Recent insights into the effect of natural and environmental estrogens on mammary development and carcinogenesis

The present work reviews recent findings related to the action of steroidal (physiological) estrogens on normal mammary gland development and carcinogenesis, as well as effects of related environmental mediators (phyto- and xeno-estrogens), the role of which remains controversial. Orchestration by estrogen receptors (i.e. ERα and ERβ) and coregulators of growth, apoptosis and differentiation of epithelial cells, directed our analysis. The bidirectional coordination between epithelium and stroma in parallel with maintenance of stemness are also investigated. The relevance of nuclear and extranuclear localization of ERs and other eventual estrogen binding sites, mediating differential actions in regard to these various topics, is critically addressed to delineate the importance of direct and indirect activation procedures and delicate feedback loops (ligand-induced or/and cross-talk activation, respectively). The inclusion of the outlined regulatory concepts in drug design programs for the prevention and treatment of breast cancer may have potent effects.     

Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells

Secretagogue-induced changes in intracellular Ca(2+) play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca(2+) are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca(2+)-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca(2+)-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.     

Dual TCR expression biases lung inflammation in DO11.10 transgenic mice and promotes neutrophilia via microbiota-induced Th17 differentiation

A commonly used mouse model of asthma is based on i.p. sensitization to OVA together with aluminum hydroxide (alum). In wild-type BALB/c mice, subsequent aerosol challenge using this protein generates an eosinophilic inflammation associated with Th2 cytokine expression. By constrast, in DO11.10 mice, which are transgenic for an OVA-specific TCR, the same treatment fails to induce eosinophilia, but instead promotes lung neutrophilia. In this study, we show that this neutrophilic infiltration results from increased IL-17A and IL-17F production, whereas the eosinophilic response could be restored upon blockade of IFN-γ, independently of the Th17 response. In addition, we identified a CD4(+) cell population specifically present in DO11.10 mice that mediates the same inflammatory response upon transfer into RAG2(-/-) mice. This population contained a significant proportion of cells expressing an additional endogenous TCR α-chain and was not present in RAG2(-/-) DO11.10 mice, suggesting dual antigenic specificities. This particular cell population expressed markers of memory cells, secreted high levels of IL-17A, and other cytokines after short-term restimulation in vitro, and triggered a neutrophilic response in vivo upon OVA aerosol challenge. The relative numbers of these dual TCR lymphocytes increased with the age of the animals, and IL-17 production was abolished if mice were treated with large-spectrum antibiotics, suggesting that their differentiation depends on foreign Ags provided by gut microflora. Taken together, our data indicate that dual TCR expression biases the OVA-specific response in DO11.10 mice by inhibiting eosinophilic responses via IFN-γ and promoting a neutrophilic inflammation via microbiota-induced Th17 differentiation.     

NLRP3/caspase-1-independent IL-1beta production mediates diesel exhaust particle-induced pulmonary inflammation

Inhalation of diesel exhaust particles (DEP) induces an inflammatory reaction in the lung; however, the mechanisms are largely unclear. IL-1β/IL-1RI signaling is crucial in several lung inflammatory responses. Typically, caspase-1 is activated within the NLRP3 inflammasome that recognizes several damage-associated molecular patterns, which results in cleavage of pro-IL-1β into mature IL-1β. In this study, we hypothesized that the NLRP3/caspase-1/IL-1β pathway is critical in DEP-induced lung inflammation. Upon DEP exposure, IL-1RI knockout mice had reduced pulmonary inflammation compared with wild-type mice. Similarly, treatment with rIL-1R antagonist (anakinra) and IL-1β neutralization impaired the DEP-induced lung inflammatory response. Upon DEP exposure, NLRP3 and caspase-1 knockout mice, however, showed similar IL-1β levels and comparable pulmonary inflammation compared with wild-type mice. In conclusion, these data show that the DEP-induced pulmonary inflammation acts through the IL-1β/IL-1RI axis. In addition, DEP initiates inflammation independent of the classical NLRP3/caspase-1 pathway, suggesting that other proteases might be involved.     

Fascin1 promotes cell migration of mature dendritic cells

Dendritic cells (DCs) play central roles in innate and adaptive immunity. Upon maturation, DCs assemble numerous veil-like membrane protrusions, disassemble podosomes, and travel from the peripheral tissues to lymph nodes to present Ags to T cells. These alterations in morphology and motility are closely linked to the primary function of DCs, Ag presentation. However, it is unclear how and what cytoskeletal proteins control maturation-associated alterations, in particular, the change in cell migration. Fascin1, an actin-bundling protein, is specifically and greatly induced upon maturation, suggesting a unique role for fascin1 in mature DCs. To determine the physiological roles of fascin1, we characterized bone marrow-derived, mature DCs from fascin1 knockout mice. We found that fascin1 is critical for cell migration: fascin1-null DCs exhibit severely decreased membrane protrusive activity. Importantly, fascin1-null DCs have lower chemotactic activity toward CCL19 (a chemokine for mature DCs) in vitro, and in vivo, Langerhans cells show reduced emigration into draining lymph nodes. Morphologically, fascin1-null mature DCs are flatter and fail to disassemble podosomes, a specialized structure for cell-matrix adhesion. Expression of exogenous fascin1 in fascin1-null DCs rescues the defects in membrane protrusive activity, as well as in podosome disassembly. These results indicate that fascin1 positively regulates migration of mature DCs into lymph nodes, most likely by increasing dynamics of membrane protrusions, as well as by disassembling podosomes.     

