Release Of Histidine Research Articles

Genotoxicity of processed food items and ready-to-eat snacks in Finland

Processed foods are an insufficiently characterized source of chemical mutagens for consumers. Here, we evaluated the genotoxicity of selected food products in Finland. Mutagenicity was determined by the standard plate incorporation assay followed by methylcellulose overlay and treat-and-wash assays, using the Salmonella strains TA 100 and 98 with and without metabolic activation. Generally, the mutagenic activity of food samples was low, but exhibited lot-wise variation. Cold cuts of cold-smoked beef, grilled turkey, and smoked chicken (a single batch of each) were mutagenic in all three assays with the TA 100 strain with and without metabolic activation, indicating the mutagenic effect was not secondary to histidine release from the food products. However, none of the food extracts showing mutagenic potential induced DNA damage in vitro using the Comet Assay. Our findings imply that in Finland today, there are still products the production methods of which should be refined to reduce the potential risk of mutagenicity to consumers.

Molecular model of a soluble guanylyl cyclase fragment determined by small-angle X-ray scattering and chemical cross-linking.

Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The β1 H-NOX and α1 PAS domains are in contact and form the core signaling complex, while the α1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.

Advancing Age Enhances AKT Activity and Expression in Skeletal Muscle Without Suppressing Contractile Protein Degradation and Atrogin‐1 Expression

The purpose of this study was to determine if sarcopenia is associated with insulin resistance, impaired AKT signaling, and increased contractile protein degradation rates. Insulin sensitivity was assessed by an insulin‐assisted glucose tolerance test (IAGTT) in middle‐aged (MA, 11–13 months) and aged (AG, 24–26 months) male C57B/6 mice. Contractile protein degradation rates were assessed by measuring 3‐methyl histidine release from isolated soleus (SOL) and extensor digitorum longus (EDL) muscles. During the IAGTT, blood glucose levels were significantly lower in AG mice compared to MA mice, indicating an improvement in insulin sensitivity with advancing age. The increase in insulin sensitivity observed in AG mice was associated with a significant increase in the phosphorylation of AKT in SOL and EDL muscles from AG compared to MA mice. The increase in AKT phosphorylation appears to be due to significant increases in both AKT1 and AKT2 expression. The phosphorylation of FOXO3a was significantly higher in EDL muscles from AG mice compared MA mice, but no age‐related differences in FOXO3a phosphorylation were observed in SOL muscles. The expression of the ubiquitin ligase, Atrogin‐1, was similar in SOL and EDL muscles from AG and MA mice. These results indicate that sarcopenia is not related to a decline in the ability of AKT to suppress Atrogin‐1 expression and contractile protein degradation.

Net flux of amino acids across the portal-drained viscera and liver of the ewe during abomasal infusion of protein and glucose12

The objective of the study was to measure net AA flux rates across the portal-drained viscera (PDV) and liver in the presence and absence of abomasal glucose infusion. Decreasing the fraction of AA metabolized by the mucosal cells may increase the fraction of AA being released into the blood. A potential mechanism to reduce AA catabolism by mucosal cells is to provide an alternative source of energy. We hypothesized that increasing glucose flow to the small intestine would increase net appearance of AA across the PDV. Eighteen mature ewes with sampling catheters were placed on study. The experimental design was a split-plot with a complete randomized design on the whole-plot and a Latin-square subplot with 5 periods and incremental levels of protein infusion. One-half of the ewes received abomasal glucose infusions (3.84 g/h), and all ewes received each of 5 protein abomasal infusion levels over 5 periods (0, 2.6, 5.2, 7.8, and 10.5 g/h). Net PDV release of isoleucine, leucine, methionine, phenylalanine, aspartate, glutamate, glutamine, proline, serine, and tyrosine increased linearly with increased protein infusion, and net PDV release of histidine, lysine, threonine, valine, alanine, and glycine did not differ with protein infusion. Net hepatic glucose release decreased with glucose infusion. With the exception of histidine, phenylalanine, and valine, net hepatic AA uptake increased linearly with increased delivery of AA to the liver. Glucose infusion increased the hepatic lysine and valine uptake and decreased phenylalanine uptake. Based on the observations in the current study, we reject our hypothesis that glucose can spare AA metabolism by PDV tissue. Our findings suggest that hepatic gluconeogenesis can be increased in the presence of increased AA delivery to the liver and that hepatic gluconeogenesis can be decreased with increased absorption of dietary glucose. Our findings support the concept that for most AA, hepatic transport of AA can be described by mass action kinetics; however, the rates of hepatic uptake of specific AA are upregulated directly or indirectly by elevated glucose.

