Ascending Paper Chromatography Research Articles

Truncation and Activation of Calcineurin A by Calpain I in Alzheimer Disease Brain

A disturbance of calcium homeostasis is believed to play an important role in the neurodegeneration of the brains of Alzheimer disease (AD) patients, but the molecular pathways by which it contributes to the disease are not well understood. Here we studied the activation of two major Ca(2+)-regulated brain proteins, calpain and calcineurin, in AD brain. We found that calpain I is activated, which in turn cleaves and activates calcineurin in AD brain. Mass spectrometric analysis indicated that the cleavage of calcineurin by calpain I is at lysine 501, a position C-terminal to the autoinhibitory domain, which produces a 57-kDa truncated form. The 57-kDa calcineurin maintains its Ca(2+)/calmodulin dependence of the phosphatase activity, but the phosphatase activity is remarkably activated upon truncation. The cleavage and activation of calcineurin correlate to the number of neurofibrillary tangles in human brains. These findings suggest that the overactivation of calpain I and calcineurin may mediate the role of calcium homeostatic disturbance in the neurodegeneration of AD.

Eye pigments of the blood-sucking insect, Triatoma infestans klug (Hemiptera, Reduviidae)

The pigmentation of black (wild) and red (mutant) eyes of Triatoma infestans was studied spectrophotometrically and compared with red-eyed (wild) and white-eyed (mutant) forms of Drosophila melanogaster. The spectral absorption profiles of the black and red eye pigments of T. infestans were similar to each other and to that of the wild-type eyes of D. melanogaster. The similarity to the wild form of D. melanogaster indicated that both eye forms of T. infestans contained ommochromes of the xanthommatin type, a finding confirmed by ascending paper chromatography. Pteridines, melanins, and ommins were not detected as eye pigments in T. infestans. The eye color difference in T. infestans was assumed to be a function of the xanthommatin concentration, with a smaller content of ommochrome in red eyes, although this probably did not affect the insect's visual acuity. These data support other findings regarding the similarities between black- and red-eyed specimens of T. infestans for other characteristics.

The Influence of Malic Acid, Phosphate Ion, and Urea on the Mobility of Metal Ions

The interactions of malic acid, phosphate ion, and urea with metal ions (aluminum, copper, iron, lead, manganese, and mercury) at different pH values (3.7, 5.0, 7.0, and 8.5) were investigated using a model system simulating natural conditions. The system consists of the ascending paper chromatography of metals in the tap water or in the solution of malic acid and mixture of malic acid, urea, or phosphate ion as developer. The chromatographic results show that malic acid increases the mobilities of all examined metals except mercury.

CHROMATOGRAPHIC MOBILITY OF METALS ON PAPER IMPREGNATED WITH SOME COMPONENTS OF PLANT CELL WALLS

ABSTRACT The interactions of various components of the plant cell walls and several essential (Cu, Fe) and toxic (Al, Cd, Hg, Pb) metals were investigated using a model system simulating natural conditions. The system consists of ascending paper chromatography of the metals on paper impregnated with different components of plant cell walls like pectin, galacturonic acid, and model compounds of lignin. The tap water was used as developer. The chromatographic results, compared with those on the free paper, show acceleration or retardation of metals and demonstrate the influence of considered ligands on their mobilities.

Polifosfatos em detergentes em pó comerciais

A didactic experiment is proposed in order to identify inorganic phosphates in home laundry detergents by ascending paper chromatography. Chemical principles related with hard water are also investigated. The role of polyphosphates in detergent formulations is discussed and the softening of hard water by sodium tripolyphosphate is illustrated. Several chemical concepts, related to the daily experience of the students, can be explored in post-lab discussions.

Evidence for a non-phosphorylated route of galactose breakdown in cell-free extracts of Aspergillus niger

Aspergillus niger could utilize D-galactose as sole source of carbon. Cell-free extracts of D-galactose-grown mycelia were able to catalyze the oxidation of D-galactose to D-galactonic acid-γ-lactone (GalA-γ-lact) in the presence of NAD, followed by the appearance of 2-keto-3-deoxy-D-galactonate (KDGal), pyruvate and glyceraldehyde. From 10 μmoles only 6.6 μmoles of GalA-γ-lact were disappeared after 60 min of reaction indicating the presence of GalA-γ-lactonase. Identification of GalA-γ-lact was achieved by ascending paper chromatography. KDGal, pyruvate and glyceraldehyde were also chromatographically identified in the reaction mixture containing D-galactonate which suggests that D-galactonate is degraded into pyruvate and glyceraldehyde via the intermediate formation of KDGal. Such reactions are supposed to be catalyzed by an inducible D-galactonate dehydratase and a constitutive KDGal aldolase. The amount of KDGal, pyruvate and glyceraldehyde were found to be almost equivalent and the equilibrium of the reaction being toward the formation of KDGal. The apparent equilibrium constant (Keq) was calculated and found to be 0.5 × 10−3 M. Results also proved the reversibility of the reaction catalyzed by KDGal aldolase of A. niger. In the light of the findings obtained from the degradation of D-galactose by cell-free extracts of A. niger grown on D-galactose and D-galactonate a nonphosphorolytic pathway was suggested to be operative for the degradation of D-galactose in extracts of A. niger.

