An examination of mercury levels in the coastal environment and fish of Cote d'Ivoire.

Artisanal and small-scale gold mining (ASGM), energy production and other industrial inputs are a major source of anthropogenic mercury (Hg) to the aquatic environment globally, and these inputs have led to environmental contamination and human exposure. While studies have documented the effects of Hg inputs to rivers and marine waters of the West African region, estuarine waters of Cote d'Ivoire have been understudied, besides the waters surrounding Abidjan. To fill this gap, and to examine the potential for human exposure to methylmercury (MeHg), we measured the concentrations of total Hg, MeHg, and ancillary parameters in water (dissolved and particulate phases), sediment and fish to determine the extent of environmental impact and the potential for MeHg exposure for people consuming these fish. Levels of Hg and MeHg in sediment were elevated in the vicinity of the urban environment (up to 0.3 ng/g dry weight (dw) MeHg and 623 ng/g dw total Hg) and lowest in the more remote estuarine environments. Measurements of Hg in tuna and other larger pelagic coastal species indicated that levels were elevated but comparable to other North Atlantic regions. However, levels of Hg in fish, even smaller estuarine species, were such that the rural and urban populations are potentially being exposed to unsafe levels of MeHg, primarily as a result of the relatively high fish consumption in Cote d'Ivoire compared to other countries. Overall, both local point sources and the transport of Hg used in interior ASGM activities are the sources for Hg contamination to these coastal waters.

DLX1 acts as a crucial target of FOXM1 to promote ovarian cancer aggressiveness by enhancing TGF-ß/SMAD4 signaling.

Recent evidence from a comprehensive genome analysis and functional studies have revealed that FOXM1 is a crucial metastatic regulator that drives cancer progression. However, the regulatory mechanism by which FOXM1 exerts its metastatic functions in cancer cells remains obscure. Here, we report that DLX1 acts as a FOXM1 downstream target, exerting pro-metastatic function in ovarian cancers. Both FOXM1 isoforms (FOXM1B or FOXM1C) could transcriptionally upregulate DLX1 through two conserved binding sites, located at +61 to +69bp downstream (TFBS1) and -675 to -667bp upstream (TFBS2) of the DLX1 promoter, respectively. This regulation was further accentuated by the significant correlation between the nuclear expression of FOXM1 and DLX1 in high-grade serous ovarian cancers. Functionally, the ectopic expression of DLX1 promoted ovarian cancer cell growth, cell migration/invasion and intraperitoneal dissemination of ovarian cancer in mice, whereas small interfering RNA-mediated DLX1 knockdown in FOXM1-overexpressing ovarian cancer cells abrogated these oncogenic capacities. In contrast, depletion of FOXM1 by shRNAi only partially attenuated tumor growth and exerted almost no effect on cell migration/invasion and the intraperitoneal dissemination of DLX1-overexpressing ovarian cancer cells. Furthermore, the mechanistic studies showed that DLX1 positively modulates transforming growth factor-ß (TGF-ß) signaling by upregulating PAI-1 and JUNB through direct interaction with SMAD4 in the nucleus upon TGF-ß1 induction. Taken together, these data strongly suggest that DLX1 has a pivotal role in FOXM1 signaling to promote cancer aggressiveness through intensifying TGF-ß/SMAD4 signaling in high-grade serous ovarian cancer cells.

The Forkhead box transcription factor FOXM1 is required for the maintenance of cell proliferation and protection against oxidative stress in human embryonic stem cells.

Human embryonic stem cells (hESCs) exhibit unique cell cycle structure, self-renewal and pluripotency. The Forkhead box transcription factor M1 (FOXM1) is critically required for the maintenance of pluripotency in mouse embryonic stem cells and mouse embryonal carcinoma cells, but its role in hESCs remains unclear. Here, we show that FOXM1 expression was enriched in undifferentiated hESCs and was regulated in a cell cycle-dependent manner with peak levels detected at the G2/M phase. Expression of FOXM1 did not correlate with OCT4 and NANOG during in vitro differentiation of hESCs. Importantly, knockdown of FOXM1 expression led to aberrant cell cycle distribution with impairment in mitotic progression but showed no profound effect on the undifferentiated state. Interestingly, FOXM1 depletion sensitized hESCs to oxidative stress. Moreover, genome-wide analysis of FOXM1 targets by ChIP-seq identified genes important for M phase including CCNB1 and CDK1, which were subsequently confirmed by ChIP and RNA interference analyses. Further peak set comparison against a differentiating hESC line and a cancer cell line revealed a substantial difference in the genomic binding profile of FOXM1 in hESCs. Taken together, our findings provide the first evidence to support FOXM1 as an important regulator of cell cycle progression and defense against oxidative stress in hESCs.

