Prolonged Acid Hydrolysis Research Articles

Long form of thymic stromal lymphopoietin of keratinocytes is induced by protein allergens

A growing body of evidence suggests that epicutaneous sensitization of protein allergens induces immediate-type hypersensitivity (IHS) following induction of Type 2 immune responses in animals and humans. Thymic stromal lymphopoietin (TSLP) derived from keratinocytes is a cytokine that can activate dendritic cells and has been implicated in development of inflammatory Type 2 helper T-cells. However, there is no direct evidence that allergens directly regulate TSLP expression in keratinocytes. This study aimed to evaluate the response of TSLP to protein allergens in cultured human keratinocytes and to identify appropriate endpoints for IHS. The transcription of long-form TSLP (loTSLP) was strongly induced by ovalbumin, wheat gluten (WG), acid-hydrolyzed WG (acid-HWG), and extracts from feces of Dermatophagoides pteronyssinus and D. farina, and trypsin, but not by rare allergens, human serum albumin (HSA), or extracts of mite bodies. In acid-HWG, loTSLP mRNA was significantly augmented by acid hydrolysis of WG for 0.5 h compared to WG. However, prolonged acid hydrolysis attenuated this induction similarly to that reported in previous animal studies. These results suggested that intense loTSLP transcriptional induction was a characteristic of a high-allergenic protein. Additionally, TSLP production was induced by exposure to ovalbumin, WG, and acid-HWG in combination with a trio of cytokines, i.e. interleukin (IL)-4, IL-13, and tumor necrosis factor (TNF)-α. However, no TSLP protein was detected following exposure to HSA, even in the presence of these cytokines. With acid-HWG, TSLP protein release was consistent with loTSLP transcription. Thus, intense loTSLP transcriptional induction and TSLP protein expression are each effective indicators that can be used for in vitro screening of IHS.

Influence of acid hydrolysis on thermal and rheological properties of amaranth starches varying in amylose content

The granules of amaranth starch are very small compared with starches from other sources. In the current work, amaranth starches with different amylose contents were treated with hydrochloric acid as a function of time in order to study the effect of acid treatments on starches. Differential scanning calorimetry and dynamic oscillation in shear were employed to analyse the thermal and rheological properties of acid-modified amaranth starch. Results showed that gelatinisation temperatures and enthalpy change of gelatinisation (ΔH) decreased steeply initially, and had a slight increase with further treatment up to 12 h then decreased, an outcome that reflected distinct resistance to acid with various amylose contents. Rheological parameters of storage and loss moduli during heating, cooling and frequency sweep of modified starches reflected the differential scanning calorimetry results by decreasing in value as the time of acid hydrolysis increased. With amylose content increase, the effects of acid hydrolysis on gelatinisation temperatures became less pronounced. Nevertheless, prolonged acid hydrolysis decreased the storage and loss moduli, with the starch pastes becoming more liquid-like.

Preparation and properties of enzymatically and chemically modified sugar beet pectins

Sugar beet pectin was extracted from fresh sugar beet pulp and further deesterified by base, plant or fungal pectin esterase or esterified in acid methanol. The modified sugar beet pectins were characterised by chemical analyses, macromolecular and rheological properties, capillary electrophoresis and enzymatic fingerprinting. The majority of the modified sugar beet pectins had the same molar mass and neutral sugar content except for loss of arabinose by prolonged acid hydrolysis. The ferulic acid content was almost stable at about 0.8%. It was found that the enzymatically modified sugar beet pectins were more inhomogeneous due to the presence of acetyl groups hindering the attack by pectin esterases.

Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways.

The ability of human keratinocytes and squamous carcinoma cell lines to attach lipid covalently to cell proteins has been examined using both palmitic and myristic acids. SDS-polyacrylamide gel analyses of the proteins labelled with these lipids demonstrated that each labelled a different set of proteins. Covalently protein bound palmitic acid could be removed from the proteins by mild alkali hydrolysis but the bound myristic acid required prolonged acid hydrolysis to release it from the associated proteins. H.p.l.c. analyses of the released lipid confirmed that both lipids were attached to proteins directly and that the labelling was not due to the lipids being catabolised. Cycloheximide could prevent the attachment of myristic acid to cell proteins, but only reduced the levels of palmitic acid incorporation. Pulse chase experiments indicated that there was little turnover of the attached myristic acid whereas this was significant for covalently bound palmitic acid. These observations show for the first time that two different protein populations are labelled by different lipids in eukaryotic cells, and that there appear to be two separate pathways for the acylation of proteins in such cells.

