Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: Insights on the origin of protoluciferase activity and bioluminescence colours

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of D-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site. Copyright © 2006 John Wiley & Sons, Ltd.

Antibody and cytokine serum levels in patients subjected to anti-rabies prophylaxis with serum-vaccination

Rabies is considered a fatal disease once clinical symptoms have developed. The aim of this study was to evaluate epidemiological aspects and immune response in patients attacked by domestic and wild animals and subjected to post-exposure rabies treatment with equine serum and associated vaccine. Thirty-three patients were evaluated; they were between 13 and 65 years old, 75.8% were male and 24.2% female, and from the Botucatu neighborhood. Twenty healthy control individuals with the same age range were also studied. Specific antibodies to equine immunoglobulins and IFN-γ, IL-2, IL-4, and IL-10 production were evaluated by ELISA. IgM, IgE, IgG and subclasses, and rabies virus antibodies serum levels were determined by nephelometry and seroneutralization methods, respectively. No anaphylactic or serum sickness allergic reactions were observed in patients after treatment. Anti-equine IgG levels were significantly higher than those of IgM after 14 and 28 days of treatment. Protective antibodies to rabies virus > 0.5 UI/ml were detected in 84.6% and 75% of patients at days 14 and 28, respectively. IFN-γ, IL-2 and IL-10 levels in patients before and 48h after treatment were significantly higher than in controls suggesting that both Th1 and Th2 cells were activated in the patients. Serum IgM levels were higher at day 14, and IgG 2 and IgE levels were higher at day 28 of treatment. These results suggest that post-exposure rabies treatment in humans induces significant alterations in patient immune response characterized by increased levels of cytokines, serum levels of specific rabies virus antibodies, and the equine serum components employed in the treatment.

Hematological and serum chemistry values for the ring-necked pheasant (Phasianus colchicus): Variation with sex and age

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Students' preferred teaching techniques for biochemistry in biomedicine and medicine courses

The aim of this study was to investigate the students' preferred teaching techniques, such as traditional blackboard, power-point, or slide-projection, for biochemistry discipline in biomedicine and medicine courses from São Paulo State University, UNESP, Botucatu, São Paulo, Brazil. Preferences for specific topic and teaching techniques were determined from questionnaires on a Liquert scale from 1 to 5 (strongly disagree; disagree; neither agree, nor disagree; agree; strongly agree) distributed at the end of biochemistry discipline to 180 biomedical students (30 students/year) and 540 medical students (90 students/year), during the years 2000-2005. Despite of the different number of hours applied to the course topics for the two groups of students, the majority of undergraduates from biomedicine and medicine preferred metabolic topics. Although the perception of a medical student is expected to be different than that of a biomedical student, as the aims of the two programs are different, 92.4% of students from each course agreed or strongly agreed with the biochemistry topics, and 92.1% thought highly on this subject. The majority of students, a number of 139 undergraduates from biomedicine and 419 from medicine course, preferred traditional blackboard teaching than slide-projection, or power-point class. In conclusion, it is imperative that the health courses reflect on sophisticated technology and data presentation with high density of information in biochemistry discipline. The traditional classes with blackboard presentation were most favored by students from biomedicine and medicine courses. The use of students' preferred teaching techniques might turn biochemistry more easily understood for biomedical and medical students. © 2007 by The International Union of Biochemistry and Molecular Biology.

Clinical and nutritional profile of individuals with Chagas disease

Chagas disease (CD), caused by the protozoan Trypanossoma cruzi, affects approximately 18 million individuals in the Americas, 5 million of which five in Brazil. Most chronic sufferers have either the indeterminate form of the disease, without organic compromise, or the cardiac or digestive forms. Despite the importance of this disease, there is no information on the effect of nutrition on CD evolution. We evaluated the clinical-nutritional profile of individuals with CD treated at the Tropical Diseases Nutrition Out-Patient Clinic of the Botucatu School of Medicine, UNESP.A retrospective cohort study was performed between 2002 and 2006, on 66 patients with serum and parasitological diagnosis of CD. Epidemiological, clinical, nutritional, and biochemical data were collected, including gender, age, skin color, smoking, alcoholism, physical activity, weight, stature, body mass index, abdominal circumference, glycemia, and lipid profile. Fifty-three percent were mate and 47% female; 96% were white skinned. Mean age was 49.6±6.36 years. The predominant form was indeterminate in 71 %; smoking and drinking were recorded in 23% and 17%, respectively. Sedentariness predominated in 83%, and 55% presented increased abdominal circumference. Most, 94%, were overweight or obese. The biochemical exam revealed hyperglycemia in 12% and dyslipidemia in 74%. These findings suggest that the Chagas population presents co-morbidities and risk factors for developing chronic non-transmissible diseases, including cardiovascular diseases, making CD evolution even worse. © 2007 by The Brazilian Journal of Infectious Diseases and Contexto Publishing. All rights reserved.

