SOX17 is expressed in regenerating oligodendrocytes in experimental models of demyelination and in multiple sclerosis.

We have previously demonstrated that Sox17 expression is prominent at developmental stages corresponding to oligodendrocyte progenitor cell (OPC) cycle exit and onset of differentiation, and that Sox17 promotes initiation of OPC differentiation. In this study, we examined Sox17 expression and regulation under pathological conditions, particularly in two animal models of demyelination/remyelination and in post-mortem multiple sclerosis (MS) brain lesions. We found that the number of Sox17 expressing cells was significantly increased in lysolecithin (LPC)-induced lesions of the mouse spinal cord between 7 and 30 days post-injection, as compared with controls. Sox17 immunoreactivity was predominantly detected in Olig2(+) and CC1(+) oligodendrocytes and rarely in NG2(+) OPCs. The highest density of Sox17(+) oligodendrocytes was observed at 2 weeks after LPC injection, coinciding with OPC differentiation. Consistent with these findings, in cuprizone-treated mice, Sox17 expression was highest in newly generated and in maturing CC1(+) oligodendrocytes, but low in NG2(+) OPCs during the demyelination and remyelination phases. In MS tissue, Sox17 was primarily detected in actively demyelinating lesions and periplaque white matter. Sox17 immunoreactivity was co-localized with NOGO-A+ post-mitotic oligodendrocytes both in active MS lesions and periplaque white matter. Taken together, our data: (i) demonstrate that Sox17 expression is highest in newly generated oligodendrocytes under pathological conditions and could be used as a marker of oligodendrocyte regeneration, and (ii) are suggestive of Sox17 playing a critical role in oligodendrocyte differentiation and lesion repair.

Distribution of pathogenic Leptospira in environmental water and soils of selected recreational forests in Perak, Malaysia.

Leptospirosis is an emerging zoonotic disease endemic in tropical regions. Aiming at assessing the potential infection risks via recreational exposure, the molecular prevalence of pathogenic Leptospira in 14 amenity forests in five selected districts of the state of Perak was determined. Water and soil samples along streams and waterfalls were subjected to culture of leptospires and the pathogenic Leptospira spp. was detected by lipL32-based polymerase chain reaction (PCR). Twenty out of 154 samples (13%) that tested positive for leptospires were mostly soils and still water recorded with tolerable temperatures (22.2- 26.5°C) and pHs (5.73-6.70). The localised prevalence was highly varied among eight positive forests (6.7-41.7%), particularly higher in Kampar and Kinta districts which are the more populated urban areas. The importance of public health surveillance should not be underrated given the high prevalence of Leptospira spp. in forests in close proximity to indigenous settlements, even where the places are clean. Overall, this study discovered a wide distribution of pathogenic Leptospira spp. in recreational areas.

Modulation of endothelial cell growth arrest and apoptosis by vascular endothelial growth inhibitor.

Vascular endothelial growth inhibitor (VEGI), a new member of the tumor necrosis factor family, is an endothelial cell-specific gene and a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth. We report here that VEGI mediates the following two activities in endothelial cells: early G(1) arrest in G(0)/G(1) cells responding to growth stimuli, and programmed death in proliferating cells. G(0)/G(1)-synchronized bovine aortic endothelial cells were treated with VEGI before and after the onset of the growth cycle. When the cells were stimulated with growth conditions but treated simultaneously with VEGI, a reversible, early-G(1) growth arrest occurred, evidenced by the lack of late G(1) markers such as hyperphosphorylation of the retinoblastoma gene product and upregulation of the c-myc gene. Additionally, VEGI treatment led to inhibition of the activities of cyclin-dependent kinases CDK2, CDK4, and CDK6. In contrast, VEGI treatment of cells that had entered the growth cycle resulted in apoptotic cell death, as evidenced by terminal deoxytransferase labeling of fragmented DNA, caspase 3 activation, and annexin V staining, all of which were lacking in nonproliferating cells treated with VEGI. Additionally, stress-signaling proteins p38 and JNK were not as fully activated by VEGI in quiescent as compared with proliferating populations. These findings suggest a dual role for VEGI, the maintenance of growth arrest and induction of apoptosis, in the modulation of the endothelial cell cycle.