The metabolism of p, p′-DDT by the feral pigeon ( Columba livia)

Male feral pigeons were dosed with ring-labeled [ 14 C]p,p′- DDT and the tissues and droppings analyzed for total 14C, extractable 14C, and metabolites. Only 16% of an intraperitoneal dose of 1.5–2.2 mg kg −1 was voided in the droppings over 28 days; the rate of loss reached a maximum on the 14th day and then fell quickly away. The rate of removal of 14C in droppings was low in comparison to that found in the rat and the Japanese quail. When pigeons were dosed with 32–38 mg kg −1 DDT per bird, and killed after 77 days, 5.4% of the dose was eliminated in droppings and 87% was recovered in the body. The tissues and droppings from this experiment were analyzed for DDT and its metabolites. Of the 14C remaining in tissues 88% was accounted for as the apolar compounds DDE, DDT, and DDD. Approximately half of the 14C in droppings was present as DDE, DDT, and DDD, whereas 27–35% was apparently in conjugated form, extractable from aqueous solutions by ethyl acetate after prolonged acid hydrolysis. Two polar metabolites were isolated from the acid-released material. One was p, p′-DDA; the other was extractable from aqueous solution at pH 8 and was tentatively identified as a monohydroxy derivative of p, p′-DDT. DDE accounted for 93% of the 14C present as metabolites in tissues and droppings, clearly indicating the importance of this intermediate in this study. The metabolism of DDT in the feral pigeon is discussed in relation to its metabolism by other species.

The hexosamine content of some seeds and pollens.

AbstractTwenty‐one Monocotyledon and 59 Dicotyledon seed samples, nine pollen samples and 12 fractions of seeds were analysed for their content of dry matter, total nitrogen and glycoprotein and total hexosamine. An estimate was made of the frequency of occurrence of hexosamine in the proteins of the samples. The dry matter contents (as g per 100 g sample) showed little variation (88.0‐95.1, seeds and 84.1‐90.9, pollens). The total nitrogen contents (as g per 100 g sample) varied from 0.84‐3.29 (Monocotyledons), 1.42‐7.13 (Dicotyledons) and 2.91‐5.43 (pollens). The glycoprotein hexosamine content (as mmol per 100 g sample) varied from 0.078‐0.32 (Monocotyledons), 0.11‐0.73 (Dicotyledons) and 0.36‐0.78 (pollens). The frequency of occurrence of hexosamine in the proteins (as hexosamine residues:amino acid residues) varied from 1:450 to 1:1940 (Monocotyledons), 1:300 to 1:3640 (Dicotyledons) and 1:330 to 1:760 (pollens). The decomposition of hexosamines during prolonged acid hydrolysis prevented accurate estimates of non‐protein hexosamine contents being obtained but the results indicated that they varied from a few percent of, to about equal to, the contents of glycoprotein hexosamine in the samples examined.

Characterization of phosphatidylthreonine in polyoma virus transformed fibroblasts

A threonine phospholipid in polyoma virus-transformed hamster embryo fibroblasts has been characterized as phosphatidylthreonine. The identification has been made by chemical and enzymatic hydrolysis. Acid hydrolysis of the phospholipid produces free threonine. Mild alcoholysis produces a water-soluble derivative having the properties of glycerophosphorylthreonine. Hydrolysis with phospholipase C produces phosphorylthreonine which on prolonged acid hydrolysis yields threonine. Phosphatidylthreonine in the cell is more accessible to reaction with fluorodinitrobenzene than is phosphatidylserine. Phosphatidylthreonine also has been found as a major aminophospholipid in two other polyoma-transformed hamster cell lines and in the BHK-21/C13 line including the PVT-3 and TS-3 lines. the latter derived from BHK cells. Only a trace amount of phosphatidylthreonine occurs in normal liver, kidney and spleen of the adult mouse, in normal liver and kidney of the adult hamster, in whole mouse and hamster embryos, and in mouse 3T3 cells and SV40-transformed 3T3 cells.

