylate with POCl(3), followed by addition of the appropriate (alkylamino)alkylhydrazines. In vitro cytotoxic activity toward sensitive and resistant leukemia cell lines: L1210, K562, K562/DX, HL-60, HL-60/VINC, and HL-60/DX, with various type of multidrug resistance (MDR and MRP) was determined. The compounds studied exhibited in comparison to the reference cytostatics (DX, MIT) desirable very low resistance indexes (RI). Variations have been observed depending upon the substituent and the type of drug exporting pump. The cytotoxic activities of examined compounds, as well as of model anthrapyridazone derivative PDZ, were lower than those of reference drugs (DX, MIT) due to their diminished affinity to DNA.     

Transmural distribution of iron in the hypoxic and reoxygenated rabbit left ventricular myocardium

Transmural distribution of low molecular weight iron (LMWI), total iron, and protein carbonyls (PC) was investigated in the perfused rabbit heart under aerobic conditions, and after 60 min hypoxia followed or not by 3 min reoxygenation. In the aerobic perfused hearts, LMWI, total iron and PC did not show significant transmural differences. Hypoxia increased LMWI and PC levels, which were significantly higher in the subendocardium than in the subepicardium; further significant changes were not observed after reoxygenation. Total iron showed no transmural difference and was not significantly affected by both hypoxia and reoxygenation. Free iron was undetectable in the myocardial effluent of all experimental groups. Thus, hypoxia favors myocardial iron decompartmentalisation and oxidative stress, which are significantly greater in the inner than in the outer ventricular layers. Such findings may add further insight into the problem of the vulnerability of the mammalian subendocardium to injury induced by oxygen deprivation.     

The <Emphasis Type="Italic">Nramp1</Emphasis>AA genotype confers susceptibility to <Emphasis Type="Italic">Brucella abortus</Emphasis> in water buffalo

The 3′ untranslated region of the water buffalo Nramp1 (natural resistance-associated macrophage protein 1) gene contains two alleles (Nramp1A and Nramp1B), as detected by the denaturing gradient gel electrophoresis (DGGE) technique. The Nramp1BB genotype is associated with resistance of water buffalo to the intracellular pathogen Brucella abortus. This article provides evidence that the Nramp1AA genotype is associated with susceptibility to the same pathogen. Susceptibility or resistance of water buffalo to B. abortus was established by agglutination, complement fixation, and skin tests. The Nramp1 genotype was established by DGGE analysis. The association between the Nramp1AA genotype and susceptibility to B. abortus was demonstrated in two independent population samples (152 cases and 281 controls; 87 cases and 124 controls, respectively). Macrophages from Nramp1AA subjects displayed a lower Nramp1 mRNA level when compared with macrophages from Nramp1BB subjects. Also, monocytes and macrophages from Nramp1AA subjects displayed a higher number of viable intracellular bacteria in comparison with monocytes and macrophages from Nramp1BB animals, providing biological significance to the results from association studies.     

Modeling the Ternary Complex TCR-Vβ/CollagenII(261–273)/HLA-DR4 Associated with Rheumatoid Arthritis

Background

It is known that genetic predisposition to rheumatoid arthritis (RA) is associated with the MHC class II allele HLA-DR4 and that residues 261–273 of type II collagen (huCollp261) represent an immunodominant T cell epitope restricted by the DR4 molecule. Despite recent advances in characterization of MHC and T cell receptor (TCR) contacts to this epitope, the atomic details of TCR/huCollp261/HLA-DR4 ternary complex are not known.

Methodology/Principal Findings

Here we have used computational modeling to get insight into this interaction. A three-dimensional model of the TCR Vβ domain from a DR4⁺ patient affected by RA has been derived by homology modeling techniques. Subsequently, the structure of the TCR Vβ domain in complex with huCollp261/HLA-DR4 was obtained from a docking approach in conjunction with a filtering procedure based on biochemical information. The best complex from the docking experiments was then refined by 20 ns of molecular dynamics simulation in explicit water. The predicted model is consistent with available experimental data. Our results indicate that residues 97–101 of CDR3β are critical for recognition of huCollp261/HLA-DR4 by TCR. We also show that TCR contacts on p/MHC surface affect the conformation of the shared epitope expressed by DR alleles associated with RA susceptibility.

Conclusions/Significance

This work presents a three-dimensional model for the ternary complex TCR-Vβ/collagenII(261–273)/HLA-DR4 associated with rheumatoid arthritis that can provide insights into the molecular mechanisms of self reactivity.