Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.     

P-31 NMR saturation transfer experiments in Chlamydomonas reinhardtii : evidence for the NMR visibility of chloroplastidic Pi

ATP synthesis and consumption in respiring cells of the green alga Chlamydomonas reinhardtii were measured with ³¹P in vivo NMR saturation transfer experiments to determine the intracellular compartmentation of inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the measured flux to these enzymes is supported by the observation, that (i) the magnetization transfer was strongly reduced by iodoacetate, an irreversible inhibitor of GAPDH and that (ii) the unidirectional flux was much greater than the net flux through the mitochondrial F₀F₁-ATPase as determined by oxygen consumption measurements. In Chlamydomonas, glycolysis is divided into a chloroplastidic and a cytosolic part with the enzymes GAPDH/PGK being located in the chloroplast stroma (Klein 1986). The ³¹P-NMR signal of inorganic phosphate must, therefore, originate from the chloroplast. The life time of the magnetic label transferred to P_i by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envelope. When the intracellular compartmentation of P_i was taken into consideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equilibrium. The assignment of most of the intracellular P_i to the chloroplast is in contradiction to earlier reports, which attributed the Pi signal to the cytosol. This is of special interest for the use of the chemical shift of the P_i signal as an intracellular pH-marker in plant cells.Abbreviations 3-PGA 3-phosphoglycerate - CW continuous wave - dG6P 2-deoxyglucose-6-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - MO equilibrium z-magnetization - M₀ instantaneous z-magnetization after selective saturation for time t - MDP methylene-diphosphonic acid - PDE phosphodiester - PGK phosphoglycerate kinase - P_i inorganic orthophosphate - polyP polyphosphate - T₁ longitudinal relaxation time - ₁ longitudinal relaxation time with chemical exchange - TCA cycle tricarboxylic acid cycle Correspondence to: A. Mayer     

Essential trace elements in humans

Serum arsenic concentrations of persons suffering from renal failure and undergoing hemodialysis treatment (n=85) and of healthy controls (n=25) were determined by hydride-generation AAS technique after microwave digestion. The results were evaluated by comparing the values of both groups, considering physiological factors and individual data, as well as comorbid conditions of the hemodialysis (HD) patients. Serum arsenic levels were diminished in the patient group compared with controls (mean values 8.5±1.8 ng/mL vs 10.6±1.3 ng/mL). Furthermore, additional diseases within the hemodialysis group, particularly injuries of the central nervous system (CNS), vascular diseases, and cancer, were correlated to occasionally markedly decreased serum arsenic concentrations. It was concluded that arsenic homeostasis is disturbed by HD treatment and certain additional diseases. Desirable arsenic concentrations in the body seem to be reasonable. This consideration results in the conclusion that arsenic could play an essential role in human health. Thus, reference arsenic concentrations in different human tissues and body fluids should be established in order to recognize not only arsenic intoxication, but also arsenic deficiency. Perhaps arsenic deficiency contributes to the increased death risk of HD patients, and therefore, arsenic supplementations for patients with extremely low serum arsenic concentrations should be taken into account.     

Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Cellular location,activity states,and macromolecular organization of glucoamylase in Clostridium thermosaccharolyticum

By application of immunocytochemical techniques at the electron microscope level, glucoamylase was localized to the cell periphery in Clostridium thermosaccharolyticum during and following growth on starch, sucrose or glucose. Levels of immunolabelling were found to be relatively independent of growth substrate and of phase of growth, whereas previous studies had demonstrated strong dependence of glucoamylase activity on growth conditions; previously high levels of glucoamylase activity had been detected after growth on starch (i.e. during the stationary phase after growth) and only very low activities detected during exponential growth and following growth on glucose. The results presented demonstrate that levels of the glucoamylase protein are independent of measurable enzyme activity, and imply that the protein is constitutive. This indicates that the protein can exist in active and inactive states in the cell. By analogy with similar systems, we consider it likely that maturation or activation of newly synthesized glucoamylase occurs during (or following) transport through the cytoplasmic membrane. Electron microscopy of individual protein molecules which had been subjected to negative staining revealed that the enzyme consists of two domains of approximately equal size which are linked by a hinge region.     

