The effects of N-methyl-N'-nitro-N-nitrosoguanidine on KB cells. II. Cell sensitivity to the lethal effect of the drug as a function of the cell age and of the nature of the culture medium

We have tested the sensitivity of KB cells to the lethal effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), measured as cell loss within the 20-h period following a 1-h drug treatment, as a function of culture age and of the medium in which treated cells were incubated after elimination of MNNG. We showed that KB cell sensitivity to the lethal effect of the drug decreased with time after seeding when the treated cells were post-incubated in drug-free medium conditioned by untreated cells of the same age as treated ones but not when they were post-incubated in fresh drug-free medium. This difference was due in part to the fact that the conditioned medium had acquired with time a protective activity for treated cells and in part to an increased competence of aging cells to be protected by this medium. By post-incubating treated stationary cells sequentially in both media, we showed that a brief (15 min) post-incubation of the cells in fresh medium was sufficient to trigger cell death even if the cells were afterwards transferred to conditioned medium. In contrast, long post-incubation in fresh medium did not cause cell death if the cells were first post-incubated in conditioned medium for about 3 h. We conclude that: the medium acted on cell sensitivity to the lethal effect of MNNG through its growth regulatory ability; quiescent cells were less sensitive to the drug than growing cells; the sensitive phase of the cell was located before S; cell hypersensitivity might be due to deficient repair of cellular lesions rather than to increased lesion formation.     

Effects of the antimutagen cinnamaldehyde on reversion and survival of selected Salmonella tester strains

The antimutagenic activity of trans-cinnamaldehyde (C6H5CH = CHCHO) on chemically induced mutagenesis has been shown in E. coli. Using the Ames Salmonella typhimurium tester strains TA1535 (hisG46 uvrB rfa) and TA100 (TA1535/pKM101), the effects of cinnamaldehyde on spontaneous reversions and reversions induced by 4-nitroquinoline-N-oxide(4NQO) and ethyl methanesulfonate (EMS) have been examined. To observe the effect of cinnamaldehyde in the absence of functional muc genes, a third strain, TA1535/pGW201 (pKM101 muc140: :Tn5) was included in the testing. Modifications of the standard Ames test procedures and direct-plating techniques were employed to study the "antimutagenic" response exerted by cinnamaldehyde. In all strains tested, concentrations of cinnamaldehyde up to 25 micrograms/ml slightly decreased the number of spontaneous reversions and induced reversions were more markedly reduced. The decreases in the numbers of 4NQO-induced revertants were greater than those decreases which occurred for EMS-induced reversions. There was no effect on viability in 1% (v/v) nutrient broth supplemented minimal medium containing 5-25 micrograms/ml of cinnamaldehyde. Cinnamaldehyde did not display any mucAB dependent or independent specificity against the mutagens used. On minimal medium supplemented with histidine and biotin, concentrations of cinnamaldehyde above 10 micrograms/ml were lethal for the strains tested. When the test medium was supplemented with 1-5% (v/v) liquid nutrient broth, viability was not affected at concentrations up to 25 micrograms/ml. For both TA100 and TA1535 the presence of 20 micrograms/ml of cinnamaldehyde in 1% (v/v) liquid nutrient broth-supplemented minimal glucose broth extended the lag phase for 2-4 h with no effect on survival. Depending on the test procedure employed, decreases in numbers of revertants may reflect lethality rather than antimutagenesis. When used to test for antimutagenesis rather than mutagenesis, modifications of the standard Ames test procedure may mimic an antimutagenic response due to a decrease in the total number of revertants seen even though enough cells survive to produce a background lawn.     

The 'antimutagenic' effect of cinnamaldehyde is due to a transient growth inhibition

Addition of cinnamaldehyde to the selective medium causes a reduction in the number of revertant colonies of S. typhimurium or E. coli when the cells have been mutagenized with 4NQO but not when they have been mutagenized with MNNG. Toxicity of the cinnamaldehyde exposure depends largely on the status of growth and/or nutrient supply of the cells. We present evidence that simple growth inhibition due to lack of nutrients mimics the effect of cinnamaldehyde in 4NQO- and MNNG-treated cells. This argues that the reduction of mutant colonies is due to a transient growth retardation caused by cinnamaldehyde exposure, which presumably allows the cells to repair 4NQO-induced damage--but not MNNG-induced damage--via a more error-free pathway.     

