Physiological responses of Sargasso Sea picoplankton to nanomolar nitrate perturbations

A study was conducted in July 1989 at three stations in thenorthern Sargasso Sea, where picoplankton (<1 µm)provided approximately half of the standing crop of chlorophyll.Temporal changes in the position of the nitracline at a singlelocation indicated that the vertical supply of nitrate was notat ‘steady-state’ and phytoplankton distributionstracked the nitracline. Our main experimental objective wasto examine the short-term effects of ecologically significantnitrate perturbations (+20 and +100 nM) on the physiologyof <1 µm communities growing at low (nanomolar)ambient nitrate concentrations. A chemiluminescent nitrate methodwas used to measure the time course (up to 4 h) of nitratedisappearance at in situ irradiance, in parallel with measurementsof photosynthetic ¹⁴CO₂ assimilation. Picoplankton growing at<60 nM nitrate rapidly responded to nanomolar nitratesupplements with luxury consumption and enhanced photosynthesisin proportion to their ambient nitrate environment. Light-saturatedSynechococcus populations from the most nitrate-depleted waters(13 nM) had doubled their cellular rate of photosynthesisafter 4 h, in response to a 20 nM nitrate pulse.     

Ovine fetal electrocortical activity and regional cerebral blood flow

Fetuses of 12 near-term sheep were prepared for microsphere determination of cerebral blood flow. Experiments were performed 5 days postsurgery. The regional blood flows were measured in successive high (HV), low (LV) and high voltage electrocorticographic states. Comparisons were made between the observations made in the LV and averaged flanking HV cycles. Total cerebral blood flow was 95 +/- 8, 119 +/- 11 and 100 +/- 9 ml/min/100 g in HV, LV and HV, respectively. Low voltage electrocortical activity increased average cerebral blood flow by 22% (P less than 0.01). Significant changes were seen in all regions except the occipital cortex. The maximum change was observed in the thalamus in which the flows were 152 +/- 23, 243 +/- 35 and 138 +/- 20 ml/min/per 100 g tissue, respectively. The increase was 68% (P less than 0.001). The percent changes seen in the cerebrum are as follows: Frontal grey + 18%, frontal white + 22%, parietal white + 22%, temporal + 18%. A + 17% change was seen in the cord (P less than 0.03). It is concluded that in low voltage electrocortical activity all of the brain, except the occipital region, shows an increase in cerebral blood flow. This is probably secondary to a variance in cerebral activity. This preparation may be useful in localizing function in the fetal brain.     

Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine

Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO₂ had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H₂SO₄ substitution for HCl and N₂-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations.     

Isolation of 3 beta,20 alpha-hydroxysteroid oxidoreductase from sheep fetal blood

3 beta,20 alpha-Hydroxysteroid oxidoreductase has been isolated from ovine fetal blood by a 2,370-fold purification scheme of ammonium sulfate fractionation, calcium phosphate gel adsorption, affinity chromatography, and fast performance liquid chromatography. A new high performance liquid chromatography-based assay for measuring 20 alpha-reductase activity is described. The enzyme is a monomer with a molecular weight of 35,000 and uses NADPH as a cofactor for reductase activity. It reduces progesterone to 4-pregnen-20 alpha-ol-3-one or 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol with kinetic characteristics of Km = 30.8 microM and Vmax = 0.7 nmol min-1 (nmol of enzyme)-1 or Km = 74 microM and Vmax = 1.3 nmol min-1 (nmol of enzyme)-1, respectively. 5 alpha-Dihydrotestosterone competitively inhibits 20 alpha-reductase activity with a Ki value of 102 microM.     

Chromosome aberration and ploidy equilibrium of Vicia faba in tissue culture

Summary Different phytohormone concentrations induced different fequencies of various chromosome aberrations in calli of Vicia faba. NAA 10 ppm plus KT 2.5 ppm produced more haploids and NAA 30 ppm plus NAA 7.5 ppm produced more tetraploids and breakage. The relationship among the aberrations was analyzed. The hypothesis of ploidy equilibrium was established. The chromosome doubling rate and reduction rate of each treated group were calculated in relation to the observed data and the hypothesis. The frequency of tetraploids and breakage are correlated with each other. The frequency of total aberrations is linearly correlated with that of micronucleus formation. The regression equation is x=31.92+ 10.67 y.     

pH-dependent structural transition in rabbit skeletal troponin C

Although the crystal structure of troponin C is known (Herzberg, O., and James, M. N. G. (1985) Nature 313, 653-659; Sundaralingam, M., Bergstrom, R., Strasburg, G., Rao, S. T., Roychowdhury, P., Greaser, M., and Wang, B. C. (1985) Science 227, 945-948), its structure in solution, particularly under physiological conditions, has not been established. We examined the conformation of troponin C under a variety of conditions by measuring the distance between sites located in the N- and C-terminal domains using the technique of resonance energy transfer. The donor was the luminescent lanthanide ion Tb3+ bound at the low affinity metal sites in the N-terminal domain. The acceptor was 4-dimethylaminophenylazophenyl-4'-maleimide attached at Cys-98 in the C-terminal domain. The distance between these sites was found to be greater than 5.2 nm at pH 5.0, 2.7 nm at pH 6.8 for uncomplexed troponin C, and 4.1 nm for troponin C complexed with troponin I at pH 6.8. These findings suggest that uncomplexed troponin C undergoes a pH-dependent transition from an elongated conformation, compatible with the crystal structure at acidic pH, to a more compact conformation at neutral pH. When complexed with troponin I, troponin C adopts a conformation of intermediate length compared to the uncomplexed molecule at pH 6.8 and 5.0.     

Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

Molecular characterization of a mucin-type antigen associated with human pancreatic cancer. The DU-PAN-2 antigen

This work describes the molecular characterization of a human pancreatic cancer-associated antigen defined by a murine monoclonal antibody (DU-PAN-2). DU-PAN-2 antigen was isolated from a pancreatic adenocarcinoma cell line (HPAF) or patient's ascitic fluid, and the antigenic activity was monitored by competitive inhibition radioimmunoassay. Affinity chromatography and CsCl/guanidine HCl density gradient centrifugation were employed to remove other populations of mucin-type glycoproteins and noncovalently associated proteins, respectively. Three electrophoretically distinct components were detected by 1% agarose gel electrophoresis and were resolved by chromatography on Sepharose CL-4B. The major fraction (FII) was subjected to carbohydrate and amino acid analyses. The sum of threonine, serine, proline, glycine, and alanine comprised more than 50% of the amino acid residues. The saccharide units, O-glycosidically linked to the peptide via GalNAc, contained fucose, galactose, GlcNAc, GalNAc, and sialic acid. The total carbohydrate content of FI and FII was 80.8% and 77.4% by weight, respectively. The molecular weight of FII antigen showed two species of molecules of 1.45 X 10(6) and 4.59 X 10(6) by analytical sedimentation equilibrium. DU-PAN-2 antigen was susceptible to neuraminidase, pepsin, Pronase, and papain digestion. These results suggest that both protein components and sialic acid residues may play important roles in the binding of DU-PAN-2 antibody.