Chromatographic mobility of some metals on paper impregnated with lignin

Interactions between lignin and several essential and toxic metals were investigated in a model system simulating natural conditions. The system consists of the ascending paper chromatography of metals on paper impregnated with lignin with water developer. The chromatographic results compared with those on free paper may indicate the behaviour in plants of the metals tested i.e. their reduced mobility by interaction with lignin.

Electrolytic reduction of carrier-free 188Re

Electrolytic reduction of 188Re 7+ as an alternative to reduction with SnCl 2 for protein labeling has been investigated. The electrolysis cell consisted of an “H” type cell with a tungsten cathode and a platinum anode, and 7 M HCl as the supporting electrolyte. Reduction of 188ReO 4 − was conducted at 25–35 V ( I = 0.05–0.5 A, current density on cathode 4 × 10 −3–4 × 10 −2 A·cm −2) for 15 min. Ascending paper chromatography on Whatman DE81 paper in 7 M HCl at 4°C confirmed that 75–77% of 188Re was reduced to the +5 oxidation state. Subsequent to evaporation of 7 M HCl under nitrogen, it was shown that the reduced 188Re 5+ as complexed with DMSA ( meso-1,2-dimercaptosuccine acid) or citrate at pH = 4.5 was stable with regard to reoxidation for up to 40 min.

Potential weekly intake of artificial food colours by 3-14-year-old children in Brazil.

The Potential Weekly Intake (PWI) of artificial food colours by 3-14-year-old children living in the District of Barão Geraldo, Campinas, São Paulo, Brazil, was estimated on the basis of average consumption data of artificially coloured food and analytically determined colour concentration in foodstuffs ingested. Coloured food consumption data were obtained through dietary recall interviews and collection of the packages and/or labels of the coloured foods consumed during a two-week period. Colours found in the individual types of foods detected through the consumption survey were identified and determined by methods that included wool dyeing and polyamide column extractions, ascending paper chromatography and spectrophotometry. The results showed that all artificial colours used in the composition of 83 commercial food products, including jellies, juices, soft drinks, syrups and 57 different candies, were permitted for use in food in Brazil the year the survey was conducted (1986), in amounts below those prescribed by law. Statistical analysis performed to compare the PWI for different population groups demonstrated that young male children, especially from lower social classes, were most exposed to artificial colours. Comparison of the estimated potential intakes with the toxicologically Acceptable Daily Intake (ADI) showed that consumption of Amaranth, Sunset Yellow, Indigotine and Tartrazine by all children in the study represented approximately 24%, 3%, 0.05% and 0.4%, of the actual ADI values, respectively.

Requirements of mutually miscible liquids for ternary chromatographic solvents with reference to monosaccharide paper chromatography

Ascending paper chromatography of three monossacharides is re-examined using ternary solvent mixtures. These solvents contain water together with a “water-immiscible” organic solvent ( n-butanol, ethyl acetate or cyclohexanol) made into a monophase with one of four mutually miscible liquids (MMLs). The requirements of such MMLs are defined as a consequence.

Autoregulation of iodide transport in the rabbit: absence of autoregulation in fetal tissue and comparison of maternal and fetal thyroid iodination products.

