A new mouse model for studying EGFR-dependent gastric polyps

Hyperactivation of the epidermal growth factor receptor (EGFR) in gastric cells due to excess of its ligand transforming growth factor-α (TGFA) is associated with hyperplastic lesions in Ménétrier's disease patients and in transgenic mice. Other EGFR ligands, however, have never been associated with stomach diseases. Here, we report that overexpression of the EGFR ligand betacellulin (BTC) results in a severe, age-dependent hyperplasia of foveolar epithelium. The stomach weight of affected mice reached up to 3g representing more than 10% of total body weight. The preexisting corpus mucosa was severely depleted, and both parietal and chief cells were replaced by proliferating foveolar epithelium. The lesions were more severe in male as compared to female transgenic mice, and partially regressed in the former after castration-mediated androgen removal. The gastric hyperplasia fully disappeared when BTC-tg mice were crossed into the Egfr(Wa5) background expressing a dominant-negative EGFR, indicating that the phenotype is EGFR-dependent. This is, to our knowledge, the first report of hyperplastic gastric lesions due to the overexpression of an EGFR ligand other than TGFA. BTC-tg mice are therefore a new, promising model for studying EGFR-dependent gastric polyps.     

Evaluation of a new model system for studying the formation of heterocyclic amines

Heterocyclic amines (HAs) are an important class of food mutagens and carcinogens, which can be found in cooked meat and fish. Increasing heating temperatures and times usually increase mutagenic activity in meat and meat extracts during cooking. We developed a model system, which allows to examine the effects of precursor composition and heating conditions (time and temperature) on the formation of HAs in meat. Homogenized and freeze dried meat samples (beef, pork chops, chicken breast and turkey breast) are heated with diethylene glycol in closed vials under stirring in a thermostated heating block. After an appropriate sample preparation (extraction and clean-up) ten different HAs were measured by HPLC analyses with gradient elution and mass selective detection. The time courses of HA-formation in the different kinds of meat at varying heating temperatures were determined up to heating times of 30 min. 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was the most abundant HA in these experiments and reached the highest concentrations in the beef meat samples, as did the other HAs (MeIQ, AalphaC) at 220 degrees C in the heating block under stirred conditions. Additionally the influence of the antioxidant TBHQ (t-butylhydroquinone) on the formation of HAs in the model system was tested. However TBHQ effected only slight reductions of HA formation in all kinds of meat.     

A model T-cell receptor system for studying memory T-cell development

When T-cell clones were first grown in long-term cell culture, each clone was considered to be capable of displaying a limited range of functional activities, constrained by the clones' coreceptor, CD4 or CD8, and the specificity of its antigen-specific receptor (TCR) for one or a few peptides in association with a class I or class II MHC molecule. Subsequent studies, especially with transgenic mice, have shown, however, that T cells expressing the same receptor can be obtained in a variety of differentiated states, including na?ve cells, activated effector cells, memory cells, and anergic or tolerant cells, as well as cells with or without a coreceptor. In each of these states T cells can display distinctly different responses to the peptide-MHC (pepMHC) complexes the TCR recognizes. Recently, memory T cells have received considerable attention, in part because of the likelihood that they confer long-term protective immunity against diverse infectious agents and possibly against some forms of cancer. Here we review some recent studies that our colleagues and we have carried out on memory CD8(+) T cells. These studies have made extensive use of cells that express the TCR called 2C. The diverse set of cells expressing the 2C TCR, in mice and in cultured clones and cell lines, are referred to as the 2C system. Before reviewing the studies of memory cells, we summarize the 2C system's main features, including evidence that a large and diverse array of pepMHC complexes, involving at least four class I MHC proteins, can stimulate TCR-mediated responses of 2C cells.     

A nonlinear model for studying oscillations in the blood pressure control system

The idea that physiological systems sometimes include oscillations as part of their control function is a relatively new idea; the paper discusses the role of such oscillations in the regulation of arterial blood pressure and how they can be studied by modelling techniques. A nonlinear model is proposed which can be related directly to the physiology. This is seen as an important step because mainly previous studies of biological oscillations have used lumped equations, e.g. the Van der Pol equation, which do not possess this property. On the basis of the model, the oscillatory behaviour of the blood pressure control system is analysed by the application of the dual input describing function technique, a powerful analytical method which has wide application to the study of nonlinear oscillatory phenomena in physiology.The final section of the paper considers the digital simulation of the model by Z transform techniques and how the simulation can be used to study the interaction of respiratory and blood pressure control systems.     

