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Dua senyawa masing-masing turunan santon lakton, yaitu artonol B (1) dan turunan furanodihidrobenzosanton, yakni Sikloartobilosanton (2), telah ditemukan pada tumbuhan Artocarpus teysmanii Miq. (Moraceae), suatu tumbuhan langka yang endemik untuk Indonesia dan dikenal dengan nama Tipulu. Struktur molekul kedua senyawa tersebut telah ditetapkan berdasarkan data fisika dan spektroskopi (MS, 1H dan 13C NMR). Artonol B (1) dan sikloartobilosanton (2) memperlihatkan toksisitas yang tinggi terhadap udang Artemia salina, dan senyawa 2 juga memperlihatkan aktivitas antimitotik yang rendah terhadap target cdc2 kinase dan cdc25 fosfatase. Artonol B and Cycloartobiloxanthone from Artocarpus teysmanii MIQ Two compounds, a xanthone lactone derivative, namely artonol B (1) and a furanodihydrobenzoxanthone derivative, namely cycloartobiloxanthone (2) had been isolated from Artocarpus teysmanii Miq. (Moraceae), locally known as Tipulu. The structures of both compounds were elucidated based on physical and spectroscopic data (MS, 1H and 13C NMR). Both artonol B (1) and cycloartobiloxanthone (2) showed strong toxicity against Artemia salina in the shrimp bioassay, and compound 2 also showed weak antimitotic activity against cdc2 kinase and cdc25 phosphatase as the targets. Bagian ke-8 dari seri Ilmu Kimia Tumbuhan Moraceae Indonesia, untuk bagian ke-6 lihat pustaka [10].

Highlights: [•] In vitro oocyte maturation (OM) in Tenualosa ilisha was induced by 17,20β-P. [•] 17,20β-P induction of OM was dose- and time-dependent. [•] PI3 kinase shown to be the initial signal molecule for 17,20β-P induced OM. [•] MAP kinase although was activated during 17,20β-P induced OM. [•] MAP kinase activation is not involved in induction of OM. [ABSTRACT FROM AUTHOR]

Abstract: Cdc2 kinase is a catalytic subunit of the maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocytes. MPF has been studied in a wide variety of animal species; however, its expression in crustaceans is poorly characterized. In this study, a complete cDNA sequence of Cdc2 kinase was cloned from the red claw crayfish, Cherax quadricarinatus, and its spatiotemporal expression profiles were analyzed. The Cdc2 cDNA (1769bp) encodes for a 299 amino acid protein with a calculated molecular weight of 34.7kDa. Quantitative real-time PCR demonstrated that Cdc2 mRNA was expressed mainly in the ovary tissue and the expression decreased as the ovaries developed. Immunohistochemistry analysis revealed that the Cdc2 protein relocated from the cytoplasm to the nucleus during oogenesis. These findings suggest that Cdc2 kinase may play an important role in the gametogenesis and gonad development in C. quadricarinatus. [Copyright &y& Elsevier]

Abstract: Previously, we observed that in vitro germinal vesicle breakdown (GVBD) in Cyprinus carpio oocytes was induced by recombinant human insulin-like growth factor-I (IGF-I) and bovine insulin (b-insulin) and this induction was steroid-independent. To investigate further the early signal transduction components involved in this process, the possible role of phosphatidylinositol 3-kinase (PI3 kinase) during oocyte maturation was examined. IGF-I- and b-insulin-induced oocyte maturation was significantly inhibited by Wortmannin and LY294002, two mechanistically different specific inhibitors of PI3 kinase. IGF-I and b-insulin were shown to activate PI3 kinase after 90min of their treatment. Both IGF-I and b-insulin were found to activate cdc2 kinase at 21h of treatment. We examined the relative involvement of PI3 kinase, MAP kinase and cdc2 kinase in IGF-I- and b-insulin-induced oocyte maturation in C. carpio. MAP kinase was rapidly phosphorylated and activated (30–150min) in response to exposure of the oocytes with IGF-I and b-insulin. This response preceded the phosphorylation and activation of cdc2 by several hours (almost 19h). A potent and selective inhibitor of MEK, PD98059, the protein kinase that phosphorylates and activate MAP kinase, blocked the phosphorylation and activation of MAP kinase and cdc2 kinase and GVBD induction. Likewise, PI3 kinase inhibitors strongly inhibited phosphorylation and activation of MAP kinase, which was increased during oocyte maturation. Taken together, these results suggest that PI3 kinase is an initial component of the signal transduction pathway which precedes MAP kinase, and MPF activation during IGF-I- and b-insulin-induced oocyte maturation in C. carpio. [Copyright &y& Elsevier]

