Postmitotic Neuronal Cells Research Articles

Differential effect of cycloheximide on neuronal and glioma cells treated with chemotherapy and radiation.

Dividing cells and non-dividing cells are distinct in their cell cycle kinetics, and react differently when facing cytotoxic stimuli. A protein synthesis inhibitor, cycloheximide (CHX), has recently been found to protect neuronal cells from oxidative stress. We investigated whether CHX exerts differential effects on dividing and non-dividing cells in the brain under cytotoxic stimuli. Mitotic C6 rat glioma cells and postmitotic neuronal cells were studied with a cytotoxic regimen combining gamma-irradiation (RT) and 1,3-bis,2-chloroethyl-1-nitrosourea (BCNU). Cells were exposed to BCNU (1 g/ml) for 15 h before gamma-irradiation and incubated with CHX (1 g/ml) from 30 min before and until 5 h after irradiation. Clonogenic assay was used to assess cytotoxic effects on C6 glioma cells. LDH assay was used for the viability of H19-7 postmitotic neuronal cells. A 2.27-3.75 fold enhancement of cytotoxicity was noticed with the addition of CHX to BCNU and 2-10 Gy of radiation. Our data demonstrated that CHX enhanced cytotoxicity of RT plus BCNU, while no additional toxicity was incurred to the postmitotic neuronal cells when CHX was added. We further studied whether the inhibition of DNA repair, assayed by single-cell DNA electrophoresis (comet assay), is a contributing factor for the enhanced cytotoxicity on C6 glioma cells. Interestingly, the initial DNA damage after RT plus BCNU was equivalent; whereas DNA repair was significantly less at 5 h after radiation in CHX-treated C6 glioma cells. Protecting non-dividing neuronal cells to avoid excessive functional deficit is an integral part of a successful brain tumor treatment regimen. Taking advantage of the differential effect of CHX on glioma and neuronal cells may improve tumor control without excessive neural toxicity.

The involvement of p53 in dopamine-induced apoptosis of cerebellar granule neurons and leukemic cells overexpressing p53.

1. The pathogenesis of the selective degeneration of the dopaminergic neurons in Parkinson's disease is still enigmatic. Recently we have shown that dopamine can induce apoptosis in postmitotic neuronal cells, as well as in other cellular systems, thus suggesting a role for this endogenous neurotransmitter and associated oxidative stress in the neuronal death process. 2. Dopamine has been shown to be capable of inducing DNA damage through its oxidative metabolites. p53 is a transcription factor that has a major role in determining cell fate in response to DNA damage. We therefore examined the possible correlation between dopamine-triggered apoptosis, DNA damage and levels of total phosphorylated p53 protein in cultured mouse cerebellar granule neurons. 3. Marked DNA damage and apoptotic nuclear condensation and fragmentation were detected within several hours of exposure to dopamine. An associated marked threefold increase in p53 phosphorylation was observed within this time window. Using a temperature-sensitive p53 activation system in leukemia LTR6 cells, were found that p53 inactivation dramatically attenuated dopamine toxicity. 4. We therefore conclude that DNA damage and p53 activation may have a role in mediating dopamine-induced apoptosis. Modulation of the p53 system may therefore have a protective role against the toxicity of this endogenous neurotransmitter and associated oxidative stress.

Adenoviruses encoding HPRT correct biochemical abnormalities of HPRT-deficient cells and allow their survival in negative selection medium.

The Lesch-Nyhan syndrome is an X-linked disorder caused by a virtually complete absence of the key enzyme of purine recycling, hypoxanthine-guanine phosphoribosyltransferase (HPRT). It is characterized by uric acid overproduction and severe neurological dysfunction. No treatment is yet available for the latter symptoms. A possible long-term solution is gene therapy, and recombinant adenoviruses have been proposed as vectors for gene transfer into postmitotic neuronal cells. We have constructed an adenoviral vector expressing the human HPRT cDNA under the transcriptional control of a short human cytomegalovirus major immediate early promoter (RAd-HPRT). Here we show that infection of human 1306, HPRT-negative cells with RAd-HPRT, expressed high enough levels of HPRT enzyme activity, as to reverse their abnormal biochemical phenotype, thus enhancing hypoxanthine incorporation and restoring purine recycling, increasing GTP levels, decreasing adenine incorporation, and allowing cell survival in HAT medium in which only cells expressing high levels of HPRT can survive. Infection of murine STO cells, increased hypoxanthine incorporation and restored purine recycling, thus allowing cell survival in HAT medium, and reduced de novo purine synthesis. Although both cells were able to survive in HAT medium post infection with RAd-HPRT, some of the biochemical consequences differed. In summary, even though adenoviral vectors do not integrate into the genome of target HPRT-deficient human or murine cells, RAd-HPRT mediated enzyme replacement corrects abnormal purine metabolism, increases intracellular GTP levels, and allows cells to survive in a negative selection medium.

