A negative test for mutagenic action of microwave radiation in Drosophila melanogaster.

Microwave radiation (2450 MHz CW) was tested for mutagenicity in Drosophila melanogaster. Embryos in water were exposed to the electromagnetic field with a mean specific absorption rate of 100 W/kg. A sensitive somatic test system was used, in which mutagenicity was measured as the frequency of somatic mutations for eye pigmentation. With the test system used, microwaves did not show any mutagenic activity.     

Antigenic variation in foot-and-mouth disease: studies based on the virus neutralization reaction

The neutralization reaction is the most appropriate in vitro reference test system for assessing intratypic antigenic variation as it involves the antigenic determinants responsible for virus strain specificity and evoking protective antibody. Antigenic relationships determined in different neutralization test systems were independent of the system used and were assumed to truly reflect antigenic variation. The two-dimensional microneutralization test was found to be appropriate for foot and mouth disease (FMD) virus strain differentiation. To minimize test to test variation, comparisons are performed as matched pairs. The pooled variance of the test system is used to assess the significance of the relationships obtained. Antisera from convalescent animals were less specific than those from vaccinates. Serum quality seemed less critical for the virus neutralization than the complement fixation reaction. A system for FMD virus strain differentiation based on the use of the virus neutralization reaction taking into account the statistical and biological significance of observed r values is recommended.     

Detection of brucella antigens with colloid metal particles as markers for specific antigens

Gold and silver sols were comparatively approved as markers of specific IgG isolated from hyperimmune Brucella antisera for the detection of brucellar antigens. The sensitivity of the test system using gold immunosol proved to be some higher (3.1-9.8 ng/ml of soluble and 2.0 x 10(4)-5.3 x 10(6) CFU/ml of corpuscular brucellar antigens) than that achieved with the use of silver immunosol (5.7-18.4 ng/ml of soluble and 6.1 x 10(4)-8.0 x 10(6) CFU/ml of corpuscular brucellar antigens). At the same time silver sol was a cheaper and more available marker. Both test systems were found to be highly specific. False positive results were observed only with Yersinia enterocolitica O:9 at high concentrations due to the fact that they had common polysaccharide haptens incorporated into lipopolysaccharides of these microorganisms. The proposed test systems with colloid metal particles used as markers of specific antibodies for the detection of Brucella antigens are technologically simple, economic, rapid, highly sensitive and specific. Their use in combination with other serological methods will make the results of analyses more informative, thus improving the quality of laboratory diagnostics of brucellosis.     

耐热DNA连接酶介导的TLCR技术在简化DNA重组实验中的应用

传统的DNA重组方法Type Ⅱ型限制酶技术受到片段纯化的限制,无法做到复杂混合体系中DNA片段的特异性连接。为解决这个问题,本研究将耐热连接酶链式反应(Thermostable ligase chain reaction, TLCR)引入DNA片段的连接与捕获。该技术利用耐热型DNA连接酶的特性,在热循环反应中配合针对目的片段末端序列设计的单链寡核苷酸连接模板--Helper,实现目的片段的特异性连接和产物数量的指数性增长。两个质粒构建实验被用于验证TLCR技术的可行性和应用效果。首先利用TLCR技术从一个未经纯化的含有7种不同大小片段的混杂体系中特异性地将一段1.5 kb的片段捕获进载体,随机抽取的克隆样品经检验准确率达到80%,验证TLCR技术在复杂混合体系中特异性连接DNA片段的可行性和准确性。在另一个质粒构建实验中,TLCR技术从λ噬菌体基因组Hind消化物中将两段总长达27 kb的片段按顺序捕获进载体,随机抽取的克隆样品经检验同样达到了80%的准确率。结果表明,TLCR技术能够简化DNA重组实验的操作,并且适用于多片段和大片段的连接,可以为生物学研究提供便利。     

Automated Procedure for Measuring Antigenicity of Extracted and Intact Influenza Virus