Alternatively polyadenylated vasoactive intestinal peptide mRNAs are differentially regulated at the level of stability.

The role of cis-acting destabilizing RNA sequences in the determination of endocrine gene expression has been investigated using a novel paradigm, in which the differential regulation of two alternatively polyadenylated RNA transcripts may be observed both in vivo and in vitro. In the rat anterior pituitary gland in vivo, we have shown that, after the termination of an estrogen stimulus, a 1.7-kilobase (kb) vasoactive intestinal peptide (VIP) RNA containing an extensive 3'-untranslated region (UTR), is preferentially down-regulated with respect to a 1.0 kb VIP transcript that is uniquely abundant in this tissue. Differential regulation of the anterior pituitary VIP transcripts can be modeled in an explant culture system in which we defined both transcriptional and posttranscriptional phases of VIP gene regulation in vitro, and showed that selective down-regulation of the 1.7-kb transcript is posttranscriptional. Inhibitors of transcription and translation have also allowed us to show in vitro that differential regulation of VIP transcripts occurs through an active process that appears to involve the synthesis of a labile, destabilizing factor. In order to confirm the role of RNA destabilization as the primary mechanism of differential posttranscriptional regulation, we have also performed cell-free stability assays in which explant extracts were incubated with 32P-labeled run-off transcripts corresponding to the two alternatively polyadenylated VIP RNAs. The resultant estimates of RNA half-life showed significantly lower values for the synthetic VIP transcript containing the 3'-UTR. Our findings demonstrate the presence of functional destabilizing sequences in the 3'-UTR of the rat VIP RNA which appear to act in the physiological control of VIP gene expression.

Regulation of ion channel expression in neural cells by hormones and growth factors.

Voltage-and ligand-gated ion channels are key players in synaptic transmission and neuron-glia communication in the nervous system. Expression of these proteins can be regulated at several levels (transcriptional, translational, or posttranslational) and by multiple extracellular factors in the developing and mature nervous system. A wide variety of hormones and growth factors have been identified as important in neural cell differentiation, which is a complex process involving the acquisition of cell-type-specific ion channel phenotypes. Much literature has already accumulated describing the structural and functional characteristics of ion channels, but relatively little is known about the factors that influence their synthesis and cell surface expression, although this area has generated considerable interest in the context of neural cell development. This article reviews several examples of regulated expression of these channels by cellular factors, namely peptide growth factors and steroid hormones, and discusses, where applicable, current understanding of molecular mechanisms underlying such regulation of voltage-and neurotransmitter-gated ion channels.

Anterior pituitary vasoactive intestinal peptide mRNA is colocalised with prolactin mRNA in hyperoestrogenised rats.

It is well established that oestrogens can stimulate prolactin (PRL) secretion as well as the expression of the vasoactive intestinal peptide (VIP) gene whose product is also a potent PRL secretagogue. Previous evidence has supported both an autocrine and a paracrine role for pituitary VIP in PRL release in vitro; however, the cellular origin of VIP in pituitary tissue still remains poorly defined. In these studies, we have demonstrated by in situ hybridisation that VIP RNA is detected in the anterior pituitaries of chronically hyperoestrogenised rats, but not in those of untreated animals. Using a double-probe labelling procedure, VIP RNA has been shown to be present in a subpopulation of PRL-producing cells, while colocalisation of VIP and GH RNA was not observed. VIP gene expression in the rat anterior pituitary gland was characterised by the presence of two alternatively polyadenylated transcripts, 1.7 kb and 1.0 kb in size. We have generated a probe specific for the 1.7 kb transcript and double-labelling studies also showed definitive colocalisation with PRL mRNA. Our results demonstrating the presence of VIP RNA in PRL-producing cells thus suggest that VIP may play an autocrine role in PRL hypersecretion under conditions of oestrogen-induced hyperplasia.

