A Common Practice of Widespread Antimicrobial Use in Horse Production Promotes Multi-Drug Resistance.

The practice of prophylactic administration of a macrolide antimicrobial with rifampin (MaR) to apparently healthy foals with pulmonary lesions identified by thoracic ultrasonography (i.e., subclinically pneumonic foals) is common in the United States. The practice has been associated epidemiologically with emergence of R. equi resistant to MaR. Here, we report direct evidence of multi-drug resistance among foals treated with MaR. In silico and in vitro analysis of the fecal microbiome and resistome of 38 subclinically pneumonic foals treated with either MaR (n = 19) or gallium maltolate (GaM; n = 19) and 19 untreated controls was performed. Treatment with MaR, but not GaM, significantly decreased fecal microbiota abundance and diversity, and expanded the abundance and diversity of antimicrobial resistance genes in feces. Soil plots experimentally infected with Rhodococcus equi (R. equi) and treated with MaR selected for MaR-resistant R. equi, whereas MaR-susceptible R. equi out-competed resistant isolates in GaM-treated or untreated plots. Our results indicate that MaR use promotes multi-drug resistance in R. equi and commensals that are shed into their environment where they can persist and potentially infect or colonize horses and other animals.

Detection of depleted uranium in biological samples from Gulf War veterans.

During the Persian Gulf War, soldiers may have inhaled, ingested, and/or experienced wound contamination by depleted uranium (DU), which is used in military projectiles and armor. DU is produced by depleting natural uranium of 234U and 235U during the uranium-enrichment process. Although the long-term effects of significant DU exposures require investigation, many veterans express fears about its impact on health. An assay by which DU exposure can be assessed would not only be a useful research tool, but the information could help mitigate the concerns of exposed individuals. In this study, urine samples from individuals enrolled in the Depleted Uranium Follow-Up Program at the Baltimore Veterans Administration Medical Center were examined for uranium content. Isotopic composition of urine uranium was determined by measuring the 235U/238U ratio, using an inductively coupled plasma mass spectrometer. Using this method, natural and depleted uranium could be readily differentiated. By demonstrating the absence of DU in soldiers who suspect exposure by inhalation or ingestion, the assay should reduce psychological stress in these individuals.

The role of magnesium in the susceptibility of soldiers to noise-induced hearing loss.

A number of studies have demonstrated that magnesium, administered prophylactically, can reduce the amount of hearing loss resulting from noise exposure. This study explored the possible role of naturally occurring body magnesium concentration in susceptibility of soldiers to noise-induced hearing loss. Participants were 68 adult males who had received extensive noise exposure over several years as a result of training with weapon systems. Each participant provided a pure-tone audiogram, blood sample, and noise-exposure history. A variety of pure-tone indices was correlated with serum magnesium levels as determined by atomic absorption spectroscopy. No significant correlations were observed between any audiometric index and body magnesium. The results of this study, therefore, do not support the hypothesis that there is a strong association between naturally occurring body magnesium and susceptibility to noise-induced hearing loss.

Content of minor and trace elements, and organic nutrients in representative mixed total diet composites from the USA.

As part of an international nutrition project sponsored by the International Atomic Energy Agency (see footnote), Vienna, Austria, a number of bench-mark mixed total diet composites from the United States were collected and analyzed for minor and trace elements. In this segment of the project, the daily dietary intakes of the minor elements Ca, Cl, K, Mg, N and P and the trace elements Al, As, Au, Br, Cd, Co, Cr, Cs, Cu, Fe, Hg, I, Mn, Mo, Ni, Pb, Rb, Sb, Sc, Se, Sn, Sr, V, Zn and W were determined in mixed total diet composites of foods collected in the FDA Total Diet Study (FDA-TDS). These diets are representative of foods consumed by 25-30-year-old males (representing the mixed population groups in the United States), the highest of eight intake groups in the TDS scheme. In order to link the US mixed diet composite results from this study group to the more comprehensive information generated by the FDA-TDS, the results are compared with the same age-sex group published by the FDA-TDS scheme. The FDA-TDS scheme is based on individual analysis of the 201 food items, with resultant calculation of the daily intake representative of various age-sex groups. The comparison shows excellent agreement for 21 elements which have been investigated by both approaches. Additional elements are reported in the US mixed diet composites from the present study which demonstrate a valuable supplement to the data obtained by FDA-TDS scheme. Further the mixed total diet composite approach has also proven useful for the assessment of dietary intake of proximates (protein, fat, carbohydrates), fiber and phytate. In addition, vitamins thiamin, riboflavin, niacin, B6, B12, pantothenic acid, folic acid and biotin were also assayed in these composites.

