Modulation of monocytic differentiation of HL-60 cells by inhibitors of protein tyrosine kinases.

We showed previously that the expressions of various src family protein tyrosine kinases (PTKs) were induced independently during the monocytic differentiation of HL-60 cells. The role of PTKs was further assessed in the present study by investigating the effects of PTK inhibitors on the differentiation. It was demonstrated that PTK inhibitors such as genistein and herbimycin A modulated monocytic differentiation of HL-60 cells; they inhibited the differentiation induced by TPA, while promoting that induced by vitamin D3 (D3). Immunoblotting analysis of protein molecules which had been phosphorylated on their tyrosine residues demonstrated that TPA induced phosphorylation of certain molecules different from those induced by D3 in HL-60 cells. PTK inhibitors blocked the phosphorylation and modulated differentiation driven by the inducers. These data suggest that PTKs are involved both promotively and suppressively in signaling events that induce monocytic differentiation of HL-60 cells.     

Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein-tyrosine kinase

Engagement of the TCR (CD3-Ti) by Ag/MHC, CD3 mAb, or lectin mitogen stimulates the very early tyrosine phosphorylation of several cellular substrates including TCR-zeta. The T cell specific protein-tyrosine kinase (PTK), p56lck, has been implicated in the tyrosine phosphorylation of TCR-zeta. However, the significance of this event with regard to CD3-Ti signal transduction remains unclear. Herein, we have investigated the effect of the selective PTK inhibitor genistein (4',5,7-trihydroxyisoflavone) on cellular events associated with activation via CD3-Ti triggering. Genistein inhibited the T cell PTK, p56lck, in a dose-dependent fashion with an ID50 = 40 microM. Genistein also inhibited CD3 mAb or PHA-induced TCR-zeta chain phosphorylation in intact peripheral blood T cells. Genistein blocked the expression of IL-2 and IL-2R (CD25) in T cells stimulated with PHA/PMA or CD3 mAb/PMA, but did not inhibit the de novo expression of the CD69 early activation Ag, which is induced primarily by a PKC-dependent pathway. IL-2 and CD25 expression induced by calcium ionophore A23187 and PMA was largely refractory to inhibition by genistein, suggesting an effect of the drug on calcium-dependent pathways stimulated via CD3-Ti triggering. In this last regard, genistein partially inhibited the CD3 mAb-induced rise in [Ca2+]i but did not inhibit PHA- or CD3 mAb-induced phosphatidylinositol hydrolysis. Consequently, protein-tyrosine phosphorylation does not appear to be a prerequisite for CD3-Ti-mediated activation of phosphatidylinositol-specific phospholipase C activity and PIP2 hydrolysis. An alternative role for PTK in CD3-Ti signal transduction is suggested.     

Cytochalasin treatment disrupts the endogenous currents associated with cell polarization in fucoid zygotes: studies of the role of F-actin in embryogenesis

We determined the distribution of F-actin in fucoid (Pelvetia, Fucus) embryos with nitrobenzoxadiazole-phallacidin, and studied the effect of cytochalasin upon the endogenous currents associated with cell polarization by using the vibrating probe. F-actin is not localized at the presumptive rhizoid immediately after experimental induction of the polar axis with a light gradient; however, a preferential distribution of F-actin develops at the presumptive rhizoid by the time the position of the polar axis is fixed. F-actin continues to be localized at the tip of the rhizoid after germination, except during cytokinesis, when the furrow is the only brightly staining region of the embryo. Incubation with cytochalasin can result in either an enhanced or a diminished pool of F-actin in the embryonic cortex (see Results). Cytochalasin D (100 micrograms/ml) significantly reduces the inward current at the rhizoid pole (n = 11) after a 2.5-h incubation. This drop is concentration dependent and occurs within approximately 30 min at 100 micrograms/ml and approximately 60 min at 10 micrograms/ml. Cytochalasin treatment eliminates the pulsatile component of the current. Preliminary results suggest that 100 micrograms/ml cytochalasin D prevents development of inward current at the presumptive rhizoid but does not completely delocalize this locus if added after photopolarization. We conclude that microfilaments are required for the establishment and maintenance of the pattern of endogenous currents observed during early embryogenesis. This suggests a new model for axis formation and fixation.     