Osmotic stimuli attenuate vasoactive intestinal peptide gene expression in the rat anterior pituitary gland.

Studies on vasoactive intestinal peptide (VIP) in the anterior pituitary gland have shown that it is synthesized locally, physiologically regulated, and may act as a paracrine/autocrine factor. We have now investigated the regulation of anterior pituitary VIP gene expression in rats during osmotic stimulation. Both salt-loading and dehydration resulted in a progressive and marked reduction in VIP mRNA levels as determined by Northern analysis, to 10% of control levels at 14 days of salt-loading. The 1.7 and 1.0 kb VIP RNA transcripts were equally affected. Since anterior pituitary VIP is partially localized in lactotrophs we also measured prolactin (PRL) mRNA levels. In contrast to VIP, PRL mRNA levels were increased during both osmotic paradigms, the mRNA levels being significantly raised after 5 days of salt-loading to 130% of controls. Further experiments, conducted to examine the mechanism by which VIP gene expression is down-regulated during osmotic stimulation, demonstrated that dopamine and angiotensin II do not appear to be involved. The results show dissociated regulation of VIP and PRL during osmotic stimulation and provide suggestive evidence of a role for anterior pituitary VIP in the animal's osmoregulatory responses. VIP may therefore be a paracrine factor with diverse functional roles.

Transcription of the vasoactive intestinal peptide gene in response to glucocorticoids: differential regulation of alternative transcripts is modulated by a labile protein in rat anterior pituitary.

Expression of the vasoactive intestinal peptide (VIP) gene is controlled by glucocorticoids in a tissue- and endocrine status-specific manner. We have investigated the molecular mechanisms that determine glucocorticoid regulation of VIP gene expression in the rat pituitary. In initial experiments, using explant cultures of rat pituitary glands, we have demonstrated that treatment with the glucocorticoid agonist dexamethasone leads to a marked increase in VIP mRNA levels. This effect was found to be selective for the larger of two alternatively polyadenylated VIP transcripts, and in addition, protein synthesis inhibitors markedly enhanced the magnitude of this response indicating that a labile pituitary protein acts to attenuate the transcript-selective response to glucocorticoids. Nuclear run-on analysis of transcription demonstrated that the effects of dexamethasone in vitro are mediated largely, if not completely, at the level of transcription. In order to investigate the role of VIP promoter sequence in the glucocorticoid response, we then demonstrated that the activity of rat VIP gene promoter/reporter constructs in GH3 pituitary cells are up-regulated by dexamethasone. This up-regulation is virtually abolished following removal of promoter sequence between -162 and -89 of the start of transcription. Using an in vitro electrophoretic mobility shift assay, we have also demonstrated that this region of the promoter binds recombinant glucocorticoid receptor protein. The results of our study therefore indicate a direct mechanism of action for the modulation of VIP gene expression by glucocorticoids, and furthermore provide evidence of a mechanism that permits selective glucocorticoid regulation of alternative VIP transcripts.

Differential use of 3'poly(a) addition sites in vasoactive intestinal Peptide messenger ribonucleic Acid of the rat anterior pituitary gland.

The description of vasoactive intestinal peptide (VIP) immunoreactivity in the rat anterior pituitary gland has stimulated much interest in possible autocrine and/or paracrine roles as well as in the regulation of its biosynthesis (1-6). The detection of two VIP transcripts in this tissue, together with the knowledge of the rat VIP cDNA sequence (7), has led us to speculate that they are products of the same gene but as a result of differential transcriptional termination, differ in the length of the 3'untranslated region. In this study, we present evidence for the differential utilization of poly(A) addition sites which accounts for the generation of two distinct VIP RNA species.

In vitro regulation of rat prolactin messenger ribonucleic acid poly(A) tail length: modulation by bromocriptine.

