Estrogen-induced alterations in amh and dmrt1 expression signal for disruption in male sexual development in the zebrafish

Dmrt1 and amh are genes involved in vertebrate sex differentiation. In this study, we cloned dmrt1 and amh cDNAs in zebrafish (Danio rerio) and investigated the effects of exposure to 17a-ethinylestradiol (EE2), during early life on their patterns of expression and impact on the subsequent gonadal phenotype. Expression of both amh and dmrt1 in embryos was detected as early as at 1 day post fertilization (dpf) and enhanced expression of amh from 25 dpf was associated with the period of early gonadal differentiation. Sex-dependent differences in enhanced green fluorescent protein transgene expression driven by the promoter of the germ cell-specific vas gene were exploited to show that at 28dpf and 56dpf both amh and dmrt1 mRNA were overexpressed in males compared with females. Exposure during early life to environmentally relevant concentrations of EE2 had a suppressive effect on the expression of both amh and dmrt1 mRNAs and this was associated with a cessation/retardation in male gonadal sex development. Our findings indicate that estrogen-induced suppression in expression of dmrt1 and amh during early life correlate with subsequent disruptive effects on the sexual phenotype in males.     

Protocell design through modular compartmentalization

De novo synthetic biological design has the potential to significantly impact upon applications such as energy generation and nanofabrication. Current designs for constructing organisms from component parts are typically limited in scope, as they utilize a cut-and-paste ideology to create simple stepwise engineered protein-signalling pathways. We propose the addition of a new design element that segregates components into lipid-bound ‘proto-organelles’, which are interfaced with response elements and housed within a synthetic protocell. This design is inspired by living cells, which utilize multiple types of signalling molecules to facilitate communication between isolated compartments. This paper presents our design and validation of the components required for a simple multi-compartment protocell machine, for coupling a light transducer to a gene expression system. This represents a general design concept for the compartmentalization of different types of artificial cellular machinery and the utilization of non-protein signal molecules for signal transduction.     

FORMI: Integrating Adaptive Fragmented Objects into Java RMI

Distributed object-oriented applications are commonly implemented atop middleware platforms such as CORBA, .NET Remoting, and Java remote method invocation (RMI). These platforms provide a simple mechanism to invoke methods of remote objects. Increasingly more applications are demanding nonfunctional properties such as fault tolerance, high availability, and adaptivity, which require extensions to distributed objects' basic interaction model. A fragmented-object model, such as the one Marc Shapiro proposed, can provide the required flexibility. It's far more generic and flexible than the traditional client-server approach. A fragmented object is a truly distributed object; it consists of multiple fragments located on multiple nodes. Such a model allows arbitrary partitioning of state and functionality on these fragments, and arbitrary internal interaction between fragments of a single object. We have investigated integrating a fragmented-object model into CORBA (AspectIX), which requires internal modifications to the CORBA object request broker. Our approach for transparently integrating fault-tolerant objects into .NET Remoting is also useful for seamlessly integrating fragmented objects. Our FORMI architecture integrates fragmented objects into Java RMI without requiring internal modifications to the RMI runtime     

Expansion of Access Tunnels and Active‐Site Cavities Influence Activity of Haloalkane Dehalogenases in Organic Cosolvents

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein–solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady‐state kinetics assays, molecular dynamics simulations, and time‐resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme–solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K_0.5 and increasing k_cat, or caused enzyme inactivation by promoting substrate inhibition and decreasing k_cat. The induced activation and inhibition of the enzymes correlated with expansion of the active‐site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active‐sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein–solvent pair and effective cosolvent concentration.     

Human blood eosinophils produce and secrete interleukin 4

Interleukin 4 (Il-4) is an immunoregulatory cytokine which induces T-cell proliferation and differentiation into a Th2 phenotype, and is of particular importance for the induction of IgE synthesis. In the present study, the capability of human peripheral blood eosinophils from allergic and non-allergic donors to produce Il-4 was examined. Using reverse transcribed polymerase chain reaction (RT-PCR), it was shown that highly purified eosinophils from allergic patients express mRNA for Il-4. Resting eosinophils also gave specific immunoreactivity with anti-Il-4 antibodies, consistent with translation of Il-4 mRNA. Light and electron microscopic immunocytochemistry revealed that Il-4 was prestored in the eosinophilic granules. These results were confirmed by Il-4 specific ELISA which showed that Il-4 production could be upregulated in the eosinophils and released from the eosinophils following stimulation with the calcium ionophore A23187. These data indicate that eosinophils may be an important source of Il-4 at sites of allergic inflammation. Thus, eosinophils may act as immunomodulatory cells enhancing the allergic response through formation of Th2-cells and inducing the isotype switching to IgE in human B-cells.     

Estimating the Effective Soil Temperature at L-Band as a Function of Soil Properties

To retrieve soil moisture from L-band microwave radiometry, it is necessary to account for the effects of temperature within both vegetation and soil media. To compute the effective soil temperature TG, several simple formulations accounting for soil temperatures at the surface and at depth and surface soil moisture have been developed. However, the effects of the soil physical properties in terms of texture, density, or structure, which all may be important variables in the modeling of TG, have never been investigated. In this paper, several simple formulations of TG at L-band, accounting for or ignoring the effects of soil texture and density, were developed and compared based on a very large simulated data set. The best configurations and parameterizations of these simple formulations were computed and could be directly used for operational applications in future soil moisture retrieval studies. For instance, we showed that the use of the surface temperature in the estimation of TG can be significantly improved by using additional information on the soil temperature at depth (the average error in the estimation of TG decreased from ~ 4 to ~ 1.8 K). On the contrary, almost no improvement was obtained if air temperature was used instead of surface temperature. Also, it is shown that the use of additional information on the soil properties, mainly the soil clay content and density, led to improved results by about 0.2 K in the estimation of TG. The improvement was found to be larger for sandy and dry soils: simplified formulations accounting for soil properties are able to represent the fact that TG is closer to the soil temperature at depth for these soil conditions.     

Design and Synthesis of α-Diketones. The Cyclobutane-1,2-dione Chromophore: Synthesis,Dienophilic Reactivity and Electronic Properties of Cyclobutenedione and Polycyclic Cyclobutanediones [1]

A new synthesis of cyclobutenedione (1) is described. It is found that the tricyclic cyclobutanedione 3 splits into 1 and cyclopentadiene if subjected to flash pyrolysis conditions. The dienophilic reactivity of 1 allows the construction of various polycyclic cyclobutanediones, such as 3–5 and 12–14. Other new polycyclic diones, like 9–11, 15 and 16 are prepared by means of the acyloin condensation. The low stereoselectivity of 1 with cyclopentadiene is compared with the selectivity of other cyclobutenes, and the results are placed in the context of secondary interactions and steric hindrance. The unusual colour properties of these diones, from colourless 1 to blue 6 are investigated by means of photoelectron spectroscopic measurements and semi-empirical calculations (MNDO, HAM/3, CNDO/S-CI).