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.     

Geographical and Temporal Variations of Chemical Constituents in Winter Precipitation Collected in the Areas along the Coast of the Sea of Japan

The geographical and temporal variations of chemical constituents in winter precipitation collected in the areas along the coast of the Sea of Japan (AASJ) were discussed by analyzing the data obtained in the 1st and 2nd National Acid Deposition Survey by Japan Environmental Laboratories Association. In western Tohoku (WT) and Hokuriku (HR) areas in AASJ, in spite of large amounts of precipitation in winter, concentrations of non sea salt (nss-)SO₄ ^2? are not as low as the other areas, and nss-Ca²⁺ in these areas is lower than the other areas. As a result, H⁺ concentrations of precipitation in these areas are somewhat higher than other areas. From the temporal analysis of daily sampled data and back trajectory analysis of air mass, it was found that the concentrations of nss-SO₄ ^2?, NO₃ ^?, NH₄ ⁺ and nss-Ca²⁺ are correlatively varied when air mass come from the Asian Continent, showing higher concentrations at the western sites in AASJ and depending on the meteorological conditions such as the direction of in flow air mass.     

Precipitation chemistry in Japan 1989–1993

Precipitation chemistry in Japan was discussed on a wet-only sample database obtained in a nationwide survey from April 1989 to March 1993. Wet-only samples were collected at 29 stations over Japan on a biweekly basis. Commonly determined chemical parameters were measured in laboratories. The volume-weighted annual mean pH at each site ranged from 4.50 to 5.83 with a mean of 4.76. Concentration ranges and means (parenthesized) on an equivalent basis for major ions were as follows: nss-SO₄ ^2–; 5.2–58.9 (38.6), NO₃ ^–; 1.8–25.0 (14.1), NH₄ ⁺; 0.55–29.8 (18.3), nss-Ca²⁺; 2.0–34.5(14.2), Na⁺; 6.4–275.3 (49.1), Cl^–; 13.7–322.4 (63.5) eq L^–1. Acid-base relationships for Phase-II records were quantitatively discussed in terms of three measures: pH, fractional acidity, and our proposed pA_i.     

Development of transferred xenogeneic vole embryos in mouse uteri

An experimental model to study interspecific pregnancy using voles, Microtus arvalis, and green fluorescent protein gene‐induced transgenic mice is presented. Xenogeneic blastocysts from the vole were transferred into the uteri of pseudopregnant mice along with allogeneic blastocysts from green fluorescent protein gene‐induced transgenic mice. The uteri containing xeno‐allo combined transfers were examined from day 6 to 13 of gestation. Although the vole embryos implanted, the uteri containing vole embryos were smaller compared with those having allogeneic mouse embryos. On day 8, the uteri containing vole embryos hemorrhaged internally and no vole embryo was found in the pregnant uterus after day 11. Allogeneic mouse embryos developed normally despite the presence and abortion of the vole embryos. In uteri implanted with vole embryos, decidua were formed and numerous blood vessels were distributed around the embryo. Maternal blood cells infiltrated into the celomic cavity of the vole embryo through the discontinuous region of trophoblast. Periodic acid‐Schiff‐positive granulated metrial gland cells were remarkably increased in the decidual sites. These findings suggest that a disorder of embryo–maternal interaction might induce the appearance of numerous granulated metrial gland cells and rejection of the embryos.     

Reliability of using reagent test strips to estimate blood urea nitrogen concentration in dogs and cats

OBJECTIVE: To evaluate the clinical accuracy of reagent test strips used to estimate BUN concentration in dogs and cats. DESIGN: Prospective study. ANIMALS: 116 dogs and 58 cats. PROCEDURE: Blood samples were collected at the time of admission to the hospital. Estimates of BUN concentration obtained with reagent test strips (category 1 [5 to 15 mg/dL], 2 (15 to 26 mg/dL], 3 [30 to 40 mg/dL], or 4 [50 to 80 mg/dL]) were compared with SUN concentrations measured with an automated analyzer. For dogs, category 1 and 2 test strip results were considered a negative result (nonazotemic) and category 3 and 4 test strip results were considered a positive result (azotemic). For cats, category 1, 2, and 3 test strip results were considered a negative result (nonazotemic) and category 4 test strip results were considered a positive result (azotemic). RESULTS: On the basis of SUN concentration, 40 of the 174 (23%) animals (20 dogs and 20 cats) were classified as azotemic. One dog and 2 cats had false-negative test strip results, and 1 dog had a false-positive result. Sensitivity and specificity were 95% (20/21) and 99% (94/95), respectively, for dogs and 87% (13/15) and 100% (43/43), respectively, for cats. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that reagent test strips are a reliable method for rapidly estimating BUN concentrations in dogs and cats. Because test strip results are only semiquantitative and there remains a potential for misclassification, especially in cats, urea nitrogen concentration should ultimately be verified by means of standard chemistry techniques.     

