Incorporation of 14C in the cyanobacterium Synechococcus PCC 6301 following salt stress

Synechococcus PCC 6301 synthesized sucrose as a compatible solute following hyperosmotic shock induced by NaCl. Initial rates of photosynthetic ¹⁴C incorporation were reduced following salt shock. Photosynthetic rates were comparable in cells enriched for glycogen (by growth in NO ₃ ^- -deficient medium) and cells grown in NO ₃ ^- -sufficient medium in the absence of osmotic shock. Incorporation of ¹⁴C was predominantly into the NaOH fraction and the residual acidic fraction in cells grown in NO ₃ ^- -sufficient medium, whereas incorporation was predominantly into the residual acidic fraction in cells grown in NO ₃ ^- -deficient medium. Following salt stress, ¹⁴C incorporation was initially into the ethanol-soluble fraction and the majority of tracer was recovered in sucrose. Carbon-14 was detected in sucrose in cells which had been enriched for [¹⁴C]glycogen prior to salt stress, inferring that glycogen can act as a carbon source for sucrose synthesis following salt stress. Changes in the specific activity of sucrose are consistent with an initial synthesis of sucrose from glycogen followed by synthesis of sucrose using newly fixed carbon, in response to salt stress.This work was supported by the Agricultural and Food Research Council.     

Products of CO2 fixation and 14C labelling pattern of alanine in Methanobacterium thermoautotrophicum pulse-labelled with 14CO2

The incorporation of ¹⁴CO₂ by an exponentially growing culture of the autotrophic bacterium Methanobacterium thermoautotrophicum has been studied. The distribution of radioactivity during 2s–120s incubation periods has been analyzed by chromatography and radioautography. After a 2 s incubation most of the radioactivity of the ethanolsoluble fraction was present in the amino acids alanine, glutamate, glutamine and aspartate, whereas phosphorylated compounds were only weakly labelled. The percentage of the total radioactivity fixed, which was contained in the principal early labelled amino acid alanine, increased in the first 20 s and only then decreased, indicating that alanine is derived from primary products of CO₂ fixation.The labelling patterns of alanine produced during various incubation times have been determined by degradation. After a 2 s ¹⁴CO₂ pulse, 61% of the radioactivity was located in C-1, 23% in C-2, and 16% in C-3. The results are consistent with the operation of a previously proposed autotrophic CO₂ assimilation pathway which involves the formation of acetyl CoA from 2 CO₂ via one-carbon unit intermediates, followed by the reductive carboxylation of acetyl CoA to pyruvate.     

Influence of growth regulator treatments on dry matter production,fruit abscission,and14C-assimilate partitioning in citrus

Experiments were performed to monitor (1) uptake and translocation of foliar-applied microdroplets of¹⁴C hormones and (2) effects of multiple growth regulator sprays on foliar and fruit growth variables and photosynthate partitioning in Valencia orange trees (Citrus sinensis (L.) Osbeck). The uptake of¹⁴C-sucrose,¹⁴C-paclobutrazol (PP333), and¹⁴C-napthaleneacetic acid (NAA) in 6-month-old greenhouse-grown trees exceeded that of¹⁴C-abscisic acid (ABA) and¹⁴C-benzyladenine (BA) 48 h after microdroplet application.¹⁴C-sucrose transport from the application site was much greater than any other source, especially¹⁴C-BA. In a second study, 2-year-old Valencia orange trees were maintained under field conditions and were sprayed to foliar runoff (3 × /week for 3 weeks) with BA, NAA, ABA, PP333, and gibberellic acid (GA₃) at 100 M during flowering and early fruit set. Select branches were then briefly exposed to¹⁴CO₂ and harvested 24 h later. Both GA₃ and BA sprays promoted foliar growth. BA also stimulated fruit growth, whereas GA₃ sharply increased fruit dry weight while fruit number decreased. BA and GA₃ enhanced¹⁴C assimilate export by the foliage to the developing fruit, and GA₃ was especially active in promoting fruit sink intensity (¹⁴C/dry wt). The other compounds (NAA, ABA, PP333) restricted foliar and fruit growth. They also inhibited transport of¹⁴C assimilate from the leaves to the fruit. Results indicate that foliar-applied growth regulators can influence source-sink relations in citrus early in reproductive development by manipulating photoassimilate production and partitioning.     

