Kinetics of biofilm nitrification

The reaction rates (r(NH(4) (+) ) and r(NO(2) (-) )) in the two-step nitrification reaction were measured in a fluidized-sand-bed biofilm reactor under a range of steady-state conditions with respect to bulk NH(4) (+), NO(2) (-), and O(2) concentrations. It was shown from theory and experiment that under low NH(4) (+) concentration conditions, if the O(2)/NH(4) (+) concentration ratio in the bulk liquid is less than the stoichiometric coefficient (3.4 mg/mg), then oxygen will be rate limiting. In all experiments r(NO(2) (-) ) decreased more than r(NH(4) (+) ) under low oxygen conditions. This resulted in high NO(2) (-) effluent concentrations under low residence time conditions. The influence of the oxygen penetration effects on the relative values of r(NH(4) (+) ) and r(NO(2) (-) ) was experimentally shown to be caused either by the Nitrobacter location in the inner biofilm regions or by a K(m) effect for oxygen. Theoretical support of these findings was provided by a differential diffusion-reaction model which was used to simulate the experimental results.     

Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.     

Size Differences Among Root-knot Nematodes on Resistant and Susceptible Alyceclover Genotypes

The influence of plant resistance on the size of individual root-knot nematodes was determined in greenhouse experiments. Five genotypes of alyceclover were inoculated with second-stage juveniles of Meloidogyne incognita race 3 or M. arenaria race 1. Plants were harvested at selected intervals and stained for detection of the nematodes, which were dissected from the roots. Length, width, and sagittal-sectional area of each animal were measured using an image-analysis system, and areas of nematodes in all stages were compared at different times and across alyceclover lines. Nematodes feeding on roots of resistant lines were consistently smaller than those on susceptible plants, with significant differences in growth detected after the final molt. Similar results were observed with both nematode species.     

Preparation and biological properties of native and recombinant ciliary neurotrophic factor.

CNTF (ciliary neurotrophic factor), purified from rabbit sciatic nerves by a relatively simple procedure, is bioactive in tissue culture at low picomolar concentration and appears as a doublet on polyacrylamide gel electrophoresis (PAGE). In these nerves, CNTF accounts for more than one-half of the survival-promoting activity on ciliary neurons. The concentration of CNTF in rabbit sciatic nerves is estimated to be 5 nmol/kg, more than 1000 times higher than would seem to be required to support neurons if the neurotrophic factor were homogeneously distributed. With recombinant DNA technology, rat CNTF has been synthesized in Escherichia coli, purified without denaturating agents, and found to be bioactive at a slightly lower concentration than CNTF extracted from rabbit sciatic nerves. After radioiodination, CNTF retains biological activity but is not specifically internalized and retrogradely transported in motor and sensory axons. In peripheral nerves, ciliary neurotrophic factor differs biologically from nerve growth factor (NGF) by its much higher tissue concentration and apparent lack of internalization by peripheral nerve axons.     

Neuroendocrine control of reduced persistence of egg-laying in domestic hens: evidence for the development of photorefractoriness.

Egg-laying in hens exposed for more than 11 months to photostimulatory daylengths was intermittent and associated with a reduction in numbers of yellow-yolky ovarian follicles. Old laying hens (105 weeks) had lower concentrations of luteinizing hormone (LH) in the pituitary gland and plasma and reduced pituitary gland responsiveness to chicken LH-releasing-hormones (LHRH-I and II) in vivo when compared with young laying hens (28 weeks). Four weeks after transfer from 14 to 8 h light/day, egg production almost stopped in old, but not in young hens, although plasma LH concentrations decreased in all birds. After transfer from 14 to 20 h light/day, plasma LH increased in young, but not in old, hens, without a change in the rate of egg production. Reproductive function was enhanced in old hens returned to long days after induction of a moult and ovarian regression by reducing daylength and dietary restriction. Moulted hens had a greater rate of egg production, higher concentrations of plasma LH and a greater pituitary-gland responsiveness to LHRH-II in vivo than unmoulted control hens. After transfer from 14 to 8 h light/day, egg-laying decreased more rapidly in unmoulted than in moulted hens; transfer to 17 h light/day increased egg production in moulted, but not in unmoulted, birds. Induction of ovarian regression in old hens by dietary restriction alone also enhanced reproductive function after the dietary restriction was relaxed. Egg-laying was more persistent in hens brought into lay for a second year by transferring them from 3 to 11 h light/day than in hens transferred from 3 to 20 h light/day. Egg production was stimulated in hens maintained on 3 or 11 h light/day for 42 weeks, after transfer to 20 h light/day. Egg production ceased in hens maintained on 20 h light/day for 46 weeks, after transfer to 3 h light/day. These observations are consistent with the view that poor persistence of laying in hens less than 2 years old and exposed continuously to long days is caused, in part, by a reduction in hypothalamic-gonadotroph function. This reduction in neuroendocrine function may be due, in part, to the development of relative photorefractoriness.     

Ultrastructural observations on the redial tegument of Paramphistomum epiclitum from the planorbid snail, Indoplanorbis exustus.

