Evidence of paternal nutrient provisioning to embryos in broad-nosed pipefish Syngnathus typhle

In two experiments, radioactively labelled nutrients (either (3)H-labelled amino-acid mixture or (14)C-labelled glucose) were tube-fed to brooding male Syngnathus typhle. Both nutrients were taken up by the males and radioactivity generally increased in the brood pouch tissue with time. Furthermore, a low but significant increase of (3)H-labelled amino acids in embryos was found over the experimental interval (48 h), whereas in the (14)C-glucose experiment the radioactivity was taken up by the embryos but did not increase over the experimental time (320 min). Uptake of radioisotopes per embryo did not differ with embryo size. A higher uptake mg(-1) tissue of both (3)H-labelled amino acids and (14)C-labelled glucose was found in smaller embryos, possibly due to a higher relative metabolic rate or to a higher surface-area-to-volume ratio compared to larger embryos. Uptake in embryos was not influenced by male size, embryonic developmental advancement or position in the brood pouch. It is concluded that brooding males provide amino acids, and probably also glucose, to the developing embryos in the brood pouch.     

A new method for the measurement of protein turnover.

A new technique for the determination of rate constants of protein degradation is described. By using the method, half-lives of total soluble protein of Lemna minor during growth on full culture medium and distilled water were measured. The method involves incubating Lemna on a growth medium containing 3H2O. After a short exposure (20 min) to 3H-labelled culture medium, 3H was found in soluble amino acids, especially aspartate, glutamate, glutamine and alanine. After transfer to a 3H-free medium for 30 min, 80% of the 3H originally present in soluble amino acids was lost. These results suggest that 3H enters and leaves amino acids at the alpha-carbon atom, a conclusion supported by the observed labelling of glutamates. The exchange of H and 3H on the alpha-carbon atom is catalysed by transaminases and the speed of this exchange ensures that when the 3H2O is removed, the 3H in free amino acids is rapidly lost, thereby eliminating problems connected with metabolic pools and recycling. After an exposure of 20 min to 3H-labelled medium all protein amino acids, except for arginine, were found to be radioactive. The loss of radioactivity from protein amino acids was used to measure protein degradation.     

Relationship between intracellular amino acids and protein synthesis in the extensor digitorum longus muscle of rats

1. The incorporation into protein, and the accumulation into the free amino acid pools, of radioactive l-leucine and glycine was studied in rat extensor digitorum longus muscle. 2. The tissue was incubated first with (14)C-labelled and then with (3)H-labelled amino acid. 3. The experimental results were consistent with a model based on the premise that the amino acids in protein were incorporated directly from the extracellular pool.     

The third ribosomal tRNA-binding site, the E site, is occupied in native polysomes

The nucleic acids of Escherichia coli cells were uniformly labelled with 32P by growing the cells in [32P]orthophosphoric acid for about four generations. The cells were harvested in the logarithmic phase, resuspended in a buffer containing 6 mM Mg2+, 150 mM NH4+ and polyamines and incubated for 3 min at 37 degrees C in the presence of 3H-labelled amino acids. This procedure preferentially labels growing peptidyl chains. Polysomes were isolated, the fraction in the post-translocational state was assessed by a puromycin reaction and the tRNA content/70S ribosome was quantified in comparison to the amount of 5S rRNA determined after separation by gel electrophoresis. The data revealed that at least 75% of post-translocational ribosomes in isolated native polysomes carry a tRNA in their E site. The results are consistent with the allosteric three-site model for the elongation cycle but disagree with the two-site model.     

Determination of isokinetic ratios necessary for equimolar incorporation of carboxylic acids in the solid-phase synthesis of mixture-based combinatorial libraries

The methods used to study the relative reaction rates of 45 different aliphatic and aromatic carboxylic acids when coupled to resin-bound amino acid amides is described. Competition experiments involving the coupling of incoming carboxylic acids to resin-bound amino acid amides were performed. The relative composition of each N-acylated amino acid amide in the resulting mixtures was compared to controls prepared by physically mixing equal aliquots of individual compounds in order to study the relative reaction rates of the incoming carboxylic acids. The ratios of the incoming carboxylic acids were then iteratively adjusted to yield as close to equimolar products as possible. As expected, the steric and electronic nature of the incoming carboxylic acids was found to influence their relative reaction rates. The steric hindrance of the resin-bound amino acid appears to have a proportional effect on the reaction rates of the incoming carboxylic acids. N-acylated amino acid amides in the final mixtures, prepared using the final isokinetic ratios, were found to be approximately equimolar.     

