Protein synthesis and RNA in tissues of the pig.

The rate of protein synthesis in 75-kg pigs was measured by continuous intravenous infusion of [14C]tyrosine. In the whole body, over 600 g of protein were synthesized each day. In pigs, rats, and man the rate of protein synthesis in the whole body was related to metabolic rate. The fractional rate of synthesis of protein in the tissues was also measured. The proteins of visceral organs (liver kidney, lung, and spleen) were renewed at rates close to 20% per day, those of brain at 8% per day, heart 7% per day, and skeletal muscle 4% per day. A significant correlation was observed between the fractional rate of protein synthesis in the tissue and RNA concentration. Calculation of the total amount of protein synthesized in skeletal muscle of the pig (fractional rate of synthesis X protein content) shows that muscle contributes 42% of whole-body synthesis. By contrast, in the rat only 19% of whole-body synthesis occurs in muscle.     

A new and versatile method for determination of thiolamines of biological importance

A method for the separation and quantitation of several important biological thiolamines is described. The procedure employs a C18 reversed-phase HPLC system to separate the dinitrophenyl derivatives of reduced and oxidized glutathione and cysteine and relies on an internal standard, Nepsilon-methyllysine, to minimize experimental error. The method was validated in three matrices (water, HepG2 cell lysates, and mouse liver homogenates) using several criteria. The detector response was linear for the dinitrophenyl derivatives of glutathione, glutathione disulfide, cysteine, and cystine in the concentrations ranging from 10 to 50 nmol/ml. Inter- and intra-day variation, percent recovery in the biological matrices, and limits of detection and quantitation were determined. For the most accurate determination, it is essential that standard curves be produced daily and in the same matrix as that being analyzed.     

Protein synthesis in the gastrointestinal tissues of the ovine fetus

An isotope dilution procedure employing an 8h continuous infusion of L-[2,3,5,6-3H] tyrosine was used to determine fractional protein synthetic rates in the gastrointestinal tissues of ovine fetuses. The infusions were made into the inferior vena cava of the fetuses at 120-130 days of gestation. Immediately following the termination of the infusion the ewes were sacrificed and fetal tissues were collected, frozen in liquid nitrogen and stored at -20 C. The specific activity of the intracellular free and protein bound amino acid pools was determined from which the fractional synthetic rates (%/day) were calculated. These rates were as follows: reticulo-rumen, 49%; omasum, 10%; abomasum, 14%; proximal duodenum, 93%; and distal colon, 15%. The contribution of duodenum to the whole body protein synthesis was 10.5% while the contribution of the entire GIT (13.9%) was very close to that of liver (14.4%). The specific activity of tyrosine in the amniotic fluid and fetal ruminal fluid averaged 22% and 36% respectively of the specific activity in the plasma. The high turnover of tissue proteins in the fetal gastrointestinal tract as well as the presence of labelled tyrosine in the ruminal fluid indicate the functional importance of gastrointestinal activity in utero preparing the gastrointestinal tract for post-natal life.     

Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse

Uptake of the neurotransmitter glutamate is effected primarily by transporters expressed on astrocytes, and downregulation of these transporters leads to seizures and neuronal death. Neurons also express a glutamate transporter, termed excitatory amino acid carrier-1 (EAAC1), but the physiological function of this transporter remains uncertain. Here we report that genetically EAAC1-null (Slc1a1(-/-)) mice have reduced neuronal glutathione levels and, with aging, develop brain atrophy and behavioral changes. EAAC1 can also rapidly transport cysteine, an obligate precursor for neuronal glutathione synthesis. Neurons in the hippocampal slices of EAAC1(-/-) mice were found to have reduced glutathione content, increased oxidant levels and increased susceptibility to oxidant injury. These changes were reversed by treating the EAAC1(-/-) mice with N-acetylcysteine, a membrane-permeable cysteine precursor. These findings suggest that EAAC1 is the primary route for neuronal cysteine uptake and that EAAC1 deficiency thereby leads to impaired neuronal glutathione metabolism, oxidative stress and age-dependent neurodegeneration.     

Astrocytic redox remodeling by amyloid beta peptide

Astrocytes are critical for neuronal redox homeostasis providing them with cysteine needed for glutathione synthesis. In this study, we demonstrate that the astrocytic redox response signature provoked by amyloid beta (Aβ) is distinct from that of a general oxidant (tertiary-butylhydroperoxide [t-BuOOH]). Acute Aβ treatment increased cystathionine β-synthase (CBS) levels and enhanced transsulfuration flux in contrast to repeated Aβ exposure, which decreased CBS and catalase protein levels. Although t-BuOOH also increased transsulfuration flux, CBS levels were unaffected. The net effect of Aβ treatment was an oxidative shift in the intracellular glutathione/glutathione disulfide redox potential in contrast to a reductive shift in response to peroxide. In the extracellular compartment, Aβ, but not t-BuOOH, enhanced cystine uptake and cysteine accumulation, and resulted in remodeling of the extracellular cysteine/cystine redox potential in the reductive direction. The redox changes elicited by Aβ but not peroxide were associated with enhanced DNA synthesis. CBS activity and protein levels tended to be lower in cerebellum from patients with Alzheimer's disease than in age-matched controls. Our study suggests that the alterations in astrocytic redox status could compromise the neuroprotective potential of astrocytes and may be a potential new target for therapeutic intervention in Alzheimer's disease.     

