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Inactivation of Escherichia coli O157:H7, Salmonella typhimurium DT104, and Listeria monocytogenes on inoculated alfalfa seeds with a fatty acid-based sanitizer.

Alfalfa seeds were inoculated with a three-strain cocktail of Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Typhimurium DT104, or Listeria monocytogenes by immersion to contain approximately 6 to 8 log CFU/g and then treated with a fatty acid-based sanitizer containing 250 ppm of peroxyacid, 1,000 ppm of caprylic and capric acids (Emery 658), 1,000 ppm of lactic acid, and 500 ppm of glycerol monolaurate at a reference concentration of 1X. Inoculated seeds were immersed at sanitizer concentrations of 5X, 10X, and 15X for 1, 3, 5, and 10 min and then assessed for pathogen survivors by direct plating. The lowest concentration that decreased all three pathogens by >5 log was 15. After a 3-min exposure to the 15X concentration, populations of E. coli O157:H7, Salmonella Typhimurium DT104, and L. monocytogenes decreased by >5.45, >5.62, and >6.92 log, respectively, with no sublethal injury and no significant loss in seed germination rate or final sprout yield. The components of this 15x concentration (treatment A) were assessed independently and in various combinations to optimize antimicrobial activity. With inoculated seeds, treatment C (15,000 ppm of Emery 658, 15,000 ppm of lactic acid, and 7,500 ppm of glycerol monolaurate) decreased Salmonella Typhimurium, E. coli O157:H7, and L. monocytogenes by 6.23 and 5.57 log, 4.77 and 6.29 log, and 3.86 and 4.21 log after 3 and 5 min of exposure, respectively. Treatment D (15,000 ppm of Emery 658 and 15,000 ppm of lactic acid) reduced Salmonella Typhimurium by >6.90 log regardless of exposure time and E. coli )157:H7 and L. monocytogenes by 4.60 and >5.18 log and 3.55 and 3.14 log after 3 and 5 min, respectively. No significant differences (P > 0.05) were found between treatments A, C, and D. Overall, treatment D, which contained Emery 658 and lactic acid as active ingredients, reduced E. coli O157:H7, Salmonella Typhimurium, and L. monocytogenes populations by 3.55 to >6.90 log and may provide a viable alternative to the recommended 20,000 ppm of chlorine for sanitizing alfalfa seeds.

Colony Count, Microbial↗

Biosynthesis of thiopeptide antibiotic A10255 in stirred reactors using a chemically defined medium supplemented with continuous nutrient feeds.

A10255 is a complex of new thiopeptide antibiotics produced by Streptomyces gardneri. When stirred reactors were operated in batch mode using a defined medium with a glucose feed, 250 micrograms/ml of A10255 were produced during a four-day fermentation cycle. The linear growth phase of S. gardneri was extended through seven days by supplementing the defined medium with continuous feeds of hydrolyzed casein and methyl caprate. With the supplementary feeds, antibiotic biosynthesis paralleled growth during the extended cycle and attained levels of 1,750 micrograms/ml. Increasing the standard glucose feed rate increased titers principally by increasing cell mass. Supplementing the standard glucose feed with lipids such as caprylate or caprate, and decyl alcohol, affected cell mass minimally but produced higher titers by increasing the specific biosynthesis of A10255 per unit of biomass.

Anti-Bacterial Agents↗

Role of the ADP/ATP and aspartate/glutamate antiporters in the uncoupling effect of fatty acids, lauryl sulfate, and 2, 4-dinitrophenol in liver mitochondria.

