The 15N amino acid dilution method allows the determination of the real digestibility and of the ileal endogenous losses of the respective amino acid in pigs

The study of cytotoxic T cell responses to measles antigens during infection and after vaccination may provide insight into the immunopathology of the infection. It will also provide a knowledge of the immunity conferred by wild or attenuated virus, which will help in the design of new vaccines. Direct cytotoxic T cell responses, which did not require in vitro restimulation, were measured from peripheral blood by a standard 51Cr-release assay in 35 patients with acute measles, using HLA class I matched allogeneic B cells as targets. 77% showed specific responses to measles fusion protein, 69% to the hemagglutinin, and 50% to the nucleoprotein. These responses, which were related to severity of disease and history of previous vaccination, had waned by 14-24 wk after measles when memory responses to the same antigens could be elicited by restimulation in 71% of the 13 patients tested. A similar pattern followed vaccination: direct cytotoxic responses to fusion and hemagglutinin proteins were shown in 70% of the 20 children tested while 50% responded to the nucleoprotein. These responses, which were mediated by both CD8(+) and CD4(+) cells, faded over 6 wk when memory responses could be restimulated. Thus, a vigorous cytotoxic T lymphocyte response to fusion, hemagglutinin, and nucleoproteins is important in both natural and vaccine-induced immunity to measles.     

The effect of dietary fat content on amino acid digestibility in young pigs

Studies were carried out with 12 pigs (Yorkshire x Landrace) to determine the effect of dietary fat content on amino acid digestibility. The pigs were weaned at 21 d of age and fitted with a simple T-cannula at the distal ileum at 27 or 28 d of age. After a 7-d recuperation period, the pigs were fed one of four isonitrogenous cornstarch-based soybean meal diets (22.5% CP) containing 3.2, 6.2, 9.2, or 12.2% canola oil according to a balanced two-period change-over design. The pigs were fed four times daily, equal amounts, at 6-h intervals. The diets were supplied at a rate of 5% of the average body weight that was determined at the initiation of the first (11.0 kg) and second (12.5 kg) experimental period. Each experimental period consisted of 10 d. Feces were collected for 48 h on d 6 and 7 and ileal digesta for 24 h during d 8, 9, and 10. Chromic oxide was used as digestibility marker. The apparent ileal digestibilities of most of the amino acids increased linearly (P < .05) with increasing dietary fat levels. There were differences (P < .05) in the ileal digestibilities of most of the amino acids between the diets containing 3.2 and 12.2% canola oil. Conversely, the dietary level of inclusion of canola oil did not affect (P > .05) the fecal amino acid digestibilities. The protein-sparing effect of additional canola oil inclusion results, in part, from an increase in ileal amino acid digestibility.     

The use of electrophoretic methods for determining modified proteins in diabetic patients

Modified proteins were determined by isoelectric focusing in borate-polyol system with subsequent colorimetry (micromethod) and electrophoresis of blood serum on paper with subsequent TCA-ethanol treatment. Increased levels of glycated hemoglobin and modified albumin and changed light absorbance of glycated albumin were detected. The levels of glycated hemoglobin assessed by the micromethod and colorimetry without calibration did not correlate.     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.     

A kinetic model for the competitive reactions of ozone with amino acid residues in proteins in reverse micelles

Lysozyme and 10 other proteins are solubilized in reverse micelles formed by 0.1 M sodium di-2-ethyl-hexylsulfosuccinate and 2.0-2.5 M water (pH 7.4) in isooctane solvent. Exposure of the protein-containing reverse micellar solutions to ozone causes oxidative damage to the proteins, as assessed by the oxidation of tryptophan residues. The oxidation product of the protein-bound tryptophan has a molar absorption coefficient of 3275 +/- 81 M-1 cm-1 (mean +/- S.D., n = 6) at 320 nm. The product is suggested to be a Criegee ozonide or a tautomer of the Criegee ozonide and not N-formylkynurenine. Ozonation of lysozyme in reverse micelles results in the formation of hydrogen peroxide in yields of only approximately 0.07 mol/mol of tryptophan residues oxidized. The recovery of hydrogen peroxide added as an internal standard to the lysozyme-containing reverse micellar solutions ranges from 84 to 88%, whether or not the samples are subjected to ozonation. This suggests that hydrogen peroxide is neither destroyed during the process of ozonation nor consumed by the protein to a significant extent in an adventitious reaction. A kinetic model for the overall reaction of ozone with the proteins is developed, taking into account the concentrations and the reactivities of individual amino acid residues toward ozone. The model predicts the fractional reaction of ozone with tryptophan residues in the proteins, despite differences in amino acid composition, molecular weight, and tertiary structures. The lack of influence of protein structure is confirmed further by the observation that the native lysozyme (with and without external S-carboxymethylcysteine) and S-carboxymethylated lysozyme give identical values of the fractional reaction of ozone with tryptophan residues. The kinetic equations for the competitive reactions of ozone with amino acid residues in proteins, with some minor modification, are applicable to ozonations on complex mixtures of lipids, proteins, and antioxidants.     