Effector controlling accumulation of N-acetylglucosaminidase during development of Dictyostelium discoideum

The accumulation of N-acetylglucosaminidase, an early developmentally regulated enzyme in Dictyostelium discoideum, is dependent upon the action of a heat-stable effector molecule secreted by the cells. Stimulation of enzyme accumulation is inhibited by cycloheximide and actinomycin, suggesting that it requires concomitant RNA and protein synthesis. The effector elutes from Sephadex columns as a molecule of 300 to 1000 daltons. It is stable to treatment with a variety of proteolytic enzymes and mild acid hydrolysis but can be inactivated by prolonged acid hydrolysis.

Studies on the lipid components of endotoxin. II. Chemical analyses and biological properties of neutral, polar-I and polar-II subfractions.

Lipid components obtained from Salmonella typhosa O-901 endotoxin by acid hydrolysis were separated into neutral, polar-I and polar-II lipid fractions by silica gel column chromatography. These lipids were further separated by silica gel column and/or thin-layer chromatography. The subfractions were analyzed by thin-layer chromatography, gas chromatography and infrared spectrophotometry. Seven subfractions obtained from the neutral lipid fraction contained lauric, myristic, palmitic, 3-OH-myristic acid, artificial products of 3-OH-myristic acid, or a small amount of two unidentified fatty acids. These fatty acids and glucosamine were commonly detected in six subfractions obtained from the polar-I lipid fraction. Fatty acids, glucosamine, and O-phosphorylethanolamine were detected in all of the 13 subfractions obtained from the polar-II lipid fraction. Chick embryo lethal activity, rabbit pyrogenicity and in vitro interferon inducing activity were found in three polar-I lipid subfractions and five polar-II lipid subfractions, but not in neutral lipids. The activities were highest in a polar-II lipid subfraction which contained smaller amounts of O-phosphorylethanolamine and glucosamine than the other subfractions. However, no particular chemical constituent(s) related to the biological activities could be found. Prolonged acid hydrolysis of the polar-II lipids gave rise to neutral and polar-I lipids. Chemical and biological aspects of the lipid constituents of endotoxin are discussed.

O-carboxamidomethyl tyrosine as a reaction product of the alkylation of proteins with lodoacetamide

Iodoacetamide-1- 14C was found to be rapidly incorporated into RBP, a protein devoid of free SH, under conditions similar to those normally employed for the derivatization of SH. The reaction extent is pH dependent and at pH 10.0 one mole of acetamide was incorporated per mole of tyrosine residue. The amino acid composition of the alkylated RBP was found to be identical to RBP except for a loss of tyrosine and the appearance of a new dicarboxylic acid peak which was converted to tyrosine by prolonged acid hydrolysis Under similar reaction conditions, phenol and p-cresol reacted rapidly to form phenoxyacetamide and p-cresoxyacetamide. These phenolic ethers as well as anisole and phenetole were found to be readily hydrolized under the conditions normally used to hydrolyze protein. The incorporation of acetamide into RBP did not effect its riboflavinbinding capacity or its immunological reactivity to RBP antibody. The 14C alkylated RBP has been found to be a convenient tool for biological half-life studies. The tyrosine residues of glucagon react with iodoacetamide in a similar fashion and the use of 14C-iodoacetamide may prove to be a convenient means of introducing 14C into proteins. Iodoacetamide in the pH 7–10.0 range will derivatize the cysteine and tyrosine groups of proteins at comparable rates.

D-alanine interference in microbiological assays of vitamin B 6 in human blood

Prolonged acid hydrolysis of whole blood prior to microbiological assay increases its apparent vitamin B 6 content for the lactic acid bacteria, L. casei and S. faecium, but not for the yeast, S. carlsbergensis. Since yeast growth reflects all three forms of vitamin B 6 while the lactic acid bacteria respond either to pyridoxal or to pyridoxamine and pyridoxal, the differential assay for vitamin B 6 gives incongruous results when applied to blood. We find the growth of S. faecium and L. casei but not that of S. carlsbergensis reflects the increase in d-alanine and peptide content of the samples with increased hydrolysis time. These substances, known to stimulate growth of the lactic acid bacteria but not yeast, suggest a possible explanation for the curious finding that the total vitamin B 6 content of whole blood estimated microbiologically appears to be many times lower than the sum of its parts.

Changes in ultra-violet absorption spectra of deoxyribonucleic acid by treatment with bromine.