Value of the Hydra model system for studying symbiosis

Green Hydra is used as a classical example for explaining symbiosis in schools as well as an excellent research model. Indeed the cosmopolitan green Hydra (Hydra viridissima) provides a potent experimental framework to investigate the symbiotic relationships between a complex eumetazoan organism and a unicellular photoautotrophic green algae named Chlorella. Chlorella populates a single somatic cell type, the gastrodermal myoepithelial cells (also named digestive cells) and the oocyte at the time of sexual reproduction. This symbiotic relationship is stable, well-determined and provides biological advantages to the algal symbionts, but also to green Hydra over the related non-symbiotic Hydra i.e. brown hydra. These advantages likely result from the bidirectional flow of metabolites between the host and the symbiont. Moreover genetic flow through horizontal gene transfer might also participate in the establishment of these selective advantages. However, these relationships between the host and the symbionts may be more complex. Thus, Jolley and Smith showed that the reproductive rate of the algae increases dramatically outside of Hydra cells, although this endosymbiont isolation is debated. Recently it became possible to keep different species of endosymbionts isolated from green Hydra in stable and permanent cultures and compare them to free-living Chlorella species. Future studies testing metabolic relationships and genetic flow should help elucidate the mechanisms that support the maintenance of symbiosis in a eumetazoan species.     

Chlamydomonas reinhardtii as a new model system for studying the molecular basis of the circadian clock

The genome of the unicellular green alga Chlamydomonas reinhardtii has both plant-like and animal-like genes. It is of interest to know which types of clock genes this alga has. Recent forward and reverse genetic studies have revealed that its clock has both plant-like and algal clock components. In addition, since C. reinhardtii is a useful model organism also called "green yeast", the identification of clock genes will make C. reinhardtii a powerful model for studying the molecular basis of the eukaryotic circadian clock. In this review, we describe our forward genetic approach in C. reinhardtii and discuss some recent findings about its circadian clock.     

A model two-component system for studying the architecture of elastin assembly in vitro

Tropoelastin is encoded by a single human gene that spans 36 exons and is oxidized in vivo by mammalian lysyl oxidase at the epsilon amino group of available lysines to give the adipic semialdehyde, which then facilitates covalent cross-link formation in an enzyme-free process involving tropoelastin association. We demonstrate here that this process is effectively modeled by a two protein component system using purified lysyl oxidase from the yeast Pichia pastoris to facilitate the oxidation and subsequent cross-linking of recombinant human tropoelastin. The oxidized human tropoelastin forms an elastin-like polymer (EL) that is elastic, shows hydrogel behavior and contains typical elastin cross-links including lysinonorleucine, allysine aldol, and desmosine. Protease digestion and subsequent mass-spectrometry analysis of multiple ELs allowed for the identification of specific intra- and inter-molecular cross-links, leading to a model of the molecular architecture of elastin assembly in vitro. Specific intra-molecular cross-links were confined to the region of tropoelastin encoded by exons 6-15. Inter-molecular cross-links were prevalent between the regions encoded by exons 19-25. We find that assembly of tropoelastin molecules in ELs are highly enriched for a defined subset of cross-links.     

Roles for the phosphatidylinositol cycle in early development

Founded on the seminal studies and writings of Hokin, Michell and Berridge, a vast body of data now exists documenting the central importance of phosphatidylinositol (PtdIns) cycle activation in transducing information of many types across the plasma membrane. The great majority of these data derive from studies of terminally differentiated somatic cells. Nevertheless, the fact that many crucial events in animal development also involve transduction of information across the plasma membrane has recently led developmental biologists to search for regulatory roles for PtdIns cycle activity in such developmental processes as oocyte maturation, fertilization, and embryogenesis, with encouraging results. In this paper I briefly review the progress of such studies, beginning with the event in which the PtdIns cycle's role is best understood (fertilization), then progressing both backwards and forwards in developmental time to explore more speculative roles for the PtdIns cycle in oocyte maturation and pattern formation during embryogenesis.     

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.     

A new model system for tomato genetics

The purpose of this study was to develop a model system for studying tomato genetics. Agronomic, genetic, and molecular data are presented which show that the miniature Lycopersicon esculentum cultivar, Micro-Tom (Micro tomato), fulfills the requirements for such a model. It grows at high density (up to 1357 plants/m⁻²); it has a short life cycle (70–90 days from sowing to fruit ripening); and it can be transformed at frequencies of up to 80% through Agrobacterium -mediated transformation of cotyledons. Moreover, it differs from standard tomato cultivars by only two major genes. Therefore, any mutation or transgene can be conveniently studied in Micro-Tom's background and, when needed, transferred into a standard background. We took advantage of Micro-Tom's features to improve the infrastructure for mutagenesis in tomato. A screening of 9000 M1 and 20 000 M2 EMS mutagenized plants is described. Mutants with altered pigmentation or modified shape of leaves, flowers and fruits were found. In addition, an enhancer trapping and a gene trapping system, based on the Ac/Ds maize transposable elements, were transformed into Micro-Tom and found to be active. In summary, Micro-Tom opens new prospects to achieve saturated mutagenesis in tomato, and facilitates the application of transposon-based technologies such as gene tagging, trapping and knockout.     