Study of the uncoupling effect of various saturated fatty acids (from caprylic to palmitic) revealed that the glutamate recoupling effect was more pronounced in the case of short chain fatty acids, whereas recoupling of mitochondria by carboxyatractylate was more effective in the case of long chain fatty acids. The overall recoupling effect, however, did not depend on the fatty acid chain length. Besides carboxyatractylate, glutamate and aspartate also exhibited a recoupling effect under uncoupling by lauryl sulfate. The uncoupling effect of lauryl sulfate was markedly weaker in the presence of DNP or laurate (but not FCCP) which were added in concentrations causing twofold increase in mitochondrial respiration. In the presence of lauryl sulfate the uncoupling action of laurate and DNP was insensitive to carboxyatractylate and glutamate. With laurate and DNP as uncouplers increasing the pH from 7.0 to 7.8 potentiated the recoupling effect of carboxyatractylate and attenuated the recoupling effect of glutamate. In the case of uncoupling by lauryl sulfate similar changes in the recoupling effect of carboxyatractylate and glutamate were observed only in the presence of 10 microM tetraphenylphosphonium. Thus, when uncoupling is induced by fatty acids, DNP, and lauryl sulfate, the ADP/ATP and aspartate/glutamate antiporters function as two parallel and independent pathways for mitochondrial membrane potential dissipation. We suggest that the role of the ADP/ATP antiporter in uncoupling includes proton capture from the intermembrane space with subsequent protonation of uncoupler anions, their transport as neutral molecules on the internal side, and deprotonation followed by proton release into the matrix and transfer of the uncoupler anion in the reverse direction. During uncoupling the aspartate/glutamate antiporter cyclically carries the uncoupler anion with simultaneous proton transfer from the intermembrane space into the matrix.

2,4-Dinitrophenol↗

Estimation of absorption enhancement by medium-chain fatty acids in rat large intestine.

The absorption enhancement by the sodium salts of several fatty acids was investigated in rat large intestine for model compounds having a wide range of molecular weight. Sodium caprylate (C8), sodium caprate (C10), sodium laurate (C12), which are categorized in medium-chain fatty acid, and sodium oleate (C18:1), long-chain unsaturated fatty acid, were employed as lipoidal adjuvants. Phenol red (MW=354.4), glycyrrhizin (822.9), fluorescein isothiocyanate-dextran-4 (FD-4, 4400), FD-10 (9400) and FD-40 (38900) were selected as model compounds for the assessment of the enhancing effect of the lipoidal adjuvants. The absorption of phenol red was promoted at the highest level, about 20 times higher by C12 and C18:1 than the control. The absorption rate - time profiles calculated by deconvolution method showed that C12 takes effect most rapidly and efficiently. In the case of glycyrrhizin, four adjuvants including C12 showed almost the same improvement of the absorption, about 30-40 times larger than the control. C8 and sodium citrate did not significantly enhance the absorption of those model compounds. For FD-4, FD-10 and FD-40, C10, C12 and C18:1 revealed almost the same enhancing effect and the absorption of FD-4, FD-10 and FD-40 was enhanced about 80 times, 1000-1800 times and about 200 times, respectively, larger than the control. The enhancement ratio, the ratio of AUC with adjuvant to AUC of control, suggests that these lipoidal adjuvants would improve most efficiently the absorption of the compound having the molecular weight of around 10000. Furthermore, C12 was suggested to be an effective adjuvant for the compounds with the wide range of molecular weight.

Adjuvants, Pharmaceutic↗

Development of an improved method for production of antiscorpion F(ab')2 fragment of IgG with high yield and potency.

The main objective of the present work was to obtain a stable and highly purified scorpion antitixon, rich in specific antibodies, free of immunologically irrelevant plasma proteins or gross proteins. Our results indicate that the most potent yielded and purified F(ab')2 antivenom preparation was obtained when the first discarded precipitate was washed with 14% ammonium sulphate saline; then after the second addition of ammonium sulphate, the mixture was stirring overnight followed by precipitation of most non-immunoglobulin proteins with the aid of caprylic acid to produce antivenom rich in specific antibodies with higher yield and potency compared to the method commonly used.

Animals↗

[Study on pulmonary delivery system of salmon calcitonin in rats].

OBJECTIVE: To study the serum calcitonin(CT) levels in vivo after pulmonary delivery to rats and the effects of absorption enhancers. METHODS: Salmon calcitonin (sCT) solution was delivered by intramuscular injection or intratracheal administration to rats. The absorption of sCT was determined by radioimmunoassay method. Changes in serum drug levels after the pulmonary administration of sCT with various absorption enhancers or in different pH conditions were investigated. RESULTS: Compared with intramuscular administration, the relative bioavailability of sCT solution (2 micrograms.kg-1) given by intratracheal route was 121.0%. Transpulmonary absorption of sCT solution, with some absorption enhancers (Brij78, sodium cholate, lecithin, chitosan and sodium caprylate) or under acidic conditions (pH3.0), was significantly greater than the values obtained in controls. CONCLUSION: sCT solution showed higher bioavailability after pulmonary administration.

