Conductance of GABAA Channels Activated by Pentobarbitone in Hippocampal Neurons from Newborn Rats

Neurons were obtained from the CA1 region of the hippocampus of newborn rats and maintained in culture. Channels were activated by pentobarbitone in cell-attached, inside-out or outside-out patches, normally by applying pentobarbitone in flowing bath solution. Currents were outwardly rectifying and blocked by bicuculline, properties of GABA_A channels in these cells. Maximum channel conductance increased as pentobarbitone concentration was increased to 500 μ m but conductance then decreased as pentobarbitone concentration was raised further. The best fit of a Hill-type equation to the relationship between maximum channel conductance and pentobarbitone concentration (up to 500 μ m ) gave an EC₅₀ of 41 μ m , a maximum conductance of 36 pS and a Hill coefficient of 1.6. Bicuculline decreased the maximum conductance of the channels activated by pentobarbitone, with an IC₅₀ of 224 μ m . Diazepam increased channel conductance, with a maximum effect being obtained with 1 μ M diazepam. Diazepam (1 μ M ) decreased the EC₅₀ of the pentobarbitone effect on channel conductance from 41 μ M to 7.2 μ M and increased maximum conductance to 72 pS. We conclude that GABA_A channel conductance is related to the concentration of the allosteric agonist pentobarbitone.     

Deficiency in Metabolic Regulators PPARγ and PTEN Cooperates to Drive Keratinizing Squamous Metaplasia in Novel Models of Human Tissue Regeneration

