Effects of vitamins on hepatic nuclear binding of L-tryptophan

Summary. This study investigated the in vitro effects of selected vitamins on nuclear L-tryptophan receptor binding of rat liver. Our results revealed that some fat-soluble vitamins, β-carotene, retinyl acetate, calciferol, α-tocopherol, and Trolox, as well as some water-soluble vitamins, thiamine and riboflavin, acted to inhibit in vitro ³H-tryptophan binding to hepatic nuclei. On the other hand, pyridoxine had little or no effect. The addition of dithiothreitol, a protective agent for sulfhydryl groups, along with each vitamin decreased the vitamin's inhibitory effect on in vitro ³H-tryptophan binding to nuclei, with the exception of riboflavin and calciferol. The addition of L-leucine, which alone had no inhibitory effect on in vitro ³H-tryptophan binding to hepatic nuclei but when added with unlabeled L-tryptophan negated the effect of unlabeled L-tryptophan, caused a markedly diminished inhibitory binding effect due to each of the following vitamins, thiamine, β-carotene, retinyl acetate, and α-tocopherol and Trolox, but no effect on riboflavin and calciferol. Received December 29, 1999 Accepted March 8, 2000     

Effect of cycloheximide on tryptophan binding to rat hepatic nuclei

Summary. This study evaluated whether cycloheximide, an inhibitor of protein synthesis, would affect the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous reports have indicated that the binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. Also, the administration of L-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we determined that the addition of cycloheximide in vitro inhibited ³H-tryptophan binding to hepatic nuclei or nuclear envelopes. Heat-treated cycloheximide failed to have this inhibitory binding effect. In vivo treatment of rats with cycloheximide diminished in vitro ³H-tryptophan binding to hepatic nuclei of treated rats compared to controls. Puromycin, another inhibitor of hepatic protein synthesis, when added in vitro did not affect ³H-tryptophan binding to hepatic nuclei but did diminish in vitro binding after in vivo treatment. Thus, cycloheximide added in vitro diminished ³H-tryptophan binding to hepatic nuclei probably by its structural effect on the receptor while cycloheximide administered in vivo may also act in part by inhibiting protein synthesis. Received March 22, 1999, Accepted May 31, 1999     

Effect of amino acid imbalances on the stimulatory effect of L-tryptophan on hepatic protein synthesis

Summary Earlier it was reported that mice or rats tube-fed a single feeding of L-tryptophan (TRP) demonstrated a stimulation of hepatic protein syn thesis. The present study was concerned with whether dietary imbalances induced by tube-feeding different ratios of L-alanine (ALA) or L-leucine (LEU) in relation to TRP would affect TRP's stimulatory effect on hepatic protein synthesis. Male Swiss mice, food-deprived overnight, were tube-fed one feeding of solution keeping TRP constant and varing ratios of ALA/TRP of 0.4, 2.1, or 4 or ratios of LEU/TRP of 4.8, 7.2, or 9.6. After 1 h, mice were killed and protein synthesis (¹⁴C-leucine incorporation into proteins in vitro using microsomes of livers) was measured. TRP alone stimulated hepatic protein synthesis by 83 % while ALA/TRP ratios of 2.1 or 4 but not of 0.4 and LEU/TRP ratios of 9.6 but not of 4.8 or 7.2 caused significant decreases in the stimulation of hepatic protein synthesis. Measurements of serum and hepatic free TRP concentrations in the experimental groups were similar in all groups tube-fed TRP alone or in combinations.This study was supported by U.S. Public Health Service Research Grant DK-45353 from the National Institute of Diabetes and Digestive and Kidney Diseases.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.     

Anticancer and DNA binding studies of potential amino acids based quinazolinone analogs: Synthesis,SAR and molecular docking

A novel series of amino acids conjugated quinazolinone-Schiff’s bases were synthesized and screened for their in vitro anticancer activity and validated by molecular docking and DNA binding studies. In the present investigations, compounds 32, 33, 34, 41, 42 and 43 showed most potent anticancer activity against tested cancer cell lines and DNA binding study using methyl green comparing to doxorubicin and ethidium bromide as a positive control respectively. The structure-activity relationship (SAR) revealed that the tryptophan and phenylalanine derived electron donating groups (OH and OCH₃) favored DNA binding studies and anticancer activity whereas; electron withdrawing groups (Cl, NO₂, and F) showed least anticancer activity. The molecular docking study, binding interactions of the most active compounds 33, 34, 42 and 43 stacked with A–T rich regions of the DNA minor groove by surface binding interactions were confirmed.     

