Self-Incompatibility Involved in the Level of Acetylcholine and cAMP

Elongation of pollen tubes in pistils after self-pollination of Lilium longiflorum cv. Hinomoto exhibiting strong gametophytic self-incompatibility was promoted by cAMP and also promoted by some metabolic modulators, namely, activators (forskolin and cholera toxin) of adenylate cyclase and inhibitors (3-isobutyl-1-methylxanthine and pertussis) of cyclic nucleotide phosphodiesterase. Moreover, the elongation was promoted by acetylcholine (ACh) and other choline derivatives, such as acetylthiocholine, L-α-phosphatidylcholine and chlorocholinechloride [CCC; (2-chloroethyl) trimethyl ammonium chloride]. A potent inhibitor (neostigmine) of acetylcholinesterase (AChE) as well as acetylcholine also promoted the elongation. cAMP enhanced choline acetyltransferase (ChAT) activity and suppressed AChE activity in the pistils, suggesting that the results are closely correlated with self-incompatibility in L. longiflorum. In short, it came to light that cAMP modulates ChAT (acetylcholine-forming enzyme) and AChE (acetylchoine-decomposing enzyme) activities to enhance the level of ACh in the pistils of L. logiflorum after self-incompatible pollination. These results indicate that the self-incompatibility on self-pollination is caused by low levels of ACh and/or cAMP.Key Words: pollen tubes, self-incompatibility, Lilium longiflorum, cAMP, acetylcholie, AChE, ChATCyclic AMP (cAMP) is an essential signaling molecule in both prokaryotes and eukaryotes.¹ The existence of cAMP in higher plants was questioned by some reviewers^2–4 in the mid 1970''s, so that many workers were discouraged from studying roles in plant biology. However, its presence was confirmed by mass spectrometry⁵ and infrared spectrometry⁶ in the early 1980''s and increasing evidence^7–12 now suggests that cAMP makes important contributions in plant cells, as in animals.Lily (Lilium longiflorum) exhibits strong gametophytic self-incompatibility.^13,14 Thus, elongation of pollen tubes in the pistil after self-incompatible pollination in L. longiflorum cv. Hinomoto stops halfway, in contrast to the case after cross-compatible pollination (cross with cv. Georgia).¹⁴ This self-incompatibility appears to be associated with the stress and self-incompatible pollination on stigmas of lilies results in activation and/or induction of enzymes such as NADH- and NADPH-dependent oxidases, xanthine oxidase, superoxide dismutase (SOD), catalase and ascorbate peroxidase in the pistils.¹⁵ The activities of NADH- and NADPH-dependent oxidases (O₂⁻-forming enzymes), however, are known to be suppressed by cAMP¹⁶ and increase in the level of cAMP in guinea pig neutrophils results in their decreased expression.¹⁷ The level of O₂⁻ reactions with SOD is also decreased by cAMP.¹⁸ In the case of the lily, inhibition of NADH- and NADPH-dependent oxidases by cAMP was found to be noncompetitive with NAD(P)H.¹⁶ We hypothesized that decrease in active oxygen species such as O₂⁻ and suppression of stress enzyme activities in self-pollinated pistils of lily by cAMP might cause elongation of pollen tubes after self-pollination and this proved to be the case. Namely, elongation of pollen tubes after self-incompatible pollination in lily was promoted by exogenous cAMP at a concentration as low as 10 nM, a conceivable physiological level.¹³ Moreover, similar elongation could be achieved with adenylate cyclase activators [forskolin(FK) and cholera toxin] and cAMP phosphodiesterase inhibitors [3-isobutyl-1-methylxanthine (IBMX) and pertussis toxin].^14,19 These phenomena led us to examine the involvement of endogenous cAMP in pistils after self-incompatible or cross-compatible pollination. As expected, the level of endogenous cAMP in pistils after self-pollination was approximately one half of that after cross-pollination. Furthermore, this was associated with a concomitant decrease in adenylate cyclase and increase in cAMP phosphodiesterase.¹⁹Many researchers in the field of plant biology have been unsuccessful in attempts to estimate the quantity of cAMP and to detect activities of adenylate cyclase and cAMP phosphodiesterase. On major difficulty is the presence of proteases and we have overcome this problem by using protease inhibitors, such as aprotinin and leupeptin.¹⁹In 1947, acetylcholine (ACh) of higher plants was first reported in a nettle (Urtica urens) found in the Himalaya mountain range.²⁰ In 1983, its existence in plants was confirmed by mass spectrometry of preparations from Vigna seedlings.²¹ In our preliminary studies, CCC (chlorocholinechloride), a plant growth retardant (specifically an anti-gibberellin), enhanced the elongation of the pollen tubes in pistils after self-incompatible pollination in lilies. This led us to investigate whether other choline derivatives cause similar effects and positive findings were obtained with ACh, acetylthiocholine and L-α-phosphatidlylcholine.²² Moreover, the elongation was also promoted by neostigmine, an inhibitor of acetylcholine esterase (AChE) activity. In line with these results, choline acetyltransferase (ChAT) demonstrated low and AChE high activity in pistils after self-incompatible pollination.The positive influence of cAMP^14,19 and ACh²² in pistils of L. longiflorum after self-incompatible pollination encouraged us to examine the involvement of these two molecules in regulation of pollen tube elongation of lily after self-incompatible and cross-compatible pollination. As a result, it was revealed that cAMP promotes ChAT and suppresses AChE activity in pistils after both self- and cross-pollination. In other words, the self-incompatibilty in pistils of L. longiflorum appears to be due to levels of ACh and/or cAMP below certain threshold values.Hitherto, these substances have not been recognized to play important roles in the metabolic systems of higher plants. However, given their conservation through evolution, it is natural that such central metabolic substances make essential contributions, regardless of the organism. We have succeeded in establishing physiological functions of cAMP and ACh in pistils of lily^14,19,22 and this points to use of plant reproductive organs such as research materials. The exact responsibilities of the two molecules may depend on differences in tissues or organs of plants and further molecular biological studies in this area are clearly warranted. This issue is currently being investigated.     

