Effect of Dietary-Resistant Starch on Inhibition of Colonic Preneoplasia and Wnt Signaling in Azoxymethane-Induced Rodent Models.

Dietary fiber has been reported to prevent preneoplastic colon lesions. The aim of this study was to determine the effect of resistant starches, novel dietary fibers, on the development of colonic preneoplasia and Wnt signaling in azoxymethane (AOM)-treated rats and mice fed resistant starches at 55% of the diet after AOM treatment. Another objective was to determine the effect of resistant starches on the development of preneoplasia in rats treated with antibiotics (Ab), administered between AOM treatment and resistant starch feeding. Diets containing resistant starches, high-amylose (HA7), high-amylose-octenyl succinic anhydride (OS-HA7), or high-amylose-stearic acid (SA-HA7) were compared with control cornstarch (CS). The resistant starch content of the diets did not alter the yield of colonic lesions but animals treated with AOM and fed the diet with the highest resistant starch content, SA-HA7 developed the highest average aberrant crypt foci (ACF) per animal. Mice fed the OS-HA7 diet had decreased expression of some upstream Wnt genes in the colonic crypts. This study suggests that further research is needed to determine if resistant starch impacts colon carcinogenesis in rodents.

Impact of dual-enzyme treatment on the octenylsuccinic anhydride esterification of soluble starch nanoparticle.

The hypothesis of improving the esterification of sugary maize soluble starch through dual-enzyme pretreatment was investigated. Native starch nanoparticle (NSP) was enzymatically pretreated using ß-amylase and transglucosidase (ESP) and then esterified with octenylsuccinic anhydride (OSA). The degree of substitution (DS), reaction efficiency (RE), molecular weight (Mw), molecular density (ρ) and in vitro digestibility were determined. Fourier transform infrared spectroscopy and confocal laser scanning microscopy were used to analyze starch particle and its OS derivatives. The emulsification properties of OS-NSP and OS-ESP were also compared. The results showed that dual-enzyme modification increased the DS and RE of OSA modified starch particle compared with the control. Enzymatic modification had a thinning effect at the surface of starch particle, resulting in lower Mw. The extent of reduction in ρ of OS-ESP was greater than that of OS-NSP. At equivalent DS, OSA modification of EPS was more effective than that of NPS in reducing digestibility. Also, there was brighter fluorescence spheres of OS-ESP in comparison to OS-NSP at equivalent DS, suggesting more OS groups were substituted on the chains near the branch points at less density areas. OS-ESP with higher DS (0.0197) had lower zeta-potential and average particle size for superior emulsion stabilization properties with high stability. The results revealed the OS-starch prepared under dual-enzyme pretreatment was a Pickering particle stabilizer for potential application in encapsulation and delivery of bioactive components.

Removal of N-terminal blocking groups from proteins.

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.

DNA microarrays based on noncovalent oligonucleotide attachment and hybridization in two dimensions.

Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10(11) molecules/mm(2), which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.

Removal of N-terminal blocking groups from proteins.

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented in this unit; one uses pyroglutamate aminopeptidase for N(alpha)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(alpha)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking can be carried out, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis. Protocols are also provided for unblocking N-terminally blocked proteins using acid-catalyzed hydrolysis or methanolysis, hydrazinolysis, and beta-elimination after acid-catalyzed N-O shift. Alternate protocols describe chemical removal of acetyl and longer-chain alkanoyl groups, as well as formyl groups to open the cyclic imide of pyrrolidone carboxylate.

The crystal structure of saporin SO6 from Saponaria officinalis and its interaction with the ribosome.