Age determination based on amino acid racemization: A new possibility

Summary A method has been developed to determine the age of fossil bone samples based on amino acid racemization (AAR). Approximately one hundred fossil bone samples of known age from Hungary were collected and analysed for D- and L-amino acids. As the racemization of amino acids is affected by temperature, pH, metal content of the soil, and time passed since death, these factors were eliminated by comparing the estimated age to age determined by the radiocarbon method. Determining the D- and L-amino acid contents in samples of known age, determining the half life of racemization and plotting the D/L ratio as a function of time, calibration curves were obtained. These curves can be used for the age estimation of samples after determining their D- and L-amino acid content. The D/L ratio for 2 to 3 amino acids was determined for each sample and the mean value of estimated ages based on calibration curves was considered to estimate age of the fossil samples.     

Alteration and localization of glycan‐binding proteins in human hepatic stellate cells during liver fibrosis

Glycan‐binding proteins (GBPs) play an important role in cell adhesion, bacterial/viral infection, and cellular signaling pathways. However, little is known about the precision alteration of GBPs referred to pathological changes in hepatic stellate cells (HSCs) during liver fibrosis. Here, the carbohydrate microarrays were used to probe the alteration of GBPs in the activated HSCs and quiescent HSCs. As a result, 12 carbohydrates (e.g. Gal, GalNAc, and Man‐9Glycan) showed increased signal, while seven carbohydrates (e.g. NeuAc, Lac, and GlcNAc‐O‐Ser) showed decreased signal in activated HSCs. Three carbohydrates (Gal, GalNAc, and NeuAc) were selected and subsequently used to validate the results of the carbohydrate microarrays as well as assess the distribution and localization of their binding proteins in HSCs and liver tissues by cy/histochemistry; the results showed that GBPs mainly distributed in the cytoplasma membrane and perinuclear region of cytoplasm. The immunocytochemistry was further used to verify some GBPs really exist in Golgi apparatus of the cells. The precision alteration and localization of GBPs referred to pathological changes in HSCs may provide pivotal information to help understand the biological functions of glycans how to exert through their recognition by a wide variety of GBPs. This study could lead to the development of new anti‐fibrotic strategies.     

Molecular structural requirements for binding and activation of L-alanine taste receptors

Summary L-Alanine binds to and activates specific taste receptors ofIctalurus punctatus, the channel catfish. In order to determine the structural requirements for receptor binding and activation in this model system, a number of analogues of L-alanine were tested using a neurophysiological assay and a competitive ligand binding assay. These assays measured the ability of analogues to activate taste receptors and to displace L-[³H]alanine from L-alanine binding sites. Of those derivatives with modifications of the sidechain, L-serine, glycine,-chloro-L-alanine and 1-amino-cyclopropane-1-carboxylic acid were the most potent analogues with IC₅₀s similar to and neural responses slightly decremented from that of L-alanine. Derivatives containing branched sidechains or sidechains of otherwise increased volume were considerably less active. All modifications of the-carboxylic acid and the-amine, including amides, esters and various isosteres, led to substantial reduction in the analogues' ability to displace L-[³H]alanine and, in most cases, very weak stimulatory capability. However, L-lactic acid was a reasonably strong stimulus, but a poor competitor, suggesting that it acts at a different receptor site. Overall, these results indicate the importance of the charged amine and carboxylic acid groups for binding to and activation of the receptor for L-alanine. Moreover, modifications around the chiral center of L-alanine support the hypothesis that receptor binding and activation are separate processes in this model taste system.     