A novel in vitro co-culture model to examine contact formation between astrocytic processes and cerebral vessels

Here, we developed a novel in vitro co-culture model, in which process-bearing astrocytes and isolated cerebral microvessels from mice were co-cultured. Astrocytes formed contacts with microvessels from both adult and neonatal mice. However, concentrated localization of the immunofluorescence signal for aquaporin-4 (AQP4) at contact sites between perivascular endfoot processes and blood vessels was only detected with neonatal mouse microvessels. Contact between astrocytic processes and microvessels was retained, whereas concentrated localization of AQP4 signal at contact sites was lost, by knockdown of dystroglycan or α-syntrophin, reflecting polarized localization of AQP4 at perivascular regions in the brain. Further, using our in vitro co-culture model, we found that astrocytes predominantly extend processes to pericytes located at the abluminal surface of microvessels, providing additional evidence that this model is representative of the in vivo situation. Altogether, we have developed a novel in vitro co-culture model that can reproduce aspects of the in vivo situation and is useful for assessing contact formation between astrocytes and blood vessels.     

Effect of gap junction-mediated intercellular communication on TGF-β induced epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is the process in which epithelial cells lose cell polarity and cell adhesion with surrounding cells to obtain migratory and invasive abilities. On the other hand, the expression of connexin is decreased or lacked in the many types of tumor cells. This study examined the effect of gap junctional intercellular communication (GJIC) on EMT induced by the transforming growth factor-β1 (TGF-β1). To investigate the effect of GJIC on EMT in U2OS cells, smooth muscle 22-α (sm22α) promoter-driven luciferase reporter gene was introduced into Cx43-expressing cells (U2OS-Luc Cx43) and into the control parental cell line (U2OS-Luc). TGF-β1 induced the expression of EMT markers and the sm22α promoter activity of U2OS-Luc cells. Sm22α promoter activity of U2OS cells was neither dependent on the expression of Cx43 nor on the establishment of GJIC among U2OS cells. Furthermore, we found that the homocellular communication among tumor cells did not affected the tumor cell growth and migration. However, we revealed that tumor cell density was an important factor for tumor cells to acquire metastatic phenotype. Interestingly, the co-culture of U2OS cells with osteoblasts revealed that sm22α promoter activity was inhibited only by the GJIC established between these two cell types. These results suggest that normal osteoblast cells negatively regulate the EMT of tumor cells, at least in part. Thus, Cx43-mediated GJIC may have anti-metastatic activity in tumor cells. Our findings provide a new insight into the role of GJIC in cancer progression and metastasis and identify potential therapeutic targets for the treatment of cancer.     

A factor of inducing IgE from a filarial parasite is an agonist of human CD40

Immune responses to parasitic helminth are usually characterized by quite mysterious phenomena: dominance of Th2-like immunity and antigen-nonspecific IgE secretion. We previously purified a factor from Dirofilaria immitis that induces antigen-nonspecific IgE in rats and named it DiAg. In the presence of IL-4, DiAg induces mouse B cells to secrete IgE, which is antigen-nonspecific polyclonal antibody. We investigated the biochemical characteristics of DiAg as a factor of inducing IgE in this study. Recombinant DiAg (rDiAg) with interleukin (IL)-4 induced IgE synthesis in highly purified human normal B cells in vitro cell culture systems. The addition of recombinant human soluble CD40 IgG fusion protein (rsCD40-Ig) inhibited induction of IgE synthesis by rDiAg with IL-4. Monocyte cells were stimulated with rDiAg and recombinant human soluble CD40L (rsCD40L); IL-12 and TNF-alpha were induced. The addition of rsCD40-Ig to THP-1 cells activated with rDiAg and rsCD40L inhibited the production of IL-12. rDiAg bound to the monocyte cell membrane fraction and recombinant human soluble CD40; this binding of rDiAg was competitively inhibited by addition of rsCD40L. Moreover, in CD40-deficient mice, IgE production and MLN-B cell proliferation by rDiAg were completely absent. Based on these results, we concluded that DiAg is an agonist of CD40.     

