Estradiol restores diabetes-induced reductions in sex steroid receptor expression and distribution in the vagina of db/db mouse model

Sex steroid hormones and receptors play an important role in maintaining vaginal physiology. Disruptions in steroid receptor signaling adversely impact vaginal function. Limited studies are available investigating the effects of diabetic complications on steroid receptor expression and distribution in the vagina. The goals of this study were to investigate type 2 diabetes-induced changes in expression, localization and distribution of estrogen (ER), progesterone (PR) and androgen receptors (AR) in the vagina and to determine if estradiol treatment ameliorates these changes. Eight-week-old female diabetic (db/db) mice (strain BKS.Cg-m+/+ Lepr^db/J) were divided into two subgroups: untreated diabetic and diabetic animals treated with pellets containing estradiol. Control normoglycemic littermates were subcutaneously implanted with pellets devoid of estradiol. At 16 weeks of age, animals were sacrificed, vaginal tissues excised and analyzed by Western blot and immunohistochemical methods. Diabetes produced marked reductions in protein expression of ER, PR, and AR. Diabetes also resulted in marked differences in the distribution, staining intensity and proportion of immunoreactive cells containing these steroid receptors in the epithelium, lamina propria and muscularis. Treatment of diabetic animals with estradiol restored receptor protein expression and distribution similar to those levels observed in control animals. This study demonstrates that type 2 diabetes markedly reduces steroid receptor protein expression and distribution in the vagina. Estradiol treatment of diabetic animals ameliorates these diabetes-induced changes.     

Role of Isoprenylcysteine Carboxylmethyltransferase-catalyzed Methylation in Rho Function and Migration

