Deposits of transforming growth factor-beta-induced protein in granular corneal dystrophy type II after LASIK.

PURPOSE: To analyze components of the deposits in the corneal flap interface of granular corneal dystrophy type II (GCD II) patients after laser in situ keratomileusis (LASIK). METHODS: Four corneal GCD II specimens displaying disease exacerbation after LASIK were analyzed. Three of these specimens included the recipient corneal button after penetrating keratoplasty or deep lamellar keratoplasty for advanced GCD II after LASIK. The fourth specimen, a similar case of GCD II after LASIK, included the amputated corneal flap. Specimens were processed for histopathologic and immunohistochemical analyses. RESULTS: Corneal stromal deposits in the LASIK flaps of all specimens were stained with 3 anti-transforming growth factor-beta-induced protein (TGFBIp) antibodies. The deposits displayed bright red color staining with Masson trichrome; however, negative staining was seen with Congo red, suggesting that hyaline is the main component localizing to the TGFBIp deposits rather than amyloid. CONCLUSIONS: Amorphous granular material deposited along the interface of the LASIK flap in GCD II corneas is composed mainly of hyaline deposits.

Regulation of catecholamine release and tyrosine hydroxylase in human adrenal chromaffin cells by interleukin-1beta: role of neuropeptide Y and nitric oxide.

Adrenal chromaffin cells synthesize and secrete catecholamines and neuropeptides that may regulate hormonal and paracrine signaling in stress and also during inflammation. The aim of our work was to study the role of the cytokine interleukin-1beta (IL-1beta) on catecholamine release and synthesis from primary cell cultures of human adrenal chromaffin cells. The effect of IL-1beta on neuropeptide Y (NPY) release and the intracellular pathways involved in catecholamine release evoked by IL-1beta and NPY were also investigated. We observed that IL-1beta increases the release of NPY, norepinephrine (NE), and epinephrine (EP) from human chromaffin cells. Moreover, the immunoneutralization of released NPY inhibits catecholamine release evoked by IL-1beta. Moreover, IL-1beta regulates catecholamine synthesis as the inhibition of tyrosine hydroxylase decreases IL-1beta-evoked catecholamine release and the cytokine induces tyrosine hydroxylase Ser40 phosphorylation. Moreover, IL-1beta induces catecholamine release by a mitogen-activated protein kinase (MAPK)-dependent mechanism, and by nitric oxide synthase activation. Furthermore, MAPK, protein kinase C (PKC), protein kinase A (PKA), and nitric oxide (NO) production are involved in catecholamine release evoked by NPY. Using human chromaffin cells, our data suggest that IL-1beta, NPY, and nitric oxide (NO) may contribute to a regulatory loop between the immune and the adrenal systems, and this is relevant in pathological conditions such as infection, trauma, stress, or in hypertension.

Glutamate exocytosis from astrocytes controls synaptic strength.

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.

D-serine increases adult hippocampal neurogenesis.

Adult hippocampal neurogenesis results in the continuous formation of new neurons and is a process of brain plasticity involved in learning and memory. The neurogenic niche regulates the stem cell proliferation and the differentiation and survival of new neurons and a major contributor to the neurogenic niche are astrocytes. Among the molecules secreted by astrocytes, D-serine is an important gliotransmitter and is a co-agonist of the glutamate, N-methyl-D-aspartate (NMDA) receptor. D-serine has been shown to enhance the proliferation of neural stem cells in vitro, but its effect on adult neurogenesis in vivo is unknown. Here, we tested the effect of exogenous administration of D-serine on adult neurogenesis in the mouse dentate gyrus. We found that 1 week of treatment with D-serine increased cell proliferation in vivo and in vitro and increased the density of neural stem cells and transit amplifying progenitors. Furthermore, D-serine increased the survival of newborn neurons. Together, these results indicate that D-serine treatment resulted in the improvement of several steps of adult neurogenesis in vivo.

Mutation spectrum of MLL2 in a cohort of Kabuki syndrome patients.

ABSTRACT: BACKGROUND: Kabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause. METHODS: Genomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools. RESULTS: We identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site. CONCLUSIONS: This study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management.

Nitric oxide reduces Cl- absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism.

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na(+) and Cl(-) in the kidney, we asked whether NO regulates net Cl(-) flux (JCl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl(-) absorption. Cl(-) absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced JCl and transepithelial voltage (VT) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N(G)-nitro-l-arginine methyl ester) increased JCl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in JCl, we examined the effect of NO on JCl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive JCl, NO modulates JCl independent of the Na(+)-dependent Cl(-)/HCO3(-) exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H(+)-ATPase and pendrin. In contrast, both total and NO-sensitive JCl and VT were abolished with application of an epithelial Na(+) channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl(-) absorption in the CCD through a mechanism that is ENaC-dependent.

