BACKGROUND: Application of green chemistry to the synthesis of nanomaterials is of vital importance in medicinal and technological aspects. Recently, synthesis of silver nanoparticles using plants and marine macro algae to adapt this approach to the environment, has become  more popular. Objectives: The purpose of this study is biological synthesis of silver nanoparticles using seaweed, Sargassum angustifolium, and determining its toxicity in common carp. Methods: First, synthesis of silver nanoparticles using Sargassum algae was conducted and then acute toxicity of these silver nanoparticles was investigated at static renewal condition during 96 hours in common carp according to standard methods (1998) OECD. Results: TEM analysis showed that the average size of the bionanoparticles was found to be 32.54 nm and spherical in shape. The toxicity results showed that the LC50 at 24, 48, 72 and 96-h after exposure was 79.54 ± 0.007, 52.17 ± 0.006, 30.62 ± 0.008 and 11.34 ± 0.016 mg/l respectively. Conclusions: Analysis related to the characterization of the properties of silver nanoparticles proves bioreduction of silver ions by sargassum seaweed extract. According to the results the mortality rates of common carp showed an increasing trend with increasing concentration and exposure time, which indicates the toxicity of this substance in high concentration for common carp.

The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples.

Middle carpal cartilage explants from 4 horses with mild osteoarthritis involving that joint were maintained in tissue culture to test the effects of a polysulfated glycosaminoglycan (PSGAG) on proteoglycan synthesis and degradation. Cultures were exposed to 0.025 or 25 mg of PSGAG/ml for 48 hours, after which the medium was replaced with medium containing similar doses of PSGAG and 35S. Subsequently, the sulfated proteoglycan content of the medium and extracts of the explants was measured. Gel filtration chromatography was used to estimate the size and to purify the principal, large proteoglycan monomer, which was further characterized by digestion, using glycosidic enzymes. In a second experiment, explants were incubated with 35S for 48 hours, and were subsequently exposed to the same concentrations of the PSGAG for an additional 48 hours. The amount of remaining labeled proteoglycan was determined for culture medium and cartilage extracts. Gel filtration chromatography was used to assess the hydrodynamic size of the large proteoglycan monomer. Aliquots of proteoglycans from the second experiment were incubated in high-molecular weight hyaluronate and chromatographed to assess reaggregation. Polysulfated glycosaminoglycan caused a significant (P < 0.04) decrease in sulfated proteoglycan synthesis by cartilage explants. Radioactive proteoglycan content in explants labeled prior to exposure to PSGAG were similar. Large proteoglycan monomer size was similar in both experiments (median partition coefficient [K(AV)] = 0.40), and was not influenced by PSGAG treatment. Prelabeled explants exposed to hyaluronate and chromatographed under associative conditions had similar proportions of the radiolabel eluting as proteoglycan aggregate. Enzymatic digestion of newly synthesized large monomer revealed a mild dose-dependent increase in the proportion of keratan sulfate substitution on core protein. It was concluded that PSGAG in vitro, at the dosages evaluated, caused a decre.

The antibiotic polymyxin B sulfate is a cationic polypeptide with a unique cyclical configuration and distinct cationic characteristics. In this investigation, polymyxin B was evaluated to determine its ability to prevent synthesis of lactic acid and tumor necrosis factor-alpha (TNF-alpha) by lipopolysaccharide-stimulated strain RAW 2647 macrophage-like cell populations. In this context, gradient concentrations of polymyxin B were formulated in the presence of fixed concentrations of lipopolysaccharide fractions from Escherichia coli (B4:0111), E. coli (J5), Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella minnesota, and S. typhimurium (Re). Quantitation of TNF-alpha was established by the application of a tissue culture-based biological assay system, using the WEHI 164 clone 13 indicator cell line. Investigations also included evaluation of the ability of gradient concentrations of lipopolysaccharide fractions from E. coli (B4:0111), E. coli (J5), K. pneumoniae, P. aeruginosa, S. minnesota, and S. typhimurium (Re) to form a complex with polymyxin B. This was established through application of high-performance thin-layer chromatography techniques. On the basis of the known molecular characteristics of lipopolysaccharide, its lipid A-core subfractions, and polymyxin B, these results imply that cytoprotective properties of polymyxin B are attributable to direct interaction and subsequent complex formation. More specifically, the mechanism by which polymyxin B exerts affinity for lipopolysaccharide fractions is proposed to occur through attractive ionic interactions established between the cationic diaminobutyric acid residues of polymyxin B and the mono- or diphosphate group(s) of the lipid A-core moiety. It is highly probable that this molecular phenomenon is accompanied by hydrophobic interactions established between the terminal methyloctanoyl or methylheptanoyl groups of polymyxin B and the saturated carbon chains of the lipid A-core subfraction of lipopolysaccharide fractions.