CXCL12 G801A polymorphism and susceptibility to glioma: a case­control study.

Previous studies have demonstrated that the CXCL12 G801A polymorphism is closely correlated with tumor susceptibility. In addition, the CXCL12/CXCR4 pathway is closely related to proliferation, metastasis, and invasion of glioma. However, the genetic effects of the CXCL12 G801A polymorphism on glioma risk in Chinese populations remain unknown. In this study, we investigated the potential associations between the CXCL12 G801A polymorphism with glioma susceptibility and its clinicopathological characteristics. Frequencies of CXCL12 G801A polymorphic variants between glioma patients (N = 750) and healthy controls (N = 750) were assessed using restriction length fragment polymorphism analysis. The association among the CXCL12 G801A polymorphism, glioma grade (WHO classification), and histological type was also evaluated. Our results showed that patients with glioma had significantly higher frequency of the CXCL12-3' A/A genotypes (P = 0.039) as compared with healthy controls. When stratified by the glioma histology, high-grade glioma patients had significantly higher frequency of the CXCL12-3' A/A genotypes (P = 0.019) as compared with low-grade glioma patients. When stratified by the WHO grade, significantly higher frequency of the CXCL12-3' A/A genotype was observed in stage IV glioma patients (P = 0.037). We conclude that the CXCL12 G801A polymorphism is a risk factor that increases susceptibility to gliomas in a subset of the general Han Chinese population.

Aberrant activation of ERK/FOXM1 signaling cascade triggers the cell migration/invasion in ovarian cancer cells.

Forkhead box M1 (FOXM1) is a proliferation-associated transcription factor essential for cell cycle progression. Numerous studies have documented that FOXM1 has multiple functions in tumorigenesis and its elevated levels are frequently associated with cancer progression. Here, we characterized the role of ERK/FOXM1 signaling in mediating the metastatic potential of ovarian cancer cells. Immunohistochemical (IHC), immunoblotting and semi-quantitative RT-PCR analyses found that both phospho-ERK and FOXM1 were frequently upregulated in ovarian cancers. Intriguingly, the overexpressed phospho-ERK (p<0.001) and FOXM1 (p<0.001) were significantly correlated to high-grade ovarian tumors with aggressive behavior such as metastasized lymph node (5 out of 6). Moreover, the expressions of phospho-ERK and FOXM1 had significantly positive correlation (p<0.001). Functionally, ectopic expression of FOXM1B remarkably enhanced cell migration/invasion, while FOXM1C not only increased cell proliferation but also promoted cell migration/invasion. Conversely, inhibition of FOXM1 expression by either thiostrepton or U0126 could significantly impair FOXM1 mediated oncogenic capacities. However, the down-regulation of FOXM1 by either thiostrepton or U0126 required the presence of p53 in ovarian cancer cells. Collectively, our data suggest that over-expression of FOXM1 might stem from the constitutively active ERK which confers the metastatic capabilities to ovarian cancer cells. The impairment of metastatic potential of cancer cells by FOXM1 inhibitors underscores its therapeutic value in advanced ovarian tumors.

Increased basal insulin secretion in Pdzd2-deficient mice.

Expression of the multi-PDZ protein Pdzd2 (PDZ domain-containing protein 2) is enriched in pancreatic islet beta cells, but not in exocrine or alpha cells, suggesting a role for Pdzd2 in the regulation of pancreatic beta-cell function. To explore the in vivo function of Pdzd2, Pdzd2-deficient mice were generated. Homozygous Pdzd2 mutant mice were viable and their gross morphology appeared normal. Interestingly, Pdzd2-deficient mice showed enhanced glucose tolerance in intraperitoneal glucose tolerance tests and their plasma insulin levels indicated increased basal insulin secretion after fasting. Moreover, insulin release from mutant pancreatic islets was found to be twofold higher than from normal islets. To verify the functional defect in vitro, Pdzd2 was depleted in INS-1E cells using two siRNA duplexes. Pdzd2-depleted INS-1E cells also displayed increased insulin secretion at low concentrations of glucose. Our results provide the first evidence that Pdzd2 is required for normal regulation of basal insulin secretion.

Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer.

BACKGROUND: Loss of growth inhibitory response to transforming growth factor-beta (TGF-beta) is a common feature of epithelial cancers. Recent studies have reported that genetic lesions and overexpression of oncoproteins in TGF-beta/Smads signalling cascade contribute to the TGF-beta resistance. Here, we showed that the overexpressed FOXG1 was involved in attenuating the anti-proliferative control of TGF-beta/Smads signalling in ovarian cancer. METHODS: FOXG1 and p21(WAF1/CIP1) expressions were evaluated by real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR), western blot and immunohistochemical analyses. The effect of FOXG1 on p21(WAF1/CIP1) transcriptional activity was examined by luciferase reporter assays. Cell lines stably expressing or short hairpin RNA interference-mediated knockdown FOXG1 were established for studying the gain-or-loss functional effects of FOXG1. XTT cell proliferation assay was used to measure cell growth of ovarian cancer cells. RESULTS: Quantitative RT-PCR and western blot analyses showed that FOXG1 was upregulated and inversely associated with the expression levels of p21(WAF1/CIP1) in ovarian cancer. The overexpression of FOXG1 was significantly correlated with high-grade ovarian cancer (P=0.025). Immunohistochemical analysis on ovarian cancer tissue array was further evidenced that FOXG1 was highly expressed and significantly correlated with high-grade ovarian cancer (P=0.048). Functionally, enforced expression of FOXG1 selectively blocked the TGF-beta-induced p21(WAF1/CIP1) expressions and increased cell proliferation in ovarian cancer cells. Conversely, FOXG1 knockdown resulted in a 20-26% decrease in cell proliferation together with 16-33% increase in p21(WAF1/CIP1) expression. Notably, FOXG1 was able to inhibit the p21(WAF1/CIP1) promoter activity in a p53-independent manner by transient reporter assays. CONCLUSION: Our results suggest that FOXG1 acts as an oncoprotein inhibiting TGF-beta-mediated anti-proliferative responses in ovarian cancer cells through suppressing p21(WAF1/CIP1) transcription.

Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis.

The Forkhead Box M1 (FOXM1) transcription factor plays a crucial role in regulating expression of cell cycle genes which are essentially involved in cell proliferation, differentiation and transformation. Recent studies have reported that aberrant expression of FOXM1 in a variety of human cancers is associated with their aggressive behaviour. However, the functional significance of FOXM1 in human cervical cancer is not known. We have shown that FOXM1 was significantly over-expressed in cervical squamous cell carcinoma (SCC) compared to normal cervical epithelium immunohistochemically (p < 0.001). In addition, intratumoural FOXM1 positivity was increased in cervical intraepithelial neoplasia (CIN) and carcinoma, compared with that in normal epithelium, indicating that FOXM1 is involved in tumour progression. Indeed, this is supported by clinicopathological analysis that the over-expression of FOXM1 was significantly associated with tumour late stage (p = 0.012) and cell proliferation marker, Ki67 (p < 0.001). Functionally, enforced expression of FOXM1c in FOXM1-deficient cervical cancer cells (C33A) remarkably enhanced cell proliferation and anchorage-independent growth ability. Conversely, depletion of FOXM1 by RNA interference in FOXM1-over-expressing cervical cancer cells (SiHa) caused significant inhibition on cell proliferation and anchorage-independent growth ability on soft agar. This inhibitory phenomenon was associated with the reduced expressions of cyclin B1, cyclinD1 and cdc25B but increased expression of p27(Kip1) and p21(Cip1). Our findings suggest a role for FOXM1 in the development and pathogenesis of human cervical SCC.

PDZ-domain containing-2 (PDZD2) is a novel factor that affects the growth and differentiation of human fetal pancreatic progenitor cells.