Effects of heat stress on the redox status in the oviduct and early embryonic development in mice

This study examined the association between redox status in the oviduct and early embryonic death in heat-stressed mice. In Experiment 1, non-pregnant mice were heat-stressed at 35 C with 60% relative humidity for 12, 24, or 36 h, and the maternal redox status was verified by measuring the levels of reactive oxygen species (ROS) and free radical scavenging activity (FRSA) in the oviduct, and thiobarbituric acid reactive substances (TBARS) and glutathione peroxidase (GSH-Px) activity in the liver. In Experiment 2, zygotes were collected from mice heat-stressed for 12 h on the day of pregnancy, and their developmental abilities were assessed in vitro, along with the intensity of DNA damage at the 2-cell stage. The TBARS value and GSH-Px activity in the liver, and ROS level in the oviduct were significantly higher in heat-stressed mice, and this increase appeared to depend on the duration of the heat stress. Maternal heat stress significantly reduced the percentage of zygotes that developed to the morula and blastocyst and the total cell number in the blastocyst. In addition, DNA damage at the 2-cell stage was significantly higher in maternally heat-stressed embryos. These results suggest that heat stress induces systemic changes in redox status in the maternal body, and the resultant increase in oxidative stress in the oviduct is possibly involved in heat stress-induced early embryonic death .     

Elevated endothelin-1 expression in dogs with heartworm disease

We explored the involvement of endothelin-1 (ET-1) in the pathophysiology of dog dirofilariasis (heartworm disease caused by Dirofilaria immitis) by analyzing mRNA levels of preproendothelin-1 (PPET-1), the precursor form of ET-1, in cardiopulmonary organs as well as ET-1 peptide levels in plasma. To determine the cDNA sequence and primary protein structure of dog PPET-1, we performed molecular cloning of the full-length cDNA. Based on the determined sequence information, comparative expression analysis of PPET-1 mRNA was carried out by real-time polymerase chain reaction on cardiopulmonary organs from healthy (n=5) and filarial (n=5) dogs. Filarial dogs showed a significantly (p<0.05) higher mRNA expression level in the heart (about one hundred times) and lung (about ten times) than healthy dogs. Analysis of plasma ET-1 levels in healthy (n=10) and filarial (n=10) dogs showed that filarial dogs (6.9+/-2.7 pg/ml) have significantly (p<0.01) increased plasma ET-1 levels compared with healthy dogs (1.4+/-0.3 pg/ml). To assess the pathophysiological significance of ET-1 in dirofilariasis relative to other cardiopulmonary disorders, plasma ET-1 levels determined in dogs diagnosed with mitral regurgitation (n=10), tricuspid regurgitation (n=5), ventricular septal defect (n=5), and patent ductus arteriosus (n=5) were compared to plasma ET-1 levels in filarial dogs. Filarial dogs, which commonly develop serious pulmonary hypertension, exhibited by far the highest ET-1 levels of the disease states examined. Based on the fact that ET-1 is a potent bioactive mediator that induces vasoconstriction and promotes vascular remodeling, these findings suggest that ET-1 plays an important role in the pathophysiology of dog dirofilariasis as an aggravating factor by inducing pulmonary hypertension.     

Establishment and characterization of eight feline mammary adenocarcinoma cell lines

Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.     

Serological Studies of African Horse-Sickness Virus with Emphasis on Neutralization Test in Tissue Culture

In place of mice, monkey kidney stable (MS) cell cultures were used successfully in serologic studies of African horse-sickness virus.

The maintenance medium containing 2% serum was chosen as the virus diluent. Maximum neutralization occurred after 1-hour incubation at 37 C., and maintained the same titer during an additional 4-hour incubation period. No significant difference was observed between neutralization titers titrated using the same antiserum mixed with two different passage levels of virus.

Rabbit and guinea pig antiserums prepared using virus grown in MS cell cultures had antibody titer as high as those prepared in the same manner using mouse brain suspension.

African horse-sickness virus strains isolated in Asia were serologically identified using a standard neutralization technique in tissue culture. All the strains were closely related to each other and all had antigenic similarity to Type 6 virus (strain 114).

     

Use of monoclonal antibodies for analyses of Coxiella burnetii major antigens

Monoclonal antibodies (MAbs) to major antigens of Coxiella burnetii were produced. Some of the MAbs to a 62-kDa protein antigen, peptidoglycan protein complex and lipopolysaccharide (LPS) O-chains reacted with other bacteria whereas none of the MAbs to outer membrane proteins and LPS outer-core did. The LPS outer-core and OMPs may be useful antigens for specifically detecting antibodies to C. burnetii.