Recommended protocol for the BALB/c 3T3 cell transformation assay

The present protocol has been developed for the BALB/c 3T3 cell transformation assay (CTA), following the prevalidation study coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and reported in this issue (Tanaka et al. [16]). Based upon the experience gained from this effort and as suggested by the Validation Management Team (VMT), some acceptance and assessment criteria have been refined compared to those used during the prevalidation study. The present protocol thus describes cell culture maintenance, the dose-range finding (DRF) experiment and the transformation assay, including cytotoxicity and morphological transformation evaluation. Use of this protocol and of the associated photo catalogue included in this issue (Sasaki et al. [17]) is recommended for the future conduct of the BALB/c 3T3 CTA.     

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 6.7. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.     

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 7.0. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.     

Prevalidation study of the BALB/c 3T3 cell transformation assay for assessment of carcinogenic potential of chemicals

The cell transformation assays (CTAs) have attracted attention within the field of alternative methods due to their potential to reduce the number of animal experiments in the field of carcinogenicity. The CTA using BALB/c 3T3 cells has proved to be able to respond to chemical carcinogens by inducing morphologically transformed foci. Although a considerable amount of data on the performance of the assay has been collected, a formal evaluation focusing particularly on reproducibility, and a standardised protocol were considered important. Therefore the European Centre for the Validation of Alternative Methods (ECVAM) decided to coordinate a prevalidation study of the BALB/c 3T3 CTA. Three different laboratories from Japan and Europe participated. In the study the following modules were assessed stepwise: test definition (Module 1) consisted of the standardisation of the protocol, the selection of the cell lineage, and the preparation of a photo catalogue on the transformed foci. The within-laboratory reproducibility (Module 2) and the transferability (Module 3) were assessed using non-coded and coded 3-methylcholanthrene. Then, five coded chemicals were tested for the assessment of between-laboratory reproducibility (Module 4). All three laboratories obtained positive results with benzo[a]pyrene, phenanthrene and o-toluidine HCl. 2-Acetylaminofluorene was positive in two laboratories and equivocal in one laboratory. Anthracene was negative in all three laboratories. The chemicals except phenanthrene, which is classified by IARC (http://monographs.iarc.fr) as group 3 "not classifiable as to its carcinogenicity to human", were correctly predicted as carcinogens. Further studies on phenanthrene will clarify this discrepancy. Thus, although only a few chemicals were tested, it can be seen that the predictive capacity of the BALB/c 3T3 CTA is satisfactory. On the basis of the outcome of this study, an improved protocol, incorporating some changes related to data interpretation, has been developed. It is recommended that this protocol be used in the future to provide more data that may confirm the robustness of this protocol and the performance of the assay itself. During the study it became clear that selecting the most appropriate concentrations for the transformation assay is crucial.     

Understanding mammalian genetic systems: the challenge of phenotyping in the mouse

Understanding mammalian genetic systems is predicated on the determination of the relationship between genetic variation and phenotype. Several international programmes are under way to deliver mutations in every gene in the mouse genome. The challenge for mouse geneticists is to develop approaches that will provide comprehensive phenotype datasets for these mouse mutant libraries. Several factors are critical to success in this endeavour. It will be important to catalogue assay and environment and where possible to adopt standardised procedures for phenotyping tests along with common environmental conditions to ensure comparable datasets of phenotypes. Moreover, the scale of the task underlines the need to invest in technological development improving both the speed and cost of phenotyping platforms. In addition, it will be necessary to develop new informatics standards that capture the phenotype assay as well as other factors, genetic and environmental, that impinge upon phenotype outcome.     