The effect of human chorionic gonadotropin on methotrexate concentration in the rat testes.

PURPOSE: We analyzed the effect of human chorionic gonadotropin (hCG) on drug concentrations in testicular interstitial fluid and whole testis tissue samples in rats receiving hCG prior to methotrexate (MTX) administration and in animals that did not receive hCG. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were injected subcutaneously with 200 i.u. hCG (Goldline Laboratories, Ft. Lauderdale, FL.). Controls were injected subcutaneously with normal saline (0.2 cc). Sixteen hours after injection, each rat was given methotrexate (Methotrexate LPF, Immunex Corp. Seattle WA.) via a carotid artery cannula in a dose of 30 mg./kg. Methotrexate (MTX) levels were collected 60 minutes post infusion time in 27 rats and 90 minutes post infusion in 27 rats. MTX levels were measured in serum, testicular interstitial fluid and testicular tissue. MTX levels were measured using high performance liquid chromatography (HPLC). RESULTS: A significantly higher concentration of MTX was found in testicular interstitial fluid (TIF) in rats injected with hCG when specimens were collected 60 minutes post infusion. MTX levels in TIF had reversed 90 minutes post infusion with higher levels found in control rats. Tissue levels of MTX demonstrated no significant difference at either 60 or 90 minutes in the hCG treated animals or controls. CONCLUSION: Our results suggest that hCG effects the tissue distribution of MTX within the testis. Human chorionic gonadotropin may have this effect on the testicular microvasculature by 1) selectively increasing capillary permeability, 2) increasing lymphatic flow within the testes or 3) increasing testicular blood flow.

Effect of deacetylation on the synergistic interaction of acetan with locust bean gum or konjac mannan.

It has been discovered that deacetylation of the bacterial polysaccharide acetan promotes synergistic interactions with either locust bean gum (LBG) or konjac mannan (KM). Acetan is similar in structure to xanthan, and adopts a similar 5-fold conformation in the solid state. Like xanthan, it shows a thermally reversible order (helix)-disorder (coil) transition in solution. Both polymers have a cellulosic backbone with charged (anionic) sidechains attached at O-3 of alternate glucosyl residues, but the sidechains in acetan are longer (pentasaccharide rather than trisaccharide) and do not contain pyruvic substituents. Acetan has two sites of acetylation, one at O-6 of the inner mannosyl residue of the carbohydrate sidechains (as in xanthan) and the other on the polymer backbone (believed to be at O-6 of the branched glucosyl residues). Solutions of acetan or deacetylated acetan were equilibrated against 10 mM potassium chloride (to stabilise the ordered conformation) and were mixed (at 25 degrees C) with solutions of LBG or KM, also equilibrated against 10 mM potassium chloride. Unlike xanthan, native acetan showed no evidence of synergistic interaction with either LBG or KM. After deacetylation, however, large enhancements were observed in dilute-solution viscosity, and thermoreversible gels were formed at higher concentrations. With KM as co-synergist, gel melting was accompanied by an intense endotherm in differential scanning calorimetry. The magnitude of this endotherm increased with storage time at 25 degrees C, reaching a final value of delta H approximately 15.9 J/g (in comparison with delta H approximately 5.0 J/g for the order-disorder transition of deacetylated acetan alone). It is suggested that interaction occurs by formation of heterotypic junctions between the acetan backbone and unsubstituted regions of the plant polysaccharide, and that the acetate groups on native acetan promote solubility and hence inhibit association.

Structure and conformation of acetan polysaccharide.

Acetan is an anionic bacterial polysaccharide. The chemical repeat unit consists of a cellobiose unit solubilised by attachment of a charged pentasaccharide sidechain to one of the glucose residues. The repeat unit contains two sites of acetylation. 1H and 13C NMR studies, coupled with both basic-methylation and mild-methylation studies, have shown that acetylation occurs at C6 on the (1,2)D-Man and the (1,34)D-Glc residues. A variety of techniques including NMR, optical rotation, circular dichroism and DSC show evidence for a thermoreversible conformational order (helix)-disorder (coil) transition for acetan in aqueous solution. The studies suggest that acetylation of the backbone does not prevent helix formation.