Elution of 111Indium from reticuloendothelial cells.

Measurement of isotope accumulation in an organ is often used to assess that organ's removal of blood cells labelled with the isotope. This technique is only valid if the isotope does not elute from the organ. Elution of 111In from the liver and spleen has been investigated in 14 subjects following intravenous injection of heat-damaged erythrocytes labelled with 111In. The elution rate from the spleen was found to be low, about 2% of the initial activity per day. The liver accumulated activity with respect to its initial uptake at a rate of about 5% per day. Bone marrow was not visualised except in two patients in whom it was identifiable in the initial scan.     

N-acetylcysteine, but not methionine or 2-oxothiazolidine-4-carboxylate, serves as cysteine donor for the synthesis of glutathione in cultured neurons derived from embryonal rat brain

The ability of neurons to metabolize sulfur-containing compounds to cysteine was investigated using as indicator the glutathione content in neuron-rich primary cultures derived from the brains of embryonal rats. The-glutathione content of these cultures was doubled during a 4-h incubation in a minimal medium containing cysteine, glutamine and glycine. In contrast, absence of cysteine or replacement of cysteine by methionine or 2-oxothiazolidine-4-carboxylate failed to increase the glutathione content of cultured neurons. Besides cysteine, N-acetylcysteine (NAC) also caused in the millimolar range, a concentration-dependent increase in the neuronal glutathione content during a 4-h incubation. These data suggest that neurons in culture, contain an acylase activity which allows them to generate from extracellular NAC as precursor intracellular cysteine in concentrations sufficient for glutathione synthesis. In contrast, generation of cysteine from 2-oxothiazolidine-4-carboxylate by the reaction of 5-oxoprolinase or from methionine by the transsulfuration pathway appears not to take place in these cultured neurons.     

alpha-Methyl tryptophan as a tracer for in vivo studies of brain serotonin system, from autoradiography to positron emission tomography.

The procedure for measuring the brain serotonin synthesis rate in living brain using labelled alpha-methyl-L-tryptophan (alpha-MTrp), a tryptophan analog, is described. This tracer could also be used for the anterograde axonal transport measurement of serotonin. One advantage of this method is that alpha-MTrp is converted in situ into a serotonin tracer, alpha-methyl serotonin, and should be distributed into the same compartments as serotonin.     

The immunosorbtion of influenza virus nucleocapsids from cell lysates as a technique for the studies on viral RNA synthesis

Radioimmunosorbtion of influenza virus nucleocapsids from lysates of the infected cells was applied for studies on virus-specific RNA synthesis. The method allowed the isolation of intact-labelled viral RNA segments. The procedure included preadsorbtion of antiviral antibodies on protein A containing sorbents. The protein A containing sorbent with attached antibodies was mixed with lysates of influenza virus-infected [3H]uridine-labelled cells. Viral nucleocapsids bound by the antibodies to the sorbent were used for RNA extraction. The isolated RNA was analysed by polyacrylamide gel electrophoresis with subsequent autoradiography. The method allows the isolation of nondegraded labelled virus-specific RNA by means of a relatively simple procedure.     

Effect of cysteine derivatives administration in healthy men exposed to intense resistance exercise by evaluation of pro-antioxidant ratio

The aim of this study was to ascertain the influence of cysteine derivatives on pro-antioxidant equilibrium and to compare the antioxidant effectiveness of N-acetylcysteine, alpha-lipoic acid, and taurine by using Loverro's coefficient (pro-antioxidant ratio) in healthy men exposed to intensity-resistance exercise. Fifty-five men were randomly assigned to one of four groups: control (CON, placebo), N-acetylcysteine (NAC 1.8 g.day(-1), 3 days), alpha-lipoic acid (LIP 1.2 g.day(-1), 3 days), or taurine (TAU 3 g.day(-1), 3 days). The erythrocyte superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, lipid peroxidation products (TBARS), and plasma protein thiol concentrations were evaluated. The P/A ratio was determined from the mean values of TBARS, SOD, GPx, and CAT. The applied exercise at maximal intensity induced the significant changes in pro-antioxidant equilibrium toward peroxidation, which was proved by a 25% increase in TBARS concentration in the CON group. The peroxidation was significantly diminished by NAC (-14%) and LIP (-16%), whereas TAU had no effect on the TBARS concentration. Cysteine derivatives administration prevented exercise-induced decline in SOD activity and increased in GPx activity during exercise. CAT activity changed only in the LIP group. The estimation of P/A ratio showed the lowest level of pro-antioxidant equilibrium after LIP administration. In the CON group, P/A ratio was directly correlated with the protein thiols level (r = 0.495, p < 0.001). These data confirm the antioxidant action of tested cysteine derivatives, particularly lipoic acid, and demonstrate the practical application of P/A ratio to evaluate the effectiveness of antioxidants in athletes.     

