Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19% cold ethanol and 0.6% chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99% purity, a yield of 25.0 ± 1.2 g/l plasma and >71.5% recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0% (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60°C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06% (w/v) glutaraldehyde and 0.1% (w/v) formaldehyde at 37°C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0% monomers, 23.6% dimers, 12.2% trimers and 8.2% polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results.

Urethroplasty using a bovine pericardium graft: an experimental study using normal urethras from dogs

The use of bovine pericardium as a urethral patch to substitute a ventral segment of canine urethras was studied. Healing, epithelial growth, urethral permeability, fistulas, and calcification were analyzed. Thirty male mongrel dogs of medium and large size underwent resection of a ventral segment of the medial urethra measuring 2.0 x 0.5 cm, which was replaced with a bovine pericardium graft, treated with buffered glutaraldehyde and preserved in formaldehyde. Two running sutures of polygalactin 5-0 were applied, one on each side of the patch. The corpus spongiosum was closed with uninterrupted suture and the skin with interrupted suture of polygalactin 5-0. Six months later, the animals were examined and sacrificed under anesthesia. Retrograde urethrograms showed that the urethral healing was complete in six of the 30 animals, without stenosis, fistulas or dilations. Microscopic examination showed complete epithelization of these six urethras. The remaining 24 animals presented urethrocutaneous fistulas without stenosis, demonstrated by urethral catheterism using a 10-Fr plastic catheter. These data show that a successful urethral reconstruction of the penile urethra was possible in only 20% of the operated animals. Infection and leakage may be the cause of the urethrocutaneous fistulas present in 80% of cases. Further studies are necessary to determine whether such fistulas are avoidable. If they are, the bovine pericardium may well be an option in the treatment of urethral lesions in dogs.

Role of Leishmania (Leishmania) amazonensis amastigote glycosphingolipids in macrophage infectivity

The role of glycosphingolipids (GSLs) present in amastigote forms of Leishmania (Leishmania) amazonensis during infection of macrophages was analyzed, with particular emphasis on GSLs presenting the terminal Galpß1-3Galpa disaccharide. Macrophage invasion by L. (L.) amazonensis amastigotes was reduced by 37% when the disaccharide Galpß1-3Galp (1 mM) was added to the culture medium. The putative macrophage receptor/lectin for ß-Gal-globotriaosylceramide (Galpß1-3Galpa1-4Galpß1-4Glc pß1-1Cer) and other structurally related GSLs from L. (L.) amazonensis amastigotes were analyzed by micelles and parasite binding assay to peritoneal macrophage proteins fractionated by SDS-PAGE under nonreducing conditions. Micelles containing purified amastigote GSLs or a suspention of L. (L.) amazonensis amastigotes fixed with 2% formaldehyde were incubated with nitrocellulose membrane containing the macrophage proteins transferred by Western blotting. Binding of micelles containing purified GSLs from amastigote forms or fixed L. (L.) amazonensis amastigotes to nitrocellulose membrane was probed using monoclonal antibody ST-3, which recognizes the glycoepitope Galpß1-3Galpa1-R present either in the micelle preparation or on the amastigote surface. Macrophage protein with molecular mass ~30 kDa bound the amastigote GSL and appeared to be a doublet on electrophoresis. The specificity of this interaction was confirmed using fixed L. (L.) chagasi amastigotes, which do not express GSLs such as ß-Galp-globotriaosylceramides, and which do not bind to 30-kDa protein.

Decomposition of trimethylamine oxide related to the use of sulfites in shrimp

Currently, sulfites are employed on board to inhibit melanosis (blackspot) on crustaceans. However, when used in excess this chemical compound not only can cause adverse reactions in SO2-sensitive individuals, but also favors the decomposition of trimethylamine oxide (TMAO) into dimethylamine (DMA) and formaldehyde (FA), thus compromising the quality of the product, which can be observed mainly through the texture change of the meat after cooking. This study was conducted to verify the increase of the contents of DMA and FA by the excessive use of sodium metabisulfite in white shrimp (Penaeus schmitti). For laboratory trials, shrimp were beheaded, washed and immersed in a 2% sodium metabisulfite solution for 10 minutes. Specimens were stored either on ice and maintained for 48 hours in refrigeration, or stored in a freezer for 48 hours. Samples were collected at intervals of 0, 24 and 48 hours, and analyzed for residual SO2, TMAO, TMA, DMA and FA. The immersion of shrimp in a 2% sodium metabisulfite for 10 minutes favored the decomposition of TMAO which greatly increased the contents of DMA and FA. The FA and DMA measured in fresh shrimp was low. Moreover, the storage of shrimp tails on ice resulted in a significant reduction of the TMA, DMA, FA and residual SO2 contents compared to the specimens under frozen storage.

Quality changes during frozen storage of blue shrimp (Litopenaeus stylirostris) with antioxidant, α-tocopherol, under different conditions

Fresh blue shrimp (Litopenaeus stylirostris) muscle was stored with antioxidants under different conditions: ANTIOX 2%, packed in bilayer film of polyamide-low density polyethylene film (PA-LDPE) with 2% α-tocopherol; ANTIOX 4%, packed in PA-LDPE film with 4% α-tocopherol; and ANTIOX-GLAZED, samples stored glazed with 2% α-tocopherol. Shrimps packed in PA-LDPE without α-tocopherol were used as CONTROL. All samples were stored at –20 °C for 120 days. As compared to the CONTROL, the shrimp stored with the antioxidant showed lower lipid oxidation (0.10-0.14 vs 1.58 mgMA/kg of muscle), lost less firmness and astaxanthin content. ANTIOX 2% and ANTIOX-GLAZED showed the lowest concentrations of formaldehyde (0.081-0.083 μM/g). There were no significant differences in color and sensory properties, but differences in the integrity of the muscle fibers were observed. The treatments with α-tocopherol maintained the shrimp muscle quality during frozen storage. However, no significant differences were found between these treatments.