Platelet-derived growth factor (PDGF) induces intranuclear protein accumulation in 3T3 fibroblasts

Quiescent 3T3 fibroblasts were grown for a short time in the presence of [3H]amino acids then treated with PDGF, and the nucleo-cytoplasmic distribution of the 3H-labelled proteins was analysed by autoradiography. There was no difference in the total amount of 3H-labelled proteins in PDGF-treated and untreated cells but PDGF induced a significant increase in intranuclear protein accumulation.     

Modeling dynamic experiments on the anaerobic degradation of molasses wastewater

The kinetics of anaerobic degradation of a molasses wastewater were measured under constant pH conditions in a laboratory scale packed bed reactor. In continuous and batch experiments the formation and degradation rates of the organic acids (butyric, propionic and acetic) have been followed. The influence of hydrogen gas on the acid degradation rates has been measured and, contrary to the literature and the thermo-dynamic calculations, no inhibition was detected, biofilm diffusional effects may be the reason. Two dynamic simulation models were tested, a heterogeneous model, which considered the biofilm diffusion-reaction phenomena and a quasihomogeneous model with the same kinetics. Except for hydrogen, the diffusion effects were found to be negligible. Otherwise both models gave essentially the same results and the time profiles of acids, hydrogen, carbon dioxide and methane agreed relatively well with dynamic startup experiments. Batch experiments showed the acid concentrations to be highly sensitive to the initial molasses concentration. This aspect was not included in the model but is being investigated further.     

Determination of limiting mobilities and dissociation constants of 21 amino acids by capillary zone electrophoresis at very low pH

The dependence of the effective electrophoretic mobility on pH of the background electrolyte was experimentally determined by capillary zone electrophoresis (CZE) for cationic forms of amino acids. The pH of the background electrolytes was in the highly acidic range, 1.6-2.6 pH units, to ensure a high degree of protonation of the amino acids. Poly(vinyl alcohol) was added to the background electrolytes to avoid possible adsorption of the analytes at the inner capillary wall. Non-linear regression of the experimental data was applied to obtain the parameters of the relevant regression functions--the actual mobilities and mixed dissociation constants corresponding to the actual ionic strength. The extended Onsager and Debye-Hückel law was used to calculate the limiting mobilities and thermodynamic dissociation constants. The comparison of the experimental electropherogram with the computer prediction by PeakMaster using the determined data is presented for the selected sample of amino acids.     

The site of amino acid addition to posttranslationally modified proteins of regenerating rat sciatic nerves

The posttranslational modification of proteins by amino acids has been described in a variety of biological systems. These reactions occur at low levels in intact sciatic nerves of rats but are increased 10-fold following nerve injury and during subsequent regeneration of the nerve. While it has been shown in brain and liver that the site of addition of Arg is to the N-terminus, there is no information on the location at which the other amino acids add on to targeted proteins nor the site of addition of Arg in regenerating nerves. In the present study, we have used manual micro-Edman degradation combined with HPLC, and digestion with carboxypeptidase A and B to determine the site of addition of various amino acids to targeted proteins. Of the 3H-labelled amino acids incorporated posttranslationally into proteins of regenerating sciatic nerves (Arg, Lys, Leu, Phe, Val, Ala, Pro and Ser), only [3H]Arg was found to be present at the N-terminus. To determine whether amino acid additions were occurring at the C-terminus, proteins modified by two of the amino acids incorporated in greatest amounts (Lys and Leu) were incubated with specific carboxypeptidases. [3H]Leucine was not liberated following incubation with carboxypeptidase, suggesting that Leu is not added at the C-terminus of modified proteins. Under similar conditions, some [3H]Lys was liberated, but in amounts not significantly different from controls incubated without carboxypeptidase, indicating a non-specific degradation of Lys modified proteins rather than a specific release of Lys from the C-terminus. These experiments show that in regenerating sciatic nerves of rats, Arg is the only amino acid added posttranslationally to the amino terminus of target proteins, and that Leu, and probably Lys, are not conjugated to proteins at the C-terminus.     

Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis

Fluxes in central carbon metabolism of a genetically engineered, riboflavin-producing Bacillus subtilis strain were investigated in glucose-limited chemostat cultures at low (0.11 h(-1)) and high (0.44 h(-1)) dilution rates. Using a mixture of 10% [U-(13)C] and 90% glucose labeled at natural abundance, (13)C-labeling experiments were carried out to provide additional information for metabolic flux balancing. The resulting labeling pattern in the proteinogenic amino acids were analyzed by two-dimensional [(13)C, (1)H] nuclear magnetic resonance (NMR) spectroscopy. To account rigorously for all available data from these experiments, we developed a comprehensive isotopomer model of B. subtilis central metabolism. Using this model, intracellular carbon net and exchange fluxes were estimated on the basis of validated physiological data and biomass composition in combination with 2D NMR data from 45 individual carbon atom spectra in the amino acids. Glucose catabolism proceeded primarily via glycolysis but pentose phosphate pathway fluxes increased with increasing growth rate. Moreover, significant back fluxes from the TCA cycle to the lower part of glycolysis via the gluconeogenic PEP carboxykinase were detected. The malic enzyme reaction, in contrast, was found to be inactive. A thorough statistical analysis was performed to prove the reliability of the isotopomer balance model and the obtained results. Specifically, a chi(2) test was applied to validate the model and the chi-square criterion was used to explore the sensitivity of model predictions to the experimental data.     

Uptake of L-leucine and L-phenylalanine across the basolateral cell surface in isolated oxyntic glands.

The time course, kinetic, specificity and sodium-dependence of L-leucine and L-phenylalanine uptake by rabbit isolated oxyntic glands were studied in order to identify the systems involved in the transport of branched-chain and aromatic neutral amino acids through the basolateral cell membrane. The uptake was measured directly in the disrupted cells after incubation of the glands with the 3H-labelled amino acid both in a sodium-containing and a sodium-free medium. The uptake of L-leucine was largely carrier-mediated whilst L-phenylalanine was taken up by either carrier-mediated and nonsaturable processes. Both amino acids were taken up by a Na(+)-independent process. The kinetic parameters of L-leucine and L-phenylalanine carrier-mediated influx were, respectively: Kt = 2.71 mM and Jmax = 1390 nmol mg-1 s-1, Kt = 1.03 mM and Jmax = 176 nmol mg-1 s-1. From cross-inhibition studies it can be inferred that L-leucine is primarily transported by a Na(+)-independent system which shows specificity for bulky side chains dipolar amino acids. The system displays similar affinities for L-phenylalanine (Ki = 2.81 mM) and L-isoleucine (Ki = 2.62 mM). Similar results were obtained from self-inhibition experiments: the Ki of the carrier-mediated uptake of L-leucine and L-phenylalanine were 2.12 and 2.40 mM (from a Hanes plot) or 3.2 and 0.8 mM (from a Dixon plot), respectively. It is concluded that a sodium-independent transport system, like Christensen's 'L' type, is shared by branched-chain and aromatic dipolar amino acids, which only shows slight differences in their affinities for the carrier.     

Studies on the coupling rates in liquid-phase peptide synthesis using competition experiments.

Competition experiments were carried out in order to determine relative reaction rates, Vrel, for the peptide-forming step. Using the liquid-phase method of peptide synthesis a model system was developed to investigate the influence of coupling method, amino component, derivatization and solvent upon Vrel. According to a standard procedure, Vrel for all 20 commonly occurring amino acids were established. A strong dependence of Vrel upon steric effects of the coupling components was found. The data obtained may serve as a guideline for optimizing the reaction conditions in peptide synthesis.     