Elevation of glutathione levels in bovine pulmonary artery endothelial cells by N-acetylcysteine.

N-Acetylcysteine (NAC), a cysteine derivative with chemoprotective and radioprotective effects, was found to elevate bovine pulmonary artery endothelial cell (EC) glutathione after in vitro incubation. The elevation in glutathione was associated with enhanced uptake of radioactivity of cystine from the medium. Because cystine in medium was converted rapidly to cysteine and cysteinyl-NAC in the presence of NAC and given that cysteine has a higher affinity for uptake by EC than cystine, we conclude that the enhanced uptake of radioactivity was in the form of cysteine and at least part of the stimulatory effect of NAC on EC glutathione was due to a formation of cysteine by a mixed disulfide reaction of NAC with cystine similar to that previously reported for Chinese hamster ovarian cells (R. D. Issels et al. 1988. Biochem. Pharmacol. 37:881-888). However, NAC was more effective than cysteine in elevating cellular glutathione at equimolar concentrations, and at higher concentrations of NAC an elevation of EC glutathione occurred even in the absence of cystine in the medium through a currently unknown mechanism. Thus, at least two mechanisms are operative in the elevation of endothelial cellular glutathione by NAC. NAC may be a useful compound for elevating glutathione of the pulmonary vasculature for protection against oxidant stress.     

Metabolism in the erythrocytes of dogs with low and high glutathione concentrations

Metabolism of the erythrocytes of high potassium and high glutathione (HK-high GSH), high potassium and low GSH (HK-low GSH) and normal low potassium and low GSH (LK-low GSH) dogs were studied with respect to the levels of enzymes associated with GSH synthesis and metabolism, and the capacity of the cells to regenerate GSH. HK-high GSH dogs were found to have significantly higher levels of hexokinase (Hx) and -glutamylcyclotransferase (GCT) than low GSH groups. The rate of GSH regeneration was much greater in high GSH dogs than in those of low GSH groups, and there is a positive correlation between GSH level and its, regeneration rate. Glutathione-S-transfer-ase (GST) activity was high in both HK-high GSH and HK-low GSH cells. There were no significant differences in the activities of -glutamyl cysteine synthetase (GCS), glutathione synthetase (GSHS) and glutathione reductase (GR) among these three groups. Our results indicate that GSH levels and Hx activity influence GSH regeneration rate, and the GSH levels also affect GST activity.     

Long-term effect of clofibrate on albumin turnover and distribution in man

The long-term effect of clofibrate (at least 6 months' treatment) on albumin metabolism was investigated in 7 subjects and the results were compared with those from 15 control subjects. Human albumin labelled with 131I was used as a tracer. A significant difference between the groups was found in the following parameters: The clofibrate-treated group had a prolonged rapid component (t1 1/2) of the disappearance curve (p less than 0.05), relatively increased albumin in the extravascular space (i.e. decreased distribution ratio, p less than 0.01) and increased extravascular albumin space when corrected for body size by calculating it as per cent of the extravascular bromide space (p less than 0.01). There was no significant difference between the groups in albumin synthesis, fractional catabolic rate or the slow component (t2 1/2) of the disappearance curve. The results suggest that long-term treatment with clofibrate causes changes in the intercellular matrix.     

Evaluation of labelled monoclonal antibodies by simultaneous estimation of the association constant, the immunoreactive fraction, and the number of effective binding sites on the specific target.

The reaction between a labelled monoclonal antibody (MoAb) and its specific target is characterised by three parameters: the association constant (Ka) of the labelled MoAb, the number (N) of effective binding sites on the specific target, and the immunoreactive fraction (F) of the labelled MoAb preparation. Immunological binding parameters are usually estimated graphically, by fitting the experimental data to linear equations derived from the first order law of mass action (FLMA) at equilibrium. However, only two parameters can be estimated simultaneously in a two-dimensional plot. Consequently, graphical estimation of Ka, F and N must be performed stepwise, using at least two different plots. The three parameters are interdependent, and therefore a stepwise estimation procedure might give suboptimal results. In order to investigate whether this is a problem of practical significance in the evaluation of labelled MoAbs, a computerised iterative nonlinear least squares (INLSQ) method was applied to estimate the three parameters simultaneously. The binding parameters in reactions between different 125I-labelled MoAbs and different types of targets were significantly changed when a graphical procedure was replaced by the computerised INLSQ method, and the goodness of fit to FLMA was improved. Hence, the nonlinear least squares method is the preferred procedure. Values were affected when only a subset of the data was included in the estimation procedure, indicating some heterogeneity even in these presumably homogeneous MoAb reactions. The radiolabelling procedure was presumed to be the main reason for this heterogeneity.     

