Galectin 3-ß-galactobiose interactions.

Force spectroscopy has been used to investigate the interaction between the disaccharide ß-galactobiose and the pro-metastatic regulatory protein galectin-3 (Gal3). The studies revealed specific interactions characterised by an off-rate dissociation constant k(off)=0.33 s(-1) and interaction distance x=0.2 nm at zero applied force. These data suggest a lifetime for the interaction of 3.0 s. The results are consistent with the hypothesis that oral consumption of modified citrus pectin controls cancer metastasis by inhibiting the role of Gal3. The modification is considered to facilitate binding of pectin-derived galactan sidechains to Gal3 and inhibition of the roles of Gal3 as a pro-metastatic regulatory protein.

Interfacial structure of sugar beet pectin studied by atomic force microscopy.

Unlike pectins from other origins, sugar beet pectin (SBP) acts as an emulsifier, a property which has been correlated to its more hydrophobic character and high protein content. In this work, we have investigated the structure of SBP at interfaces by atomic force microscopy (AFM). Three situations were studied: the mica/water, graphite/water, and air/water interface. For the latter, the interfacial film was transferred onto mica using the Langmuir-Blodgett method. While the adsorption of individual pectin chains on mica requires the addition of divalent cations, on graphite a thin layer containing amorphous areas and rodlike chains forms spontaneously. We suggest that the layer contains proteins and pectin chains which are bound to the graphite via CH-pi interactions. SBP adsorbed at the air/water interface forms an elastic layer, as evidenced by pendant drop and surface shear rheology measurements. AFM Images reveal the layer is crippled with holes and contains rodlike chains, suggesting that the pectin chains prevent the formation of a densely packed protein layer. Nevertheless, we show that the interfacial pectin film is more resistant to displacement by surfactants than a pure protein film, possibly because of the formation of linkages between the pectin chains. In contrast, alkali treatment of the pectin appears to remove the pectin chains from the air/water interface and leaves a film that behaves similarly to pure protein. This work gives a new insight into the nanoscale organization of polysaccharides and polysaccharide-protein mixtures at macroscopic surfaces. The results gathered from the different interfaces studied permit a better understanding of the likely structure of SBP at the interface of emulsion droplets. Such knowledge might be used to modify rationally the pectin in order to improve its emulsifying properties, leading to broader commercial applications.

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin.

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

Mortality in mice infected with an amyocarditic coxsackievirus and given a subacute dose of mercuric chloride.

An amyocarditic strain of coxsackievirus B3 (CVB3/0) induces heart damage when inoculated into selenium (Se)-deficient mice. Mercury (Hg), an Se antagonist, is known to aggravate viral infections. The experiments reported here assessed the effect of prior Hg treatment in mice subsequently inoculated with an amyocarditic strain of coxsackievirus. A pilot study showed that under our conditions the maximum tolerated dose of HgCl2 in uninfected mice was 6 mg HgCl2/kg body weight. In the main study, doses of 0, 3 or 6 mg HgCl2/kg body weight were administered intraperitoneally (ip) to 7-wk-old male mice fed a standard chow diet. Two hours later, half the mice were inoculated ip with CVB3/0. Ten days postinoculation, no mortality was observed in mice given only virus. In mice not given virus, 10% injected with 6 mg HgCl2/kg body weight died. On the other hand, 64% of the mice given both virus and 6 mg HgCl2/kg body weight died. Fifteen percent of the hearts from virus-infected mice given 3 mg HgCl2/kg body weight and 33% of the hearts from virus-infected mice given 6 mg HgCl2/kg body weight exhibited a higher incidence of lesions than hearts from mice-given virus alone. Moreover, viral heart titers were elevated in infected mice injected with 6 mg HgCl2/kg body weight compared to infected mice receiving no Hg. Thus, an amyocarditic coxsackievirus given to mice after a nonlethal subacute dose of Hg results in mortality, increased incidence of heart lesions, and elevated viral heart titers. These results demonstrate the important role of toxic elements in determining the severity of viral infections.

Investigating the nature of branching in pectin by atomic force microscopy and carbohydrate analysis.

