Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.     

Elucidating mechanisms underlying robustness of morphogen gradients

Morphogen gradients play a pivotal role in most phases of developmental patterning. To ensure proper patterning, reproducible gradients are established under diverse environmental conditions and genetic backgrounds. We refer to the capacity to buffer fluctuations in gene dosage or environmental conditions as 'robustness'. By theoretical analysis of mechanisms that facilitate robustness, it is possible to unravel the machinery responsible for generating the spatial distribution of morphogens.     

In silico screening of a saturated mutation library of tomato

A comprehensive mutant population is a basic resource for exploring gene function. We developed an isogenic tomato 'mutation library' in the genetic background of the inbred variety M82. A total of 13 000 M(2) families, derived from EMS (ethyl methane sulfonate) and fast-neutron mutagenesis, were visually phenotyped in the field and categorized into a morphological catalog that includes 15 primary and 48 secondary categories. Currently, 3417 mutations have been cataloged; among them are most of the previously described phenotypes from the monogenic mutant collection of The Tomato Genetics Resource Center, and over a thousand new mutants, with multiple alleles per locus. The phenotypic database indicates that most mutations fall into more than a single category (pleiotropic), with some organs such as leaves more prone to alterations than others. All data and images can be searched and accessed in the Solanaceae Genome Network (SGN) on a site called 'The Genes That Make Tomatoes' (http://zamir.sgn.cornell.edu/mutants/).     

Prostaglandins regulate melanoma-induced cytokine production in macrophages

Tumor-secreted products can affect macrophage cytokine expression and in that way alter the immune response. Prostaglandins (PGs) are found in the tumor microenvironment and have been associated with local and regional immunosuppression. We investigated whether tumor-secreted factors could induce PG synthesis in macrophages and whether these PGs could alter macrophage production of immunoregulatory cytokines. In both murine and human models, melanoma conditioned medium (MCM) induced macrophage production of PGE(2), IL-6, and TNF-alpha. PGE(2) production increased over 24 h and was accompanied by an increase in cyclooxygenase-2 (COX-2) expression, while COX-1 expression remained unchanged. In the presence of 10 microM NS398, a selective COX-2 inhibitor, MCM-stimulated PGE(2) synthesis was almost completely suppressed, while production of IL-6 and TNF-alpha proteins and mRNA also was partially abrogated. In the murine model, 200 microM NS398 resulted in more significant inhibition of cytokine protein and mRNA production. Although MCM induced NFkappaB and NF-IL-6 activation, neither dose of NS398 altered this effect. We conclude that melanoma-secreted products stimulate COX-2 expression and PGE(2) synthesis in macrophages and that inhibition of COX-2-derived PG synthesis results in partial abrogation of macrophage cytokine production.     

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.     

Are Biological Effects of Desert Shrubs More Important than Physical Effects on Soil Microorganisms?

Vegetation cover plays a major role in providing organic matter and in acting as a physical barrier, with both together contributing to the formation of “fertile islands,” which play an active role in prolonging biological activity in desert ecosystems. By undertaking this study, a long-term research, we designed an experiment to separate the two components—the physical and biotic parts of the perennial plants—and to identify the factor that contributes the most to the ecosystem. The study site was located in the northern Negev Desert, Israel, where 50 Hammada scoparia shrubs and 50 artificial plants were randomly marked. Soil samples were collected monthly over 3 years of research at three locations: under the canopy of H. scoparia shrubs, in the vicinity of the artificial plants, and between the shrubs (control). The contribution to microbial activity was measured by evaluation of the microbial community functions in soil. The functional aspects of the microbial community that were measured were CO₂ evolution, microbial biomass, microbial functional diversity, and the physiological profile of the community. The results of this study are presented in two ways: (1) according to the three locations/treatments; and (2) according to the phenological situation of the vegetation (annual and perennial plants) in the research field: the growing phase, the drying process, and the absence of annual plants. The only parameters that were found to affect microbial activity were the contribution of the organic matter of perennial shrubs and the growth of vegetation (annual and perennial) during the growing seasons. The physical component was found to have no effect on soil microbial functional diversity, which elucidates the important contribution of the desert shrub in enhancing biological multiplicity and activity.