ST8Sia IV mRNA corresponds with the biosynthesis of alpha2,8sialyl polymers but not oligomers in rat oligodendrocytes.

As oligodendrocytes mature they progress through a series of distinct differentiation steps characterized by the expression of specific markers. One such marker, polysialic acid found on the neural cell adhesion molecule (NCAM), is detected by antibodies and is present on progenitor oligodendrocytes, but is not detected to the same extent on mature oligodendrocytes. Two closely related polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST) have been cloned previously and shown to synthesize polysialic acid on NCAM and other glycoproteins. To determine whether or not polyalpha2,8sialyltransferases are downregulated during the differentiation of oligodendrocytes, the enzyme activity and expression of ST8Sia II and ST8Sia IV mRNA at two stages of maturation in JS12/1 and JS3/16 oligodendrocytes were examined. Differentiation in both oligodendroglial cell lines was accompanied by more than a 50% reduction in the biosynthesis of polymers of alpha2,8sialic acid when fetuin was used as substrate. Most interestingly, extracts of JS12/1 mature cells synthesized 60% more short oligomers of alpha2,8sialic acid than the progenitor cells, whereas JS3/16 mature cells synthesized barely detectable amounts of the short oligomers. Transcripts for ST8Sia IV mRNA were present in both JS12/1 and JS3/16 and were reduced when the biosynthesis was markedly reduced. In contrast ST8Sia II mRNA was barely detectable in JS3/16 cells and although detectable in JS12/1 cells, there was no clear modulation with maturation. These results were supported by the examination of the brains of rats from embryonic to Day 21 ages. The enzyme activity and mRNA experiments show that polyalpha2,8sialyltransferase itself is down regulated to cause the reduction in sialyl polymers on mature oligodendrocytes. Moreover, ST8Sia IV is responsible for the polysialylation of NCAM in oligodendrocytes.

Caspase-3 activation in oligodendrocytes from the myelin-deficient rat.

The myelin-deficient (MD) rat has a point mutation in its proteolipid protein (PLP) gene that causes severe dysmyelination and oligodendrocyte cell death. Using an in vitro model, we have shown that MD oligodendrocytes initially differentiate similarly to wild-type cells, expressing galactocerebroside, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin basic protein. However, at the time when PLP expression would normally begin, the MD oligodendrocytes die via an apoptotic pathway involving caspase activation. The active form of caspase-3 was detected, along with the cleavage products of poly-(ADP-ribose) polymerase (PARP) and spectrin, major targets of caspase-mediated proteolysis. A specific inhibitor of casapse-3, Ac-DEVD-CMK, reduced apoptosis in MD oligodendrocytes, but the rescued cells did not mature fully or express myelin-oligodendrocyte glycoprotein. These results suggest that mutant PLP affects not only cell death but also oligodendrocyte differentiation.

Stage-specific effects of bone morphogenetic proteins on the oligodendrocyte lineage.

Oligodendrocyte maturation is regulated by multiple secreted factors present in the brain during critical stages of development. Whereas most of these factors promote oligodendrocyte proliferation and survival, members of the bone morphogenetic protein family (BMPs) recently have been shown to inhibit oligodendrocyte differentiation in vitro. Oligodendrocyte precursors treated with BMPs differentiate to the astrocyte lineage. Given that cells at various stages of the oligodendrocyte lineage have distinct responses to growth factors, we hypothesized that the response to BMP would be stage-specific. Using highly purified, stage-specific cultures, we found that BMP has distinct effects on cultured oligodendrocyte preprogenitors, precursors, and mature oligodendrocytes. Oligodendrocyte preprogenitors (PSA-NCAM+, A2B5-) treated with BMP2 or BMP4 developed a novel astrocyte phenotype characterized by a morphological change and expression of glial fibrillary acidic protein (GFAP) but little glutamine synthetase expression and no labeling with A2B5 antibody. In contrast, treating oligodendrocyte precursors with BMPs resulted in the accumulation of cells with the traditional type 2 astrocyte phenotype (GFAP+, A2B5+). However, many of the cells with an astrocytic morphology did not express GFAP or glutamine synthetase unless thyroid hormone was present in the medium. The addition of fibroblast growth factor along with BMP to either oligodendrocyte preprogenitor or the oligodendrocyte precursor cells inhibited the switch to the astrocyte lineage, whereas platelet-derived growth factor addition had no effect. Treatment of mature oligodendrocytes with BMP elicited no change in morphology or expression of GFAP. These data suggest that as cells progress through the oligodendrocyte lineage, they show developmentally restricted responses to the BMPs.

Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB.

The glial glutamate transporter GLT-1 may be the predominant Na(+)-dependent glutamate transporter in forebrain. Expression of GLT-1 correlates with astrocyte maturation in vivo and increases during synaptogenesis. In astrocyte cultures, GLT-1 expression parallels differentiation induced by cAMP analogs or by coculturing with neurons. Molecule(s) secreted by neuronal cultures contribute to this induction of GLT-1, but little is known about the signaling pathways mediating this regulation. In the present study, we determined whether growth factors previously implicated in astrocyte differentiation regulate GLT-1 expression. Of the six growth factors tested, two [epidermal growth factor (EGF) and transforming growth factor-alpha] induced expression of GLT-1 protein in cultured astrocytes. Induction of GLT-1 protein was accompanied by an increase in mRNA and in the V(max) for Na(+)-dependent glutamate transport activity. The effects of dibutyryl-cAMP and EGF were additive but were independently blocked by inhibitors of protein kinase A or protein tyrosine kinases, respectively. The induction of GLT-1 in both EGF- and dibutyryl-cAMP-treated astrocytes was blocked by inhibitors targeting phosphatidylinositol 3-kinase (PI3K) or the nuclear transcription factor-kappaB. Furthermore, transient transfection of astrocyte cultures with a constitutively active PI3K construct was sufficient to induce expression of GLT-1. These data suggest that independent but converging pathways mediate expression of GLT-1. Although an EGF receptor-specific antagonist did not block the effects of neuron-conditioned medium, the induction of GLT-1 by neuron-conditioned medium was completely abolished by inhibition of PI3K or nuclear factor-kappaB. EGF also increased expression of GLT-1 in spinal cord organotypic cultures. Together, these data suggest that activation of specific signaling pathways with EGF-like molecules may provide a novel approach for limiting excitotoxic brain injury.

Ectopic bone morphogenetic proteins 5 and 4 in the chicken forebrain lead to cyclopia and holoprosencephaly.

Proper dorsal-ventral patterning in the developing central nervous system requires signals from both the dorsal and ventral portions of the neural tube. Data from multiple studies have demonstrated that bone morphogenetic proteins (BMPs) and Sonic hedgehog protein are secreted factors that regulate dorsal and ventral specification, respectively, within the caudal neural tube. In the developing rostral central nervous system Sonic hedgehog protein also participates in ventral regionalization; however, the roles of BMPs in the developing brain are less clear. We hypothesized that BMPs also play a role in dorsal specification of the vertebrate forebrain. To test our hypothesis we implanted beads soaked in recombinant BMP5 or BMP4 into the neural tube of the chicken forebrain. Experimental embryos showed a loss of the basal telencephalon that resulted in holoprosencephaly (a single cerebral hemisphere), cyclopia (a single midline eye), and loss of ventral midline structures. In situ hybridization using a panel of probes to genes expressed in the dorsal and ventral forebrain revealed the loss of ventral markers with the maintenance of dorsal markers. Furthermore, we found that the loss of the basal telencephalon was the result of excessive cell death and not a change in cell fates. These data provide evidence that BMP signaling participates in dorsal-ventral patterning of the developing brain in vivo, and disturbances in dorsal-ventral signaling result in specific malformations of the forebrain.

Cell cycle arrest induced by ectopic expression of p27 is not sufficient to promote oligodendrocyte differentiation.

