Loss of glial glutamate transporters and induction of neuronal expression of GLT-1B in the hypoxic neonatal pig brain

The homeostasis of glutamate is critical to normal brain function; deficiencies in the regulation of extracellular glutamate are thought to be a major determinant of damage in hypoxic brains. Extracellular levels of glutamate are regulated mainly by plasmalemmal glutamate transporters. We have evaluated the distribution of the glutamate transporter GLAST and two splice variants of GLT-1 in the hypoxic neonatal pig brain using this as model of neonatal humans. In response to severe hypoxic insults, we observe a rapid loss of two glial glutamate transporters from specific brain regions, such as the CA1 region of the hippocampus, but not the dentate gyrus. The spatial distribution of loss accords with patterns of damage in these brains. Conversely, we demonstrate that hypoxia evokes the expression of a splice variant of GLT-1 in neurons. We suggest that this expression may be induced in response to elevated extracellular glutamate around these neurons, and that this splice variant may represent a useful marker for direct quantification of the extent of likely neuronal damage in hypoxic brains. © 2004 Elsevier B.V. All rights reserved.

Expression of the tudor-related gene Tdrd5 during development of the male germline in mice

Homologues of Drosophila germ cell determinant genes such as vasa, nanos and tudor have recently been implicated in development of the male germline in mice. In the present study, the mouse gene encoding Tudor domain containing protein 5 (TDRD5) was isolated from a 12.5-13.5 days post coitum (dpc) male-enriched subtracted cDNA library. Whole-mount in situ hybridization analysis of Tdrd5 expression in the mouse embryonic gonad indicated that this gene is upregulated in the developing testis from 12.5 dpc, with expression levels remaining higher in testis than ovary throughout embryogenesis. Expression of Tdrd5 was absent in testes isolated from W-e/W-e embryos, which lack germ cells. In situ hybridization (ISH) on cryosectioned 13.5 dpc testes suggests that expression of Tdrd5, like that of Oct4, is restricted to germ cells. Northern hybridization analysis of expression in adult tissues indicated that Tdrd5 is expressed in the testis only, implying that expression of this gene is restricted to the male germline throughout development to adulthood. (C) 2004 Elsevier B.V. All rights reserved.

Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs

Neurons in pelvic ganglia receive nicotinic excitatory post-synaptic potentials (EPSPs) from sacral preganglionic neurons via the pelvic nerve, lumbar preganglionic neurons via the hypogastric nerve or both. We tested the effect of a range of calcium channel antagonists on EPSPs evoked in paracervical ganglia of female guinea-pigs after pelvic or hypogastric nerve stimulation. omega-Conotoxin GVIA (CTX GVIA, 100 nM) or the novel N-type calcium channel antagonist, CTX CVID (100 nM) reduced the amplitude of EPSPs evoked after pelvic nerve stimulation by 50-75% but had no effect on EPSPs evoked by hypogastric nerve stimulation. Combined addition of CTX GVIA and CTX CVID was no more effective than either antagonist alone. EPSPs evoked by stimulating either nerve trunk were not inhibited by the P/Q calcium channel antagonist, omega-agatoxin IVA (100 nM), nor the L-type calcium channel antagonist, nifedipine (30 muM). SNX 482 (300 nM), an antagonist at some R-type calcium channels, inhibited EPSPs after hypogastric nerve stimulation by 20% but had little effect on EPSPs after pelvic nerve stimulation. Amiloride (100 muM) inhibited EPSPs after stimulation of either trunk by 40%, while nickel (100 muM) was ineffective. CTX GVIA or CTX CVID (100 nM) also slowed the rate of action potential repolarization and reduced afterhyperpolarization amplitude in paracervical neurons. Thus, release of transmitter from the terminals of sacral preganglionic neurons is largely dependent on calcium influx through N-type calcium channels, although an unknown calcium channel which is resistant to selective antagonists also contributes to release. Release of transmitter from lumbar preganglionic neurons does not require calcium entry through either conventional N-type calcium channels or the variant CTX CVID-sensitive N-type calcium channel and seems to be mediated largely by a novel calcium channel. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Functional expression and characterization of Eehinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage

A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible. (C) 2003 Elsevier B.V. All rights reserved.

Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat 1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals. (c) 2005 Elsevier B.V. All rights reserved.