14C-Studies on Apple Trees. VII. The Early Seasonal Growth in Leaves, Flowers and Shoots as Dependent upon Current Photosynthates and Existing Reserves

The young leaves' consumption proper of photosynthates and their contribution to the growth of flowers, fruits and shoots by exposing spurs and shoots to ¹⁴CO₂ at the earliest stages of the growth period in apple trees (Malus X domestica) were studied. By a parallel determination of the growth intensity in various organs an attempt is made to evaluate their relative dependence on current photosynthates and on reserves from inside the tree. The proper fixation of ¹⁴C by growth in the exposed leaves is high in the earliest phases of growth. The fixation of ¹⁴C is considerable in the flowers, including the petals, immediately prior to flowering, in intensely growing fruits, and in the woody parts of the current year's shoots, when the main part of the terminal growth has been completed. Under conditions of high intensity of growth in an organ, the total fixation by growth in the parts studied may amount to as much as 80–90% of the ¹⁴C absorbed. Only in the very earliest phases of development does the growth of flowers and shoots appear to be based to a greater extent on materials supplied from reserves than from current photosynthesis. Quantitatively the greater part by far of the total new growth in fruits and shoots appears to be based on materials from current photosynthesis.     

A novel, convenient assay of lanosterol 14α-demethylase

A rapid and sensitive kinetic assay of lanosterol 14α-demethylation has been developed and analyzed. Three substrates, [32-³H]-24,25-dihydrolanosterol, [32-³H]lanost-8-en-3β,32-diol, and [32-³H]lanost-7-en-3β-32-diol, were studied. In all cases, the rate of tritium released into aqueous solution provided a simple and direct assay of 14α-demethylase activity. The kinetic parameters of K_m and V_max for each substrate have been determined in a reconstituted system from rat liver. The percentage of turnover monitored by the novel tritium release assay was comparable to that observed by conventional GC methods. Separation of unreacted sterol from tritiated formate and water via reverse-phase chromatography permitted several samples to be analyzed at once.     

Biosorption of Malathion by immobilized cells of Bacillus sp. S14

Abstract

Biosorption is potentially an attractive technology for the treatment of wastewater by removing pesticide molecules from dilute solutions. This study investigated the feasibility of an isolated Bacillus sp. S₁₄ immobilized in calcium alginate that was used as a biosorbent for Malathion removal from aqueous solutions in batch mode. The highest value of Malathion uptake by isolated Bacillus sp. S14 (1.33g L^?1, dry basis) immobilized in 3% calcium alginate was 64.4% at 25°C and pH7.0 when the initial Malathion concentration was 50 mg L^?1. Equilibrium was attained at 8h. The sorption data conformed well to the Fruendlich isotherm model.     

Chromosome 14 in B10.A(18R) mice is recombinant and includes Tcra-V a alleles

Analysis of mouse Tcr genes has previously defined at least five different Tcra-V haplotypes among inbred strains of mice. For mice of the Tcra-V ^b haplotype, including C57BL/10 (B10), T-cell expression of the Tcra-V11 gene subfamily can be detected with a monoclonal antibody, 1.F2. In the course of further characterizing the specificity of 1.F2, we found that it fails to recognize Tcra-V11-expressing T-cell hybrids derived from the B10 congenic strain, B10.A(18R)/SgIcr. Moreover, staining analysis indicated that the Va11 epitope recognized by 1.F2 is not expressed by peripheral T cells from several different B10.A(18R) colonies with the exception of that at the Research Institute of Scripps Clinic. Nucleotide sequences were determined for cDNA representing rearranged Tcra-V11 genes from two independent, B10.A(18R)/SgIcr derived T-cell hybrids. The two Tcra-V11 gene segments were identical and the predicted amino acid sequence differed by at least five residues from Tcra-V11 sequences previously obtained from B10.A mice. Southern blot analysis of restriction fragment length polymorphisms (RFLP) associated with Tcra-V11, as well as Tcra-V ₁ , subfamily genes revealed that the B10.A(18R) mouse has inherited Tcra-V ^a alleles rather than the expected Tcra-V ^b alleles from the B10 strain. RFLP analysis of the Rib-1 locus, located in close proximity to the Tcra locus on chromosome 14, showed that B10.A(18R) carries the Rib-1 ^b allele from B10. These results indicate that the B10.A(18R) mouse has inherited a recombinant chromosome 14 with a recombination event having occured between the Rib-1 locus and the Tcra-V ^a gene subfamilies examined. Inheritance of Tcra-V ^a alleles in B10.A(18R) probably originated from strain 129/J which breeding records show was used in the first cross with B10.A in the production of B10.A(18R) and which we found exhibits Tcra-V11 ^a RFLPs.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M55634 and M55635. Address correspondence and offprint requests to: P. B. Nakajima.     

