Depolarization and Agonist-Stimulated Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulation in Rat Cerebral Cortex

Muscarinic receptor stimulation or depolarization with elevated extracellular K+ induced rapid and sustained increases in mass accumulations of myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] in cerebral cortex slices. Synergistic but transient responses of both inositol polyphosphate second messengers were observed when slices were stimulated with carbachol under depolarizing conditions; this synergy was observed as an increase in the maximal responsiveness, with no significant change in EC50 values for carbachol. Omission of buffer Ca2+ ([Ca2+]e 10-20 microM) reduced basal Ins(1,4,5)P3 and Ins(1,3,4,5)P4 concentrations; the relative stimulatory effects of muscarinic receptor stimulation were maintained, but the effects of depolarization were markedly attenuated under these conditions. A component of the response to depolarization appeared to be indirectly mediated by the release of acetylcholine, because the K(+)-evoked increase in Ins(1,3,4,5)P4 was enhanced by the cholinesterase inhibitor physostigmine, and was partially attenuated by atropine. An additive suppression by nitrendipine suggests that entry of Ca2+ through L-type Ca2+ channels may serve to accelerate phosphorylation of Ins(1,4,5)P3 by 3-kinase. Norepinephrine did not significantly increase Ins(1,4,5)P3 or Ins(1,3,4,5)P4 accumulation; however, in the presence of depolarizing K+, norepinephrine caused a dramatic increase in Ins(1,3,4,5)P4 mass accumulation. In contrast, the excitatory amino acid quisqualate caused significant increases in the mass accumulations of both inositol polyphosphates measured, with no further increase being observed under depolarizing conditions. The results are discussed with respect to the interactive effects of agonist and depolarization stimuli on inositol polyphosphate accumulation which might more accurately reflect the conditions pertaining in vivo.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.     

Vanadyl Causes Hydroxyl Radical Mediated Degradation of Deoxyribose

Vanadyl caused a time- and dose-dependent degradation of deoxyribose to carbonyl products detectable with thiobarbituric acid. This process was inhibited by catalase, ethanol or HEPES; whereas superoxide dismutase was without effect. Vanadate did not substitute for vanadyl even in the presence of a source of O₂^- plus H₂ O ₂; but it did so in the presence of reductants such as thiols or NADH. It appears that hydrogen peroxide, generated by the autoxidation of vanadyl, is reduced by vanadyl to the hydroxyl radical; which, in turn, was responsible for the degradation of deoxyribose. A similar process might contribute to the toxic and pharmacological effects of vanadium salts.     

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.     

Survival of phage M13 with uracils on one or both DNA strands

Summary The survival of M13 DNA was studied after partial replacement of thymine by uracil in the bacteriophage. Uracils carry the same genetic information as the thymines. Nevertheless in Escherichia coli wild-type cells, uracils in DNA are replaced by thymines by excision repair initiated by uracil-DNA glycosylase (UDG). Thus inactivation of uracil-containing phage DNA is solely due to repair initiated by UDG. Incorporation of uracils was achieved in one or in both strands, either randomly or site-specifically using differently uracylated oligonucleotides. The results show that up to 580 uracils can be repaired without a significant decrease in survival if the uracils are localized in the (–) strand only. Incorporation of 246 uracils into the (+) strand leads to 30% or 10% survival when expressed in Escherichia coli strains CMK and JM103, respectively. However, when uracils are distributed over both strands a sharp decrease in survival occurs. This shows that the repair of two uracils localized in close proximity on opposite strands of the DNA by the excision repair mechanism is difficult, whereas uracils occurring in one strand are repaired more efficiently, irrespective of their number.     

Auxin-induced H+-pump stimulation does not depend on the presence of epidermal cells in corn coleoptiles

Cell-wall acidification and electrical reactions (depolarization and hyperpolarization) are typical auxin responses in maize (Zea mays L.) coleoptiles. In an attempt to test the role of the outer epidermis in these responses, they have been measured and compared in intact and peeled coleoptile fragments. To exclude interactions between parenchymal and epidermal cells, the coleoptile pieces were completely stripped of their outer epidermis. This preparation was monitored by means of a scanning electron microscope. When externally applied indole-3-acetic acid was tested, we found that neither cell-wall acidification nor the electrical membrane responses depended on the presence of intact epidermal cells.Abbreviations IAA Indole-3-acetic acid - MES 2-[N morpholino-ethane-sulfonic acid - TRIS 2-Amino-2-hydroxymethyl-1,3-propanediol We thank Kuki Kaethner for her excellent technical assistance. This work was supported by the Hessische Graduiertenförderung and the Deutsche Forschungsgemeinschaft.     