Renal and hepatic accumulation of cadmium and lead in the expression of CYP4F2 and CYP2E1

The present study examined accumulation of the metal toxins cadmium (Cd) and lead (Pb) in relation to the abundance of cytochrome P450 4F2 (CYP4F2), CYP2E1 and concentrations of zinc and copper in liver and kidney samples using immunoblotting coupled with metal analysis. The post mortem liver and kidney cortex samples were from 23 males and 8 females aged 3-89 years. All were Caucasians who had not been exposed to metals in the workplace. Average kidney cortex Cd load of 17.4 mu g/g w.w. was 17 times greater than average liver Cd load (1.1 mu g/g w.w.). In contrast, average kidney cortex Ph load of 0.09 mu g/g w.w. was two times lower than liver Pb load of 0.19 mu g/g w.w. Average Zn and Cu concentrations in the kidney cortex samples were 67% and 33% lower than those in the liver. Liver and kidney Cd loads, but not liver or kidney Ph loads, correlated positively with donors' age. After controlling for liver Cd load, an inverse correlation was seen between Zn and age (partial r = -0.39, P = 0.02), suggesting reduction in liver Zn levels in old age. Liver CYP2E1 protein abundance correlated with age-adjusted Cd load (partial r = 0.37, P = 0.02) whereas kidney CYP4172 protein abundance showed a positive correlation with age-adjusted Cd loads (partial r = 0.40, P = 0.02). These findings suggest that Cd may be an inducer of renal CYP4172 and hepatic CYP2E1 and that increased renal CYP4172 expression may implicate in Cd-linked renal tubular dysfunction and high blood pressure, involving CYP4F2-dependent arachidonic acid metabolism. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Sequence, circulating levels, and expression of C-type natriuretic peptide in a euryhaline elasmobranch, Carcharhinus leucas

The present study has examined expression and circulating levels of C-type natriuretic peptide (CNP) in the euryhaline bull shark, Carcharhinus leucas. Complementary DNA and deduced amino acid sequence for CNP in C leucas were determined by RACE methods. Homology of CNP amino acid sequence in C. leucas was high both for proCNP and for mature CNP when compared with previously identified elasmobranch CNPs. Mature CNP sequence in C. leucas was identical to that in Triakis seyllia and Seyliorhinus canicula. Levels of expression of CNP mRNA were significantly decreased in the atrium but did not change in either the brain or ventricle following acclimation to a SW environment. However, circulating levels of CNP significantly increased from 86.0 +/- 7.9 fmol ml(-1) in FW to 144.9 +/- 19.5 fmol ml(-1) in SW. The results presented demonstrate that changes in environmental salinity influences both synthesis of CNP from the heart and also circulating levels in C. leucas. Potential stimulus for release and modes of action are discussed. (c) 2005 Elsevier Inc. All rights reserved.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

Co-expression of SOX9 and SOX10 during melanocytic differentiation in vitro

Investigations into pigment cell biology have relied on the ability to culture both murine and human melanocytes, numerous melanoma cell lines and more recently, murine and human melanoblasts. Melanoblast culture requires medium supplemented with a range of growth factors including Stem Cell Factor, Endothelin-3 and Fibroblast Growth Factor-2, withdrawal of which causes the cells to differentiate into melanocytes. Using the human melanoblast culture system, we have now examined the expression and/or DNA binding activity of several transcription factors implicated in melanocytic development and differentiation. Of these, the POU domain factor BRN2 and the SOX family member SOX10 are both highly expressed in unpigmented melanocyte precursors but are down-regulated upon differentiation. In contrast, the expression levels of the previously described MITF and PAX3 transcription factors remain relatively constant during the melanoblast-melanocyte transition. Moreover, BRN2 ablated melanoma cells lack expression of SOX10 and MITF but retain PAX3. A novel finding implicates a second SOX protein, SOX9, as a potential melanogenic transcriptional regulator, as its expression level is increased following the down-regulation of BRN2 and SOX10 in differentiated melanoblasts. Our results suggest that a complex network of transcription factor interactions requiring proper temporal coordination is necessary for acquisition and maintenance of the melanocytic phenotype. (c) 2005 Elsevier Inc. All rights reserved.

Expression analysis of a human hepatic cell line in response to palmitate

Saturated fat plays a role in common debilitating diseases such as obesity, type 2 diabetes, and coronary heart disease. It is also clear that certain fatty acids act as regulators of metabolism via both direct and indirect signalling of target tissues. As the molecular mechanisms of saturated fatty acid signalling in the liver are poorly defined, hepatic gene expression analysis was undertaken in a human hepatocyte cell line after incubation with palmitate. Profiling of mRNA expression using cDNA microarray analysis revealed that 162 of approximately 18,000 genes tested were differentially expressed after incubation with palmitate for 48 h. Altered transcription profiles were observed in a wide variety of genes, including genes involved in lipid and cholesterol transport, cholesterol catabolism, cell growth and proliferation, cell signalling, P-oxidation, and oxidative stress response. While palinitate signalling has been examined in pancreatic beta-cells, this is the first report showing that palmitate regulates expression of numerous genes via direct molecular signalling mechanisms in liver cells. (C) 2005 Elsevier Inc. All rights reserved.