Animals↗

The effect of a glucocorticoid on olfactory response of isolated mouse olfactory cells.

Oral administration and nasal instillation of glucocorticoids are currently being conducted and are producing satisfactory results for the treatment of olfactory dysfunctions. However, there are still many unanswered questions about the actions of glucocorticoids on the olfactory cells. In the present study, the effects of glucocorticoids on the olfactory response in mouse olfactory cells were examined by measuring changes in the intracellular Ca2+ concentration. The olfactory cells isolated from the BALB/c female mouse mucosa were stained with a Fura-2/AM and the changes in the intracellular Ca2+ concentration were observed by using a fluorescent microscopic image processing device, ARGUS50. The cells were exposed to the following olfactory stimuli, 3-ethoxy-4-hydroxy-benzaldehyde, caprylic acid, nonanoic acid, phenethyl alcohol and n-amyl acetate. When 0.1 and 0.01% betamethasone was applied to the olfactory cells for short periods(1 or 3 minutes), no changes were observed. However, long-term(7 minutes) application of 0.1 and 0.01% (but not 0.001 and 0.0001%) betametathone significantly decreased intracellular Ca2+ concentration, which was increased by olfactory stimulation. The inhibitory effect of betamethasone on Ca2+ influx into olfactory cells was attenuated by pre-treatment olfactory cells with RU486, a glucocorticoid receptor antagonist.

Animals↗

Studies on purification of methamidophos monoclonal antibodies and comparative immunoactivity of purified antibodies.

OBJECTIVE: To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. METHOD: Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies. RESULTS: Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 microg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 microg/mL and 1.03 microg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 microg/mL, and the linear working range and LOD were 10.91-11412.29 microg/mL and 3.42 microg/mL, respectively. CONCLUSION: Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.

Antibodies, Monoclonal↗

An improved gas chromatographic determination of valproic acid and valpromide in plasma.

A newly, improved analytical method for determination of valproic acid (VPA) and valpromide (VPD) in human plasma was developed. The method is based on gas chromatographic determination with flame ionization detection, after chloroform extraction of the drugs from plasma. Caprylic acid was used as an external standard. With the extraction procedure chosen, high recoveries for both VPA and VPD were achieved (98-102%), with correlation coefficient of 0.9998 for VPA and 0.9996 for VPD. Sensitivity of the method was also high (2 ng or 2 mg/L), while the linearity was obtained over the range of 5-150 mg/L, with high correlation for both drugs (0.9997 and 0.9995 for VPA and VPD, respectively). Reproducibility of the method was documented with low values of coefficients of variation, both inter-assay and day-to-day values (1.4-4.4 and 1.7-3.7 for VPA and 2.1-5.0 and 2.6-5.1 for VPD, respectively). The method has been used to follow VPA plasma levels in adult epileptic patients on sodium valproate therapy.

Adult↗

[The role of ketone bodies in the development of postalcoholic-intoxication heart damage in rats].

Experiments on rats subjected to forced alcoholization for 5.5 days were made to measure the content of ethanol, acetaldehyde and ketone bodies in the blood during intoxication and 2 days after ethanol withdrawal and to estimate the intensity of postintoxication disorders in heart activity on the third day after alcoholization withdrawal. A positive correlation was discovered between depression of left ventricular contractility and the blood content of acetaldehyde and ketone bodies. The magnitude of the threshold of heart fibrillation did not correlate well with the concentration of ethanol during alcoholization. However, it agreed well with ethanol concentration in the postintoxication period. Additional administration to the animals of beta-hydroxybutyrate or caprylic acid in the postintoxication period intensified heart contractility depression. The conclusion is drawn that elimination of ketosis in ethanol withdrawal as well as a progressive taking out of alcoholic patients from dipsomania can prevent the development or attenuate the intensity of postintoxication heart injury.

Alcoholic Intoxication↗

[Studies on the chemical constituents of Cynanchum thesioides].

Fourteen constituents were isolated from Cynanchum thesioides (Freyn) K. Schum. On the basis of chemical and spectral data, seven of them have been identified including two new compounds: glyceride-1,3-dipalmito-2-sorbate (4), and thesoideaside (7); and five known compounds: beta-amyrin acetate (I), lupeol acetate (2), alpha-amyrin caprylate (3), tamarixetin (5) and tamarixetin-3-O-beta-D-galacto-pyranoside (6).