Role of protein kinase activation in the induction of B cell adhesion by MHC class II ligands.

Engagement of MHC class II (Ia) molecules on B cells induces tyrosine phosphorylation, phosphoinositide turnover, elevation of intracellular calcium concentrations, and a rise in cAMP levels. However, a role for these biochemical signals in mediating functional responses induced by Ia ligands remains largely undefined. In this study, we utilized the induction of B cell adhesion by Ia ligands to demonstrate a role for signals transduced via Ia molecules in the generation of a functional response. Ia ligands that induced B cell aggregation induced tyrosine phosphorylation, whereas Ia ligands that did not induce B cell aggregation failed to induce any detectable tyrosine phosphorylation. Ia-induced B cell aggregation and tyrosine phosphorylation were inhibited by genistein and by herbimycin A, inhibitors of tyrosine kinases (PTK). Sphingosine and calphostin C, inhibitors of protein kinase C (PKC), also inhibited Ia-induced adhesion whereas HA1004, an inhibitor of cyclic nucleotide-dependent kinases, did not. Ia ligands induced both LFA-1-dependent and LFA-1-independent B cell adhesion. These two pathways of cell adhesion differed in their requirement for activation signals. PKC activation was sufficient for LFA-1-dependent adhesion, whereas LFA-1-independent adhesion required independent phosphorylation events mediated by PKC and by PTK. These results provide functional relevance for biochemical signals transduced via Ia molecules by demonstrating that Ia-induced B cell adhesion is mediated by the activation of PKC and by one or more PTK.     

Dual role of ATP in supporting volume-regulated chloride channels in mouse fibroblasts

The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl(-) current (I(Cl(vol))) through volume-regulated anion/chloride (VRAC) channels whilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-filling solution prevented activation of the current during osmotic cell swelling and when the volume of patched cells was increased by the application of positive pressure through the patch pipette to achieve rates exceeding 100%/min. Equimolar substitution of ATP in the pipette solution with its non-hydrolyzable analogs, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or adenylyl-(beta,gamma-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibitors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2,4-dienonitrile++ + and genistein (all at 100 microM), inhibited I(Cl(vol)) in a time-dependent manner. Tyrphostin A1, which does not inhibit PTK activity, did not affect the current amplitude. The PTK inhibitors also inhibited I(Cl(vol)) under conditions where ATP in the pipette was substituted with ATPgammaS or AMP-PCP. We conclude that in mouse fibroblasts ATP has a dual role in the regulation of the current: it is required for protein phosphorylation to keep VRAC channels operational and, through non-hydrolytic binding, determines the magnitude of I(Cl(vol)). We also suggest that tyrosine-specific protein kinases and phosphatases exhibit an interdependent involvement in the regulation of VRAC channels.     

Essential role of growth factor receptor-mediated signal transduction through the mitogen-activated protein kinase pathway in early embryogenesis of the echinoderm

In this study it was shown that growth factor receptors (GFR) play a crucial role in early embryogenesis of the echinoderms Hemicentrotus pulcherrimus and Clypeaster japonicus by transmitting signals to the mitogen-activated protein kinase (MAPK) pathway. The phosphorylation ratio of extracellular signal-regulated kinase 1 (ERK1) changed dynamically during early embryogenesis and showed a peak at the swimming blastula (sBl) stage. Suramin, an inhibitor of GFR, when applied during the sBl stage perturbed morphogenesis, including primary mesenchyme cell (PMC) migration, cell proliferation, archenteron elongation, spiculogenesis, pigment cell differentiation and phosphorylation of myosin light chains (MLC). Genistein, a receptor-type protein tyrosine kinase inhibitor, severely inhibited PMC migration, gastrulation and the phosphorylation of MLC. Manumycin A, a Ras inhibitor, inhibited spiculogenesis and invagination. PD98059, a MAPK/ERK kinase inhibitor, perturbed early PMC migration and pigment cell differentiation, but not spiculogenesis and gastrulation (although these two events were significantly delayed). PMC ingression was not perturbed by genistein, suramin, manumycin A or PD98059. All of the inhibitors perturbed the phosphorylation of ERK1, which was completely restored by exogenous platelet-derived growth factor (PDGF)-AB. PDGF-AB also partially restored elongation of the archenteron by restoring cell proliferation that had been perturbed by suramin.     