Analysis of the antimutagenic effect of cinnamaldehyde on chemically induced mutagenesis in Escherichia coli

The antimutagenic effect of cinnamaldehyde on mutagenesis was investigated using ten kinds of chemical mutagen in Escherichia coli WP2s (uvr A-). In addition, the frequency of mutation induction by each mutagen in an SOS repair deficient (umuC-) strain was compared with that in a wild-type (umuC+) strain. Cinnamaldehyde greatly suppressed the umuC-dependent mutagenesis induced by 4-nitroquinoline 1-oxide (4-NQO), furylfuramide or captan. However, cinnamaldehyde was less effective against the umuC-independent mutagenesis by alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine and ethylmethanesulfonate. On the other hand, no inhibitory effect of cinnamaldehyde was observed on prophage induction or tif-mediated filamentous growth. These results suggest that a cinnamaldehyde does not prevent the induction of the SOS functions. Despite the decrease in the number of revertants, a remarkable increase was observed in the survival of 4-NQO-treated WP2s cells after exposure to cinnamaldehyde. The reactivation of survival suggests the promotion of some DNA repair system by cinnamaldehyde. This enhancement of survival was also observed in uvr B, polA, recF or umuC mutants and less in lexA or recB, C mutants. However, it was not observed in recA mutants. Therefore, we assume that cinnamaldehyde may enhance an error-free recombinational repair system by acting on recA-enzyme activity.     

Nb2 cell mitogenesis: effect of lactogens on cAMP and protein phosphorylation

Rat lymphoma cells (Nb2) are exquisitely sensitive to lactogenic hormones and are an ideal system to study receptor-mediated signal transduction. The effect of human growth hormone (hGH) on macromolecular synthesis, intracellular cAMP concentrations and protein phosphorylation was investigated in Nb2 cells maintained in serum-free medium. hGH stimulated the incorporation of radiolabeled precursors into protein, RNA and DNA in a time-dependent manner. The concentration of hGH inducing half-maximal DNA synthesis was 11 pM, indicating that Nb2 cells cultured in serum-free medium maintain the same sensitivity to lactogen as cells in horse serum-containing medium. hGH over a period of 4 h had no effect on intracellular cAMP regardless of the presence or absence of isobutylmethylxanthine (IBMX). IBMX (250 microM), increased intracellular cAMP levels 2-fold indicating that the cAMP assay was sufficiently sensitive to detect relatively small changes in intracellular cAMP. Cyclic AMP had no effect on protein phosphorylation. However, hGH, prolactin and placental lactogen enhanced phosphorylation of many protein targets, as well as that of a specific protein (Mr = 29,000). Rat growth hormone, which is not mitogenic, had no effect on protein phosphorylation. These results suggest that lactogen-mediated Nb2 mitogenesis does not involve modulation of intracellular cAMP concentration and that cAMP-independent protein phosphorylation may play a role.     

Effect of sodium monensin and cinnamaldehyde on the growth and phenotypic characteristics of Prevotella bryantii and Prevotella ruminicola

Two representative strains of Gram-negative rumen bacteria from the genus Prevotella were used as model organisms in order to evaluate the effect of cinnamaldehyde (the secondary metabolite found in extracts of the Cinnamomum family) vs. sodium monensin on growth, cell size and cell protein production. Prevotella bryantii B(1)4 was found to be remarkably more resistant to the action of both compounds than Prevotella ruminicola 23. The approximate IC(50) concentrations of sodium monensin influenced the increase in cell size of both strains during growth, which was much more pronounced in the case of the B(1)4 strain. A similar effect was observed in strain B(1)4 when 1.438 mmol/L cinnamaldehyde was added to the growth medium, indicating a possible interference with cell division. The action of cinnamaldehyde on P. bryantii B(1)4 was concentration-dependent, in contrast to the effect observed on P. ruminicola 23.     