Abstract: Cdc2 kinase is a catalytic subunit of maturation-promoting factor (MPF), a central factor for inducing the meiotic maturation of oocyte. To understand the role of Cdc2 kinase on the oocyte maturation in crustacean, a complete cDNA sequence of Cdc2 kinase was cloned from Chinese mitten crab Eriocheir sinensis and its spatial-temporal expression profiles were analyzed during oogenesis at RNA and protein levels. The crab Cdc2 cDNA (1364 bp) encodes for a 299 amino acids protein with calculated molecular weight of 34.7 kDa. The Cdc2 mRNAs level showed no significant change in the ovary during oogenesis, whereas higher protein level was found at previtellogenesis, late vitellogenesis and germinal vesicle breakdown (GVBD) stages. Two forms (35 kDa and 34 kDa) of Cdc2 proteins were simultaneously identified in ovary at all stages. Immunocytochemistry analysis revealed that Cdc2 proteins locate exclusively in ooplasm of previtellogenic oocyte, and then relocate into germinal vesicle at vitellogenesis stage and accumulate on meiotic spindle at oocyte maturation. These findings suggest that Cdc2 kinase has essential roles in inducing GVBD and generating meiotic apparatus during the crab oocyte maturation. [Copyright &y& Elsevier]

Abstract: In the present study, we investigated the effects of specific cdc2 kinase inhibitor, butyrolactone I (BL I) on the prevention of germinal vesicle breakdown, changes of microtubular structures, and development of porcine oocytes after removal of the drug. In Experiment 1, cumulus–oocyte complexes (COCs) were cultured (44h) in NCSU-23 medium containing different concentrations of BL I. The percentages of oocytes remaining at GV stage were 0, 0, 32, 80, and 84% (P <0.05), and the maturation rates were 86, 63, 30, 0, and 0% (P <0.05) for oocytes treated with 0, 10, 20, 40, and 80μM of BL I, respectively. When oocytes were released from BL I incubation (Experiment 2) and cultured for an additional 44h, 79, 84, and 83% of oocytes resumed meiosis, but only 52, 38 and 17% of oocytes reached normal metaphase II (MII) stage in the groups treated with 20, 40 and 80μM BL I, respectively. In Experiments 3–5, reversibility and development of oocytes and embryos were evaluated after removal of the inhibitor. A reduced duration of BL I incubation (22h) at 20μM increased the percentage of oocytes remaining at the GV stage compared to the control group (85% versus 9%, P <0.05). Blastocyst rates were lower in treatment groups than in the control (44h) group (0–14% versus 24%; P <0.05). However, all developing blastocysts possessed similar cell numbers, regardless of the drug-treated or non-treated controls. Taken together, treatment with 20–80μM of BL I effectively prevented the resumption of meiosis and polymerization of periooplasmic microtubules. Furthermore, reversibility of the oocytes after reduced duration of BL I treatment was satisfactory. [Copyright &y& Elsevier]