Ca2+ and Calmodulin Dynamics during Photopolarization in Fucus serratus Zygotes

The role of Ca2+ in zygote polarization in fucoid algae (Fucus, Ascophyllum, and Pelvetia species) zygote polarization is controversial. Using a local source of Fucus serratus, we established that zygotes form a polar axis relative to unilateral light (photopolarization) between 8 and 14 h after fertilization (AF), and become committed to this polarity at approximately 15 to 18 h AF. We investigated the role of Ca2+, calmodulin, and actin during photopolarization by simultaneously exposing F. serratus zygotes to polarizing light and various inhibitors. Neither removal of Ca2+ from the culture medium or high concentrations of EGTA and LaCl3 had any effect on photopolarization. Bepridil, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester, nifedipine, and verapamil, all of which block intracellular Ca2 release, reduced photopolarization from 75 to 30%. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-L-naphthalenesulfonamide and trifluoperazine inhibited photopolarization in all zygotes, whereas N-(6-aminohexyl)-L-naphthalenesulfonamide had no effect. Cytochalasin B, cytochalasin D, and latrunculin B, all of which inhibit actin polymerization, had no effect on photopolarization, but arrested polar axis fixation. The role of calmodulin during polarization was investigated further. Calmodulin mRNA from the closely related brown alga Macrocystis pyrifera was cloned and the protein was expressed in bacteria. Photopolarization was enhanced following microinjections of this recombinant calmodulin into developing zygotes. Confocal imaging of fluorescein isothiocyanate-labeled recombinant calmodulin in photopolarized zygotes showed a homogenous signal distribution at 13 h AF, which localized to the presumptive rhizoid site at 15 h AF.     

Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein

Rod photoreceptor cyclic nucleotide–gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.     

Differential induction of tumor necrosis factor alpha and manganese superoxide dismutase by endotoxin in human monocytes: role of protein tyrosine kinase, mitogen-activated protein kinase, and nuclear factor kappaB

A mutant Escherichia coli lipopolysaccharide (LPS) lacking myristoyl fatty acid markedly stimulates the activity of manganese superoxide dismutase (MnSOD) without inducing tumor necrosis factor alpha (TNFalpha) production by human monocytes (Tian et al., 1998, Am J Physiol 275:C740.), suggesting that induction of MnSOD and TNFalpha by LPS are regulated through different signal transduction pathways. The protein tyrosine kinase (PTK)/mitogen-activated protein kinase (MAPK) pathway plays an important role in the LPS-induced TNFalpha production. In the current study, we determined the effects of PTK inhibitors, genistein and herbimycin A, on the induction of MnSOD and TNFalpha in human monocytes. Genistein (10 microg/ml) and herbimycin A (1 microg/ml) markedly inhibited LPS-induced protein tyrosine phosphorylation, phosphorylation and nuclear translocation of MAPK (p42 ERK, extracellular signal-regulated kinase), and increases in the steady state level of TNFalpha mRNA as well as TNFalpha production. In contrast, at similar concentrations, genistein and herbimycin A had no effect on the LPS-induced activation of nuclear factor kappaB (NFkappaB) and induction of MnSOD (mRNA and enzyme activity) in human monocytes. In addition, inhibition of NFkappaB activation by gliotoxin and pyrrodiline dithiocarbamate, inhibited LPS induction of TNFalpha and MnSOD mRNAs. These results suggest that (1) while PTK and MAPK are essential for the production of TNFalpha, they are not necessary for the induction of MnSOD by LPS, and (2) while activation of NFkappaB alone is insufficient for the induction of TNFalpha mRNA by LPS, it is necessary for the induction of TNFalpha as well as MnSOD mRNAs.     

