thin-sectioning-of-slice-preparations-for-immunohistochemistry

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Expression of the norepinephrine transporter (NET), which mediates the reuptake of norepinephrine into presynaptic nerve terminals, is restricted to noradrenergic (NA) neurons. We have demonstrated previously that the 9.0 kb upstream sequences and the first intron residing in the 5' untranslated area are critical for high-level and NA cell-specific transcription. Here, using transient transfection assays, we show that 4.0 kb of the 5' upstream sequences contains sufficient genetic information to drive reporter gene expression in an NA cell type-specific manner. Three functional domains appear to be potentially important for the regulation of human NET (hNET) gene transcription: an upstream enhancer region at -4.0 to -3.1 kb, a proximal domain at -133 to -75 bp, and a middle silencer region between these two domains. DNase I footprinting analysis of the proximal promoter region shows that a subdomain at -128 to -80 bp is protected in a cell-specific manner. We provide evidence that multiple protein factors interact with the proximal promoter domain to critically regulate the transcriptional activity of the hNET gene. In the middle of this proximal subdomain resides a homeodomain (HD)-binding core motif, which interacts with HD factors, including Phox2a and HoxA5, in an NA-specific manner. Cotransfection analyses suggest that HoxA5 and Phox2a may transactivate the hNET gene promoter. Together with previous studies indicating direct activation of dopamine beta-hydroxylase transcription by Phox2a/2b, the present results support a model whereby Phox2 proteins may coordinately regulate the phenotypic specification of NA neurons by activating both NA biosynthetic and reuptake genes.

A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-specific transcription of the human norepinephrine transporter gene.

Ein proximaler Promotor-Dom&#xE4;ne enth&#xE4;lt eine Homeodomain-bindenden Kernmotiv interagiert mit vielen Transkriptionsfaktoren, einschlie&#xDF;lich HoxA5 und Phox2 Proteine &#x200B;&#x200B;und kritisch reguliert Zelltyp-spezifische Transkription des menschlichen Noradrenalin-Transporter-Gens.

Gentechnische Ver&#xE4;nderung von embryonalen Stammzellen der Maus durch Nurr1 verbessert die Differenzierung und Reifung in dopaminergen Neuronen.

Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor-1alpha promoter. The Nurr1-expression in ES cells lead to up-regulation of all DA neuronal markers tested, resulting in about a 4- to 5-fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4-fold increase in the number of DA neurons in Nurr1-expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic L-amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate.

Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons.

To induce differentiation of embryonic stem cells (ESCs) into specialized cell types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ESCs by overexpressing the transcription factor Nurr1 and coculturing with PA6 stromal cells. Nurr1-expressing ESCs (N2 and N5) differentiated into a higher number of neurons (approximately twofold) than the naÃ¯ve ESCs (D3). In addition, N2/N5-derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)+ neurons than D3 (<30%) and an even greater proportion of TH+ neurons (approximately 90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8, and ascorbic acid. N2/N5-derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein-regulated inwardly rectifying K+ channel 2) and produce and release a higher level of dopamine, compared with D3-derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH+ neurons after implantation into mouse striatum. Taken together, our results show that the combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may pave the way for future cell transplantation therapy in Parkinson's disease.

Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells.

Stromal cell-derived induzierende Aktivit&#xE4;t, Nurr1, und Signalmolek&#xFC;len synergistisch zu induzieren dopaminergen Neuronen aus embryonalen Stammzellen.

Silicon is the second-most abundant element on the surface of the earth, and has been considered important for plant growth and development. As for its role in enhanced plant disease resistance, silicon has been reported to reinforce the physical barrier against the penetration and colonization of pathogens. Rice leaves of silicon-treated plants and control plants at the eight- and twelve-leaf growth stages were analyzed by 29Si solid-state nuclear magnetic resonance spectroscopy to characterize the silicon-induced, cell wall fortification of rice leaves, which demonstrated an ability to counter a pathogen attack.

Solid-state NMR spectroscopy of silicon-treated rice with enhanced host resistance against blast.