Atomic force microscopy (AFM) has been used to investigate the nature of the long branches attached to pectin which were described in a previous report [Round, A. N.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Carbohydr. Res. 1997, 303, 251-253]. Analysis of the AFM images and comparison with neutral sugar and linkage analyses of the two pectin fractions suggest that the distribution and total amount of branches observed do not correspond with the pattern of neutral sugar distribution. It is thus postulated that the long chains consist of polygalacturonic acid, attached via an as yet undetermined linkage to the pectin backbone, with the neutral sugars present as short, undetected branches. This explanation would have important implications for the nature of 'in situ' pectin networks within plant cell walls and models of gelation in commercial extracted pectin, and the existence of significant branching will markedly influence the viscosity of extracted pectins.

Rapid determination of glutathione peroxidase and thioredoxin reductase activities using a 96-well microplate format: comparison to standard cuvette-based assays.

Gluthatione peroxidase and thioredoxin reductase are selenocysteine-containing enzymes that are constituents of the cellular antioxidant defense system. Conventional cuvette-based assays for glutathione peroxidase and thioredoxin reductase enzymes are laborious and time consuming. The ability to assay their activities rapidly in multiple samples would aid efforts focused on understanding the impact of these enzymes on the cellular antioxidant defense system. High throughput can be achieved with assays adapted to work in a clinical analyzer but require expensive equipment. Assays designed to work in a 96-well microplate reader provide an alternative methodology for high throughput with reduced instrumentation cost. However, due to differences in the light pathlength when using a 96-well format, the values obtained cannot be compared directly with those obtained using a 1-cm cuvette. Described here are assays for glutathione peroxidase and thioredoxin reductase modified to work in a 96-well format that incorporates light pathlength determinations into the assays. The values obtained using a high throughput 96-well format in conjunction with pathlength determinations are in agreement with those obtained using a standard 1-cm cuvette. While spectrophotometrically derived pathlengths are the most accurate, calculated pathlengths based on assay volume and well size can be used with only a small amount of error introduced. This method can also be applied to many other enzyme assays, thus allowing the rapid analysis of large numbers of samples without the need for expensive equipment.

Assay of topically administered ibuprofen using a model of post-injury hypersensitivity. A randomised, double-blind, placebo-controlled study.

OBJECTIVE: To develop a reliable assay for quantifying the analgesic efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) using a model that is accepted as a paradigm of clinical pain. SUBJECTS: Fifteen normal subjects, all of whom were volunteers from medical school staff, took part in the study. METHODS: Capsaicin (20 microl) in solution (0.03 mg/ml) was applied to the volar surface of the forearm, and the skin was maintained at a constant temperature using a thermal stimulator. The magnitude of the surrounding area of mechanical allodynia to a brush stimulus (i.e. a clinical correlate of tenderness to touch) was assessed. Under double-blind, placebo-controlled conditions, the test was repeated using skin previously treated with ibuprofen gel or placebo. RESULTS: A close linear relationship was observed between skin temperature over a range of 30 degrees C to 40 degrees C and the area of capsaicin-induced allodynia. Ibuprofen gel significantly reduced (P < 0.004) the area of touch-evoked allodynia at a constant skin temperature of 40 degrees C. CONCLUSIONS: The thermal-facilitated adaptation of the capsaicin model described in this study represents an inexpensive and reliable assay for the effects of topical formulations of NSAID upon mechanical sensitivity. As such, it is a potential alternative to many clinical studies in which inherent confounding and bias can preclude a meaningful conclusion.

Observations on the crystallization of oligogalacturonates.

Oligogalacturonates were produced by the limited enzymic hydrolysis of polygalacturonic acid and purified by ion-exchange chromatography. The fractions obtained were of limited polydispersity, determined by analytical ion-exchange chromatography. Oligomers with an average degree of polymerization of 10-15 were readily crystallized from aqueous salt solutions at neutral pH as single crystals. Crystal morphology of the salts examined, Na+, K+ and Ca2+ were characteristic of the salt. The wide-angle X-ray diffraction patterns obtained for the sodium salt were consistent with published fibre diffraction data of this salt form.