Glutamate induces phosphorylation of Elk-1 and CREB, along with c-fos activation, via an extracellular signal-regulated kinase-dependent pathway in brain slices.

In cell culture systems, the TCF Elk-1 represents a convergence point for extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) subclasses of mitogen-activated protein kinase (MAPK) cascades. Its phosphorylation strongly potentiates its ability to activate transcription of the c-fos promoter through a ternary complex assembled on the c-fos serum response element. In rat brain postmitotic neurons, Elk-1 is strongly expressed (V. Sgambato, P. Vanhoutte, C. Pagès, M. Rogard, R. A. Hipskind, M. J. Besson, and J. Caboche, J. Neurosci. 18:214-226, 1998). However, its physiological role in these postmitotic neurons remains to be established. To investigate biochemically the signaling pathways targeting Elk-1 and c-fos in mature neurons, we used a semi-in vivo system composed of brain slices stimulated with the excitatory neurotransmitter glutamate. Glutamate treatment leads to a robust, progressive activation of the ERK and JNK/SAPK MAPK cascades. This corresponds kinetically to a significant increase in Ser383-phosphorylated Elk-1 and the appearance of c-fos mRNA. Glutamate also causes increased levels of Ser133-phosphorylated cyclic AMP-responsive element-binding protein (CREB) but only transiently relative to Elk-1 and c-fos. ERK and Elk-1 phosphorylation are blocked by the MAPK kinase inhibitor PD98059, indicating the primary role of the ERK cascade in mediating glutamate signaling to Elk-1 in the rat striatum in vivo. Glutamate-mediated CREB phosphorylation is also inhibited by PD98059 treatment. Interestingly, KN62, which interferes with calcium-calmodulin kinase (CaM-K) activity, leads to a reduction of glutamate-induced ERK activation and of CREB phosphorylation. These data indicate that ERK functions as a common component in two signaling pathways (ERK/Elk-1 and ERK/?/CREB) converging on the c-fos promoter in postmitotic neuronal cells and that CaM-Ks act as positive regulators of these pathways.

A Critical Role for DNA End-Joining Proteins in Both Lymphogenesis and Neurogenesis

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

The C. elegans MDL-1 and MXL-1 proteins can functionally substitute for vertebrate MAD and MAX.

The genes of the myc/max/mad family play an important role in controlling cell proliferation and differentiation. We have identified the first homologues of the mad and max genes in the nematode C. elegans, which we have named mdl-1 and mxl-1 respectively. Like the vertebrate MAD proteins, MDL-1 binds an E-box DNA sequence (CACGTG) when dimerized with MXL-1. However, unlike vertebrate MAX, MXL-1 can not form homodimers and bind to DNA alone. Promoter fusions to a GFP reporter suggest that these genes are coexpressed in posterior intestinal and post-mitotic neuronal cells during larval development. The coexpression in the posterior intestinal cells occurs before their final division at the end of the L1 stage and persists afterwards, demonstrating that mad and max expression can be correlated directly to the cell cycle state of an individual cell type. These data also show that mxl-1 is an obligate partner for mdl-1 in vivo and in vitro and indicate that these genes may play an important role in post-embryonic development. Finally, MDL-1 can suppress activated c-MYC/RAS-induced focus formation in a rat embryo fibroblast transformation assay. Like the vertebrate MAD protein, MDL-1 activity in suppressing transformation is dependent on a functional SIN3 interaction domain.

The identification and characterization of expression of Pftaire-1, a novel Cdk family member, suggest its function in the mouse testis and nervous system.