18F-FLT and 125I-IdUrd uptake increase in human tumour cell lines induced by the thymidylate synthase inhibitor FdUrd.

Aim: 5-fluoro-2'-deoxyuridine (FdUrd) depletes the endogenous 5'-deoxythymidine triphosphate (dTTP) pool. We hypothesized whether uptake of exogenous dThd analogues could be favoured through a feedback enhanced salvage pathway and studied the FdUrd effect on cellular uptake of 3'-deoxy-3'-18F-fluorothymidine (18F-FLT) and 5-125I-iodo-2'-deoxyuridine (125I-IdUrd) in different cancer cell lines in parallel. Methods: Cell uptake of 18F-FLT and 125I-IdUrd was studied in 2 human breast, 2 colon cancer and 2 glioblastoma lines. Cells were incubated with/without 1 µmol/l FdUrd for 1 h and, after washing, with 1.2 MBq 18F-FLT or 125I-IdUrd for 0.3 to 2 h. Cell bound 18F-FLT and 125I-IdUrd was counted and expressed in % incubated activity (%IA). Kinetics of 18F-FLT cell uptake and release were studied with/without FdUrd modulation. 2'-3H-methyl-fluorothymidine (2'-3H-FLT) uptake with/without FdUrd pretreatment was tested on U87 spheroids and monolayer cells. Results: Basal uptake at 2 h of 18F-FLT and 125I-IdUrd was in the range of 0.8-1.0 and 0.4-0.6 Bq/cell, respectively. FdUrd pretreatment enhanced 18F-FLT and 125I-IdUrd uptake 1.2-2.1 and 1.7-4.4 fold, respectively, while co-incubation with excess thymidine abrogated all 18F-FLT uptake. FdUrd enhanced 18F-FLT cellular inflow in 2 breast cancer lines by factors of 1.8 and 1.6, respectively, while outflow persisted at a slightly lower rate. 2'-3H-FLT basal uptake was very low while uptake increase after FdUrd was similar in U87 monolayer cells and spheroids. Conclusions: Basal uptake of 18F-FLT was frequently higher than that of 125I-IdUrd but FdUrd induced uptake enhancement was stronger for 125I-IdUrd in five of six cell lines. 18F-FLT outflow from cells might be an explanation for the observed difference with 125I-IdUrd.

Impact of biological and environmental variabilities on biological monitoring: an approach using toxicokinetic models

Biological monitoring of occupational exposure is characterized by important variability, due both to variability in the environment and to biological differences between workers. A quantitative description and understanding of this variability is important for a dependable application of biological monitoring. This work describes this variability,using a toxicokinetic model, for a large range of chemicals for which reference biological reference values exist. A toxicokinetic compartmental model describing both the parent compound and its metabolites was used. For each chemical, compartments were given physiological meaning. Models were elaborated based on physiological, physicochemical, and biochemical data when available, and on half-lives and central compartment concentrations when not available. Fourteen chemicals were studied (arsenic, cadmium, carbon monoxide, chromium, cobalt, ethylbenzene, ethyleneglycol monomethylether, fluorides, lead, mercury, methyl isobutyl ketone, penthachlorophenol, phenol, and toluene), representing 20 biological indicators. Occupational exposures were simulated using Monte Carlo techniques with realistic distributions of both individual physiological parameters and exposure conditions. Resulting biological indicator levels were then analyzed to identify the contribution of environmental and biological variability to total variability. Comparison of predicted biological indicator levels with biological exposure limits showed a high correlation with the model for 19 out of 20 indicators. Variability associated with changes in exposure levels (GSD of 1.5 and 2.0) is shown to be mainly influenced by the kinetics of the biological indicator. Thus, with regard to variability, we can conclude that, for the 14 chemicals modeled, biological monitoring would be preferable to air monitoring. For short half-lives (less than 7 hr), this is very similar to the environmental variability. However, for longer half-lives, estimated variability decreased. [Supplementary materials are available for this article. Go to the publisher's online edition of Journal of Occupational and Environmental Hygiene for the following free supplemental resource: tables detailing the CBTK models for all 14 chemicals and the symbol nomenclature that was used.] [Authors]

Determination of plasma levels of citalopram and its demethylated and deaminated metabolites by gas chromatography and gas chromatography-mass spectrometry.