Molecular basis of the optochin-sensitive phenotype of pneumococcus: characterization of the genes encoding the F0 complex of the Streptococcus pneumoniaeStreptococcus oralis H+-ATPases

The gene responsible for the optochin-sensitive (Opt^S) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F₀ complex of an H⁺-ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin-sensitive resistant (Opt^S/Opt^R) phenotype. Our results also show that natural as well as laboratory Opt^R isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F F₀ complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opt^S) and M222 (an Opt^R strain produced by inter-species recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane-bound ATPase activity of the S. pneumoniae wild-type (Opt^S) strains, and found a 100-fold difference between Opt^S and Opt^R strains, both in growth inhibition and in membrane ATPase resistance.     

Microcalorimetric studies of the action of Er3+ on Halobacterium halobium R1 growth

A microcalorimetric technique was used to evaluate the influence of Er³⁺ on Halobacterium halobium R1 growth. By means of a LKB-2277 Bioactivity Monitor ampoule method, we obtained the thermogenic curves of H. halobium R1 growth at 37°C. In order to analyze the results, the relationship between k and C was obtained. The addition of Er³⁺ in low concentration cause a decrease of the maximum heat production P _max and growth rate constants k; however, Er³⁺ in a high concentration might promote growth of H. halobium R1. When Er³⁺ is in a much higher concentration, the growth of H. halobium R1 is inhibited completely. For comparison, the shapes of H. halobium R1 cells were observed by means of transmission electron microscope (TEM). According to the thermogenic curves and TEM photos of H. halobium R1 under different conditions, it is clear that the metabolic mechanism of H. halobium R1 growth has been changed with the addition of Er³⁺.     

Microcalorimetric Studies of the Biological Effects of Ho(III) on Halobacterium halobium R1

The biological effect of Ho³⁺ on Halobacterium halobium R1 growth was analyzed using the microcalorimetric method. Using the LKB-2277 Bioactivity Monitor with the ampoule method at 37°C, the thermogenic curves of the growth of H. halobium R1 were obtained. Then, the maximum power (P _m) and the growth rate constants (k) were determined, and the values of P _m and k were linked to the concentration of Ho³⁺. In all, the addition of Ho³⁺ cause a decrease in the maximum heat production and growth rate constants. To confirm the results, the shapes of H. halobium R1 cell addition with Ho³⁺ using a transmission electron microscope (TEM) were observed. According to the thermogenic curves and TEM photos of H. halobium R1 under different conditions, it is clear that the metabolic mechanism of H. halobium R1 growth has been changed with the addition of Ho³⁺.     

Burn-induced Oxidative Stress is Altered by a Low Zinc Status: Kinetic Study in Burned Rats Fed a Low Zinc Diet

The biological effect of Ho³⁺ on Halobacterium halobium R1 growth was analyzed by a microcalorimetric technique. By means of LKB-2277 Bioactivity Monitor, ampoule method at 37°C, we obtained the thermogenic curves of H. halobium R1 growth. To analyze the results, the maximum power (P _m) and the growth rate constants (k) were determined, which show that values of P _m and k are linked to the concentration of Ho³⁺. In all, the addition of Ho³⁺ causes a decrease of the maximum heat production and growth rate constants. For comparison, we observed the shapes of H. halobium R1 cell by means of transmission electron microscope (TEM). According to the thermogenic curves and TEM photos of H. halobium R1 under different conditions, it is clear that metabolic mechanism of H. halobium R1 growth has been changed with the addition of Ho³⁺.     

Modulation of Mouse Rod Photoreceptor Responses by Grb14 Protein

Previous experiments have indicated that growth factor receptor-bound protein 14 (Grb14) may modulate rod photoreceptor cGMP-gated channels by decreasing channel affinity for cGMP; however, the function of Grb14 in rod physiology is not known. In this study, we examined the role of Grb14 by recording electrical responses from rods in which the gene for the Grb14 protein had been deleted. Suction-electrode recordings from single mouse rods showed that responses of dark-adapted Grb14^−/− mice to brief flashes decayed more rapidly than strain-controlled wild type (WT) rods, with decreased values of both integration time and the exponential time course of decay (τ_REC). This result is consistent with an increase in channel affinity for cGMP produced by deletion of Grb14. However, Grb14^−/− mouse rods also showed little change in dark current and a large and significant decrease in the limiting time constant τ_D, which are not consistent with an effect on channel affinity but seem rather to indicate modulation of the rate of inactivation of cyclic nucleotide phosphodiesterase 6 (PDE6). Grb14 has been reported to translocate from the inner to the outer segment in bright light, but we saw effects on response time course even in dark-adapted rods, although the effects were somewhat greater after rods had been adapted by exposure to bleaching illumination. Our results indicate that the mechanism of Grb14 action may be more complex than previously realized.     