We have isolated a murine cDNA encoding for a novel putative Cdk-related protein kinase, which has been named Pftaire-1, by screening a testis cDNA library for new serine/threonine kinases. Pftaire-1 showed 50% and 49% amino acid identity with Cdk5 and Pctaire-3, respectively, and contains the eleven subdomains characteristic of the protein kinases. By northern blot analysis we detected two transcripts of approximately 5.5 and 4.9 kb in size. These transcripts were expressed at low level in all murine tissues tested, except in the brain, testis and embryo, where high expression was detected. Cellular localization of the mRNAs by in situ hybridization analysis shows that Pftaire-1 is expressed in late pachytene spermatocytes in the testis and in post mitotic neuronal cells both in the brain and the embryo, suggesting a role of Pftaire-1 both in the process of meiosis as well as neuron differentiation and/or function.

Human bZIP Transcription Factor GeneNRL:Structure, Genomic Sequence, and Fine Linkage Mapping at 14q11.2 and Negative Mutation Analysis in Patients with Retinal Degeneration

TheNRLgene encodes an evolutionarily conserved basic motif-leucine zipper transcription factor that is implicated in regulating the expression of the photoreceptor-specific gene rhodopsin.NRLis expressed in postmitotic neuronal cells and in lens during embryonic development, but exhibits a retina-specific pattern of expression in the adult. To understand regulation ofNRLexpression and to investigate its possible involvement in retinopathies, we have determined the complete sequence of the humanNRLgene, identified a polymorphic (CA)nrepeat (identical to D14S64) within theNRL-containing cosmid, and refined its location by linkage analysis. Since a locus for autosomal recessive retinitis pigmentosa (arRP) has been linked to markers at 14q11 and since mutations in rhodopsin can lead to RP, we sequenced genomic PCR products of theNRLgene and of the rhodopsin-Nrl response element from a panel of patients representing independent families with inherited retinal degeneration. The analysis did not reveal any causative mutations in this group of patients. These investigations provide the basis for delineating the DNA sequence elements that regulateNRLexpression in distinct neuronal cell types and should assist in the analysis ofNRLas a candidate gene for inherited diseases/syndromes affecting visual function.

Reduction of connexin43 expression and dye-coupling during neuronal differentiation of human NTera2/clone D1 cells.

Gap junctions are plasma membrane specializations that allow direct communication among adjoining cells. We used a human pluripotential teratocarcinoma cell line, NTera-2/clone D1 (NT2/D1), as a model to study gap junctions in CNS neurons and their neuronal precursors. These cells were differentiated following retinoic acid (RA) treatment for 4 weeks and antiproliferative agents for 3 weeks, respectively, to yield post-mitotic CNS neuronal (NT2-N) cells. The cytoplasmic RNA was isolated from NT2/D1 cells both before and during RA treatment and from differentiated neurons (NT2-N cells). These RNA samples were examined using Northern blot analysis with cDNA probes specific for connexin26, -32, and -43. Connexin26 and -32 mRNAs were absent in NT2/D1 and NT2-N cells. Connexin43 mRNA was expressed at high levels in NT2/D1 cells before RA treatment, but it decreased significantly during RA induction. There was no detectable connexin43 mRNA in NT2-N cells. Western blot analysis confirmed the expression of connexin43 protein in NT2/D1 cells before and during RA treatment. The protein profile detected in Western blot analysis indicated two bands representing different phosphorylation states of connexin43. Our immunocytochemistry results did not show connexin26 and -32 immunoreactivity in NT2/D1 and NT2-N cells. However, we detected connexin43 immunoreactivity in NT2/D1 cells with a decreasing pattern upon RA induction. Both Western blotting and immunocytochemistry confirmed the absence of connexin43 protein in NT2-N cells. NT2/D1 cells passed calcein readily to an average of 18 cells, confirming the functionality of gap junctions in these cells. The extent of dye-coupling decreased about 78% when NT2/D1 cells were RA treated for 4 weeks. NT2-N differentiated neurons did not pass dye to the adjacent cells. We conclude that both connexin43 expression and dye coupling capacity decrease during neuronal differentiation of NT2/D1 cells.

CD59 expression and complement susceptibility of human neuronal cell line (NTera2).