Vitamin E-deficient diets enriched with fish oil suppress lethal Plasmodium yoelii infections in athymic and scid/bg mice.

Mice fed vitamin E-deficient diets containing omega-3 fatty acids survive infection with lethal Plasmodium yoelii. The current study sought to determine if antimalarial T- and B-cell responses were required for such dietary-mediated protection. In the first set of experiments, nu/nu mice (which lack alphabeta T-cell-receptor-positive T cells and do not produce antimalarial antibody) and nu/+ mice were fed casein-based diets containing 4% menhaden oil, with or without vitamin E supplementation, for 4 weeks prior to infection with lethal P. yoelii. All mice fed diets containing vitamin E developed fulminating parasitemias and quickly died, whereas both nu/nu and nu/+ mice fed diets deficient in vitamin E controlled their parasitemias for the first 18 days of infection. Thereafter, the nu/nu mice became anemic and died, whereas the nu/+ mice produced antimalarial antibodies and survived. In the second set of experiments, scid/scid.bg/bg mice (which lack B cells and alphabeta and gammadelta T cells and have reduced NK-cell activity) were fed the experimental diet for 6 weeks and then infected with the less virulent 17XNL strain of P. yoelii. Mice fed vitamin E-containing diets quickly died, whereas those fed the vitamin E-deficient diet survived without developing detectable parasitemias. Results from these experiments show that under prooxidant dietary conditions, mice were able to control and even survive malaria in the absence of malaria-primed T cells and antimalarial antibody. These results emphasize the importance of cellular oxidative processes in parasite elimination.

Association of lowered plasma carotenoids with protection against cecal coccidiosis by diets high in n-3 fatty acids.

A series of four experiments was run to assess the effectiveness of diets containing high amounts of n-3 fatty acids in reducing the pathological effects of cecal coccidiosis in chickens caused by Eimeria tenella. To determine whether the dietary effects were related to development of oxidative stress, plasma samples were analyzed for tocopherols and carotenoids. Plasma vitamin E (alpha-tocopherol) values were not consistent between experiments. Total plasma carotenoids, however, were significantly decreased by 2.5 to 20% diet supplementation with fish oil in several experiments. These decreases coincided with significant reductions in lesion scores. Under the experimental conditions, total plasma carotenoid concentrations may serve as a sensitive indicator for oxidative stress, which may be a factor in reducing cecal lesions in E. tenella infections.

Rapid genomic evolution of a non-virulent coxsackievirus B3 in selenium-deficient mice results in selection of identical virulent isolates.

Previous work from our laboratory demonstrated that selenium deficiency in the mouse allows a normally benign (amyocarditic) cloned and sequenced Coxackievirus to cause significant heart damage. Furthermore, Coxsackievirus recovered from the hearts of selenium-deficient mice inoculated into selenium-adequate mice still induced significant heart damage, suggesting that the amyocarditic Coxsackievirus had mutated to a virulent phenotype. Here we report that sequence analysis revealed six nucleotide changes between the virulent virus recovered from the selenium-deficient host and the avirulent input virus. These nucleotide changes are consistent with known differences in base composition between virulent and avirulent strains of Coxsackievirus. To the best of our knowledge, this is the first report of a specific nutritional deficiency driving changes in a viral genome, permitting an avirulent virus to acquire virulence due to genetic mutation.

Protection against murine cerebral malaria by dietary-induced oxidative stress.

Feeding 20% (w/w) menhaden-fish oil in a standard laboratory chow diet for 4 wk partially protected CBA/CaJ mice from the central nervous system consequences of infection with Plasmodium berghei (ANKA). Full protection (complete survival for 14 days postinfection) could be obtained by feeding a purified pro-oxidant vitamin E-deficient diet containing 4% (w/w) menhaden oil (MO - VE diet). The purified pro-oxidant MO - VE diet also exerted a pronounced suppressive effect against the parasite (depressed 6-day parasitemias). The anitmalarial effect of the MO - VE diet could be prevented by supplementing the diet with vitamin E or with either of 2 synthetic antioxidants, N,N'-diphenyl-p-phenylenediamine or probucol. These results suggest that the fish oil exerts its antimalarial effect by imposing a dietary-induced oxidative stress on the infected host erythrocyte, the parasite, or both. Nutritional manipulation of host oxidative stress status may be a useful adjunct therapy in patients undergoing treatment with pro-oxidant antimalarials such as drugs of the qinghaosu family.

