Genetic effects of photoactivated psoralens during meiosis in DNA repair mutantpso3-1 ofSaccharomyces cerevisiae

The influence of the DNA repair genePSO3 on photoactivated psoralen-induced meiotic recombination, gene conversion, reverse mutation, and on survival, was assayed in diploid strains ofSaccharomyces cerevisiae homozygous for the wild-type or thepso3-1 mutant allele. Sporulation was normal in thepso3-1 diploid. Wild-type and mutant strains had the same sensitivity to photoactivated monofunctional psoralen (3-CPs+UVA) in meiosis-uncommitted and meiosis-committed stages. The mutant showed higher sensitivity to photoactivated bifunctional psoralen (8-MOP+UVA) during all stages of the meiotic cycle. Mutation induction by 3-CPs+UVA or 8-MOP+UVA in meiosis-committed cells revealed no significant differences between wild-type and thepso3-1 mutant. The status of thePSO3 gene has no influence on the kinetics of induction of gene conversion and crossing-over after 3-CPs+UVA treatment in meiosis-committed cells: gene conversion was blocked while recombination was induced. After treatment with 8-MOP+UVA gene conversion was also blocked in both strains while crossing-over could only be observed in meiosis-committed wild-type cells.     

Regionally defective colonization of the terminal bowel by the precursors of enteric neurons in lethal spotted mutant mice

In order to gain insight into the process of colonization of the bowel by the neural crest-derived precursors of enteric neurons, the development of the enteric nervous system was examined in lethal spotted mutant mice, a strain in which a segment of bowel is congenitally aganglionic. In addition, nerve fibers within the ganglionic and aganglionic zones of the gut of adult mutant mice were investigated with respect to their content of acetylcholinesterase, immunoreactive substance P, vasoactive intestinal polypeptide and serotonin, and their ability to take up [³Hserotonin. In both the fetal gut of developing mutant mice and in the mature bowel of adult animals abnormalities were limited to the terminal 2 mm of colon. The enteric nervous system in the proximal alimentary tract was indistinguishable from that of control animals for all of the parameters examined. In the terminal bowel, the normal plexiform pattern of the innervation and ganglion cell bodies were replaced by a coarse reticulum of nerve fibers that stained for acetylcholineserase and were continuous with extrinsic nerves running between the colon and the pelvic plexus. These coarse nerve bundles contained greatly reduced numbers of fibers that displayed substance P- and vasoactive intestinal polypeptide-like immunoreactivity, but a serotonergic innervation was totally missing from the aganglionic bowel. During development, acetylcholineserase and uptake of [³Hserotonin appeared in neural elements in the foregut of mutant mice on the 12th day of embryonic life (E12), about the same time these markers appeared in the forgut in normal mice. By day E14, neurons expressing one or the other marker were recognizable as far distally as about 2 mm from the anus. The appearance of neurons in segments of gut grown for 2 weeks as expiants in culture was used as an assay for the presence of neuronal progenitor cells in the segments of fetal bowel at the time of explantation. Both acetyl- cholinesterase activity and uptake of [³Hserotonin developed in neuronsin vitro in expiants of proximal bowel between days E10 and E17. At all times, however, the terminal 2mm of mutant but not normal fetal gut gave rise to aneuronal cultures. In some mutant mice rare, small, ectopically-situated pelvic ganglia were found just outside aganglionic segments of fetal colon. Uptake of [³Hserotonin, normally a marker for intrinsic enteric neurites, was found in these ganglia.The experiments suppport the hypothesis that the terminal 2 mm of the gut in lethal spotted mutant mice is intrinsically abnormal and thus cannot be colonized by the precursors of enteric neurons. The defect seems to be specific in that both cells and processes of intrinsic enteric neurons, including all serotonergic and most peptidergic neurites, seem to be excluded from the abnormal region while extrinsic nerve fibers, including sympathetic and sensory axons, are able to enter the aganglionic zones. Since examination of neural progenitor cells has failed to reveal a significant proximo-distal displacement of these cells through the enteric tube during development of the murine bowel, a defect in the migration of precursor cells down the alimentary tract to the terminal gut seems unlikely to be substantially involved in the pathogenesis of aganglionosis. This conclusion is supported by the normal enteric nervous system in proximal regions of the mutant gut and the presence of enteric type neurons outside of, but at the same level as the aganglionic region.     