Abstract: Oocytes undergo spontaneous germinal vesicle breakdown (GVBD) after being released from the follicular environment; this potentially prevents manipulation of the oocyte at the germinal vesicle (GV) stage. The objectives of this study were to investigate the effects of indirubin, a potent cdc2 kinase inhibitor, on GVBD and microtubular structure of porcine oocytes. Cumulus-oocyte-complexes (COCs) were collected from abattoir-derived ovaries and were randomly allocated to different concentrations of indirubin treatments (0, 10, 25, 50, and 100μM in Experiment 1 and 0, 50, 75, and 100μM in Experiment 2) during 44h of IVM. The influences on the GVBD, microtubules, and maturation rates were evaluated using epifluorescence microscopy. The percentages of oocytes remaining at the GV stage were 0, 16, 26, 69, and 85% for oocytes treated with 0, 10, 25, 50, and 100μM of indirubin, respectively, which differed among treatment groups (P <0.05). However, there were no significant differences between the oocytes treated with 75 and 100μM (79 and 81%). The cytoplasmic microtubules were fragmented in oocytes maintained at the GV stage and the chromatin became condensed or aggregated. When COCs were incubated with indirubin (50–75μM) for 22h and then transferred to maturation medium for 44h (Experiments 3–5), the percentages of oocytes reaching the metaphase II stage were generally higher than when the COCs were cultured in the presence of the drug for 44h (62–65% versus 44–46%). However, the parthenogenetic development of the oocytes in Experiment 6 was reduced significantly in drug-treated oocytes. In summary, treatment with 50–75μM of indirubin effectively prevented GVBD in porcine oocytes, but the developmental competence of the oocytes was compromised. [Copyright &y& Elsevier]

Recent data on the association between growth hormone (GH) and male and female reproductive processes, as well as the effects of GH on these processes and on some reproductive and non-reproductive disorders, and possible extra- and intracellular mediators of its action are reviewed. The available data suggest that GH is an important endocrine and autocrine/paracrine regulator of reproduction. It controls proliferation, apoptosis, growth and differentiation and the secretory and generative activities of different reproductive organs. It also regulates their response to gonadotrophin-releasing hormone (GnRH) and gonadotropins. Despite the effects of GH on the IGF/IGFBP (insulin-like growth factor binding protein) system, oxytocin, steroids, activin, gonadotropin and gonadotropin receptors, the majority of GH's actions on the reproductive processes are probably mediated not by these substances but by specific GH receptors acting through cAMP/protein kinase A, protein kinase G, tyrosine kinase-, MAP kinase and CDC2 kinase-dependent intracellular mechanisms. Although GH treatments can increase the risk of some reproductive and non-reproductive disorders, they may be useful in improving gonadal function, inducing superovulation and in embryo production. [ABSTRACT FROM AUTHOR]

Genistein, a natural isoflavone phytoestrogen present in soybeans, has been extensively studied as a chemopreventive or therapeutic agent in several types of cancer. The traditional Asian diet is rich in soy products may explain in part why the incidence of breast cancer in Asian women is relatively low. To improve therapeutic benefits, we investigated the combination of genistein with chemotherapeutic agents in phenotypically dissimilar human breast cancer cells, MCF-7 and MDA-MB-231, in which estrogen receptor expression is positive and negative, respectively. In the present study, genistein significantly decreased cell apoptosis induced by tubulin-binding agents, paclitaxel and vincristine. FACScan analysis revealed that genistein also diminished the accumulation of the G2/M phase in the cell cycle caused by tubulin-binding agents. In situ staining of microtubules revealed that genistein could decrease paclitaxel-induced tubulin polymerization. However, in vivo tubulin polymerization assay revealed that simultaneous treatment of genistein did not change the tubulin/microtubule dynamic. Genistein reduced Bcl-2 phosphorylation triggered by paclitaxel and vincristine without changing Bax protein expression. p53 and p21 expression, monitored by Western blotting, was not altered by genistein. However, the expression of cyclin B1 and CDC2 kinase was markedly decreased in combination with genistein. In conclusion, genistein inversely affected tubulin-binding agent-induced apoptosis via down-regulation of cyclin B1/CDC2 kinase expression resulting in reduced Bcl-2 phosphorylation. [Copyright &y& Elsevier]