Taurine: Protective properties against ethanol-induced hepatic steatosis and lipid peroxidation during chronic ethanol consumption in rats

Summary Alcohol was administered chronically to female Sprague Dawley rats in a nutritionally adequate totally liquid diet for 28 days. This resulted in hepatic steatosis and lipid peroxidation. Taurine, when co-administered with alcohol, reduced the hepatic steatosis and completely prevented lipid peroxidation. The protective properties of taurine in preventing fatty liver were also demonstrated histologically. Although alcohol was found not to affect the urinary excretion of taurine (a non-invasive marker of liver damage), levels of serum and liver taurine were markedly raised in animals receiving alcohol + taurine compared to animals given taurine alone. The ethanol-inducible form of cytochrome P-450 (CYP2E1) was significantly induced by alcohol; the activity was significantly lower than controls and barely detectable in animals fed the liquid alcohol diet containing taurine. In addition, alcohol significantly increased homocysteine excretion into urine throughout the 28 day period of ethanol administration; however, taurine did not prevent this increase. There was evidence of slight cholestasis in animals treated with alcohol and alcohol + taurine, as indicated by raised serum bile acids and alkaline phosphatase (ALP). The protective effects of taurine were attributed to the potential of bile acids, especially taurine conjugated bile acids (taurocholic acid) to inhibit the activity of some microsomal enzymes (CYP2E1). Thesein vivo findings demonstrate for the first time that hepatic steatosis and lipid peroxidation, occurring as a result of chronic alcohol consumption, can be ameliorated by administration of taurine to rats.     

Determination of amino acid tissue concentrations by microdialysis: method evaluation and relation to plasma values

Summary. Microdialysis is an in vivo technique to monitor tissue concentrations of low molecular weight substances by means of a continuously perfused artificial capillary with a semipermeable membrane placed into the region of interest. The suitability of microdialysis to determine tissue concentrations of amino acids was evaluated in vitro by placing the catheter into Ringer buffer or into a plasma protein (50 g/l) solution containing 32 different amino acids (150 μmol/l each). All amino acids tested crossed freely the microdialysis membrane with recoveries close to 100%. Microdialysis fluid was sampled from subcutaneous tissue of five newborns and amino acid content analysed. Total and non protein bound amino acids were determined in the patients plasma by acid precipitation or ultrafiltration, respectively. Mean subcutaneous tissue concentrations were lower as compared to plasma for taurine, serine, alanine, aspartate, glutamate and ornithine and higher for valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and arginine, indicating net uptake or release of amino acids from subcutaneous tissue. Thus, microdialysis offers a convenient and minimal invasive way to study tissue amino acid composition and appears to be a promising analytical tool for the study of amino acid metabolism in vivo. Received August 7, 2000 Accepted January 7, 2001     

CRF receptors in the rodent and human cardiovascular systems: species differences

CRF has powerful receptor-mediated cardiovascular actions. To evaluate the precise distribution of CRF receptors, in vitro CRF receptor autoradiography with ¹²⁵I-[Tyr⁰, Glu¹, Nle¹⁷]-sauvagine or [¹²⁵I]-antisauvagine-30 was performed in the rodent and human cardiovascular system. An extremely high density of CRF₂ receptors was detected with both tracers in vessels of rodent lung, intestine, pancreas, mesenterium, kidney, urinary bladder, testis, heart, brain, and in heart muscle. In humans, CRF₂ receptors were detected with ¹²⁵I- antisauvagine-30 at low levels in vessels of kidneys, intestine, urinary bladder, testis, heart and in heart muscle, while only heart vessels were detected with ¹²⁵I-[Tyr⁰, Glu¹, Nle¹⁷]-sauvagine. This is the first extensive morphological study reporting the extremely wide distribution of CRF₂ receptors in the rodent cardiovascular system and a more limited expression in man, suggesting a species-selective CRF receptor expression.     

Comparative digestibility of energy and ileal amino acids in yeast extract and spray-dried porcine plasma fed to pigs

Two experiments were conducted to evaluate the digestible (DE) and metabolisable energy (ME), apparent (AID) and standardised ileal digestibility (SID) of amino acids (AA) in yeast extract (YE) and spray-dried porcine plasma (SDPP). In Experiment 1, 18 barrows (25.1 ± 1.2 kg body weight [BW]) were randomly allotted to three treatments with six replicates per treatment. The DE and ME of YE was 20.64 and 19.31 MJ/kg, respectively, which were not significantly different with the DE and ME of SDPP (18.74 and 18.05 MJ/kg, respectively). In Experiment 2, six barrows (20.6 ± 2.6 kg BW) fitted with ileal T-cannulas were fed three diets in a repeated 3-period Latin square design. For Met and Glu, the AID tended to be, while the SID was significantly higher (p < 0.05), in YE than in SDPP. The AID of Cys tended to be lower in YE (p = 0.07), while the SID of Phe tended to be higher in YE than in SDPP (p = 0.06). Accordingly, YE could be a potential substitute for SDPP as a superior protein ingredient in diets for pigs in terms of the available energy and AA digestibility.     

