A novel and ancient group of type I keratins with members in bichir, sturgeon and gar-2

<p><b>Copyright information:</b></p><p>Taken from "A novel and ancient group of type I keratins with members in bichir, sturgeon and gar"</p><p>http://www.frontiersinzoology.com/content/4/1/16</p><p>Frontiers in Zoology 2007;4():16-16.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1896152.</p><p></p>nce. *For three type I keratin sequences the Ensembl database gene IDs are given

A novel and ancient group of type I keratins with members in bichir, sturgeon and gar-0

<p><b>Copyright information:</b></p><p>Taken from "A novel and ancient group of type I keratins with members in bichir, sturgeon and gar"</p><p>http://www.frontiersinzoology.com/content/4/1/16</p><p>Frontiers in Zoology 2007;4():16-16.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1896152.</p><p></p> and gar. Thick black lines mark the four helical subdomains (coils 1A to 2B), which are typical for the central rod domain of all known IF-proteins. Asterisks indicate identical amino acids; double dots indicate a high and single dots a lower degree of amino acid conservation. Pse, (bichir); Aba, (sturgeon); Loc, (gar). Note that only AbaK14 comprises the complete amino acid sequence. From PseK14a at least a section and from LocK14 the complete head sequence is still missing. From PseK14b we still have to recover both, the complete head and tail sequence

A novel and ancient group of type I keratins with members in bichir, sturgeon and gar-4

<p><b>Copyright information:</b></p><p>Taken from "A novel and ancient group of type I keratins with members in bichir, sturgeon and gar"</p><p>http://www.frontiersinzoology.com/content/4/1/16</p><p>Frontiers in Zoology 2007;4():16-16.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1896152.</p><p></p> and gar. Thick black lines mark the four helical subdomains (coils 1A to 2B), which are typical for the central rod domain of all known IF-proteins. Asterisks indicate identical amino acids; double dots indicate a high and single dots a lower degree of amino acid conservation. Pse, (bichir); Aba, (sturgeon); Loc, (gar). Note that only AbaK14 comprises the complete amino acid sequence. From PseK14a at least a section and from LocK14 the complete head sequence is still missing. From PseK14b we still have to recover both, the complete head and tail sequence

A novel and ancient group of type I keratins with members in bichir, sturgeon and gar-3

<p><b>Copyright information:</b></p><p>Taken from "A novel and ancient group of type I keratins with members in bichir, sturgeon and gar"</p><p>http://www.frontiersinzoology.com/content/4/1/16</p><p>Frontiers in Zoology 2007;4():16-16.</p><p>Published online 6 Jun 2007</p><p>PMCID:PMC1896152.</p><p></p>14 sequences from bichir, sturgeon and gar to the other type I keratins known from vertebrates. The tree was rooted with the lancelet type I keratin sequences. It clearly shows that the K14 sequences form a separate branch (boxed in violet) close to the sequences we cloned from the river lamprey. They even branch off prior to the twig formed by the gnathostomian K18 sequences (boxed in green) that apparently emerged before the separation of cartilaginous and bony fish. The tree, furthermore, suggests that most of the ray-finned fish type I keratins (boxed in blue) evolved independently from those present in lungfish, frog or man and that early in actinopterygian evolution gene duplications already gave rise to at least two different type I keratin groups with members in both, ancient and modern ray-finned fish. Importantly, within the tetrapod lineage the Bayesian analysis revealed four highly supported keratin subgroups, each with members in both, frog and man (encircled by dotted lines and coloured orange). Bar, 0.1 substitutions per site

The siRNA-mediated knockdown of GluN3A in 46C-derived neural stem cells affects mRNA expression levels of neural genes, including known iGluR interactors