Physiology in the mirror of systematic catalogue of Russian Academy of Sciences Library

Representation of general human and animal physiology publications in the systematic catalogue of the Library of the Russian Academy of Sciences is considered. The organization of the catalogue as applied to the problems of physiology, built on the basis of library-bibliographic classification used in the Russian universal scientific libraries is described. The card files of the systematic catalogue of the Library contain about 8 million cards. Topics that reflect the problems of general physiology contain 39 headings. For the full range of sciences including physiology the tables of general types of divisions were developed. They have been marked by indexes using lower-case letters of the Russian alphabet. For further detalizations of these indexes decimal symbols are used. The indexes are attached directly to the field of knowledge index. With the current relatively easy availability of network resources value and relevance of any catalogue are reduced. However it concerns much more journal articles, rather than reference books, proceedings of various conferences, bibliographies, personalities, and especially the monographs contained in the systematic catalogue. The card systematic catalogue of the Library remains an important source of information on general physiology issues, as well as its magistral narrower sections.     

A proteomic approach for unraveling the oncogenic H-Ras protein networks in NIH/3T3 mouse embryonic fibroblast cells

To elucidate the oncogenic H-Ras network, we have established various stable and inducible oncogenic H-Ras-expressing NIH/3T3 mouse embryonic fibroblast cell clones, which express G12V H-Ras and G12R H-Ras proteins under the influence of a strong cytomegalovirus promoter and under the tight control of expression by an antibiotic, doxycycline, respectively. Here we provide a catalogue of proteome profiles in total cell lysates derived from oncogenic H-Ras-expressing NIH/3T3 cells. In this biological context, we compared total proteome changes by the combined methods of 2-DE, quantitative image analysis and MALDI-TOF MS analysis using both a stable expression system as well as an inducible expression system. There were a large number of common targets for oncogenic H-Ras, which were identified in both cell lines and consisted of 64 proteins (36 up-regulated and 28 down-regulated). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, the results presented here show that comparative analysis of the proteome from the oncogenic H-Ras-expressing cells yielded interpretable data to elucidate protein networks directly and/or indirectly.     

Antigenicity of the carbohydrate moiety of ganglioside GM3 having 3-O-acetyl ceramide

To elucidate the effect of a modification of ceramide on antigenicity of the carbohydrate of ganglioside, the reactivity of O-acetyl GM3 having 3-O-acetyl ceramide, which has been characterized as a gliomarelated ganglioside, with monoclonal antibody M2590 was examined in comparison to that of non-acetylated GM3, by means of quantitative enzyme-linked immunosorbent assay, TLC-immunostaining and liposome immune lysis assay. In all these assay systems, O-acetyl GM3 showed less activity than GM3 as follows: GM3 was detected till 0.1 nmol in TLC-immunostaining, whereas O-acetyl GM3 could not be detected even at 0.25 nmol; the GM3 reaction was approximately twofold that of O-acetyl GM3 at each diluted point in the enzyme-linked immunosorbent assay; and 20% of the liposomes containing GM3 were lysed at 6 mol%, while liposomes containing O-acetyl GM3 did not lyse at that concentration. The lesser antigenicity of the sugar moiety of O-acetyl GM3 could be ascribed to the presence of an acetyl group in the ceramide at the 3-position of sphingosine.     

A catalogue of abnormalities in the division spindles of higher plants

This catalogue collates observations on meiotic division in a large number of plant forms with abnormal meiosis, including mutants, wide hybrids, haploids, polyploids, aneuploids, and alloplasmics. The process of division spindle formation remains relatively unexplored in the "centrosomeless" world of higher plant cells. Thus, analysis of abnormal spindles, each of which is a result of an aberration of a distinct prometaphase stage, is informative. A catalogue of spindle abnormalities is also useful for analysing the morphological phenotype of the corresponding mutations, especially insertional ones. It is particularly worthwhile for those organisms that are less than ideal for cytological analysis, e.g. Arabidopsis. In the catalogue, abnormal spindles are listed in relation to the time of the manifestation of aberrations that caused them.     