IN 1947, a procedure was developed1 for the determination of pyrimidine nucleoside in which bromination and prolonged acid hydrolysis precede the performance of Bial reaction. More recently, it was also shown that marked changes in ultra-violet absorption spectra, particularly disappearance of selective absorption of cytosine, uracil and their nucleosides, occurred before and after the bromination of the compounds2. While studying the effects of bromine on the chemical nature of nucleic acids, we have also found that the maximal absorption of deoxyribonucleic acid shows a decrease with increasing bromination period. In further experiments, it has also been demonstrated that acid hydrolysis of brominated deoxyribonucleic acid results in a loss of selective absorption without significant loss of phosphorus.

Accumulation of a stable phosphorus compound in glycolysing insect muscle.

IN isolated metathoraxes of the cockroach Periplaneta americana, 85 per cent of the mass of which is formed by the thoracic muscles, changes in various fractions of acid-soluble phosphorus compounds were followed during exposure of the preparations to anoxia. It was found that in such conditions there is a rapid breakdown of the labile phosphorus compounds, namely, arginine phosphate and adenosine triphosphate1. At the same time, however, the level of inorganic phosphate did not increase significantly; on the contrary, in many cases it showed a tendency to decrease. As the total acid-soluble phosphorus did not change at all, these facts indicated an accumulation of some stable phosphorus compound during anaerobiosis: this compound was found to be stable even to prolonged acid hydrolysis (three hours in normal acid).

A POLYSACCHARIDE FROM THE BLUE-GREEN ALGA, ANABAENA CYLINDRICA

A complex polysaccharide has been isolated from the fresh-water alga, Anabaena cylindrica, grown in a synthetic culture medium. Prolonged acid hydrolysis yielded glucose, xylose, glucuronic acid, galactose, rhamnose, and arabinose in a molar ratio of 5: 4: 4: 1: 1: 1. Chemical fractionations of the polysaccharide material from solution in cupriethylenediamine, and of its acetate from organic solvents indicated chemical homogeneity.

The Effect of Mucin on the Growth of Rats Fed Simulated Human Milk

Hog gastric mucin, a potent source of a microbiological growth factor also present in human milk, when fed with a basal diet of a composition approximating that of human milk resulted in increased growth and, in some cases, increased survival time of weanling rats. The biological effect of mucin was demonstrable with a modified skim cow's milk ration, with a semi-synthetic diet composed of casein, lactose, hydrogenated cottonseed oil and salts, and with milk rations containing iodinated casein. The destruction of the microbiological growth activity of mucin by prolonged acid hydrolysis also resulted in a loss of the rat growth activity.

Contributions to the biochemistry of growth.—On the lipoids of transplantable tumours of the mouse and the rat

The following observations were carried out with the object of obtaining information concerning the presence of lipoid substances in rapidly growing cells. Our observations were made on transplantable tumours of mice and rats, which, as we have pointed out in previous papers of this series, are especially suitable for the study of the biochemical problems of growth. So far as we are aware, neither qualitative nor quantitative observations on the presence of lipoids in these tumours have as yet been made. The analytical method employed was the same as the one used in our observations on normal and degenerating nerves. The fact that by means of this method 99 to 101 per cent. of the total lipoids present in normal nervous tissue—which is free from ordinary non-phosphorised fat—can be accounted for is an indication of the accuracy of the method. Details of the method will be published in a separate paper. Here it may be sufficient to indicate the principle of the method, which consists in extracting the total lipoids by chloroform, and estimating in aliquot portions of the extracts: (1) the cholesterin and cholesterin esters by Windaus’ method, using certain precautions which are essential for the accuracy of the results; (2) the phosphatides by a phosphorus estimation; (3) the cerebrosides by estimating the galactose liberated after prolonged acid hydrolysis; (4) the ordinary non-phosphorised fats (glycerides of fatty acids) by difference. In calculating the phosphatides and cerebrosides from the data obtained by this method, it is assumed that the average phosphorus percentage of the phosphatides and the galactose content of the cerebrosides present in the tumour cells do not differ essentially from the figures known for the phosphatides and cerebrosides of nervous tissue. It is, of course, possible that this assumption is not correct. In that case a recalculation of the figures given in this paper for the phosphatides and cerebrosides would be necessary. But since our arguments are based on a comparison between the figures obtained for different strains of tumours, such a correction would not materially affect our conclusions. In fact, in the case of the cerebrosides, for which the differences between different tumour-strains are—as will be seen—so great as to become qualitative, this possible source of error can be disregarded.