Serum biochemical parameters in the ring-necked pheasant (Phasianus colchicus) on breeding season

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - Part I: Clinical evaluation, hematology and myelogram

The present study aimed at evaluating clinical and laboratory aspects during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. The clinical signs of ataxia, sedation, flaccid paralysis, mydriasis, eyeball paralysis, mandible ptosis, sialorrhea, vomiting and diarrhea observed in the dogs were very similar to those observed in humans. The decrease in hemoglobin, hematocrit, erythrocyte, platelet and fibrinogen levels, prolongation of clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT), as well as hypocellularity in the bone marrow characterized anemia, thrombocytopenia and blood incoagulability, as well as hypofibrinogenemia and decreased bone-marrow activity. Important bleeding was not observed. Increased numbers of leukocytes and neutrophils and decreased numbers of lymphocytes and eosinophils characterized an acute inflammatory response and stress caused by generalized pain. The employed antiophidic serum was effective and all animals survived.

Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - Part II: Laboratory aspects, electrocardiogram and histopathology

The present work shows laboratory aspects, electrocardiogram and histopathology results during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. Urinalysis showed brown urine, proteinuria, occult blood and myoglobinuria. Respiratory acidosis and hypotension were also observed. At the venom inoculation site, there was discreet edema, popliteal lymph node response, musculature presenting whitish areas and necrotic myositis with myoregenerative activity. There was not evidence of electrocardiographical and biochemical alterations.

Cryopreservation of dog spermatozoa: Effect of bovine serum albumin on acrosomal integrity and pregnancy rates after artificial insemination

The objective of the present study was to compare the in vitro and in vivo profile of frozen dog semen with Tris-bovine serum albumin (TB) and Tris-egg yolk (TE) extenders. Twenty dogs were used as donors. Each dog was stimulated by penile massage and only the sperm-rich fraction was collected weekly until 40 ejaculates were obtained. After macroscopic and microscopic analysis, equal parts of each ejaculate were diluted with TB and TE by the one-step method at 37 °C. The semen was added to 0.5-mL French straws which presented normal characteristics before freezing and after thawing. Acrosomal integrity was evaluated by double Trypan blue-Giemsa staining, in which alive intact (LI), alive reacted (LR), dead intact (DI) and dead reacted (DR) spermatozoa, were identified by the time of thawing and up to 4 h of incubation at 39 °C, the TE being significantly superior to TB (P<0,01) in the LI and LR variables. The TB being significantly superior to TE (P<0,01) in the DR variable. Female dogs in natural heat were submitted to artificial insemination, 20 receiving TE-semen and 20 receiving TB-semen with the Osiris probe (IMV, L'Aigle, France) and the numbers indicate that TE was significantly better than TB (P<0,01) to pregnancy rate and number of puppies/delivery. We concluded from this study, that TE was better than TB, because this, induced an eady acrossome reaction in dog's sperm.

Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G1 (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 × 10 4 VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T. gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T. gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T. gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) × IFA (ah) (r = 0.62, P = 0.05), MAT(s) × MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) × r-ELISA (ah) (r = 0.14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. © 2007 Elsevier Ltd. All rights reserved.

Application of a serum/protein-free medium for the growth of mammalian cell lines and high level production of classical, variant and very virulent strain of infectious bursal disease virus (IBDV)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of ring-necked pheasant (Phasianus colchicus) serum protein concentrations by refractometry and the biuret method

The purpose of this study was to evaluate the accuracy of hand-held refractometer in determining serum protein concentrations in ring-necked pheasants (Phasianus colchicus) as compared with the standard biuret method. The results indicated that serum protein values may be accurately determined in ring-necked pheasants with a hand-held refractometer. © Asian Network for Scientific Information, 2008.

Serum protein profiles of juvenile ring-necked pheasants vaccinated or not against newcastle disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serum proteinogram of Chinese goose (Anser cygnoides) vaccinated or not against Newcastle disease

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Experimental infection of commercial layers using a Salmonella enterica serovar Gallinarum strain: Leukogram and serum acute-phase protein concentrations

The aim of the present study was to evaluate white blood cell counts and serum protein profiles of commercial layers experimentally infected with Salmonella Gallinarum (SG) in order to better understand the pathophysiology of the disease caused by this bacterium. 180 five-day-old commercial layers were divided into 3 groups (G); G1 and G2 received 0.2 mL of inoculate containing 3.3x10 8 CFU or 3.3×10 5 CFU SG resistant to nalidix acid (Nal r)/mL, respectively, directly into their crops. G3 group did not receive the inoculum. Birds were sacrificed 24 hours before (T1) and 24 hours after the infection (T2), and three (T3), five (T4), seven (T5), and ten (T6) days after the administration of the inoculum. White blood cell counts were carried out in a Neubauer hemocytometer and in blood smears. Serum protein concentrations, including acute-phase proteins, were determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Data were submitted to analysis of variance, and means were compared by Tukey's test (P <0.05). G1 and G2 groups presented higher leukocyte counts on T4 and T5, respectively, due to the increase of circulating lymphocytes and heterophils, with a significant difference relative to G3. In electrophoresis, an increase in the serum levels of ceruloplasmin, haptoglobin, and hemopexin and a decrease in transferrin, which are acute-phase proteins, was verified. IgA serum levels did not change; however, IgG concentration increased during the infection. In conclusion, the results provide information for the better understanding of the pathophysiology of fowl typhoid.