Improved methods for the assay and activation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

A simple and rapid mixed-phase method for the quantitative assay of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and a procedure for the efficient reactivation of Mg-ATP-inactivated microsomal HMG-CoA reductase by potato acid phosphatase are described. The mixed-phase assay entails the direct addition of the acidified, deproteinized incubation mixture to a toluene-based scintillation fluor. The enzymatic reaction product [3H]-mevalonolactone partitions into the toluene while unreacted 3H-labeled HMG-CoA substrate remains in the aqueous phase and is not detected on scintillation counting. The accuracy and reproducibility of this method are compared to a thin-layer chromatographic assay for HMG-CoA reductase. Microsomal and solubilized HMG-CoA reductase inactivated by incubation with Mg-ATP is reactivated by purified potato acid phosphatase. Under appropriate conditions quantitative reactivation of HMG-CoA reductase is achieved, indicating that endogenous inhibitory and activating proteins regulate HMG-CoA reductase via a kinase-phosphatase system.     

Metagenomic Data Utilization and Analysis (MEDUSA) and Construction of a Global Gut Microbial Gene Catalogue

Metagenomic sequencing has contributed important new knowledge about the microbes that live in a symbiotic relationship with humans. With modern sequencing technology it is possible to generate large numbers of sequencing reads from a metagenome but analysis of the data is challenging. Here we present the bioinformatics pipeline MEDUSA that facilitates analysis of metagenomic reads at the gene and taxonomic level. We also constructed a global human gut microbial gene catalogue by combining data from 4 studies spanning 3 continents. Using MEDUSA we mapped 782 gut metagenomes to the global gene catalogue and a catalogue of sequenced microbial species. Hereby we find that all studies share about half a million genes and that on average 300 000 genes are shared by half the studied subjects. The gene richness is higher in the European studies compared to Chinese and American and this is also reflected in the species richness. Even though it is possible to identify common species and a core set of genes, we find that there are large variations in abundance of species and genes.     

The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells

To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells.     

A radioisotopic method for the determination of acetoacetyl Coenzyme A with 3-hydroxy-3-methylglutaryl Coenzyme A synthase

A simple and sensitive assay for the quantitative determination of acetoacetyl-CoA (AcAc-CoA) in liver and heart is described. The method is based on incorporation of [¹⁴C]acetyl-CoA into acid-stable nonvolatile material in the presence of avian HMG-CoA synthase. The specificity of this procedure for the measurement of AcAc-CoA was demonstrated by pretreating tissue extracts with 3-hydroxyacyl-CoA dehydrogenase or CoA transferase from Escherichia coli to deplete. AcAc-CoA prior to assay. Acid-stable nonvolatile ¹⁴C activity measured in the assay was proportional to the amount of tissue extract added. Satisfactory recovery of AcAc-CoA added at the initial extraction step further validated this procedure. This radioactive assay for acetoacetyl-CoA using a highly purified avian 3-hydroxy-3-methylglutaryl-CoA synthase has the advantages of both extreme specificity for AcAc-CoA as substrate and high sensitivity, facilitating the determination of this metabolite under a variety of physiological conditions.     

Antibodies to interleukin 3 as probes for the interaction of interleukin 3 with its receptor.

A series of antibodies, directed against murine interleukin-3 (IL-3) or synthetic peptides corresponding to portions of the IL-3 sequence, has been used to detect receptor-bound IL-3 on the surface of cells. An assay was developed in which the bound primary antibody was detected using a biotinylated secondary antibody and fluorescein isothiocyanate-labeled streptavidin, followed by analysis on a fluorescence-activated cell sorter. The fluorescence signal was shown to be specific for cells known to express IL-3 receptors and was dependent on the preincubation of cells with IL-3 under conditions that did not allow internalization of receptors. Antisera raised against full-length synthetic IL-3 or to the N-terminal 29 residues were found to give equivalent signals. On the other hand, antibodies to residues 91-118 showed no signal in this assay, despite being able to bind to IL-3 in solution and neutralize IL-3 bioactivity. When peptides corresponding to residues 30-43 and 123-140 were incubated with the anti-IL-3 antiserum, the majority of the fluorescence signal was abolished, indicating that these two peptides contained the primary epitopes being recognized by the antiserum in this assay. This antiserum also bound to the 91-118 peptide, but the corresponding peptide was not able to reduce the fluorescence signal in a similar competition assay. These results suggest that the 91-118 region is not accessible to antibody when IL-3 is bound to its receptor, whereas at least portions of epitopes 1-29, 30-43, and 123-140 remain accessible to antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contributions to the knowledge of the Noctuoidea (Lepidoptera) of the Republic of Belarus