一类大尺度系统中昆虫种群时空动态模拟方法

本文介绍了一类描述大尺度作物系统害虫种群时空动态的模型,该模型包含了寄主作物,昆虫密度以及天敌间的相互作用,并简要介绍了该模型的一些模拟结果在IPM中的应用。     

A model system for studying the adsorption of a hydrophobic mutagen to dietary fibre

The adsorption of 1,8-dinitropyrene (DNP) to alpha-cellulose has been studied as a model system for examining the adsorption of a hydrophobic mutagen to dietary fiber. Most of the DNP rapidly disappeared from an aqueous solution and partitioned between the glass wall of the test tube and the alpha-cellulose. Factors affecting DNP distribution included (i) the time of incubation, (ii) the final concentration of the solvent, dimethyl sulphoxide, in which the DNP has been dissolved, and (iii) the relative concentrations of DNP and alpha-cellulose. We suggest that this model system could be applied to other mutagens, and that alpha-cellulose would provide a useful standard fiber to permit inter-laboratory comparisons.     

The human erythrocyte ghost: A new experimental model for studying adenosine transport

Previous work on adenosine transport has always had problems with the interference of adenosine metabolism, due to its high metabolic rate and because the enzymes involved are consistently present in most tissues. A new experimental model for studying adenosine transport in human erythrocyte ghosts is presented in this work: Human erythrocyte ghosts were sealed in the presence of erythro-3(2-hydroxynonyl)adenine and P1-P5-di(adenosine)5′-pentaphosphate, inhibitors of adenosine deaminase and adenosine kinase, respectively. These ghosts proved to lack adenosine metabolism when incubated in [U-¹⁴C]adenosine at 10 μm concentration at 37 °C for 60 min. Ghosts were 99.4% sealed in the correct orientation and had constant intracellular water volume. With these characteristics, the erythrocyte ghost preparation has many advantages for studying adenosine transport without adenosine metabolism interference. Adenosine transport was studied following the technique of 24., 25. Experiments to study Zero-trans influx and efflux, equilibrium exchange, and infinite-trans influx and efflux are presented. Adenosine transport did not behave linearly in any of these experimental procedures. Adenosine basic kinetic constants, calculated according to the procedure of Lieb and Stein, were R_1→-2 = 4.1 × 10⁻⁴, R_2→-1 = 3.97 × 10⁻⁴, R_ee = 1.94 × 10⁻⁴, R_oo = 6.08 × 10⁻⁴, K_1→-2 = 125.67 μm, andK_2→-1 = 84.36 μm. Lieb and Stein rejection criteria were used to distinguish a simple pore from a simple carrier. The data accumulated indicate that adenosine transport is carried out by a system that satisfies the criteria used for the simple carrier model. Asymmetric behavior was observed indicating lower affinity of the carrier for adenosine influx, although V_max values for influx and efflux were similar.     

A new flow co-culture system for studying mechanobiology effects of pulse flow waves

Artery stiffening is known as an important pathological change that precedes small vessel dysfunction, but underlying cellular mechanisms are still elusive. This paper reports the development of a flow co-culture system that imposes a range of arterial-like pulse flow waves, with similar mean flow rate but varied pulsatility controlled by upstream stiffness, onto a 3-D endothelial-smooth muscle cell co-culture. Computational fluid dynamics results identified a uniform flow area critical for cell mechanobiology studies. For validation, experimentally measured flow profiles were compared to computationally simulated flow profiles, which revealed percentage difference in the maximum flow to be <10, <5, or <1% for a high, medium, or low pulse flow wave, respectively. This comparison indicated that the computational model accurately demonstrated experimental conditions. The results from endothelial expression of proinflammatory genes and from determination of proliferating smooth muscle cell percentage both showed that cell activities did not vary within the identified uniform flow region, but were upregulated by high pulse flow compared to steady flow. The flow system developed and characterized here provides an important tool to enhance the understanding of vascular cell remodeling under flow environments regulated by stiffening.

Electronic supplementary material

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