Hepatic betaine-homocysteine methyltransferase activity in the chicken is influenced by dietary intake of sulfur amino acids, choline and betaine

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine [hyperhomocyst(e)inemia] is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.     

The evolution of proteins from random amino acid sequences. I. Evidence from the lengthwise distribution of amino acids in modern protein sequences

We examine in this paper one of the expected consequences of the hypothesis that modern proteins evolved from random heteropeptide sequences. Specifically, we investigate the lengthwise distributions of amino acids in a set of 1,789 protein sequences with little sequence identify using the run test statistic (ro) of Mood (1940, Ann. Math. Stat. 11, 367-392). The probability density of ro for a collection of random sequences has mean = 0 and variance = 1 [the N(0,1) distribution] and can be used to measure the tendency of amino acids of a given type to cluster together in a sequence relative to that of a random sequence. We implement the run test using binary representations of protein sequences in which the amino acids of interest are assigned a value of 1 and all others a value of 0. We consider individual amino acids and sets of various combinations of them based upon hydrophobicity (4 sets), charge (3 sets), volume (4 sets), and secondary structure propensity (3 sets). We find that any sequence chosen randomly has a 90% or greater chance of having a lengthwise distribution of amino acids that is indistinguishable from the random expectation regardless of amino acid type. We regard this as strong support for the random-origin hypothesis. However, we do observe significant deviations from the random expectation as might be expected after billions years of evolution. Two important global trends are found: (1) Amino acids with a strong alpha-helix propensity show a strong tendency to cluster whereas those with beta-sheet or reverse-turn propensity do not. (2) Clustered rather than evenly distributed patterns tend to be preferred by the individual amino acids and this is particularly so for methionine. Finally, we consider the problem of reconciling the random nature of protein sequences with structurally meaningful periodic "patterns" that can be detected by sliding-window, autocorrelation, and Fourier analyses. Two examples, rhodopsin and bacteriorhodopsin, show that such patterns are a natural feature of random sequences.     

The role of endogenous acid in the development of acute gastric ulcer induced by ischemia-reperfusion in the rat

We investigated the role of endogenous gastric acid in the development of gastric ulcer from erosion induced by ischemia-reperfusion of the celiac artery in the rat. A half-hour clamping of the celiac artery (ischemia) caused acute gastric erosions 1 hour after reperfusion and such acute injuries progressed to ulcers 48-72 hours after reperfusion without any necrotizing agents. Gastric acid secretion decreased immediately after ischemia and didn't recover until 12 hours after reperfusion. Intraperitoneal administrations of cimetidine (100 mg/kg, every 12 hours) or omeprazole (30 mg/kg, every 24 hours) were started at 1, 6, or 12 hours after reperfusion. When administrations were started 1 hour after reperfusion, both drugs significantly decreased the total damaged area and prevented the progression of gastric erosions to ulcers. However, administrations started 6 or 12 hours after reperfusion failed to inhibit the total damaged area and to prevent ulcer formation. These results suggest that endogenous gastric acid may play an important role in the progression of gastric erosions to ulcers although ischemia itself reduces acid secretion. Furthermore, treatment with anti-acid-secretory drugs in the early stage of mucosal damage may be important for the prevention of ulcer.     

The key to the antiestrogenic mechanism of raloxifene is amino acid 351 (aspartate) in the estrogen receptor

The crystallization of the ligand-binding domain (LBD) of the estrogen receptor (ER) with 17beta-estradiol and raloxifene [A. M. Brzozowski et al., Nature (Lond.), 389: 753-758, 1997] now provides a molecular basis for the biological activity of complexes as either agonists or antagonists. It is well established that the critical structural feature of antiestrogens is a correctly positioned alkylaminoethoxy side chain. The X-ray crystallography clearly shows that the alkylaminoethoxy side chain of raloxifene causes a specific and inappropriate molecular perturbation of the LBD and that the nitrogen in the side chain must hydrogen bond with aspartate 351 in the LBD of ER. We previously identified and characterized a naturally occurring mutation in the ER from a tamoxifen-stimulated transplantable human breast tumor line. The mutation is at AA351 of LBD, where the aspartate is changed to tyrosine (Asp351Tyr). In this report, we compared and contrasted the pharmacology of raloxifene to block or induce E2-stimulated increase in TGF-alpha mRNA in stable transfectants of ER-negative human breast cancer cells with the cDNAs from wild-type, mutant-amino acid (AA) 400 ER and mutant-AA 351 ER. Our results show that the mutation at AA 351 that replaces aspartate by tyrosine specifically changes the pharmacology of raloxifene from an antiestrogen to an estrogen. By contrast, a mutation at AA 400 does not, and the antiestrogenic properties of raloxifene are retained. These data and the fact that the nitrogen in the side chain must specifically interact with aspartate 351 makes this the key to the antiestrogenic activity of raloxifene.     