Selected-fit versus induced-fit protein binding: kinetic differences and mutational analysis

The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.     

Plasma protein binding of ketoprofen enantiomers in man: method development and its application.

The protein binding of ketoprofen enantiomers was investigated in human plasma at physiological pH and temperature by ultrafiltration. 14C-labelled (RS)-ketoprofen was synthesized and purified by high-performance liquid chromatography and utilized as a means of quantifying the unbound species. In vitro studies were conducted with plasma obtained from six healthy volunteers. The plasma was spiked with (R)-ketoprofen alone, (S)-ketoprofen alone, and (RS)-ketoprofen in the enantiomeric concentration range of 1.0 to 19.0 micrograms/ml. The plasma protein binding of ketoprofen was nonenantioselective. At a racemic drug concentration of 2.0 micrograms/ml the mean (+/- SD) percentage unbound of (R)-ketoprofen was 0.80 (+/- 0.15)%. The corresponding value for (S)-ketoprofen, 0.78 (+/- 0.18)%, was not statistically different (P greater than 0.05). At this racemic drug concentration (2.0 micrograms/ml) the percentage unbound of each enantiomer was unaffected (P greater than 0.05) by the presence of the glucuronoconjugates of ketoprofen (10 micrograms/ml) in plasma. At clinically relevant concentrations, the plasma binding of ketoprofen did not exhibit enantioselectivity or concentration dependence nor was the binding of either enantiomer influenced by its optical antipode (P greater than 0.05).     

Domains of the human androgen receptor and glucocorticoid receptor involved in binding to the nuclear matrix

Steroid receptors have been reported to bind to the nuclear matrix. The nuclear matrix is operationally defined as the residual nuclear structure that remains after extraction of most of the chromatin and all soluble and loosely bound componnets. To obtain insight in the molecular mechanism of the interaction of steroid receptors with the nuclear matrix, we studied the binding of several deletion mutants of the human androgen receptor (hAR) and the human glucocorticoid receptor (hGR) to the nuclear matrix. Receptor binding was tested for two different nuclear matrix preparations: complete matrices, in which most matrix proteins are retained during the isolation procedure, and depleted matrices, which consist of only a subset of these proteins. The results show that the C-terminal domain of the hAR binds tightly to both depleted and complete matrices. In addition, at least one other domain of the hAR binds to complete matrices but not to depleted matrices. In contrast to the hAR, the hGR binds only to complete matrices. For this interaction both the DNA-binding domain and the C-terminal domain of the hGR are required, whereas the N-terminal domain is not. We conclude that specific protein domains of the hAR and the hGR are involved in binding to the nuclear matrix. In addition, our results indicate that the hAR and the hGR are attached to the nuclear matrix through different molecular interactions.     

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Age and sex differences in human skeletal muscle: Role of reactive oxygen species

Previous studies, conducted on experimental animals, have indicated that reactive oxygen species (ROS) are involved in the aging process. The objective of this work was to study the relationship between oxidative damage and human skeletal muscle aging, measuring the activity of the main antioxidant enzymes superoxide dismutase (total and MnSOD), glutathione peroxidase (GPx) and catalase in the skeletal muscle of men and women in the age groups: young (17–40 years), adult (41–65 years) and aged (66–91 years). We also measured glutathione and glutathione disulfide (GSH and GSSG) levels and the redox index; lipid peroxidation and protein carbonyl content. Total SOD activity was lower in the 66–91 year-old vs. the 17–40 year-old men; MnSOD activity was significantly greater in 66–91 year-old vs. 17–40 year-old women. GPx activity remained unchanged. The activity of catalase was lower in adults than in young men but higher in the aged. We observed no changes in GSH levels and significantly higher GSSG levels only in aged men vs. adult men, and a significant decrease in aged women vs. aged men. The protein carbonyl content increased significantly in the 41–65 and 66–91 year-old vs. the 17–40 year-old men. Finally, young women have lower lipid peroxidation levels than young men. Significantly higher lipid peroxidation levels were observed in aged men vs. both young and adult men, and the same trend was noticed for women. We conclude that oxidative damage may play a crucial role in the decline of functional activity in human skeletal muscle with normal aging in both sexes; and that men appear to be more subject to oxidative stress than women.