Effect of two broad-spectrum antibiotics on activity and stability of continuous nitrifying system

A 13 amino acid sequence, CRVARGDWNDNYC, originated from disintegrin eristostatin, was introduced into an inactive human proinsulin molecule between the B29 and A2 sites to replace proinsulin C-peptide by molecular cloning techniques. The constructed Arg-Gly-Asp (RGD)-proinsulin gene was cloned into a temperature-inducible vector pBV220 and expressed in Escherichia coli. The expressed RGD-proinsulin was refolded and purified by Sephadex G50 and DEAE-Sephadex A25 separations. The chemical identity was confirmed by both amino acid composition and mass spectrometry analyses. This RGD-proinsulin showed an inhibitory activity of adenosine 5′-diphosphate-induced human platelet aggregation with an IC₅₀ value of 200 nM. Its insulin receptor binding activity remained as low as 0.03% with native insulin as a control, and its insulin immune activity retained 27.6% compared with proinsulin.     

Thermodynamic studies of interaction between cetylpyridinium bromide and lysozyme by means of calorimetric titrations

In this study, we have investigated the micellization characteristics of n-cetylpyridinium bromide (CPB) and lysozyme–CPB system using isothermal titration calorimetry (ITC) technique. The ITC operates in a stepwise addition mode, providing an excellent method of determination of critical micelle concentration (CMC) and enthalpy of demicellization (and hence micellization). The micellization characteristics of CPB have been investigated by microcalorimetric technique at 25, 30, 35, and 40 °C in a buffer solution of Tris(hydroxymethyl)aminomethane and pH of 7.3. A scheme to describe lysozyme–CPB interaction has also been proposed on the basis of ITC technique in a buffer solution of Tris(hydroxymethyl)aminomethane at 30 °C, pH of 7.3, 0 mM NaCl, and 1 mM NaCl. The enthalpy changes associated with micelle dissociation were temperature and lysozyme concentration dependent, indicating the importance of hydrophobic interactions. The ΔG _mic was found to be negative, implying that, micellization, as expected, occurs spontaneously once the CMC has been reached. The values of ΔG _mic were found to become more negative with increasing temperature (in case of micellization of CPB) and with increasing the lysozyme concentration (in the case of lysozyme–CPB). The ΔS _mic was also found to decrease with increasing temperature in both cases. The presence of NaCl (1 mM) in the solutions decreased the CMC of CPB.     

人胰岛素半合成方法的改进

本文对森原等人合成人胰岛素的方法进行了改进。用羧肽酶A和胰酶成功地实施了人胰岛素的半合成。精品的总收率为70％,高于1986年森原等报道的水平(50％)。 苏氨酸叔丁酯是半合成人胰岛素的主要原料之一。我们改进了它的合成方法。采用N端未加保护的苏氨酸一步法合成了     

Lipogenesis stimulatory and inhibitory activities of the insulin mediators

Incubating plasma membranes prepared from pig liver with varying concentrations of insulin (50-1000 microU/ml) resulted in the release of at least two insulin chemical mediators. One of them was fraction 1 of insulin mediator (M. W. 3700-4000 daltons) which had a significant lipogenesis-stimulating activity. The other was fraction 2 of insulin mediator (M. W. about 1000 daltons) which exhibited a lipogenesis-inhibitory activity. The ratio of yield between the two mediators produced from the membranes was not only dependent on the concentration but also on the potency of insulin and its analogs added. The result showed that there was more production of fraction 2 than fraction 1 with the inducer at low concentration (100 microU/ml), while the production of fraction 1 from the plasma membranes incubated with high concentration of insulin (300 microU/ml) was higher than fraction 2. On the other hand, insulin and its analogs which have different biological activities and receptor binding activities have been used to induce the insulin mediators. The results obtained were similar to those mentioned above. This suggested that the generation of the mediators was dependent on the biological potences but not the binding activities.     