The amyloid peptide induces early genotoxic damage in human preneuron NT2

The antimutagenic activities of benzalacetone (4-phenyl-3-buten-2-one) and its structurally-related compounds were evaluated through their use as post-treatments for the UV-induced mutagenesis in Escherichia coli WP2s (uvrA) and the gamma-induced mutagenesis in Salmonella typhimurium TA2638, the latter of which is sensitive to oxidants. Structure-activity relationships were studied between IC(50) activity values, i.e. the dose (micromol/ml) at which the mutation frequency is reduced to 50% of the control, and electronic and hydrophobicity properties of the studied molecules. Benzalacetone and benzalacetone analogs, cinnamaldehyde and trans-1,1,1-trifluoro-4-phenyl-3-buten-2-one (TF), inhibited both forms of mutagenesis, but methyl cinnamate, cinnamic acid and cinnamamide did not. The IC(50) values of TF, for UV-induced mutagenesis and gamma-induced mutagenesis, were 0.028 and 0.045 micromol/ml, respectively, and one order of magnitude lower than those of cinnamaldehyde and benzalacetone. The three antimutagenic analogs listed in order of decreasing activity are: TF>cinnamaldehyde>benzalacetone. This order is proportional to the electron-withdrawing property of the terminal group attached to an alpha,beta-unsaturated carbonyl moiety in the side chain that is known to play an important role in the antimutagenicities of benzalacetone and related compounds. In UV-induced mutagenesis in E. coli WP2s, mono-substituted benzalacetones - the ring-substituents of which have electron-withdrawing properties - showed antimutagenic activity that correlated with their electronic property. In gamma-induced mutagenesis in S. typhimurium TA2638, the antimutagenic activities of mono-substituted benzalacetones were proportional to the substituent hydrophobicities (pi). The different effects on both the mutation-induced systems is suggested to be related to the relative permeability of the cell membranes and the different sensitivities to mutagens between E. coli WP2s and S. typhimurium TA2638. In addition, the antimutagenic activity against gamma-induced mutagenesis could be due to the ability of parent compounds or their derivatives to scavenge long-lived organic radicals; the radicals have been described to be generated as a result of the X-irradiation of cells by Koyama et al. [Mutat. Res. 421 (1998) 45].     

UV induces nucleolar translocation of ING1 through two distinct nucleolar targeting sequences

The ING1 candidate tumor suppressor is downregulated in a variety of primary tumors and established cancer cell lines. Blocking its expression experimentally promotes unregulated growth in vitro and in vivo, using cell and animal models. Alternative splicing products encode proteins that localize to the nucleus, inhibit cell cycle progression and affect apoptosis in different model systems. Here we show that ING1 proteins translocate to the nucleolus 12–48 h after UV-induced DNA damage. When a small 50 amino acid portion of ING1 was fused to green fluorescent protein, the fusion protein was efficiently targeted to the nucleolus, indicating that ING1 possesses an intrinsic nucleolar targeting sequence (NTS). We mapped this activity to two distinct 4 amino acid regions, which individually direct fused heterologous proteins to the nucleolus. Overexpression of ING1 induced apoptosis of primary fibroblasts in the presence and absence of UV exposure. In contrast, NTS mutants of ING1 that were not targeted to the nucleolus did not efficiently induce apoptosis when overexpressed and instead protected cells from UV-induced apoptosis. Taken together, these results indicate that UV induces ING1 to translocate to the nucleolus and that this translocation may facilitate apoptosis.     

Antimutagenesis by factors affecting DNA repair in bacteria

The term 'antimutagen' was originally used to describe an agent that reduces the apparent yield of spontaneous and/or induced mutations, regardless of the mechanisms involved. The 'antimutagens' include 'desmutagens' and 'bio-antimutagens'. In this article, our attention was focused on the bio-antimutagens affecting DNA repair in bacteria. Cobaltous chloride reduced the frequency of mutations in Escherichia coli induced by MNNG. The possibility that metal compound inhibits the growth of mutagen-treated cells was examined. The results clearly showed that the antimutagen surely reduces the mutation rate. The target of cobaltous chloride was found to be cellular factors including Rec A. Vanillin and cinnamaldehyde had strong antimutagenic activities against UV, 4NQO and AF-2. They stimulated Rec A-dependent recombination repair functions in the mutagen-treated cells. Among plant materials, tannins possess antimutagenic activity against UV-induced mutations in E. coli. It has been found that tannic acid stimulates the excision repair encoded by the uvrA gene thereby reducing the yield of mutants. Substances which are antimutagenic in bacterial systems also had antimutagenic activity in cultured mammalian cell systems. Vanillin reduced the frequency of mutagen-induced chromosomal aberrations.     

Activation of a ribosomal protein S6 kinase in mouse fibroblasts during infection with herpesvirus

If confluent fibroblasts are infected with the swine alpha-herpes virus, pseudorabies virus, ribosomal protein S6 becomes phosphorylated after a lag of approximately 2 h. When cell-free extracts were prepared from such cells in the presence of glycerol 2-phosphate and EGTA, a ribosomal protein S6 kinase activity was found to appear at approximately the same time as the phosphorylation in vivo. This protein kinase was similar to that activated in the same cells by replenishing the nutrient medium, and in other quiescent cells by the action of growth factors and mitogens. It was distinct from the previously described pseudorabies virus protein kinase, which is unique to infected cells. When medium from cells infected with pseudorabies virus was freed of virus and added to confluent fibroblasts, rapid activation of the ribosomal protein S6 kinase activity occurred. A similar, although more limited, effect could be seen when the pH of the medium was increased. These results suggest that the phosphorylation of ribosomal protein S6 in cells infected with herpes virus is a consequence of the production of a factor which initiates the metabolic programme for cellular growth. The possible function of this effect in the infective strategy of herpes viruses is discussed in relation to requirements for the replication of viral DNA.     