Recent analysis of endocrine gene transcripts has revealed that several hormone mRNAs exhibit regulated size changes (due to alterations in length of the 3' poly(A) tail) which may function as an additional level of control in the determination of gene expression. We have now shown, through the novel application of an organ culture technique, that prolactin mRNA exhibits a similar regulated change in poly(A) tail length when rat anterior pituitary glands are explanted. The effect is observed in glands of either male or female rats and is specific with respect to growth hormone and alpha-tubulin mRNAs. Furthermore, we have also found that the size change in prolactin mRNA is attenuated in the presence of bromocriptine, indicating regulation through a dopaminergic pathway.

Estradiol induces vasoactive intestinal peptide and prolactin gene expression in the rat anterior pituitary independently of plasma prolactin levels.

It is well established that estrogens are potent stimulators of prolactin (PRL) secretion. It has also been demonstrated that estradiol (E2) can increase the expression and the anterior pituitary levels of the vasoactive intestinal peptide (VIP), a peptide which also acts as a potent PRL-releasing factor. It thus remained unknown whether the effects on pituitary VIP were due to E2 itself or to E2-induced hyperprolactinemia (HPRL). In order to test this hypothesis, various plasma PRL levels were induced in rats either with ectopic pituitary grafts, PRL secreting tumours or E2 implants, and VIP mRNA expression in the anterior pituitary was measured by in situ hybridization and Northern blot analyses. Whereas decreases in VIP mRNA can be observed in pituitaries of rats with pure HPRL, a 6-fold increase in VIP mRNA can be seen in E2-treated rats. E2 increased both 1.0 and 1.7 Kb VIP mRNA species. The presence of the graft in E2-treated rats significantly reduced the increase in VIPmRNA observed following E2. The direct stimulation by E2 of VIP mRNA expression was further demonstrated by the fact that statistical analysis of the data indicated that both E2 and graft were acting independently of each other, and that a new selective antiestrogen, RU 58668, almost totally blocked the effect of E2. Moreover, under similar experimental conditions, pituitary PRL mRNA levels were reduced in the graft group and a marked up-regulation was observed similarly in both E2 and in E2 rats bearing ectopic grafts.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of pathogenic Leptospira in environmental water and soils of selected recreational forests in Perak, Malaysia

@#Leptospirosis is an emerging zoonotic disease endemic in tropical regions. Aiming at assessing the potential infection risks via recreational exposure, the molecular prevalence of pathogenic Leptospira in 14 amenity forests in five selected districts of the state of Perak was determined. Water and soil samples along streams and waterfalls were subjected to culture of leptospires and the pathogenic Leptospira spp. was detected by lipL32-based polymerase chain reaction (PCR). Twenty out of 154 samples (13%) that tested positive for leptospires were mostly soils and still water recorded with tolerable temperatures (22.226.5°C) and pHs (5.73-6.70). The localised prevalence was highly varied among eight positive forests (6.7-41.7%), particularly higher in Kampar and Kinta districts which are the more populated urban areas. The importance of public health surveillance should not be underrated given the high prevalence of Leptospira spp. in forests in close proximity to indigenous settlements, even where the places are clean. Overall, this study discovered a wide distribution of pathogenic Leptospira spp. in recreational areas.

Identification of nuclear orphan receptors as regulators of expression of a neurotransmitter receptor gene.