Initial soil organic carbon stocks govern changes in soil carbon: Reality or artifact?

Changes in soil organic carbon (SOC) storage have the potential to affect global climate; hence identifying environments with a high capacity to gain or lose SOC is of broad interest. Many cross-site studies have found that SOC-poor soils tend to gain or retain carbon more readily than SOC-rich soils. While this pattern may partly reflect reality, here we argue that it can also be created by a pair of statistical artifacts. First, soils that appear SOC-poor purely due to random variation will tend to yield more moderate SOC estimates upon resampling and hence will appear to accrue or retain more SOC than SOC-rich soils. This phenomenon is an example of regression to the mean. Second, normalized metrics of SOC change—such as relative rates and response ratios—will by definition show larger changes in SOC at lower initial SOC levels, even when the absolute change in SOC does not depend on initial SOC. These two artifacts create an exaggerated impression that initial SOC stocks are a major control on SOC dynamics. To address this problem, we recommend applying statistical corrections to eliminate the effect of regression to the mean, and avoiding normalized metrics when testing relationships between SOC change and initial SOC. Careful consideration of these issues in future cross-site studies will support clearer scientific inference that can better inform environmental management.     

Membrane inlet—ion mobility spectrometry with automatic spectra evaluation as online monitoring tool for the process control of microalgae cultivation

The cultivation of algae either in open raceway ponds or in closed bioreactors could allow the renewable production of biomass for food, pharmaceutical, cosmetic, or chemical industries. Optimal cultivation conditions are however required to ensure that the production of these compounds is both efficient and economical. Therefore, high-frequency analytical measurements are required to allow timely process control and to detect possible disturbances during algae growth. Such analytical methods are only available to a limited extent. Therefore, we introduced a method for monitoring algae release volatile organic compounds (VOCs) in the headspace above a bioreactor in real time. This method is based on ion mobility spectrometry (IMS) in combination with a membrane inlet (MI). The unique feature of IMS is that complete spectra are detected in real time instead of sum signals. These spectral patterns produced in the ion mobility spectrum were evaluated automatically via principal component analysis (PCA). The detected peak patterns are characteristic for the respective algae culture; allow the assignment of the individual growth phases and reflect the influence of experimental parameters. These results allow for the first time a continuous monitoring of the algae cultivation and thus an early detection of possible disturbances in the biotechnological process.     

GENETIC EVIDENCE FOR MULTIPLE ORIGINS OF DIOECY IN THE HAWAIIAN SHRUB WIKSTROEMIA (THYMELAEACEAE)

Dioecy is unusually common in the Hawaiian Islands, yet little is known about the evolutionary biology of this breeding system. A native shrub, Wikstroemia, has an unusually diverse array of breeding systems: two forms of dioecy, cryptic and morphological dioecy, as well as hermaphroditism (perfect flowers). The existence of two forms of dioecy is significant for three reasons: 1) the presence of cryptic unisexuals that are functionally unisexual, but retain the appearance of hermaphroditism in both sexes, is strong evidence for the ancestral status of hermaphroditism; 2) the production of nonfunctional pollen, by female cryptic unisexuals, is a new instance of a phenomenon which has previously been reported for a few other species; 3) the two forms of dioecy are morphological markers which are useful in hybridization studies for tracing the genetic basis of their inheritance. Crosses were made between cryptically unisexual individuals (C), between morphologically unisexual individuals (M), and between the two types of unisexuality. The offspring of crosses between individuals with the same sex type usually resulted in offspring with that sex type, but most of the progeny of between-sex type crosses were, unexpectedly, perfect-flowered hermaphrodites. These results show that genetic control of sex determination is not homologous in all populations, suggesting that dioecy has evolved at least twice in Hawaiian Wikstroemia. The genetic data further suggest that males are the heterozygous sex.