The binding of the PDZ tandem of syntenin to target proteins

PDZ domains are among the most abundant protein modules in the known genomes. Their main function is to provide scaffolds for membrane-associated protein complexes by binding to the cytosolic, C-terminal fragments of receptors, channels, and other integral membrane proteins. Here, using both heteronuclear NMR and single crystal X-ray diffraction, we show how peptides with different sequences, including those corresponding to the C-termini of syndecan, neurexin, and ephrin B, can simultaneously bind to both PDZ domains of the scaffolding protein syntenin. The PDZ2 domain binds these peptides in the canonical fashion, and an induced fit mechanism allows for the accommodation of a range of side chains in the P(0) and P(-)(2) positions. However, binding to the PDZ1 domain requires that the target peptide assume a noncanonical conformation. These data help explain how syntenin, and perhaps other PDZ-containing proteins, may preferentially bind to dimeric and clustered targets, and provide a mechanistic explanation for the previously reported cooperative ligand binding by syntenin's two PDZ domains.     

Polymorphisms in methyl-group metabolism genes and risk of sporadic colorectal cancer with relation to the CpG island methylator phenotype

Background: The CpG island methylator phenotype (CIMP), together with extensive promoter methylation, is regarded as one of the mechanisms involved in colorectal carcinogenesis. The mechanisms underlying CIMP in sporadic colorectal cancer are poorly understood. Genes involved in methyl-group metabolism are likely to affect DNA methylation and thereby influence an individual's risk of CIMP. The aim of the present study was to evaluate whether polymorphisms in the genes encoding methyl-group metabolism pathway predispose to CIMP+ and/or CIMP? CRC. Methods: We examined the potential association between the polymorphisms of MTHFR 677C>T, TS 5′UTR 2R/3R, TS 3′UTR 1494del6, ΔDNMT3B ?149C>T and DNMT3B ?283T>C in a group of 46 CIMP+ CRC cases, 140 CIMP? CRC cases and 140 healthy controls. The CIMP status of the CRC cases was determined by MS-PCR in tumor tissue by a panel of five markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), which was also followed by analyzing hMLH1 methylation and BRAF V600E mutation. Results: The variant allele homozygote genotype for the ΔDNMT3B ?283T>C polymorphism was associated with a decreased risk for CIMP+ CRC (OR: 0.31, 95%CI: 0.09–0.73, p = 0.009). Individuals with TS 3R/3R had an increased risk of CIMP? CRC (OR: 2.21, 95%CI: 1.23–4.91, p = 0.01). Moreover, the carriers of 3R allele had an increased risk of CIMP? CRC (OR: 1.45, 95%CI: 1.10–2.13, p = 0.01). Conclusion: This study provides support to the hypothesis that methyl-group metabolism plays a role in the etiology of both CIMP+ and CIMP? colorectal cancers but has a different impact on a distinct molecular subgroups of colorectal cancer.     

Identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host

Brucella species are gram-negative bacteria which belong to alpha-Proteobacteria family. These organisms are zoonotic pathogens that induce abortion and sterility in domestic mammals and chronic infections in humans known as Malta fever. The virulence of Brucella is dependent upon its ability to enter and colonize the cells in which it multiplies. The genetic basis of this aspect is poorly understood. Signature-tagged mutagenesis (STM) was used to identify potential Brucella virulence factors. PCR amplification has been used in place of DNA hybridization to identify the STM-generated attenuated mutants. A library of 288 Brucella melitensis 16M tagged mini-Tn5 Km2 mutants, in 24 pools, was screened for its ability to colonize spleen, lymph nodes and liver of goats at three weeks post-i.v. infection. This comparative screening identified 7 mutants (approximately 5%) which were not recovered from the output pool in goats. Some genes were known virulence genes involved in biosynthesis of LPS (lpsA gene) or in intracellular survival (the virB operon). Other mutants included ones which had a disrupted gene homologous to flgF, a gene coding for the basal-body rod of the flagellar apparatus, and another with a disruption in a gene homologous to ppk which is involved in the biosynthesis of inorganic polyphosphate (PolyP) from ATP. Other genes identified encoded factors involved in DNA metabolism and oxidoreduction metabolism. Using STM and the caprine host for screening, potential virulence determinants in B. melitensis have been identified.     