Recovery of photosynthesis and phtosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light

Recovery from 60 min of photoinhibitory treatment at photosynthetic photon flux densities of 500, 1400 and 2200 μMmol m^?2 s^? was followed in cells of the green alga Chlamydomonas reinhardtii grown at 125 μMmol m^?2 s^?1. These light treatments represent photoregulation, moderate photoinhibition and strong photoinhibition, respectively. Treatment in photoregulatory light resulted in an increased maximal rate of oxygen evolution (P_max) and an increased quantum yield (Φ), but a 15% decrease in F_v/F_M. Treatment at moderately photoinhibitory light resulted in a 30% decrease in F_v/F_M and an approximately equal decrease in Φ. Recovery in dim light restored F_v/F_M within 15 and 45 min after high light treatment at 500 and 1400 μMmol m^?2 s^?1, respectively. Convexity (Θ), a measure of the extent of co-limitation between PS II turnover and whole-chain electron transport, and Φ approached, but did not reach the control level during recovery after exposure to 1400 μMmol m^?2 s^?1, whereas P_max increased above the control. Treatment at 2200 μMmol m^?2 s^?1 resulted in a strong reduction of the modeled parameters Φ, Θ and P_max. Subsequent recovery was initially rapid but the rate decreased, and a complete recovery was not reached within 120 min. Based on the results, it is hypothesized that exposure to high light results in two phenomena. The first, expressed at all three light intensities, involves redistribution within the different aspects of PS II heterogeneity rather than a photoinhibitory destruction of PS II reaction centers. The second, most strongly expressed at 2200 μmol m^?2 s^?1, is a physical damage to PS II shown as an almost total loss of PS II_α and PS II Q_B-reducing centers. Thus recovery displayed two phase, the first was rapid and the only visible phase in algae exposed to 500 and 1400 μmol m^?2 s^?1. The second phase was slow and visible only in the later part of recovery in cells exposed to 2200 μmol m^?2 s^?1.     

A Pseudomonas strain accumulating polyesters of 3-hydroxybutyric acid and medium-chain-length 3-hydroxyalkanoic acids

Summary A citronellol-utilizing bacterium was isolated that accumulated a polyester consisting of 3-hydroxybutyric acid (3HB) and of medium-chain-length 3-hydroxyalkanoic acids (3HA_MCL) from various carbon sources up to approximately 70% of the cellular dry matter if the cells were cultivated in ammineral salts medium under nitrogen limitation. In octanoate-grown cells, for instance, the polyester consisted of 87.5 mol% 3HB and 12.5 mol% 3-hydroxyoctanoic acid (3HO), whereas it consisted of 10.3 mol% 3HB, 16.7 mol% 3HO and 73.0 mol% 3-hydroxydecanoic acid (3HD) in gluconate-grown cells. However, the results of various experiments indicated that a blend rather than a copolyester was synthesized in the cell. It was the only strain among 45 different recently isolated citronellol-utilizing bacteria that accumulated such a polyester. All other citronellol-utilizing bacteria behaved like Pseudomonas aeruginosa with respect to their polyhydroxyalkanoic acid (PHA) biosynthetic capabilities and accumulated PHA consisting of 3HA_MCL with 3HO and 3HD as the main constituents from octanoate or gluconate, respectively, whereas 3HB was never present. None of 232 different heavy-metal-resistant bacteria was able to accumulate PHA composed of 3HB plus, for example, 3HO. Only 20.3% did not accumulate any PHA at all, 44.8% accumulated PHB from gluconate, and 34.9% behaved like P. aeruginosa. Many bacteria belonging to the latter group were distinguished from the other by rapid growth in nutrient broth and in gluconate mineral salts medium and by their ability to grow in the presence of a high concentration (up to 1.5%, w/v) of octanoate. Correspondence to: A. Steinbüchel     

Floral display and pollination success in Achillea ptarmica (Asteraceae)

In a natural population of the self-incompatible Achillea ptarmica , many-headed inflorescences attracted most pollinators (flies), and each pollinator visited more heads on large inflorescences than on smaller ones. The visitation rate did not increase disproportionately with inflorescence size, as would be expected if clusters of heads had a synergetic effect on pollinator attraction. Both the calculated visitation rate per head and the percentage seed set increased slightly as a function of head number. The removal of rays from all heads in the inflorescence reduced the approach rate by 51%, but had only a small negative effect on seed set (12%). Hence, ray removal probably affected male fertility (pollen donation) to a greater extent than female fertility. The decline in pollination success was similar for all inflorescence sizes, suggesting that ray and inflorescence size have independent effects on the total display, and thus represent separate targets for pollinator-mediated selection. Residual variation in seed set was strongly correlated with patch identity, indicating clonal or environmentally induced differences in female reproductive success.