The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes

Summary The elemental compositions of chloragosome granules in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1=uncharacterised; Pool 2=P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the control chloragosomes. In Zn-contaminated chloragosomes, most (70%) was bound by P-containing ligand(s) but some (<1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.     

Action of phenazine methyl sulfate,inhibitors, and uncouplers on the light-induced proton transport by cells ofRhodospirillum rubrum

Summary Suspensions of log phase cells ofRhodospirillum rubrum at pH 5.5 show a light-induced decrease in the pH of the medium which is reversed during the subsequent dark period. The velocity and magnitude of the pH change were the same whether the cells were bubbled with air, CO₂-free air or N₂ during experimentation. The pH response is temperature dependent. Phenazine methyl sulfate (PMS) at concentrations above 0.05mm stimulates the light-induced pH change. PMS at 1mm gives a 2-fold increase in the initial rate upon illumination and a 1.5-fold increase in the total change in pH after 2 min of illumination. The inhibition of the proton transport by 10 g/ml antimycin A or 20 m 2-n-heptyl-4-hydroxyquinoline-N-oxide can be partially relieved by PMS. However, inhibition of the light-induced proton transport with 0.5mm 2,4-dinitrophenol or 3 m carbonylcyanide-m-chlorophenylhydrazone (CCCP) cannot be overcome by addition of PMS. Valinomycin, at a concentration of 3 m, caused a slight stimulation of the light-induced proton transport in the presence of 200mm KCl. The inhibition of proton transport by 3 m CCCP was partially relieved with 3 m valinomycin in the presence of 200mm KCl, but the antibiotic was without effect when the cells were suspended in 200mm NaCl. The results are discussed in terms of current theories of the action of PMS, antimycin A, valinomycin, and uncouplers on the light-induced electron flow and photophosphorylation inR. rubrum.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

The Biometric Analysis of Bacterial Cells in Soil

The biometric analysis of bacterial cells in soil by light, fluorescence, and scanning electron microscopy showed that their average size is 0.8 m in diameter, 1.4 m in length, and 0.7 m³ in volume. In soil loci with enhanced microbiological activity (the rhizoplane of plants and the intestinal tract of soil invertebrates), the average size of bacterial cells was found to be 40% smaller than that of cells occurring in other parts of soil. This is the first experimental evidence showing that the metabolic activity of soil bacteria, their concentration, and allometric parameters are related.     

Isospora lutrae n. sp. (Apicomplexa: Eimeriidae), a new coccidium from the European otter Lutra lutra (L.) (Carnivora: Mustelidae) from Spain

Parasitological examination of European otter originating from Extremadura, Spain revealed the presence of a new isosporan species. Oöcysts of Isospora lutrae n. sp. are spherical to subspherical, 31.2 (27.5–32) × 29.6 (28–31) m and have a smooth wall c. 1 m thick. Sporocysts are ellipsoidal, 18.2 (17–19) × 14.4 (14–16) m and lack Stieda and substieda bodies. A spherical sporocyst residuum is present, consisting of granules scattered among the sporozoites. Sporozoites are spindle-shaped, 12.4 × 2.5 m and have anterior and posterior refractile bodies. Based on its unique morphologic structure and host, I. lutrae is considered to be new.     

Ultrastructural analysis ofSpinacia oleracea sperm cells isolated from mature pollen grains

Summary In isolated condition, the sperm cells ofSpinacia oleracea are no longer arranged in pairs as in the pollen grain. The vegetative membrane, which surrounds a sperm cell pair in a mature pollen grain, is lost during the isolation procedure. The sperm cells become spherical in shape.The isolated sperm cell is surrounded by an intact plasma membrane. The heterochromatic or euchromatic sperm cell nucleus is located in the cell center. Mitochondria are round to oval and have distinct cristae. Often they are clustered in groups of 5 to 10 mitochondria. Dictyosomes are present in the cytoplasm and consist of 4 to 5 cisterns. Endoplasmatic reticulum is mostly situated at the sperm cell periphery, as single cisterns very near the plasma membrane.From diameters of sectioned sperm cells in electron micrographs, it is possible to calculate the average diameter of the whole sperm cell. This average diameter is 3.66 m with a variation of 3.0 m to 4.2 m, resulting in an average volume of 25.6 m³. The nuclear volume is 12.8 m³ (50.0% of the whole cell) and the mitochondrial volume is 0.7 m³ (2.5% of the whole cell). The frequency distribution of the isolated sperm cells diameters shows only one peak with a normal distribution, indicating that there is no dimorphism in volume.     

