Meningitic Escherichia coli K1 penetration and neutrophil transmigration across the blood-brain barrier are modulated by alpha7 nicotinic receptor

Alpha7 nicotinic acetylcholine receptor (nAChR), an essential regulator of inflammation, is abundantly expressed in hippocampal neurons, which are vulnerable to bacterial meningitis. However, it is unknown whether α7 nAChR contributes to the regulation of these events. In this report, an aggravating role of α7 nAChR in host defense against meningitic E. coli infection was demonstrated by using α7-deficient (α7(-/-)) mouse brain microvascular endothelial cells (BMEC) and animal model systems. As shown in our in vitro and in vivo studies, E. coli K1 invasion and polymorphonuclear neutrophil (PMN) transmigration across the blood-brain barrier (BBB) were significantly reduced in α7(-/-) BMEC and α7(-/-) mice. Stimulation by nicotine was abolished in the α7(-/-) cells and animals. The same blocking effect was achieved by methyllycaconitine (α7 antagonist). The tight junction molecules occludin and ZO-1 were significantly reduced in the brain cortex of wildtype mice infected with E. coli and treated with nicotine, compared to α7(-/-) cells and animals. Decreased neuronal injury in the hippocampal dentate gyrus was observed in α7(-/-) mice with meningitis. Proinflammatory cytokines (IL-1β, IL-6, TNFα, MCP-1, MIP-1alpha, and RANTES) and adhesion molecules (CD44 and ICAM-1) were significantly reduced in the cerebrospinal fluids of the α7(-/-) mice with E. coli meningitis. Furthermore, α7 nAChR is the major calcium channel for nicotine- and E. coli K1-increased intracellular calcium concentrations of mouse BMEC. Taken together, our data suggest that α7 nAChR plays a detrimental role in the host defense against meningitic infection by modulation of pathogen invasion, PMN recruitment, calcium signaling and neuronal inflammation.     

A conserved peptide in West Nile virus NS4A protein contributes to proteolytic processing and is essential for replication

The West Nile virus strain Kunjin virus (WNV(KUN)) NS4A protein is a multifunctional protein involved in membrane proliferation, stimulation of cellular pathways, and evasion of host defense and is a major component of the WNV(KUN) RNA replication complex. We identified a highly conserved region ((120)P-E-P-E(123)) upstream of the viral protease dibasic cleavage site and investigated whether this motif was required for WNV(KUN) replication. Single point mutations to alanine and a PEPE deletion mutation were created in a full-length infectious WNV(KUN) molecular clone. All mutations drastically impaired viral replication and virion production, except that of the P122A mutant, which was slightly attenuated. These mutations were subsequently transferred to a WNV(KUN) replicon to specifically assess effects on RNA replication alone. Again, all mutants, except P122A, showed severely reduced negative-sense RNA production as well as decreased viral protein production. Correspondingly, immunofluorescence analyses showed a lack of double-stranded RNA (dsRNA) labeling and a dispersed localization of the WNV(KUN) proteins, suggesting that replication complex formation was additionally impaired. Attempts to rescue replication via conservative mutants largely failed except for substitution of Asp at E121, suggesting that a negative charge at this residue is equally important. Analysis of viral protein processing suggested that cleavage of the 2K peptide from NS4A did not occur with the mutant constructs. These observations imply that the combined effects of proline and negatively charged residues within the PEPE peptide are essential to promote the cleavage of 2K from NS4A, which is a prerequisite for efficient WNV replication.     

Growth line deposition and variability in growth of two circumpolar bivalves (Serripes groenlandicus, and Clinocardium ciliatum)

Growth patterns of two common circumpolar bivalves, the Greenland cockle (Serripes groenlandicus), and the hairy cockle (Clinocardium ciliatum) have been used in previous studies to reconstruct environmental conditions in the arctic. To date, there has been no direct determination that growth lines in either species are deposited periodically, and there has been no examination of factors affecting growth. We placed calcein-marked individuals of both species on oceanographic moorings in two fjords (Rijpfjord and Kongsfjord) in the Svalbard archipelago for one and two (Kongsfjord only) years. Growth patterns were compared with concurrent in situ temperature and fluorescence data in order to assess environmental controls on growth. Dark growth lines are evident on the outer shell surface and internally in shell cross section in both S. groenlandicus and C. ciliatum, and both species deposited only one line per year, unequivocally confirming that internal lines are deposited annually. Growth line deposition in both species began in late summer to early fall, before the seasonal decline in temperature. There was no difference in growth of S. groenlandicus between the two fjords despite differences in water temperature (3°C), fluorescence (nearly threefold) and the onset and duration of the winter season. C. ciliatum, however, grew approximately 2.8 times faster in the warmer, more food-rich Kongsfjord than in Rijpfjord. Subannual lines were counted in two individuals of each species from each fjord, but deposition of these lines was not clearly related to number of growing days estimated by temperature and fluorescence.     

Normal and expanded Huntington's disease gene alleles produce distinguishable proteins due to translation across the CAG repeat.

BACKGROUND: An expanded CAG trinucleotide repeat is the genetic trigger of neuronal degeneration in Huntington's disease (HD), but its mode of action has yet to be discovered. The sequence of the HD gene places the CAG repeat near the 5' end in a region where it may be translated as a variable polyglutamine segment in the protein product, huntingtin. MATERIALS AND METHODS: Antisera directed at amino acid stretches predicted by the DNA sequence upstream and downstream of the CAG repeat were used in Western blot and immunohistochemical analyses to examine huntingtin expression from the normal and the HD allele in lymphoblastoid cells and postmortem brain tissue. RESULTS: CAG repeat segments of both normal and expanded HD alleles are indeed translated, as part of a discrete approximately 350-kD protein that is found primarily in the cytosol. The difference in the length of the N-terminal polyglutamine segment is sufficient to distinguish normal and HD huntingtin in a Western blot assay. CONCLUSIONS: The HD mutation does not eliminate expression of the HD gene but instead produces an altered protein with an expanded polyglutamine stretch near the N terminus. Thus, HD pathogenesis is probably triggered by an effect at the level of huntingtin protein.     

Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells requires protein kinase C-alpha activation

Pathogenic fungus Cryptococcus neoformans has a predilection for the central nervous system causing devastating meningoencephalitis. Traversal of C. neoformans across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of C. neoformans . Our previous studies have shown that the CPS1 gene is required for C. neoformans adherence to the surface protein CD44 of human brain microvascular endothelial cells (HBMEC), which constitute the BBB. In this report, we demonstrated that C. neoformans invasion of HBMEC was blocked in the presence of G109203X, a protein kinase C (PKC) inhibitor, and by overexpression of a dominant-negative form of PKCα in HBMEC. During C. neoformans infection, phosphorylation of PKCα was induced and the PKC enzymatic activity was detected in the HBMEC membrane fraction. Our results suggested that the PKCα isoform might play a crucial role during C. neoformans invasion. Immunofluorescence microscopic images showed that induced phospho-PKCα colocalized with β-actin on the membrane of HBMEC. In addition, cytochalasin D (an F-filament-disrupting agent) inhibited fungus invasion into HBMEC in a dose-dependent manner. Furthermore, blockage of PKCα function attenuated actin filament activity during C. neoformans invasion. These results suggest a significant role of PKCα and downstream actin filament activity during the fungal invasion into HBMEC.     

A Computer-Assisted 3D Model for Analyzing the Aggregation of Tumorigenic Cells Reveals Specialized Behaviors and Unique Cell Types that Facilitate Aggregate Coalescence

We have developed a 4D computer-assisted reconstruction and motion analysis system, J3D-DIAS 4.1, and applied it to the reconstruction and motion analysis of tumorigenic cells in a 3D matrix. The system is unique in that it is fast, high-resolution, acquires optical sections using DIC microscopy (hence there is no associated photoxicity), and is capable of long-term 4D reconstruction. Specifically, a z-series at 5 μm increments can be acquired in less than a minute on tissue samples embedded in a 1.5 mm thick 3D Matrigel matrix. Reconstruction can be repeated at intervals as short as every minute and continued for 30 days or longer. Images are converted to mathematical representations from which quantitative parameters can be derived. Application of this system to cancer cells from established lines and fresh tumor tissue has revealed unique behaviors and cell types not present in non-tumorigenic lines. We report here that cells from tumorigenic lines and tumors undergo rapid coalescence in 3D, mediated by specific cell types that we have named “facilitators” and “probes.” A third cell type, the “dervish”, is capable of rapid movement through the gel and does not adhere to it. These cell types have never before been described. Our data suggest that tumorigenesis in vitro is a developmental process involving coalescence facilitated by specialized cells that culminates in large hollow spheres with complex architecture. The unique effects of select monoclonal antibodies on these processes demonstrate the usefulness of the model for analyzing the mechanisms of anti-cancer drugs.     

Population impacts of collecting sea anemones and anemonefish for the marine aquarium trade in the Philippines

Tropical marine ornamentals comprise an increasingly important fishery worldwide. Although the potential for overexploitation of marine ornamentals is great, few studies have addressed the population-level impacts of ornamental exploitation and few ornamental fisheries are managed. Analysis of catch records obtained from collectors over a four-month period in the vicinity of Cebu, Philippines, showed that anemonefish and anemones comprised close to 60% of the total catch. Underwater visual census surveys revealed that both anemone and anemonefish densities were significantly lower in exploited areas than in protected areas. The low density of anemones on exploited reefs accounted for over 80% of the reduced density of anemonefish at those sites. There were similar numbers of anemonefish per unit area of anemone in protected and exploited sites; however, biomass of anemonefish per unit area of anemone was lower in exploited areas. Reduction of anemone removals is recommended to support the sustainable harvest of anemonefish from this region.     

Hormonal regulation of oil accumulation in Brassica seeds: metabolism and biological activity of ABA, 7'-, 8'- and 9'-hydroxy ABA in microspore derived embryos of B. napus

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Simultaneous detection of four nitrofuran metabolites in honey using a multiplexing biochip screening assay

A chemiluminescence-based biochip array sensing technique has been developed and applied to the screening of honey samples for residues of banned nitrofuran antibiotics. Using a multiplex approach, metabolites of the four main nitrofuran antibiotics could be simultaneously detected. Individual antibodies specific towards the metabolites were spotted onto biochips. A competitive assay format, with chemiluminescent response, was employed. The method was validated in accordance with EU legislation (2002/657/EC, 2002), and assessed by comparison with UHPLC-MS/MS testing of 134 honey samples of worldwide origin. A similar extraction method, based on extraction of the analytes on Oasis? SPE cartridges, followed by derivatisation with nitrobenzaldehyde and partition into ethyl acetate, was used for both screening and LC-MS/MS methods. The biochip array method was capable of detecting all four metabolites below the reference point for action of 1 μg kg(-1). The detection capability was below 0.5 μg kg(-1) for the metabolites AHD, AOZ and AMOZ; it was below 0.9 μg kg(-1) for SEM. IC(50) values ranged from 0.14 μg kg(-1) (AMOZ) to 2.19 μg kg(-1) (SEM). This biosensor method possesses the potential to be a fit-for-purpose screening technique in the arena of food safety technology.