Cap and capture-release techniques applied to solid-phase synthesis of oligosaccharides

This paper reports a new strategy for oligosaccharide synthesis by combining solid-phase methods with cap and capture-release separation techniques, using the p-(5-(ethoxycarbonyl)pentyloxy)benzyl group (CPB) as a tag for the capture of desired oligosaccharides. After a complex carbohydrate mixture was obtained by solid-phase synthesis, the desired oligosaccharide containing a free carboxyl group derived from CPB was attached to an amino resin. The loaded resin was readily separated from side products by filtration and finally treated with acid to release the pure oligosaccharide product.     

牛胰岛素去折叠过程的高效液相色谱法分析

建立了反相高效液相色谱法动态监测牛胰岛素在二硫苏糖醇存在下去折叠的过程。牛胰岛素在二硫苏糖醇作用下,首先发生构象变化,形成稳定的中间体后进一步断裂分子间的二硫键,形成Ａ链和Ｂ链。去折叠过程通过基质辅助激光解吸附质谱得到了鉴定。     

Partial purification and some properties of a hemolymph lectin from panstrongylus megistus (Hemiptera,Reduviidae)

Hemagglutinating activity was studied in homogenates of three embryonic stages, and in the hemolymph of most instar larvae and in adult insects of Panstrongylus megistus, an important Chagas' disease vector in Brazil. A hemolymph lectin from the 5th instar larvae of P. megistus was purified through a biospecific adsorption by using formaldehyde-treated erythrocytes. The lectin fraction was desorbed with 0.2M D-galactose in 0.15M NaCl. The lectin fraction activity was inhibited by L-rhamnose, D-lactose, raffinose, D-galactose, and D-fucose. The electrophoretic pattern to native and acidic proteins resolved lectin fraction in two main bands with lectin activity. These bands were considered as multiple molecular forms or isoforms of P. megistus lectin. Under denaturating conditions, isoform 1 showed one band with apparent mol wt (MW) of 64 kDa while isoform 2 was resolved in two bands with MW of 64 and 33 kDa.     

Electrospray mass and tandem mass spectrometry identification of ozone oxidation products of amino acids and small peptides

Aqueous ozonation of the 22 most common amino acids and some small peptides were studied by electrospray mass (ESI-MS) and tandem mass spectrometry. After 5 min of ozonation only His, Met, Trp, and Tyr form oxidation products clearly detectable by ESI-MS. For His, the main oxidation product is formed by the addition of three oxygen atoms, His + 30; for Met and Tyr by the addition of one oxygen atom, Met + O and Tyr + O, and for Trp by the addition of two oxygen atoms, Trp + 20. Ozone oxidation occurs rapidly, products are already detected after 30 s of ozonation, and the reactivity order is Met > Trp > Tyr > His. The structures of the oxygen addition products were investigated by electrospray product ion mass spectra, and by comparing these spectra to those of protonated intact amino acids, and when available, to those of model compounds. His + 30 was assigned as 2-amino-4-oxo-4-(3-formylureido)butanoic acid (1) formed by oxidation of the His imidazole ring, Met + O as methionine sulfoxide (2), Trp + 20 as N-formylkynurenine (4), and Tyr + O as a mixture of dihydroxyphenylalanines (7 and 8). Ozonation of peptides show that the same number of oxygen atoms are added as expected from the ozonation of the free amino acids. The product ion mass spectra of both the protonated intact peptides, MH+, and the main ozonation products (M + nO)H+ (n = 1-3) revealed b and y type ions as the main fragments, which allow one to assign the type and location of modified amino acid in the model peptides.     