The effect of a novel bovine mastitis trivalent vaccine, containing Staphylococcus aureus capsular polysaccharide type 5 (T5), 8 (T8), and 336 (T336), on lymphocyte subpopulations, antibody production, and neutrophil phagocytosis was evaluated. Twenty pregnant heifers were immunized with either the trivalent alone, trivalent emulsified in Freund's incomplete adjuvant (FICA), trivalent in aluminum hydroxide, or adjuvant only (FICA). Immunization was done 30 d before the expected calving date followed by 2 boosts in a 2-week interval. Compared to FICA, serum antigen-specific immunoglobulin (Ig)G1 and IgG2 were significantly increased in all the vaccinated groups before parturition and sustained until 3 wk postpartum. In comparison with the trivalent alone, formulation with either adjuvant enhanced production of IgG2, but not IgG1. Immune sera, which contained the highest amount of antibodies, slightly increased neutrophil phagocytosis to the 3 serotypes of killed S. aureus, but most of the differences were not significant due to large variation between the cows. The percentage of CD4+ lymphocyte was significantly higher in vaccinated groups than that of FICA 4 wk after the primary immunization. In comparison with FICA, cows inoculated with trivalent vaccine and adjuvants had an increased percentage of CD8+ lymphocytes at 2 time points, 2 wk before and after calving. Our results indicated that the whole cell trivalent vaccine, with or without adjuvants, is able to elicit antibody responses specific to the 3 capsular polysaccharide antigens. The increase of T8-specific IgG2 was more noticeable when the vaccine was emulsified with adjuvants.

In the present study we have analyzed the characteristics and distribution of the mu-opioid receptor(MOR) by raising anti-peptide antisera to the C-terminal peptide of MOR. The antisera against MOR was produced in New Zealand White rabbit against 15 residue corresponding to amino acids, 384-398of the cloned rat MOR. The antigenic peptide was synthesized using an Applied Biosystems 432 solid-phase peptide aynthesizer. The specificity and identification fo the antisera were tested by analysisi fo transfected cells, epitope mapping and immunohistochemical method. COS-7 cells electroporated with MOR cDNA were used to evaluate the characteristics and subcellular distribution of MOR.MOR immunoreactivity was prodominent in the plasmalemma and subcellular compartments such as encoplasmicreticulum, Golgi apparatus and vesicle like structure. Furthermore, both tissue sections and transfected cell lines could be immunostained with these antisera and the immunoreactivity ws abolished when anti-MOR sera were preincubated with the peptide against which they wer raised. Based on epitope mapping analysis, all antisera appeared to have a similar epitope, which ws detemined to be within the last amino acid,391-398. Moreover, immunohistochemistry showed that MOR immunoreactivity was ovserved in many brain areas including cerebral cortex, striatum, hippocampus, locus coeruleus and the superficial laminae of the dorsal horn. These stained spinal cord and brain aras showed themirrored pattern observed in autoradiographic studies of mu-opioid binding as well as a pattern similar to that seen by in situ hybridization for MOR. Thus, several lines of evidence support the conclusion that the antisera produced in the present study most likely recognize mu-opioid receptor. These results suggest that MOR antisera may be utilized as useful tool to analyze the physiological and pharmacological studies for mu-opioid receptor in the future