A number of proteins that play key roles in biological regulatory events undergo a process of post-translational modifications termed prenylation. The prenylation pathway consists of three enzymatic steps; the final processed protein is isoprenoid-modified and methylated on the C-terminal cysteine. This protein modification pathway plays a significant role in cancer biology because many oncogenic proteins undergo prenylation. Methylation of the C terminus by isoprenylcysteine carboxylmethyltransferase (Icmt) is the final step in the prenylation pathway. Cysmethynil, a specific Icmt inhibitor discovered in our laboratory, is able to inhibit Ras-mediated signaling, cell growth, and oncogenesis. We sought to examine the role of Icmt-mediated methylation on the behaviors of cancer cells associated with metastatic potential. Our results indicate that inhibition of methylation reduces migration of the highly metastatic MDA-MB-231 breast cancer cell line. In addition, cell adhesion and cell spreading are also significantly impacted by cysmethynil. To examine the mechanism of Icmt-dependent migration we focused on RhoA and Rac1, prenylated proteins that are important mediators of cell migration through their control of the actin cytoskeleton. Inhibition of Icmt significantly decreases the activation of both RhoA and Rac1; an increase in Rho GDP-dissociation inhibitor (RhoGDI) binding in the absence of methylation appears to contribute to this effect. Furthermore, in the absence of Icmt activity the addition of exogenous RhoA or Rac1 is able to partially rescue directed and random migration, respectively. These findings establish a role for Icmt-mediated methylation in cell migration and advance our understanding of the biological consequences of Rho methylation.Post-translational modifications of proteins play vital roles in many aspects of cell biology. Hence, identifying and understanding the biological impact of these processes is crucial to furthering our basic understanding of how cells function. Numerous proteins that control important biological regulatory events undergo a complex series of post-translational modifications that are directed by the presence of a so-called CaaX motif at their C terminus. This post-translational pathway, termed protein prenylation, is initiated by the attachment of an isoprenoid lipid to an invariant cysteine residue, the C of the CaaX motif (1, 2). Either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid is covalently attached to this cysteine by protein farnesyltransferase (FTase)² or protein geranylgeranyltransferase-I (GGTase-I), respectively (3). The prenylation step is followed by cleavage of the three C-terminal amino acids (the -AAX) by an endoplasmic reticulum (ER)-bound protease termed Rce1. Finally, the prenylated cysteine, which is now located at the C terminus, is methylated by isoprenylcysteine carboxylmethyltransferase (Icmt), another integral ER membrane protein (4, 5). The final result of these modifications is a protein that contains a prenylated and methylated cysteine at its C terminus. Numerous studies have demonstrated that this post-translational processing not only facilitates protein association with cellular membranes, but also can play important roles in protein-protein interactions and protein stability (1, 6, 7). Thus, it is clear that CaaX processing is necessary for the biological activities of these proteins.The prenylation pathway has been targeted for potential anticancer therapy because most members of the Ras superfamily, which contains many known oncogenes, undergo CAAX processing. The Ras superfamily consists of five large subfamilies; the two most well-characterized are the Ras and Rho subfamilies (8). Both Ras and Rho proteins are processed by the CaaX pathway; Ras family members are farnesylated, while most Rho family members are geranylgeranylated. These monomeric GTPases cycle between a GDP-bound inactive state and a GTP-bound active state. In their active states, Ras and Rho subfamily members control numerous cell signaling pathways that are involved in cell proliferation, differentiation, migration, polarity, and morphology (9).Abnormally high activity of Ras and Rho signaling pathways contribute to initiation and progression of many types of cancer (10, 11). For example, many breast cancers that are highly metastatic express abnormally high levels of Rho proteins (12). Rho proteins control migration and invasion of cells by tightly coordinating changes in the actin and microtubule cytoskeletons. Acting through their effectors, Rho proteins rearrange the actin cytoskeleton to respond to chemo-attractant gradients, polarize cells, and control migration and invasion. While cell migration is necessary for development, leukocyte function, and other normal cell biologies, dysregulation of migration and invasion results in cancer metastasis (13). Metastasis is an important and deadly progression of cancer and understanding the biology of migrating cancer cells is crucial for therapeutic targeting of this aspect of cancer.Pharmacologic targeting of the enzymes involved in the CaaX-processing pathway has emerged as a promising anticancer strategy. In particular, there has been much effort in designing inhibitors against the protein prenyltransferases, most notably FTase (14, 15). There is also recent evidence that inhibition of geranygeranylation of Rho proteins also impacts oncogenesis and metastasis (16–18). However, the overall success of the FTase inhibitors (FTIs) in the clinical setting has been somewhat disappointing. One possible reason is a phenomenon termed “alternate prenylation” in which some FTase substrates, most notably K- and N- Ras, are modified by GGTase and escape inhibition by FTIs (19–21). Because the Rce1 protease and Icmt methyltransferase act on all CaaX proteins, problems such as alternate prenylation would not arise if these enzymes were targeted. Hence, while protein prenyltransferase inhibitors still show some promise as anticancer agents, the emerging view that global attenuation of CaaX protein function may be advantageous in blocking cancer cell growth has increased interest in studying the two downstream enzymes involved in CaaX processing.While the biological consequences of prenylation are fairly well understood, the precise roles of C-terminal methylation in CaaX protein function are still elusive. Depending on the CaaX protein, methylation has been ascribed to roles in localization, protein-protein interactions and protein stability (11). The development of an Icmt knock-out mouse model has furthered our understanding of Icmt function (22, 23). Localization studies conducted in cells with genetically deleted Icmt have shown that methylation is important for proper membrane association of Ras proteins. However, the localization of Rho proteins in the absence of Icmt activity appears to be more complicated and may vary depending on family member and activation status (24–26). Importantly, inhibition of CaaX protein methylation via either genetic or pharmacologic targeting has shown a clear impact on oncogenic transformation and tumor growth (23, 27, 28).Defining the role of Icmt-mediated methylation in complex cellular behaviors such as migration and invasion is crucial for furthering our understanding of the impact of CaaX protein methylation on the biology of normal and cancer cells. In the current study, we have assessed the impact of Icmt inhibition on cell biological processes associated with the function of Rho proteins, specifically cell adhesion, morphology, and migration. We found that inhibition of Icmt results in a disruption of the actin cytoskeleton and impairs ligand-mediated activation of RhoA and Rac1, a potential consequence of increased RhoGDI binding to both RhoA and Rac1 when their methylation is impaired. Further, we show that the impact of Icmt inhibition on cell migration is due at least in part to impairment of RhoA and Rac1function. These findings establish a role for Icmt-mediated methylation in cell migration and further elucidate the role that methylation plays in the function of Rho GTPases.     

