Regulation of the chitin degradation and utilization system by the ChiX small RNA in <i>Serratia marcescens</i> 2170

<div><p><i>Serratia marcescens</i> 2170 produces three different types of chitinases and chitin-binding protein CBP21. We found that transposon insertion into the 5′ untranslated region (5′ UTR) of <i>chiPQ</i>-<i>ctb</i> led to defective chitinase and CBP21 production. ChiX small RNA possessed the complementary sequence of the 5′ UTRs of the <i>chiPQ</i>-<i>ctb</i> and <i>chiR</i> and repressed the expression of <i>chiP</i> and <i>chiR</i>. ChiX was detected in a medium containing glucose, glycerol, GlcNAc, and (GlcNAc)₂, but the expression of both <i>chiP</i> and <i>chiR</i> was only observed in a medium containing (GlcNAc)₂. ∆<i>chiX</i> mutant produced chitinases, CBP21, and chitobiase without induction. <i>chiP</i> transcripts were more abundant than those of <i>chiR</i> or <i>chiX</i> in a medium containing (GlcNAc)₂. These results suggest that the constitutively expressed ChiX binds to the highly abundant <i>chiP</i> 5′ UTR, thereby leading to the induction of <i>chiR</i> mRNA translation and the subsequent expression of chitinases and CBP21.</p></div

Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells

<div><p>The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. <i>In vitro</i> BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals.</p></div

Effects of inhibitors of the canonical Wnt pathway on the differentiation of hiPS-ECs into BMEC-like cells by treatment of C6CM.

<p>(A) Expression of <i>Axin-2</i> mRNA was examined in iPSECs, iPSEC-mono and iPSEC-C6CM by qRT-PCR analysis. (B, C) TEER values and permeability coefficients of iPSEC-mono, iPSEC-C6CM or Dkk1- or XAV939-treated iPSEC-C6CM were measured. All results shown are the mean of three independent experiments with the indicated standard deviations (S.D.).</p

Primer list used in quantitative real-time PCR.

<p>Primer list used in quantitative real-time PCR.</p

Differentiation of human iPSCs into ECs.

<p>(A) EC differentiation of human iPSCs. The detailed differentiation procedure is described in the Materials and Methods section. (B) Human iPSC-derived EBs (day 6, day 9 and day 12) were stained with anti-CD34 and anti-CD144 antibodies, and were then subjected to flow cytometric analysis. Representative results from one of the three independent experiments are shown. (C) Total RNA was extracted from undifferentiated human iPSCs (day 0) and human iPSC-derived EBs (day 6 and day 9). Then, qRT-PCR analysis was performed. Results shown are the mean of three independent experiments with the indicated standard deviations (S.D.). * p < 0.05, ** p < 0.01. (D-G) Sorted CD34⁺ cells were cultured with FGF2, ECGS, and heparin on fibronectin-coated plates. They showed an endothelial-like morphology under these culture conditions (D). These cells were stained positive for CD31 and vWF (E). They were also capable of forming tube-like structures on Matrigel (F) and demonstrated acetylated-LDL uptake (G). The scale bar indicates 300 μm (D) or 100 μm (E, F, G).</p

Induction of BMEC-like properties of hiPS-ECs by treatment with C6CM.

<p>(A) hiPS-EC monolayers were transferred onto non-cell culture (iPSEC-mono), C6-cell culture (iPSEC-C6) or C6CM (iPSEC-C6CM) in 24-well plates. Then, the TEER value of each hiPS-EC monolayer was measured at indicated days. (B) The permeability coefficient for FD was measured in hiPS-ECs before (iPSECs) and after 5-day mono-culture (iPSEC-mono), 5-day co-culture with C6 cells (iPSEC-C6) or 5-day culture in C6CM (iPSEC-C6CM). (C) Expressions of tight junction-related genes (<i>Claudin-5</i>, <i>Occuludin</i> and <i>ZO-1</i>) were examined in iPSECs, iPSEC-mono, iPSEC-C6 and iPSEC-C6CM. (D) Expressions of transporter genes (<i>P-gp</i>, <i>Bcrp</i>, <i>Mrp-4</i> and <i>Glut1</i>) were examined in iPSECs, iPSEC-mono, iPSEC-C6 and iPSEC-C6CM by RT-PCR analysis. (E) The iPSEC-C6 was treated with CSA or MK571, and then the permeability coefficient for Rhodamin 123 was investigated in them. All results shown are the mean of three independent experiments with the indicated standard deviations (S.D.). * p < 0.05.</p

Induction of BMEC-like properties in hiPS-ECs by co-culture with C6 cells.

