Chemoreception in the green algaDunaliella tertiolecta

A capillary assay was employed to quantify positive chemotactic responses in the motile, unicellular, marine algaDunaliella tertiolecta. Among a wide range of inorganic and organic compounds tested, only ammonium ion,l-tyrosine,l-tryptophan, andl-phenylalanine were found to be major atractants for the chlorophyte.l-Methionine andl-cysteine weakly attracted the alga at 10⁻³ M. The minimum concentration of the major attractants needed to elicit an observable chemotactic response was approximately 10⁻⁶ M. The maximum response occurred when the capillaries contained 10⁻⁵ Ml-tyrosine orl-tryptophan, 10⁻⁴ Ml-phenylalanine, and 10⁻³ M ammonium chloride. The other amino acids, carbohydrates, B-vitamins, urea, and nitrate were among the chemicals that failed to attractD. tertiolecta. The alga apparently possesses one chemoreceptor that binds ammonium ion only, and another chemoreceptor that binds the three aromatic amino acids.     

Molecular and biochemical characterization of an extracellular serine-protease from <Emphasis Type="Italic">Vibrio metschnikovii</Emphasis> J1

A protease-producing bacterium was isolated from an alkaline wastewater of the soap industry and identified as Vibrio metschnikovii J1 on the basis of the 16S rRNA gene sequencing and biochemical properties. The strain was found to over-produce proteases when it was grown at 30°C in media containing casein as carbon source (14,000 U ml⁻¹). J1 enzyme, the major protease produced by V. metschnikovii J1, was purified by a three-step procedure, with a 2.1-fold increase in specific activity and 33.3% recovery. The molecular weight of the purified protease was estimated to be 30 kDa by SDS-PAGE and gel filtration. The N-terminal amino acid sequence of the first 20 amino acids of the purified J1 protease was AQQTPYGIRMVQADQLSDVY. The enzyme was highly active over a wide range of pH from 9.0 to 12.0, with an optimum at pH 11.0. The optimum temperature for the purified enzyme was 60°C. The activity of the enzyme was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The kinetic constants K _m and K _cat of the purified enzyme using N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide were 0.158 mM and 1.14 × 10⁵ min⁻¹, respectively. The catalytic efficiency (K _cat /K _m) was 7.23 × 10⁸ min⁻¹ M⁻¹. The enzyme showed extreme stability toward non-ionic surfactants and oxidizing agents. In addition, it showed high stability and compatibility with some commercial liquid and solid detergents. The aprJ1 gene, which encodes the alkaline protease from V. metschnikovii J1, was isolated, and its DNA sequence was determined. The deduced amino acid sequence of the preproenzyme differs from that of V. metschnikovii RH530 detergent-stable protease by 12 amino acids, 7 located in the propeptide and 5 in the mature enzyme.     

Histidine Absorption across Apical Surfaces of Freshwater Rainbow Trout Intestine: Mechanistic Characterization and the Influence of Copper

The essential amino acid histidine performs critical roles in health and disease. These functions are generally attributed to the amino acid itself, but could also be mediated by a positive effect on trace element bioavailability. Mechanistic information regarding the absorption of histidine across the gastrointestinal tract is essential for understanding the interplay between amino acid and mineral nutrients and the implications of these interactions for nutrition and toxicology. Using intestinal brush-border membrane vesicles obtained from freshwater rainbow trout, absorption of histidine over the range 0.78–780 μm was found to be saturable, with a maximal transport rate (J _max) of 9.1 ± 0.8 nmol mg protein⁻¹ min⁻¹ and a K _m (histidine concentration required to reach 50% of this level) of 339 ± 68 μm. Histidine uptake was highly specific as 10-fold elevated levels of a variety of amino acids with putative shared transporters failed to significantly inhibit uptake. Elevated levels of d-histidine, however, impaired uptake of the natural l-isomer. The presence of “luminal” copper (8.3 μm) significantly increased both the J _max and K _m of histidine transport. This suggests that chelated copper–histidine species cross the brush-border epithelium through transport pathways distinct from those used by histidine alone.     

Amino acid transport inRhodotorula glutinis

The yeastRhodotorula glutinis was found to transport amino acids against a concentration gradient (100∶1 for 10⁻⁶ m l-lysine and 1500∶1 for 10⁻⁶ m α-aminoisobutyric acid). Anaerobically, the concentration gradients of free amino acids were occasionally higher than aerobically. The influx is saturable with an apparentK _m of 1mm forl-lysine and 2mm for α-aminoisobutyric acid. The pH optimum for AIB uptake was 5.0, the apparent activation energy between 5° and 30° was 13,200 cal/mole. Competition of an asymmetric nature among various amino acids for uptake was observed. Intracellular amino acids did not leave the cell under any conditions of incubation, short of breaking up the plasma membrane, but they showed a powerful “trans” inhibitory effect on the uptake of amino acids.     