Effect of Spark Discharge Plasma on Water,Physiological Saline,and Hanks’ Solution

The effect of the duration of a current pulse of spark discharge in air on the composition of products formed in liquid both by the action of plasma radiation and with the participation of species formed in the discharge itself has been studied. The products formed in water, 0.9% NaCl, and in Hanks’ solution have been determined. It has been that in all the cases, nitrous acid is one of the primary products. The yield of nitrous acid is the same in all the three solutions. With a decrease in the current pulse duration, the influence of the species formed in the discharge itself on the yield of nitrous acid increases. The products formed in water decompose within up to 13 days. Peroxynitrite and N₂O₃ were identified as degradation products.     

Investigation of vanadocene(IV) alpha-amino acid complexes: synthesis, structure, and behavior in physiological solutions, human plasma, and blood

This work is focused on investigating the interaction of antitumor active metallocene vanadocene dichloride (Cp2VCl2) and amino acids in aqueous solution at physiological pH. Sixteen vanadocene amino acid complexes [Cp2V(aa)][X] (aa = gly, ala, val, leu, ile, phe, his, and trp; X = Cl, PF6) were prepared and characterized on the basis of spectral measurements (EPR, MS, IR, Raman). Amino acids are coordinated to the vanadocene fragment through the oxygen atom of the carboxylic group and the nitrogen of the amino group, resulting in a five-membered chelate ring. Complexes [Cp2V(val)][PF6] and [Cp2V(ile)][PF6] have been characterized by X-ray structure analyses. It was evidenced that all prepared complexes are stable in both aqueous solutions with physiological pH and in therapeutic NaCl solutions. EPR spectra of vanadocene amino acid complexes in Krebs-Ringer solution in human blood plasma and in whole blood showed that these complexes react with the hydrogen carbonate anion present forming complex Cp2V(O2CO).     

Micellar catalysis on the redox reaction of glycolic acid with chromium(VI)

Chromium(VI) oxidation of glycolic acid in the absence and presence of cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB) followed the same mechanism as shown by kinetic study. The reaction followed second‐order kinetics, first‐order in each reactant. The oxidation is strongly catalyzed by manganese(II) and cationic micelles of CTAB or CPB. The catalytic effect of micelles can be fitted to a model in which the reaction rate depends upon the concentration of both reactants in the micellar pseudophase. Some added inorganic salts (NaCl, NaBr, NaNO₃, and Na₂SO₄) reduce the micellar catalysis by excluding glycolic acid from the reaction site. The reactivity of glycolic acid towards chromium(VI) has been discussed and also compared with those obtained previously for the reaction between chromium(VI) and the reductants oxalic and lactic acids. On the basis of the observed results, probable mechanisms have been proposed. © 2001 John Wiley & Sons, Inc. Int J Chem Kinet 33: 377–386, 2001     

Kinetics and mechanism of the dehydration reaction of sarcosine to a bislactame through diacyclperoxide intermediate in strong acidic medium

The influence of substitution on the amine functional group of glycine in the permanganic oxidation of such an α‐amino acid in moderately concentrated sulfuric acid medium has been investigated. Reaction products analysis has revealed that contrary to the usual α‐amino acid oxidation product, which is an aldehyde species, a valuable compound, namely 1,4‐dimethylpiperazine‐2,5‐dione, has been obtained as the main product via a cheap, simple, efficient, and novel method. Sarcosine has been chosen as a substituted derivative of glycine, and the kinetics and mechanism of its permanganic oxidation have been investigated using a spectrophotometric technique. Conclusive evidence has proven delayed autocatalytic activity for Mn(II) in this reaction, analogous to some α‐amino acids. It has been revealed that such activity can show up when a certain concentration ratio of Mn(II) to sarcosine is built up in the medium, which we call the “critical ratio.” The magnitude of the latter ratio depends on the sulfuric acid concentration. Considering the “delayed autocatalytic behavior” of Mn(II) ions, rate equations satisfying observations for both catalytic and noncatalytic routes have been presented. The reaction shows first‐order dependence on permanganate ions and sarcosine concentrations in both catalytic and noncatalytic pathways, and apparent first‐order dependence on Mn²⁺ ions in catalytic pathways. The correspondence of pseudo‐order rate constants of the catalytic and noncatalytic pathways to Arrhenius and Eyring laws has verified “critical ratio” as well as “delayed autocatalytic behavior” concepts. The activation parameters associated with both pathways have been computed and discussed. Mechanisms for both catalytic and noncatalytic routes involving radical intermediates as well as a product having a diketopiperazine skeleton have been reported for the first time. © 2009 Wiley Periodicals, Inc. Int J Chem Kinet 41: 689–703, 2009     

Preparation of biologically active and site specifically radioiodinated recombinant human insulin-like growth factor-I.

