Identification of the coiled-coil domains of Enterococcus faecalis DivIVA that mediate oligomerization and their importance for biological function

Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10-12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVA(Ef) self-interacts and forms a 10-12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVA(Ef) self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVA(Ef) did not affect DivIVA(Ef) oligomerization. The introduction of DivIVA(Ef) mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVA(Ef) in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVA(Ef) are indispensable for proper biological function. Overexpression of wild-type DivIVA(Ef) in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVA(Ef) mutations failed to induce such alterations.     

Cowpea mosaic virus nanoscaffold as signal enhancement for DNA microarrays

Previous studies have shown that a functionalized viral nanoparticle can be used as a fluorescent signal-generating element and enhance detection sensitivity for immunoassays and low density microarrays. In this study, we further tested this ability in commercial DNA microarrays, including Affymetrix high density resequencing microarray. Optimum conditions for NeutrAvidin and dye coupling to a double-cysteine mutant of cowpea mosaic virus (CPMV) were found to be comparable to the commonly used streptavidin-phycoerythrin (SAPE) for high density resequencing microarray. A 3-fold signal enhancement in comparison to Cy5-dCTP controls was obtained when using nanoparticles on control scorecard expression microarrays. Hybridization results from commercially available 8000 rat expression arrays indicate an increment of 14% on the detected features when the virus complex was used as the staining reagent in comparison to Cy5-dCTP controls. The current work shows the utility of the CPMV-dye nanoparticles as a detection reagent in well-established detection platforms.     

Spherezymes: A novel structured self-immobilisation enzyme technology

Background  

Enzymes have found extensive and growing application in the field of chemical organic synthesis and resolution of chiral intermediates. In order to stabilise the enzymes and to facilitate their recovery and recycle, they are frequently immobilised. However, immobilisation onto solid supports greatly reduces the volumetric and specific activity of the biocatalysts. An alternative is to form self-immobilised enzyme particles.     

Initial results for urban metal distributions in house dusts of Syracuse, New York, USA

A program of house dust sample collection and analysis has begun in Syracuse,New York, USA, in order to determine the feasibility of a geography-based exposure assessment for urban metals. The sampling program, and the protocols it employs, is described for two different types of wipe media, Ghost Wipes and Whatman Filters. Preliminary results show that strong spatial patterns of floor dust loading (mg dust per square foot) can be observed for data aggregated at a spatial scale of about 1600 m (～2.5 kin2). Floor dust metal concentrations were similar to those found in other urban environments, with some regional variation. The median floor dust Pb concentration was ～108 mg· kg-1 for this initial data set of ～264 sampled residential locations, and varied from 50 to 1100 mg Pb · kg-1.     

Initial results for urban metal distributions in house dusts of syracuse,New York,USA

A program of house dust sample collection and analysis has begun in Syracuse, New York, USA, in order to determine the feasibility of a geography-based exposure assessment for urban metals. The sampling program, and the protocols it employs, is described for two different types of wipe media, Ghost Wipes and Whatman Filters. Preliminary results show that strong spatial patterns of floor dust loading (mg dust per square foot) can be observed for data aggregated at a spatial scale of about 1600 m (∼2.5 km²). Floor dust metal concentrations were similar to those found in other urban environments, with some regional variation. The median floor dust Pb concentration was ∼108 mg · kg⁻¹ for this initial data set of ∼264 sampled residential locations, and varied from 50 to 1100 mg Pb · kg⁻¹.

     

Dispersal of epithelial cancer cell colonies by lysophosphatidic acid (LPA)

We describe a model system in which cancer cell colonies disperse into single, highly migratory cells in response to lysophosphatidic acid (LPA). Though LPA is known to stimulate chemotaxis and chemokinesis, a colony dispersal effect has not been reported, to our knowledge. Cancer colony dispersal by LPA is comprised of an ordered sequence of events: (1) stimulation of membrane ruffling and formation of lamellipodia, (2) dissolution of adherens junctions, (3) single cell migration in a mesenchymal-like morphology we term "ginkgo-leaf." The net result is dispersal of carcinoma cells from a compact colony. We analyzed these three steps using live-cell imaging and computer-assisted quantification and measured the following parameters: onset of lamellipodia formation, lamellipodia velocity, colony dispersal, trans-epithelial resistance, migrating cell number and speed. Because hepatocyte growth factor (HGF) was described as an epithelial scatter factor, we compared it to LPA in our system and found that HGF has no epithelial colony dispersal properties and that this effect is strictly related to LPA. Given its striking similarity to tumor cell budding observed in patients, we propose that LPA-colony dispersal may provide a cellular mechanism underlying cancer invasion and as such deserves further studies.     

