Disaccharide Molecules Research Articles

Kinetic studies of the sucrose adsorption onto an alumina interface

An account is given of an experimental kinetic study of adsorption of analar reagent sucrose (ARS) onto an alumina interface spectrometrically ( λ max=570 nm) at pH 8.0 and at room temperature. The adsorption isotherm is a typical Langmuirian isotherm (S-type) and adsorption parameters have been deduced according to the Langmuir’s model. The adsorption coefficient evaluated from the Langmuir’s equation was found to be 2.52×10 2 l mol −1. Adsorption mechanism has been interpreted on the basis of metal–saccharide interaction as found in organometallic compounds and interaction due to negatively charged ends on the disaccharide molecules and positively charge groups on the surface on alumina which depends on the pH value. The effects of variation in experimental conditions of the adsorption system have also been investigated. The adsorption exhibited a typical response to the pH effect and on going towards the PZC the net charge decreases and any reaction making dependence on charge and maximum adsorption (amount) was found near the isoelectric point of alumina (pH 9.0). The presence of ions like Cl −, SO 4 2− and PO 4 3− affect the adsorbed amount quantitatively and it seems that these anions compete with sucrose for the positively charged surface sites. The addition of similar concentration of cations was found to reduce the adsorbed amount. The temperature was found to have an inverse effect on adsorption. The additions of catonic and anionic detergents influence both the adsorbed amount and the adsorption rate. The thermodynamics of the titled adsorption model indicates the spontaneous and exothermic nature. The negative value of entropy is an indication of probability of favorable and complex nature of the adsorption.

Heparin‐derived disaccharides modulate proliferation and Erb‐B2–mediated signal transduction in colon cancer cell lines

Organ-specific extracellular matrix (ECM) determines metastasis formation by regulating tumor cell proliferation. Hepatocyte-derived ECM enhances proliferation of colon cancer cell lines by increasing expression of tyrosine kinase receptors of the erb-B family. The active components in the ECM are the heparan sulfates, which are highly heterogeneous in their chemistry and size. We determined the effect of heparan sulfate disaccharides, of defined chemistry and present in high amounts in the liver heparan sulfate chains, on the proliferation of colon cancer cell lines and investigated the mechanism involved. The low-metastatic cell line KM12 was stimulated to proliferate by a highly sulfated disaccharide, found in the highest amounts in hepatocyte-derived heparan sulfate. Growth of the highly metastatic cell line KM12SM was inhibited by the second most common disaccharide in hepatocyte-derived heparan sulfate. The effect of both disaccharides was not accompanied by changes in the expression of erb-B1, erb-B2, erb-B3 or heregulin-alpha. We determined whether the disaccharides modified the signal-transduction pathways mediated by the erb-B receptors. The erb-B2-specific tyrosine kinase inhibitor AG825 abolished the enhancement of KM12 cell proliferation by the stimulatory disaccharide. This disaccharide increased tyrosine phosphorylation of erb-B1 and erb-B2 receptors, effects that were abolished by AG825. Moreover, the disaccharide caused increased expression of cyclin D1 and of activated MAP kinase, again reduced in the presence of the inhibitor AG825. The growth-inhibitory disaccharide reduced phosphorylation of erb-B1, but not of erb-B2, receptors in KM12SM cells. In conclusion, not only hepatocyte-derived heparan sulfate but also disaccharide molecules derived from heparan sulfate can affect colon cancer cell proliferation. Their effect is mediated by modulation of the erb-B signal transduction.

Template-imprinted nanostructured surfaces for protein recognition

Synthetic materials capable of selectively recognizing proteins are important in separations, biosensors and the development of biomedical materials. The technique of molecular imprinting creates specific recognition sites in polymers by using template molecules. Molecular recognition is attributed to binding sites that complement molecules in size, shape and chemical functionality. But attempts to imprint proteins have met with only limited success. Here we report a method for imprinting surfaces with protein-recognition sites. We use radio-frequency glow-discharge plasma deposition to form polymeric thin films around proteins coated with disaccharide molecules. The disaccharides become covalently attached to the polymer film, creating polysaccharide-like cavities that exhibit highly selective recognition for a variety of template proteins, including albumin, immunoglobulin G, lysozyme, ribonuclease and streptavidin. Direct imaging of template recognition is achieved by patterning a surface at the micrometre scale with imprinted regions.

Heparin disaccharides inhibit tumor necrosis factor-alpha production by macrophages and arrest immune inflammation in rodents.

Inflammation is the clinical expression of chemical mediators such as the pro-inflammatory cytokine tumor necrosis factor (TNF-)-alpha produced by macrophages and other cells activated in the immune response. Hence, agents that can inhibit TNF-alpha may be useful in treating arthritis and other diseases resulting from uncontrolled inflammation. We now report that the cleavage of heparin by the enzyme heparinase I generates sulfated disaccharide (DS) molecules that can inhibit the production of TNF-alpha. Administration of nanogram amounts of the sulfated DS molecules to experimental animals inhibited delayed-type hypersensitivity to a skin sensitizer and arrested the joint swelling of immunologically induced adjuvant arthritis. Notably, the sulfated DS molecules showed a bell-shaped dose-response curve in vitro and in vivo: decreased effects were seen using amounts of the DS molecules higher than optimal. Thus, molecular regulators of inflammation can be released from the natural molecule heparin by the action of an enzyme.

Structural requirements of lipid A species in activation of clotting enzymes from the horseshoe crab, and the human complement cascade.