Oligodendrocyte differentiation is accompanied by dramatic changes in gene expression as well as cell cycle arrest. To determine whether cell cycle arrest is sufficient to induce the changes in cell phenotype associated with differentiation, we inhibited oligodendrocyte precursor proliferation in vitro by overexpressing p27, a cyclin kinase inhibitor, using a recombinant adenovirus. Ectopic expression of p27 efficiently inhibited oligodendrocyte precursor cell division, even in the presence of exogenous mitogens, by blocking the activity of the cyclin-dependent kinase, cdk2. Although the cells had stopped dividing, they did not express galactocerebroside (GalC) or myelin basic protein (MBP), changes associated with oligodendrocyte differentiation, suggesting that they had not differentiated. After removal of exogenous mitogens, however, adenovirus-expressing oligodendrocyte precursors differentiated with a temporal profile similar to that of control, uninfected oligodendrocytes, as indicated by expression of GalC and MBP. We conclude that cell cycle arrest is not sufficient to induce differentiation of dividing oligodendrocyte precursors, and that modulation of additional, as yet unknown, signaling pathways is required for this to occur.

Maturation-dependent apoptotic cell death of oligodendrocytes in myelin-deficient rats.

Mutations in the proteolipid protein gene (PLP/plp), which encodes the major intrinsic membrane protein in central nervous system (CNS) myelin, cause inherited dysmyelination in mammals. One of these mutants, the myelin-deficient (md) rat, has severe dysmyelination that is associated with oligodendrocyte cell death. Using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay, which labels apoptotic cells, we find that cell death is increased in multiple white matter tracts of md rats. The tracts that myelinate the earliest show the earliest increase in cell death, and cell death persists for at least 22 days, the lifespan of these mutant animals. In all tracts, and at all developmental ages examined, apoptotic cells expressed the markers of mature oligodendrocytes, such as myelin basic protein, myelin-associated glycoprotein, and the Rip antigen, but not chondroitin sulfate proteoglycan, a marker of oligodendrocyte precursors. Mature oligodendrocytes fail to accumulate in md brain because they die before they fully mature.

The growth response to tumour necrosis factor alpha of human gynaecological cancer cell lines.

The cytokine tumour necrosis factor alpha (TNF-alpha) is implicated in the regulation of diverse gynaecological cell types, its biological activity being potentially mediated by two distinct cell surface receptors (TNFR) of molecular weight 55 and 75 kDa, respectively. In this study the sensitivity to the growth regulatory properties of TNF-alpha of a panel of human cervical, endometrial and ovarian cancer cell lines was investigated in relation to the expression and biological activity of the 55- and 75-kDa receptor. There was no evidence of expression or function of the 75-kDa receptor in any of the cell lines tested. The expression and biological activity of the 55-kDa receptor was demonstrated in each TNF sensitive cell line, with one exception, the HOG-1 cervical cancer cell line. The data suggest that the 55-kDa receptor mediates the cellular response to TNF-alpha in sensitive gynaecological cancer cell lines but raises the possibility of the presence of a distinct receptor in HOG-1 cells.

Increased platinum-DNA damage tolerance is associated with cisplatin resistance and cross-resistance to various chemotherapeutic agents in unrelated human ovarian cancer cell lines.

We have examined a panel of 12 unrelated human ovarian cancer cell lines derived from patients who were either untreated or treated with platinum-based chemotherapy to determine whether a relationship is present between cisplatin sensitivity and: (a) cellular platinum accumulation; (b) glutathione levels; (c) platinum-DNA adduct formation; (d) platinum-DNA adduct removal; and (e) platinum-DNA damage tolerance. Multiple regression and correlation analysis revealed that of these resistance mechanisms, platinum-DNA damage tolerance correlates strongly with cisplatin sensitivity (r = 0.84, P = 0.001), whereas platinum accumulation (r = -0.11), cellular glutathione levels (r = 0.13), and platinum-DNA adduct removal (r = 0.44) correlate insignificantly. The correlation of platinum-DNA damage tolerance to cisplatin sensitivity (IC50s) is derived from the clustering of platinum-DNA adduct formation into three distinct groups spanning a 3-fold range, which is narrow relative to the corresponding 43-fold range in sensitivity. Adduct formation itself is not associated with cisplatin sensitivity (r = -0.38). Strong correlations were also observed between platinum-DNA damage tolerance and sensitivity to Adriamycin (r = 0.80, P = 0.002), paclitaxel (r = 0.87, P = 0.0002), etoposide (r = 0.78, P = 0.003), and mitomycin C (r = 0.73, P = 0.007). These results suggest that the failure of pathways that are involved in recognizing and processing platinum-DNA damage and other types of drug-induced damage that culminate in cell death may result in a broad resistance phenotype.