Antimutagenicity of cinnamaldehyde and vanillin in human cells: Global gene expression and possible role of DNA damage and repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary flavorings that exhibit antimutagenic activity against mutagen-induced and spontaneous mutations in bacteria. Although these compounds were antimutagenic against chromosomal mutations in mammalian cells, they have not been studied for antimutagenesis against spontaneous gene mutations in mammalian cells. Thus, we initiated studies with VAN and CIN in human mismatch repair-deficient (hMLH1(-)) HCT116 colon cancer cells, which exhibit high spontaneous mutation rates (mutations/cell/generation) at the HPRT locus, permitting analysis of antimutagenic effects of agents against spontaneous mutation. Long-term (1-3 weeks) treatment of HCT116 cells with VAN at minimally toxic concentrations (0.5-2.5mM) reduced the spontaneous HPRT mutant fraction (MF, mutants/10(6) survivors) in a concentration-related manner by 19-73%. A similar treatment with CIN at 2.5-7.5microM yielded a 13-56% reduction of the spontaneous MF. Short-term (4-h) treatments also reduced the spontaneous MF by 64% (VAN) and 31% (CIN). To investigate the mechanisms of antimutagenesis, we evaluated the ability of VAN and CIN to induce DNA damage (comet assay) and to alter global gene expression (Affymetrix GeneChip) after 4-h treatments. Both VAN and CIN induced DNA damage in both mismatch repair-proficient (HCT116+chr3) and deficient (HCT116) cells at concentrations that were antimutagenic in HCT116 cells. There were 64 genes whose expression was changed similarly by both VAN and CIN; these included genes related to DNA damage, stress responses, oxidative damage, apoptosis, and cell growth. RT-PCR results paralleled the Affymetrix results for four selected genes (HMOX1, DDIT4, GCLM, and CLK4). Our results show for the first time that VAN and CIN are antimutagenic against spontaneous mutations in mammalian (human) cells. These and other data lead us to propose that VAN and CIN may induce DNA damage that elicits recombinational DNA repair, which reduces spontaneous mutations.     

Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production

The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.     

肉桂醛抗人宫颈癌相关机制的研究

目的探讨不同浓度的肉桂醛对HeLa细胞P21、CDK4蛋白表达的影响及意义。方法不同浓度的经过纯度鉴定的肉桂醛处理体外培养的HeLa细胞,培养24 h后免疫组织化学和Western blotting法检测HeLa细胞P21、CDK4蛋白表达的变化。结果肉桂醛纯度〉96.24%;肉桂醛能显著增高P21和降低CDK4蛋白在HeLa细胞中的表达,各浓度肉桂醛处理组的P21、CDK4蛋白表达与溶剂对照组相比差异均有统计学意义（P〈0.01,P〈0.05）。结论肉桂醛能上调宫颈癌HeLa细胞P21蛋白表达和下调CDK4蛋白表达,可能是促进HeLa细胞凋亡的机制之一。     

Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli

Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE-. Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.     

Changes in inducibility of ornithine decarboxylase activity in differentiating human neuroblastoma cells

Human SH-SY5Y neuroblastoma cells could be induced to differentiate morphologically and biochemically in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA), retinoic acid (RA), or a combination of these two substances. The phenotypical changes induced by these substances differed, but one effect of both was an inhibition of the cell growth. Addition of TPA or RA to non-treated cells had no effect on the activation of ornithine decarboxylase (ODC, EC 4.1.1.17.), while a change to fresh medium stimulated the ODC to maximum activity after 4-6 h. The activity was not altered by the presence of RA in the fresh medium, but TPA partially inhibited the medium-stimulated ODC activity. Cells treated for 4 or 8 days with TPA or a combination of TPA and RA had a low ODC activity which could not be induced by fresh medium. However, RA-treated (and thus growth-inhibited) cells still responded to a change of medium by exhibiting an ODC activity of the same magnitude and duration as in medium-stimulated control cells. The results seem to suggest that the growth inhibition induced by TPA and RA, respectively, is mediated by different mechanisms.     

Immune Suppressive Effect of Cinnamaldehyde Due to Inhibition of Proliferation and Induction of Apoptosis in Immune Cells: Implications in Cancer

Background

Besides its anti-inflammatory effects, cinnamaldehyde has been reported to have anti-carcinogenic activity. Here, we investigated its impact on immune cells.