The present study analysed the susceptibility of neuronal cells to human complement. A human neuronal precursor cell line (NTera2) which can be induced to differentiate into post-mitotic neuronal cells was employed. Using immunocytochemistry, NTera2 neurones, but not their precursors (stem cells), were shown to activate complement in the absence of antibody and to lack CD59, an inhibitor of the terminal stages of the complement cascade. Following exposure to human serum, neurones but not stem cells were lysed by complement, as demonstrated by a cell viability assay and consistent rise in intracellular calcium. This response was abrogated when cells were treated with complement-depleted serum by heat inactivation or cobra venom factor. Protection from lysis was observed following incorporation of CD59 with its glycolipid anchor in the neuronal cell membrane.

Inhibitors of Cyclin-dependent Kinases Promote Survival of Post-mitotic Neuronally Differentiated PC12 Cells and Sympathetic Neurons

Previous studies have demonstrated that multiple agents that promote survival of PC12 cells and sympathetic neurons deprived of trophic support also block cell cycle progression. Presently, we address whether inhibition of cell cycle-related cyclin-dependent kinases (CDKs) prevents neuronal cell death. We show that two distinct CDK inhibitors, flavopiridol and olomoucine, suppress the death of neuronal PC12 cells and sympathetic neurons. In addition, we demonstrate that inhibitor concentrations required to promote survival correlate with their ability to inhibit proliferation. Promotion of survival, however, does not correlate with inhibition of extracellular signal-regulated kinase or c-Jun kinase activities or with interference with the activation of c-Jun kinase that accompanies serum/nerve growth factor deprivation. In contrast to their actions on nerve growth factor-differentiated PC12 cells, the CDK inhibitors do not prevent the death of proliferation-competent PC12 cells and, in fact, promote their cell death. These findings support the hypothesis that post-mitotic neuronal cells die after removal of trophic support due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner. We speculate that cycling PC12 cells are not saved by these agents due to a signaling conflict between an inherent oncogenic signal and the inhibition of CDK activity.

Cell cycle blockers mimosine, ciclopirox, and deferoxamine prevent the death of PC12 cells and postmitotic sympathetic neurons after removal of trophic support

In the present study, we tested whether apoptotic neuronal death caused by withdrawal of trophic support might be prevented by agents that block cell cycle progression. We used three complementary model systems that exhibit apoptotic death: dividing PC12 cells deprived of nerve growth factor (NGF); and primary cultures of postmitotic sympathetic neurons deprived of NGF. We show that cell death in each case can be suppressed by treatment with the G1/S blockers mimosine, ciclopirox, and deferoxamine at concentrations that correlate with their abilities to block PC12 cell proliferation. In contrast, agents that block cell cycle progression in the S-, G2-, or M-phase do not prevent cell death. These observations support the hypothesis that removal of trophic support from dividing or postmitotic neuronal cells provokes their apoptotic death by causing them either to proceed through or to attempt to re-enter an uncoordinated and consequently fatal cell cycle. Moreover, the data suggest that simply blocking the cycle at any point is not protective but, rather, that it is necessary to block at specific "safe" points. This study defines a safe point in the cell cycle before the G1/S transition that is demarcated by the action of these three agents.

Transcription factors: potential roles in drug-induced neuroplasticity.

Transcription factors act to regulate gene expression. Many transcription factor families have been discovered based on their roles in cell cycle events involved in development and oncogenesis. In post-mitotic neuronal cells, however, many transcription factor genes are "trans-synaptically" regulated: their patterns of expression can be dramatically altered by extracellular stimuli. Transcription factor proteins can then potently influence expression of other genes, whose products can directly alter neuronal function. The central nervous system (CNS) displays varying degrees of neuroplasticity in adult life. Flexible neurochemical pathways that link extracellular stimuli to long-term modifications in neuronal functions are likely to contribute substantially to this neuroplasticity. This review summarizes evidence supporting central roles for transcription factors in such neurochemical cascades. It furthermore illustrates how drugs of abuse can trigger and modulate neuroadaptive processes that could conceivably contribute to clinically relevant addiction phenomena such as craving, tolerance, sensitization, and withdrawal.