Development of an L6 myoblast in vitro model of moniliformin toxicosis

L6 myoblasts were used as an in vitro model to investigate the role of moniliformin and its interaction with monensin in turkey knockdown syndrome and sudden death syndromes in poultry. Cell viability and microscopic and ultrastructural alterations noted in L6 myoblasts cultured in the presence of moniliformin (0.0–0.3 g/l) were compared to those observed in parallel cultures also containing one of the following compounds: selenium (0–0.004 ng/l), thiamine (0–0.3 g/l), or pyruvate (0–0.46 g/l). Marked dilation of the RER, membranous whorls, glycogen deposition, membrane-bound cytoplasmic inclusions and necrosis were observed in myoblasts exposed to 0.03/2-0.30 g moniliformin/l medium. Supplementation of medium with thiamine and pyruvate, or selenium, provided significant protection to cells exposed to 0.0–0.3 g/l or 0.0–0.15 g moniliformin/l, respectively. Dose-dependent differences in protein and ATP production were not detected. Myoblasts grown in medium containing 0–0.15 g moniliformin/l and 7.5–50.0 M A23187, beauvericin or monensin had degrees of cytotoxicity similar to parallel cultures receiving only an ionophore. L6 myoblasts were a useful model of moniliformin toxicosis. The findings of this study suggest cytotoxicity due to moniliformin in L6 myoblasts may be due in part to oxidative damage and altered pyruvate metabolism, and that moniliformin does not predispose myoblasts to ionophore toxicosis. This study supports the results of in vivo investigations in poultry that moniliformin and monensin do not act synergistically to induce knockdown or monensin toxicosis.     

Pretraining but not Preexposure to the Task Apparatus Prevents the Memory Impairment Induced by Blockade of Protein Synthesis,PKA or MAP Kinase in Rats

Adult male Wistar rats were trained and tested in a step-down inhibitory avoidance task (0.4 mA footshock, 24 h training-test interval). Fifteen minutes before or 0, 1.5 or 3 hours after training, animals received a 0.8 l intrahippocampal infusion of the protein synthesis inhibitor anisomycin (80 g), the PKA inhibitor Rp-cAMP (0.05 g), the MAPK kinase inhibitor PD 098059 (50 M solution) or vehicle (phosphate buffer in saline, pH 7.4). Anisomycin, Rp-cAMP and PD 098059 impaired retention test performance in animals injected at different times, prior and after training. Pretraining with a low footshock intensity (0.2 mA) 24 h before training prevented the amnestic effect of all drugs studied. However, simple preexposure to the inhibitory avoidance apparatus did not alter the amnestic effects of all drugs. The results suggest that memory processing requires hippocampal mechanisms dependent on protein synthesis, PKA and MAPK kinase at different times after training. These findings suggest that weak training must be sufficient to produce some lasting cellular expression of the experience so that the enhancement of consolidation of a previously acquired memory is not dependent on protein synthesis, PKA or MAPK.     