Ascorbate offsets the inhibitory effect of inositol phosphates on iron uptake and transport by Caco-2 cells.

Differentiated monolayer cultures of Caco-2 human intestinal cells were used as a model to examine interactions between various dietary factors related to the intestinal uptake and absorption of nonheme Fe. Caco-2 cells accumulated 91-98 pmol Fe/mg protein from uptake buffer containing 12 nmol of Fe(III)-nitrilotriacetate during a 1-hr incubation at 37 degrees C. Addition of a 10-fold molar excess of inositol hexaphosphate (IP6) and its lesser phosphorylated derivatives (IP3, IP4, and IP5) decreased cellular uptake and transport of Fe from the lumenal compartment. Addition of ascorbic acid (AA) to the solution containing IPs stimulated Fe uptake and transport in a manner dependent upon the ratio of AA to IP and inversely proportional to the degree of phosphorylation of inositol (i.e., IP3 > IP4 > IP5 > IP6). A mixture of essential amino acids had minimal impact on Fe uptake in either the absence or presence of IPs. Cellular acquisition of Fe from solutions containing IPs was further enhanced by simultaneous addition of essential amino acids and AA. The stimulatory influence of ascorbic acid on Fe uptake from solutions containing IP6 was associated with an increase in the level of ferrous ion. These data further support the usefulness of Caco-2 cells as a model for investigating the effects of various dietary factors on mineral bioavailability.

Reduction of Fe(III) is required for uptake of nonheme iron by Caco-2 cells.

Differentiated cultures of Caco-2 human colonic cells were used to examine the importance of reduction of nonheme ferric iron, Fe(III), for transport across the brush border surface. Cultures accumulated approximately 100 pmol Fe/(h.mg protein) when 10 mumol Fe(III) as the nitrilotriacetic acid complex (1Fe:2NTA) was added to the apical compartment. Ascorbic acid enhanced cellular acquisition of iron in a dose-dependent manner, with a concentration as low as 8 mumol/L ascorbate increasing iron uptake by 50%. Similarly, the rate of iron transport from the apical to the basolateral compartment increased 5.6- and 30-fold when 100 and 1000 mumol/L ascorbic acid, respectively, were present in the apical chamber. Ascorbate-mediated stimulation of iron uptake was temperature dependent and required the reduction of Fe(III) to Fe(II), because it was inhibited by ascorbate oxidase and chelators of Fe(II). Moreover, Caco-2 cells recycled dehydroascorbic acid to ascorbic acid. Ferricyanide and Fe(II) chelators also partially inhibited iron uptake from a medium devoid of ascorbic acid. Intact Caco-2 cells exhibited a ferrireductase activity on the apical surface that accounted for the majority of iron accumulated by cells incubated in the absence of exogenous reductant. These data suggest that reduction of Fe(III) within the lumen or at the cell surface is required for transfer of this essential micronutrient across the intestinal brush border surface.

Intra- and extracellular amyloid fibrils are formed in cultured pancreatic islets of transgenic mice expressing human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is the constituent peptide of amyloid deposits found in the islets of non-insulin-dependent diabetic patients. Formation of islet amyloid is associated with a progressive destruction of insulin-producing beta cells. Factors responsible for the conversion of IAPP into insoluble amyloid fibrils are unknown. Both the amino acid sequence of human IAPP (hIAPP) and hypersecretion of hIAPP have been implicated as factors for amyloid fibril formation in man. We have generated transgenic mice using rat insulin promoter-hIAPP or rat IAPP (rIAPP) gene constructs. No fibrillar islet amyloid was detectable in vivo in these normoglycemic mice, although small amorphous perivascular accumulations of IAPP were observed in hIAPP mice only. To determine the effects of glucose on IAPP secretion and fibrillogenesis, pancreatic islets from transgenic and control mice were examined in vitro. Islet IAPP secretion and content were increased in transgenic islets compared with control islets. IAPP-immunoreactive fibrils were formed at both intra- and extracellular sites in isolated hIAPP islets cultured with glucose at 11.1 and 28 mM for only 7 days. At 28 mM glucose, fibrils were present in deep invaginations of beta cells as observed in non-insulin-dependent diabetic patients. No fibrils were present at low glucose concentrations in hIAPP islets or at any glucose concentration in rIAPP or control islets. Thus, glucose-induced expression and secretion of hIAPP in transgenic mouse islets can lead to formation of amyloid fibrils similar to that found in non-insulin-dependent diabetes mellitus.

