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.

Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.

A nested polymerase chain reaction (PCR) test was developed to examine infection with the bovine lentivirus, bovine immunodeficiency-like virus (BIV), in cattle. Primers were designed to amplify 2 separate regions of the pol and env segments of the BIV genome. Two calves were experimentally infected with an isolate derived from the original strain of BIV, R29, or with a recent field isolate, FL491. Serial blood samples were collected and examined by virus isolation, protein immunoblot, and nested PCR. The nested PCR test detected BIV infection by 3 days after inoculation, earlier than the other 2 methods, and continued to identify infected cattle 9 to 15.5 months after inoculation, even when results from virus isolation and serology became negative. Nested PCR also detected multiple-size env products in samples obtained later in the infection from the calf that received FL491, giving evidence that viral quasispecies were selected during in vivo replication of the virus. Results indicated that the nested PCR test is more sensitive than virus isolation or serology for the detection of BIV infection in cattle.

A murine monoclonal antibody (MAB) 6G7 generated against tachyzoites of Neospora caninum recognized 8 major and several minor antigens, as observed by western blot analysis. Relative rate of migration of the 8 major antigens ranged from 31 to 97.4 kd. In addition, MAB 6G7 recognized a Toxoplasma gondii tachyzoite antigen with a relative rate of migration of 107 kd. Immunogold labeling of N caninum tachyzoites grown in human foreskin fibroblast cells indicated that MAB 6G7 binds to micronemes, dense granules, basal portions of rhoptries, and intravacuolar tubules within the parasitophorous vacuole. Monoclonal antibody 6G7 also bound to micronemes and basal portions of rhoptries within tachyzoites of T gondii. Monoclonal antibody 6G7 did not significantly inhibit development of tachyzoites in vitro.

Histomorphologic/morphometric evaluation, leukocyte scintigraphy, and myeloperoxidase activity were used to determine whether neutrophils accumulate in the large colon of horses during low-flow ischemia and reperfusion. Twenty-four adult horses were assigned to 1 of 3 groups: group 1, sham-operated (n = 6); group 2, 6 hours of ischemia (n = 9); and group 3, 3 hours of ischemia and 3 hours of reperfusion (n = 9). Low-flow ischemia of the large colon was induced in horses of groups 2 and 3 by reducing colonic arterial blood flow to 20% of baseline. Radiolabeled (99mTc) autogenous neutrophils were injected at 175 minutes, which corresponded to 5 minutes prior to reperfusion in group-3 horses. Full-thickness biopsy specimens of the left ventral colon were collected at baseline and at 30-minute intervals for 6 hours; a portion of the biopsy specimen was placed in formalin for histologic examination, and the remainder was used to measure mucosal radioactivity and myeloperoxidase activity. There were no differences in baseline mucosal neutrophil index, mucosal neutrophil numbers, submucosal venular neutrophil numbers, mucosal radioactivity, or mucosal myeloperoxidase activity among groups, or over time in group-1 horses. Neutrophils accumulated in the colonic mucosa during ischemia and further increased at reperfusion, as indicated by neutrophil index (morphology) and mucosal neutrophil numbers (morphometry); mucosal neutrophil index was significantly (P < 0.05) greater in group-3 horses during reperfusion than at the corresponding periods of ischemia in group-2 horses. Neutrophil numbers were significantly (P < 0.05) increased in submucosal venules at 10 minutes of reperfusion in group-3 horses and were significantly (P < 0.05) greater in group-3 than in group-2 horses during the interval from 3 to 6 hours. Mucosal radioactivity significantly (P < 0.05) increased at reperfusion in group-3 horses; there was a trend (P = 0.076) toward greater mucosal radioactivity in group-3, compared with group-2 horses, throughout the 3- to 6-hour interval. There were no differences in mucosal myeloperoxidase activity among or within any of the 3 groups over time. Neutrophils accumulated in the large colon of horses during low-flow ischemia and reperfusion. Neutrophil infiltration was detected by histologic examination and leukocyte scintigraphy, but not by measurement of myeloperoxidase activity. The accumulation of neutrophils during ischemia and the further neutrophil infiltration during reperfusion indicate that neutrophils may contribute to reperfusion injury of the large colon.

Binding of endogenous and exogenous homologous IgG, and IgM to bovine neutrophils before and after in vitro migration through micropore filters, and in vivo migration through mammary tissues after intramammary injection of endotoxin was evaluated by use of flow cytometry. Immunoglobulin binding to neutrophils at 4 and 37 C was also evaluated. Before and after in vitro migration, neutrophils with endogenously bound IgG, and IgM averaged 1 and 2% and 23 and 7%, respectively. Before and after in vivo migration, IgG2 and IgM binding averaged 1 and 7% and 26 and 15%, respectively. Before and after in vitro migration, binding of purified IgG2 and IgM averaged 75 and 67% and 8 and 24%, respectively. Before and after in vivo migration, percentage of neutrophils binding purified IgG2 and IgM averaged 92 and 98% and 54 and 70%, respectively. When serum was used as a source of exogenous immunoglobulins, binding of total Igg after in vitro migration increased from 5% to 28% and of IgM from 4% to 20%. After in vivo migration, binding increased from 21% to 47% and from 24% to 56%, respectively. Exogenous binding of IgG2 at 4 and 37 C averaged 75 and 84%, and binding of IgM averaged 8% at either temperature. Endogenous IgG2 was unaffected by temperature; however, binding of IgM decreased from 23% at 4 C to 2% at 37 C. These data indicate that endogenous binding was higher for IgM before migration than after migration, in vitro and in vivo. Furthermore, migration in vivo through cellular matrices induced receptor upregulation for IgG and IgM. Source and concentration of ligand and serum components, other than immunoglobulins, appeared to contribute to receptor expression and availability. Neutrophils that were exposed to endotoxin and migrated into milk expressed more receptors than did unstimulated and nonmigrating neutrophils. The association of IgM with its receptor was temperature-dependent.