Antioxidant Properties of Carnosine Re-Evaluated with Oxidizing Systems Involving Iron and Copper Ions

Carnosine has antioxidant properties and is efficient in the treatment of chemically-induced inflammatory lesions in animals. However, some studies question its biological significance as antioxidant and show lack of protection and even pro-oxidant effect of carnosine in systems containing nickel and iron ions. The ability of carnosine to: (1) reduce Fe(3+) into Fe(2+) ions; (2) protect deoxyribose from oxidation by Fe(2+)-, Fe(3+)-, and Cu(2+)-H(2)O(2)-EDTA systems; (3) protect DNA from damage caused by Cu(2+)-, and Fe(2+)-H(2)O(2)-ascorbate systems; (4) inhibit HClO- and H(2)O(2)-peroxidase-induced luminol dependent chemiluminescence was tested in vitro. At concentration 10 mM carnosine reduced 16.6+/-0.5 nmoles of Fe(3+) into Fe(2+) ions during 20 min. incubation and added to plasma significantly increased its ferric reducing ability. Inhibition of deoxyribose oxidation by 10 mM carnosine reached 56+/-5, 40+/-11 and 30+/-11% for systems containing Fe(2+), Fe(3+) and Cu(2+) ions, respectively. The damage to DNA was decreased by 84+/-9 and 61+/-14% when Cu(2+)-, and Fe(2+)-H(2)O(2)-ascorbate systems were applied. Combination of 10 mM histidine with alanine or histidine alone (but not alanine) enhanced 1.3 and 2.3 times (P<0.05) the DNA damage induced by Fe(2+)-H(2)O(2)-ascorbate. These amino acids added to 10 mM carnosine decreased 3.1-fold (P<0.05) its protective effect on DNA. Carnosine at 10 and 20 mM decreased by more than 90% light emission from both chemiluminescent systems. It is concluded that carnosine has significant antioxidant activity especially in the presence of transition metal ions. However, hydrolysis of carnosine with subsequent histidine release may be responsible for some pro-oxidant effects.

17beta-estradiol effect on the extracellular concentration of amino acids in the glutamate excitotoxicity model in the rat.

Estrogen has demonstrated a neuroprotective role in a rat model of glutamate excitotoxicity and other neurodegenerative disorders. We studied the effect of 17beta-estradiol on glutamate-induced increases in amino acids levels (aspartate, histidine, taurine and GABA) in the rat cortex. Local perfusion of glutamate produced a transient increase of aspartate, histidine, taurine and GABA in the extracellular fluid. Pretreatment with 17beta-estradiol significantly reduced the increases of taurine and moderately attenuated that of histidine, whereas aspartate and GABA releases were not modified. The effect of 17beta-estradiol on histidine release was reversed by the antiestrogen tamoxifen, suggesting a receptor-dependent mechanism. Good correlations between the volumes of the glutamate-induced lesions and the extracellular concentrations of taurine and aspartate were observed. These findings suggest that the attenuation of the glutamate-induced release of taurine by 17beta-estradiol may participate in the neuroprotective effects of 17beta-estradiol and that increased levels of aspartate and taurine are markers for the severity of the glutamate-induced cortical lesions.

The effect of lead on dopamine, GABA and histidine spontaneous and KCI-dependent releases from rat brain synaptosomes

The effect of lead on the release of [14C]GABA, [3H]dopamine and [14C]histidine (as a precursor of histamine) was studied in synaptosomes obtained from chronically lead-treated rats and in synaptosomes with in vitro lead added. In vivo treatment of rats with lead acetate results in a decrease in the K(+)-depolarization-dependent release of GABA and dopamine and histidine. Lead given in vitro itself (independently of depolarizing condition) stimulated the release of previously accumulated neurotransmitters in synaptosomes (GABA and dopamine). This effect depends on lead acetate concentration. On the other hand lead, in different concentrations, did not cause changes in the histidine release. The results show that lead can attack the synaptic neurotransmission in two ways: by depressing the Ca-KCl-evoked release of GABA, dopamine and histidine and by a selective stimulation of a spontaneous release (independent of depolarization conditions) of GABA and dopamine but not histidine.