Delayed Sry and Sox9 expression in developing mouse gonads underlies B6-Y-DOM sex reversal

The phenomenon of B6-Y-DOM sex reversal arises when certain variants of the Mus domesticus Y chromosome are crossed onto the genetic background of the C57BL/6J (136) inbred mouse strain, which normally carries a Mus musculus-derived Y chromosome. While the sex reversal has been assumed to involve strain-specific variations in structure or expression of Sry, the actual cause has not been identified. Here we used in situ hybridization to study expression of Sry, and the critical downstream gene Sox9, in strains containing different chromosome combinations to investigate the cause of B6-Y-DOM sex reversal. Our findings establish that a delay of expression of Sry(DOM) relative to Sry(B6) underlies B6-Y-DOM sex reversal and provide the first molecular confirmation that Sry must act during a critical time window to appropriately activate Sox9 and effect male testis determination before the onset of the ovarian-determining pathway. (C) 2004 Elsevier Inc. All rights reserved.

Peroxisome proliferator-activated receptor alpha expression is regulated by estrogen receptor alpha and modulates the response of MCF-7 cells to sodium butyrate

Peroxisome proliferator-activated receptors are ligand-activated transcription factors with a potential role in cancer. We investigated peroxisome proliferator-activated receptor alpha expression in breast cancer cell lines and showed a relationship between mean peroxisome proliferator-activated receptor alpha and estrogen receptor alpha mRNA levels in estrogen receptor alpha positive breast cancer cells. Transfection of estrogen receptor alpha into the estrogen receptor alpha negative cell line, MDA-MB-231 decreased peroxisome proliferator-activated receptor a mRNA and conversely inhibition of estrogen receptor alpha by ICI-182 780 in estrogen receptor a positive, MCF-7 cells increased peroxisome proliferator-activated receptor a mRNA levels. Estrogen receptor alpha levels can be modulated by histone deacetylase inhibitors and such agents are in clinical trials for cancer treatment. We found the histone deacetylase inhibitor, sodium butyrate, increased peroxisome proliferator-activated receptor alpha mRNA levels within 4 h of treatment. Peroxisome proliferator-activated receptor a modulation was independent of estrogen receptor alpha, as a similar increase was observed in the estrogen receptor a negative MDA-MB-231 cells. To further investigate the relationship between sodium butyrate and peroxisome proliferator-activated receptor alpha expression, we created an MCF-7 cell line that conditionally over-expresses human peroxisome proliferator-activated receptor alpha. Over-expression of the peroxisome proliferator-activated receptor protected MCF-7 cells from sodium butyrate-mediated inhibition of proliferation and attenuated sodium butyrate-mediated induction of histone deacetylase 3 mRNA, indicating that elevated levels of peroxisome proliferator-activated receptor alpha may reduce the sensitivity of cells to histone deacetylase inhibitors. The estrogen receptor alpha dependence of peroxisome proliferator-activated receptor alpha levels may be significant since estrogen receptor alpha negative breast cancer cells are associated with a more aggressive phenotype. Our studies also suggest that peroxisome proliferator-activated receptor alpha levels may be a marker of breast cancer cell sensitivity to histone deacetylase inhibitors. (c) 2004 Elsevier Ltd. All rights reserved.

Dissection of peripheral and central endogenous opioid modulation of systemic interleukin-1 beta responses using c-fos expression in the rat brain

In opiate addicts or patients receiving morphine treatment, it has been reported that the immune system is often compromised. The mechanisms responsible for the adverse effects of opioids on responses to infection are not clear but it is possible that central and/or peripheral opioid receptors may be important. We have utilised an experimental immune challenge model in rats, the systemic administration of the human pro-inflammatory cytokine interleukin-1 beta (IL-1 beta) to study the effects of selectively blocking peripheral opioid receptors only (using naloxone methiodide) or after blocking both central and peripheral opioid receptors (using naloxone). Pre-treatment with naloxone methiodide decreased (15%) IL-1 beta-induced Fos-immunoreactivity (Fos-IR) in medial parvocellular paraventricular nucleus (mPVN) corticotropin-releasing hormone (CRH) neurons but increased responses in the ventrolateral medulla (VLM) C1 (65%) and nucleus tractus solitarius (NTS) A2 (110%) catecholamine cell groups and area postrema (136%). However no effect of blocking peripheral opioid receptors was detected in the central nucleus of the amygdala (CeA) or dorsal bed nucleus of the stria terminalis (BNST). We next determined the effect of blocking both central and peripheral opioid receptors with naloxone and, when compared to the naloxone methiodide pre-treated group, a further 60% decrease in Fos-IR mPVN CRH neurons induced by IL-1 beta was detected, which was attributed to block of central opioid receptors. Similar comparisons also detected decreases in Fos-IR neurons induced by IL-1 beta in the VLM A1, VLM C1 and NTS A2 catecholamine cell groups, area postrema, and parabrachial nucleus. In contrast, pre-treatment with naloxone increased Fos-IR neurons in CeA (98%) and dorsal BNST (72%). These results provide novel evidence that endogenous opioids can influence central neural responses to systemic IL-1 beta and also suggest that the differential patterns of activation may arise because of actions at central and/or peripheral opioid receptors that might be important in regulating behavioural, hypothalamic-pituitary-adrenal axis and sympathetic nervous system responses during an immune challenge. (c) 2005 Elsevier Ltd. All rights reserved.