Improvement of l-valine production at high temperature in Brevibacterium flavum by overexpressing ilvEBNrC genes

Brevibacterium flavum ATCC14067 was engineered for l-valine production by overexpression of different ilv genes; the ilvEBN^rC genes from B. flavum NV128 provided the best candidate for l-valine production. In traditional fermentation, l-valine production reached 30.08 ± 0.92 g/L at 31°C in 72 h with a low conversion efficiency of 0.129 g/g. To further improve the l-valine production and conversion efficiency based on the optimum temperatures of l-valine biosynthesis enzymes (above 35°C) and the thermotolerance of B. flavum, the fermentation temperature was increased to 34, 37, and 40°C. As a result, higher metabolic rate and l-valine biosynthesis enzymes activity were obtained at high temperature, and the maximum l-valine production, conversion efficiency, and specific l-valine production rate reached 38.08 ± 1.32 g/L, 0.241 g/g, and 0.133 g g⁻¹ h⁻¹, respectively, at 37°C in 48 h fermentation. The strategy for enhancing l-valine production by overexpression of key enzymes in thermotolerant strains may provide an alternative approach to enhance branched-chain amino acids production with other strains.     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Uptake of amino acids by actidione-treated yeast cells

Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10⁻⁴ m for glycine, 2.5×10⁻⁴ m forl-cysteine, 6×10⁻⁵ and 4×10⁻⁴ m forl-lysine, 3×10⁻⁵ and 6×10⁻⁴ m forl-methionine, 7–18×10⁻⁵ and 1.6×10⁻³ m forl-aspartic acid and 6×10⁻⁵ and 2×10⁻³ m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan (and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine, arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Overproduction and characterization of a recombinant <Emphasis Type="SmallCaps">D</Emphasis>-amino acid oxidase from <Emphasis Type="Italic">Arthrobacter protophormiae</Emphasis>

A screening of soil samples for d-amino acid oxidase (d-AAO) activity led to the isolation and identification of the gram-positive bacterium Arthrobacter protophormiae. After purification of the wild-type d-AAO, the gene sequence was determined and designated dao. An alignment of the deduced primary structure with eukaryotic d-AAOs and d-aspartate oxidases showed that the d-AAO from A. protophormiae contains five of six conserved regions; the C-terminal type 1 peroxisomal targeting signal that is typical for d-AAOs from eukaryotic origin is missing. The dao gene was cloned and expressed in Escherichia coli. The purified recombinant d-AAO had a specific activity of 180 U mg protein⁻¹ for d-methionine and was slightly inhibited in the presence of l-methionine. Mainly, basic and hydrophobic d-amino acids were oxidized by the strictly enantioselective enzyme. After a high cell density fermentation, 2.29 × 10⁶ U of d-AAO were obtained from 15 l of fermentation broth.     

Block of a Ca<Superscript>2+</Superscript>-activated Potassium Channel by Cocaine

The primary target for cocaine is believed to be monoamine transporters because of cocaine’s high-affinity binding that prevents re-uptake of released neurotransmitter. However, direct interaction with ion channels has been shown to be important for certain pharmacological/toxicological effects of cocaine. Here I show that cocaine selectively blocks a calcium-dependent K⁺ channel in hippocampal neurons grown in culture (IC₅₀ = ∼30 μM). Single-channel recordings show that in the presence of cocaine, the channel openings are interrupted with brief closures (flicker block). As the concentration of cocaine is increased the open-time is reduced, whereas the duration of brief closures is independent of concentration. The association and dissociation rate constants of cocaine for the neuronal Ca²⁺-activated K⁺ channels are 261 ± 37 μM⁻¹s⁻¹ and 11451 ± 1467 s⁻¹. The equilibrium dissociation constant (K_B) for cocaine, determined from single-channel parameters, is 43 μM. The lack of voltage dependence of block suggests that cocaine probably binds to a site at the mouth of the pore. Block of Ca²⁺-dependent K⁺ channels by cocaine may be involved in functions that include broadening of the action potential, which would facilitate transmitter release, enhancement of smooth muscle contraction particularly in blood vessels, and modulation of repetitive neuronal firing by altering the repolarization and afterhyperpolarization phases of the action potential.     