Arachidonic acid release and inositol lipid metabolism in response to bradykinin and related peptides in human endometrial cells in vitro.

Prostaglandins of the 2 series are known to play a role in the regulation of menstruation and implantation but, more recently, other vasoactive peptides have been considered as potential regulators of these endometrial processes. The aim of the present study was to investigate the action of the potent vasoactive peptide bradykinin and the structurally related peptide, kallidin, on endometrial function by examining their effect on phosphoinositide hydrolysis and arachidonic acid release from endometrial cells in vitro. Primary cultures of endometrial glands and stromal cells were prelabelled with [14C]-arachidonic acid (AA) or [3H]-inositol to monitor arachidonic acid release and inositol phosphate accumulation respectively. Bradykinin and kallidin stimulated a dose and time-dependent release of arachidonic acid from stromal cells which, with 100 nmol/L bradykinin, was 30-150% above basal release and maximal at 5 min. Glands were less responsive; 100 nmol/L bradykinin (at 5 min) caused a release of AA of 30-69% above basal level. Bradykinin also stimulated a dose dependent increase in inositol monophosphate production. The maximum response with stromal cells was 8- to 10-fold and with glands, 2-fold (1 and 100 nmol/L bradykinin, respectively). Kallidin was equipotent to bradykinin with respect to both AA and inositol phosphate accumulation. The bradykinin analogue des Arg bradykinin (which acts through the B1 receptor) released AA from stromal cells but did not alter phosphoinositide hydrolysis, suggesting that these two cellular responses are mediated by different receptors (B1 and B2 respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Endometrial phospholipases A2, polycystic ovaries and pelvic pain.

OBJECTIVE: To compare the activity of calcium independent phospholipase A2 in the endometrium of women with polycystic ovaries and in women with normal ovaries, and to investigate the influence of chronic pelvic pain on phospholipase A2 activity. DESIGN: A prospective descriptive study. SETTING: The Samaritan Hospital for Women and the Unit of Metabolic Medicine, St Mary's Hospital Medical School, London. SUBJECTS: 73 women attending the Samaritan Hospital for Women for clip sterilization, infertility, early recurrent miscarriage or pelvic venous congestion. 45 of these women had no pelvic pain, 22 had normal ovaries and 23 had polycystic ovaries diagnosed by ultrasound. The other 28 women had chronic pelvic pain, 14 of them had normal ovaries and 14 polycystic ovaries. INTERVENTIONS: Endometrial tissue was obtained at curettage or from the excised uterus in the proliferative or secretory phase of the menstrual cycle. The activities of calcium dependent (type 1) and calcium independent (type 2) phospholipase A2 isoenzymes were measured in all endometrial samples. RESULTS: In all the women phospholipase A2 type 1 activity, was significantly higher in the secretory phase than in the proliferative phase (P less than 0.001). There was no difference between women with normal ovaries and those with polycystic ovaries at either stage of the cycle irrespective of whether or not chronic pelvic venous congestion was present. In women with normal ovaries, both with and without chronic pelvic pain, phospholipase A2 type 2 activity was significantly higher in the secretory phase than in the proliferative phase (P less than 0.02 and P less than 0.05 respectively) but in women with polycystic ovaries, with and without chronic pelvic pain, there was no significant difference in activity between the two phases of the cycle. Women with polycystic ovaries had markedly higher proliferative phase type 2 activity than women with normal ovaries (P less than 0.001). Secretory phase type 2 activity was similar in all the women investigated. CONCLUSION: These data suggest that phospholipase A2 type 2 activity is increased in proliferative phase endometrium of women with polycystic ovaries but that the increase is not associated with chronic pelvic venous congestion.