Localization of Actin mRNA during the Establishment of Cell Polarity and Early Cell Divisions in Fucus Embryos

Localization of mRNA is a well-described mechanism to account for the asymmetric distribution of proteins in polarized somatic cells and embryos of animals. In zygotes of the brown alga Fucus, F-actin is localized at the site of polar growth and accumulates at the cell plates of the first two divisions of the embryo. We used a nonradioactive, whole-mount in situ hybridization protocol to show the pattern of actin mRNA localization. Until the first cell division, the pattern of actin mRNA localization is identical to that of total poly(A)+ RNA, that is, a symmetrical distribution in the zygote followed by an actin-dependent accumulation at the thallus pole at the time of polar axis fixation. At the end of the first division, actin mRNA specifically is redistributed from the thallus pole to the cell plates of the first two divisions in the rhizoid. This specific pattern of localization in the zygote and embryo involves the redistribution of previously synthesized actin mRNA. The initial asymmetry of actin mRNA at the thallus pole of the zygote requires polar axis fixation and microfilaments but not microtubules, cell division, or polar growth. However, redistribution of actin mRNA from the thallus pole to the first cell plate is insensitive to cytoskeletal inhibitors but is dependent on cell plate formation. The F-actin that accumulates at the rhizoid tip is not accompanied by the localization of actin mRNA. However, maintenance of an accumulation of actin protein at the cell plates of the rhizoid could be explained, at least partially, by a mechanism involving localization of actin mRNA at these sites. The pattern and requirements for actin mRNA localization in the Fucus embryo may be relevant to polarization of the embryo and asymmetric cell divisions in higher plants as well as in other tip-growing plant cells.     

Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg

The aminoguanide, methylglyoxal bis(guanylhydrazone) (MGBG), was shown to stimulate phosphorylation of RR-SRC, a synthetic protein tyrosine kinase (PTK) substrate, and different levels of tyrosyl phosphorylation of endogenous proteins in a sea urchin egg membrane-cortex preparation. Stimulating protein tyrosine kinase activity in the sea urchin egg stimulated intracellular Ca2+ release, because microinjection of 1-5 mM of MGBG into unfertilized eggs triggered a transient rise in intracellular Ca2+ activity ([Ca2+]i) after a brief latent period. Pretreating eggs with PTK-specific inhibitors, genistein or tyrphostin B42, significantly inhibited the MGBG-induced rise in [Ca2+]i. Methylglyoxal bis(guanylhydrazone) stimulation of PTK activities in the unfertilized sea urchin egg appeared to trigger Ca2+ release through phospholipase C (PLC)-dependent inositol 1,4,5-trisphosphate (InsP3) production. The MGBG-induced Ca2+ response could be suppressed in eggs preloaded with the InsP3 receptor antagonist, heparin, and was reduced in eggs pretreated with U73122, a PLC inhibitor. However, the response was unchanged in eggs treated with nicotinamide, an inhibitor of ADP-ribosyl cyclase, or nifedipine, an inhibitor of nicotinic acid adenine dinucleotide phosphate activity. These results suggest that MGBG may be useful as a chemical agonist of PTK in sea urchin eggs and allow direct testing of the PTK requirement for the transient rise in [Ca2+]i in sea urchin eggs during fertilization. Although genistein was observed to significantly delay the onset, the sperm-induced Ca2+ response in PTK inhibitor-loaded eggs otherwise appeared normal. Therefore, it was concluded that sea urchin eggs contain a PTK-dependent pathway that can mediate intracellular Ca2+ release, but PTK activity does not appear to be required for the fertilization response.     

Inhibition of Kv4.3 by genistein via a tyrosine phosphorylation-independent mechanism