Hindgut-derived endoderm can differentiate into rectal, prostatic, and bladder phenotypes. Stromal-epithelial interactions are crucial for this development; however, the precise mechanisms by which epithelium responds to stromal cues remain unknown. We have previously reported ectopic expression of peroxisome proliferator-activated receptor-γ2 (PPARγ2) increased androgen receptor expression and promoted differentiation of mouse prostate epithelium. PPARγ is also implicated in urothelial differentiation. Herein we demonstrate that knockdown of PPARγ2 in benign human prostate epithelial cells (BHPrEs) promotes urothelial transdifferentiation. Furthermore, in vitro and in vivo heterotypic tissue regeneration models with embryonic bladder mesenchyme promoted urothelial differentiation of PPARγ2-deficient BHPrE cells, and deficiency of both PPARγ isoforms 1 and 2 arrested differentiation. Because PTEN deficiency is cooperative in urothelial pathogenesis, we engineered BHPrE cells with combined knockdown of PPARγ and PTEN and performed heterotypic recombination experiments using embryonic bladder mesenchyme. Whereas PTEN deficiency alone induced latent squamous differentiation in BHPrE cells, combined PPARγ and PTEN deficiency accelerated the development of keratinizing squamous metaplasia (KSM). We further confirmed via immunohistochemistry that gene expression changes in metaplastic recombinants reflected human urothelium undergoing KSM. In summary, these data suggest that PPARγ isoform expression provides a molecular basis for observations that adult human epithelium can be transdifferentiated on the basis of heterotypic mesenchymal induction. These data also implicate PPARγ and PTEN inactivation in the development of KSM.Bidirectional communication between epithelial and stromal tissue compartments is essential for normal organ development and tissue differentiation. In the context of developmental biology, mesenchyme has been historically classified as having the ability to act in either a permissive or instructive manner.¹ Permissive mesenchyme enables the epithelium to follow a default preprogramed developmental fate, and instructive mesenchyme induces the epithelium to adopt an alternative developmental fate. The ability of mesenchyme to act either permissively or instructively is often tested through implementation of tissue recombination experiments, which can be homotypic (matched mesenchyme and epithelium) or heterotypic.² Landmark tissue recombination studies using embryonic urogenital sinus mesenchyme (UGM) and either embryonic urogenital epithelium or adult bladder urothelium have demonstrated that the urothelium could be instructed to form prostate-like acini.^3–7 Studies have also shown that prostatic differentiation is dependent on expression of a functional androgen receptor (AR) in the stromal compartment.⁸Although important discoveries have been made insofar as the molecular program required for prostate development and differentiation, insights into epithelial and stromal factors important for bladder development and urothelial differentiation have been generally lacking. The identification of pathways important for normal bladder development and urothelial differentiation has important implications for developmental biology, tissue regeneration, and malignancy.^9,10 Recent studies have identified specific molecular pathways in normal bladder development and urothelial response to injury; however, tissue regeneration using genetically manipulated epithelial and stromal components has rarely been applied to determine their importance in bladder development.The peroxisome proliferator-activated receptor-γ (PPARγ) and phosphatase and tensin homologue deleted on chromosome ten (PTEN) pathways are important modulators of cellular energy balance that have been implicated repeatedly in urothelial and prostate differentiation and malignancy. PPARγ is expressed as two isoforms, which are tissue specific.¹¹ PTEN is commonly lost or mutated in prostate and bladder disease,^12,13 which suggests that it has an important role in the maintenance of differentiation. When combined with inhibition of epidermal growth factor receptor, a pathway repressed by PTEN, the PPARγ agonist troglitazone is associated with increased expression of markers of urothelial differentiation in normal human urothelial cells.¹⁴ An understanding of PPARγ activity in urothelial differentiation is potentially important clinically because use of the PPARγ agonist pioglitazone is associated with an increased risk of bladder cancer.¹⁵ These studies strongly identified PPARγ as an important regulator of the urothelial phenotype but warrant a deeper understanding of the molecular control.^14–20 Inactivation of PTEN has also been implicated in the development of bladder cancer and seems to cooperate with other important genetic changes to drive tumorigenesis.^21,22The objective of the present study was to determine the importance of PPARγ isoforms and PTEN activity in urogenital differentiation. We adapted three-dimensional (3D) co-culture and in vivo tissue regeneration models using a benign human prostate epithelial cell line (BHPrE) that expresses both PPARγ isoforms and regenerates CK14⁺/CK18⁺/AR⁺ prostate tissue in vivo,²³ Knockdown experiments demonstrated that PPARγ2 is essential for induction of prostate epithelial differentiation by urogenital mesenchyme and that PPARγ2-deficient prostate epithelium can be induced by fetal bladder mesenchyme to transdifferentiate into urothelium. Knockdown of both PPARγ isoforms 1 and 2 arrested even urothelial differentiation of BHPrE cells. Furthermore, we demonstrated that tissue regeneration of PTEN-deficient BHPrE cells induced focal metaplasia and hyperplasia at 3 months, followed by squamous metaplasia at 6 months. Deficiency of both PTEN and PPARγ1/2 cooperated to drive keratinizing squamous metaplasia (KSM) by 3 months, which exhibited a similar deregulation of genes implicated in human KSM. In summary, we used novel tissue regeneration models with genetically manipulated human prostate epithelial cells to reproduce and characterize differentiation to urothelial and KSM phenotypes.     

Late anti‐apoptotic effect of KATP channel opening in skeletal muscle

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Amplitude Changes of the Electrically Evoked Compound Action Potential in Children with Cochlear Implants: Preliminary Results

Objective

Use of electrical instead of acoustical stimulation has made much objective electrophysiological evaluation possible. This is useful for management process of young children before and after the cochlear implant. These evaluations have been used for assessment of neuronal survival before cochlear implant and for monitoring of prosthesis function during and after the surgery. Electrically evoked compound action potential is one of these tests which makes a valid and reliable objective evaluation possible. The aim of this study was to evaluate the potential''s amplitude changes three months after receiving the device in pediatric cochlear implant recipients.

Methods

In this longitudinal study, changes of the potential''s amplitude in four given electrodes in four sessions after receiving the device are evaluated by approximately one month intervals in children implanted in Amir Alam and Hazrat-e-Rasoul hospitals, Tehran in July to December 2007.

