A mutation in the pericentrin gene causes abnormal interneuron migration to the olfactory bulb in mice

Precise control of neuronal migration is essential for proper function of the brain. Taking a forward genetic screen, we isolated a mutant mouse with defects in interneuron migration. By genetic mapping, we identified a frame shift mutation in the pericentrin (Pcnt) gene. The Pcnt gene encodes a large centrosomal coiled-coil protein that has been implicated in schizophrenia. Recently, frame shift and premature termination mutations in the pericentrin (PCNT) gene were identified in individuals with Seckel syndrome and microcephalic osteodysplastic primordial dwarfism (MOPD II), both of which are characterized by greatly reduced body and brain sizes. The mouse Pcnt mutant shares features with the human syndromes in its overall growth retardation and reduced brain size. We found that dorsal lateral ganglionic eminence (dLGE)-derived olfactory bulb interneurons are severely affected and distributed abnormally in the rostral forebrain in the mutant. Furthermore, mutant interneurons exhibit abnormal migration behavior and RNA interference knockdown of Pcnt impairs cell migration along the rostal migratory stream (RMS) into the olfactory bulb. These findings indicate that pericentrin is required for proper migration of olfactory bulb interneurons and provide a developmental basis for association of pericentrin function with interneuron defects in human schizophrenia.     

Shear-induced Interleukin-6 Synthesis in Chondrocytes: ROLES OF E PROSTANOID (EP) 2 AND EP3 IN cAMP/PROTEIN KINASE A- AND PI3-K/Akt-DEPENDENT NF-κB ACTIVATION*

Mechanical overloading of cartilage producing hydrostatic stress, tensile strain, and fluid flow can adversely affect chondrocyte function and precipitate osteoarthritis (OA). Application of high fluid shear stress to chondrocytes recapitulates the earmarks of OA, as evidenced by the release of pro-inflammatory mediators, matrix degradation, and chondrocyte apoptosis. Elevated levels of cyclooxygenase-2 (COX-2), prostaglandin (PG) E₂, and interleukin (IL)-6 have been reported in OA cartilage in vivo, and in shear-activated chondrocytes in vitro. Although PGE₂ positively regulates IL-6 synthesis in chondrocytes, the underlying signaling pathway of shear-induced IL-6 expression remains unknown. Using the human T/C-28a2 chondrocyte cell line as a model system, we demonstrate that COX-2-derived PGE₂ signals via up-regulation of E prostanoid (EP) 2 and down-regulation of EP3 receptors to raise intracellular cAMP, and activate protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3-K)/Akt pathways. PKA and PI3-K/Akt transactivate the NF-κB p65 subunit via phosphorylation at Ser-276 and Ser-536, respectively. Binding of p65 to the IL-6 promoter elicits IL-6 synthesis in sheared chondrocytes. Selective knockdown of EP2 or ectopic expression of EP3 blocks PKA- and PI3-K/Akt-dependent p65 activation and markedly diminishes shear-induced IL-6 expression. Similar inhibitory effects on IL-6 synthesis were observed by inhibiting PKA, PI3-K, or NF-κB using pharmacological and/or genetic interventions. Reconstructing the signaling network regulating shear-induced IL-6 expression in chondrocytes may provide insights for developing therapeutic strategies for arthritic disorders and for culturing artificial cartilage in bioreactors.     

Preparation and characterization of lyophilized liposomes with incorporated quercetin

Liposomes composed of egg-phosphatidylcholine (EPC) incorporating quercetin (QR) were prepared by the thin-film hydration method (TFHM) and the monophase solution method (MSM). A rapid and slow freeze-drying process was applied for both laboratory and industrial scales. The purpose of this study was to compare the two methods of liposome preparation, and further determine whether the lyophilization process affects the liposome physicochemical characteristics (size, polydispersity index, and ?-potential) and incorporation of quercetin.     