The use of N-succinyl derivatives in the study of amino acids and peptides by mass spectrometry

The terminal amino group of amino acids and peptides is blocked as the N-succinyl derivative by reaction with succinic anhydride. The product is then converted to the N,O-permethyl derivative in order to increase its volatility for use in mass spectrometry. The permethylated N-succinyl derivative retains the advantages of the permethylated N-acetyl derivative in regard to ease of preparation on a small scale, volatility and the presence of characteristic fragmentation patterns in their mass spectra. However, peaks in the high mass region are more abundant due to loss of CH3O-from the N-succinyl carbomethoxyl group as well as from the C-terminal carbomethoxyl group. Ions characteristic of the sequence and of individual amino acids are observed, and molecular weight can be determined from the relatively abundant ion at [M--CH3O]+ and from the weak molecular ion.     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The effects of systemic administration of muscle extract on normal muscle were studied. Male Wistar rats received intraperitoneal injections of normal and denervated muscle extract over 5 consecutive days. Soleus muscles were then submitted to histological, histochemical and quantitative-morphometric analysis. The group receiving denervated muscle extract showed considerable muscle fiber hypertrophy, together with the formation of new fibers suggestive of hyperplasia. The systemic administration of denervated muscle extract was shown to have a considerable myotrophic effect on normal muscle, evident in the hypertrophy and hyperplasia of muscle fibers.     

The amino acid sequences of carboxypeptidases I and II from Aspergillus niger and their stability in the presence of divalent cations

The amino acid sequences of serine carboxypeptidase I (CPD-I) and II (CPD-II), respectively, from Aspergillus niger have been determined by conventional Edman degradation of the reduced and vinylpyridinated enzymes and peptides hereof generated by cleavage with cyanogen bromide, iodobenzoic acid, glutamic acid cleaving enzyme, AspN-endoproteinase and EndoLysC proteinase. CPD-I consists of a single peptide chain of 471 amino acid residues, three disulfide bridges and nine N-glycosylated asparaginyl residues, while CPD-II consists of a single peptide chain of 481 amino acid residues, has three disulfide bridges, one free cysteinyl residue and nine glycosylated asparaginyl residues. The enzymes are closely related to carboxypeptidase S3 from Penicillium janthinellum. Both Ca2+ and Mg2+ stabilize CPD-I as well as CPD-II, at basic pH values, Ca2+ being most effective, while the divalent ions have no effect on the activity of the two enzymes.     

The prolongation of survival in mice by dietary antioxidants depends on their age by the start of feeding this diet

The effect of daily dietary supplements of an antioxidant mixture (AM) consisting of beta carotene, alpha tocopherol, ascorbic acid, rutin, selenium, and zinc on the survival of male C57BL/6 mice starting at 2, 9, 16, and 23 months of age was investigated. The survival of mice given AM starting at 2 and 9 months of age was found to increase significantly (from 86 to 108 days) compared to the control. The times, of 50, 90, and 100% mortality in mice given AM starting at 2 and 9 months of age increased by 16-9.5% compared to the control, whereas in mice given AM, starting at 16 and 23 months of age, no effect was observed.     

The efficacy of Aspergillus niger phytase in rendering phytate phosphorus available for absorption in pigs is influenced by pig physiological status

We performed an experiment with 112 piglets, 32 growing-finishing pigs, and 12 sows during pregnancy and lactation to test the hypotheses that apparent total tract digestibilities of P and Ca as well as the efficacy of Aspergillus niger phytase in rendering phytate P available in pigs depend on pig physiological status. All pigs were fed diets with identical feedstuff composition either without or with added microbial phytase (Natuphos, 500 FTU/kg diet). The diets contained 6.2 g Ca, 4.8 g total P, and 3.7 g phytate P per kilogram, and intrinsic phytase activity of 120 FTU/kg. The digestibility of P increased by 8.3 percentage units when BW of pigs increased from 30 to 60 kg and then remained stable until 100 kg. Pregnant sows had a lower efficiency of P absorption than piglets and growing-finishing pigs. During lactation, the efficiency of P absorption was 3.4 percentage units higher than during pregnancy but was still 6.6 percentage units lower than for growing-finishing pigs. The efficacy of the phytase in generating digestible P decreased in the order or lactating sows, growing-finishing pigs, sows at the end of pregnancy, piglets, and sows at midpregnancy; the average amounts of generated digestible P were 1.03, .83, .74, .66, and .32 g/kg diet, respectively. The addition of phytase to the diet raised apparent Ca digestibility in the piglets and growing-finishing pigs (by 4.6 and 4.0 percentage units, respectively) but not in the sows. We conclude that in the formulation of swine diets the amount of phytase to be added should be tailored to the target category.     

Potentiometric methods for determining boron, hydrogen cyanide and hydrogen chloride compounds and their use in controlling the quality of manufacturing and natural environments

The authors present basic science for, elaborate and set as methodic recommendations some potentiometric methods using ion-selective electrodes. Efficiency of those methods was proved in experiments with such substances as boron compounds, hydrogen cyanide, hydrogen chloride. High sensitivity, selectivity, accuracy and rapidity of the methods with handy technique make the facilities available for analysis of air at workplace, ambient atmosphere, water and biologic materials, detecting wide range of concentrations. Practical application of the methods enabled to specify measures to preserve health of workers and general population, helped in hygienic evaluation of work conditions in composition materials production, high-temperature soldering, optic glass industry.