Polysulfone affinity membranes for the treatment of amino acid mixtures

Affinity membranes for the treatment of solutions containing amino acids were obtained via lithiating polysulfone that was subsequently converted with glycidylether. From this polymer asymmetric ultrafiltration membranes were cast. The membranes were reacted with iminodiacetic acid yielding membranes fitted out with bidentate chelates. The same reaction path was applied to commercially available symmetric microfiltration membranes. The chelate-bearing membranes were complexed with Cu, Ni, and Zn ions. For the experiments with amino acids only the Cu-complexed membranes were used. The complexation constants for histidine and tryptophan for six different membranes were determined. Because of the affinity of these two amino acids for the complexed Cu ions, they could easily be separated from solutions containing amino acids such as alanine, glycine, and valine. Also, concentrating very dilute amino acid solutions was carried out successfully. (c) 1995 John Wiley & Sons, Inc.     

Evaporation cycle experiments — A simulation of salt-induced peptide synthesis under possible prebiotic conditions

Evaporation cycles applied to dilute solutions of amino acids, Cu(II) and NaCl lead to peptides within 1–3 days. This simulation of possible coastal or laguna processes in a primitive earth environment gives further indications towards the relevance of the salt-induced peptide formation reaction in chemical evolution. The experiments were successfully applied to glycine, alanine, aspartic and glutamic acid. Besides isolated amino acids, also their mixtures with glycine as reaction partner were studied, leading to peptides for all of the aforementioned substances, as well as for valine and proline, which do not dimerize alone. Sequence preferences and some conservation of optical purity were observed.     

ETA: Robust software for determination of cell specific rates from extracellular time courses

Accurate quantification of cell specific rates and their uncertainties is of critical importance for assessing metabolic phenotypes of cultured cells. We applied two different methods of regression and error analysis to estimate specific metabolic rates from time‐course measurements obtained in exponentially growing cell cultures. Using simulated data sets to compute specific rates of growth, glucose uptake, and lactate excretion, we found that Gaussian error propagation from prime variables to the final calculated rates was the most accurate method for estimating parameter uncertainty. We incorporated this method into a MATLAB‐based software package called Extracellular Time‐Course Analysis (ETA), which automates the analysis workflow required to (i) compute cell specific metabolic rates and their uncertainties; (ii) test the goodness‐of‐fit of the experimental data to the regression model; and (iii) rapidly compare the results across multiple experiments. ETA was used to estimate the uptake or excretion rate of glucose, lactate, and 18 different amino acids in a B‐cell model of c‐Myc‐driven cancer. We found that P493‐6 cells with High Myc expression increased their specific uptake of glutamine, arginine, serine, lysine, and branched‐chain amino acids by two‐ to threefold in comparison to low Myc cells, but exhibited only modest increases in glucose uptake and lactate excretion. By making the ETA software package freely available to the scientific community, we expect that it will become an important tool for rigorous estimation of specific rates required for metabolic flux analysis and other quantitative metabolic studies. Biotechnol. Bioeng. 2013; 110: 1748–1758. © 2013 Wiley Periodicals, Inc.     

Diethyl pyrocarbonate inactivates swine pepsinogen by reaction at amino groups

One of the histidine residues in swine pepsinogen can be modified in a rapid reaction with diethyl pyrocarbonate (DEP). At the same time, the potential proteolytic activity of the zymogen is reduced to 40% of its initial value. Reaction at the histine residue is reversed by neutral hydroxylamine, but the loss in activity is not. Fractionation of DEP-pepsinogen activation mixtures gave fully active pepsin in reduced yield, not modified enzyme with reduced specific activity. This was taken to indicate that the reaction had produced a mixture of products. Irreversible incorporation of [¹⁴C]DEP indicates that carbethoxy groups are incorporated into the molecule at amino acids other than histidine. The positions of these carbethoxy groups were determined by tryptic digestion and hplc. Modification was found to have occurred, in nonstoichiometric amounts, at lysine residues 3 and 9 and at leucine 1. Control experiments showed that activation was not affected by reaction at leucine 1, indicating that inactivation is caused by reaction at one or more of the lysine residues.     