Sampling DNA from the rhizosphere of Brassica napus to investigate rhizobacterial community structure

Variations in genotype rankings among screenings for Al tolerance in hydroponics may be related to differences in the composition of the solutions. In the present study, we investigated the involvement of Mg ions in modifying Al rhizotoxicity in soybeans. Root elongation was strongly inhibited by Al in a simple, 800 M CaSO₄ solution, but elongation increased noticeably when the solutions also contained Mg. Amelioration of Al rhizotoxicity was not associated with an increase in ionic strength of treatment solutions because Al³⁺ activities were kept constant. Concentration series experiments indicated that the Mg effect occurred in the M range, while Ca amelioration of Al toxicity occurred at mM concentrations. The positive effect of Mg on root elongation was greatest for Al-sensitive genotypes and minimized genotypic differences for Al-tolerance. The Mg protection against Al rhizotoxicity apparently does not occur with all species, because it was not observed in Atlas and Scout 66 wheat varieties. The ability of Mg to ameliorate Al toxicity in soybean at M levels suggests the involvement of distinct physiological factors.     

The nutritional status of the apical meristem of Lactuca sativa as affected by NaCl salinization: An electron-probe microanalytic study

A volume of tissue of lettuce (Lactuca sativa L.) plants extending 2 mm basipetally from the apical meristem and including leaf primordia and young expanding leaves was surveyed using electron-probe microanalysis (EPMA) on both frozen-hydrated and freeze-dried samples. This analysis was carried out either 2 or 5 d following NaCl salinization of the medium from the 10 mol · m^–-3 control level up to 80 mol · m^–-3. The objective was the investigation of possible changes in the nutritional status of the apical meristem that might account for some aspects of salt-induced growth inhibition. Sodium and chloride increased significantly in tissues basal to the apical meristem, while both phosphorus and potassium decreased in the same region. These changes were evident in specimens collected just 2 d after the commencement of salinization (20 h after completion of the salinization) and were not exacerbated by an additional 3 d of treatment; they were present in tissue as close as 100 m to the meristem and extending down to 500 m. The apical 10–50 m were relatively protected from both the increase in sodium and chloride and the decrease in phosphorus and potassium that occurred in more basal regions. Young leaves (up to 1.5 mm in length) appear to control their own mineral nutrient levels when challenged by salinization of the medium, presumably because of altered growth. A decrease in the concentration of total Ca as a result of salinization was significant in cells 500 m basal to the meristem, but was evident as a tendency in the data even within the first 50 m. Using an improved automatic method for the analysis of calcium by EPMA, it was found that total Ca was reduced by salinization, especially in basal regions (500 m below the apex) and also in young leaves (1–1.5 mm in length). We suggest that the nutrition of the shoot apical meristem may be disturbed soon after salinization and that the shoot meristem might be the source of a signal to expanding leaves, as well as exerting its own direct influence over leaf emergence.Abbreviation EPMA electron-probe microanalysis This work was supported by U.S. Department of Agriculture grant 87-CRCR-1-2462.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Establishment and characterization of Rubus tissue culture systems for in vitro bioassays against phytotoxins from Rubus fungal pathogens

The Rubus species R. parviflorus, R. spectabilis and R. strigosus interfere with conifer seedling establishment on forest regeneration sites in Canada and the United States. As a first step towards microbial metabolite-based control, callus and cell suspension cultures of the Rubus species were developed as a bioassay system to detect phytotoxic compounds that may have relevance in a vegetation control context. Rapidly growing friable callus and suspension cultures were obtained from leaf disks of the three weedy Rubus species using similar culture media conditions (modified Murashige and Skoog) but required different plant growth regulators (R. parviflorus, 4.5 M 2,4-D; R. spectabilis, 26.9 M NAA/0.5 M zeatin; and R. strigosus, 12.4 M picloram). Cell growth and health attributes including callus circumference, degree of browning and suspension culture cell viability as measured by the TTC vital stain assay were developed and were rapid and convenient to use. We have established Rubus tissue culture systems that will make it possible for large scale screening of phytotoxic metabolites.     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

A novel cilia-based feature within the food grooves of the ctenophore Mnemiopsis mccradyi Mayer

