Technetium and rhenium complexes of mercapto-containing peptides 1. Tc(V) and Re(V) complexes with mercaptoacetyl diglycine (MAG2) and X-ray structure of AsPh4[TcO(MAG2)]·C2H5OH

The reaction of mercaptoacetyl diglycine (MAG₂) with technetium(V) gluconate in aqueous solution produced [TcO(MAG₂)]⁻. A single X-ray structure determination was carried out for the tetraphenylarsonium salt. The dark brown crystals are monoclinic, space group P2(1)/n, with a=12.478(5), b=14.922(5), c=17.183(9) Å and Z=4. The [TcO(MAG₂)]⁻ ion has a square pyramidal geometry with the technetium atom displaced by 0.756 Å towards the oxo ligand from the plane formed by the equatorial S,N,N,O atoms. The rhenium complex AsPh₄[ReO(MAG₂)] was prepared analogously starting from Re(V) gluconate and characterized.     

Long-term changes in the rotifer fauna after biomanipulation in Haussee (Feldberg,Germany, Mecklenburg-Vorpommern) and its relationship to the crustacean and phytoplankton communities

In Feldberg Haussee, an anthropogenic eutrophicated lake, biomanipulation was executed for restoration. To increase the biomass of crustaceans, fish grazing on zooplankton was reduced by catching small fishes and introducing pike-perch. After biomanipulation rotifer biomass from a wide range of species decreased to a small spring maximum with three dominant species. The development of food in spring and food competition between crustaceans probably controlled the rotifer development.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Neither Moderate Hypoxia nor Mild Hypoglycaemia Alone Causes Any Significant Increase in Cerebral [Ca2±i: Only a Combination of the Two Insults Has This Effect.: A 31P and 19F NMR Study

(1) The energy state and free intracellular calcium concentration ([Ca^2±_i) of super-fused cortical slices were measured in moderate hypoxia (～65 μM O₂), in mild hypoglycaemia (0.5 mM glucose), and in combinations of the two insults using ¹⁹F and ³¹P NMR spectroscopy. (2) Neither hypoxia nor hypoglycaemia alone caused any significant change in [Ca^2±_i. Hypoxia caused a 40% fall in phosphocreatine (PCr) content but not in ATP level, and hypoglycaemia produced a slight fall in both (as expected from previous studies). These changes in the energy state recovered on return to control conditions. (3) A combined sequential insult (hypoxia, followed by hypoxia plus hypoglycaemia) produced a 100% increase in [Ca^2±, and a decrease in PCr level to ～25% of control. The reverse combined sequential insult (hypoglycaemia, followed by hypoglycaemia plus hypoxia) had the same effect. On return to control conditions there was some decrease in [Ca^2±_i and a small increase in PCr content, but neither recovered to control levels. (4) Exposure of the tissue to the combined simultaneous insult (hypoxia plus hypoglycaemia) immediately after the control spectra had been recorded resulted in a fivefold increase in [Ca^2±_i and a similar decrease in PCr level to 20–25% of control. There was little if any change of [Ca^2±_i or PCr level on return to control conditions. (5) These results are discussed in terms of metabolic adaptation of some but not all of the cortical cells to the single type of insult, which renders the tissues less vulnerable to the combined insult.     

Endothelin-Mediated Calcium Response and Inositol 1,4,5-Trisphosphate Release in Neuroblastoma-Glioma Hybrid Cells (NG108-15): Cross Talk with ATP and Bradykinin

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108-15) induced increases in cytosolic free Ca²⁺ ([Ca²⁺]_i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. The increases in [^Ca2+]_i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca²⁺ in the medium by adding excess EGTA decreased the ET-mediated Ca²⁺ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca²⁺]_i was due to influx from an extracellular source. However, the increase in [Ca²⁺]_i was not affected by verapamil or nifedipine (10^?5M). A rank order potency of ET-1 ET-2 ET-3 is shown for the stimulated increase in [Ca²⁺]_i, as well as labeled inositol phosphates, in these cells. ATP (10^?4M) and bradykinin (10^?7M) also induced the increases in [Ca²⁺]_i and Ins(1,4,5)P₃ in NG108-15 cells, albeit to a different extent. When compared at 10^?7M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P₃, but less than a twofold higher increase in [Ca²⁺]_i than those induced by ET-1. Additive increases in both Ins(1,4,5)P₃ and [Ca²⁺]_i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization to the Ca²⁺ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Effect of Inhibitors of Eicosanoid Metabolism on Release of [3H]Noradrenaline from the Human Neuroblastoma, SH-SY5Y