Recombinant human insulin-like growth factor-I (rhIGF-I) was iodinated using a lactoperoxidase-catalyzed labeling method. The labeled products were separated into more than five fractions by ion-paired reverse-phase high performance liquid chromatography (HPLC). A fraction (peak 1), which showed the highest yield and radioactivity, was found to be biologically active in the BALB/c 3T3 cell proliferating system. The site of the iodination was investigated by S-pyridylethylation followed by trypsinization and separation with HPLC using reverse phase columns. From the amino acid analysis of the peaks which were radioactive, the iodination site of peak 1 was revealed to be Tyr-24 and Tyr-60. This is the first report of the biological activity of radioactive peptide hormone with a defined labeled site.     

Column separation using Bio-Gel P100 for the characterization of the products of human lung elastin degradation by leucocyte elastase and cathepsin G

The solubilization of human lung elastin by leucocyte elastase and cathepsin G is described. Elastolysis kinetic studies clearly show that leucocyte elastase is more efficient in solubilizing elastin fibres than is cathepsin G. Cathepsin G can degrade elastin but at a much slower rate. Characterization of elastase and cathepsin G soluble elastin fragments, obtained after 24 h of digestion (enzyme-substrate ratio, 1:100), was first performed by isoelectric focusing. Whole digests were focused as 6 bands in a pH range 4.2 to 4.7 and were found to have no significant differences in amino acid compositions. Biogel P-100 gel filtration of the elastase digested fragments separated a major excluded fraction (Mr's: 80,000 to 30,000) and a small retained one (Mr's: 6000 to 4000). Conversely, cathepsin G digests were eluted as a minor excluded fraction and a more important retarded one (Mr's: 6000 to 4000). Only the high molecular weight fractions of both enzymes digests contain crosslinked amino acids; this assigns a role for desmosines in the resistance of elastin to these proteases. These results are discussed in comparison with the data obtained by others.     

Identification and isolation of human insulin A and B chains by high-performance liquid chromatography

A method for the isolation, identification and quantification of human insulin A and B chains by high-performance liquid chromatography (HPLC) is described. These chains were isolated from a peptide mixture produced by E. coli with modified genes obtained by genetic engineering. The method is based on the use of hydrophilic reagents, forming ion pairs in a reversed-phase column. Because some undesirable effects resulting from the use of phosphoric acid were observed, especially with the B chain, a new HPLC method was developed for each of the two human insulin chains. The use of trifluoroacetic acid as a counter ion for the A chain and of formic acid for the B chain led to the rapid isolation and purification of each chain by HPLC. The advantage of this method is that it provides a highly pure product, which was identified by polyacrylamide gel electrophoresis and amino acid analysis.     

The combination effect of L-arginine and NaCl on bitterness suppression of amino acid solutions