Detection of mutagenic activity in textiles with Salmonella typhimurium

A hundred and ninety-six textile samples were tested in a modified version of the Salmonella/microsome assay for release of mutagenic contaminants. As heat sterilization of the samples can result in reduction of mutagenic activity, tests were performed with streptomycin resistant derivatives of Salmonella tester strains TA98 and TA100. Textile samples were preincubated in buffered saline (PBS), DMSO or ethanol. Subsequently, the fabrics were placed on streptomycin supplemented selective agar plates. In total, 18 samples (9.2%) exerted mutagenic activity. DMSO was the most effective solvent (15 positives) followed by ethanol (9 positive samples) and PBS (7 positives). Most fabrics (16) caused mutagenic effects only upon metabolic activation with liver S9 mix. Chemical analysis indicates that the positive results obtained with PBS are not due to release of histidine or formaldehyde. Three directly active samples gave negative results in strain TA98NR which is devoid of classical nitroreductase. With one exception all other textiles were negative in strain TA98/1,8-DNP 6 (which lacks O-acetyltransferase). These findings indicate that nitroaromatics and amines might be responsible for the mutagenic effects of the textiles.

Enhanced proteolysis and changes in membrane-associated calpain following phenylhydrazine insult to human red cells

Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins. The association of the Ca 2+-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis. HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis. Phenylhydrazine (4 m m) increased the rate of leucine, lysine, and histidine release by ∼12-, 7-, and 5-fold, respectively. N-acetylcysteine (20 m m), dithiothreitol (50 m m), and dimethylthiourea (50 m m) decreased the rate of phenylhydrazine-stimulated amino acid release by ∼30–50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 m m) and dimethyl sulfoxide (50 m m) were without significant effect. The calcium chelator, EGTA (10 m m), inhibited phenylhydrazine-stimulated proteolysis by ∼30%. Phenylhydrazine (4 m m) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of ∼28 kDa. Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE. Immunoblot analysis of spectrin confirmed these results. Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control. Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed. Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control. The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms. In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain. In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control. These forms were absent when EGTA (10 m m) was included in the incubation and the level of proenzyme was decreased. These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca 2+-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).

Calcium-dependent histidine and histamine release from superfused synaptosomes

The release of radioactive histidine and histamine was studied in a superfusion system by using guinea pig synaptosomes which were preloaded with 3H-histidine. Depolarization by 50 mM K+ induced a simultaneous release of histidine and histamine. The stimulated release of both of these substances was diminished when calcium was removed. The calcium-dependency of histamine release, previously demonstrated in brain slices, was confirmed using synaptosomes. It was also demonstrated that potassium-induced histidine release from guinea pig synaptosomes under superfusion conditions is at least partially calcium-dependent.

Catalysis of Slow C‐Terminal Processing Reactions by Carboxypeptidase H

A hypothesis was examined that carboxypeptidase H (CpAse H), which is known to catalyse the release of lysine and arginine from the C-terminus of peptides, can also release histidine, tyrosine, and phenylalanine. Synthetic peptides terminating in -His-Lys or -Tyr-Lys were used as model substrates for the enzyme and amino acid analysis was employed to detect release of the terminal amino acids. With N-acetyl-beta-Ala-Asn-Ala-His-Lys and N-acetyl-beta-Ala-Asn-Ala-Tyr-Lys, which correspond to intermediates in the processing of porcine and human beta-endorphin, lysine was removed rapidly and quantitatively but no release of histidine or tyrosine could be detected. To allow more sensitive analysis, radiolabelled substrates were employed and the amounts of the products formed on incubation with CpAse H were determined after separation by ion-exchange chromatography. With 125I-D-Tyr-Ala-His-Lys-Lys as substrate at pH 5.7, very small amounts of D-Tyr-Ala were released; the main product was D-Tyr-Ala-His. At pH 5.0 the release of histidine from 125I-D-Tyr-Ala-His took place 6,000 times more slowly than the release of lysine from 125I-D-Tyr-Ala-Lys. When the tripeptides were incubated at pH 5 with porcine pituitary secretory granules, the lysine was released rapidly but no release of histidine could be detected. The results demonstrate that CpAse H catalyses the release of C-terminal histidine with great difficulty.(ABSTRACT TRUNCATED AT 250 WORDS)

Histidine release from brain slices and synaptosomes.