Findings

The mean amplitude of the electrodes did not significantly change in different sessions, while there was significant difference between the first and the other electrodes’ responses in every session (P<0.05).

Conclusion

Due to high reliability of the responses, the clinician can fit the speech processor for a long time. Better responses in apical electrodes may lead to develop an effective coding strategy.     

Parkinson��s disease-linked LRRK2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures

Sequence variants at or near the leucine-rich repeat kinase 2 (LRRK2) locus have been associated with susceptibility to three human conditions: Parkinson's disease (PD), Crohn's disease and leprosy. As all three disorders represent complex diseases with evidence of inflammation, we hypothesized a role for LRRK2 in immune cell functions. Here, we report that full-length Lrrk2 is a relatively common constituent of human peripheral blood mononuclear cells (PBMC) including affinity isolated, CD14(+) monocytes, CD19(+) B cells, and CD4(+) as well as CD8(+) T cells. Up to 26% of PBMC from healthy donors and up to 43% of CD14(+) monocytes were stained by anti-Lrrk2 antibodies using cell sorting. PBMC lysates contained full-length (>260 kDa) and higher molecular weight Lrrk2 species. The expression of LRRK2 in circulating leukocytes was confirmed by microscopy of human blood smears and in sections from normal midbrain and distal ileum. Lrrk2 reactivity was also detected in mesenteric lymph nodes and spleen (including in dendritic cells), but was absent in splenic mononuclear cells from lrrk2-null mice, as expected. In cultured bone marrow-derived macrophages from mice we made three observations: (i) a predominance of higher molecular weight lrrk2; (ii) the reduction of autophagy marker LC3-II in (R1441C)lrrk2-mutant cells (<31%); and (iii) a significant up-regulation of lrrk2 mRNA (>fourfold) and protein after exposure to several microbial structures including bacterial lipopolysaccharide and lentiviral particles. We conclude that Lrrk2 is a constituent of many cell types in the immune system. Following the recognition of microbial structures, stimulated macrophages respond with altered lrrk2 gene expression. In the same cells, lrrk2 appears to co-regulate autophagy. A pattern recognition receptor-type function for LRRK2 could explain its locus' association with Crohn's disease and leprosy risk. We speculate that the role of Lrrk2 in immune cells may also be relevant to the susceptibility of developing PD or its progression.     

α-Synuclein in human cerebrospinal fluid is principally derived from neurons of the central nervous system

The source of Parkinson disease-linked α-synuclein (aSyn) in human cerebrospinal fluid (CSF) remains unknown. We decided to measure the concentration of aSyn and its gradient in human CSF specimens and compared it with serum to explore its origin. We correlated aSyn concentrations in CSF versus serum (Q(aSyn)) to the albumin quotient (Q(albumin)) to evaluate its relation to blood-CSF barrier function. We also compared aSyn with several other CSF constituents of either central or peripheral sources (or both) including albumin, neuron-specific enolase, β-trace protein and total protein content. Finally, we examined whether aSyn is present within the structures of the choroid plexus (CP). We observed that Q(aSyn) did not rise or fall with Q(albumin) values, a relative measure of blood-CSF barrier integrity. In our CSF gradient analyses, aSyn levels decreased slightly from rostral to caudal fractions, in parallel to the recorded changes for neuron-specific enolase; the opposite trend was recorded for total protein, albumin and β-trace protein. The latter showed higher concentrations in caudal CSF fractions due to the diffusion-mediated transfer of proteins from blood and leptomeninges into CSF in the lower regions of the spine. In postmortem sections of human brain, we detected highly variable aSyn reactivity within the epithelial cell layer of CP in patients diagnosed with a range of neurological diseases; however, in sections of mice that express only human SNCA alleles (and in those without any Snca gene expression), we detected no aSyn signal in the epithelial cells of the CP. We conclude from these complementary results that despite its higher levels in peripheral blood products, neurons of the brain and spinal cord represent the principal source of aSyn in human CSF.