Increased virulence of a human enterovirus (coxsackievirus B3) in selenium-deficient mice.

Coxsackievirus B3 (CVB3/20)-induced myocarditic lesions occurred more quickly and were more severe and virus titers in heart and liver were higher in selenium (Se)-deficient than Se-adequate mice. NK cell activity and serum neutralizing antibody titers were similar in both Se-adequate and -deficient CVB3/20-infected mice; however, lymphocyte proliferation to both mitogen and antigen was decreased in Se-deficient mice. CVB3/20 isolated from Se-deficient donor mice and inoculated into Se-adequate recipient mice induced severe myocarditis. In contrast, CVB3/20 isolated from Se-adequate donor mice and inoculated into Se-adequate recipient mice induced only moderate myocarditis, similar to that caused by the original virus stock. Thus, the general population of CVB3/20 virions, as a consequence of replicating in an Se-deficient host, underwent a phenotypic change to increased virulence. These results have important implications for the emergence of virulent viruses.

Benign human enterovirus becomes virulent in selenium-deficient mice.

Coxsackieviruses have been implicated as possible co-factors in the etiology of the selenium (Se)-responsive cardiomyopathy known as Keshan disease. Here we report that a cloned and sequenced amyocarditic coxsackievirus B3 (CVB3/0), which causes no pathology in the hearts of Se-adequate mice, induces extensive cardiac pathology in Se-deficient mice. CVB3/0 recovered from the hearts of Se-deficient mice inoculated into Se-adequate mice induced significant heart damage, suggesting mutation of the virus to a virulent genotype. We demonstrate the important role of host nutritional status in determining the severity of a viral infection.

Vitamin E deficiency intensifies the myocardial injury of coxsackievirus B3 infection of mice.

Feeding a vitamin E-deficient diet increases pathology in hearts of mice infected with a myocarditic coxsackievirus B3 (CVB3/20). Hearts from infected mice fed a vitamin E-deficient diet rich in highly unsaturated fat (menhaden oil) exhibited more severe pathology than hearts from infected mice fed a vitamin E-deficient diet based largely on saturated fat (lard). Furthermore, a cloned and sequenced amyocarditic coxsackievirus B3 (CVB3/0), which caused little or no pathology in the hearts of vitamin E-supplemented mice, induced extensive cardiac pathology in vitamin E-deficient mice. In infected mice, both mitogen and antigen responses were depressed by vitamin E deficiency, although neutralizing antibody responses were unaffected. Natural killer cell responses were comparable in infected mice fed a lard-based diet with or without supplemented vitamin E. However, a menhaden oil-based diet, whether supplemented with vitamin E or not, significantly depressed natural killer cell activity in infected mice compared with mice fed the lard-based diet. Coxsackievirus B3/0 recovered from the heart of a vitamin E-deficient donor mouse, passaged one time onto HeLa cells, caused significant heart damage when passed back into vitamin E-supplemented recipient mice, demonstrating that the amyocarditic CVB3/0 had changed to a virulent phenotype. Enhanced virulence was also seen with CVB3/20 virus similarly passaged in a vitamin E-deficient donor. Our work demonstrates the important role of host nutritional antioxidant status in determining the severity of certain viral infections.

The urinary excretion of thiobarbituric acid reactive substances and malondialdehyde by normal adult males after consuming a diet containing salmon.

