Role of Abca7 in Mouse Behaviours Relevant to Neurodegenerative Diseases

ATP-binding cassette transporters of the subfamily A (ABCA) are responsible for the translocation of lipids including cholesterol, which is crucial for neurological function. Recent studies suggest that the ABC transporter ABCA7 may play a role in the development of brain disorders such as schizophrenia and Alzheimer’s disease. However, Abca7’s role in cognition and other behaviours has not been investigated. Therefore, we characterised homozygous Abca7 knockout mice in a battery of tests for baseline behaviours (i.e. physical exam, baseline locomotion and anxiety) and behaviours relevant to schizophrenia (i.e. prepulse inhibition and locomotor response to psychotropic drugs) and Alzheimer’s disease (i.e. cognitive domains). Knockout mice had normal motor functions and sensory abilities and performed the same as wild type-like animals in anxiety tasks. Short-term spatial memory and fear-associated learning was also intact in Abca7 knockout mice. However, male knockout mice exhibited significantly impaired novel object recognition memory. Task acquisition was unaffected in the cheeseboard task. Female mice exhibited impaired spatial reference memory. This phenomenon was more pronounced in female Abca7 null mice. Acoustic startle response, sensorimotor gating and baseline locomotion was unaltered in Abca7 knockout mice. Female knockouts showed a moderately increased motor response to MK-801 than control mice. In conclusion, Abca7 appears to play only a minor role in behavioural domains with a subtle sex-specific impact on particular cognitive domains.     

The Chlamydomonas Fus1 protein is present on the mating type plus fusion organelle and required for a critical membrane adhesion event during fusion with minus gametes

The molecular mechanisms of the defining event in fertilization, gamete fusion, remain poorly understood. The FUS1 gene in the unicellular, biflagellated green alga Chlamydomonas is one of the few sex-specific eukaryotic genes shown by genetic analysis to be essential for gamete fusion during fertilization. In Chlamydomonas, adhesion and fusion of the plasma membranes of activated mt+ and mt- gametes is accomplished via specialized fusion organelles called mating structures. Herein, we identify the endogenous Fus1 protein, test the idea that Fus1 is at the site of fusion, and identify the step in fusion that requires Fus1. Our results show that Fus1 is a approximately 95-kDa protein present on the external surface of both unactivated and activated mt+ gametes. Bioassays indicate that adhesion between mating type plus and mating type minus fusion organelles requires Fus1 and that Fus1 is functional only after gamete activation. Finally, immunofluorescence demonstrates that the Fus1 protein is present as an apical patch on unactivated gametes and redistributes during gamete activation over the entire surface of the microvillous-like activated plus mating structure, the fertilization tubule. Thus, Fus1 is present on mt+ gametes at the site of cell-cell fusion and essential for an early step in the fusion process.     

Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre‐mRNAs of serine/arginine‐rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis‐elements and trans‐acting proteins involved in regulating AS. Using a splicing reporter (GFP–intron–GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis‐elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis‐elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP–intron–GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto‐regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis‐element involved in AS of a plant SR gene, and elucidated a mechanism for auto‐regulation of AS of this intron.     

A Review on Potential Footprints of Ferulic Acid for Treatment of Neurological Disorders

Ferulic acid is being screened in preclinical settings to combat various neurological disorders. It is a naturally occurring dietary flavonoid commonly found in grains, fruits, and vegetables such as rice, wheat, oats, tomatoes, sweet corn etc., which exhibits protective effects against a number of neurological diseases such as epilepsy, depression, ischemia-reperfusion injury, Alzheimer’s disease, and Parkinson’s disease. Ferulic acid prevents and treats different neurological diseases pertaining to its potent anti-oxidative and anti-inflammatory effects, beside modulating unique neuro-signaling pathways. It stays in the bloodstream for longer periods than other dietary polyphenols and antioxidants and easily crosses blood brain barrier. The use of novel drug delivery systems such as solid-lipid nanoparticles (SLNs) or its salt forms (sodium ferulate, ethyl ferulate, and isopentyl ferulate) further enhance its bioavailability and cerebral penetration. Based on reported studies, ferulic acid appears to be a promising molecule for treatment of neurological disorders; however, more preclinical (in vitro and in vivo) mechanism-based studies should be planned and conceived followed by its testing in clinical settings.

     

Incidence and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

Rho-ROCK-Myosin Signaling Meditates Membrane Type 1 Matrix Metalloproteinase-induced Cellular Aggregation of Keratinocytes

Membrane type 1-matrix metalloproteinase (MT1-MMP, MMP14), which is associated with extracellular matrix (ECM) breakdown in squamous cell carcinoma (SCC), promotes tumor formation and epithelial-mesenchymal transition. However, in this report we demonstrate that MT1-MMP, by cleaving the underlying ECM, causes cellular aggregation of keratinocytes and SCC cells. Treatment with an MMP inhibitor abrogated MT1-MMP-induced phenotypic changes, but decreasing E-cadherin expression did not affect MT1-MMP-induced cellular aggregation. As ROCK1/2 can regulate cell-cell and cell-ECM interaction, we examined its role in mediating MT1-MMP-induced phenotypic changes. Blocking ROCK1/2 expression or activity abrogated the cellular aggregation resulting from MT1-MMP expression. Additionally, blocking Rho and non-muscle myosin attenuated MT1-MMP-induced phenotypic changes. Moreover, SCC cells expressing only the catalytically active MT1-MMP protein demonstrated increased cellular aggregation and increased myosin II activity in vivo when injected subcutaneously into nude mice. Together, these results demonstrate that expression of MT1-MMP may be anti-tumorigenic in keratinocytes by promoting cellular aggregation.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.