Sensitive and specific methods based on gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) for the determination of levels of citalopram, desmethylcitalopram and didesmethylcitalopram in the plasma of patients treated with citalopram are presented, as well as a GC-MS procedure for the assay of the citalopram propionic acid derivative. After addition of a separate internal standard for each drug, liquid-solvent extraction is used to separate the basic compounds from the acid compounds. The demethylated amines are derivatized with trifluoroacetic anhydride, and the acid metabolite with methyl iodide. GC-MS is performed in the electron impact mode, as mass spectrometry by the (positive-ion) chemical ionization mode (methane and ammonia) appeared to be unsuitable. The limits of quantification were 1 ng/ml for citalopram and desmethylcitalopram and 2 ng/ml for the other metabolites. The correlation coefficients for the calibration curves (range 10-500 ng/ml) were > or = 0.999 for all compounds, whether determined by GC or GC-MS.

Nitric oxide activity through guanylate cyclase and phosphodiesterase modulation is impaired in fetal lambs with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension and death. Administration of nitric oxide (NO) alone remains ineffective in CDH cases. We investigated in near full-term lambs with and without CDH the role of guanylate cyclase (GC), the enzyme activated by NO in increasing cyclic 3'-5'-guanylosine monophosphate, and the role of phosphodiesterase (PDE) 5, the enzyme-degrading cyclic 3'-5'-guanylosine monophosphate. METHODS: Congenital diaphragmatic hernia was surgically created in fetal lambs at 85 days of gestation. Pulmonary hemodynamics were assessed by means of pressure and blood flow catheters (135 days). In vitro, we tested drugs on rings of isolated pulmonary vessels. RESULTS: In vivo, sodium nitroprusside, a direct NO donor, and methyl-2(4-aminophenyl)-1,2-dihydro-1-oxo-7-(2-pyridinylmethoxy)-4-(3,4,5 trimethoxyphenyl)-3-isoquinoline carboxylate sulfate (T-1032) and Zaprinast, both PDE 5 blockers, reduced pulmonary vascular resistance in CDH and non-CDH animals. The activation of GC by sodium nitroprusside and the inhibition of PDE 5 by T-1032 were less effective in CDH animals. In vitro, the stimulation of GC by 3(5'hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1) (a benzyl indazole derivative) and the inhibition of PDE 5 by T-1032 were less effective in pulmonary vascular rings from CDH animals. The YC-1-induced vasodilation in rings from CDH animals was higher when associated with the PDE 5 inhibitor T-1032. CONCLUSIONS: Guanylate cyclase and PDE 5 play a role in controlling pulmonary vascular tone in fetal lambs with or without CDH. Both enzymes seem to be impaired in fetal lambs with CDH.

Ventricular but not atrial electro-mechanical delay of the embryonic heart is altered by anoxia-reoxygenation and improved by nitric oxide.

BACKGROUND/AIM: Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent. METHODS: Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor). RESULTS: Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values. CONCLUSION: This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.

Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis.

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.

Extent and patterns of MGMT promoter methylation in glioblastoma- and respective glioblastoma-derived spheres.

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255-66. (C)2010 AACR.

Extent and Patterns of MGMT Promoter Methylation in Glioblastoma- and Respective Glioblastoma-Derived Spheres.

PURPOSE: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. EXPERIMENTAL DESIGN: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. RESULTS: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. CONCLUSIONS: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255-66. ©2010 AACR.

Glucose represses connexin36 in insulin-secreting cells.

The gap-junction protein connexin36 (Cx36) contributes to control the functions of insulin-producing cells. In this study, we investigated whether the expression of Cx36 is regulated by glucose in insulin-producing cells. Glucose caused a significant reduction of Cx36 in insulin-secreting cell lines and freshly isolated pancreatic rat islets. This decrease appeared at the mRNA and the protein levels in a dose- and time-dependent manner. 2-Deoxyglucose partially reproduced the effect of glucose, whereas glucosamine, 3-O-methyl-D-glucose and leucine were ineffective. Moreover, KCl-induced depolarization of beta-cells had no effect on Cx36 expression, indicating that glucose metabolism and ATP production are not mandatory for glucose-induced Cx36 downregulation. Forskolin mimicked the repression of Cx36 by glucose. Glucose or forskolin effects on Cx36 expression were not suppressed by the L-type Ca(2+)-channel blocker nifedipine but were fully blunted by the cAMP-dependent protein kinase (PKA) inhibitor H89. A 4 kb fragment of the human Cx36 promoter was identified and sequenced. Reporter-gene activity driven by various Cx36 promoter fragments indicated that Cx36 repression requires the presence of a highly conserved cAMP responsive element (CRE). Electrophoretic-mobility-shift assays revealed that, in the presence of a high glucose concentration, the binding activity of the repressor CRE-modulator 1 (CREM-1) is enhanced. Taken together, these data provide evidence that glucose represses the expression of Cx36 through the cAMP-PKA pathway, which activates a member of the CRE binding protein family.