Release of arachidonic acid from human endometrial cells in culture mediated by calcium ionophore (A23187) or fluoride.

Primary cultures of endometrial glands and stromal cells were labelled with [14C]-arachidonic acid for 4 h before exposure to either the calcium ionophore, A23187 (which activates phospholipase A2 (PLA2) by increasing intracellular calcium concentrations) or sodium fluoride (which activates a G-protein). Calcium ionophore (0.5-50 mumol/l) stimulated a dose- and time-dependent release of arachidonic acid from endometrial glands. Incubation with ionophore (10 mumol/l) for 1 h released 22% of the incorporated arachidonic acid. There was a corresponding decrease in phospholipids and no loss from triglycerides. Stromal cells were unresponsive to ionophore. Fluoride (10 mmol/l) stimulated a release of arachidonic acid from stromal cells and endometrial glands (6.5% of the total arachidonic acid incorporated). In stromal cells, arachidonic acid was released from triglycerides in Day-1 cultures and from phospholipids in Day-2 cultures. In both Day-1 and Day-2 cultures of endometrial glands, arachidonic acid was released from phospholipids, but not from triglycerides. Among the phospholipids, phosphatidylcholine was always the major source of arachidonic acid. Arachidonic acid release from endometrial glands and stromal cells may be mediated by activation of PLA2 (or phospholipase C) via a G-protein, but in glands calcium ionophore may have a direct effect on PLA2. The response to calcium ionophore may reflect the differences in calcium requirements of the two endometrial PLA2 isoenzymes.

Phospholipase activity in the endometrium of women with normal menstrual blood loss and women with proven ovulatory menorrhagia.

The activity of phospholipase A2 types 1 and 2 and phospholipase C was measured in the endometrium of women with ovulatory menorrhagia and in those with normal menstrual blood loss. In both groups of subjects phospholipase A2 type 1 activity was significantly higher in the secretory phase than in the proliferative phase (P less than 0.001). The median activity (pmol/mg protein/min) for the proliferative phase was 27.6 in normal subjects and 40.4 in women with ovulatory menorrhagia and for the secretory phase the median activity was 144.5 in normal women and 138.1 in women with ovulatory menorrhagia. There was no difference between the two groups of women at either stage of the cycle. Phospholipase A2 type 2 activity was also higher in the secretory phase than in the proliferative phase (P less than 0.05 for normal subjects and P less than 0.001 for women with menorrhagia). The median activity (pmol/mg protein/min) for the proliferative phase was 94.4 (normal subjects) and 56.6 (women with menorrhagia) and for the secretory phase 148.3 (normal subjects) and 142.5 (women with menorrhagia). The activity of phospholipase A2 type 2 was significantly lower in the proliferative phase of women with ovulatory menorrhagia compared with normal subjects (P less than 0.05). Phospholipase C activity (nmol/mg protein/min) was significantly higher in women with ovulatory menorrhagia (median 8.2) compared with women with normal blood loss (median 5.5) (P less than 0.01).

Molluscicidal properties of organotin and organolead compounds.

Triphenyltin acetate has proved successful as a molluscicide in the laboratory and in small-scale field trials. However, in view of its cost and its toxicity to swamp rice at molluscicidal concentrations, an effort has been made to explore the molluscicidal value of other organotin and organolead compounds. Those that proved most toxic to snails were screened in the laboratory against swamp rice in order to obtain some measure of their phytotoxicity. This study yielded 6 compounds that were relatively non-toxic to rice. One of these, triphenyllead acetate, has been formulated as an emusifiable concentrate for use in the field and the results of trials in two parts of Tanzania are reported. Chemical analyses of water treated with triphenyllead acetate are discussed, and the reasons for the disappearance of the compound in the field are investigated. Some preliminary information on the toxicity of triphenyllead and triphenyltin derivatives to other organisms is also given.