<Emphasis Type="Italic">In vitro</Emphasis> selection of NaCl-tolerant callus lines and regeneration of plantlets in a bamboo (<Emphasis Type="Italic">Dendrocalamus strictus</Emphasis> Nees)

Summary Sodium chloride-tolerant plantlets of Dendrocalamus strictus were regenerated successfully from NaCl-tolerant embryogenic callus via somatic embryogenesis. The selection of embryogenic callus tolerant to 100 mM NaCl was made by exposing the callus to increasing (0–200 mM) concentrations of NaCl in Murashige and Skoog medium having 3% (w/v) sucrose, 0.8% (w/v) agar, 3.0 mg l⁻¹ (13.6 μM) 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.5mg l⁻¹ (2.3μM) kinetin (callus initiation medium). The tolerance of the selected embryogenic callus to 100 mM NaCl was stable through three successive transfers on NaCl-free callus initiation medium. The tolerant embryogenic callus had high levels of Na⁺, sugar, free amino acids, and proline but a slight decline was recorded in K⁺ level. The stable 100 mM NaCl-tolerant embryogenic callus differentiated somatic embryos on maintenance medium [MS medium +3% sucrose +0.8% agar +2.0 mg l⁻¹ (9.0 μM) 2,4-D+0.5 mg l⁻¹ (2.3 μM) kinetin] supplemented with different (0–200 mM) concentrations of NaCl. About 39% of mature somatic embryos tolerant to 100 mM NaCl germinated and converted into plantlets in germination medium [half-strength MS+2% sucrose+0.02 mg l⁻¹ (0.1 μM) α-naphthaleneacetic acid +0.1 mg l⁻¹ (0.49 μM) indole-3-butyric acid] containing 100 mM NaCl. Of these plantlets about 31% established well on transplantation into a garden soil and sand (1:1) mixture containing 0.2% (w/w) NaCl.     

Putative ClC-2 Chloride Channel Mediates Inward Rectification in <Emphasis Type="Italic">Drosophila </Emphasis>Retinal Photoreceptors

We report that Drosophila retinal photoreceptors express inwardly rectifying chloride channels that seem to be orthologous to mammalian ClC-2 inward rectifier channels. We measured inwardly rectifying Cl⁻ currents in photoreceptor plasma membranes: Hyperpolarization under whole-cell tight-seal voltage clamp induced inward Cl⁻ currents; and hyperpolarization of voltage-clamped inside-out patches excised from plasma membrane induced Cl⁻ currents that have a unitary channel conductance of ∼3.7 pS. The channel was inhibited by 1 mM Zn²⁺ and by 1 mM 9-anthracene, but was insensitive to DIDS. Its anion permeability sequence is Cl⁻ = SCN⁻> Br⁻>> I⁻, characteristic of ClC-2 channels. Exogenous polyunsaturated fatty acid, linolenic acid, enhanced or activated the inward rectifier Cl⁻ currents in both whole-cell and excised patch-clamp recordings. Using RT-PCR, we found expression in Drosophila retina of a ClC-2 gene orthologous to mammalian ClC-2 channels. Antibodies to rat ClC-2 channels labeled Drosophila photoreceptor plasma membranes and synaptic regions. Our results provide evidence that the inward rectification in Drosophila retinal photoreceptors is mediated by ClC-2-like channels in the non-transducing (extra-rhabdomeral) plasma membrane, and that this inward rectification can be modulated by polyunsaturated fatty acid. G. Ugarte and R. Delgado contributed equally to this work.     

Signaling Pathways in the Biphasic Effect of ANG II on Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchanger in T84 Cells

The effect of ANG II on pH_i, [Ca²⁺]_i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pH_i via the Na⁺/H⁺ exchanger was examined in the first 2 min following the acidification of pH_i with a NH₄Cl pulse. In the control situation, the pH_i recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10⁻¹² M or 10⁻⁹ M) increased this value (by 106% or 32%, respectively) but ANG II (10⁻⁷ M) decreased it to 47%. The control [Ca²⁺]_i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pH_i recovery and [Ca²⁺]_i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na⁺/H⁺ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10⁻¹² – 10⁻⁷ M ANG II) and by increases of [Ca²⁺]_i in the lower range (at 10⁻¹² M ANG II) and 2) inhibition of the exchanger at high [Ca²⁺]_i levels (at 10⁻⁹ – 10⁻⁷ M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.     