Early-trimester human fetal pancreas is a promising potential source of pancreatic progenitor cells. However, the ethical controversy associated with the source of these cells, and technical difficulties associated with their differentiation into insulin-producing cells have limited both their availability and utility. This study aimed to characterize a population of pancreatic progenitor cells (PPCs) isolated from human fetus and describe the effects of a novel factor, PDZ-domain containing-2 (PDZD2), and its secreted form (sPDZD2), on PPC proliferation and differentiation. In particular, we examined and characterized the expression of several stem cell (nestin, ABCG2, c-kit), growth and differentiation markers (GLP-1R, c-met, erbB1), and PDZD2 in PPCs by RT-PCR, Western blot, and immunocytochemistry. We also examined the effects of sPDZD2 on PPC proliferation and differentiation by examining BrdU incorporation, MTT, cell number, and real-time PCR as well as ELISA. PPCs were isolated, cultured and characterized from human fetal pancreas. PDZD2 and sPDZD2 were detected at high levels in both human fetal pancreas and in PPCs. sPDZD2 acted as a potent mitogen on PPCs, and inhibited the differentiation of PPC-derived islet-like cell-clusters (ICCs), evidenced by the downregulation of Isl-1, Pdx-1, and insulin mRNA levels. sPDZD2 treatment also reduced levels of C-peptide in ICCs. These results show that a novel pancreatic developmental factor, PDZD2, is sufficient to promote the proliferation of human fetal PPCs while limiting differentiation of ICCs into islet/endocrine cells. Findings from this study will contribute to the development of improved methods for islet transplantation therapy in the treatment of diabetes.

Inhibition of prostate cancer cell growth by human secreted PDZ domain-containing protein 2, a potential autocrine prostate tumor suppressor.

A possible role of the PDZ domain-containing protein 2 (PDZD2) in prostate tumorigenesis has been suggested. Besides, PDZD2 is posttranslationally cleaved by a caspase-dependent mechanism to form a secreted PDZ domain-containing protein 2 (sPDZD2) with unknown functions in humans. In this study, we demonstrate the endogenous expression of PDZD2 and secretion of sPDZD2 in cancerous DU145, PC-3, 22Rv1, LNCaP, and immortalized RWPE-1 prostate epithelial cells. Inhibition of endogenous sPDZD2 production and secretion by DU145, PC-3, 22Rv1, and RWPE-1 cells via the caspase-3 inhibitor Z-DEVD-FMK resulted in increased cell proliferation, which was abrogated by treatment with exogenous recombinant sPDZD2. Whereas sPDZD2-induced antiproliferation in DU145, PC-3, and 22Rv1 cells, it induced apoptosis in LNCaP cells. The data suggest that endogenous sPDZD2, produced by caspase-3-mediated cleavage from PDZD2, may function as a novel autocrine growth suppressor for human prostate cancer cells. The antiproliferative effect of sPDZD2 was apparently mediated through slowing the entry of DU145, PC-3, and 22Rv1 cells into the S phase of the cell cycle. In DU145 cells, this can be attributed to stimulated p53 and p21(CIP1/WAF1) expression by sPDZD2. On the other hand, the apoptotic effect of sPDZD2 on LNCaP cells was apparently mediated via p53-independent Bad stimulation. Together our results indicate the presence of p53-dependent and p53-independent PDZD2/sPDZD2 autocrine growth suppressive signaling pathways in human prostate cancer cells and suggest a novel therapeutic approach of harnessing the latent tumor-suppressive potential of an endogenous autocrine signaling protein like sPDZD2 to inhibit prostate cancer growth.

Over-expression of FoxM1 stimulates cyclin B1 expression.

FoxM1 (previously named WIN, HFH-11 or Trident) is a Forkhead box (Fox) transcription factor widely expressed in proliferating cells. Various findings, including a recent analysis of FoxM1 knockout mice, suggest that FoxM1 is required for normal S-M coupling during cell cycle progression. To study the regulatory role of FoxM1 and its downstream regulatory targets, three stably transfected HeLa lines that display doxycycline (dox)-inducible FoxM1 expression were established. Over-expression of FoxM1 by dox induction facilitates growth recovery from serum starvation. Quantitation of cyclin B1 and D1 levels using flow cytometric, Western and Northern analyses reveals that elevated FoxM1 levels lead to stimulation of cyclin B1 but not cyclin D1 expression. Transient reporter assays in the dox-inducible lines and upon co-transfection with a constitutive FoxM1 expression plasmid suggest that FoxM1 can activate the cyclin B1 promoter.