In the ovary, mammalian oocytes resume meiosis and mature to the second metaphase when they are stimulated with gonadotrophins. Similarly, oocytes can mature in vitro when they are liberated from ovarian follicles and cultured under appropriate conditions. Early in the process of maturation, oocytes undergo dramatic but well-ordered changes at the G2/M transition in the cell cycle including: (i) chromosome condensation; (ii) nucleolus disassembly; (iii) germinal vesicle breakdown (GVBD); and (iv) spindle formation in the first metaphase (MI-spindle). These events have been thought to be induced by MPF (maturation-promoting factor or M-phase promoting factor), now known as Cdc2 kinase or Cdk1 kinase, which consists of a catalytic subunit, Cdc2, and a cyclin B regulatory subunit. In fact, nuclear lamins are phosphorylated by Cdc2 kinase, and nuclear membrane breakdown occurs concomitantly with the activation of Cdc2 kinase in the M-phase of both somatic cells and oocytes. Based on the classical and recent studies of the pig oocyte, however, the chromosomes start to condense and the nucleolus disassembles before full activation of Cdc2 kinase, and the MI-spindle is formed after activation of both Cdc2 kinase and MAP kinase; another kinase known to become activated during oocyte maturation. These findings suggest that chromosome condensation and nucleolus disassembly in oocytes are induced by either some kinase(s) other than Cdc2 kinase and MAP kinase or some phosphatase(s). The accumulation of new results regarding the molecular nature of oocyte maturation is important for improving the reproductive technologies in domestic animals as well as in humans. (Reprod Med Biol 2003; 2: 91–99) [ABSTRACT FROM AUTHOR]

Cytoplasmic dynein, a large minus-end-directed microtubule motor, performs multiple functions during the cell cycle. In interphase, dynein moves membrane organelles, while in mitosis it moves chromosomes and helps to form the mitotic spindle. The cell-cycle regulation of dynein activity may be controlled, at least in part, by the phosphorylation of its light intermediate chains (DLIC), since a 10-fold increase in light intermediate chain phosphorylation correlates with a decrease in dynein-based membrane transport of similar magnitude in mitosis. In this study, we sought to identify the kinase responsible for this potentially important phosphorylation event. We show that bacterially-expressed chicken light intermediate chain (chDLIC) will undergo mitosis-specific phosphorylation when added to Xenopus egg extracts. Mutation of a conserved cdc2 kinase consensus site (Ser197) abolishes this phosphorylation event, and mass spectroscopy analysis confirms that the wild-type DLIC is stoichiometrically phosphorylated at this site when incubated with metaphase but not interphase extracts. We also show that purified cdc2 kinase phosphorylates purified DLICs at Ser197 in vitro and that Ser197 phosphorylation is dramatically reduced in metaphase extracts depleted of cdc2 kinase. These results indicate that cdc2 kinase directly phosphorylates dynein and thus may be an important regulator of dynein activity in the cell cycle. [ABSTRACT FROM AUTHOR]

The cell division cycle in several pelagic dinoflagellate species has been shown to be phased with the diurnal cycle, suggesting that their cell cycle may be regulated by a circadian clock. In this study, we examined the cell cycle of an epibenthic dinoflagellate. Gambierdiscus toxicus Adachi and Fukuyo (Dinophyceae), and found that cell division was similarly phased to the diurnal cycle. Cell division occurred during a 3-h window beginning 6 h after the onset of the dark phase. Cell cycle progression in higher eukaryotes is regulated by a cell cycle regulatory protein complex consisting of cycline and the cyclin-dependent kinase CDC2. In this report, we identified a CDC2-like kinase in G. toxicus that displays activity in vitro against a known substrate of CDC2 kinase, histone H1. As in higher eukaryotes, CDC2 kinase was expressed constitutively in G. toxicus throughout the cell cycle, but it was activated only late in the dark phase, concurrent with the presence of mitotic cells. These results indicate that cell division in G. toxicus is regulated by molecular controls similar to those found in higher eukaryotes. [ABSTRACT FROM AUTHOR]

Recombinant isolated β-subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (K1 = 1.7 μM, Vmax = 20 nmol · min-1 · mg -1). Two synthetic peptides reproducing the 170–215 and the 206–215 C-terminal fragments of the β-subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 μM, respectively). In contrast, both the β-subunit associated with the α-subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 β-subunit phosphorylation observed in intact cells occurs prior to β-subunit incorporation into the holoenzyme. The isolated CK2 α-subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with pcdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the β-subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to β-subunit, p34cdc2/cyclin B also protects the α-subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated α-subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated β-subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the β or the α-subunit. These data indicate that in vitro CK2 α-subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2. [ABSTRACT FROM AUTHOR]