Ca2+-dependence of the evoked release from guinea pig cerebral cortex slices of endogenous 14C-labelled amino acids, labelled via D-[U-14C]glucose.

Spontaneous and electrically evoked release of exogenous labelled amino acids and endogenous amino acids labelled from D-[U-14C]glucose were compared in control and Ca2+-free medium using guinea pig cerebral cortex slices. Spontaneous release of all labelled amino acids, except that of endogenous 14C-labelled threonine-serine-glutamine (unseparated) and exogenous [14C]aspartate, was doubled in Ca2+-free medium. The major portion of the electrically evoked release of endogenous [14C]glutamate, [14C]aspartate, gamma-amino[14C]butyrate (14C-labelled GABA) and exogenous 3H-labelled GABA was Ca2+-inpendent. More than half of the evoked release of the other labelled amino acids was Ca2+-independent. As the pattern of Ca2+-dependence of the evoked release concurred with the selectivity of the evoked release for endogenous [14C]-glutamate, [14C]aspartate, and 14C-labelled GABA, it was concluded that these labelled amino acids were probably released from the amino acid 'transmitter pool'.     

Thermophilic anaerobic amino acid degradation: deamination rates and end-product formation

Anaerobic thermophilic degradation of several amino acids was studied in batch cultures using an inoculum from a steady-state semicontinuous enrichment culture. Experiments were done in the presence and absence of methanogenesis and known electron acceptors in the Stickland reaction. Methanogenesis was found to be crucial for the degradation of amino acids known to be oxidatively deaminated (leucine, valine and alanine). Other amino acids (serine, threonine, cysteine and methionine) were degraded under both methanogenic and non-methanogenic conditions. Degradation rates for these four amino acids were 1.3 to 2.2 times higher in cases where methanogenesis was active. The degradation rates of serine, threonine, cysteine and methionine were about twice as high as the rates of leucine, valine and alanine under methanogenic conditions. Inclusion of different electron acceptors, known to work in the Stickland reaction, did not enhance the degradation rates of any amino acid used nor did they alter the degradation patterns. Glycine was oxidatively deaminated to acetate, carbon dioxide, hydrogen and ammonium.     

Nuclear binding of oestradiol-17β and induction of protein synthesis in the rat uterus during postnatal development

1. The uterine response to a single injection of oestradiol-17beta during postnatal development of the rat was studied with respect to (i) nuclear binding of oestradiol-17beta; (ii) induction of the synthesis of a specific cytoplasmic protein (;induced protein' of Gorski); (iii) rate of incorporation of (3)H-labelled amino acids into total protein and into nuclear acid-soluble and acid-insoluble protein; and (iv) rate of [(3)H]thymidine incorporation into DNA. 2. Specific nuclear binding of oestradiol-17beta could be demonstrated even at birth. Administration of oestradiol-17beta in vivo caused a significant increase in the number of nuclear binding sites in rats aged 10 days or older. 3. A rapid method is described for the detection of the ;induced protein', based on cellulose acetate electrophoresis. Induction of this protein could be demonstrated at the age of 10, 15 and 20 days, but not in 5-day-old rats. 4. In 20-day-old rats the rate of (3)H-labelled amino acid incorporation into protein increased by 3h after oestradiol administration. Incorporation into the different protein fractions reached peak values asynchronously: at 3-4h for acid-insoluble nuclear protein, at 6h for total protein and at about 12h for acid-soluble protein. 5. Treatment with oestradiol failed to stimulate amino acid incorporation into protein in 5- or 10-day-old rats; at the age of 15 to 30 days the hormone caused a significant increase in incorporation into total protein and into both types of nuclear protein. 6. Since the capacity for nuclear binding of oestradiol and for synthesis of the induced protein is demonstrable in the rat uterus before it acquires the ability to respond to the hormone with enhanced general protein synthesis and DNA synthesis, it appears that nuclear binding and the synthesis of the induced protein may be necessary but not sufficient conditions for the trophic action of oestradiol.