Inhibition of malignant trophoblastic cell proliferation in vitro and in vivo by melatonin.

Melatonin inhibited thymidine incorporation into human choriocarcinoma JEG-3 cells at physiological and pharmacological concentrations in the present study. Gene expression of MT2 receptor, but not that of mt1 receptor, was detected in JEG-3 cells by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression profile of the two human melatonin receptor subtypes in JEG-3 cells was identical to that previously reported for JAr cells, whose proliferation had also been shown to be similarly inhibited by physiological and pharmacological concentrations of melatonin. In contrast, melatonin had no effect on thymidine incorporation into 3A-Sub-E cells (a transformed trophoblast cell line), in which gene expression of both receptor subtypes could not be detected. The data suggest that in human placental trophoblasts, a correlation may exist between MT2 receptor gene expression and the direct anti-proliferative action of melatonin. Although melatonin has been reported to induce G1/S delay in cell cycle progression of JAr cells, no significant changes in the percentages of JEG-3 cells in different cell cycle phases upon melatonin treatment was recorded by flow cytometric analysis. This indicates that G1/S transition delay is probably not an important cellular mechanism in the direct anti-proliferative action of melatonin on human JEG-3 cells in vitro. Furthermore, melatonin inhibited the growth of both JAr and JEG-3 xenograft tumors in athymic nude mice, and prolonged the survival of those animals that developed choriocarcinoma. While the number of apoptotic tumor cells was not increased by melatonin, the pineal hormone induced significant decreases in the numbers of JAr and JEG-3 cells expressing proliferating cell nuclear antigen (PCNA) and cyclin A in the tumors. Taking into account both the in vitro and in vivo findings, it is likely that the inhibitory effect of melatonin on choriocarcinoma JAr and JEG-3 cell proliferation in vivo is largely a direct action of the hormone on the tumor cells.

Bridge-1, a novel PDZ-domain coactivator of E2A-mediated regulation of insulin gene transcription.

Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neurogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancreatic beta cell, E2A proteins, in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for beta-cell function. By yeast two-hybrid screening of a cDNA library prepared from rat insulinoma (INS-1) cells, we identified a novel protein, Bridge-1, that interacts with E2A proteins and functions as a coactivator of gene transcription mediated by E12 and E47. Bridge-1 contains a PDZ-like domain, a domain known to be involved in protein-protein interactions. Bridge-1 is highly expressed in pancreatic islets and islet cell lines and the expression pattern is primarily nuclear. The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro-translated Bridge-1 with E12 or E47 and by mammalian two-hybrid studies. The PDZ-like domain of Bridge-1 is required for interaction with the carboxy terminus of E12. In both yeast and mammalian two-hybrid interaction studies, Bridge-1 mutants lacking an intact PDZ-like domain interact poorly with E12. An E12 mutant (E12DeltaC) lacking the carboxy-terminal nine amino acids shows impaired interaction with Bridge-1. Bridge-1 has direct transactivational activity, since a Gal4 DNA-binding domain-Bridge-1 fusion protein transactivates a Gal4CAT reporter. Bridge-1 also functions as a coactivator by enhancing E12- or E47-mediated activation of a rat insulin I gene minienhancer promoter-reporter construct in transient-transfection experiments. Substitution of the mutant E12DeltaC for E12 reduces the coactivation of the rat insulin I minienhancer by Bridge-1. Inactivation of endogenous Bridge-1 in insulinoma (INS-1) cells by expression of a Bridge-1 antisense RNA diminishes rat insulin I promoter activity. Bridge-1, by utilizing its PDZ-like domain to interact with E12, may provide a new mechanism for the coactivation and regulation of transcription of the insulin gene.

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

Molecular analysis of a novel winged helix protein, WIN. Expression pattern, DNA binding property, and alternative splicing within the DNA binding domain.

