Epstein-barr virus-induced resistance to drugs that activate the mitotic spindle assembly checkpoint in Burkitt's lymphoma cells

Epstein-Barr virus (EBV) is associated with a number of human cancers, and latent EBV gene expression has been reported to interfere with cell cycle checkpoints and cell death pathways. Here we show that latent EBV can compromise the mitotic spindle assembly checkpoint and rescue Burkitt's lymphoma (BL)-derived cells from caspase-dependent cell death initiated in aberrant mitosis. This leads to unscheduled mitotic progression, resulting in polyploidy and multi- and/or micronucleation. The EBV latent genes responsible for this phenotype are expressed from the P3HR1 strain of virus and several viruses with similar genomic deletions that remove the EBNA2 gene. Although EBNA2 and the latent membrane proteins are not expressed, the EBNA3 proteins are present in these BL cells. Survival of the EBV-positive cells is not consistently associated with EBV lytic gene expression or with the genes that are expressed in EBV latency I BL cells (i.e., EBNA1, EBERs, and BARTs) but correlates with reduced expression of the cellular proapoptotic BH3-only protein Bim. These data suggest that a subset of latent EBV gene products may increase the likelihood of damaged DNA being inherited because of the impaired checkpoint and enhanced survival capacity. This could lead to greater genetic diversity in progeny cells and contribute to tumorigenesis. Furthermore, since it appears that this restricted latent EBV expression interferes with the responses of Burkitt's lymphoma-derived cells to cytotoxic drugs, the results of this study may have important therapeutic implications in the treatment of some BL.     

Rare coexistence of keratinizing squamous metaplasia with xanthogranulomatous pyelonephritis. Report of a case with the role of immunocytochemistry in the differential diagnosis

BACKGROUND: Xanthogranulomatous pyelonephritis (XPN), a rare form ofchronicpyelonephritis, is commonly associated with lithiasis and rarely leads to keratinizing squamous metaplasia. Its manifestations closely mimic those of a renal neoplasm, leading to misdiagnosis of malignancy, often resulting in radical nephrectomy. The role of immunocytochemistry in the preoperative cytologic diagnosis is assessed in the present case report. CASE: A 20-year-old male presented with fever and an enlarging mass in the right renal angle. Ultrasonography revealed a heterogeneous mass in the renal pelvis. Fine needle aspiration cytology was advised to rule out malignancy. Aspiration smears from the mass showed many dissociated cells and clusters of them with abundant vacuolated cytoplasm, vesicular nuclei and prominent nucleoli in some cells. Many desquamated metaplastic squamous cells were also seen. The background was predominantly necrotic, with inflammatory cells. The cytologic possibility of XPN with squamous metaplasia vs. renal cell carcinoma was considered. Immunocytochemical markers, epithelial membrane antigen (EMA) and CD68 (histiocytic marker) were used to determine the nature of the suspicious vacuolated cells; these cells were immunoreactive for CD68 and negative for EMA, thus confirming the cytologic diagnosis of XPN with keratinizing squamous metaplasia. CONCLUSION: The case highlights the presence of metaplastic squamous cells in XPN in smears for the first time. Immunocytochemistry is an essential tool in the preoperative cytologic diagnosis of XPN. The patient can be managed conservatively with antibiotics.     

The structural topology of wild-type phospholamban in oriented lipid bilayers using 15N solid-state NMR spectroscopy

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13 degrees+/-6 degrees with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.     

Synthesis and evaluation of antifungal properties of a series of the novel 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and its analogues

A series of 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and various analogues have been synthesized in excellent isolated yields starting from various arylidenemalononitrile and 3-amino-2-cyclohexen-1-one in 1-propanol as solvent at reflux temperature in the absence of any added catalyst. All the synthesized compounds were evaluated for their antifungal activity. The relationship between functional group variation and biological activity of the evaluated compounds is discussed in the article.     

Application of response surface methodology to cell immobilization for the production of palatinose

Response surface methodology (RSM), based on multivariate non-linear model, was applied to study the interactions and optimization of the immobilization parameters for cell entrapment, namely alginate concentration, cell loading and bead diameter using Erwinia rhapontici NCPPB 1578 that produced palatinose. ANOVA analysis and statistical parameters calculations showed that RSM could be used effectively to model and improve a complex system like cell immobilization. Palatinose yield was increased by 40%. The maximum yield of 140 mg/ml was achieved in a batch of 1h at alginate concentration of 5% w/v, cell loading of 5 g l(-1) and 2.25 mm bead diameter. Thus, the E. rhapontici NCPPB 1578 immobilization in alginate bead and subsequent palatinose yield was successfully improved by application of RSM technique.     