Silizium ist die Sekunde-am meisten vorkommende Element auf der Oberfl&auml;che der Erde und wurde als wichtig f&uuml;r Wachstum und Entwicklung. F&uuml;r seine Rolle in erweiterte Anlage Krankheitsresistenz hat Silizium berichtet, die physische Barriere gegen das Eindringen und die Kolonisation von Krankheitserregern zu verst&auml;rken. Reis verl&auml;sst der Silizium-behandelten Pflanzen und Kontrolle Pflanzen zu den acht zw&ouml;lf-Blatt-Entwicklungsstadien von 29Si Solid-State KERNRESONANZSPEKTROSKOPIE charakterisieren die Silizium-induzierte analysiert wurden, Zellwand Anreicherung von Reis l&auml;sst, demonstrierte die ihre um einen Pathogen-Angriff zu begegnen.

Festk&ouml;rper-NMR-Spektroskopie von Silizium-behandelten Reis mit erweiterten Host Widerstand gegen Explosion.

This study was conducted to evaluate prognostic factors and cancer-specific survival (CSS) in a cohort of 41 patients with urachal carcinoma by use of a Bayesian model-averaging approach.

Prognostic factors for urachal cancer: a bayesian model-averaging approach.

Most intraoperative provocative tests previously reported were performed after mesh adjustment to confirm the absence of urine leakage. Instead, our test was performed before adjustment of the mesh to control the tape tension after observing the pattern of the urine leakage. We studied whether this method had an effect on the success rate of transobturator tape (TOT) procedures. A total of 96 patients were included: 47 patients underwent TOT procedures without intraoperative testing (Group I) and 49 patients underwent TOT procedures with testing (Group II). Bladder filling was performed with at least 300 ml of normal saline during the test. After observing the pattern of the urine leakage before adjustment of the mesh by coughing or manual pressure on the suprapubic area, we controlled the mesh tension. In Group I, which did not undergo the intraoperative test, the Valsalva leak-point pressure, cough leak-point pressure, preoperative and postoperative peak flow velocity (Qmax), and postvoiding residual urine (PVR) were 86.46 cmH2O, 101.91 cmH2O, 20.82 ml/s, 22.74 ml/s, 19.77 ml, and 45.98 ml, respectively. Changes in the postoperative and preoperative Qmax and PVR were 1.92 ml/s and 26.21 ml, respectively. In Group II, in which the test was applied, the corresponding results were 85.50 cmH2O, 100.45 cmH2O, 25.60 ml/s, 26.90 ml/s, 17.16 ml, and 29.67 ml, respectively. Changes in the postoperative and preoperative Qmax and PVR were 1.3 ml/s and 12.51 ml, respectively. The two groups showed no significant differences in any of the variables. In Group I, the cure and improvement rates were 70.2% and 27.7%, respectively. In Group II, the rates were 91.8% and 8.2%, respectively. Group II had a significantly higher success rate than Group I (p value= 0.011). In the univariable logistic regression analysis, Group II exhibited a higher odds ratio (4.771) than Group I in terms of cure rate, and Group II had a higher success rate than Group I (p value=0.011). In the multivariable logistic regression analysis, Group II exhibited a higher odds ratio (4.700) than Group I in terms of cure rate under calculation of the variables (namely, age, hypertension, preoperative Qmax, and PVR), and the cure rate of Group II was verified to be significantly higher than that of Group I (p value=0.019). We suggest that our test is an effective method to confirm whether adequate tension is being applied to the tape. Our method presents some advantages in that surgeons can control and adjust the tension of the mesh after observing the degree and pattern of the urine leakage.

Article	Year
A proximal promoter domain containing a homeodomain-binding core motif interacts with multiple transcription factors, including HoxA5 and Phox2 proteins, and critically regulates cell type-s... The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 11923423	2002
Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons. The European journal of neuroscience\| PubMed ID: 12453046	2002
Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells. Stem cells (Dayton, Ohio)\| PubMed ID: 16123386	2006
Solid-state NMR spectroscopy of silicon-treated rice with enhanced host resistance against blast. Analytical sciences : the international journal of the Japan Society for Analytical Chemistry\| PubMed ID: 16760615	2006
Prognostic factors for urachal cancer: a bayesian model-averaging approach. Korean journal of urology\| PubMed ID: 25237458	2014
Intraoperative Observation of the Degree and Pattern of Urine Leakage before Adjustment of the Mesh during a Transobturator Tape Procedure. Chonnam medical journal\| PubMed ID: 25568844	2014

Jae-Joon Park

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