Maternal and fetal rabbit thyroid glands were compared as to their ability to respond to excess iodide in vitro with a reduction in subsequent iodide transport activity. Preincubation of maternal thyroid tissue slices for 2 h with excess iodide (30 microM) resulted in a 31% reduction in the subsequently measured thyroid-medium radioiodide concentration ratio. In contrast, similar iodide pretreatment had no significant effect on fetal iodide transport. In all other respects, fetal iodide transport, although it was 10 times higher, did not differ significantly from maternal transport activity. Combined radiolabeled maternal (125I) and fetal (131I) rabbit thyroid tissue was eluted on Sephadex G-25 columns. Fractions were analyzed for both 125I and 131I activity, and the maternal to fetal ratios (125I/131I) were determined for each fraction. The majority of iodoproteins eluted with the void volume, and the 125I/131I ratio was constant in these fractions. Thereafter, two peaks of elevated 125I/131I activity could be observed. Peak A eluted below lysozyme (Mr = 14,300) and above insulin (Mr = 6,000), with an apparent mol wt of 8,000 to 10,000. A second peak, peak B, eluted from the column at a site similar to that of MIT or a protein of Mr of 2,000. Ascending paper chromatography of this latter peak of 125I/131I activity and MIT was carried out in two solvent systems. In both systems, peak B comigrated with MIT. These findings suggest that the failure of fetal thyroid tissue to exhibit autoregulation may be associated with the reduced iodination of a compound with an approximate Mr of 8,000 to 10,000. The role of this substance in iodide transport remains to be identified. The reason for the apparent increase in the labeling of MIT observed in maternal vs. fetal tissue is unknown.

Radioiodination of human calcitonin using the Iodogen reagent

A method is described for the radioiodination of human calcitonin using the Iodogen reagent. The tracer was purified by 2 successive gel filtrations. The first, using Sephadex G-10, resulted in the complete removal of free 125iodide as assessed by ascending paper chromatography. The second, involving Sephadex G-75, permitted the separation of the tracer from high molecular weight aggregates. Analytical RP-HPLC showed a slight proportion (13%) of a contaminating product, probably (sulfoxyl-methionine 9)calcitonin. The final yield, after radioiodination, was found to be 42.7±5.5% and the tracer displayed a specific activity of 850±110 Ci/mmol. The effects of long-term storage of the frozen tracer were studied.

Paper Chromatographic Behavior of Some Tertiary Amine Pollutants and an Attempt at Structure-Activity-Correlation

Abstract Ascending paper chromatography of some pesticides and toxicants containing a tertiary amino group has been performed to record their mobilities in acidic, basic and saline waters and some common organic solvents, Several binary separations of these pollutants have been achieved. The important separations are summarized below: Amitrole from azobenzene, bavistin, calixin, 2,4-Lutidine, 2,6-Lutidine, N-ethylmorpholine, β-picoline, γ-picoline and quinoline; Azobenzene from bavistin, calixin, 2,4-Lutidine, 2,6-Lutidine, β-picoline, γ-picoline, pyridine and quinoline; Bavistin from 2,4-Lutidine, 2,6-Lutidine, β-picoline, γ-picoline and quinoline; and Quinoline from 2,4-Lutidine, 2,6-Lutidine, β-picoline, γ-picoline and N-ethylmorpholine from binary mixtures. An attempt at structure-activity-correlation reveals that the physical forces responsible for both the chromatographic behaviour as well as the toxicity are almost identical and the most important common physical interaction seems to be the hydroge...

A straightforward radiometric technique for measuring IMP dehydrogenase

[2- 3H]Inosinic acid ([2- 3H]IMP) has been biosynthesized in good yield from [2- 3H]hypoxanthine and PRPP via the action of a partially purified preparation of hypoxanthine/guanine phosphoribosyl transferase from mouse brain. The product was purified in one step by ascending paper chromatography, and used to assess the activity of IMP dehydrogenase. To conduct the assay, tritiated substrate is admixed with enzyme in a final volume of 10 μl; NAD is present to serve as cofactor for the reaction, and allopurinol to inhibit the oxidation of any hypoxanthine generated as a consequence of side reactions. After an appropriate period of incubation, the 3H 2O arising from the oxidation of tritiated IMP via [ 3H]NAD is isolated by quantitative microdistillation. Performed as described, the assay is facile, sensitive, and accurate, with the capability of detecting the dehydrogenation of as little as 1 pmol of [ 3H]IMP. Using it, measurements have been made of IMP dehydrogenase in a comprehensive array of mouse organs. Of these, pancreas contained the enzyme at the highest specific activity.

Chromatographic assay of steroids on immuno-affinity paper strips; a rapid method for the quantitation of digoxin and oestriol-16α-glucuronide concentrations

The combination of affinity chromatography and immunoassay provides a novel method for the quantitation of steroids in biological fluids. Anti-digoxin and anti-oestriol-16α-glucuronide antibodies were immobilised to cellulose strips, and used for ascending paper chromatography. Labelled digoxin and oestriol-16α-glucuronide were selectively retarded on strips containing the appropriate immobilised antibodies. The degree of retardation was quantitatively affected by the addition of non-labelled steroid to the sample. Calibration curves could, therefore, be constructed. The sensitivity of the assay system can be altered by changing the concentration of immobilised antibody on the cellulose strip. Calibration curves over the range 50–800 pg for digoxin and 50–250 pg for oestriol-16α-glucuronide are presented.

