Light pulse-induced heme and iron-associated transcripts in mouse brain: a microarray analysis.

Synchronization of circadian oscillators with the outside world is achieved by the acute effects of light on the levels of one or more clock components. In mammals the PAS transcription factors Clock, NPAS2, and BMAL1 regulate gene expression as a function of the day-night cycle. Both PAS domains of NPAS2 were found to bind heme as a prosthetic group, form a gas-regulated sensor, and exert heme-status control of DNA binding in vitro.

Species differences in the effects of prostanoids on MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in bovine and cat iris sphincter smooth muscle.

There is evidence from our own laboratory and that of others that EP-receptor ligands are strong contractile agonists in bovine iris sphincter and that FP-receptor agonists are strong contractile agonists in cat iris sphincter. Here, we have investigated the effects of prostaglandin (PG) receptor agonists of the FP-, EP-, TP- and DP-class on myosin light chain (MLC) phosphorylation, p42/p44 MAP kinase phosphorylation and contraction in the iris sphincter of bovine and cat. Using three signal transduction mechanism assays, namely MLC phosphorylation, MAP kinase phosphorylation and contraction, we demonstrated that in bovine iris sphincter the rank order of potency of the PG agonists in the contractile and MLC phosphorylation assays is as follows: E2>U46619>F2alpha>D2, and in cat F2alpha>D2>E2>U46619.

Bcl-2 family regulation of neuronal development and neurodegeneration.

Neuronal cell death is a key feature of both normal nervous system development and neuropathological conditions. The Bcl-2 family, via its regulation of both caspase-dependent and caspase-independent cell death pathways, is uniquely positioned to critically control neuronal cell survival. Targeted gene disruptions of specific bcl-2 family members and the generation of transgenic mice overexpressing anti- or pro-apoptotic Bcl-2 family members have confirmed the importance of the Bcl-2 family in the nervous system.

Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection.

Conventional immunofluorescence detection of biologically relevant proteins and antigens in tissue sections is often limited by relatively weak signals that fade rapidly on illumination. We have developed an immunohistochemical protocol that combines the sensitivity of tyramide signal amplification with the photostability of quantum dots to overcome these limitations. This simple method provides a sensitive and stable fluorescence immunohistochemical alternative to standard chromogen detection.

Branded versus generic clozapine for treatment of schizophrenia.

Objective: To report clinical findings resulting from a switch from branded to generic clozapine.

Methods: Twenty patients diagnosed with schizophrenia were followed in this naturalistic outpatient study. The Positive and Negative Syndrome Scale (PANSS), Beck Anxiety Inventory (BAI), Abnormal Involuntary Movement Scale, and the Movement Disorder Assessment were used to assess differences in the clinical status of patients before and after switching from Clozaril to generic clozapine (Mylan Pharmaceuticals).

Neurokinin B induces oedema formation in mouse lung via tachykinin receptor-independent mechanisms.

The tachykinin neurokinin B (NKB) has been implicated in the hypertension that characterises pre-eclampsia, a condition where tissue oedema is also observed. The ability of NKB, administered intradermally or intravenously, to induce oedema formation (assessed as plasma extravasation) was examined by extravascular accumulation of intravenously injected (125)I-albumin in wild-type and tachykinin NK(1) receptor knockout mice. Intradermal NKB (30-300 pmol) caused dose-dependent plasma extravasation in wild-type (P < 0.

Circadian cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is driven by the suprachiasmatic nucleus.

Background: Genes encoding the circadian pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) of mammals have recently been identified, but the molecular basis of circadian timing in peripheral tissue is not well understood. We used a custom-made cDNA microarray to identify mouse liver transcripts that show circadian cycles of abundance under constant conditions.

Results: Using two independent tissue sampling and hybridization regimes, we show that approximately 9% of the 2122 genes studied show robust circadian cycling in the liver.

Non-ahr gene susceptibility Loci for porphyria and liver injury induced by the interaction of 'dioxin' with iron overload in mice.