We describe a novel compound ciliary structure (g-cilium) from the food groove of the lobate ctenophore Mnemiopsis mccradyi. G-cilia are small, flat compound ciliary organelles that are oriented with their tips pointing toward the mouth. Typically three to four rows of g-cilia line the inner surface of the tentacular groove, which together with the transport groove, make up the food groove. G-cilium cells are 11.4 m long and 4.2 m wide at the g-cilium base. The g-cilium itself is 3.4 m long and tapers to a flat, sharp tip. G-cilia are not motile but are surrounded by many hundreds of smaller, actively motile cilia that beat with orally-directed effective strokes. G-cilia contain 50 conventional `9+2' cilia embedded in a fibrous core that arises from the cell body. In addition, g-cilia contain mitochondria, thousands of small membrane-bounded vesicles and rod bacteria. G-cilia basal bodies are anchored by large, strongly-banded rootlets that extend approximately the entire length of the cell. G-cilia may have organizational, sensory and/or secretory function within the feeding apparatus. Their placement strongly suggests that they play critical roles in feeding. They may enhance the efficiency of prey capture and so contribute to M. mccradyi's well-known voracious appetite. By enhancing prey capture they probably play a critical role in the capacity of this organism to follow prey dynamics, so contributing to dense blooms in mid-late summer in coastal regions.     

The Effect of Phenylalanine on DOPA Synthesis in PC12 Cells

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The K_m for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the K_m for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

In vitro comparison of ceftazidime,aztreonam, cefpirome,gentamicin, imipenem,enoxacin, and ticarcillin plus clavulanic acid against 108 strains ofPseudomonas maltophilia

Pseudomonas maltophilia is an uncommon cause of hospital-acquired infection and is resistant to most of the antimicrobial agents used in the treatment of gram-negative infections. Susceptibility of 108 isolates ofP. maltophilia to ceftazidime, aztreonam, defpirome, gentamicin, imipenem, enoxacin, and ticarcillin plus clavulanic acid was determined by an agar dilution method. The isolates were in general resistant to the antibiotics. Imipenem and cefpirome were not active at clinically achievable levels. Of the isolates, 20% were susceptible to 16 g/ml ceftazidime, 53% were susceptible to 4 g/ml enoxacin, 10% were susceptible to 4 g/ml gentamicin, and 25% were susceptible to 64 g/ml ticarcillin plus 2 g/ml clavulanic acid.     

Metals and phosphate in the chloragosomes of Lumbricus terrestris and their possible physiological significance

Summary The chloragog cells of the earthworm Lumbricus terrestris contain numerous granules, chloragosomes, which were analyzed for metals and phosphate by histochemistry, by use of an electron microscope X-ray microprobe (EMMA), and by chemical analysis of chloragosome preparations. Inorganic and organic phosphate each accounts for about 3% of the chloragosome dry mass, Ca for 2–3%, Zn for 1–3% and Mg for 0.2–0.4%. Carbonate is not present in chloragosomes. The average molar ratios CaMgZntotal PO₄ are 10.10.31. The CaPO₄ ratio is fairly constant (correlation coefficient 0.99), while the ZnPO₄ ratio varies considerably. It is concluded that Ca is bound in the form of inorganic CaHPO₄ and organic ROPO ₃Ca (or possibly Ca-polyphosphate complexes). Mg may also be phosphate-bound, while Zn probably is not. Chemical analysis of the calciferous glands revealed a high concentration of Ca, small amounts of Mg and phosphate, but no Zn. It is concluded that Zn is not excreted through the calciferous gland.Storage of Ca in the chloragocytes and excretion of CaCO₃ by the calciferous gland may be physiologically linked. Regulation of the concentrations of Ca and HCO₃ ^–ions in the blood and coelomic fluid may assist in equalization of osmotic pressures during dehydration and rehydration. This regulation may be a major function of the chloragosomes.The chloragosomes were discussed in relation to the spherites of various arthropods and molluscs and to the cytosomes of anoxia-tolerating molluscs.I wish to thank Theodore A. Hall for the use of the EMMA-4 microprobe facility, T.C. Norrnann for help in performing the microprobe analysis, Bruno Hansen for the improved zinc determination method and Annette Prentø for performing the chemical analyses. This work was supported by the Danish Natural Science Research Council (grant 511-3522). The EMMA-4 facility is supported by a grant from the British Science Research Council