The paper contains a detailed overview of the history of Noctuoidea study in Belarus since the middle of the XIX century. Critical analysis of the Noctuoidea species list in the Catalogue of Lepidoptera of Belarus (Merzheyevskaya et al., 1976) is made. A list of species missing from this catalogue but recorded in preceding publications is compiled, supplemented with records of the species found in Belarus after publication of the catalogue. Data on the examined material for 19 species new to the fauna of Belarus are given.     

Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect

betaig-h3 is an extracellular matrix protein that mediates adhesion and migration of several cell types through interaction with integrins. In the present study, we tested whether betaig-h3 mediates endothelial cell adhesion and migration, thereby regulating angiogenesis. In this study, we demonstrate that not only betaig-h3 itself but also all four fas-1 domains of betaig-h3 mediate endothelial cell adhesion and migration through interaction with the alphavbeta3 integrin. We found that the alphavbeta3 integrin-interacting motif of the four fas-1 domains of betaig-h3 is the same YH motif that we reported previously to interact with alphavbeta5 integrin. The YH peptide inhibited endothelial cell adhesion and migration in a dose-dependent manner. We demonstrate that the YH peptide has anti-angiogenic activity in vitro and in vivo using an endothelial cell tube formation assay and a Matrigel plug assay, respectively. Our results reveal that betaig-h3 bears alphavbeta3 integrin-interacting motifs that mediate endothelial cell adhesion and migration and, therefore, may regulate angiogenesis.     

皮金龟科的分类研究进展

皮金龟科Trogidae对分解动物尸体具有重要作用,全球已知3属320种。作者对其分类地位、分类研究历史和现状进行了概要总结;还就世界分类研究文献、分类系统和种类情况做了介绍,并对该科昆虫的地理分布特点进行了初步分析。     

Binding of the Tn3 transposase to the inverted repeats of Tn3

The transposase protein and the inverted repeat sequences of Tn3 are both essential for Tn3 cointegrate formation and transposition. We have developed two assays to detect site-specific binding of transposase to the inverted repeats: (1) a nitrocellulose filter binding assay in which transposase preferentially retains DNA fragments containing inverted repeat sequences, and (2) a DNase 1 protection assay in which transposase prevents digestion of the inverted repeats by DNase 1. Both assays show that transposase binds directly to linear, duplex DNA containing the inverted repeats. The right inverted repeat of Tn3 binds slightly more strongly than the left one. Site-specific binding requires magnesium but does not require a high energy cofactor.     

Potentiometric and colorimetric methods for the assay of 2',3'-cyclic nucleotide 3'-phosphohydrolase

Abstract— A potentiometric titration method for the assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase is presented. Progress curves of the reaction were recorded automatically by pH-stat. 2-Mercaptoethanol was added to the reaction mixture to maintain a linear rate of reaction. The method is suitable for obtaining kinetic parameters and can be used for the rapid assay of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in nervous tissues. An improved colorimetric method for estimation of 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity at the optimum pH is described. This method employs the two-step procedure in which decyclization by 2′,3′-cyclic nucleotide 3′-phosphohydrolase and dephosphorylation by Escherichia coli alkaline phosphatase (EC 3.1.3.1) are carried out separately under the optimum conditions for each enzyme. The method is sensitive and most convenient for routine assays.