An automated serum-blocking (S-B) technique was developed in an attempt to find an in vitro test for the determination of the antigenicity of extracted influenza vaccines. The S-B test depends on the ability of an antigen to combine with (or block) specific (in this case, hemagglutination-inhibiting) antibodies. After mixing the test virus with a constant amount of specific antiserum and hemagglutinating virus in an Auto Analyzer, chicken erythrocytes were pumped into the system and the mixture was incubated by passing through coils. The hemagglutinated cells were removed and the residual cells were lysed. The optical density was read and recorded automatically. The S-B test was much more reproducible than the chicken cell agglutination (CCA) test. There was good correlation between the S-B and CCA titers of intact influenza virus, but not of ether-extracted influenza virus. The CCA titer of influenza strains of type A was reduced significantly during ether-extraction. The S-B titers indicated that there was no significant loss in specific antigenicity when influenza strains of types A and B were extracted with ether and Tween-80 according to the described procedure. The S-B test seemed to be a true measurement of the total antigens present in influenza vaccines.     

Detection of Renibacterium salmoninarum by the enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection and identification of Renibacterium salmoninarum. The immune γ-globulin used in the assay was absorbed with two species of cross-reacting bacteria to make a specific test system. R. salmoninarum could be detected in clinically-diseased fish within 30 minutes of preparing a kidney sample, and thus because of its ease of use, the ELISA could be employed as a rapid field test for bacterial kidney disease (BKD), although isolation of R. salmoninarum was more sensitive than the ELISA for detecting individual carrier fish.     

Validation of a high-throughput in vitro alkaline elution/rat hepatocyte assay for DNA damage

In vitro alkaline elution is a sensitive and specific short term assay which measures DNA strand breakage in a mammalian test system (primary rat hepatocytes). This lab has previously demonstrated the performance of the assay with known genotoxic and non-genotoxic compounds. The methodology employed has relatively low sample throughput and is labor-intensive, requiring a great deal of manual processing of samples in a format that is not amenable to automation. Here, we present an automated version of the assay. This high-throughput alkaline elution assay (HT-AE) was made possible through 3 key developments: (1) DNA quantitation using PicoGreen and OliGreen fluorescent DNA binding dyes; (2) design and implementation of a custom automation system; and (3) reducing the assay to a 96-well plate format. The assay can now be run with 5-50mg of test compound. HT-AE was validated in a similar manner as the original assay, including assessment of non-genotoxic and non-carcinogenic compounds and evaluation of cytotoxicity to avoid confounding effects of toxicity-associated DNA degradation. The validation test results from compounds of known genotoxic potential were used to set appropriate criteria to classify alkaline elution results for genotoxicity.     

Monoclonal antibodies specific for human IgE and their clinical applications

By applying the hybridoma technique, two mouse anti-human Immunoglobulin E (IgE) monoclonal antibodies, designated as E17-58 and E20-62, were generated and characterized. E17-58 was a murine IgG2b with an affinity constant of 4 x 10(8)l/mole. E20-62 was a murine IgG1 with an affinity constant of 1 x 10(8) l/mole. These two antibodies recognized different antigenic determinants specific to the IgE molecule. They were used in combination to quantify the total serum IgE level of forty-nine persons. Data obtained correlated highly with that obtained by using the Pharmacia PRIST Kit (r = 0.91). E17-58 was also used to detect the anti-Aspergillus specific IgE of twenty-one atopic patients by a radioimmunosorbent test. The positive rate detected correlated very well with the skin test (p less than 0.05). In addition, in the Western blot system, these monoclonal antibodies were capable of identifying IgE binding components of crude allergen extracts. Extracts from pollens of Bermuda grass were evaluated, and a new major allergenic component with a molecular weight of 40 kd was identified.     