Among the actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) in mice is the induction of hepatic porphyria. This is similar to the most common disease of this type in humans, sporadic porphyria cutanea tarda (PCT). Evidence is consistent with the actions of dioxin being mediated through binding to the aryl hydrocarbon receptor (AHR) with different Ahr alleles in mouse strains apparently accounting for differential downstream gene expression and susceptibility.

Upregulation of phospholipase Cgamma1 activity during EGF-induced proliferation of corneal epithelial cells: effect of phosphoinositide-3 kinase.

Purpose: Previously, the authors showed that epidermal growth factor (EGF) stimulates phospholipase Cgamma1 (PLCgamma1) and phosphoinositide-3 kinase (PI3K) activities in confluent rabbit corneal epithelial cells (RCECs). The purpose of this study was to investigate whether PLCgamma1 activity is upregulated during EGF-induced proliferation of RCECs and to determine whether there is any cross-talk between PLCgamma1 and PI3K in these cells.

Methods: Simian virus (SV)-40-immortalized RCECs were cultured in the presence and absence of EGF and other agents.

Effects of atrial natriuretic peptide and sodium nitroprusside on epidermal growth factor-stimulated wound repair in rabbit corneal epithelial cells.

Purpose: Treatment of rabbit corneal epithelial cells (RCEC) with epidermal growth factor (EGF) stimulates cell proliferation and wound repair in a cell culture model system. Studies have also shown that atrial natriuretic peptide (ANP) and sodium nitroprusside (SNP), a nitric oxide-generating agent, inhibit proliferation of a variety of cell types. The aim of the present work was to examine whether ANP or SNP has any effect on EGF-stimulated proliferation of RCEC involved in wound repair.

Genetic variation of basal iron status, ferritin and iron regulatory protein in mice: potential for modulation of oxidative stress.

Toxic and carcinogenic free radical processes induced by drugs and other chemicals are probably modulated by the participation of available iron. To see whether endogenous iron was genetically variable in normal mice, the common strains C57BL/10ScSn, C57BL/6J, BALB/c, DBA/2, and SWR were examined for major differences in their hepatic non-heme iron contents. Levels in SWR mice were 3- to 5-fold higher than in the two C57BL strains, with intermediate levels in DBA/2 and BALB/c mice.

Epidermal growth factor stimulates phospholipase cgamma1 in cultured rabbit corneal epithelial cells.

Phospholipase Cgamma1 (PLCgamma1) catalyses hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate (IP(3)), two second messengers which play important roles in cell proliferation and differentiation. The purpose of the current study was to identify PLCgamma1 in corneal epithelial cells and investigate whether epidermal growth factor (EGF) stimulates the activity of this enzyme. Addition of EGF to [(3)H]myo-inositol-labeled, cultured corneal epithelial cells stimulated production of IP(3), indicating activation of PLC.

Expression of phosphatidylinositol 3-kinase during EGF-stimulated wound repair in rabbit corneal epithelium.

Purpose: To investigate the effect of epidermal growth factor (EGF) on the induction of phosphatidylinositol 3-kinase (PI 3- kinase) gene expression during rabbit corneal epithelial wound repair.

Methods: Epithelial wounds (6 mm in size) were created in rabbit corneas and EGF (2 microg) applied every 8 hours to one eye, and the other eye served as a control. The wound repair was monitored by staining the tissue with fluorescein followed by photography.

Low levels of p53 are associated with resistance to tetrachlorodibenzo-p-dioxin toxicity in DBA/2 mice.

We show here that DBA/2 strain mice have a complex mutation/polymorphism in the promoter region of the Trp53 locus (the mouse p53 locus). This region has previously been shown to be essential for p53 expression. We further show that the DBA/2 mutation is associated with approximately fourfold lower p53 levels in thymocytes treated with the DNA-damaging agent etoposide in-vitro, and with relative resistance of these thymocytes to apoptosis induced by the DNA-damaging agent etoposide compared with C57BL/6 mice.

Chromosomal linkage analysis of porphyria in mice induced by hexachlorobenzene-iron synergism: a model of sporadic porphyria cutanea tarda.

Genetic susceptibility to toxic chemicals is of major importance but most studies concentrate on candidate genes and searches for unknown susceptibility genes are uncommon. Human sporadic porphyria cutanea tarda is usually precipitated by alcohol, oestrogens, hepatitis viruses, HIV or haemodialysis. The mechanism is not known but there is a role for iron metabolism and an underlying genetic predisposition is suspected.

