Detection and quantification of protein residues in food grade amino acids and nucleic acids using a dot-blot fluorescent staining method

Food allergies represent an important health problem in industrialized countries, such that detection and quantitative analysis of the protein considered to be the main allergen is crucial. A dot-blot fluorescent staining method for the detection and quantitative analysis of protein residues in food grade amino acids and nucleic acids is presented. This method combines fluorescence staining with dot-blotting onto PVDF membrane. Several standard proteins, such as bovine serum albumin (66 kDa), lysozyme (14 kDa), ubiquitin (8.6 kDa), bovine insulin (5.7 kDa), and oxidized insulin B chain (3.5 kDa), were detectable at 0.1 ppm using SYPRO Ruby blot stain. Twenty-five different amino acids and two different nucleic acids of food grade were analyzed using this method combined with an internal standard addition method using BSA as an internal standard. All amino acids and nucleic acids were dissolved in 3.6% aqueous HCl and dot-blotted onto PVDF membrane before a large amount of amino acids and nucleic acid were removed. Protein residues and the internal standard protein immobilized on the membrane were stained using SYPRO ruby blot stain. The internal standard in all samples was detectable at 0.1 ppm. Samples were dissolved at 120 or 70 mg/mL, according to their solubility under acidic conditions. The detection limit of protein residues per weight was 0.8-1.4 ppm in amino acids and nucleic acids; residual protein was not detected in any sample.     

ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors

Cells must amplify external signals to orient and migrate in chemotactic gradient fields. We find that human neutrophils release adenosine triphosphate (ATP) from the leading edge of the cell surface to amplify chemotactic signals and direct cell orientation by feedback through P2Y2 nucleotide receptors. Neutrophils rapidly hydrolyze released ATP to adenosine that then acts via A3-type adenosine receptors, which are recruited to the leading edge, to promote cell migration. Thus, ATP release and autocrine feedback through P2Y2 and A3 receptors provide signal amplification, controlling gradient sensing and migration of neutrophils.     

Changes in density of under-yearling masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis> in a natural stream after release

ABSTRACT:   The relation between the density of fish and parameters such as body weight, condition factor and the hepatic triglyceride (TG) content of masu salmon Oncorhynchus masou juveniles after release into a natural stream were studied. Hatchery-reared juveniles of different sizes and numbers were released in May 1999, 2000 and 2001, and investigations were carried out from July to September each year. The density of fish was relatively high in 1999, when juveniles of the largest size and number were released, but then continued to decrease during the investigation. The hepatic TG content remained considerably low in 1999, and there was a significant negative correlation between the density of fish and the hepatic TG content of the juveniles caught over three years. The initial fish size and number at release as well as the nutritional condition of the juveniles may affect the density of fish after release.     

Expression of P-glycoprotein and breast cancer resistance protein in three cases of canine lymphoma showing drug resistance

In canine lymphoma, drug resistance is the major factor hindering treatment. In this study, we performed immunohistochemical examination of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), which are considered as transporters related to multidrug resistance in three recurrent canine lymphomas. All cases were negative for both transporters before anticancer drug administration, but became positive after this administration. The expression was confirmed in capillary endothelial cells, such as in brain capillaries acting as the blood-brain barrier (BBB). It is suggested that both transporters expressed on capillary endothelial cells in lymphoma tissue may inhibit the spread of anticancer drugs into tumor tissues from blood, the same as the BBB. Therefore, capillary endothelial cells could act as a blood-tumor barrier, which might be involved in drug resistance in canine lymphoma.     

Comparison of N-terminal pro-atrial natriuretic peptide and three cardiac biomarkers for discriminatory ability of clinical stage in dogs with myxomatous mitral valve disease

Plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) concentration increases with progression of myxomatous mitral valve disease (MMVD) in dogs. This multicentre, prospective study compared plasma NT-proANP, N-terminal pro-brain natriuretic peptide (NT-proBNP), ANP, and cardiac troponin I (cTnI) concentrations in dogs with MMVD for their characteristics and discriminatory ability to detect cardiac dilatation and congestive heart failure (CHF). Thirty-six healthy dogs and 69 dogs with MMVD were included. Clinical variables were obtained via physical examination, thoracic radiography, and echocardiography. The discriminatory ability of each cardiac biomarker (CB) to determine the presence or absence of cardiac dilatation (event 1) and CHF (event 2) was evaluated using the receiver operating characteristic curves. Plasma NT-proANP, NT-proBNP, and ANP concentrations showed a significant association with the left atrium/aorta ratio (P<0.01). The area under the curve of plasma NT-proANP and NT-proBNP concentrations were 0.72 and 0.75, respectively in event1 and 0.72 and 0.76, respectively in event2. Plasma NT-proANP and NT-proBNP concentrations showed sensitivity 80.0 and 80.0%; specificity 67.6 and 64.7% in event1 (cutoff value; 8,497.81 pg/ml and 1,453.00 pmol/l, respectively) and sensitivity 85.7 and 81.0%; specificity 60.4 and 64.6% in event2 (cutoff value; 8,684.33 pg/ml and 1,772.00 pmol/l, respectively). In dogs with MMVD, plasma NT-proANP, NT-proBNP, and ANP concentrations increase with left atrial enlargement. Particularly, plasma NT-proANP and NT-proBNP concentrations appeared to be equally useful in the discriminatory ability to detect cardiac dilatation and CHF.     

Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.     

Circadian variations in salivary chromogranin a concentrations during a 24-hour period in dogs

The purpose of this study was to determine if salivary chromogranin a secretion in dogs exhibits a circadian rhythm. Saliva sampling was performed during three different sessions occurring in three nonconsecutive 24-h periods. Sixteen healthy adult beagle dogs (8 males and 8 females) were moved to a sampling room and housed individually in cages. Saliva samples were obtained every 4 h from 12:00 p.m. to 12:00 p.m. the following day. In the interest of habituation, saliva was obtained hourly from each dog 3 h before the experiment was started. Salivary chromogranin A concentrations were measured using an enzyme-linked immunosorbent assay. No circadian rhythm was detected for salivary chromogranin A secretion, and no differences in salivary chromogranin A concentrations measured every 4 h were demonstrated during the 24-h cycle in dogs.