Antimicrobial susceptibility recording and reporting system for veterinary medicine

Objectives: To develop a system to (1) provide automated interpretation of antimicrobial susceptibility results using animal species, specimen site and bacterial isolate identification criteria, (2) report antimicrobial susceptibility results, according to recommended use category and animal species approval status, (3) allow for changes in antimicrobial agent being tested and result interpretations without need for programming changes and (4) allow for intuitive data entry process without need for reference material. Design: Tables are used to match results and reporting categories to a test method, specific tests, animal species, specimen sites and bacterial isolates. Information used for interpretation of test readings is maintained in tables for user reference while entering results. Results: A table driven system was developed to account for variations in reporting due to antimicrobial susceptibility method used, animal species, bacterial isolate tested and the site from which bacteria isolate was recovered. Conclusions: This process provides for accurate reporting of antimicrobial susceptibility results and is easily adaptable to changes in reporting requirements. The system minimizes reporting criteria and decision-making requirements for technical laboratory personnel, while improving antimicrobial susceptibility reports generated for veterinary practitioners.     

Generalization of a prototype intelligent hybrid system for hard gelatin capsule formulation development

The aim of this project was to expand a previously developed prototype expert network for use in the analysis of multiple biopharmaceutics classification system (BCS) class II drugs. The model drugs used were carbamazepine, chlorpropamide, diazepam, ibuprofen, ketoprofen, naproxen, and piroxicam. Recommended formulations were manufactured and tested for dissolution performance. A comprehensive training data set for the model drugs was developed and used to retrain the artificial neural network. The training and the system were validated based on the comparison of predicted and observed performance of the recommended formulations. The initial test of the system resulted in high error values, indicating poor prediction capabilities for drugs other than piroxicam. A new data set, containing 182 batches, was used for retraining. The percent of the test batches were used for cross-validation, resulting in models with R²≥70%. The comparison of observed performance to the predicted performance found that the system predicted succcessfully. The hybrid network was generally able to predict the amount of drug dissolved within 5% for the model drugs. Through validation, the system was proven to be capable of designing formulations that met specific drug performance criteria. By including parameters to address wettability and the intrinsic dissolution characteristics of the drugs, the hybrid system was shown to be suitable for analysis of multiple BCS class II drugs. Published: October 22, 2005     

Viewer-centered object representation in the human visual system revealed by viewpoint aftereffects

Are there neurons representing specific views of objects in the human visual system? A visual selective adaptation method was used to address this question. After visual adaptation to an object viewed either 15 or 30 degrees from one side, when the same object was subsequently presented near the frontal view, the perceived viewing directions were biased in a direction opposite to that of the adapted viewpoint. This aftereffect can be obtained with spatially nonoverlapping adapting and test stimuli, and it depends on the global representation of the adapting stimuli. Viewpoint aftereffects were found within, but not across, categories of objects tested (faces, cars, wire-like objects). The magnitude of this aftereffect depends on the angular difference between the adapting and test viewing angles and grows with increasing duration of adaptation. These results support the existence of object-selective neurons tuned to specific viewing angles in the human visual system.     

酒酒球菌的快速特异性PCR鉴定

以Oenococcus oeni苹果酸-乳酸酶基因(mleA)为目标基因,设计了1对特异性引物PmleaL/PmleaR进行酒酒球菌的快速鉴定研究。结果表明,直接以O.oeni的菌落为模板,通过引物对PmleaL/PmleaR的PCR扩增,可得到mleA基因的特异性条带;用此特异性引物进行供试乳酸菌的PCR鉴定,所有O.oeni菌系均得到特异性条带,而供试的其它种类乳酸菌未扩增出目标带。PmleaL/PmleaR可用于O.oeni的快速PCR鉴定。     

Effects of myostatin and other growth factors on cultured human cells

A dedicated cell-based biological test system was used for studying specific effects of myostatin and other human growth factors on the proliferation of cultured myoblasts and fibroblasts. Myostatin inhibited myoblast growth without affecting human fibroblasts. In this test system, human growth hormone and insulin-like growth factor I acted as antagonists of myostatin, which indicates that these agents have a potential for blocking its effects in vivo.     

Effects of Myostatin and Growth Factors on Cultured Human Cells

A dedicated cell-based biological test system was used to study specific effects of myostatin and other human growth factors on the proliferation of cultured myoblasts and fibroblasts. Myostatin inhibited myoblast growth without affecting human fibroblasts. In this test system, human growth hormone and insulin-like growth factor I acted as antagonists of myostatin, which indicates that these agents have a potential for blocking its effects in vivo.     