The 2.0 A resolution crystal structure of the ribosome inactivating protein saporin (isoform 6) from seeds of Saponaria officinalis is presented. The fold typical of other plant toxins is conserved, despite some differences in the loop regions. The loop between strands beta7 and beta8 in the C-terminal region which spans over the active site cleft appears shorter in saporin, suggesting an easier access to the substrate. Furthermore we investigated the molecular interaction between saporin and the yeast ribosome by differential chemical modifications. A contact surface inside the C-terminal region of saporin has been identified. Structural comparison between saporin and other ribosome inactivating proteins reveals that this region is conserved and represents a peculiar motif involved in ribosome recognition.

Clostridium difficile recombinant toxin A repeating units as a carrier protein for conjugate vaccines: studies of pneumococcal type 14, Escherichia coli K1, and Shigella flexneri type 2a polysaccharides in mice.

Unlike the native protein, a nontoxic peptide (repeating unit of the native toxin designated rARU) from Clostridium difficile toxin A (CDTA) afforded an antigen that could be bound covalently to the surface polysaccharides of pneumococcus type 14, Shigella flexneri type 2a, and Escherichia coli K1. The yields of these polysaccharide-protein conjugates were significantly increased by prior treatment of rARU with succinic anhydride. Conjugates, prepared with rARU or succinylated (rARUsucc), were administered to mice by a clinically relevant dosage and immunization scheme. All conjugates elicited high levels of serum immunoglobulin G both to the polysaccharides and to CDTA. Conjugate-induced anti-CDTA had neutralizing activity in vitro and protected mice challenged with CDTA, similar to the rARU alone. Conjugates prepared with succinylated rARU, therefore, have potential for serving both as effective carrier proteins for polysaccharides and for preventing enteric disease caused by C. difficile.

Low-density lipoprotein modification and arterial wall accumulation in a rabbit model of atherosclerosis.

Chemically or enzymatically modified low-density lipoproteins (LDL), with and without changes in surface charge, were studied in vivo in the healing, balloon catheter-deendothelialized rabbit aorta to determine the effect of LDL modification on its accumulation in arterial lesions. In this model, in which healing (reendothelialization) proceeds radially outward from individual aortic branch arteries, it was previously shown by autoradiography that two kinetically distinct compartments accumulated 125I-labeled LDL. In aortic regions which were still deendothelialized, accumulation was diffuse and labile. In contrast, at the edges of the islands of regenerating endothelium, LDL accumulation was intensely focal, as it is in human atherosclerotic lesions, and persisted for at least 40 h after injection in spite of falling levels of radiolabeled LDL in plasma [Chang, M. Y., et al. (1992) Arterioscler. Thromb. 12, 1088-1098]. In the present study, modified LDLs with gradations in charge change were prepared to clarify the role of changes in surface charge on focal aortic LDL accumulation. Oxidized LDL (weakly anionized), desialated LDL (weakly cationized), and reductively methylated LDL (no change in net charge) all accumulated focally. Focal accumulation of native LDL also occurred in ballooned rabbits fed probucol to inhibit LDL oxidation. Strongly anionized succinylated and diazobenzenearsonylated LDL and strongly cationized dimethylpropanediamine LDL did not accumulate focally. The results support the concept that focal sequestration of LDL in arterial lesions is mediated by specific, oxidation-independent patterns of charge and polarity on LDL which are disrupted by major changes in LDL surface charge.

Binding of suprofen to human serum albumin. Role of the suprofen carboxyl group.