Epidermal growth factor stimulates phospholipase D independent of phospholipase C, protein kinase C or phosphatidylinositol-3 kinase activation in immortalized rabbit corneal epithelial cells.

Purpose: Activation of phospholipase D (PLD) is believed to be an important signaling pathway involved in cell growth and differentiation in several tissues, in response to a variety of mitogens. The aim of the present study was to investigate the effect of epidermal growth factor (EGF) on PLD activity in rabbit corneal epithelial cells (RCEC). We have also examined whether the EGF effect is dependent on concurrent activation of phospholipase C (PLC), protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI-3-kinase) in these cells.

Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents.

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively.

Activated-protein-C resistance in cancer patients.

Background: Resistance to activated protein C (aPC) is usually linked to factor V Leiden, but may occur in other disorders associated with hypercoagulability. In this study, we investigated the frequency of resistance to aPC in patients with advanced cancer and examined the relationship of aPC resistance to other markers of coagulation activation.

Methods: Patients (n = 39) had established diagnosis of advanced cancer; controls (n = 20) were healthy persons.

Epidermal growth factor stimulation of phosphatidylinositol 3-kinase during wound closure in rabbit corneal epithelial cells.

Purpose: To determine whether there is an association between epidermal growth factor (EGF)-induced activation of phosphatidylinositol 3-kinase (PI 3-kinase) and stimulation of wound closure in rabbit corneal epithelial cells.

Methods: Immortalized rabbit corneal epithelial cells were cultured in 24-well plates until they became confluent. Circular wounds were created in confluent cultures by cell denudation and then incubated in the absence and presence of EGF for varying intervals.

Uroporphyria induced by 5-aminolaevulinic acid alone in Ahrd SWR mice.

In mice, depression of hepatic uroporphyrinogen decarboxylase (UROD) leading to porphyrin accumulation (uroporphyria) occurs with chlorinated ligands of the aryl hydrocarbon (AH) receptor especially after iron overload. However, in the absence of chlorinated ligands, iron itself will eventually cause uroporphyria, but this response is not associated with the Ahr genotype. These effects are potentiated by administration of the haem precursor 5-aminolaevulinate (ALA).

Effect of epidermal growth factor on phosphatidylinositol 3-kinase activity in rabbit corneal epithelial cells.

We investigated the effect of epidermal growth factor (EGF) on phosphatidylinositol 3-kinase (PI 3-kinase) activity in SV-40 transformed rabbit corneal epithelial cells (RCEC) and in normal corneal epithelial cells grown in primary culture. The enzyme products, 3-phosphorylated phosphoinositides, were identified by TLC and by deacylation followed by separation on HPLC. Addition of 30 ng ml-1 EGF to 32P-labeled SV-40 transformed or primary epithelial cell culture, increased the radioactivity in phosphatidylinositol 3,4,5-trisphosphate (PIP3) by about 96% over that of control incubations.

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells.

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.

Effect of carbachol on phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate hydrolysis, and its modulation by isoproterenol in rabbit corneal epithelial cells.

The effects of carbachol (CCh) on phospholipase C(PLC)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and its modulation by isoproterenol were investigated in SV40-adenovirus transformed rabbit corneal epithelial cells (RCEC). When examined under light microscope, these cells exhibited a cobblestone-like appearance typical of the corneal epithelial cells grown in primary culture. Addition of CCh (0.

Purification and properties of D-myo-inositol 1,4,5-trisphosphate 3-kinase from bovine iris sphincter smooth muscle: effects of protein phosphorylation in vitro and in intact muscle.

Stimulation of bovine iris sphincter muscle with carbachol (10 microM) increased accumulation of Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) by 86 and 32% respectively. Addition of isoproterenol (5 microM) to muscle pretreated with carbachol reduced the 3H-radioactivity in InsP3 by 30% and increased that of InsP4 by 41%. InsP3 3-kinase was predominantly localized in the soluble fraction (110,000 g supernatant) of the iris sphincter.