Drugs, Chinese Herbal↗

Ester synthesis catalyzed by Mucor miehei lipase immobilized on magnetic polysiloxane-polyvinyl alcohol particles.

Mucor miehei lipase was immobilized on magnetic polysiloxane-polyvinyl alcohol particles by covalent binding with high activity recovered. The performance of the resulting immobilized biocatalyst was evaluated in the synthesis of flavor esters using heptane as solvent. The impact on reaction rate was determined for enzyme concentration, molar ratio of the reactants, carbon chain length of the reactants, and alcohol structure. Ester synthesis was maximized for substrates containing excess acyl donor and lipase loading of 25 mg/mL. The biocatalyst selectivity for the carbon chain length was found to be different concerning the organic acids and alcohols. High reaction rates were achieved for organic acids with 8 or 10 carbons, whereas increasing the alcohol carbon chain length from 4 to 8 carbons gave much lower esterification yields. Optimal reaction rate was determined for the synthesis of butyl caprylate (12 carbons). Esterification performance was also dependent on the alcohol structure, with maximum activity occurring for primary alcohol. Secondary and tertiary alcohols decreased the reaction rates by more than 40%.

Alcohols↗

[Preparation and characterization of a McAb against tissue factor pathway inhibitor].

OBJECTIVE: To prepare monoclonal antibody (McAb) against tissue factor pathway inhibitor (TFPI). METHODS: Twice intrasplenic immunization of BALB/c mice with TFPI-binded nitrocellulose paper followed by an intraperitoneal boost with TFPI raised two hybridoma cell lines, 4F8 and 4F4, by hybridoma technique, 4F8 was studied in detail. The mode value of its chromosome is 93. The Ig class of McAb 4F8 was IgG1. The McAb was purified from ascites by a simple procedure of caprylic acid pre cipitation. The purified McAb was conjugated with horseradish peroxidase (HRP) by a modified periodate oxidation procedure. RESULTS: Immunoblotting indicated that the McAb recognized a single band of TFPI with a molecular weight of 34800. The McAb was also proved to shorten markedly the dilute thromboplastin coagulation time of factor IX-deficient and normal plasma. The results of Sandwich ELISA using the HRP labeled McAb showed that the mean TFPI in normal human plasma is 103.2 +/- 11.5 microg/L, being similar to that measured with TFPI ELISA kit from America Diagnostica Inc. (98.4 +/- 10.3 microg/L) (r=0.92). CONCLUSION: McAb 4F8 may be a useful reagent for TFPI research.

Animals↗

Quantitation of model digestive mixtures by 13C NMR.

13C nuclear magnetic resonance (NMR) spectra were obtained at 50.3 and 100.5 MHz for methanolic and aqueous mixtures of sodium taurocholate, 1-monocapryloyl-rac-glycerol, and caprylic acid. Distortionless Enhancement by Polarization Transfer (DEPT) was used to improve spectral sensitivity and resolution, and to generate calibration curves for quantitative determinations of each lipid in methanol. Alternatively, the heights for nonoverlapping peaks in a 13C NMR spectrum acquired with inverse-gated decoupling provide reliable quantitative estimates for each component of the mixture, particularly when the data are obtained in methanol. These experiments also demonstrate the feasibility of detailed NMR structural investigations in model systems for glyceride digestion.

Carbon Isotopes↗

[Production of polyclonal antibody against HBD-2 by immunization with recombinant GST-HBD-2 fusion protein].

For the purpose of detecting the HBD-2 expression at protein level, the recombinant prokaryotic expression vector pGEX-1lambdaT-HBD-2 was constructed and the E. coli-based product of GST-HBD-2 fusion protein was prepared. When rabbit was immunized with the fusion protein, the anti-serum against HBD-2 was produced. After caprylic acid and ammonium sulfate precipitation, high titer of specific polyclonal antibody against HBD-2, which was detected by ELISA and Western blot, was obtained. This result suggests that recombinant peptide fusion protein could be used instead of the conjugate of peptide-albumin or peptide-thyroid globulin to produce antibody. The obtained antibodies could be used for revealing the tissue distribution of HBD-2 and the regulation of its gene expression.