2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole: a novel powerful and selective inhibitor of protein kinase CK2

Protein kinase CK2 is a highly pleiotropic enzyme whose high constitutive activity is suspected to be instrumental to the enhancement of the tumour phenotype and to the propagation of infectious diseases. Here we describe a novel compound, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), which is superior to the commonly used specific CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) in several respects. DMAT displays the lowest K(i) value ever reported for a CK2 inhibitor (40 nM); it is cell permeable and its efficacy on cultured cells, both in terms of endogenous CK2 inhibition and induction of apoptosis, is several fold higher than that of TBB. The selectivity of DMAT assayed on a panel of >30 protein kinases is comparable to that of TBB, with the additional advantage of being ineffective on protein kinase CK1 up to 200 microM. These properties make DMAT the first choice CK2 inhibitor for in vivo studies available to date.     

Radiative decay engineering 4. Experimental studies of surface plasmon-coupled directional emission

Fluorescence is typically isotropic in space and collected with low efficiency. In this paper we describe surface plasmon-coupled emission (SPCE), which displays unique optical properties and can be collected with an efficiency near 50%. SPCE occurs for fluorophores within about 200 nm of a thin metallic film, in our case a 50-nm-thick silver film on a glass substrate. We show that fluorophore proximity to this film converts the normally isotropic emission into highly directional emission through the glass substrate at a well-defined angle from the normal axis. Depending on the thickness of the polyvinyl alcohol (PVA) film on the silver, the coupling efficiency of sulforhodamine 101 in PVA ranged from 30 to 49%. Directional SPCE was observed whether the fluorophore was excited directly or by the evanescent field due to the surface plasmon resonance. The emission is always polarized perpendicular to the plane of incidence, irrespective of the polarization of the incident light. The lifetimes are not substantially changed, indicating a mechanism somewhat different from that observed previously for the effects of silver particles on fluorophores. Remarkably, the directional emission shows intrinsic spectral resolution because the coupling angles depend on wavelength. The distances over which SPCE occurs, 10 to 200 nm, are useful because a large number of fluorophores can be localized within this volume. The emission of more distant fluorophores does not couple into the glass, allowing background suppression from biological samples. SPCE can be expected to become rapidly useful in a variety of analytical and medical sensing applications.     

Fluorescence properties of labeled proteins near silver colloid surfaces

The fluorescence properties of a monolayer of labeled avidin molecules were studied near silver island films. We first adsorbed a monolayer of biotinylated-BSA as a base that was used to capture labeled avidin molecules. For labeled avidin on silver island films, we observed an increase of the fluorescence intensity of between 18 and 80 with one-photon excitation and up to several hundredfold or larger with two-photon excitation. The probes were moderately more photostable in the presence of silver islands. There was also a dramatic decrease in the lifetimes with the amplitude-weighted values decreasing from 7- to 35-fold. The data suggest that these spectral changes are due to both increased rates of excitation near the metallic particles and increases in the rates of radiative decay. Because these silver island surfaces are very heterogeneous, we are hopeful that larger increases in intensity and photostability can be obtained for probes situated at an optimal distance from the ideal island surfaces.     

Structure of the N-acetyl-L-rhamnosamine-containing O-polysaccharide of Proteus vulgaris TG 155 from a new Proteus serogroup, O55

The O-polysaccharide of the lipopolysaccharide (LPS) of Proteus vulgaris TG 155 was found to contain 2-acetamido-2,6-dideoxy-L-mannose (N-acetyl-L-rhamnosamine, L-RhaNAc), a monosaccharide that occurs rarely in Nature. The following structure of the O-polysaccharide was established by NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H,13C HSQC experiments, along with chemical methods: [carbohydrate structure in text] Rabbit polyclonal O-antiserum against P. vulgaris TG 155 reacted with both core and O-polysaccharide moieties of the homologous LPS but showed no cross-reactivity with other LPS from the complete set of serologically different Proteus strains. Based on the unique O-polysaccharide structure and the serological data, we propose classifying P. vulgaris TG 155 into a new, separate Proteus O-serogroup, O55.