Conditional expression of MCM7 increases tumor growth without altering DNA replication activity

The minichromosome maintenance (MCM) 2-7 complex is a putative DNA helicase complex that facilitates the initiation of DNA replication. Here, we generated a cell line MCM7(+/-)/MCM7-FLAG, in which one allele of MCM7 is mutated whereas a tetracycline-repressible promoter could manipulate the expression of exogenous MCM7 protein. Overexpressed MCM7 protein supports efficient DNA replication of Epstein-Barr virus oriP and rapid formation of tumors in nude mice without altering the activity of cellular DNA replication. This system provides a unique setting for studying the function of MCM7 and for screening for potential therapeutics for malignant tumors.     

Successful contemporary reverse controlled antegrade and retrograde subintimal tracking without contrast medium: a case report

Background

Contrast-induced acute kidney injury is one of the common adverse events related to percutaneous coronary intervention and a predictor for worse outcome. In the setting of percutaneous coronary intervention for chronic total occlusion, large amounts of contrast medium, more than 200–400?mL, are generally injected. A higher dose of contrast medium causes contrast-induced acute kidney injury more frequently. Therefore, patients who undergo chronic total occlusion-percutaneous coronary intervention are at risk for contrast-induced acute kidney injury.

Case presentation

We present the case of a 77-year-old Japanese man with post-acute myocardial infarction angina pectoris, heart failure, and chronic kidney disease who underwent percutaneous coronary intervention for chronic total occlusion in his right coronary artery. In the procedure, the retrograde wire was a visible penetration mark that made contrast medium unnecessary. Contemporary reverse controlled antegrade and retrograde subintimal tracking was successfully achieved and stents were implanted without contrast medium. Contrast medium was injected two times after stent implantation to confirm coronary flow and no perforation. The total amount of contrast medium was only 8?mL for chronic total occlusion-percutaneous coronary intervention.

Conclusion

Chronic total occlusion-percutaneous coronary intervention with contemporary reverse controlled antegrade and retrograde subintimal tracking without contrast medium may be safe and feasible in selected patients.

     

Quantitative analysis of membrane components in a highly active O2-evolving photosystem II preparation from spinach chloroplasts

Stoichiometry of membrane components associated with Photosystem II was determined in a highly active O₂-evolving Photosystem II preparation isolated from spinach chloroplasts by the treatment with digitonin and Triton X-100. From the analysis with sodium dodecyl sulfate polyacrylamide gel electrophoresis and Triton X-114 phase partitioning, the preparation was shown to contain the reaction center protein (43 kDa), the light-harvesting chlorophyll-protein complex (the main band, 27 kDa), the herbicide-binding protein (32 kDa) and cytochrome b-559 (10 kDa) as hydrophobic proteins, and three proteins (33, 24 and 18 kDa) which probably constitute the O₂-evolution enzyme complex as hydrophilic proteins. These proteins were associated stoichiometrically with the Photosystem II reaction center: one Photosystem II reaction center, approx. 200 chlorophyll, one high-potential form of cytochrome b-559, one low-potential form of cytochrome b-559, one 33 kDa protein, one (to two) 24 kDa protein and one (to two) 18 kDa protein. Measurement of fluorescence induction showed the presence of three electron equivalents in the electron acceptor pool on the reducing side of Photosystem II in our preparation. Three molecules of plastoquinone A were detected per 200 chlorophyll molecules with high-performance liquid chromatography. The Photosystem II preparation contained four managanese atoms per 200 chlorophyll molecules.     

Synergism between stem cell factor and granulocyte-macrophage colony-stimulating factor on cell proliferation by induction of cyclins

Synergism between stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be essential for hematopoietic cell proliferation. Since HML-2 cells proliferate exponentially in the presence of SCF and GM-CSF together, we analyzed the molecular mechanism of the interaction between these two factors in the cells. An immediate-early gene product, c-myc, was additively upregulated in HML-2 cells by addition of a combination of SCF and GM-CSF. c-myc antisense oligonucleotides effectively suppressed cell proliferation and downregulated the induction of D3, E, A, and B cyclins in HML-2 cells stimulated with the two-factor combination. HML-2 cells arrested at the G0/G1 phase with SCF alone and expressed modest amounts of c-myc and cyclin D3, but not cyclin E. With GM-CSF treatment alone, the cells could not progress to the G2/M phase and expressed c-myc, cyclin D3 and cyclin E but not cyclins A or B. The addition of the counterpart cytokine resulted in cell cycle completion by induction of the deficient cyclins. Taken together, it appears that the induction of c-myc is an indispensable event in the proliferation of HML-2 cells and that the cytokines SCF and GM-CSF interact reciprocally for expression of all cyclins required for cell cycle progression.     