Inositol phosphates inhibit uptake and transport of iron and zinc by a human intestinal cell line.

To examine the influence of inositol phosphates on the uptake and absorption of Fe and Zn, Caco-2 cells were grown on either plastic (uptake studies) or porous membranes in bicameral chambers (transport/absorption studies). Caco-2, a human colon adenocarcinoma cell line, was selected as the test cell because it spontaneously differentiates into polarized enterocyte-like cells at confluency. Uptake of Fe (added as Fe-nitrilotriacetate complex) from a calcium-free solution by fully differentiated cells was 37 pmol/cm2. Addition of 10-fold molar excess of individual inositol phosphates (IP3, IP4, IP5 or IP6) decreased Fe solubility by 13 to 25% and reduced Fe uptake by 50 to 65%. The rate of transport of Fe from the apical solution into the basolateral chamber [1.4 +/- 0.1 pmol/(h.cm2)] decreased (34-96%) in proportion to the degree of phosphorylation of the inositol derivative in the apical compartment. Uptake and transepithelial transport of Zn were 246 +/- 5 pmol/cm2 and 23 +/- 1 pmol/(h.cm2), respectively. The solubility, uptake and rate of transport of Zn also decreased in proportion to the degree of phosphorylation of inositol. These results demonstrate the inhibitory influence of IP3-IP6 on the uptake and transport of Fe and Zn and support the usefulness of the Caco-2 human cell line as an appropriate model for evaluating the effects of specific dietary factors on trace metal bioavailability.

Native ordered structure of welan polysaccharide: conformational transitions and gel formation in aqueous dimethyl sulphoxide.

Welan, in aqueous solution, has "weak gel" properties analogous to those of ordered xanthan but, unlike xanthan, shows no evidence of conformational change between 0 and 100 degrees C. When the polymer is dissolved in dimethyl sulphoxide (Me2SO) rather than in water, however, there is a massive decrease in viscosity and total loss of gel-like character. In mixtures of the two solvents, the change in rheology occurs over a narrow range of composition (approximately 85-90% v/v Me2SO for 0.5% welan). On heating and cooling in a solvent close to the lower end of the critical range (86% Me2SO), the polymer shows typical order-disorder and disorder-order transitions [as monitored by optical rotation, differential scanning calorimetry, and temperature-course of rheological change]. When solutions of disordered welan in Me2SO are poured into excess water they form cohesive strings of gel. We interpret these results as showing that: (1) the stable conformation of welan in water is the double helix structure identified by X-ray fibre diffraction in the solid state; (2) in native welan, as biosynthesised, the strands are perfectly paired, and ordered along their full length; (3) on exposure to high concentrations of Me2SO, the native structure is dissociated into disordered coils; (4) rapid renaturation from the disordered state gives shorter helices, with exchange of partners to form a stable cross-linked network.

Enzyme-linked immunosorbent assays for gliadin and ovalbumin and their application in normal subjects.

Two quick, sensitive and direct sandwich enzyme-linked immunosorbent assays (ELISAs) for the measurement of gliadin and ovalbumin in serum have been developed. The ovalbumin assay has a sensitivity of 0.14 ng/ml (2 SD from zero). Cross-reactivities with ovomucoid and conalbumin were 58% and 0.2%, respectively. No cross-reactivity was observed with bovine serum albumin (BSA), human serum albumin (HSA), gliadin and beta-lactoglobulin. The gliadin assay did not cross-react with BSA, HSA, ovalbumin and beta-lactoglobulin and had a sensitivity of 7 pg/ml. In an acute feeding study, of 6 h duration, serum ovalbumin levels were measured in five non-food-allergic subjects who had been given raw eggs (5 g ovalbumin) after an overnight fast. Following a period of at least 1 week the same subjects were given 132 g wholemeal bread (5 g gliadin) after an overnight fast, and serum gliadin levels were measured. After the consumption of raw eggs ovalbumin levels peaked between 210 and 300 min with maximum serum concentration between 0.52 and 31.08 ng/ml. After the consumption of bread, gliadin levels reached a maximum between 180 and 300 min, reaching peak levels between 1.12 and 12.2 ng/ml. We conclude that levels of detected ovalbumin were higher than those for gliadin in this group of individuals.