The binding of suprofen (SP), a non-steroidal anti-inflammatory drug of the arylpropionic acid class, and its methyl ester derivative (SPM) to human serum albumin (HSA) was studied by dialysis and spectroscopic techniques. In spite of the remarkable differences in the physicochemical properties of SP and SPM, the binding of each molecule to HSA was quantitatively very similar. Thermodynamic analysis suggests that the interaction of SP with HSA may be caused by electrostatic as well as hydrophobic forces, whereas the interactions with SPM may be explained by hydrophobic and van der Waals forces. Similarities in the difference UV absorption spectra between ligand-detergent micelle and -HSA systems indicate that the SP and SPM molecules are inserted into a hydrophobic crevice on HSA. The same studies suggest that the carboxyl group of SP interacts with a cationic sub-site which is closely associated with the SP binding site. Proton relaxation rate measurements indicate that the thiophen ring and propanoate portion of the SP molecule is the major binding site for HSA. The locations of SP and SPM binding sites were identified by using fluorescence probes which bind to a known site on HSA. The displacement data implied that SP primarily binds to Site II, while the high affinity site of SPM as well as low affinity site of SP are at the warfarin binding site in the Site I area. From binding data with chemically modified HSA derivatives, it is likely that highly reactive tyrosine (Tyr) and lysine (Lys) residues, which may be Tyr-411 and Lys-195, are specifically involved in SP binding. In contrast, these two residues are clearly separated from the SPM binding site. The binding of SP and SPM is independent of conformational changes on HSA that accompany N-B transition. There is evidence that the carboxyl group may play a crucial role in the high affinity binding processes of SP to HSA.

Effect of chemical modification of lysine amino groups on redox and protonmotive activity of bovine heart cytochrome c oxidase reconstituted in phospholipid membranes.

A study is presented of the effect of chemical modification of lysine amino groups on the redox and protonmotive activity of bovine heart cytochrome c oxidase. Treatment of soluble oxidase with succinic acid anhydride resulted in succinylation of lysines in all the subunits of the enzyme. The consequent change of surface charges from positive to negative resulted in inversion of the orientation of the reconstituted enzyme from right-side-out to inside-out. Reconstitution of the oxidase in phospholipid vesicles prevented succinylation of subunits III and Vb and depressed that of other subunits with the exception of subunits II and IV which were predominantly labeled in a concentration-dependent manner by succinic acid anhydride. This modification of lysines produced a decoupling effect on redox-linked proton ejection, which was associated with a decrease of the respiratory control exerted by the delta pH component of PMF. The decoupling effect was directly shown to be exerted at the level of the pH-dependent rate-limiting step in intramolecular electron flow located on the oxygen side of heme a.

Degradation of cartilage proteoglycans by elastase is dependent on charge-mediated interactions.

We investigated the mechanism of cartilage degradation by pancreatic elastase as a model system for the action of cationic proteases such as leukocyte elastase and cathepsin-G. It is shown that the cationic properties of elastase contribute to its proteolytic potential with respect to cartilage degradation. Elastase preparations with neutral or negative charge, obtained by chemical modification were far less effective in cartilage degradation than the cationic, native enzyme. Modification of elastase did not affect the active site of the enzyme as shown by kinetic studies, nor did it alter the complexing with alpha-1-proteinase inhibitor. Quantitative and autoradiographic studies indicate that the positive charge of the enzyme favors the penetration in cartilage and interaction with the polyanionic proteoglycan substrate. It is concluded that the potent cartilage-degrading activity of elastase is dependent on charge-mediated interactions with its substrate.

The interaction of anions with native and phenylglyoxal-modified human serum transferrin.

The interaction of various anions with human serum transferrin was investigated due to the concomitant binding of iron and a synergistic anion to form the transferrin-anion-iron complex. Two tetrahedral oxyanion oxidizing agents, periodate and permanganate, were found to partially inactivate transferrin when used at equimolar ratios of oxidizing agent to protein active sites. Hypochlorite, a strong oxidizing agent with little structural similarity to periodate and permanganate, had little effect on iron-binding activity when used at similar low molar ratios of reagent to transferrin active sites. Transferrin treated with a 3:1 molar ratio of periodate or permanganate to active sites lost 74 or 67% of its iron-binding capacity, respectively. The composition of the buffer affected the extent of transferrin inactivation by periodate and permanganate; for example, the extent of inactivation by periodate was threefold greater in a borate buffer than in a phosphate buffer. Comparative oxidations in buffer systems suggest the following order of affinity of three buffer anions for the apotransferrin metal-binding center: phosphate greater than bicarbonate greater than borate. The interaction of phosphate ions with the iron-transferrin complex was also examined due to the increased susceptibility to periodate inactivation of iron-saturated transferrin in phosphate buffer (M. H. Penner, R. B. Yamasaki, D. T. Osuga, D. R. Babin, C. F. Meares, and R. E. Feeney (1983) Arch. Biochem. Biophys. 225, 740-747). The apparent destabilization of the iron-transferrin complex in phosphate buffer was found to be due to the competitive removal of iron by phosphate from the iron-protein complex. We found that phenylglyoxal-modified Fe-transferrin, with no loss of bound iron, was much more resistant to iron removal by phosphate and other competitive chelators.