Stage specific protein and nucleic acid synthesis during the asexual cycle of the rodent malaria Plasmodium chabaudi

The asexual intraerythrocytic stage of Plasmodium chabaudi develops synchronously in CBA mice. This in vivo synchrony has been exploited in in vitro pulse-labelling experiments to investigate the stage specificity of macromolecular synthesis by malaria parasites. Groups of mice were infected on day 0 with P. chabaudi and on day 3, individual mice were killed at three hour intervals, and the parasitised blood labelled in vitro for 2 h with radioactive precursors of protein or nucleic acid synthesis. By taking 11 samples covering one and one-third parasite division cycles, it was shown that the synthesis of many parasite polypeptides was restricted to defined morphological stages of parasite development. Other polypeptides were synthesised more or less continuously during the growth cycle. The synthesis of at least 6 polypeptides was confined to schizont or merozoite differentiation. RNA synthesis was shown to increase in rate steadily during parasite growth and to fall sharply during merozoite invasion. Approximately 40% of DNA synthesis was shown to occur during trophozoite growth, but the majority (60%) was confined to a short 4–6 h period at or just before schizogony.     

C-labeling of synthetic peptides

Two methods are described for the labeling of synthetic peptides using iodo[¹⁴C]acetic acid. The first procedure may be employed when the synthetic fragment contains a cysteine with a free sulfhydryl group. Alternatively, a commercial amino-protected cysteine may be carboxymethylated using radioactive iodoacetic acid. This derivative can be added to the growing peptide chain in the manual or automatic solid-phase synthesis of the fragment.     

Myoinositol turnover in the intact brain of conscious and anesthetized rabbits

Turnover of myo-inositol in the intact brain of the conscious or anesthetized rabbit was estimated from the rate of appearance of endogenous myo-inositol in the cerebroventricular compartment. The rate of appearance of brain-produced myoinositol was determined by isotope dilution during ventriculo-cisternal perfusion with artificial cerebrospinal fluid containing [14C]myoinositol. Removal of the labelled compound from the perfusate was also determined. A mean value of 5.6 nmol/min was obtained for the rate of appearance of cerebral myoinositol in conscious rabbits. Pentobarbital anesthesia caused a 30% decrease in the rate of appearance of myoinositol in the perfusate without significantly altering its fractional rate of removal.     

Interrelated effects of cysteine,thyroxin, and substrate on growth rate of influenza virus in tissue culture

Summary In cultures of chorioallantoic membrane in simple balanced salt solution, cysteine at a concentration of 0.5 to 1.0 mg/ml. interferes with the utilization of pyruvate and glutamate for tissue metabolism and growth of influenza virus. Thyroxin stimulates growth of virus and causes partial or complete reversal of the inhibitory effect of cysteine on virus reproduction. When glucose is employed as the substrate, cysteine is much less inhibitory to tissue respiration and virus, and thyroxin is found to be less effective in accelerating virus reproduction. Thyroxin was also found to increase slightly the rate of virus synthesis in tissue cultures partially inhibited with 2,4-dinitrophenol or malonate.This work was aided by research grant C1657 from the National Cancer Institute, U. S. Public Health Service, and by funds from the Eugene Higgins Trust.     

Immunoglobulins in myasthenia gravis. Kinetic properties of the acetylcholine-receptor antibody studied during lymph drainage.

A specific immunoglobulin, the receptor antibody, can be found in most patients with myasthenia gravis. In order to study the kinetic properties of this antibody, serial determinations of receptor antibody, total IgG and IgG 3 were made during drainage of thoracic duct lymph in three patients. The values obtained were used in a mathematical model to calculate some kinetic parameters. Values for T 1/2 and fractional rates of synthesis and catabolism obtained for total IgG and IgG 3 by this method were shown to agree with those found with other techniques. Most of the receptor antibody activity was found in the IgG 3 fraction but the receptor antibody had a shorter T 1/2 and higher fractional rates of synthesis and catabolism than IgG 3. These kinetic characteristics are consistent with rapid variations in plasma concentration of the receptor antibody. The cause of this rapid turnover could be strong antigenic stimuli and rapid elimination by the antigen, the cholinergic receptor protein.