We have cloned a novel winged helix factor, WIN, from the rat insulinoma cell line, INS-1. Northern blot analysis demonstrated that WIN is highly expressed in a variety of insulinoma cell lines and rat embryonic pancreas and liver. In adults, WIN expression was detected in thymus, testis, lung, and several intestinal regions. We determined the DNA sequences bound in vitro by baculovirus-expressed WIN protein in a polymerase chain reaction-based selection procedure. WIN was found to bind with high affinity to the selected sequence 5'-AGATTGAGTA-3', which is similar to the recently identified HNF-6 binding sequence 5'-DHWATTGAYTWWD-3' (where W = A or T, Y = T or C, H is not G, and D is not C). We have isolated human WIN cDNAs by library screening and 5'-rapid amplification of cDNA ends. Sequence analysis indicates that the carboxyl terminus of human WIN has been previously isolated as a putative phosphorylation substrate, MPM2-reactive phosphoprotein 2 (MPP2); WIN may be regulated by phosphorylation. Alignment of the rat and human WIN cDNAs and their comparison with mouse genomic sequence revealed that the WIN DNA binding domain is encoded by four exons, two of which (exons 4 and 6) are alternatively spliced to generate at least three classes of mRNA transcripts. These transcripts were shown by RNase protection assay to be differentially expressed in different tissues. Alternative splicing within the winged helix DNA binding domain might result in modulation of DNA binding specificity.

Neural specificity of elav expression: defining a Drosophila promoter for directing expression to the nervous system.

The Drosophila melanogaster vital gene, embryonic lethal abnormal visual system (elav), is required for the postdeterminative development of the nervous system. Its gene product encodes an RNA binding protein that was found to be expressed in all neurons right after their birth. This specific, ubiquitous, and continuous pattern of neural expression has led to the increasingly popular use of ELAV protein as a neural-specific marker. To understand the molecular basis of this neural-specific expression, we have defined and analyzed the structure of the elav promoter. Cis-acting sequences important for conferring the neural specificity of elav expression were identified by analyzing the reporter gene expression in transformants carrying different elav-beta-galactosidase fusion genes. This analysis delimits a 333-bp region (-92 to +241) that is necessary for specifying the elav pattern of nervous system expression. A 3.5-kb promoter fragment encompassing this region was designed for targeting gene expression specifically to the nervous system and would be a useful tool for the analysis of nervous system function.

Gene elav of Drosophila melanogaster: a prototype for neuronal-specific RNA binding protein gene family that is conserved in flies and humans.

Regulated gene activity is crucial to the formation and function of the nervous system. It is well known that gene regulation can occur at the transcriptional, post-transcriptional, translational, and post-translational levels. In this review our focus has been on the post-transcriptional regulation in neurons and on neural-specific RNA binding proteins that may be involved in post-transcriptional modulation of gene activity. We have taken advantage of this opportunity to review our work on the elav gene of Drosophila melanogaster which encodes a neural-specific RNA binding protein and relate it to other members of this elav-like gene family. We report new data that suggests that elav is post-transcriptionally regulated and we demonstrate that below-threshold levels of ELAV protein severely affects neuronal differentiation.

Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

The elav gene product of Drosophila, required in neurons, has three RNP consensus motifs.

A sequence of developmental events transforms neurons from their immature state to their mature, terminally differentiated state. The elav locus is one of the first examples of a gene that is expressed in neurons early during this developmental sequence. This gene has been shown to be required for the proper development of young neurons and for the maintenance of mature neurons. DNA sequence data presented in this report suggest that the elav gene product is an RNA binding protein, based on the presence of RNP (ribonucleoprotein) consensus sequences. This leads to the proposal that this protein is involved in the RNA metabolism of neurons.

Ornithine decarboxylase from Tetrahymena thermophila: purification, reversible modification and multiple forms.

T. thermophila ornithine decarboxylase was purified 1100-fold by an improved procedure to a specific activity of 2.31 x 10(4) nmol/hr/mg protein with a 28% yield. The purified enzyme was a monomer of Mr = 68,000 and had two isoelectric forms with pl's of 5.14 and 5.17 respectively. The Mr = 68,000 enzyme was reversibly modified by a cellular factor of approx. Mr = 10,000 to give rise to two additional forms with decreased DEAE-cellulose affinity.