Evaluation of the efficacy of the methyl bromide fumigation schedule against Mexican fruit fly (Diptera: Tephritidae) in citrus fruit

Methyl bromide fumigation is widely used as a phytosanitary treatment. Mexican fruit fly, Anastrepha ludens (Loew) (Diptera: Tephritidae), is a quarantine pest of several fruit, including citrus (Citrus spp.), exported from Texas, Mexico, and Central America. Recently, live larvae have been found with supposedly correctly fumigated citrus fruit. This research investigates the efficacy of the previously approved U.S. Department of Agriculture-Animal and Plant Health Inspection Service treatment schedule: 40 g/m3 methyl bromide at 21-29.4 degrees C for 2 h. Tolerance ofA. ludens to methyl bromide in descending order when fumigated in grapefruit (Citrus X paradisi Macfad.) is third instar > second instar > first instar > egg. Two infestation techniques were compared: insertion into fruit of third instars reared in diet and oviposition by adult A. ludens into fruit and development to the third instar. Inserted larvae were statistically more likely to survive fumigation than oviposited larvae. When fruit were held at ambient temperature, 0.23 +/- 0.12% of larvae were still observed to be moving 4 d postfumigation. Temperatures between 21.9 and 27.2 degrees C were positively related to efficacy measured as larvae moving 24 h after fumigation, pupariation, and adult emergence. Coating grapefruit with Pearl Lustr 2-3 h before fumigation did not significantly affect the proportion of third instars moving 24 h after fumigation, pupariating, or emerging as adults. In conclusion, fumigation with 40 g/m3 methyl bromide for 2 h at fruit temperatures >26.7 degrees C is not found to be inefficacious for A. ludens. Although a few larvae may be found moving >24 h postfumigation, they do not pupariate.     

Phytosanitary irradiation of Liriomyza trifolii (Diptera: Agromyzidae)

Agromyzid leafminers are economic and quarantine pests of a variety of vegetables, flowers, and ornamental foliage. Methyl bromide fumigation is often used as a phytosanitary treatment when quarantined agromyzids are found in shipped commodities; alternative treatments are sought. Ionizing radiation is a viable alternative that is increasing in use worldwide. A dose of 400 Gy is accepted by USDA-APHIS for all insects (except Lepidoptera pupae and adults) on all commodities. Efforts to lower this dose and make it acceptable to other countries involve determining radiotolerance of families of major quarantine pests. Agromyzidae is one such family for which no useful information on radiotolerance exists. This research sought to determine the dose required to control a major agromyzid pest, Liriomyza trifolii (Burgess) and was performed on L. trifolii collected in Weslaco, TX, reared on Phaseolus vulgaris L. and Capsicum annuum L. and irradiated in the late puparial stage. The measure of efficacy was prevention of F1 mine formation. Puparia collected from Gossypium hirsutum L. and reared on P. vulgaris were more radiotolerant than those collected and reared on C. annuum. A dose of 214 Gy may prevent F1 mine formation of L. trifolii. This research used a variation of probit analysis where the direct response of the treated individual is not measured, but the response of the F1 generation is. This type of analysis is useful in phytosanitary irradiation research where the measure of efficacy often involves a response of the F1 generation.     

PKN-1, a homologue of mammalian PKN, is involved in the regulation of muscle contraction and force transmission in C. elegans

To examine the in vivo functions of protein kinase N (PKN), one of the effectors of Rho small guanosine triphosphatases (GTPases), we used the nematode Caenorhabditis elegans as a genetic model system. We identified a C. elegans homologue (pkn-1) of mammalian PKN and confirmed direct binding to C. elegans Rho small GTPases. Using a green fluorescent protein reporter, we showed that pkn-1 is mainly expressed in various muscles and is localized at dense bodies and M lines. Overexpression of the PKN-1 kinase domain and loss-of-function mutations by genomic deletion of pkn-1 resulted in a loopy Unc phenotype, which has been reported in many mutants of neuronal genes. The results of mosaic analysis and body wall muscle-specific expression of the PKN-1 kinase domain suggests that this loopy phenotype is due to the expression of PKN-1 in body wall muscle. The genomic deletion of pkn-1 also showed a defect in force transmission. These results suggest that PKN-1 functions as a regulator of muscle contraction-relaxation and as a component of the force transmission mechanism.     

The novel anticytomegalovirus compound AIC246 (Letermovir) inhibits human cytomegalovirus replication through a specific antiviral mechanism that involves the viral terminase

Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.     

Design principles for robust biochemical reaction networks: what works, what cannot work, and what might almost work

We bring together recent results that connect the structure of a mass-action reaction network to its capacity for concentration robustness — that is, its capacity to keep the concentration of a critical bio-active species within narrow limits, even against large fluctuations in the overall supply of the network’s constituents.