The release of radioactive histidine was studied in a superfusion system by using preloaded rat cerebral cortical slices and guinea pig synaptosomes. ATP/ADP ratios in both preparations were used to assess the energy levels and thus the oxygenation of brain slices and synaptosomes. ATP/ADP ratios similar to the intact brain and nerve terminals were found in slices and synaptosomes even after hours of superfusion with oxygenated buffer. Depolarization by 30 or 50 mM K+ induced release of histidine from slices and synaptosomes. The results confirm that under physiological energetic conditions, histidine is released both from slices and synaptosomes after depolarization with potassium.

Hypoxia and ischemia modifies histamine metabolism and transport in brain synaptosomes.

Histamine level (HA), the activities of the HA synthetizing enzyme--histidine decarboxylase (HD) and HA metabolizing enzyme--histamine methyltransferase (HMT) and the uptake and release of histidine and histamine were analyzed in synaptosomal preparations obtained from rats with brain hypoxia and ischemia. Hypoxia produced only non-significant changes in all the parameters studied, whereas ischemia induced increase of both enzyme activities and histidine release, with simultaneous decreased of histidine uptake and HA level. The effect of ischemia appeared to be reversible; the changes retreated within 1 h of resuscitation together with the vital functions of rats.

Leg Metabolism of Amino Acids and Ammonia in Patients with Chronic Renal Failure

Leg metabolism of amino acids and ammonia in the postabsorptive state was evaluated in 10 patients with chronic renal failure (CRF) and in 10 patients with normal renal function (controls) by measuring the arterial-femoral venous (A-FV) differences for free amino acids and ammonia. Total amino acid release from the leg and alanine and glutamine release, which accounts for the greatest amount of the total amino acid release, are similar in patients and controls. Total amino acid uptake from the arterial blood and glutamate uptake, which is the amino acid extracted at the highest rate, are comparable in both groups. Taken together these data, in addition to the similarity of A-FV differences for proteolytic markers, namely tyrosine, phenylalanine and histidine, suggest that protein breakdown in peripheral tissues is not increased in patients with CRF. In CRF selective metabolic abnormalities for some amino acids are evident. Whilst only the A-FV differences for valine, leucine and isoleucine are decreased, additional alterations are observed by relating the A-FV difference for each amino acid to that of proteolytic markers. Such a procedure demonstrates that in CRF histidine release relative to that of proteolytic markers is reduced, whereas proline and arginine release is increased. In CRF the reduced release of some amino acids, mainly branched-chain amino acids, by the leg probably affects the pattern of circulating amino acids. Finally, both in patients and in controls a significant uptake of ammonia is observed; the ammonia uptake is related to arterial levels of this metabolite, confirming the role of peripheral tissues in removing ammonia from circulation.

Net release or uptake of histidine and carnosine in kidney of dogs.

Previous studies are equivocal as to whether the dog kidney produces histidine. Because one possible source of renal histidine is carnosine (beta-alanyl-L-histidine), we investigated net renal production (release) or utilization (uptake) (Qmet) of histidine and carnosine in 19 female dogs after they were fed histidine-free (9 dogs) or histidine-containing diets (10 dogs). Diets were fed in short-(2-11 days) or long-term (52-57 days) studies. Dogs were infused with half-normal saline for 120 min followed by an infusion of half-normal saline containing carnosine, 50 mumol/min. Renal Qmet histidine, calculated from either plasma or whole blood values, was positive during infusion of half-normal saline. During carnosine infusion, Qmet histidine increased markedly, and there was net renal uptake of carnosine. The Qmet histidine and carnosine were not different in the dogs fed histidine-free vs. histidine-containing diets. Thus there is net renal release of histidine in female dogs that increases when carnosine is administered. Qmet histidine or carnosine do not change adaptively when dogs are fed histidine-free diets.