Structure-activity relationship of alginate oligosaccharides in the induction of cytokine production from RAW264.7 cells

Guluronate and mannuronate oligomers with various degree of polymerization were prepared from polyguluronate (PG) and polymannuronate (PM) with an alginate lyase from a Pseudoalteromonas sp., and their activities to induce cytokine secretion from mouse macrophage cell line RAW264.7 cells were examined. Enzymatically depolymerized unsaturated alginate oligomers induced tumor necrosis factor (TNF)-alpha secretion from RAW264.7 cells in a structure-depending manner, while the activities of saturated alginate oligomers prepared by acid hydrolysis were fairly low or only trace levels. These results suggest that unsaturated end-structure of alginate oligomers was important for the TNF-alpha-inducing activity. Among the unsaturated guluronate (G3-G9) and mannuronate (M3-M9) oligomers, G8 and M7 showed the most potent activity, respectively. Bio-Plex assay revealed that interleukin (IL)-1alpha, IL-1beta, and IL-6 secretion from RAW264.7 cells were also induced by unsaturated alginate oligomers with similar structure-activity relationship profiles as seen in TNF-alpha, and the most potent activities were observed with G8 and M7. These results suggest that G8 and M7 may have the most suitable molecular size or entire structural conformation as stimulant for cytokine secretion. Since antibodies to Toll-like receptor (TLR)2 and TLR4 effectively inhibited the G8- and M7-induced production of TNF-alpha, these alginate oligomers may stimulate innate immunity through the pattern recognition receptors on macrophages similar to microbial products.     

Nonglandular silicified trichomes are essential for rice defense against chewing herbivores

Interspecific New Rice for Africa (NERICA) varieties have been recently developed and used in Sub-Saharan Africa but herbivore resistance properties of these plants remain poorly understood. Here we report that, compared to a local Japanese cultivar Nipponbare, NERICA 1, 4 and 10 are significantly more damaged by insect herbivores in the paddy fields. In contrast to high levels of leaf damage from rice skippers and grasshoppers, constitutive and induced volatile organic compounds for indirect plant defense were higher or similar in NERICAs and Nipponbare. Accumulation of direct defense secondary metabolites, momilactones A and B, and p-coumaroylputrescine (CoP) was reduced in NERICAs, while feruloylputrescine accumulated at similar levels in all varieties. Finally, we found that Nipponbare leaves were covered with sharp nonglandular trichomes impregnated with silicon but comparable defense structures were virtually absent in herbivory-prone NERICA plants. As damage to the larval gut membranes by Nipponbare silicified trichomes that pass intact through the insect digestive system, occurs, and larval performance is enhanced by trichome removal from otherwise chemically defended Nipponbare plants, we propose that silicified trichomes work as an important defense mechanism of rice against chewing insect herbivores.     

Integrating Transgenic Vector Manipulation with Clinical Interventions to Manage Vector-Borne Diseases

Many vector-borne diseases lack effective vaccines and medications, and the limitations of traditional vector control have inspired novel approaches based on using genetic engineering to manipulate vector populations and thereby reduce transmission. Yet both the short- and long-term epidemiological effects of these transgenic strategies are highly uncertain. If neither vaccines, medications, nor transgenic strategies can by themselves suffice for managing vector-borne diseases, integrating these approaches becomes key. Here we develop a framework to evaluate how clinical interventions (i.e., vaccination and medication) can be integrated with transgenic vector manipulation strategies to prevent disease invasion and reduce disease incidence. We show that the ability of clinical interventions to accelerate disease suppression can depend on the nature of the transgenic manipulation deployed (e.g., whether vector population reduction or replacement is attempted). We find that making a specific, individual strategy highly effective may not be necessary for attaining public-health objectives, provided suitable combinations can be adopted. However, we show how combining only partially effective antimicrobial drugs or vaccination with transgenic vector manipulations that merely temporarily lower vector competence can amplify disease resurgence following transient suppression. Thus, transgenic vector manipulation that cannot be sustained can have adverse consequences—consequences which ineffective clinical interventions can at best only mitigate, and at worst temporarily exacerbate. This result, which arises from differences between the time scale on which the interventions affect disease dynamics and the time scale of host population dynamics, highlights the importance of accounting for the potential delay in the effects of deploying public health strategies on long-term disease incidence. We find that for systems at the disease-endemic equilibrium, even modest perturbations induced by weak interventions can exhibit strong, albeit transient, epidemiological effects. This, together with our finding that under some conditions combining strategies could have transient adverse epidemiological effects suggests that a relatively long time horizon may be necessary to discern the efficacy of alternative intervention strategies.     

Assessing the Feasibility of Controlling Aedes aegypti with Transgenic Methods: A Model-Based Evaluation

Suppression of dengue and malaria through releases of genetically engineered mosquitoes might soon become feasible. Aedes aegypti mosquitoes carrying a conditionally lethal transgene have recently been used to suppress local vector populations in small-scale field releases. Prior to releases of transgenic insects on a wider scale, however, most regulatory authorities will require additional evidence that suppression will be effective in natural heterogeneous habitats. We use a spatially explicit stochastic model of an Ae. aegypti population in Iquitos, Peru, along with an uncertainty analysis of its predictions, to quantitatively assess the outcome of varied operational approaches for releases of transgenic strains with conditional death of females. We show that population elimination might be an unrealistic objective in heterogeneous populations. We demonstrate that substantial suppression can nonetheless be achieved if releases are deployed in a uniform spatial pattern using strains combining multiple lethal elements, illustrating the importance of detailed spatial models for guiding genetic mosquito control strategies.