<div><p>For years, GluN3A was solely considered to be a dominant-negative modulator of NMDARs, since its incorporation into receptors alters hallmark features of conventional NMDARs composed of GluN1/GluN2 subunits. Only recently, increasing evidence has accumulated that GluN3A plays a more diversified role. It is considered to be critically involved in the maturation of glutamatergic synapses, and it might act as a molecular brake to prevent premature synaptic strengthening. Its expression pattern supports a putative role during neural development, since GluN3A is predominantly expressed in early pre- and postnatal stages. In this study, we used RNA interference to efficiently knock down GluN3A in 46C-derived neural stem cells (NSCs) both at the mRNA and at the protein level. Global gene expression profiling upon GluN3A knockdown revealed significantly altered expression of a multitude of neural genes, including genes encoding small GTPases, retinal proteins, and cytoskeletal proteins, some of which have been previously shown to interact with GluN3A or other iGluR subunits. Canonical pathway enrichment studies point at important roles of GluN3A affecting key cellular pathways involved in cell growth, proliferation, motility, and survival, such as the mTOR pathway. This study for the first time provides insights into transcriptome changes upon the specific knockdown of an NMDAR subunit in NSCs, which may help to identify additional functions and downstream pathways of GluN3A and GluN3A-containing NMDARs.</p></div

Expression of NMDAR and NSC marker mRNAs in transfected 46C-derived NSCs.

<p><b>A:</b> NMDARs. <b>B:</b> NSC markers. 46C-derived NSCs were either transfected with a mixture of scrambled siRNA1/scrambled siRNA2 or with a mixture of GluN3A siRNA1/GluN3A siRNA2. The expression of NMDARs and NSC markers did not change significantly in 46C-derived NSCs upon transfection with siRNA against GluN3A. Data represent means +/- SEM; statistical significances were assigned by unpaired Student’s <i>t</i>-test. hk gene = housekeeping gene; n.s. = not significant. n = 3-8 independent experiments.</p

siRNA-mediated knockdown of GluN3A mRNA in 46C-derived NSCs.

<p>46C-derived NSCs were either transfected with scrambled siRNA or siRNA directed against GluN3A. <b>A:</b> qRT-PCRs were performed to analyse the knockdown of GluN3A at the mRNA level. GluN3A was significantly downregulated upon transfection with siRNA against GluN3A. There were no statistically significant differences in GluN3A expression between non-transfected NSCs and NSCs transfected with either scrambled siRNA1 or scrambled siRNA2. <b>B:</b> Agarose gel electrophoresis after qRT-PCR. The band of GluN3A is much weaker in 46C-derived NSCs transfected with siRNA1 or siRNA2 directed against GluN3A than in 46C-derived NSCs transfected with scrambled siRNA1 or scrambled siRNA2, respectively. Non-transfected 46C-derived NSCs show weak GluN3A expression as well. Expected fragment sizes: 368 bp (<i>β</i>-actin), 240 bp (ubiquitin), and 417 bp (GluN3A). <b>C:</b> Melting point analysis was performed after the qRT-PCRs to determine the melting points of <i>β</i>-actin and GluN3A in transfected 46C-derived NSCs. The melting points of the GluN3A fragments differed in cDNA isolated from 46C-derived NSCs either transfected with scrambled siRNA1 or with siRNA1 directed against GluN3A, whereas the melting points of <i>β</i>-actin were identical in both NSC populations. Data represent means +/- SEM; statistical significances were assigned by unpaired Student’s <i>t</i>-test. **<i>p</i> < 0.01; ***<i>p</i> < 0.001. n = 6-9 independent experiments.</p

Primers used in qRT-PCRs.

<p>Primers used in qRT-PCRs.</p

Hierarchical clustering of transcriptome data.

<p>Hierarchically clustered biological and technical replicates of the transcriptome data using the Euclidean Distance of the signals as a distance metric. scrambl = scrambled siRNA; siGluN3 = siRNA directed against GluN3A.</p

Cytotoxicity and transfection rate of various lipofection reagents for 46C-derived NSCs.

<p>46C-derived NSCs were transfected with an eGFP plasmid using various lipofection reagents. If the corresponding transfection protocol allowed to freely choose the transfection time point, NSCs were transfected 4 hrs after passaging. Otherwise, cells were transfected as suggested by the reagent’s manual (0 h or 24 hrs after passaging). X-tremeGENE siRNA (Roche) was used to transfect 46C-derived NSCs with synthetic siRNAs in the following experiments.</p