The effects of genistein, a protein tyrosine kinase (PTK) inhibitor, on voltage-dependent K(+) (Kv) 4.3 channel were examined using the whole cell patch-clamp techniques. Genistein inhibited Kv4.3 in a reversible, concentration-dependent manner with an IC(50) of 124.78 μM. Other PTK inhibitors (tyrphostin 23, tyrphostin 25, lavendustin A) had no effect on genistein-induced inhibition of Kv4.3. Orthovanadate, an inhibitor of protein phosphatases, did not reverse the inhibition of Kv4.3 by genistein. We also tested the effects of two inactive structural analogs: genistin and daidzein. Whereas Kv4.3 was unaffected by genistin, daidzein inhibited Kv4.3, albeit with a lower potency. Genistein did not affect the activation and inactivation kinetics of Kv4.3. Genistein-induced inhibition of Kv4.3 was voltage dependent with a steep increase over the channel opening voltage range. In the full-activation voltage range positive to +20 mV, no voltage-dependent inhibition was found. Genistein had no significant effect on steady-state activation, but shifted the voltage dependence of the steady-state inactivation of Kv4.3 in the hyperpolarizing direction in a concentration-dependent manner. The K(i) for the interaction between genistein and the inactivated state of Kv4.3, which was estimated from the concentration-dependent shift in the steady-state inactivation curve, was 1.17 μM. Under control conditions, closed-state inactivation was fitted to a single exponential function, and genistein accelerated closed-state inactivation. Genistein induced a weak use-dependent inhibition. These results suggest that genistein directly inhibits Kv4.3 by interacting with the closed-inactivated state of Kv4.3 channels. This effect is not mediated via inhibition of the PTK activity, because other types of PTK inhibitors could not prevent the inhibitory action of genistein.     

Tyrosine phosphorylation is required for mast cell activation by Fc epsilon RI cross-linking.

We investigated the possible role of tyrosine phosphorylation in the activation process of mast cells by cross-linking of cell-bound IgE antibodies. Bone marrow-derived mouse mast cells (BMMC) were sensitized with mouse IgE antiDNP mAb and then challenged with multivalent Ag DNP conjugates of human serum albumin. Analysis of phosphotyrosine-containing proteins in their lysates by SDS-PAGE and immunoblotting revealed that cross-linking of cell-bound IgE antibodies induced a marked increase in tyrosine phosphorylation of several proteins. To obtain direct evidence for activation of protein-tyrosine kinases (PTK), phosphotyrosine-containing proteins in lysates of mast cells were affinity purified, and kinase activity of the immunoprecipitates was assessed by an in vitro kinase assay. The results clearly showed activation of PTK upon cross-linking of Fc epsilon RI. Activation of PTK was not detected by the same assay when the sensitized BMMC were challenged with monovalent DNP-lysine. Treatment of sensitized BMMC with either Ca2+ ionophore or PMA failed to induce the activation of PTK. A representative IgE-independent secretagogue, thrombin, induced histamine release from BMMC but failed to induce activation of PTK. The results excluded the possibility that PTK activation is the consequence of an increase in intracellular Ca2+ or activation of protein kinase C. Addition of genistein, a PTK inhibitor, to sensitized BMMC before Ag challenge inhibited not only Ag-induced PTK activation, but also inositol 1,4,5-trisphosphate production, and histamine release in a similar dose-response relationship. Other PTK inhibitors, such as lavendustin A and tyrphostin RG50864, also inhibited the Ag-induced activation of PTK and histamine release. The results collectively suggest that activation of PTK is an early event upstream of the activation of phospholipase C, and is involved in transduction of IgE-dependent triggering signals to mediator release.     

Asymmetric cell division of rice zygotes located in embryo sac and produced by in vitro fertilization

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes.     

Modulation of arsenic trioxide-induced apoptosis by genistein and functionally related agents in U937 human leukaemia cells. Regulation by ROS and mitogen-activated protein kinases

The proved radio- and chemo-sensitizing capacity of genistein supports the potential use of this isoflavone in antitumour therapies. In this regard, we recently reported that genistein potentiates apoptosis induction by the anti-leukaemic agent arsenic trioxide (ATO) via reactive oxygen species (ROS) generation and p38-MAPK activation. In the present study we analyze the action of agents sharing functional similarities with the isoflavone, namely 17-β-estradiol, the DNA topoisomerase II poison etoposide, and the tyrosine kinase (PTK) inhibitors herbimycin A, epigallocatechin-3-gallate (EGCG) and adaphostin, in U937 and other human acute myeloid leukaemia cell lines. Co-treatment with 17-β-estradiol or etoposide failed to stimulate ROS production and potentiate ATO-provoked apoptosis, although etoposide caused G₂/M cycle arrest, in the same manner as genistein. By contrast, all PTK inhibitors increased ATO-provoked apoptosis, with similar efficacy as genistein. Daidzein, a genistein analogue without PTK-inhibiting activity, failed to potentiate apoptosis, and co-treatment with orthovanadate attenuated the sensitizing capacity of genistein. Apoptosis potentiation by PTK inhibitors was associated to ROS over-accumulation and stimulation of p38-MAPK phosphorylation, was mimicked by conventional pro-oxidant agents (exogenous H₂O₂ and the glutathione-depleting agent dl-buthionine-(S,R)-sulfoximine), and was attenuated by the antioxidant agent N-acetyl-l-cysteine, and by the p38-MAPK inhibitor SB203580 or p38-MAPK-directed siRNAs. On the other hand, the PTK inhibitors caused disparate effects on ERK phosphorylation, and co-treatment with the MEK/ERK inhibitor PD98059 enhanced the pro-apoptotic capacity of the PTK inhibitors. These results suggest that PTK inhibition, together with ROS generation and p38-MAPK activation, are responsible for the chemo-sensitizing action of genistein and functionally related agents in leukaemia cells.     