The purpose of the present study was to quantify the degree of suppression of the bitterness of two amino acids (L-isoleucine (L-Ile), and L-phenylalanine (L-Phe)) which could be achieved by the addition of various test chemicals, and to examine the mechanism of this bitterness suppression. The test chemicals used were two sweeteners (sucrose, aspartame), NaCl, various acidic (L-aspartic acid, L-glutamic acid), or basic (L-histidine, L-lysine and L-arginine) amino acids, tannic acid and phosphatidic acid. The combination of L-arginine (L-Arg) and NaCl together was the most effective in reducing the bitterness of 100 mM L-Ile and L-Phe solutions in human gustatory sensation tests. Even in bitterness of 0.1 mM quinine solution, L-Arg was also successful in reducing the bitterness. This bitterness-suppression effect was specific to L-Arg and not to the other basic amino acids. No comparable taste-masking effect was observed for the acidic amino acids. The artificial taste sensor failed to predict completely the bitterness-suppressing effect of L-Arg. It seems likely that the bitterness-suppressing effect of L-Arg is mediated not only by binding at the receptor site, but also elsewhere in the process of bitterness perception, such as a direct effect on the sodium channel. It is conjectured that the guanidinium group of L-Arg may interact with sodium channels in taste bud membranes.     

ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

-Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acid analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm² total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid or in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. The incubation of lens proteins with dehydroascorbic acid or l -threose, but not fructose, produced equivalent glycation, protein crosslinking and sensitizer activity. The relative sensitizer activity of the 4 week glycated sample was quantitatively very similar to that of a water-insoluble fraction from aged human lenses. These data are consistent with the hypothesis that the protein-bound brunescence in the lens may be advanced glycation endproducts, which are formed in large part by the oxidation products of ascorbic acid, and that these compounds may contribute significantly to the UVA sensitizer activity present in aged human lenses.     

Separation and some characteristics of two factors from rat liver particulate fraction which stimulate and inhibit the low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase of rat fat cells.

Two factors were separated from rat liver particulate fraction treated with insulin, one of them having a stimulating effect on low-Km adenosine 3',5' cyclic monophosphate (cAMP) phosphodiesterase activity of crude microsomal fraction (P-2 fraction) and the other having an inhibiting effect on the activity of low-Km cAMP phosphodiesterase solubilized with 0.3% Brij 58 from P-2 fraction. Trypsin and heat treatments had essentially no effect on these two factors. The stimulating factor did not significantly change the apparent Km value of enzyme in P-2 fraction but increased the maximal velocity of the reaction. The inhibiting factor raised the Km value of solubilized enzyme without affecting the maximal velocity of the reaction. The stimulating factor level in diabetic rat was larger than that in normal rat while the inhibiting factor level in diabetic rat was smaller than that in normal rat. Possible participation of both factors in insulin action is discussed.     

Electrical conductivity of cation-and anion-exchange membranes in ampholyte solutions

Several laws governing ampholyte transport through ion-exchange membranes are established by a comparative analysis of the concentration dependence of electrical conductivity for homogeneous (CMX, AMX) and heterogeneous (MK-40, MA-41) membranes in NaCl, LysHCl, and NaH₂PO₄ solutions. The increase in the electrical conductivity of membranes in ampholyte solutions as the solutions become more dilute is explained by the increased fraction of divalent ions of the amino acid (cation-exchange membrane) or from phosphoric acid (anion-exchange membrane) in the membrane as a result of Donnan exclusion of hydrolysis products (hydroxide ions or protons, respectively).     

The desalination of a mixed solution of an amino acid and an inorganic salt by means of electrodialysis with charge-mosaic membranes

A new electrodialysis with charge-mosaic membranes was proposed to achieve efficient desalination of a mixed solution of an amino acid and an inorganic salt. For such a mixed solution, the conventional electrodialytic desalination with both cation-and anion-exchange membranes had resulted in a considerable loss of the amino acid through the membranes. In this method, however, the amino acid in the desalination channel of the electrodialyzer migrates away from the membranes so that the permeation loss of the amino acid through the membrane can be prevented.

Batchwise desalination experiments by this method were carried out with a glutamic acid or arginine solution including NaCl under the condition of constant electric current density. Similar experiments by the conventional method were also carried out. As a result of comparing both methods, the amino acid loss in this method became much smaller than that in the conventional one. It was confirmed that this method was very useful for the desalination of an amino acid solution. The effects of operating conditions on the desalination process are also discussed.