Separation of polonium species in nitrate solutions by paper chromatography

Ascending paper chromatography using Toyo No. 50 paper and n-BuOH saturated with 1.0M HNO3 as a developing solvent, was useful to examine the separation of polonium species in (H,NH4)NO3 solutions. A neutral mononuclear species (Po(OH)4) appeared in the vicinity of Rf=0.6. The Rf values of each species decreased depending on the increasing cationic tendency and polymerization of the polonium species.

Preflowering Levels of Phytohormones in Sorghum. I. Analytical Procedure1

Procedures are described for the analysis of indole‐3‐acetic acid (IAA), abscisic acid (ABA), and gibberellin (GA)‐like compounds in sorghum, Sorghum bicolor (L.) Moench. Shoots of seedlings were harvested, frozen in liquid N, lyophilized, ground in a Wiley mill, and replicate samples extracted with methanol/water. After vacuum removal of methanol the residual was fractionated by partitioning against petroleum ether and ethyl acetate. Free, acidic forms of the phytohormones were recovered in the final ethyl acetate fraction, and a portion of the conjugated forms were obtained by mild acid hydrolysis of the aqueous residue followed by partitioning into ethyl acetate. Fractions containing IAA‐, ABA‐, or GA‐like compounds were further purified by ascending paper chromatography. Chromatograms of purified extracts were cut into various Rf intervals, eluted with methanol, and analyzed with high performance liquid chromatography (HPLC) operating in the reverse‐phase mode (µ Bondapak C18) for IAA or ABA and with the lettuce hypocotyl bioassay for GA‐like activity. Substantial quantities of IAA and ABA were obtained from sorghum tissue samples; recovery averaged 40 ± 10%. The identity of IAA and ABA was tentatively confirmed by: (a) identical retention times as authentic standards on gas‐liquid chromatography, (b) co‐chromatography with their respective standards on HPLC operated at different flow rates or solvent compositions. Gibberellin‐like compounds were tentatively identified as GA3, GA4, and/or GA7, by comparison to standards chromatographed on paper and bioassay ed. The putative GA3, was the most abundant GA‐like compound present. Additional quantities of putative IAA, ABA, and GA3, were obtained by mild acid hydrolysis of the final aqueous residue.

The mechanisms of methionine oxidation concomitant with hormone radioiodination comparative studies of various oxidants using a simple new method

The mechanism of oxidation of methionine concomitant with iodination was studied by ascending paper chromatography using l -[Me- 14 C] methionine . The ability of the principal iodination reactants to oxidize free methionine was measured in order to predict in situ methionyl oxidation during radioiodination of peptides. Iodide, oxidant, and tyrosine were tested individually and in combination. Dilute chloramine-T and H 2O 2 effectively oxidized free methionine, whereas electric current at the low levels used for iodination did not. Whereas KI 3 did not cause significant methionine oxidation, iodine in statu nascendi was a potent oxidant. Oxidation caused by chemical oxidants was markedly reduced by the addition of iodide, whereas the opposite effect was seen with electric current. In our in vitro system, tyrosine inhibited the effect of the chemical oxidants to different degrees, in the absence of iodide. Under conditions simulating actual ‘mild’ radioiodination conditions, with added tyrosine, none of the oxidants studied caused methionine oxidation; the presence of tyrosine and iodide appeared to preclude the oxidation of methionine. Both excess chloramine-T and high electric current resulted in the formation of a new compound, possibly a sulphinic acid derivative of methionine. The important finding was that the release of free iodine and its uptake by tyrosine were the dominant factors to be considered in the prevention of methionine oxidation. Our new chromatographic technique for the estimation of methionine oxidation in peptides is based on the observed similarity between the reactivity of free methionine and that of accessible methionyl residues in peptides. The method is simple, sensitive and reproducible.

The preparation and control of pyrrolidino-methyl tetracycline labelled with 99mTc

An instant technique for labelling lyophilized pyrrolidino-methyl tetracycline with 99 m Tc is described. Labelling yield of 96–99% is obtained at pH values of 6.9–7.2. The final product is controlled by ascending paper chromatography, and biodynamic investigations are performed on white rats. Maximum uptake in kidney of 25–35% is achieved in time intervals of 60–90 min. The preparation is applied in clinical investigations on patients with renal pathologies.

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. II. Cyanogen bromide peptides.

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).