Methods

Activation of nuclear factor-κB by cinnamaldehyde (0–10 µg/ml) alone or in combination with lipopolysaccharide was assessed in THP1XBlue human monocytic cell line and in human peripheral blood mononuclear cells (PBMCs). Proliferation and secretion of cytokines (IL10 and TNFα) was determined in primary immune cells and the human cell lines (THP1, Jurkat E6-1 and Raji cell lines) stimulated with cinnamaldehyde alone or in conjunction with lipopolysaccharide. Nitric oxide was determined in mouse RAW264.7 cells. Moreover, different treated PBMCs were stained for CD3, CD20 and AnnexinV.

Results

Low concentrations (up to 1 µg/ml) of cinnamaldehyde resulted in a slight increase in nuclar factor-kB activation, whereas higher concentrations led to a dose-dependent decrease of nuclear factor-kB activation (up to 50%) in lipopolysachharide-stimulated THP1 cells and PBMCs. Accordingly, nitric oxide, interleukin 10 secretion as well as cell proliferation were reduced in lipopolysachharide-stimulated RAW264.7 cells, PBMCs and THP1, Raji and Jurkat-E6 immune cells in the presence of cinnamaldehyde in a concentration-dependent manner. Flow cytometric analysis of PBMCs revealed that CD3+ were more affected than CD20+ cells to apopotosis by cinnamaldehyde.

Conclusion

We attribute the anti-inflammatory properties of cinnamaldehyde to its ability to block nuclear factor-κB activation in immune cells. Treatment with cinnamaldehyde led to inhibition of cell viability, proliferation and induced apoptosis in a dose-dependent manner in primary and immortalized immune cells. Therefore, despite its described anti-carcinogenic property, treatment with cinnamaldehyde in cancer patients might be contraindicated due to its ability to inhibit immune cell activation.     

In vivo studies on the interaction of RecBCD enzyme and lambda Gam protein.

The interaction between the RecBCD enzyme of Escherichia coli and the lambda Gam protein was investigated. Two types of experiments were done. In one type, Gam protein was produced by transient induction of the cells lysogenic for lambda cI857gam+. The presence of Gam protein, which inhibits RecBCD nuclease, enabled these cells to support the growth of a gene 2 mutant of bacteriophage T4 (T4 2). The lysogens overproducing the RecB subunit of RecBCD enzyme could titrate Gam protein and thus prevent the growth of T4 2. In contrast, the lysogens overproducing either RecC or RecD retained their capacity for growth of T4 2. It is therefore concluded that the RecB subunit is capable of binding Gam protein. In the second type of experiments, Gam protein was provided by derepressing the gamS gene on the plasmid pSF117 (S. A. Friedman and J. B. Hays, Gene 43:255-263, 1986). The presence of this protein did not interfere with the growth of wild-type cells (which were F-). Gam protein had a certain effect on recF mutants, whose doubling time became significantly longer. This suggests that the recF gene product plays an important role in maintenance of viability of the Gam-expressing cells. Gam protein exerted the most striking effect on growth of Hfr bacteria. In its presence, Hfr bacteria grew extremely slowly, but their ability to transfer DNA to recipient cells was not affected. We showed that the effect on growth of Hfr resulted from the interaction between the RecBCD-Gam complex and the integrated F plasmid.     

Effect of substance P on nerve fiber regeneration in tissue culture

Explants of the ganglion trigeminale from chick embryos (PNS) and of the hippocampus from fetal rats (CNS) were cultivated in maximow chambers with growth medium or maintanance medium. Varied concentrations of substance P (SP . 3 CH3COOH . 4 H2O) were added. 1. The effect of substance P (SP) is related to concentration. In the presence of 10(-7)M SP in the growth medium and of 10(-4)M SP in the maintanance medium the cultivation of PNS cultures indicates positive results. These doses are suitable. 2. Within the first 24 hours in vitro SP stimulates the index of area in PNS cultures. The index of characterizes the relation of the outgrowth zone to the explant. In CNS cultures a significant difference of this effect was not observed. 3. The index of growth of nerve fibers may compare the test cultures with the control cultures. SP significantly increases the index of fiber growth in PNS cultures. A stimulation of CNS cultures was observed, significance was not found. 4. From the beginning of the cultivation with SP up to 48 hours in vitro the growth of nerve fibers significantly increases in the treated cultures in comparison with the control cultures. After this time the growth of nerve fibers decreased and a morphological conformity of test cultures and controls was observed. 5. The role of SP is discussed in specific activity on PNS tissue in vitro. The reactive neurons may be from the medio dorsal group of cells of the sensible ganglion.