NMDA receptor mediated dendritic plasticity in cortical cultures after oxygen-glucose deprivation

Dendrites and spines undergo dynamic changes in physiological and pathological conditions. Dendritic outgrowth has been observed in surviving neurons months after ischemia, which is associated with the functional compensation. It remains unclear how dendrites in surviving neurons are altered shortly after ischemia, which might reveal the mechanisms underlying neuronal survival. Using primary cortical cultures, we monitored the dendritic changes in individual neurons after oxygen-glucose deprivation (OGD). Two to four hours of OGD induced approximately 30–50% cell death in 24 h. However, the total dendritic length in surviving neurons was significantly increased after OGD with a peak at 6 h after re-oxygenation. The increase of dendritic length after OGD was mainly due to the sprouting rather than the extension of the dendrites. The dendritic outgrowth after 2 h of OGD was greater than that after 4 h of OGD. Application of NMDA receptor blocker MK-801 abolished OGD-induced dendritic outgrowth, whereas application of AMPA receptor antagonist CNQX had no significant effects. These results demonstrate a NMDA receptor-dependent dendritic plasticity shortly after OGD, which provides insights into the early response of surviving neurons after ischemia.     

Cloning and spatial and temporal expression of the zebrafish dopamine D1 receptor.

Dopamine plays important roles in the regulation of central nervous system (CNS) development and functions. In vertebrates, two families of dopamine receptors, collectively known as dopamine D1 and D2 receptors, have been identified. Recently, dopamine receptors have been targeted by pharmacological and therapeutic studies of neurological disorders, such as Parkinson's disease. Here, we report a study on the molecular characterization of dopamine D1 receptor in zebrafish (Danio rerio). We cloned the full-length cDNA of a zebrafish dopamine D1 receptor, designated as drd1. The sequence of drd1 shares high homology to the sequences of dopamine D1 receptors in mammalian, amphibian, and other fish species. drd1 is expressed in the CNS. The first drd1 expression was observed at approximately 30 hours postfertilization, at which time the expression was seen in the developing diencephalon and hindbrain. In developing retinas, the expression of drd1 was detected in the inner nuclear layer with the exception of the marginal zones. In adult retinas, drd1 expression was detected in most cell types in the inner and outer nuclear layers as well as ganglion cell layer. Differential expression of drd1 in developing and adult retinas may play various roles in regulating visual system functions.     

Use of seminiferous tubule segments to study stage specificity of unscheduled DNA synthesis in rat spermatogenic cells

DNA repair in spermatogenic cells at various stages of maturity was determined by quantitation of unscheduled DNA synthesis (UDS). Male F-344 rats were exposed (i.p.) to methyl methanesulfonate (MMS, 35 mg/kg); 1 hr later, segments of seminiferous tubules corresponding to spermatogenesis stages II, IV-V, VI, VII, VIII, IX-X, XII, and XIV were isolated with the transillumination pattern of the tubules as a guide. Intact tubule segments were cultured 24 hr in the presence of [3H]thymidine, and UDS was quantitated by autoradiography as net grains/nucleus (NG). In primary spermatocytes from treated rats, NG count increased with increasing maturity from leptotene primary spermatocytes (3.5 NG) up through stage VIII and IX-X pachytene spermatocytes (22 NG), after which NG decreased in stage-XII pachytene and diplotene spermatocytes (to 16 NG and 8 NG, respectively). Round spermatids of steps 2-8 of spermiogenesis all exhibited approximately the same UDS response (8 NG). Elongating spermatids as mature as step 14 underwent UDS after exposure to MMS, but step-15 and later-step spermatids did not. The DNA repair response of pachytene spermatocytes cultured within segments of seminiferous tubule corresponding to stages VIII and IX-X was 4 to 25 times greater, depending on the dose of MMS, than pachytene spermatocytes isolated by enzymatic digestion and cultured in suspension [Bentley and Working, Mutat Res 203:135-142, 1988]. Thus, the use of segments of seminiferous tubule both increased the sensitivity of UDS as an indicator of DNA damage in rat germ cells and enabled the study of UDS in spermatogenic cells at different stages of maturity.     

Deoxyribonucleic acid turnover in immature neurons of the rat cerebral cortex

To test for metabolic deoxyribonucleic acid (DNA) turnover in differentiating neurons, [methyl-3H]thymidine was injected into the lateral cerebral ventricles of newly born rats, and after 6, 24 and 96 h, neuronal nuclei were prepared from the immature cerebral cortex. Enzymatic treatment converted virtually all of the DNA into soluble deoxynucleosides which were fractionated by high-performance liquid chromatography for determination of specific activity. The specific activity of thymidine was found to decline rapidly with time. The rate of this loss correlated with the radioactivity initially incorporated into the DNA. This suggested that DNA was being replaced by DNA repair as a consequence of radiation damage, rather than by spontaneous metabolic DNA turnover.     