Hypertrophy and/or Hyperplasia: Dynamics of Adipose Tissue Growth

Adipose tissue grows by two mechanisms: hyperplasia (cell number increase) and hypertrophy (cell size increase). Genetics and diet affect the relative contributions of these two mechanisms to the growth of adipose tissue in obesity. In this study, the size distributions of epididymal adipose cells from two mouse strains, obesity-resistant FVB/N and obesity-prone C57BL/6, were measured after 2, 4, and 12 weeks under regular and high-fat feeding conditions. The total cell number in the epididymal fat pad was estimated from the fat pad mass and the normalized cell-size distribution. The cell number and volume-weighted mean cell size increase as a function of fat pad mass. To address adipose tissue growth precisely, we developed a mathematical model describing the evolution of the adipose cell-size distributions as a function of the increasing fat pad mass, instead of the increasing chronological time. Our model describes the recruitment of new adipose cells and their subsequent development in different strains, and with different diet regimens, with common mechanisms, but with diet- and genetics-dependent model parameters. Compared to the FVB/N strain, the C57BL/6 strain has greater recruitment of small adipose cells. Hyperplasia is enhanced by high-fat diet in a strain-dependent way, suggesting a synergistic interaction between genetics and diet. Moreover, high-fat feeding increases the rate of adipose cell size growth, independent of strain, reflecting the increase in calories requiring storage. Additionally, high-fat diet leads to a dramatic spreading of the size distribution of adipose cells in both strains; this implies an increase in size fluctuations of adipose cells through lipid turnover.     

Synthesis of benz[d]indeno[1,2-b]pyran-5,11-diones: versatile intermediates for the design and synthesis of topoisomerase I inhibitors

A method has been developed that relies on a two-step, one-pot condensation between phthalide and 2-carboxybenzaldehydes to provide benz[d]indeno[1,2-b]pyran-5,11-diones in a multi-gram fashion. Treatment of these compounds with a primary amine allows rapid access to various N-substituted indenoisoquinolines, whose in vitro anticancer activity and topoisomerase I inhibition have been evaluated.     

Genomic approaches to plant stress tolerance

Past efforts to improve plant tolerance to drought, high salinity and low-temperature through breeding and genetic engineering have had limited success owing to the genetic complexity of stress responses. Progress is now anticipated through comparative genomics studies of an evolutionarily diverse set of model organisms, and through the use of techniques such as high-throughput analysis of expressed sequence tags, large-scale parallel analysis of gene expression, targeted or random mutagenesis, and gain-of-function or mutant complementation. The discovery of novel genes, determination of their expression patterns in response to abiotic stress, and an improved understanding of their roles in stress adaptation (obtained by the use of functional genomics) will provide the basis of effective engineering strategies leading to greater stress tolerance.     

Assessing multi-taxa sensitivity to the human footprint,habitat fragmentation and loss by exploring alternative scenarios of dispersal ability and population size: a simulation approach

Quantifying the effects of landscape change on population connectivity is compounded by uncertainties about population size and distribution and a limited understanding of dispersal ability for most species. In addition, the effects of anthropogenic landscape change and sensitivity to regional climatic conditions interact to strongly affect habitat fragmentation and loss. To further develop conservation theory and to understand the interplay between all of these factors, we simulated habitat fragmentation and loss across the Western United States for several hypothetical species associated with four biome types, and a range of habitat requirements and dispersal abilities. We found dispersal ability and population size of the focal species to be equally sensitive to habitat extent, while dispersal ability is more sensitive to habitat fragmentation. There were also strong critical threshold effects where habitat connectivity decreased disproportionately to decreases in life-history traits making these species near these thresholds more sensitive to changes in habitat loss and fragmentation. Overall, grassland and forest associated species are also most at risk from habitat loss and fragmentation driven by human related land-use. These two largest biome types were most sensitive at large contiguous patch sizes which is often considered most important for metapopulation viability and biodiversity conservation. Hypothetical simulation studies such as this can be of great value to scientists in further conceptualizing and developing conservation theory, and evaluating spatially-explicit scenarios of habitat connectivity. Our results are available for download in a web-based interactive mapping prototype useful for accessing the results of this study.     

In vivo desensitization of a high affinity PGF2 alpha receptor in the ovine corpus luteum.

The corpus luteum (CL) of the sheep exhibits a differential sensitivity to PGF2 alpha in vivo in terms of an increase in oxytocin (OT) secretion and a decrease in progesterone secretion, pointing to the presence in vivo of both high and low affinity receptors for PGF2 alpha. The presence of the high affinity PGF2 alpha receptor was assessed by monitoring the secretion rate of OT from the ovine CL in response to subluteolytic infusions of PGF2 alpha. Rapid desensitization to PGF2 alpha occurred after only one hour of infusion, while a minimum rest period of six hours was required to restore sensitivity. The possibility that these findings could be explained by the depletion and resynthesis of OT was excluded by demonstrating an increase in OT secretion rate with supra-physiological levels of PGF2 alpha two hours after desensitization. Collectively, these results indicate the presence of a high affinity receptor for PGF2 alpha in the ovine CL which exhibits desensitization and recovery in vivo. The temporal nature of the desensitization and recovery of the high affinity PGF2 alpha receptor controlling luteal OT secretion may contribute to the pulsatile nature of PGF2 alpha release from the ovine uterus.