Gene replacement therapy in the central nervous system: Viral vector-mediated therapy of global neurodegenerative disease

AbstractThis target article describes the current state of global gene replacement in the brain using viral vectors and assesses possible solutions to some of the many problems inherent in gene therapy of the central nervous system (CNS). Gene replacement therapy in the CNS is a potential means of producing a stable expression of normal human proteins in deficient cells and thus curing certain genetically inherited enzyme deficiencies and metabolic diseases as well as cancers. The two major issues to be addressed in CNS gene replacement are the delivery of genetic material to the brain and the expression of recombinant genetic material in target cells within the CNS. Focal inoculation of recombinant virions or other genetic vectors has limitations in global CNS disease. A new approach is the blood-brain barrier (BBB) disruption technique developed in this laboratory, in which hypertonic mannitol transiently shrinks the BBB endothelium, allowing passage of high molecular weight compounds and even viruses. Gene therapy of the CNS will require a viral vector system that allows long-term, nontoxic gene expression in neurons or glial cells. Retroviral vectors have limitations in CNS gene replacement, although they are suitable for expressing recombinant genes in intracerebral grafts, or toxic genes in brain tumors. Using mutant neurotropic viruses with reduced neurotoxicity (such as defective herpes simplex virus type I [HSV-1], the HSV-1 amplicon vector system we have developed, or adenovirus mutants) has potential for direct treatment of neurons. Injecting these vectors into rodent brains can lead to stable expression of foreign genetic material in postmitotic neuronal cells. We discuss our BBB disruption delivery technique, our defective HSV-1 amplicon vector system, and our feline model for the neuronal lysosomal storage disorder Gm2-gangliosidosis (Sandhoff disease), which may prove to be a useful model system for CNS gene therapy.

MN20, a D2 cyclin found in brain, is implicated in neural differentiation

Cyclins are regulatory proteins that promote the progression of dividing cells through the cell cycle. D-type cyclins are important mediators of the transition from G1 into S phase of the cell cycle and are thought to be widely expressed in mitotically active tissues (Matsushime et al., 1991b; Inaba et al., 1992; Xiong et al., 1992). We report the isolation of a cDNA clone, MN20, which represents a D2 cyclin message form whose expression pattern is highly restricted to brain. MN20 is not ubiquitous, but rather it is expressed only in restricted neuronal precursor populations, for example, in proliferating granule neuroblasts of the cerebellum but not hippocampus. Strikingly, MN20 expression is also found in postmitotic neuronal precursor cells of the embryonic cerebral cortex, but not in the dividing cortical neuroblasts. These observations suggest that the D2 cyclin gene serves regionally specific functions in neuronal differentiation, some of which may be distinct from the promotion of cell cycle progression and which act at the interface between mitosis and the assumption of mature neuronal morphology.

Activation of mitogen-activated protein kinase by epidermal growth factor in hippocampal neurons and neuronal cell lines.

Epidermal growth factor (EGF) functions in a bimodal capacity in the nervous system, acting as a mitogen in neuronal stem cells and a neurotrophic factor in differentiated adult neurons. Thus, it is likely that EGF signal transduction, as well as receptor expression, differs among various cell types and possibly in the same cell type at different stages of development. We used hippocampal neuronal cell lines capable of terminal differentiation to investigate changes in EGF receptor expression, DNA synthesis, and stimulation of mitogen-activated protein (MAP) kinase by EGF before and after differentiation. H19-7, the line that was most representative of hippocampal neurons, was mitogenically responsive to EGF only before differentiation and increased in EGF binding after differentiation. MAP kinase was stimulated by EGF in both undifferentiated and differentiated cells, as well as in primary hippocampal cultures treated with either EGF or glutamate. These results indicate that the activation of MAP kinase by EGF is an early signaling event in both mitotic and postmitotic neuronal cells. Furthermore, these studies demonstrate the usefulness of hippocampal cell lines as a homogeneous neuronal system for studies of EGF signaling or other receptor signaling mechanisms in the brain.

Expression of transfected genes by differentiated, postmitotic neurons and photoreceptors in primary cell cultures

We report here that differentiated, primary, postmitotic neurons and photoreceptors in cultures obtained from embryonic chicks can express foreign genes after transfection by the calcium phosphate method. A variety of viral promoters were tested by using either beta-galactosidase or chloramphenicol acetyltransferase as reporter genes. Histochemical and immunocytochemical analysis showed beta-galactosidase expression by both neurons and photoreceptors. As commonly observed with dividing cells, transfection efficiencies showed inter-experimental variability, with efficiencies ranging from 2% to 20% using the same plasmid. On the other hand, intra-experimental variability between replicate dishes was much smaller. Analysis using the highly sensitive enzymatic assay for chloramphenicol acetyl transferase (CAT) showed that all of the promoter/enhancers-CAT constructs tested, with the exception of a construct containing the Maloney sarcoma virus promoter, led to the expression of detectable activity when transfected into cultured retinal cells. The calcium phosphate treatment used for cell transfection did not show detectable effects on overall cell survival, although it caused selective decreases in some metabolic activities of the cells. The studies demonstrate that it is possible to obtain expression of genes transfected into primary, postmitotic neuronal cells.

