Neuroprotective Effect of D-Fructose-1,6-Bisphosphate against beta-Amyloid Induced Neurotoxicity in Rat Hippocampal Organotypic Slice Culture : Involvement of PLC and MEK/ERK Signaling Pathways

D-fructose-1,6-bisphosphate (FBP) is an endogenous intermediate of glycolyticpathway which has potent neuroprotective effect against various neurotoxic insults.This study examined whether FBP could antagonize the neurotoxicity induced byamyloid β-peptide (Aβ) in rat hippocampal organotypic slice cultures, and the possiblemechanism was also explored. Treatment with FBP (concentration ranges from 1.7 mMto 10 mM) significantly decreased the cell death in hippocampal slices in the presence ofAβ at 24h, 48h and 72h, and this neuroprotective effect of FBP against Aβ was not in adose-dependent manner, FBP 3.5 mM has better neuroprotective effect than that ofother FBP concentration groups. Treatment with FBP slightly but significantlyincreases the ATP levels in hippocampal slices in the presence of Aβ. However, theincrement of ATP levels was similar among various FBP concentration groups.Neuroprotective effect of FBP 3.5 mM against Aβ induced neurotoxicity inhippocampal slices was attenuated by addition of phospholipase C (PLC) inhibitor,U73122, mitogen activated extracellular signal protein kinase (MEK) inhibitor, U0126,or extracellular signal activated protein kinase (ERK) inhibitor, PD98059 at 24h, 48hand 72h. However, co-treatment with these three kinds of inhibitors did not change theFBP's effect on ATP levels. Our results suggested FBP has neuroprotective effectagainst Aβ induced neurotoxicity in hippocampal slice cultures, and FBP plays role notonly as an alternative energy source, but also a modulator of PLC and MEK/ERKpathways to regulate the cellular response and survival

Impaired response of perforating arteries to hypercapnia in chronic hyperglycemia

Diabetes mellitus increases the risk of cerebrovascular disease, the effects ofhypercapnia on CBF (cerebral blood flow) and cerebrovascular reactivity duringdiabetes are still inconsistent. Here, we have established a new microangiographictechnique using synchrotron radiation (SPring-8, Japan), which enabled us to visualizerat cerebral vessels with high spatial resolution in real time. The goal of the studypresented here was to identify the effects of chronic hyperglycemia onhypercapnia-induced vascular responses (endothelium-dependent vasodilatation) andnitric oxide (NO) donor- induced vascular responses (endothelium-independent) ofperforating arteries and of the deeply located large cerebral arteries. We found asignificant vasodilatation of rat perforating arteries after hypercapnia with a maximumdiameter of approximately 140% of baseline in normal Wistar rats. Chronichyperglycemia impaired vasodilatation of perforating arteries in genetically diabeticGK rats. SNP (sodium nitroprusside) caused a similar vasodilatation of perforatingvessels in normal and chronic hyperglycemia, indicating that endothelium-dependentvasodilatation of perforating arteries may be specifically impaired in chronichyperglycemia. Possible impairment of endothelium-dependent vasodilatation inperforating vessels during chronic hyperglycemia may cause decreased vascularreserve capacity of perforating artery, resulting in the increased ischemic insults andcerebrovascular diseases in diabetes

CD271 Defines a Stem Cell-Like Population in Hypopharyngeal Cancer

<div><p>Cancer stem cells contribute to the malignant phenotypes of a variety of cancers, but markers to identify human hypopharyngeal cancer (HPC) stem cells remain poorly understood. Here, we report that the CD271⁺ population sorted from xenotransplanted HPCs possesses an enhanced tumor-initiating capability in immunodeficient mice. Tumors generated from the CD271⁺ cells contained both CD271⁺ and CD271⁻ cells, indicating that the population could undergo differentiation. Immunohistological analyses of the tumors revealed that the CD271⁺ cells localized to a perivascular niche near CD34⁺ vasculature, to invasive fronts, and to the basal layer. In accordance with these characteristics, a stemness marker, <i>Nanog</i>, and <i>matrix metalloproteinases (MMPs)</i>, which are implicated in cancer invasion, were significantly up-regulated in the CD271⁺ compared to the CD271<b>⁻</b> cell population. Furthermore, using primary HPC specimens, we demonstrated that high CD271 expression was correlated with a poor prognosis for patients. Taken together, our findings indicate that CD271 is a novel marker for HPC stem-like cells and for HPC prognosis.</p></div

In vivo tumorigenicity and differentiation capacity of CD271⁺ cells.

<p>(A) Representative tumors in mice into which the indicated number of cells were transplanted. Red arrowheads indicate CD271⁺ cell injection sites (right side), and blue arrowheads indicate CD271⁻ cell injection sites (left side). Circles and dashed circles indicate transplantation locations resulting in success and failure of tumor formation, respectively. (B) Flow cytometry analysis of the sorted cells (96.8∼99.5% CD271⁺ or CD271⁻ cells). (C) Thirty CD271⁺ cells (right side) and CD271⁻ cells (left side) were transplanted into a mouse, and the generated tumors were resected. Tumor growth was plotted using the average value. (D) Flow cytometry analysis of the CD271 expression of tumor cells resulting from the injection of CD271⁺ cells. Tumor sections were stained with hematoxylin and eosin (H&E). Scale bar: 100 µm.</p

Expression profile of Nanog and MMPs in CD271⁺ and CD271⁻ cells.

<p><i>Nanog</i> expression (A) and <i>MMP1</i>, <i>MMP2</i>, and <i>MMP10</i> expression (B) in CD271⁺ and CD271⁻ cells derived from the three HPC lines were analyzed by real-time RT-PCR. The transcript levels were normalized to those for <i>GAPDH</i>, and the fold change in the MMP expression level in CD271⁺ cells versus CD271⁻ cells was calculated for each sample. Values are the mean±SD of triplicate experiments.</p

Distribution of CD271⁺ cells in HPC.

<p>(A) Cells derived from three HPC xenotransplanted lines were stained for CD271 and analyzed by FACS. (B) Tumor tissues dissected from mice transplanted with the three HPC lines were analyzed for CD271 expression by IHC. Immunopositivity appears brown (a, b, and c). High-magnification images are linked to their respective boxed areas by arrows. Scale bar: 100 µm. Red line indicates the border of the tumor and stroma (d). Red line indicates the border of a CD271⁺ cell cluster and CD271⁻ cells, and red and blue arrowheads show CD271⁺ cells and CD271⁻ cells, respectively (e). (C) Representative results of IHC for CD271 in clinical specimens. Normal mucosa (a), carcinoma <i>in situ</i>. (b). Arrowheads indicate an invasive front, and strongly positive CD271 expression (c, d). (D) IHC for CD34 with New Fuchsin substrate (a), and double staining for CD34 (New Fuchsin) and CD271 (DAB) (b-e). CD34 immunopositivity appears red. Insets show high-magnification images of the boxed areas. Red arrowheads indicate CD34-positive microvessels, and brown arrowheads indicate CD271⁺ cells. (E) Sphere-forming cells of HPCM1, and elevated CD271 expression in the FACS analysis. Scale bar: 25 µm. (F) IHC for the double staining of Ki-67 (New Fuchsin) and CD271 (DAB). Ki-67 immunopositivity appears red in the nucleus. Red arrowheads indicate Ki-67-positive cells. Scale bar: 100 µm.</p

Tumor initiation capability of CD271⁺ cells vs, CD271⁻ cells.

<p>Tumor initiation capability of CD271⁺ cells vs, CD271⁻ cells.</p