<p>(A) hiPS-ECs and HUVECs were cultured on fibronectin-coated inserts. When confluent, these inserts were transferred onto non-cultured 24-well plates (iPSEC-mono or HUVEC-mono) or C6 cell-cultured 24-well plates (iPSEC-C6 or HUVEC-C6). Then, TEER values for hiPS-ECs and HUVECs were measured at indicated days. (B, C) Expressions of tight junction-related genes (<i>Claudin-5</i>, <i>Occludin</i> and <i>ZO-1</i>) were examined by qRT-PCR analysis before (iPSECs, HUVECs) and after (iPSEC-mono, iPSEC-C6, HUVEC-mono, HUVEC-C6) 5 days of culture. (D) The permeability coefficient for FD was investigated in 5-day co-cultured hiPS-ECs and HUVECs. (E) Expressions of transporter genes (<i>P-gp</i>, <i>Bcrp</i>, <i>Mrp-4</i> and <i>Glut1</i>) were examined in 5-day mono- or co-cultured hiPS-ECs by RT-PCR analysis. (F) The 5-day co-cultured hiPS-ECs were treated with CSA (an inhibitor of P-gp) or MK571 (an inhibitor of MRP-4), and then the permeability coefficient for Rhodamin 123 (a specific substance of P-gp) was measured. All results shown are the mean of three independent experiments with the indicated standard deviations (S.D.). * p < 0.05.</p

Primer list used in Semi-quantitative PCR.

<p>Primer list used in Semi-quantitative PCR.</p

The brain-specific RasGEF very-KIND is required for normal dendritic growth in cerebellar granule cells and proper motor coordination

<div><p>Very-KIND/Kndc1/KIAA1768 (v-KIND) is a brain-specific Ras guanine nucleotide exchange factor carrying two sets of the kinase non-catalytic C-lobe domain (KIND), and is predominantly expressed in cerebellar granule cells. Here, we report the impact of v-KIND deficiency on dendritic and synaptic growth in cerebellar granule cells in v-KIND knockout (KO) mice. Furthermore, we evaluate motor function in these animals. The gross anatomy of the cerebellum, including the cerebellar lobules, layered cerebellar cortex and densely-packed granule cell layer, in KO mice appeared normal, and was similar to wild-type (WT) mice. However, KO mice displayed an overgrowth of cerebellar granule cell dendrites, compared with WT mice, resulting in an increased number of dendrites, dendritic branches and terminals. Immunoreactivity for vGluT2 (a marker for excitatory presynapses of mossy fiber terminals) was increased in the cerebellar glomeruli of KO mice, compared with WT mice. The postsynaptic density around the terminals of mossy fibers was also increased in KO mice. Although there were no significant differences in locomotor ability between KO and WT animals in their home cages or in the open field, young adult KO mice had an increased grip strength and a tendency to exhibit better motor performance in balance-related tests compared with WT animals. Taken together, our results suggest that v-KIND is required for compact dendritic growth and proper excitatory synaptic connections in cerebellar granule cells, which are necessary for normal motor coordination and balance.</p></div

No apparent gross abnormality in the cerebellar lobules or layers, or in the densely-packed granule cell layer in Nissl-stained sections of v-KIND KO cerebella.

<p>Sagittal sections of KO and WT mice cerebella (8-week-old) were analyzed by Nissl staining. (A) The lobular structure of the whole cerebellum. Scale bar, 1 mm. (B) The layer structure of the cerebellar cortex. Scale bar, 100 μm. (C) Magnified view of the granular layer indicated by the red square in (B). Cerebellar lobules II–X. ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer; WM, white matter. (D) Number of Nissl-stained puncta/1,000 μm² in the granule cell layer of WT and KO mice. 5~10 random areas per section, 2 sections per animal, and three mice for each genotype (<i>N</i> = 3) was statistically analyzed. Data are shown as mean ± SEM. Two sample Student’s <i>t</i>-test assuming equal variances showed no statistical significance (<i>p</i> > 0.5).</p