Expression of c-myc is not critical for cell proliferation in established human leukemia lines

Background

A study was undertaken to resolve preliminary conflicting results on the proliferation of leukemia cells observed with different c-myc antisense oligonucleotides.

Results

RNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotides targeting bases 1147–1166 of c-myc mRNA downregulated c-Myc protein and induced apoptosis and cell cycle arrest respectively in cultures of MOLT-4 and KYO1 human leukemia cells. In contrast, an RNase H-inactive, morpholino antisense oligonucleotide analogue 28-mer, simultaneously targeting the exon 2 splice acceptor site and initiation codon, reduced c-Myc protein to barely detectable levels but did not affect cell proliferation in these or other leukemia lines. The RNase H-active oligodeoxynucleotide 20-mers contained the phosphodiester linked motif CGTTG, which as an apoptosis inducing CpG oligodeoxynucleotide 5-mer of sequence type CGNNN (N = A, G, C, or T) had potent activity against MOLT-4 cells. The 5-mer mimicked the antiproliferative effects of the 20-mer in the absence of any antisense activity against c-myc mRNA, while the latter still reduced expression of c-myc in a subline of MOLT-4 cells that had been selected for resistance to CGTTA, but in this case the oligodeoxynucleotide failed to induce apoptosis or cell cycle arrest.

Conclusions

We conclude that the biological activity of the chimeric c-myc antisense 20-mers resulted from a non-antisense mechanism related to the CGTTG motif contained within the sequence, and not through downregulation of c-myc. Although the oncogene may have been implicated in the etiology of the original leukemias, expression of c-myc is apparently no longer required to sustain continuous cell proliferation in these culture lines.     

Helicobacter anseris sp. nov. and Helicobacter brantae sp. nov., Isolated from Feces of Resident Canada Geese in the Greater Boston Area

Numbers of nonmigratory Canada geese have increased substantially in the past decade, and they have become a nuisance in some urban areas. Because of their close contact with humans in parks and areas adjacent to surface waterways, contact with their feces poses a zoonotic risk. A total of 97 geese from 10 separate geographic locales in the greater Boston area had their feces sampled for detection of Helicobacter spp. Identification of Helicobacter spp. based on 16S rRNA genus-specific helicobacter primers was noted in 39 of 97 (40.2%) DNA fecal extracts. Twenty-seven (27.8%) of these geese had helicobacters isolated from their feces. A urease-positive novel species, Helicobacter anseris, based on phenotypic, biochemical, and 16S rRNA analyses, was isolated from 20 geese from seven different flocks. A second, novel, urease-negative Helicobacter sp., H. brantae, was identified in seven geese. Four geese had both novel Helicobacter spp. cultured from their feces. Whether these two novel helicobacters pose a zoonotic risk, similar to other enteric helicobacters (e.g., H. canadensis, previously isolated from diarrheic and bacteremic humans and from geese in Europe), will require further studies.     

Specific and sensitive combined high-performance liquid chromatographic—flow fluorometric assay for intracellular 6-thioguanine nucleotide metabolites of 6-mercaptopurine and 6-thioguanine

A new non-radioisotopic technique is described for measuring rates of intracellular formation by human leukemic blasts of 6-thioguanine nucleotide metabolites, obligatory intermediates in the antineoplastic action of both 6-mercaptopurine and 6-thioguanine itself. The method is both specific and sensitive, and involves combined high-performance liquid chromatography and flow fluorometric detection of oxidized 6-thioguanine nucleotides in alkaline permanganate-treated cell extracts. Non-metabolized 6-thioguanine and 6-thioxanthine are also separated and quantitated in this system, permitting complementary in vivo pharmacokinetic analysis. The assay may be applied to detect resistant disease at an early stage in therapy, and thereby provides the opportunity for alternative treatments to be instituted.