Animals↗

[Production of HMGN2 polyclonal antibody by immunization with recombinant GST-HMGN2 fusion protein and its application to analysis of HMGN2 distribution in human monocytes].

OBJECTIVE: To prepare high mobility group chromosomal protein N2 (HMGN2) polyclonal antibodies and determine the subcellular localization of HMGN2 in human monocytes. METHODS: The recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was constructed and E. coli-based product of GST-HMGN2 fusion protein was prepared and used to immunize rabbit for producing the anti-serum against HMGN2. The polyclonal antibodies were partially purified by caprylic acid and ammonium sulfate precipitation. The titter of specific polyclonal antibodies against HMGN2 was detected by ELISA. The immunocytochemical staining was performed to determine the distribution of HMGN2 in THP-1 cells. RESULTS: Gel electrophoresis of the enzyme-digested recombinant plasmid and the DNA sequencing confirmed that the recombinant prokaryotic expression vector pGEX-1lambdaT-HMGN2 was correctly constructed. After IPTG induction, the recombinant-transformed E. coli produced a bulk of GST-HMGN2 fusion protein. The polyclonal antibodies to HMGN2 was obtained from the serum of rabbit immunized with GST-HMGN2 fusion protein and its ELISA titer was 1:2000. The immunocytochemistry staining indicated that when stimulated with LPS, HMGN2 was present not only in THP-1 nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant. CONCLUSION: This result suggests that recombinant peptide fusion protein could be used to produce peptide antibody. HMGN2 could be present in the cytoplasm of monocytes and release to the extracellular environment when stimulated with lipopolysaccharide (LPS).

Animals↗

Deposition of insulin powders for inhalation in vitro and pharmacodynamic evaluation of absorption promoters in rats.

AIM: To prepare insulin powder for inhalation by spray-drying technology, determine the deposition of the insulin powder formulation in vitro and preliminarily investigate hypoglycemic response of the dry powder with/without absorption promoters. METHODS: The depositions of the insulin powder for inhalation were determined by the China Pharmacopoeia 2000 version addenda XH and hypoglycemic effects were evaluated by testing serum glucose with glucose oxidase-peroxidase (GOD-PAP) method. RESULTS: The depositions of the spray-dried insulin powder for inhalation were more than 40% under various humidity and their changes were not significant when air flow was no less than 18 L x min(-1). The coadministration of insulin with 8 mmol x L(-1)/dose sodium taurocholate [PA = 59.91%, Cnadir = (33 +/- 6) %] and 10 mmol x L(-1)/dose sodium deoxycholate [PA = 47.46% , Cnadir = (32 +/- 7)%] induced a significantly greater decline in blood glucose levels, while coadministration with 1% sodium caprylate, 1% sodium dodecyl sulfate, 250 microg/dose lecithin, 10 mmol x L(-1)/dose EDTA appeared to have no significant effect (P > 0.05). CONCLUSION: Insulin powder for inhalation was relatively stable under various humidity conditions and different flow current. The use of 8 mmol x L(-1)/dose sodium taurocholate and 10 mmol x L(-1)/dose sodium deoxycholate could be able to potentially improve the bioavailability of insulin by pulmonary route.

Administration, Inhalation↗

[Profile-effect on quality control of Houttuynia cordata injection].

AIM: To find corresponding relationship between the fingerprint of Houttuynia cordata injections from different factories and their effects. METHODS: Houttuynia cordata injections from six different factories were determined by gas chromatography (GC) and gas chromatography-mass spectra (GC-MS), and GC fingerprints were classified by hierarchical clustering. The anti-inflammatory activity of Houttuynia cordata injections was characterized through the rat pleurisy model induced by carrageenin and the mice ear edema model by dimethylbenzene. RESULTS: The anti-inflammatory effect of the injections from the first class factories on the two model was significant, while those from the second class not. GC-MS analysis result indicated that main effect compounds in Houttuynia cordata injections are methyl n-nonyl ketone, decanoylacetaldehyde, lauryl aldehyde, capryl aldehyde, beta-pinene, beta-linalool, 1-nonanol, 4-terpineol, alpha-terpineol, bornyl acetate, n-decanoic acid and acetic acid, geraniol ester etc. CONCLUSION: There is corresponding relationship between the fingerprint of Houttuynia cordata injections and effect to a certain extent.

Animals↗