Nuclear orphan receptors are known to be important mediators of neurogenesis, but the target genes of these transcription factors in the vertebrate nervous system remain largely undefined. We have previously shown that a 500-base pair fragment in the first intron of the GRIK5 gene, which encodes the kainate-preferring glutamate receptor subunit KA2, down-regulates gene expression. In our present studies, mutation of an 11-base pair element within this fragment resulted in a loss of nuclear protein binding and reverses negative regulation by the intron. Using yeast one-hybrid screening, we have identified intron-binding proteins from rat brain as COUP-TFI, EAR2, and NURR1. Gel shift studies with postnatal day 2 rat brain extract indicate the presence of COUP-TFs, EAR2, and NURR1 in the DNA-protein complex. Competition assays with GRIK5-binding site mutations show that the recombinant clones exhibit differential binding characteristics and suggest that the DNA-protein complex from postnatal day 2 rat brain may consist primarily of EAR2. The DNA binding activity was also observed to be enriched in rat neural tissue and developmentally regulated. Co-transfection assays showed that recombinant nuclear orphan receptors function as transcriptional repressors in both CV1 cells and rat CG4 oligodendrocyte cells. Direct interaction of the orphan receptors with and relief of repression by TFIIB indicate likely role(s) in active and/or transrepression. Our findings are thus consistent with the notion that multiple nuclear orphan receptors can regulate the transcription of a widely expressed neurotransmitter receptor gene by binding a common element in an intron and directly modulating the activity of the transcription machinery.

Characterization of the rat GRIK5 kainate receptor subunit gene promoter and its intragenic regions involved in neural cell specificity.

The GRIK5 (glutamate receptor ionotropic kainate-5) gene encodes the kainate-preferring glutamate receptor subunit KA2. The GRIK5 promoter is TATA-less and GC-rich, with multiple consensus initiator sequences. Transgenic mouse lines carrying 4 kilobases of the GRIK5 5'-flanking sequence showed lacZ reporter expression predominantly in the nervous system. Reporter assays in central glial (CG-4) and non-neural cells indicated that a 1200-base pair (bp) 5'-flanking region could sustain neural cell-specific promoter activity. Transcriptional activity was associated with the formation of a transcription factor IID-containing complex on an initiator sequence located 1100 bp upstream of the first intron. In transfection studies, deletion of exonic sequences downstream of the promoter resulted in reporter gene activity that was no longer neural cell-specific. When placed downstream of the GRIK5 promoter, a 77-bp sequence from the deleted fragment completely silenced reporter expression in NIH3T3 fibroblasts while attenuating activity in CG-4 cells. Analysis of the 77-bp sequence revealed a functional SP1-binding site and a sequence resembling a neuron-restrictive silencer element. The latter sequence, however, did not display cell-specific binding of REST-like proteins. Our studies thus provide evidence for intragenic control of GRIK5 promoter activity and suggest that elements contributing to tissue-specific expression are contained within the first exon.

Growth factor-induced transcription of GluR1 increases functional AMPA receptor density in glial progenitor cells.

We analyzed the effects of two growth factors that regulate oligodendrocyte progenitor (O-2A) development on the expression of glutamate receptor (GluR) subunits in cortical O-2A cells. In the absence of growth factors, GluR1 was the AMPA subunit mRNA expressed at the lowest relative level. Basic fibroblast growth factor (bFGF) caused an increase in GluR1 and GluR3 steady-state mRNA levels. Platelet-derived growth factor (PDGF) did not modify the mRNA levels for any of the AMPA subunits but selectively potentiated the effects of bFGF on GluR1 mRNA (4.5-fold increase). The kainate-preferring subunits GluR7, KA1, and KA2 mRNAs were increased by bFGF, but these effects were not modified by cotreatment with PDGF. Nuclear run-on assays demonstrated that PDGF+bFGF selectively increased the rate of GluR1 gene transcription (2.5-fold over control). Western blot analysis showed that GluR1 protein levels were increased selectively (sixfold over control) by PDGF+bFGF. Functional expression was assessed by rapid application of AMPA to cultured cells. AMPA receptor current densities (pA/pF) were increased nearly fivefold in cells treated with PDGF+bFGF, as compared with untreated cells. Further, AMPA receptor channels in cells treated with PDGF+bFGF were more sensitive to voltage-dependent block by intracellular polyamines, as expected from the robust and selective enhancement of GluR1 expression. Our combined molecular and electrophysiological findings indicate that AMPA receptor function can be regulated by growth factor-induced changes in the rate of gene transcription.