Okadaic Acid as a Probe to Analyse the Cell Cycle Progression in Plant Cells

Previously we have demonstrated the dynamic change of microtubules (MTs) during cell cycle progression using highly synchronized tobacco BY-2 cells and characterized the specific transition points of MT organization (Hasezawa and Nagata, 1991). In this study the effect of okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, on such changes of MTs during cell cycle was examined. These experiments revealed that cell cycle was arrested before the formation of the preprophase band (PPB), at anaphase and at the border of M/G₁. Although the block at the anaphase seemed to be analogous to that observed in animal cells (Yamashita et al., 1990), the other two blocks were specific to plant cells. It is interesting that these two blocks coincided with the transition points of MT organization, as revealed in the previous study (Hasezawa and Nagata, 1991). Thus it is proposed that phosphorylation is involved in MT organization, although the effect of OA has been shown mainly to be the activation of cdc-2/histone H1 kinase in animal cells. Another inhibitor of protein phosphatase 1 and 2A, calyculin A (CLA), showed very similar effects on the cell cycle progression. The use of such inhibitors to dissect the cell cycle progression of plant cells is discussed.     

Toxoplasma gondii: Purine synthesis and salvage in mutant host cells and parasites

Toxoplasma gondii, growing exponentially in heavily infected mutant Chinese hamster ovary cells that had a defined defect in purine biosynthesis, did not incorporate [U-¹⁴C]glucose or [¹⁴C]formate into the guanine or adenine of nucleic acids. Intracellular parasites therefore must be incapable of synthesizing purines and depend on their host cells for them. Extracellular parasites, which are capable of limited DNA and RNA synthesis, efficiently incorporated adenosine nucleotides, adenosine, inosine, and hypoxanthine into their nucleic acids; adenosine 5′-monophosphate was the best utilized precursor. Extracellular parasites incubated with ATP labeled with ³H in the purine base and ³²P in the α-phosphate incorporated the purine ring 50-fold more efficiently than they did the α-phosphate. Thus, ATP is largely degraded to adenosine before it can be used by T. gondii for nucleic acid synthesis. Two pathways for the conversion of adenosine to nucleotides appear to exist, one involving adenosine kinase, the other hypoxanthine—guanine phosphoribosyl transferase. In adenosine kinase-less mutant parasites, the efficiency of incorporation of ATP or adenosine was reduced by 75%, which indicates the adenosine kinase pathway was predominant. Extracellular parasites incorporated ATP into both the adenine and the guanine of their nucleic acids, so ATP from the host cell could supply the entire purine requirement of T. gondii. However, ATP generated by oxidative phosphorylation in the host cell is not essential for parasites because they grew normally in a cell mutant that was deficient in aerobic respiration and almost completely dependent upon glycolysis.     

Intrinsic Protein Phosphorylation in Synaptosomal Plasma Membrane Fragments: A Comparison of Cerebral Cortex Tissue from Several Species, Including Human Biopsy Specimens

Intrinsic protein phosphorylation was studied in synaptosomal membrane fragments made from cerebral cortex tissue taken from the following species: human (biopsy specimens), ox, rat, rabbit, guinea pig and mouse. Membrane fragments from all species exhibited a qualitatively similar range of protein acceptors phosphorylated by cyclic AMP-dependent protein kinase activity; contrary to a previous report, no evidence for cyclic GMP-dependent protein kinase activity was found in the human material. With the exception of membrane fragments prepared from ox brain, all the preparations exhibited the same range of Ca2+-dependent protein kinase activity. Ox brain obtained from a slaughterhouse yielded membranes containing no Ca2+-dependent protein kinase activity, but this may have been due to unavoidable postmortem losses.     

Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder

Summary The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive currentI ^a and the rate of suprabasal CO₂ productionJ _CO2 ^sb were observed in 10 toad urinary bladders at short-circuit and after clamping at 100 mV, serosa positive. Following perturbation of (0100mV),I ^a declined sharply within 1/2 min, remained near constant 15 min, and then increased slightly.J _CO2 ^sb declined more gradually, remained near constant at 4–7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy ofJ _CO2 ^sb starting at 2.9±1.1 (sd) min and lasting 4.7 ±1.8 (sd) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state,I ^a was also nearly constant. Considering that in steady statesI ^a/FJ _Na ^a , the rate of transepithelial active Na transport, during the early quasi-steady state mean values ±se ofJ _Na ^a ,J _CO2 ^sb and (J _Na ^a /J _CO2 ^sb ) were, respectively, 29.9±1.7%, 59.4 ±3.2%, and 56.4±5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4±6.0%, 38.2±6.1%, and 111.3±8.6%. Thus the flow ratioJ _Na ^a /J _CO2 ^sb was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements ofI ^a andJ _CO2 ^sb in three hemibladders were qualitatively similar. In terms of a phenomenological black-box treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.     

Calcium Binding Sites of Synaptosomes from Insect Nervous System as Probed by Trivalent Terbium Ions

Terbium binding to synaptosomes from the central nervous system of the locust was studied by fluorescence spectroscopy and electron microscopy. The protein-sensitized fluorescence of terbium was used to characterize the calcium binding sites of synaptosomes. As judged by electron microscopy and x-ray analysis, terbium ions produced electron-dense patches in regular arrays on the outer surface of synaptosomal membranes and induced marked aggregation of synaptic vesicles in isolated terminals.     