Biodegradation of Crude Oil by Thermophilic Bacteria Isolated from a Volcano Island

One-hundred and fifty different thermophilic bacteria isolated from a volcanic island were screened for detection of an alkane hydroxylase gene using degenerated primers developed to amplify genes related to the Pseudomonas putida and Pseudomonas oleovorans alkane hydroxylases. Ten isolates carrying the alkJ gene were further characterized by 16s rDNA gene sequencing. Nine out of ten isolates were phylogenetically affiliated with Geobacillus species and one isolate with Bacillus species. These isolates were able to grow in liquid cultures with crude oil as the sole carbon source and were found to degrade long chain crude oil alkanes in a range between 46.64% and 87.68%. Results indicated that indigenous thermophilic hydrocarbon degraders of Bacillus and Geobacillus species are of special significance as they could be efficiently used for bioremediation of oil-polluted soil and composting processes.     

Stimulation of human endothelium with IL-3 induces selective basophil accumulation in vitro

Basophils have been shown to accumulate in allergic airways and other extravascular sites. Mechanisms responsible for the selective recruitment of basophils from the blood into tissue sites remain poorly characterized. In this study, we characterized human basophil rolling and adhesion on HUVECs under physiological shear flow conditions. Interestingly, treatment of endothelial cells with the basophil-specific cytokine IL-3 (0.01-10 ng/ml) promoted basophil and eosinophil, but not neutrophil, rolling and exclusively promoted basophil adhesion. Preincubation of HUVECs with an IL-3R-blocking Ab (CD123) before the addition of IL-3 inhibited basophil rolling and adhesion, implicating IL-3R activation on endothelial cells. Incubation of basophils with neuraminidase completely abolished both rolling and adhesion, indicating the involvement of sialylated structures in the process. Abs to the beta(1) integrins, CD49d and CD49e, as well as to P-selectin and P-selectin glycoprotein ligand 1, inhibited basophil rolling and adhesion. Furthermore, blocking chemokine receptors expressed by basophils, such as CCR2, CCR3, and CCR7, demonstrated that CCR7 was involved in the observed recruitment of basophils. These data provide novel insights into how IL-3, acting directly on endothelium, can cause basophils to preferentially interact with blood vessels under physiological flow conditions and be selectively recruited to sites of inflammation.     

Multiple epigenetic maintenance factors implicated by the loss of Mll2 in mouse development

Epigenesis is the process whereby the daughters of a dividing cell retain a chromatin state determined before cell division. The best-studied cases involve the inheritance of heterochromatic chromosomal domains, and little is known about specific gene regulation by epigenetic mechanisms. Recent evidence shows that epigenesis pivots on methylation of nucleosomes at histone 3 lysines 4, 9 or 27. Bioinformatics indicates that mammals have several enzymes for each of these methylations, including at least six histone 3 lysine 4 methyltransferases. To look for evidence of gene-specific epigenetic regulation in mammalian development, we examined one of these six, Mll2, using a multipurpose allele in the mouse to ascertain the loss-of-function phenotype. Loss of Mll2 slowed growth, increased apoptosis and retarded development, leading to embryonic failure before E11.5. Using chimera experiments, we demonstrated that Mll2 is cell-autonomously required. Evidence for gene-specific regulation was also observed. Although Mox1 and Hoxb1 expression patterns were correctly established, they were not maintained in the absence of Mll2, whereas Wnt1 and Otx2 were. The Mll2 loss-of-function phenotype is different from that of its sister gene Mll, and they regulate different Hox complex genes during ES cell differentiation. Therefore, these two closely related epigenetic factors play different roles in development and maintain distinct gene expression patterns. This suggests that other epigenetic factors also regulate particular patterns and that development entails networks of epigenetic specificities.     

Depth estimation using the compound eye of dipteran flies

In the neural superposition eye of a dipteran fly every ommatidium has eight photoreceptors, each associated with a rhabdomere, two central and six peripheral, which altogether result in seven functional light guides. Groups of eight rhabdomeres in neighboring ommatidia have largely overlapping fields of view. Based on the hypothesis that the light signals collected by these rhabdomeres can be used individually, we investigated the feasibility of estimating 3D scene information. According to Pick (Biol Cybern 26:215–224, 1977) the visual axes of these rhabdomeres are not parallel, but converge to a point 3–6 mm in front of the cornea. Such a structure theoretically could estimate depth in a very simple way by assuming that locally the image intensity is well approximated by a linear function of the spatial coordinates. Using the measurements of Pick (Biol Cybern 26:215–224, 1977) we performed simulation experiments to find whether this is practically possible. Our results indicate that depth estimation at small distances (up to about 1.5–2 cm) is reasonably accurate. This would allow the insect to obtain at least an ordinal spatial layout of its operational space when walking.     