Identification and characterization of a novel nitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Pf-5

Nitrile groups are catabolized to the corresponding acid and ammonia through one-step reaction involving a nitrilase. Here, we report the use of bioinformatic and biochemical tools to identify and characterize the nitrilase (NitPf5) from Pseudomonas fluorescens Pf-5. The nitPf5 gene was identified via sequence analysis of the whole genome of P. fluorescens Pf-5 and subsequently cloned and overexpressed in Escherichia coli. DNA sequence analysis revealed an open-reading frame of 921 bp, capable of encoding a polypeptide of 307 amino acids residues with a calculated isoelectric point of pH 5.4. The enzyme had an optimal pH and temperature of 7.0°C and 45°C, respectively, with a specific activity of 1.7 and 1.9 μmol min⁻¹ mg protein⁻¹ for succinonitrile and fumaronitrile, respectively. The molecular weight of the nitrilase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography was 33,000 and 138,000 Da, respectively, suggesting that the enzyme is homotetrameric. Among various nitriles, dinitriles were the preferred substrate of NitPf5 with a K _m = 17.9 mM and k _cat/K _m = 0.5 mM⁻¹ s⁻¹ for succinonitrile. Homology modeling and docking studies of dinitrile and mononitrile substrate into the active site of NitPf5 shed light on the substrate specificity of NitPf5. Although nitrilases have been characterized from several other sources, P. fluorescens Pf-5 nitrilase NitPf5 is distinguished from other nitrilases by its high specific activity toward dinitriles, which make P. fluorescens NitPf5 useful for industrial applications, including enzymatic synthesis of various cyanocarboxylic acids.     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.     

Olfactory sensitivity to amino acids in the blackspot sea bream (<Emphasis Type="Italic">Pagellus bogaraveo</Emphasis>): a comparison between olfactory receptor recording techniques in seawater

The current study investigated the olfactory sensitivity of the blackspot sea bream to amino acids, odorants associated with food detection in fish, and compared the efficacy of two different experimental methods: multi-unit recording from the olfactory nerve and the electro-olfactogram (EOG). Twenty essential amino acids plus l-DOPA evoked clear, concentration-dependent olfactory responses using both methods, with estimated thresholds of 10^−8.5–10^−6.2 M (nerve recording) and 10^−7.5–10^−4.8 M (EOG). The most potent amino acids were l-cysteine, l-methionine (both sulphur-containing), l-alanine, l-leucine (both neutral), l-glutamine (amide-containing) and l-serine (hydroxyl-containing). The least potent were l-proline (secondary α-amino group), the aromatic amino acids and glycine (simplest). Although the rank order of olfactory potency was similar for the two methods used, and the calculated thresholds given by the two methods were positively correlated, the sensitivity of the EOG was consistently lower than multi-unit recording by approximately one order of magnitude, presumably due to the electrical shunting effect of seawater. As in freshwater, the EOG could be a valid method for comparing olfactory potency of different odorants in stenohaline marine fish; however, for absolute ‘biological’ thresholds, a more invasive recording technique, such as multi-unit recording from the olfactory nerve, should be used.     

Plasma free amino acid kinetics in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) using a bolus injection of <Superscript>15</Superscript>N-labeled amino acids

To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of ¹⁵N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R _d), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R _d values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R _d values ranged from 0.9 μmol 100 g⁻¹ h⁻¹ (lysine) to 22.1 μmol 100 g⁻¹ h⁻¹ (threonine) with most values falling between 2 and 6 μmol 100 g⁻¹ h⁻¹. There was a significant correlation between R _d and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) synthesis from glucose

l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS⁻ gluc⁺ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q _l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q _l-Tyr in the PTS⁺ and the PTS⁻ gluc⁺ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS⁻ gluc⁺ tyrR ⁻ strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS⁻ gluc⁺ tyrR ⁻ phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.     

Oxygen consumption rate and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in early developmental stages of the sea urchin <Emphasis Type="Italic">Paracentrotus lividus</Emphasis> Lam.

Changes in oxygen consumption rate and Na⁺/K⁺-ATPase activity during early development were studied in the sea urchin Paracentrotus lividus Lam. The oxygen consumption rate increased from 0.12 μmol O₂ mg protein⁻¹ h⁻¹ in unfertilized eggs to 0.38 μmol O₂ mg protein⁻¹ h⁻¹ 25 min after fertilization. Specific activity of the Na⁺/K⁺-ATPase was significantly stimulated after fertilization, ranging up to 1.07 μmol P_i h⁻¹ mg protein⁻¹ in the late blastula stage and slightly lower values in the early and late pluteus stages.