Differential labeling of the subunits of respiratory complex III with [3H]succinic anhydride, [14C]succinic anhydride, and p-diazobenzene-[35S]sulfonate.

Exposure of antimycin-treated Complex III (ubiquinol-cytochrome c reductase) purified from bovine heart mitochondria to [3H]succinic anhydride plus [35S]p-diazobenzenesulfonate (DABS) resulted in somewhat uniform relative labeling of the eight measured subunits of the complex by [3H]succinic anhydride. In contrast, relative labeling by [35S]DABS was similar to [3H]succinic anhydride for the subunits of high molecular mass, i.e., core proteins, cytochromes, and the iron-sulfur protein, but greatly reduced for the polypeptides of molecular mass below 15 kDa. With Complex II depleted in the iron-sulfur protein the relative labeling of core protein I by exposure of the complex to [3H]succinic anhydride was significantly enhanced, whereas labeling of the polypeptides represented by SDS-PAGE bands 7 and 8 was significantly inhibited. Dual labeling of the subunits of Complex III by 14C- and 3H-labeled succinic anhydride before and after dissociation of the complex by sodium dodecyl sulfate, respectively, was measured with the complex in its oxidized, reduced, and antimycin-inhibited states. Subunits observed to be most accessible or reactive to succinic anhydride were core protein II, the iron-sulfur protein, and polypeptides of SDS-PAGE bands 7,8, and 9. Two additional polypeptides of molecular masses 23 and 12kDa, not normally resolved by gel-electrophoresis, were detected. Reduction of the complex resulted in a significant change of 14C/3H labeling ratio of core protein only, whereas treatment of the complex with antimycin resulted in decreases in 14C/3H labeling ratios of core proteins I and II, cytochrome c1, and a polypeptide of molecular mass 13kDa identified as an antimycin-binding protein.

Succinylated bovine heart mitochondrial cytochrome c oxidase.

Cytochrome c oxidase was prepared by sequential extraction of bovine heart muscle submitochondrial particles with sodium deoxycholate, followed by fractional precipitation with ammonium sulfate and chromatography on Sephadex G-75. The resulting preparation had typical absorption spectra, an activity of 1.28 sec(-1) (mg protein)(-1) (3 ml)(-1) in deoxycholate or 4.13 sec(-1) (mg protein)(-1) (3 ml)(-1) in 0.5% Tween 80, and a minimum molecular weight of 120,000 daltons as calculated from the heme content and the total protein. Amino acid analyses of nine preparations yielded a molecular weight per heme of 86,500 daltons. The net charge was calculated to be +8.7 at pH 7.0. Succinylation of cytochrome c oxidase in the presence of 500 molar excess of succinic anhydride produced a soluble preparation having a negative charge at neutral pH. The modified enzyme was highly autoxidizable and had little or no activity toward ferrocytochrome c as a substrate. Its average s20,w was 5.8 and its apparent D was 4.0 x 10(-7) cm2 sec(-1), from which a molecular weight of 126,000 daltons was calculated. This size of enzyme is considered to be that of the monomer, because the value is practically the same as the minimum molecular weight reported herein, and since it is approximately one-half the value obtained in our laboratory (and in others) for the unmodified enzyme.