Toward a more robust assessment of intraspecies diversity, using fewer genetic markers

Phylogenetic sequence analysis of single or multiple genes has dominated the study and census of the genetic diversity among closely related bacteria. It remains unclear, however, how the results based on a few genes in the genome correlate with whole-genome-based relatedness and what genes (if any) best reflect whole-genome-level relatedness and hence should be preferentially used to economize on cost and to improve accuracy. We show here that phylogenies of closely related organisms based on the average nucleotide identity (ANI) of their shared genes correspond accurately to phylogenies based on state-of-the-art analysis of their whole-genome sequences. We use ANI to evaluate the phylogenetic robustness of every gene in the genome and show that almost all core genes, regardless of their functions and positions in the genome, offer robust phylogenetic reconstruction among strains that show 80 to 95% ANI (16S rRNA identity, >98.5%). Lack of elapsed time and, to a lesser extent, horizontal transfer and recombination make the selection of genes more critical for applications that target the intraspecies level, i.e., strains that show >95% ANI according to current standards. A much more accurate phylogeny for the Escherichia coli group was obtained based on just three best-performing genes according to our analysis compared to the concatenated alignment of eight genes that are commonly employed for phylogenetic purposes in this group. Our results are reproducible within the Salmonella, Burkholderia, and Shewanella groups and therefore are expected to have general applicability for microevolution studies, including metagenomic surveys.     

LC-MS/MS based proteomic analysis and functional inference of hypothetical proteins in Desulfovibrio vulgaris

High efficiency capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to examine the proteins extracted from Desulfovibrio vulgaris cells across six treatment conditions. While our previous study provided a proteomic overview of the cellular metabolism based on proteins with known functions [W. Zhang, M.A. Gritsenko, R.J. Moore, D.E. Culley, L. Nie, K. Petritis, E.F. Strittmatter, D.G. Camp II, R.D. Smith, F.J. Brockman, A proteomic view of the metabolism in Desulfovibrio vulgaris determined by liquid chromatography coupled with tandem mass spectrometry, Proteomics 6 (2006) 4286-4299], this study describes the global detection and functional inference for hypothetical D. vulgaris proteins. Using criteria that a given peptide of a protein is identified from at least two out of three independent LC-MS/MS measurements and that for any protein at least two different peptides are identified among the three measurements, 129 open reading frames (ORFs) originally annotated as hypothetical proteins were found to encode expressed proteins. Functional inference for the conserved hypothetical proteins was performed by a combination of several non-homology based methods: genomic context analysis, phylogenomic profiling, and analysis of a combination of experimental information, including peptide detection in cells grown under specific culture conditions and cellular location of the proteins. Using this approach we were able to assign possible functions to 20 conserved hypothetical proteins. This study demonstrated that a combination of proteomics and bioinformatics methodologies can provide verification of the expression of hypothetical proteins and improve genome annotation.     

Defective blood vessel development and pericyte/pvSMC distribution in alpha 4 integrin-deficient mouse embryos

Blood vessel development is in part regulated by pericytes/presumptive vascular smooth muscle cells (PC/pvSMCs). Here, we demonstrate that interactions between PC/pvSMCs and extracellular matrix play a critical role in this event. We show that the cranial vessels in alpha4 integrin-deficient mouse embryos at the stage of vessel remodeling are increased in diameter. This defect is accompanied by a failure of PC/pvSMCs, which normally express alpha4beta1 integrin, to spread uniformly along the vessels. We also find that fibronectin but not VCAM-1 is localized in the cranial vessels at this stage. Furthermore, cultured alpha4 integrin-null PC/pvSMCs plated on fibronectin display a delay in initiating migration, a reduction in migration speed, and a decrease in directional persistence in response to a polarized force of shear flow. These results suggest that specific motile activities of PC/pvSMCs regulated by mechanical signals imposed by the interstitial extracellular matrix may also be required in vivo for the distribution and function of the PC/pvSMCs during blood vessel development.