Growth yields and the efficiency of oxidative phosphorylation of Paracoccus denitrificans during two- (carbon) substrate-limited growth

Paracoccus denitrificans was grown aerobically during two-(carbon)substrate-limitation on mannitol and methanol in chemostat cultures. Theoretical growth parameters were calculated based on the presence of 2 or 3 sites in the electron-transport chain of Paracoccus denitrificans. Experimental growth parameters determined during two-(carbon)substrate growth were conform to the presence of 3 sites of oxidative phosphorylation, while cells grown only on mannitol possessed 2 sites. The maximum growth yield on adenosine triphosphate (ATP), corrected for maintenance requirements, determined in chemostat experiments in which the methanol concentration is less than 2.11 times the mannitol concentration was 8.6 g of biomass. When the methanol concentration was more than 2.11 times the mannitol concentration the maximum growth yield on adenosine triphosphate decreased due to the more energy consuming process of CO₂-assimilation. Cells use methanol only as energy source to increase the amount of mannitol used for assimilation purposes. When the methanol concentration in chemostat experiments was more than 2.11 times the mannitol concentration, all mannitol was used for assimilation and excess energy derived from methanol was used for CO₂-assimilation via the ribulose-bisphosphate cycle. The synthesis of ribulosebisphosphate carboxylase was repressed when the methanol concentration in chemostat experiments was less than 2.11 times the mannitol concentration or when Paracoccus denitrificans was grown in batch culture on both methanol and mannitol. When in chemostat experiments the methanol concentration was more than 2.11 times the mannitol concentration ribulose-bisphosphate carboxylase activity could be demonstrated and CO₂-assimilation will occur. It is proposed that energy produced in excess activates or derepresses the synthesis of the necessary enzymes of the ribulose-bisphosphate cycle in Paracoccus denitrificans. Consequently growth on any substrate will be carbonas well as energy-limited. When methanol is present in the nutrient cells of Paracoccus denitrificans synthesize a CO-binding type of cytochrome c, which is essential for methanol oxidase activity.The reason for the increase in efficiency of oxidative phosphorylation from 2 to 3 sites is most probably the occurrence of this CO-binding type of cytochrome c in which presence electrons preferentially pass through the a-type cytochrome region of the electron-transport chain.Non Standard Abbreviations X prosthetic group of methanol dehydrogenase - q _substrate specific rate of consumption of substrate (mol/g biomass. h.) - Y _substrate, Y _substrate ^MAX are respectively the growth yield and the maximum growth yield corrected for maintenance requirements (g biomass/mol) - m _substrate maintenance requirement (mol substrate/g biomass) - specific growth rate (h^-1) - M [methanol]/[mannitol] ratio in the nutrient - N part of mannitol that is assimilated when M=o - R _m amount of methanol-equivalents that has the same energy content as 1 mannitol-equivalent - P/O _N , P/O _F , P/O _X is the amount of ATP produced during electron-transport of two electrons from respectively NADH+H⁺, FADH₂ and XH₂ to oxygen     

Endogenous phosphorylated proteins during maturation of Xenopus laevis oocytes

During the course of maturation of Xenopus laevis oocyte a burst of phosphorylation occurs around germinal vesicle breakdown. At the same time a relative drop in a unique phosphoprotein (protein I; mot wt ～40,000) is observed. Enucleation of [³²P] labeled oocytes has shown the cytoplasmic localization of protein I. Methylxanthines and cholera toxin, which inhibit progesterone-induced maturation, block the burst of phosphorylation and do not change the amount or the distribution of [³²P] phosphoproteins.     

STE20 phosphorylation of AMPK-related kinases revealed by biochemical purifications combined with genetics

We have recently purified mammalian sterile 20 (STE20)–like kinase 3 (MST3) as a kinase for the multifunctional kinases, AMP-activated protein kinase–related kinases (ARKs). However, unresolved questions from this study, such as remaining phosphorylation activities following deletion of the Mst3 gene from human embryonic kidney cells and mice, led us to conclude that there were additional kinases for ARKs. Further purification recovered Ca²⁺/calmodulin-dependent protein kinase kinases 1 and 2 (CaMKK1 and 2), and a third round of purification revealed mitogen-activated protein kinase kinase kinase kinase 5 (MAP4K5) as potential kinases of ARKs. We then demonstrated that MST3 and MAP4K5, both belonging to the STE20-like kinase family, could phosphorylate all 14 ARKs both in vivo and in vitro. Further examination of all 28 STE20 kinases detected variable phosphorylation activity on AMP-activated protein kinase (AMPK) and the salt-inducible kinase 3 (SIK3). Taken together, our results have revealed novel relationships between STE20 kinases and ARKs, with potential physiological and pathological implications.     

3,3′,4,5′-Tetramethoxy-trans-stilbene Improves Insulin Resistance by Activating the IRS/PI3K/Akt Pathway and Inhibiting Oxidative Stress

The potential anti-diabetic effect of resveratrol derivative, 3,3′,4,5′-tetramethoxy-trans-stilbene (3,3′,4,5′-TMS) and its underlying mechanism in high glucose (HG) and dexamethasone (DXMS)-stimulated insulin-resistant HepG2 cells (IR-HepG2) were investigated. 3,3′,4,5′-TMS did not reduce the cell viability of IR-HepG2 cells at the concentrations of 0.5–10 µM. 3,3′,4,5′-TMS increased the potential of glucose consumption and glycogen synthesis in a concentration-dependent manner in IR-HepG2 cells. 3,3′,4,5′-TMS ameliorated insulin resistance by enhancing the phosphorylation of glycogen synthase kinase 3 beta (GSK3β), inhibiting phosphorylation of insulin receptor substrate-1 (IRS-1), and activating phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in IR-HepG2 cells. Furthermore, 3,3′,4,5′-TMS significantly suppressed levels of reactive oxygen species (ROS) with up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) expression. To conclude, the beneficial effect of 3,3′,4,5′-TMS against insulin resistance to increase glucose consumption and glycogen synthesis was mediated through activation of IRS/PI3K/Akt signaling pathways in the IR-HepG2 cells, accomplished with anti-oxidative activity through up-regulation of Nrf2.