Abstract: Nordihydroguaiaretic acid (NDGA; a lipoxygenase inhibitor), LY-270766 (an inhibitor of 5-lipoxygenase), and the diacylglycerol lipase inhibitor RG 80267 completely eliminated potassium-evoked release of [³H]noradrenaline ([³H]NA) from the human neuroblastoma clone SH-SY5Y with IC₅₀ values of 10, 15, and 30 μ M , respectively. In contrast, these inhibitors only partially inhibited carbachol-evoked release and had little effect on the calcium ionophore A23187-evoked release of NA in this cell line. Arachidonic acid partially inhibited potassium- and A23187-evoked release but did not reverse the inhibition of potassium-evoked release observed in the presence of RG 80267. These studies suggest that arachidonic acid (or its lipoxygenase products) are not important intermediates in the regulation of exocytosis in SH-SY5Y. This conclusion is strengthened by our studies in which SH-SY5Y cells were grown in medium supplemented with bovine serum albumin-linoleic acid (50 μ M ). Under these conditions there was a selective increase in content of membrane polyunsaturated fatty acids of the ω6 series, including arachidonic acid; however, these changes did not effect potassium-, veratridine-, carbachol-, or calcium ionophoreevoked release of [³H]NA.     

Phosphorylation of Neuronal Kinesin Heavy and Light Chains In Vivo

Abstract: The microtubule-based motor protein kinesin is thought to drive anterograde organelle transport in axons, but nothing is known about how its force-generating activity or organelle-binding properties are regulated. Studies in other motility systems suggest that protein phosphorylation is a reasonable candidate for this function. I report here that the kinesin heavy chain (HC) and light chain (LC), as well as the 160-kDa kinesin-associated protein kinectin, are phosphorylated in vivo in cultures of chick sympathetic neurons and PC12 cells labeled metabolically with ³²P. In neurons, both kinesin chains are phosphorylated exclusively on serine residues, and limiting tryptic digestion demonstrated that the phosphorylation sites are clustered in a region of ˜5 kDa for the HC and ˜14 kDa for the LC. Partial tryptic digestion of ³²P-labeled HC followed by immunoblotting with SUK4 monoclonal anti-HC and fluorography showed that the sites of HC phosphorylation are outside the globular N-terminal head region where kinesin's microtubulebinding and mechanochemical activities reside. Treatment of metabolically labeled neurons with forskolin, phorbol esters, or calcium ionophore did not alter the extent of phosphorylation, the phosphoamino acid composition, or the V8 protease phosphopeptide maps of the HC, LC, and 160-kDa protein, with one exception: treatment with calcium ionophore reduced the specific activity of the LC. In addition, when kinesin from PC12 cells was compared with that from PC12-derived cell lines lacking protein kinase A activity, neither the extent of phosphorylation nor the phosphopeptide maps were altered for either chain. Phosphopeptide mapping experiments also showed that postlysis kinase activity can phosphorylate both the neuronal HC and LC at sites not phosphorylated in vivo.     

PHYSIOLOGICAL ACCLIMATION OF MARINE PHYTOPLANKTON TO DIFFERENT NITROGEN SOURCES1

We examined the energetic dependency of the biochemical and physiological responses of Thalassiosira pseudonana Hasle and Heimdal. Chaetoceros gracilis Schütt, Dunaliella tertiolecta Butcher, and Gymnodinium sanguineum Hirasaka to NH₄⁺, NO₃^?, and urea by growing them at subsaturating and saturating photon flux (PF). At subsaturating PF, when energy was limiting, NO₃^? and NH₄⁺ grown cells had similar growth rates and C and X quotas. Therefore, NO₃^? grown cells used up to 48% more energy than NH₄⁺ grown cells to assimilate carbon and nitrogen. Based on our measurements of pigments, chlorophyll-a-specific in vivo absorption cross-section, and fluorescence-chlorophyll a^?1, we suggest that NO₃^?, grown cells do not compensate for the greater energy requirements of NO₃^? reduction by trapping more light energy. At saturating PF, when energy is not limiting, the utilization of NO₃^?, compared to NH₄⁺ resulted in lower growth rates and N quotas in Thalassiosira pseudonana and lower N quotas in Chaetoceros gracilis, suggesting enzymatic rather than energetic limitations to growth. The utilization of urea compared to Nh₄⁺ resulted in lower growth rates in Chaetoceros gracilis and Gymnodinium sanguineum (saturating PF) and in lower N quotas in all species tested at both subsaturating and saturating PF. The high C:N ratios observed in all urea-grown species suggest that nitrogen assimilation may be limited by urea uptake or deamination and that symptoms of N limitation in microalgae may be induced by the nature of the N source in addition to the N supply rate. Our results provide new eridence that the maximum growth rates of microalgae may be limited by enzymatic processes associated with the assimilation of NO₃^?, or urea.