The structure/activity relationship of lipid A, a bioactive center of endotoxic lipopolysaccharides, in the activation of the clotting enzyme cascade of a horseshoe crab amoebocyte lysate (Limulus activity) and the complement system in human serum, was examined using synthetic lipids A and related compounds. Regarding Limulus activity, a newly developed colorimetric method, which utilizes a mixture of recombined clotting factors and a chromogenic substance, was much more sensitive for detecting changes in the chemical structure of test compounds than the conventional gelation method using the amoebocyte whole lysate. (beta 1-6)-D-Glucosamine disaccharide bisphosphates, which had neither 3-hydroxyacyl nor 3-acyloxyacyl groups, and acylglucosamine phosphates, which in structure correspond or are analogous to the non-reducing or reducing moieties of lipids A and biosynthetic disaccharide lipid A precursors showed only negligible activity in the colorimetric tests, but they exhibited a distinct though much weaker gelation activity than the parent disaccharide molecules. The assay results obtained by the colorimetric Limulus test correlate better with the pyrogenicity of the test synthetic compounds than those given by the gelation method, although the dependence of pyrogenicity on chemical structure is greater. The presence of 3-hydroxyacyl groups on the bisphosphorylated (beta 1-6)-D-glucosamine disaccharide backbone is the prerequisite for effective activation of the clotting enzyme cascade of horseshoe crab amoebocyte lysate, while the presence of an adequate number (one or two) of 3-acyloxyacyl groups on the disaccharide bisphosphate backbone is needed for full pyrogenicity. Complement activation, on the other hand, showed structural requirements quite different from those for the colorimetric Limulus activity and the pyrogenicity. The disaccharide compounds that had only non-hydroxylated acyl groups, acylated glucosamine phosphates that had the structure of the non-reducing portion of lipids A and biosynthetic disaccharide precursors, which were scarcely active in the colorimetric Limulus test, caused complement activation comparable to or sometimes stronger than that of the parent disaccharide molecules. Acylglucosamine phosphates, corresponding in structure to the reducing moiety of disaccharide compounds, however, showed little activity.

Structural studies on the phosphate-free lipid A of Rhodomicrobium vannielii ATCC 17100.

The structure of the free lipid A from Rhodomicrobium vannielii ATCC 17100 was elucidated. It consists of a central beta-1',6-linked glucosamine disaccharide which is not substituted by phosphate. About 30% of the disaccharide molecules are substituted with mannopyranose in beta-1,4'-linkage to the non-reducing glucosamine. The reducing glucosamine can be directly reduced with NaBH4, indicating either that this glucosamine is not substituted at C1 or its substituent has been removed during the preparation of free lipid A or is removed during reduction with NaBH4. The following formula shows the 'backbone' structure of the free lipid A from Rm. vannielii ATCC 17100: beta-Manp(1-- leads to 4)-beta-GlcpN(1 leads to 6)GlcpN. 3-(R)-Hydroxyhexadecanoic acid is linked to the amino group of the reducing glucosamine. The residue at the amino group of the non-reducing glucosamine has not been identified. The hydroxyl groups of the central disaccharide are acylated with 3-(tetradecanoyloxy)-tetradecanoic acid, 3-hydroxytetradecanoic acid, delta 14-docosenoic acid (delta 14-C22:1) and acetyl groups. The hydroxyl groups of the mannose are not substituted.

Relationships between disaccharide hydrolysis and sugar transport in amphibian small intestine.

1. A study is described of the relationships which exist between disaccharide hydrolysis and glucose transport in the small intestine of Rana pipiens and Bufo vulgaris. The experiments were undertaken on the intestine perfused in vitro through the vascular system and with fluid circulating through the intestinal lumen. For this system it was found that, with [U-(14)C]glucose in the intestinal lumen, the apparent specific activity of the glucose appearing in the vascular effluent was not significantly different from that in the lumen.2. Changes of ionic composition of vascular and luminal fluids, and the presence of phloridzin or strophanthin, had little effect upon the maltase activity in situ in R. pipiens, although this activity was somewhat reduced when the sodium of the intestinal lumen was replaced by lithium. In contrast, in all cases a marked reduction was found in the rate of glucose translocation in the vascular effluent.3. With Tris substituted for the luminal sodium, there was evidence of a competitive inhibition of the maltase activity in situ by the buffer cation. At the same time, the rate of glucose translocation into the vascular effluent was but little affected and there was an apparent increase in the efficiency with which the cellular systems responsible for the translocation were able to capture the glucose liberated.4. It was found that competition for transepithelial translocation occurred between the glucose initially present in the intestinal lumen, and glucose derived from either maltose or trehalose. There was no evidence for competition for hydrolysis between maltose and trehalose, yet the glucose units derived from these two disaccharides competed with each other for translocation.5. The significance is discussed of the finding that it is possible to dissociate the processes of disaccharide hydrolysis from those underlying the translocation of hexose units into the vascular effluent. It is suggested that monosaccharide units released by the hydrolysis of disaccharide molecules have access to a pool of glucose which is equally accessible to glucose free in the intestinal lumen. It is also suggested that the rate of transport of glucose from the pool into the vascular effluent (i.e. glucose translocation) is determined by the concentration of glucose within the pool. From consideration of the properties of a model operating upon these principles, it is possible to predict the relative contribution of disaccharides and monosaccharides in the intestinal lumen to the glucose appearing in the vascular effluent. The experimentally determined contributions of the two sources are very similar to those predicted from the model. The implications of possible sites for the postulated pool are discussed.