Yeast pheromones block cell cycle progression in G1 in order to prepare mating partners for conjugation. We have investigated the mechanism underlying pheromone-induced G1 arrest in the fission yeast Schizosaccharomyces pombe. We find that the G1-specific transcription factor p65cdc10­p72res1/sct1 which controls the expression of S-phase genes is fully activated in pheromone, unlike the analogous control in budding yeast. In contrast, the G1 function of p34cdc2 acting after activation of the G1-specific transcription is blocked. Pheromone inhibits the p34cdc2 kinase associated with both the G1-specific B-type cyclin p45cig2 and the B-type cyclin p56cdc13 and overexpression of p45cig2 or p47cdc13Δ90 overcomes the pheromone-induced G1 arrest. G1 arrest is compromised in enlarged cells. We suggest that onset of S-phase is controlled by pheromone inhibiting the B-cyclin-associated kinase in G1, and that increasing cell size contributes to the mechanism for pheromone adaptation. Thus, pheromone in fission and budding yeast acts similarly in inhibiting the G1 cyclin-dependent kinase (CDK), but differs in its effects on the G1/S transcriptional control, suggesting that inhibition of CDKs may be a more general mechanism for the control of G1 progression compared with G1/S transcriptional control. [ABSTRACT FROM AUTHOR]

This chapter is written as a contribution to a volume commemorating the work of Krebs and Fischer that led to awarding of the Nobel Prize in 1992. This award was made because of their fundamental discovery in the mid-1950s that protein phosphorylation was the underlying mechanism that accounted for the reversible modification of activity of glycogen phosphorylase in mammalian skeletal muscle. Although it could not be anticipated at the time that phosphorylation would turn out to be such a ubiquitous regulator of cellular functions, it is now evident that phosphorylation controls virtually every important reaction in cells and provides the basis for understanding how integrated cellular behavior is regulated by both extracellular signals and internal control mechanisms. This chapter relates the historical development in biochemical terms of protein phosphorylation as a regulator of the cell cycle in Xenopus oocytes and eggs. [ABSTRACT FROM AUTHOR]

Recently, a cDNA was isolated from hydra with extensive homology to a mammalian and invertebrate gene which codes for a protein called laminin binding protein (LBP). In this paper we describe the protein expression of the hydra LBP in Escherichia coli. On SDS gels the recombinant hydra LBP displayed an apparent molecular mass of 43 kDa, although the calculated mass, including six additional histidines, is 33.7 kDa. Polyclonal antibodies were produced against the hydra recombinant LBP. The antiserum reacted with a 42-kDa and a 43-kDa protein from Hydra vulgaris and from a multiheaded mutant of Chlorohydra viridissima, respectively. In hydra, LBP RNA and protein were highly expressed in cells with short cell cycles, such as all cells of the interstitial cell lineage, less in slowly cycling epithelial cells, and at very reduced levels or not at all in differentiated cells. Higher expression in the multiheaded mutant of C. viridissima than in H. vulgaris, the cells of which differ in doubling time, hint at a function in cell proliferation. This is supported by the finding that in vitro hydra LBP is a substrate for the cell-cycle-specific kinase CDC2. [ABSTRACT FROM AUTHOR]

The implication of histone H1 kinase activity for the G2/M transition during the cell cycle was investigated using Dictyostelium discoideum Ax-2. Histone H1 kinase with its activity was purified from cell extracts by the use of p13suc1 affinity gel. In the vegetative cell cycle, the activity of histone H1 kinase including Cdc2 kinase was found using synchronized Ax-2 cells to be highest just before the entry into mitosis. The activity also was markedly enhanced just prior to the M phase from which developing cells (possibly prespore cells) reinitiate their cell cycle at the mound-tipped aggregate stage. These results strongly suggest the importance of Cdc2 kinase activity in the G2 to M phase transition during the cell cycle, as the case for other eukaryotic cells. [ABSTRACT FROM AUTHOR]