In this study we investigated the output of thiobarbituric acid reactive substances (TBARS) and malondialdehyde (MDA), as thiobarbituric acid (TBA)-MDA adduct, in the urine from subjects eating a diet in which the only source of n-3 long-chain, polyunsaturated fatty acids was fresh salmon. Nine healthy men, ages 30-65, were confined in the United States Department of Agriculture Western Human Nutrition Research Center, San Francisco, CA, for 100 d; food intake and exercise levels were controlled. All subjects were placed on a stabilization diet (StD) for 20 d, then six were fed the salmon diet for 40 d. The others remained on the StD. The groups switched diets for the last 40 d. Both diets were isocaloric (16% protein, 54% CHO and 30% fat by energy %). The salmon diet contained 7.5% of calories from n-6 fatty acids (FAs) and 2% from n-3 FAs, primarily eicosapentaenoic acid and docosahexaenoic acid in a 50:60 ratio, while the StD contained 7.5% from n-6 FAs and < 0.3% n-3 FAs (with presumably no significant amounts of C20 or C22 n-3 FAs). Twenty-four hour urinary output was collected, and 2% 3-d pool samples prepared for analysis of urinary TBARS and the TBA-MDA adduct. The total urinary output of each individual varied considerably, and on a daily basis the concentration of autoxidation products in an individual's urine varied also.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasmodium yoelii: comparative antimalarial activities of dietary fish oils and fish oil concentrates in vitamin E-deficient mice.

Feeding vitamin E-deficient diets containing either fish oils such as menhaden, salmon, or anchovy oil or fish oil concentrates based on n-3 ethyl esters or free fatty acids protected mice against Plasmodium yoelii as indicated by decreased parasitemia and improved survival. The fish oil concentrates depressed plasma tocopherol levels more strongly in vitamin E-supplemented mice than the menhaden oil. The free fatty acid concentrate appeared to suppress parasitemia in vitamin E-deficient mice better than the menhaden oil, although ultimate survival was similar in both groups. Dietary manipulation of host antioxidant status offers promise as a possible means of malaria control.

Menhaden-fish oil in a vitamin E-deficient diet: protection against chloroquine-resistant malaria in mice.

Feeding a vitamin E-deficient diet containing 5% menhaden oil to mice affords significant protection against both a chloroquine-sensitive and a chloroquine-resistant line of the malarial parasite. Nutritional manipulation may offer a new approach to the problem of drug-resistant malaria, a rapidly emerging global threat to public health.

Qinghaosu, dietary vitamin E, selenium, and cod-liver oil: effect on the susceptibility of mice to the malarial parasite Plasmodium yoelii.

Young female mice were fed torula-yeast-based diets deficient in vitamin E or selenium or supplemented with cod-liver oil to determine the effect of host antioxidant status on the therapeutic efficacy of the Chinese traditional antimalarial drug qinghaosu (QHS), a sesquiterpene endoperoxide. Vitamin E deficiency enhanced the antimalarial action of QHS against Plasmodium yoelii, both in terms of decreased parasitemia and improved survival but Se deficiency did not. A vitamin E-deficient diet containing 5% cod-liver oil had such strong antimalarial activity in itself that no additional therapeutic benefit of QHS could be demonstrated. Hematocrit values in parasitized mice treated with QHS or fed the cod-liver-oil-supplemented, vitamin E-deficient diet were normal. Nutritional manipulation of host antioxidant status may provide a promising prophylactic and/or therapeutic tool for the control of malaria.

Effect of dietary selenium on the development of Fusarium-induced tibial dyschondroplasia in broiler chickens.

A trial was conducted to determine the effects of dietary level of selenium on the pathogenesis of Fusarium-induced tibial dyschondroplasia (FITD) in broiler chicks, and to assess the applicability of FITD as an animal model of Kashin-Beck disease of humans. Day-old female broilers were fed diets that were deficient in selenium (0.02 ppm Se), adequate in selenium (0.15 ppm Se), or generous in selenium (0.50 ppm Se). TDP-1, the toxic component of the fungus, was administered to 15 of 26 chicks in each dietary group starting at 1 week of age and continuing until the chicks were killed at 24-30 days of age. Plasma selenium levels and hepatic glutathione peroxidase activity were significantly lower in the selenium-deficient group than in other dietary groups; these parameters were not affected by treatment with TDP-1. The mortality rate of the TDP-1-treated selenium-generous group was significantly less than that in the other TDP-1-treated groups, but there were no differences in the incidence, severity, or character of the FITD lesions among the groups. Thus, the interaction of selenium and TDP-1 did not include an effect on FITD.