Passive protection provided by sows inoculated with the virulent Miller strain of transmissible gastroenteritis virus (TGEV), or the ISU-1 strain of porcine respiratory coronavirus (PRCV), or both was evaluated in nursing pigs challenge exposed with virulent TGEV. Four sows (group B) were inoculated with PRCV oronasally twice at 4 and 2 weeks before parturition; 1 sow (group C) was inoculated similarly, but in 2 subsequent pregnancies; and 2 sows (group D) were oronasally primed with PRCV at 4 weeks before parturition, and 2 weeks later were administered a booster inoculation of virulent TGEV. Two additional sows (group E) remained uninoculated and served as seronegative controls, and 1 sow (group A) that had been naturally infected with TGEV served as a seropositive control. The degree of passive immunity transferred by these sows to their litters was assessed by challenge exposing the pigs of sows in groups BE (only the second litter of group C) with virulent TGEV at 3 to 5 days of age. After challenge exposure, clinical signs of infection and mortality were noted and fecal and nasal shedding of virus was assessed by ELISA. The IgA, IgG, and IgM antibody titers to TGEV were quantified in colostrum and milk of the sows by use of an isotype-specific monoclonal antibody-capture ELISA, using biotinylated monoclonal antibodies against each porcine isotype as detecting reagents. A plaque-reduction assay was used to quantify neutralizing antibody titers in serum, colostrum, milk, and fractionated whey (IgG and IgA/IgM). In the sow naturally infected with TGEV (group A), there was a pronounced decrease in IgG antibody titers to TGEV in the transition from colostrum to milk, and IgA TGEV antibodies became predominant, with high titers maintained throughout lactation. The 4 group-B sows partially protected their pigs after TGEV challenge exposure; mean mortality was 67%, compared with 100% in pigs suckling the 2 TGEV seronegative control sows (group-E litters). Although IgA TGEV antibodies were detected in colostrum and milk of group-B sows, IgG TGEV antibodies were the most abundant. The sow of group C had a marked increase in IgA TGEV antibody titers in colostrum and milk after reinoculation with PRCV during the second pregnancy, before TGEV challenge exposure of the litter. Its pigs were passively protected to a high degree after TGEV challenge exposure (27% litter mortality). The sows in group D, primed with PRCV and boosted with TGEV, provided the best passive protection after TGEV challenge exposure of their pigs. Not only litter mortality (27%) but also morbidity was reduced, compared with those factors for the other challenge exposed litters, and the sows did not become ill. In these swine, the high degree of passive protection observed could not be associated with the presence of only IgA TGEV antibodies in the milk, but high IgM TGEV antibody titers also were detected in colostrum and milk. Results of this study suggest that PRCV-inoculated sows are able to partially protect their pigs from TGEV challenge exposure and, on the basis of preliminary data, the degree of protection may increase after multiple PRCV exposures or after secondary exposure to TGEV during pregnancy. Also, an IgA respiratory tract-mammary gland link may exist as evident by the low titer of IgA TGEV antibodies in the milk of PRCV-inoculated sows, but may not be as efficient in inducing lactogenic IgA immunity as is the gastrointestinal tract-mammary gland link.

Thirty-five heifers were allotted to 3 groups. Group 1 (contro]) consisted of 10 heifers that were not vaccinated and were challenge exposed by breeding to infected bulls. Group 7 (natural challenge exposure) consisted of 10 heifers that were vaccinated and challenge exposed by breeding to infected bulls. Group 3 (experimental challenge exposure) consisted of 15 heifers that were vaccinated and challenge exposed by breeding to infected bulls and by intravaginal inoculation with 10(7) Tritrichomonas foetus. Total immunoglobulin concentrations and specific trichomonal antibodies were determined in serum and vaginal secretions of heifers, using radial immunodiffusion and ELISA procedures. Control heifers remained infected for a mean of 10.6 weeks (range, 0 to 18 weeks), and heifers of the natural and experimental challenge-exposure groups remained infected for 3.2 and 5.0 weeks, respectively (range, 0 to 12 weeks). Total serum and cervicovaginal mucus concentrations of IgM, IgA, IgG1, and IgG2 did not change significantly after vaccination or challenge exposure. However, ELISA titers of total trichomonal antibodies increased up to 1:10,000 (range, 1:400 to 1:10,000) in serum after vaccination, and increased approximately tenfold above background in cervicovaginal mucus. In serum, the predominant trichomonal antibody isotype was IgG1, although trichomonal IgA and IgM antibodies also increased. The predominant trichomonal antibody detected in cervicovaginal mucus was IgA. Antibody titers in serum and cervicovaginal mucus of vaccinated heifers were not increased by infection. However, in control heifers, the total local trichomonal antibody response increased three- to fivefold after infection. In these heifers, specific antibodies in serum were predominantly IgG1 and local (cervicovaginal) antibodies were predominantly IgA.

Systemic administration of dexamethasone led to a significant reduction in the size of the tuberculin reaction in response to intradermal injection of bovine purified protein derivative in 18 cattle experimentally sensitized to Mycobacterium bovis (P < 0.01) and 8 cattle naturally infected with M bovis (P < 0.001). The reaction in 6 of the 7 M bovis-infected cattle that received dexamethasone was classified as negative for the standard interpretation of the single intradermal comparative tuberculin test. Significantly fewer BoCD2+ (P < 0.05) and BoCD4+ T cells (P < 0.001) were present at the reaction site and in blood of dexamethasone-treated cattle, compared with untreated control cattle. Significantly fewer cells expressing the interleukin-2 receptor and WC1+ gamma delta T cells (P < 0.001), and a significantly greater number of cells expressing the ACT2 antigen (P < 0.05) were found at the reaction site in dexamethasone-treated cattle than in controls. The number of BoCD8+ T cells at the reaction site and in blood was not significantly affected by administration of dexamethasone. In vitro production of interferon-gamma by lymphocytes incubated with bovine purified protein derivative also was significantly lower (P < 0.01) in the dexamethasone-treated cattle.