pH gradients and cell polarity inPelvetia embryos

Summary Endogenous pH profiles were measured around single fertilized eggs of the brown algaPelvetia during the earliest stages of development. Profiles were constructed by measuring the pH near the cell surface at several positions using a pH sensitive microelectrode. Transcellular pH differences in the medium surrounding zygotes were detected soon after fertilization, as the developmental axis was being formed. The future rhizoid end of the cell was relatively alkaline and the presumptive thallus was acidic. At germination and throughout the first 5 d of embryogenesis, the apex of the elongating rhizoid was alkaline with respect to more distal regions. However, conditions that dissipated or reversed this extracellular pH gradient had little or no effect on polarization or growth, indicating that the gradient was not essential for early development.Inhibition of respiratory electron transport by cyanide and antimycin A eliminated the pH gradient, while uncouplers of oxidative phosphorylation [2,4-dinitrophenol (DNP) and carbonylcyanide m-chlorophenylhydrazone (CCCP)] stimulated acidification of the thallus regions. Proton ATPase inhibitors had no effect. Acidification, therefore, is not generated by ATP-dependent proton pumps in the plasma membrane, and instead probably reflects secretion of metabolic acids. Localized metabolism may establish an internal pH gradient that controls regional differentiation, and we are presently investigating this possibility.Abbreviations ASW artificial seawater - CCCP carbonylcyanide m-chlorophenylhydrazone - CD cytochalasin D - DNP 2,4-dinitrophenol     

Genistein and tyrphostin AG556 inhibit inwardly-rectifying Kir2.1 channels expressed in HEK 293 cells via protein tyrosine kinase inhibition

Previous studies reported the controversial effects that protein tyrosine kinase (PTK) inhibition could induce an up-regulation or down-regulation of Kir2.1 current. The present study investigates how the recombinant human Kir2.1 channels are regulated by PTKs using whole-cell patch voltage-clamp, immunoprecipitation and Western blot, and mutagenesis approaches. We found that hKir2.1 current was reversibly inhibited by the broad spectrum PTK inhibitor genistein and the highly selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 in a concentration-dependent manner. The inhibition of hKir2.1 channels by genistein or AG556 was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of Kir2.1 channels was reduced by genistein or AG556, and the reduction was significantly antagonized by orthovanadate. The mutation of Y242 dramatically reduced the inhibitory response to AG556. The results obtained in this study demonstrate that hKir2.1 channels are down-regulated by PTK inhibition, suggesting that EGFR kinase participates in the modulation of human cardiac excitability.     

五唇兰合子与原胚的分离

分离合子和原胚可以为植物受精和胚胎发生提供很好的研究材料, 因此具有重要意义。用酶解-振荡法分离出五唇兰(Doritis pulcherrima)受精后胚囊, 然后以显微解剖获得合子和原胚。酶解液组成为0.7%–1.3%纤维素酶、0.6%–1.0%果胶酶和10%甘露醇, pH值为5.8, 酶解时间0.5–3.0小时。在分离的早期原胚和接近成熟的球形胚中, 珠孔端均有较发达的胚柄吸器。实验获得了对五唇兰胚胎发育的新认识, 合子和原胚的成功分离也为进一步的细胞学和分子生物学研究打下基础。