The morphology of the tegument in the redia of Paramphistomum epiclitum (Digenea: Paramphistomidae) resembles that shown by most larval and adult digeneans; an outer surface syncytium is in continuity with the cytoplasm of in-sunken, nucleated cytons. Although tegumental cytons usually contain a single nucleus, some display up to six nuclei. The tegumental syncytium lining the pharynx of P. epiclitum rediae lack underlying cytons. The apical membrane of the tegument is elaborated by folds and microvilli, which presumably facilitate uptake of nutrients and/or exchange of ions involved in osmoregulation. A single type of secretory body, resulting from the fusion of smaller vesicles produced at Golgi complexes in the cytons, occurs throughout the tegument. Uniciliate sensory receptors occur in the surface syncytium particularly around the oral opening.     

Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle

Although great strides have been made in understanding the genetics of Duchenne muscular dystrophy (DMD), uncertainty still remains as to the metabolic changes which are associated with the disease. We have used the recently discovered animal model of DMD, the mdx mouse, to study aspects of high energy phosphate metabolism and metabiolic control indices in dystrophic muscle. This model of DMD has the dual advantage of having a genetic defect which is homologous to that in human DMD, and it lacks the fatty infiltration and ncecrosis which makes biochemical analysis of DMD so difficult. We have used nuclear magnetic resonance sperctroscopy (NMR) to monitor developmental changes in high energy phosphates and pH. No differences were observed between young (< 40–50 days old) control and mdx mice. The pH increase and alterations in phosphate ratios (i.e., decline in PCr/ATP) observed in adult mdx vs. control mice are quantilatively similar to those observed in humans. Biochemical analysis showed a small decline in ATP and PCr content and a decline in some indices of energy status in adult mdx mice. As young mdx mice appeared to be normal, the lack of dystrophin does not correlate with metabolic changes. The changes which were observed were small enough that alterations in fibre composition could be the major contributory factor.     

A contribution to the biology ofBangasternus fausti [Col.: Curculionidae], a potential biological control agent of diffuse knapweed,Centaurea diffusa,and its effect on the host plant

The biology of the weevilBangasternus fausti (Reitter), the larvae of which live in the flowerheads ofCentaurea diffusa (De Lamarck) and some relatedCentaurea spp., was studied in the field and in the laboratory in northern Greece and in the laboratory in Rome, Italy. The species is univoltine and overwinters as adults. Under laboratory conditions adults lived up to 90 days and up to 359 eggs were deposited by a single female (n=18). The maximum number of eggs laid by a female per day was 12 and the maximum number of days a female laid eggs was 73. Generally, there is only one larva per seedhead. One-hundred percent seed destruction was recorded in 18.7% of 502 field collected seedheads, each with oneB. fausti egg. The average mortality of the species, from egg to adult, was 94.4% in the field during 1988 (n=200) and 95.8% during 1987 (n=502).      

Sequence-specific 1H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin

The aromatic region of the 1H-FT-NMR spectrum of the biologically fully-potent, monomeric human insulin mutant, B9 Ser----Asp, B27 Thr----Glu has been investigated in D2O. At 1 to 5 mM concentrations, this mutant insulin is monomeric above pH 7.5. Coupling and amino acid classification of all aromatic signals is established via a combination of homonuclear one- and two-dimensional methods, including COSY, multiple quantum filters, selective spin decoupling and pH titrations. By comparisons with other insulin mutants and with chemically modified native insulins, all resonances in the aromatic region are given sequence-specific assignments without any reliance on the various crystal structures reported for insulin. These comparisons also give the sequence-specific assignments of most of the aromatic resonances of the mutant insulins B16 Tyr----Glu, B27 Thr----Glu and B25 Phe----Asp and the chemically modified species des-(B23-B30) insulin and monoiodo-Tyr A14 insulin. Chemical dispersion of the assigned resonances, ring current perturbations and comparisons at high pH have made possible the assignment of the aromatic resonances of human insulin, and these studies indicate that the major structural features of the human insulin monomer (including those critical to biological function) are also present in the monomeric mutant.     

The R-state proinsulin and insulin hexamers mimic the carbonic anhydrase active site

The cobalt(II)-substituted proinsulin and insulin hexamers have been studied in solution via electronic absorption spectroscopy. Hexameric proinsulin is shown to undergo the phenol-induced T6 to R6 conformational transition in a manner analogous to that previously established for insulin. In the absence of coordinating anions, the coordination spheres of the Co(II) ions in the proinsulin and insulin R6 hexamers comprise identical pseudotetrahedral arrangements of 3 histidine residues and 1 hydroxide ion. At alkaline pH, the visible absorption spectrum of the phenol-induced R6 Co(II) center is strikingly similar to the distinctive spectrum of the alkaline form of Co(II)-carbonic anhydrase. Exogenous ligands may coordinate to the Co(II) ions of the R6 proinsulin and insulin hexamers via replacement of the hydroxide ion, forming pseudotetrahedral adducts possessing characteristic spectra. The binding affinity of such ligands is shown to be strongly pH-dependent. The data presented establish that, although the Co(II)-substituted proinsulin and insulin R6 hexamers lack enzyme-like activity, these species duplicate spectrochemical characteristics of the Co(II)-carbonic anhydrase active site that are believed to be important signatures of carbonic anhydrase catalytic function.