An immunoenzyme method for demonstrating Coxiella burnetii antigens

Materials on the development of an enzyme immunoassay (EIA) system for the detection of the antigens of C. burnetii, the causative agent of Q rickettsiosis, are presented. The system is highly specific and effective with respect to both corpuscular antigens of phases 1 and 2 and soluble antigen (lipopolysaccharide). The sensitivity of this method varies within the range 5-100 ng/ml. The effectiveness of EIA as a quantitative (semiquantitative) control test used in the process of the production of Coxiella preparations has been demonstrated.     

Rapid and specific identification of Shiga toxin-producing Escherichia coli in faeces by multiplex PCR

AIMS: The object of this study was to develop a multiplex PCR system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) in faeces. METHODS AND RESULTS: A multiplex PCR (mPCR) protocol was developed using a primer pair specific for genes that are involved in the biosynthesis of the O157 E. coli antigen, and primers that identify the sequences of Shiga toxin 1 and 2 (stx 1 and stx1) and the intimin protein (eaeA). The mPCR assay was used for amplification of STEC genes in bacteria directly (after enrichment) in faeces. The test was very sensitive and could detect between 9 and 1 bacterial cells per gram of faeces. The mPCR was used for the examination of 69 bovine faecal samples derived from healthy cattle. The results indicated that 62 x 3% of the samples were positive, generating at least one PCR amplicon of the expected size. CONCLUSIONS: The method can be applied for rapid and specific identification of STEC bacteria in faecal samples, and for differentiation of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect Shiga toxin-producing E. coli directly in faeces within a short time represents a considerable advancement over more time-consuming and less sensitive methods for identification and characterization of STEC bacteria.     

Countermeasures for long-term space flights, lessons learned from the Russian space program

Physical exercises (PE) constitute the most important part of the Russian countermeasure system which is proved to be highly successful in fighting negative effects of weightlessness on the physiological systems of the body. To control the intensity and volume of the workload and also specific features of the regimens used by cosmonauts during SF there were provided monthly 3-days recordings of cosmonaut training sessions with the subsequent downlinking of the obtained information to the ground control center. Additionally once a month cosmonauts performed a fitness test that was composed of 11 minutes of locomotion on the passive treadmill including slow, middle range and maximal running. The results of 3-days recordings and fitness test allowed to evaluate correctly effectiveness of PE protocols used by cosmonauts and to form recommendations for their corrections. The effectiveness of the onboard exercise program after SF was evaluated based on results of detailed studies of alterations that were recorded in different parts of sensory-motor system.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence micro cytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Serological and genetic identification of a bovine B lymphocyte alloantigen system

A two-colour fluorescence microcytotoxicity test was used to screen antisera for antibodies specific for bovine B lymphocytes. A total of 114 cattle alloantisera were screened against peripheral blood lymphocytes from 100 unrelated individuals. Anti-B lymphocyte activity was detected in 47 antisera. Cytotoxic antibodies to antigens other than B lymphocyte specific antigens were removed by absorbing the antisera with buffy coat cells or platelets isolated from whole blood. Selected antisera were used to type paternal half-sib families. The presence of a polymorphic, MHS-linked antigen system on B lymphocytes was demonstrated. The tissue distribution and MHS linkage of these antigens suggests this system is analogous to the class II or Ia antigens of other species.     

Distribution of legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates

A hospital warm water system was monitored for the presence and distribution of legionellae. Subtyping of ten selected Legionella pneumophila isolates, originating from four different sites in the system by using serogroup specific antisera in an indirect immunofluorescence test, revealed that nine of the ten isolates belong to serogroup 6, while the remaining one was serogroup 10. Two monoclonal antibodies (mAbs) specific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reacted with these mAbs. Genome analysis by elaborating NotI profiles using the pulsed field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates derived from different sites, including a new building connected by a ring pipe, were identical according to restriction fragment patterns. The patterns were distinguishable from those of the two L. pneumophila serogroup 6 reference strains, and from that of the L. pneumophila serogroup 10 isolate. These data argue for a relatively homogeneous L. pneumophila serogroup 6 population in the entire water system.