In vivo and in vitro expression of gangliosides in chick retina Müeller cells.

The expression of gangliosides of the lactosylceramide (LC) and of the gangliotetraosylceramide (GTC) series on the surface of cells from the chick neural retina was investigated by double-color indirect immunofluorescence. GD3 was assumed to be representative of LC and was detected using a specific monoclonal antibody. GM1 was assumed to be representative of GTC and was detected using the binding of cholera toxin followed by the binding of cholera toxin antibodies. The expression of polysialosylated GTC (polysialosyl-GTC) was detected using the cholera toxin-cholera toxin antibody experimental approach, after conversion of polysialosyl-GTC to GM1 by treatment of the cells with neuraminidase. In retinas from 6-day-old embryos (R6), most cells (approximately 80%) expressed GD3 but not GTC. After culturing for 7 days, (R6+7), the expression of GTC was found confined to neuron-like cells; flat cells derived from Müller cells expressed GD3 but were negative for GTC expression. On the other hand, postmitotic Müller cells obtained from 13-day-old embryo (R13) or 1-day-old hatched chick retina (RP1) expressed GD3, GM1, and polysialosyl-GTC but were unable to maintain the expression of these GTCs when kept in culture for several days. According to these results, retinal cells can be defined on the basis of their ganglioside expression as follows: (a) retinoblasts, by the expression of GD3; (b) postmitotic neuronal cells, by the expression of GTC; and (c) postmitotic Müller cells, by the expression of GD3 and GTC.(ABSTRACT TRUNCATED AT 250 WORDS)

Vinyl chloride: An assessment of the risk of occupational exposure

There is little doubt that exposure to high levels of VCM as a consequence of occupation can result in an increased incidence of ASL. A review of 20 epidemiological studies involving about 45,000 workers occupationally exposed to VCM showed that neoplasms of the liver showed an increase in incidence in the majority of studies. For brain cancer the association between exposure to VCM and an increased incidence was less clear because of the lower relative risk. Neoplasms of the respiratory tract, digestive system, lymphatic and haemopoietic system, buccal cavity, and pharynx, cardiovascular system and colon/stomach were reported to show an increased incidence in one or more studies, but to show no increase, or in some cases a decrease, in incidence in other studies. In view of the increased incidence of breast neoplasms in rodents exposed to VCM, the studies of Chaizze et al. (1980), who did not confirm these findings in humans, are of importance. The register of ASL cases now contains records of 99 persons with confirmed ASL and occupational exposure to VCM. The average latent period between first exposure to VCM and death from ASL is 21.9 years. The majority of cases occurred in autoclave workers, who are recognized as having been exposed to extremely high levels. Although precise estimates of exposure are not available for the periods of most interest, the pattern of cases roughly suggests that extremely high exposures were necessary for the induction of ASL. For example, ASL cases tended to occur in larger numbers in some plants than in others, a finding that can be explained most easily by differences in exposure patterns. There is an extensive series of animal studies on the carcinogenicity of VCM. Some of these precede the epidemiological studies confirming the association between VCM exposure and ASL in man. ASL and neoplasms of a number of other organs have been induced in laboratory rodents by VCM. Estimation of the exposure levels likely to cause a lifetime risk of ASL of 10(-6) on the basis of these data give extremely low levels (down to 3.9 X 10(-7) ppb) which appear to be unrealistic estimates for man. Part of the reason for this is that laboratory studies have shown that VCM is metabolized in the liver (and elsewhere in the body) to the reactive metabolites chloroethylene oxide and chloroacetaldehyde. The rate of conversion is limited at high levels of exposure giving inaccurate estimates of the slope of the dose-response relationship.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuronal acquisition of tetanus toxin binding sites: Relationship with the last mitotic cycle

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Dev. Brain Res. 5, 139–147) . Using a combined approach of immunocytochemistry and [ 3H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 ± 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.