IgG subclasses to food proteins in atopic and normal individuals.

IgG subclass antibodies to ovalbumin (OVA), gliadin (GLI) and beta-lactoglobulin (beta-lac) were determined in 18 atopic subjects and 18 age- and sex-matched controls. IgG antibodies to OVA and GLI were found predominantly as IgG4 subclass, with the IgG antibodies to beta-lac distributed evenly between IgG2, IgG3 and IgG4 in both groups. The proportion of anti-OVA IgG antibodies in the IgG3 subclass was significantly higher in the atopic group (22%) than in the nonatopic group (8%: p < 0.001). The proportion of anti-GLI IgG3 antibodies was also higher in the atopic group (43%) than the nonatopic group (24%: p < 0.001). OVA subclass antibodies in the atopic and nonatopic subjects were mainly IgG4, and GLI subclass antibodies were mainly IgG3 and IgG4. This study provides information on the distribution of IgG subclasses to food proteins, and in particular those related to protein GLI. The importance of IgG3 subclasses in atopic disease is highlighted; this phenomenon, to our knowledge, has not previously been reported.

Physical properties of dietary fiber that influence physiological function: a model for polymers along the gastrointestinal tract.

The quantitative measurement of dietary fiber does not recognize its diverse actions on nutrient absorption, sterol metabolism, fermentation in the colon, and stool weight. These differences in action are more likely due to differences in physical characteristics along the gastrointestinal tract. This paper explores such physical characteristics and attempts to classify dietary fiber in a more physical manner. This approach recognizes the diverse and variant action of each dietary fiber, which may be modified as a result of processing and cooking without changing in any way the quantitative measurement of dietary fiber. The general principles developed in this paper could also be applied to other polymeric materials passing along the gastrointestinal tract.

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.

Blood glucose, plasma insulin and sensory responses to guar-containing wheat breads: effects of molecular weight and particle size of guar gum.

The effectiveness of guar gum in reducing post-prandial blood glucose and plasma insulin levels in human subjects seems to depend mainly on its ability to increase the viscosity of digesta in the small intestine. However, the precise relationship between the rheological properties of guar gum (either in vitro or in vivo) and the changes in blood metabolites and hormones is unknown. The aim of the present study, therefore, was to investigate the effects of wheat breads containing guar gum samples varying in molecular weight (Mw) and particle size (characteristics that strongly influence the rheological properties of guar gum) on post-prandial blood glucose and plasma insulin levels in healthy subjects. The sensory qualities of breads containing guar-gum flours of different Mw were also evaluated using a hedonic scoring technique. No significant differences in the post-prandial blood glucose responses were found between the control and guar breads. However, all the guar breads elicited significant (P less than 0.05) decreases in the post-prandial rise in plasma insulin, an effect that did not appear to be influenced by large variations in Mw or particle size of guar gum. Moreover, the sensory qualities of guar bread were markedly improved by using low Mw grades of guar gum.

What are people really eating? The relation between energy intake derived from estimated diet records and intake determined to maintain body weight.

Two hundred sixty-six free-living human volunteers, 21-64 y old, were trained by dietitians to record daily their food intake for at least 7 d. Subsequently, they were fed diets of conventional foods adjusted in amounts to maintain their body weight for greater than or equal to 45 d. Comparing their estimated energy intake with the intake determined to maintain weight yielded mean differences of 2365 and 1792 kJ (565 and 428 kcal) in men and women, respectively, representing an underreporting of 18%. Twenty-two individuals (8%) overestimated and 29 (11%) were accurate to within 419 kJ (100 kcal) of their maintenance requirement. The remaining 215 individuals (81%) reported their habitual intake at 2930 +/- 1586 kJ (700 +/- 379 kcal) below that subsequently determined as their maintenance requirement. These findings suggest caution in the interpretation of food-consumption data.