Studies on the interaction between ribosomes and 14 CH 3 -F 3 initation factor

Summary Initiation factor F₃ has been purified fromEscherichia coli and labelledin vitro by reductive alkylation. The¹⁴CH₃–F₃ so obtained had a specific activity of about 1 000 cpm/g and was shown to have retained its biological activity. Labelled F₃ binds to 30S ribosomal subunits ofEscherichia coli andBacillus stearothermophilus, but does not bind to either 70S ribosomes or 50S ribosomal subunits. The stoichiometry of the binding indicates that one molecule of¹⁴CH₃–F₃ is bound to each 30S ribosomal subunit. Several antibiotics, known to interact with 30S subunits, inhibit the binding. Functional studies indicate that F₃ is released from 30S ribosomes as a result of the formation of the 70S initiation complex.     

PHOTOASSIMILATION OF 14C-ACETATE BY ULVA LACTUCA1

The effect of light on the uptake of ¹⁴C-labeled acetate, glucose, α-ketoglutarate, mannitol, and glycine was investigated in Ulva lactuca var. rigida. Uptake in the light over that in the dark of ¹⁴C-acetale (8-fold) was far higher than that of the other compounds tested. Further study of the phenomenon showed that (1) an increase in light intensity from 60 through 1000 ft-c results in increased ¹⁴C-acetate uptake, the kinetics of which differ from that of ¹⁴CO₂ uptake versus light intensity over the same range: (2) the action spectrum of acetate uptake, between 450 and 730 nm conforms to the action spectrum for photosynthesis in Ulva; (3) the acetate uptake process is sensitive to photosynthetic poisons including N'- (3-4, dichlorophenyl)-N, N-dimethyl urea (DCMU), and phenazine methosulfate (PMS), and inhibitors of the Krebs cycle including sodium monofluoroacetate (MFA) and malonate; (4) uptake of acetate is favored by low CO₂ concentrations; (5) uptake of acetate is not sensitive to 10^?4 M uranyl nitrate; (6) continuous white light, with and without far-red radiation, indicates no significant phytochrome involvement in the process. These results point to an intracellular dependence of the assimilation of acetate on the Krebs cycle in some manner (possibly in cooperation with the glyoxylate cycle) other than simply releasing CO₂ from the acetate moiety with subsequent fixation via the Calvin cycle, and on some product(s) of photo-system 2, eg, NADPH⁺, reduced ferredoxin, or O₂.     

ALGAL EXCRETION OF 14C-LABELED COMPOUNDS AND MICROBIAL INTERACTIONS IN CYANIDIUM CALDARIUM MATS1

Acid spring effluents are often covered with mats of the eucaryotic phycocyanin-containing alga. Cyanidium caldarium. The primary bacterial component of such mats is an acidophilic strain of Bacillus coagulans, and the primary fungal component is Dactylaria gallopava. Because of the limited species diversity, C. caldarium mats appeared to be an excellent system for studying algal excretion and various microbial interactions in nature. From 2 to 6% of the NaH¹⁴CO₃ taken up by natural or laboratory populations of the alga was excreted as ¹⁴C-labeled materials. The maximum excretion occurred at temperature, light, and pH values optimum for NaH¹⁴CO₃ uptake. However, when excretion was expressed as a percentage of NaH14 CO₃ uptake, a higher percentage of the radioactivity was excreted at nonoptimal conditions for NaH¹⁴CO₃ uptake. Fungal biomass was directly proportional to algal density, but bacterial numbers varied widely and did not correlate with algal numbers. The bacterial and fungal components could be grown in mixed culture with either growing C. caldarium cultures or in an extract prepared, by healing algal cells.     

Pharmacokinetics of anti-ganglioside GD2 mAb 14G2a in a phase I trial in pediatric cancer patients

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with at ₁ ^2/ of 2.8±2.8 h and at ₁ ^2/ of 18.3±11.8 h. In general,t ₁ ^2/ was dose-dependent with a level of significance ofP=0.036, and it reached a plateau at doses of 250 mg/m² or more. Overall the peak serum levels were dose-dependent atP<0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m² and 250 mg/m². The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. Thet ₁ ^2/ of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease int ₁ ^2/ following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.This work was supported by grants from FDA, FD-R-000377 and NIH U10 CA 28439 and in part by a grant from the general Clinical Research Center program, MOI RR00827, of the National Center for Research Resources, National Institutes of Health. M. M. U.-F. and C.-S. H. were supported in part by a grant from the Children's Cancer Research Foundation, and R. A. R. was supported in part by NIH grant CA 42508     

Flow of 14C-methanol via assimilatory and dissimilatory sequences with yeast in presence of glucose