Comparison of topological clustering within protein networks using edge metrics that evaluate full sequence,full structure,and active site microenvironment similarity

The development of accurate protein function annotation methods has emerged as a major unsolved biological problem. Protein similarity networks, one approach to function annotation via annotation transfer, group proteins into similarity-based clusters. An underlying assumption is that the edge metric used to identify such clusters correlates with functional information. In this contribution, this assumption is evaluated by observing topologies in similarity networks using three different edge metrics: sequence (BLAST), structure (TM-Align), and active site similarity (active site profiling, implemented in DASP). Network topologies for four well-studied protein superfamilies (enolase, peroxiredoxin (Prx), glutathione transferase (GST), and crotonase) were compared with curated functional hierarchies and structure. As expected, network topology differs, depending on edge metric; comparison of topologies provides valuable information on structure/function relationships. Subnetworks based on active site similarity correlate with known functional hierarchies at a single edge threshold more often than sequence- or structure-based networks. Sequence- and structure-based networks are useful for identifying sequence and domain similarities and differences; therefore, it is important to consider the clustering goal before deciding appropriate edge metric. Further, conserved active site residues identified in enolase and GST active site subnetworks correspond with published functionally important residues. Extension of this analysis yields predictions of functionally determinant residues for GST subgroups. These results support the hypothesis that active site similarity-based networks reveal clusters that share functional details and lay the foundation for capturing functionally relevant hierarchies using an approach that is both automatable and can deliver greater precision in function annotation than current similarity-based methods.     

Design and synthesis of 3-(azol-1-yl)phenylpropanes as microbicidal spermicides for prophylactic contraception

We designed a series of 25 3-(azol-1-yl)phenylpropanes which yielded 10 compounds (3, 4, 7, 8, 13, 14, 19, 21, 23, 26) that irreversibly immobilized 100% human sperm at 1% (w/v) concentration in 60 s; 12 compounds (8, 9, 15, 16, 19-21, 23-25, 27, 28) that showed potent microbicidal activity at 12.5-50 μg/mL against Trichomonas vaginalis; and 17 compounds (3-11, 13, 15, 19, 21, 23, 26, 28, 30) that exhibited potent anticandida activity with minimum inhibitory concentration (MIC) of 12.5-50 μg/mL. Almost all the compounds exhibited high level of safety towards normal vaginal flora (Lactobacillus) and human cervical (HeLa) cells in comparison to the marketed spermicide nonoxynol-9 (N-9). All the biological activities were evaluated in vitro. Two compounds (4, 8) with good safety profile exhibited multiple (spermicidal, antitrichomonas and anticandida) activities, warranting further lead optimization for furnishing a prophylactic vaginal contraceptive.     

Novel diarylheptanoids as inhibitors of TNF-α production

Synthesis and anti-inflammatory activity of novel diarylheptanoids [5-hydroxy-1-phenyl-7-(pyridin-3-yl)-heptan-3-ones and 1-phenyl-7-(pyridin-3-yl)hept-4-en-3-ones] as inhibitors of tumor necrosis factor-α (TNF-α) production is described in the present article. The key reactions involve the formation of a β-hydroxyketone by the reaction of substituted 4-phenyl butan-2-ones with pyridine-3-carboxaldehyde in presence of LDA and the subsequent dehydration of the same to obtain the α,β-unsaturated ketones. Compounds 4i, 5b, 5d, and 5g significantly inhibit lipopolysaccharide (LPS)-induced TNF-α production from human peripheral blood mononuclear cells in a dose-dependent manner. Of note, the in vitro TNF-α inhibition potential of 5b and 5d is comparable to that of curcumin (a naturally occurring diarylheptanoid). Most importantly, oral administration of 4i, 5b, 5d, and 5g (each at 100 mg/kg) but not curcumin (at 100 mg/kg) significantly inhibits LPS-induced TNF-α production in BALB/c mice. Collectively, our findings indicate that these compounds may have potential therapeutic implications for TNF-α-mediated auto-immune/inflammatory disorders.     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.