Influence of Various Immunomodulators on the Induction of Experimental Autoimmune Encephalomyelitis in Lewis Rats

The effect of various immunomodulators on the induction of experimental autoimmune encephalomyelitis (EAE) is evaluated in the Lewis rat. Bordetella pertussis (BP) is the optimal inductor of EAE in this rat strain. Treatment of the animals with BP either before or after or simultaneously with guinea-pig spinal cord preparation (GpSC) resulted in an EAE about two weeks thereafter. Additional injection of living BCG, of CFA, IFA (incomplete Freund's adjuvant) or Vibrio cholerae neuraminidase (VCN) did not augment or mitigate the effect induced by BP or GpSC. Living BCG, IFA, VCN or Corynebacterium parvum (CP) did not induce EAE when given in combination with GpSC but without BP. CFA combined with GpSC only occasionally induced EAE. However, EAE could be induced by the combination of CFA and GpSC or IFA and GpSC in a part of the animals tested if they had been pretreated or simultaneously been injected with living BCG by intravenous route. EAE could not be enhanced by the additional injection of VCN. Surprisingly, most of the animals peracutely died after injection of CFA and BP in combination with GpSC when they had been pretreated with CP. This effect was most pronounced when pretreatment was done on day -4. No acute effect could be seen when CP was given simultaneously to CFA, BP and GpSC. Animals which did not peracutely succumb developed EAE similarly as those in the positive control groups. CP treatment simultaneously with BP but without CFA resulted in a reduction of the EAE specific mortality. This reduction could not be seen if treatment with CP was done after injection of GpSC and BP.     

A fluorescence histochemical and biochemical evaluation of the effect of p-chloroamphetamine on individual serotonergic nuclei in the rat brain.

The serotonin (5-hydroxytryptamine) content of eight raphe nuclei was measured 9 days after injection of p-chloroamphetamine. 5-Hydroxytryptamine levels were reduced in only the B-9 cell group.The histofluorescence of serotonergic neurons in the B-9 cell group was also studied at two time intervals after a single injection of p-chloroamphetamine. Nine days after the administration of p-chloroamphetamine there was a small decrease both in the intensity of fluorescence and in the number of yellow fluorescent serotonergic cells. After 2 months the drug caused a more marked decrease in the number of viable serotonergic cells. The results are discussed in relation to the mechanism of action of p-chloroamphetamine.It is suggested that following the administration of p-chloroamphetamine retrograde destruction may occur in the B-9 cell group, in contrast to those of the other raphe nuclei, whose cell bodies may possess a sufficient number of collateral processes to resist the slow neurotoxic destruction of their cell bodies.     

Control of feeding rhythm in the terrestrial slug Incilaria bilineata

A modulatory neuron of feeding rhythm was newly identified in the buccal ganglia of the isolated central nervous system (CNS) of the terrestrial slug Incilaria bilineata. This neuron was termed the “feeding rhythm modulator” (FRM). Its morphological and electrical properties were compared with those of the MGC (metacerebral giant cell, a cerebral modulatory neuron of feeding rhythm). There was no direct connection between FRM and MGC. In order to investigate the control mechanism of the buccal central pattern generator, feeding rhythm was observed by varying the activities of MGC and FRM simultaneously. At a lower level of activity of MGC, feeding rhythm was not only sensitive to the activity of MGC but also to that of FRM. As the level of activity of MGC increased, feeding rhythm was exclusively controlled by the activity of MGC, and became unaffected by the activity of FRM. This indicates that cerebral neurons such as MGC primarily control feeding rhythm and modulate the contribution of FRM in a hierarchical manner.     

Stimulation of DNA repair synthesis of rat thymocytes by novobiocin and nalidixic acid in vitro without detectable DNA damage

Scheduled (SDS) and unscheduled (UDS) DNA synthesis as well as nucleoid sedimentation was investigated in vitro under the influence of novobiocin (NB) and nalidixic acid (NA) using intact thymic (T-cells) and splenic (S-cells) rat cells and cells which were exposed to X-rays, UV irradiation, methyl methanesulfonate (MMS), and DNA polymerase inhibitors. At concentrations of 56.25 (S-cells) and 225 g/ml (T-cells), respectively, NB inhibited SDS in a dose-dependent manner. Within a concentration range of 225–900 g NB/ml, UDS of S-cells decreased to values far below the tracer ([³H-methyl]-thymidine) incorporation of control cells, whereas UDS of T-cells increased by at least 200%. Within a concentration range of 450–1800 g/ml, NA enhanced SDS and UDS by about 30% in S-cells and by 100% in T-cells. The stimulating activity of NB and/or NA could be eliminated specifically by the DNA polymerase inhibitor 2',3'-dideoxythymidine. Enhanced nucleoid sedimentation was observed at NB concentrations 750 g/ml; S-cells revealed a higher sedimentation rate than T-cells. It is suggested that NB (and NA) influence DNA topology in a rather cell specific manner, stimulating UDS of T-cells by a DNA polymerase — dependent repair-like mechanism.