Experiments were performed to reveal the extent to which individual heterotrophic substrates of a mixture contribute to the overall carbon and energy metabolism. For this reason Hansenula polymorpha MH 20 was chemostatically (C-limited) cultivated at different growth rates on mixtures of methanol and glucose fed at proportions of 3:1 and 1:3 (in weight units), respectively. The distributions of ¹⁴C-carbon from methanol in biomass as well as carbon dioxide (and supernatant) fractions were determined. From these results it followed, firstly, that energy derived from methanol dissimilation was used in part for the incorporation of glucose carbon, resulting in carbon conversion efficiencies for this substrate equivalent to yield coefficients of 0.61–0.69 g/g. Secondly, the growth yield data revealed that the efficiency of methanol conversion had to be increased in order to account for the experimentally determined yield figures. This was further confirmed by theoretical treatment of the growth yield data which showed that these could only be obtained if P/O-quotients for methanol conversion similar to those for glucose, i.e. 2.0–2.5, were considered. The latter property was regarded as the main reason for the observed improvement of growth yield accompanying the simultaneous utilization of methanol and glucose in this yeast.Abbreviations ATP_M,a ATP required for incorporation of assimilated methanol at a given P/O-quotient - ATP_M,d ATP generated from dissimilated methanol at a given P/O-quotient - G and M glucose and methanol; respectively (the indices u, a, d and e mean utilized, assimilated, dissimilated and incorporated by excess energy, respectively) - PGA 3-phosphoglyceric acid - Y _G ^app apparent growth yield on glucose in presence of methanol - Y _G ^P/O theoretical growth yield on glucose at a given P/O-quotient     

Degradation of 14C-labelled lignin and dehydropolymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi

The ability of ericoid and ectomycorrhizal fungi to utilize ¹⁴C-labelled lignin and O¹⁴CH₃-labelled dehydropolymer of coniferyl alcohol as sole C sources has been assessed in pure culture studies. The results indicate that ericoid mycorrhizal fungi are more effective in degrading lignin than ectomycorrhizal fungi. Amongst the ectomycorrhizal fungi the facultative mycorrhizal fungus Paxillus involutus degraded lignin more readily than those which are normally considered to be obligately mycorrhizal fungi such as Suillus bovinus and Rhizopogon roseolus. The importance of these lignin degrading capabilities is discussed in relation to the predominance of specific mycorrhiza forms along a gradient of increasing organic matter and hence lignin content of soil.     

Inositol 1,4,5‐trisphosphate receptor regulates replication,differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi

In animals, inositol 1,4,5‐trisphosphate receptors (IP₃Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca²⁺ release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP₃R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP₃R genes expressing T. cruzi IP₃R (TcIP₃R) exhibited IP₃‐mediated Ca²⁺ release from the ER, and demonstrated receptor binding to IP₃. TcIP₃R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP₃R gene loci led to the death of the parasite, suggesting that IP₃R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP₃R displayed defects in growth, transformation and infectivity, indicating that TcIP₃R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP₃R exhibited a reduction of disease symptoms, indicating that TcIP₃R is an important virulence factor. Combined with the fact that the primary structure of TcIP₃R has low similarity to that of mammalian IP₃Rs, TcIP₃R is a promising drug target for Chagas disease.     

Growth and water use of the mangroves Rhizophora apiculata and R. stylosa in response to salinity and humidity under ambient and elevated concentrations of atmospheric CO2

Two mangrove species, Rhizophora apiculata and R. stylosa, were grown for 14 weeks in a multifactorial combination of salinity (125 and 350 mol m^?3 NaCl), humidity (43 and 86% relative humidity at 30°C) and atmospheric CO₂ concentration (340 and 700 cm³ m^?3). Under ambient [CO₂], growth responses to different combinations of salinity and humidity were consistent with interspecific differences in distribution along natural gradients of salinity and aridity in northern Australia. Elevated [CO₂] had little effect on relative growth rate when it was limited by salinity but stimulated growth when limited by humidity. Both species benefited most from elevated [CO₂] under relatively low salinity conditions in which growth was vigorous, but relative growth rate was enhanced more in the less salt-tolerant and more rapidly growing species, R. apiculata. Changes in both net assimilation rate and leaf area ratio contributed to changes in relative growth rates under elevated [CO₂], with leaf area ratio increasing with decrease in humidity. Increase in water use efficiency under elevated [CO₂] occurred with increase, decrease or no change in evaporation rates; water use characteristics which depended on both the species and the growth conditions. In summary, elevated [CO₂] is unlikely to increase salt tolerance, but could alter competitive rankings of species along salinity × aridity gradients.