Isolation of a new potent cytotoxic pigment along with indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune

An indole derivative, schizocommunin, was isolated along with indigotin (indigo), indirubin, isatin, and tryptanthrin, from the liquid culture medium in which a culture of Schizophyllum commune, isolated from the bronchus of a human patient with allergic bronchopulmonary mycosis, had been grown. The structure of schizocommunin was established by spectroscopic investigation. Schizocommunin showed the strong cytotoxicity against murine lymphoma cells. The assignments of the ¹H- and ¹³C-NMR signals of indigotin were also listed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Field survey of sex-reversals in the medaka, Oryzias latipes: genotypic sexing of wild populations

The medaka, Oryzias latipes, has an XX/XY sex determination mechanism. A Y-linked DM domain gene, DMY, has been isolated by positional cloning as a prime candidate for the sex-determining gene. Furthermore, the crucial role of DMY during male development was established by studying two wild-derived XY female mutants. In this study, to find new DMY and sex-determination related gene mutations, we conducted a broad survey of the genotypic sex (DMY-negative or DMY-positive) of wild fish. We examined 2274 wild-caught fish from 40 localities throughout Japan, and 730 fish from 69 wild stocks from Japan, Korea, China, and Taiwan. The phenotypic sex type agreed with the genotypic sex of most fish, while 26 DMY-positive (XY) females and 15 DMY-negative (XX) males were found from 13 and 8 localities, respectively. Sixteen XY sex-reversals from 11 localities were mated with XY males of inbred strains, and the genotypic and phenotypic sexes of the F(1) progeny were analyzed. All these XY sex-reversals produced XY females in the F(1) generation, and all F(1) XY females had the maternal Y chromosome. These results show that DMY is a common sex-determining gene in wild populations of O. latipes and that all XY sex-reversals investigated had a DMY or DMY-linked gene mutation.     

The solubilization and biological activities of Aspergillus beta-(1 --> 3)-D-glucan

We have recently demonstrated that the cell wall beta-glucan of Candida albicans could be solubilized by sodium hypochlorite, followed by dimethylsulfoxide-extraction (NaClO-DMSO method). In this study, applying this method to Aspergillus spp., we prepared mycelial cell wall beta-glucan and examined its physical properties and immunotoxicological activity. The acetone-dried mycelia of Aspergillus spp. were oxidized by the NaClO-DMSO method. An analysis of (13)C NMR spectra revealed the preparations to be composed of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan. Also, the proportion of alpha-(1 --> 3) and beta-(1 --> 3)-D-glucan varied. Furthermore, a solubilized Aspergillus beta-glucan (ASBG) was prepared from OX-Asp by urea-autoclave treatment. ASBG showed limulus activity similar to Candida solubilized beta-glucan (CSBG), and there was little difference in the activity of ASBG between various Aspergillus spp. ASBG affected the production of IL-8 by human peripheral blood mononuclear cells (PBMC). ASBG should be useful for analyzing the clinical role of beta-glucan.     

Inhibition of sphingomyelinase activity helps to prevent neuron death caused by ischemic stress

Magnesium-dependent neutral sphingomyelinase (N-SMase) present in plasma membranes is an enzyme that can be activated by stress in the form of inflammatory cytokines, serum deprivation, and hypoxia. The design of small molecule N-SMase inhibitors may offer new therapies for the treatment of inflammation, ischemic injury, and cerebral infarction. Recently, we synthesized a series of difluoromethylene analogues (SMAs) of sphingomyelin. We report here the effects of SMAs on the serum/glucose deprivation-induced death of neuronally differentiated pheochromocytoma (PC-12) cells and on cerebral infarction in mice. SMAs inhibited the enhanced N-SMase activity in the serum/glucose-deprived PC-12 cells, and thereby suppressed the apoptotic sequence: ceramide formation, c-Jun N-terminal kinase phosphorylation, caspase-3 activation, and DNA fragmentation in the nuclei. Administration of SMA-7 (10 mg/kg i.v.) with IC50= 3.3 microM to mice whose middle cerebral arteries were occluded reduced significantly the size of the cerebral infarcts, compared to the control mice. These results suggest that N-SMase is a key component of the signaling pathways in cytokine- and other stress-induced cellular responses, and that inhibiting or stopping N-SMase activity is an important strategy to prevent neuron death from ischemia.