Lumican is a major proteoglycan component of the bone matrix.

MC3T3-E1 mouse calvaria cells are a clonal population of committed osteoprogenitors that in the presence of appropriate supplements form a mineralized bone matrix. The development of the MC3T3-E1 cells can be divided into three major stages, namely, proliferation, differentiation, and mineralization. Recently, using the cDNA microarray technology we found lumican to be abundantly expressed during the mineralization and differentiation stages of the MC3T3-E1 development and not during the proliferation stage. Lumican has been shown to play essential roles in regulating collagen fibril formation in different extracellular matrices but its expression in the developing bone matrix remains elusive. By examining the expression profile of this gene during the different stages of MC3T3-E1 development, utilizing the 'real-time' PCR technology, we observed that the expression of lumican increases as the osteoblast culture differentiates and matures, suggesting that lumican may be involved in regulating collagen fibrillogenesis in bone matrices. Using immunostaining, we observed that during the early embryonic development of mouse (E11 to E13), lumican is mainly expressed in the cartilaginous matrices. However, in the older embryos (E14 to E16), the expression of lumican is more prominent in the developing bone matrices. Our data suggest that lumican is a significant proteoglycan component of bone matrix, which is secreted by differentiating and mature osteoblasts only and therefore it can be used as a marker to distinguish proliferating pre-osteoblasts from the differentiating osteoblasts.     

Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis.

Elevated expression of plasminogen activator inhibitor-1 (PAI-1) in tumors is associated with a poor prognosis in many cancers. Reduced tumor growth and angiogenesis have also been reported in mice deficient in PAI-1. These results suggest that PAI-1 may be required for efficient angiogenesis and tumor growth. In the present study, we demonstrate that PAI-1 can both enhance and inhibit the growth of M21 human melanoma tumors in nude mice and that this appears to be due to PAI-1 regulation of angiogenesis. Quantitative analysis of angiogenesis in a Matrigel implant assay indicated that in PAI-1 null mice angiogenesis was reduced approximately 60% compared with wild-type mice, while in mice overexpressing PAI-1, angiogenesis was increased nearly 3-fold. Furthermore, addition of PAI-1 to implants in wild-type mice enhanced angiogenesis up to 3-fold at low concentrations but inhibited angiogenesis nearly completely at high concentrations. Together, these data demonstrate that PAI-1 is a potent regulator of angiogenesis and hence of tumor growth and suggest that understanding the mechanism of this activity may lead to the development of important new therapeutic agents for controlling pathologic angiogenesis.     

Measurement of steroid sex hormones in serum: a comparison of radioimmunoassay and mass spectrometry

Concern has been raised about the adequacy of radioimmunoassays to measure steroid sex hormones in population studies. We compared steroid sex hormone measurements in serum by radioimmunoassay with mass spectrometry. Four male and four female serum pools with known relative concentrations of steroid sex hormones were measured multiple times by both methods. Because measurements are expected to increase linearly with concentration for each sex, we examined whether the linear regressions of hormone measurements on concentration were the same for radioimmunoassay and mass spectrometry. Estradiol, estrone, androstenedione, testosterone, and dehydroepiandrosterone sulfate were measured in female pools; testosterone, dihydrotestosterone, androstenedione, and dehydroepiandrosterone sulfate were measured in male pools. Regression slopes for radioimmunoassay and mass spectrometry measurements were comparable for all hormones except androstenedione, which had a steeper slope when measured by mass spectrometry (P < or = 0.02). Intercepts for radioimmunoassay and mass spectrometry were similar and close to zero for estradiol, androstenedione, dehydroepiandrosterone sulfate, and in male samples, testosterone. For testosterone in female samples, estrone, and dihydrotestosterone, radioimmunoassay and mass spectrometry intercepts differed significantly. Standard deviations of individual measurements by radioimmunoassay and mass spectrometry differed by hormone and serum concentration; neither method consistently measured hormone concentrations with less variability. Our findings suggest that although absolute concentrations may differ for some hormones, radioimmunoassay and mass spectrometry can yield similar estimates of between subject differences in serum concentrations of most steroid sex hormones commonly measured in population studies. Relative power of studies using radioimmunoassay and mass spectrometry will depend on the hormones measured and their serum concentrations.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.     

Migration stopover ecology of Cinnamon Teal in western North America

Identifying migration routes and fall stopover sites of Cinnamon Teal (Spatula cyanoptera septentrionalium) can provide a spatial guide to management and conservation efforts, and address vulnerabilities in wetland networks that support migratory waterbirds. Using high spatiotemporal resolution GPS‐GSM transmitters, we analyzed 61 fall migration tracks across western North America during our three‐year study (2017–2019). We marked Cinnamon Teal primarily during spring/summer in important breeding and molting regions across seven states (California, Oregon, Washington, Idaho, Utah, Colorado, and Nevada). We assessed fall migration routes and timing, detected 186 fall stopover sites, and identified specific North American ecoregions where sites were located. We classified underlying land cover for each stopover site and measured habitat selection for 12 land cover types within each ecoregion. Cinnamon Teal selected a variety of flooded habitats including natural, riparian, tidal, and managed wetlands; wet agriculture (including irrigation ditches, flooded fields, and stock ponds); wastewater sites; and golf and urban ponds. Wet agriculture was the most used habitat type (29.8% of stopover locations), and over 72% of stopover locations were on private land. Relatively scarce habitats such as wastewater ponds, tidal marsh, and golf and urban ponds were highly selected in specific ecoregions. In contrast, dry non‐habitat across all ecoregions, and dry agriculture in the Cold Deserts and Mediterranean California ecoregions, was consistently avoided. Resources used by Cinnamon Teal often reflected wetland availability across the west and emphasize their adaptability to dynamic resource conditions in arid landscapes. Our results provide much needed information on spatial and temporal resource use by Cinnamon Teal during migration and indicate important wetland habitats for migrating waterfowl in the western United States.     

Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography

Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.     

Inhibition of mono-ADP-ribosyltransferase activity during the execution phase of apoptosis prevents apoptotic body formation

The objective of this study was to understand factors responsible for apoptotic body formation and release during apoptosis. We have found that inhibition of mono-ADP ribosylation after ultraviolet (UV) light induction of apoptosis in HL-60 cells does not block caspase-3 activation, gelsolin cleavage, or endonucleolytic DNA fragmentation. However, the cytoskeletal features of apoptosis leading to apoptotic body formation and release were inhibited by meta-iodobenzylguanidine (MIBG) and novobiocin, potent inhibitors of arginine-specific mono-ADP-ribosyltransferases (mono-ADPRTs). Suppression of mono-ADP ribosylation as late as 120 min following UV irradiation blocked the depolymerization of actin and release of apoptotic bodies. This suggested that the cytoskeletal changes of apoptosis may be decoupled from the caspase cascade and that there may be a biochemical event either distal to or independent of caspase-3 that regulates apoptotic body formation. To test the hypothesis that ADP ribosylation of actin may occur with the induction of apoptosis, an in vivo assay of mono-ADPRT activity using an antibody against ADP-ribosylarginine was used. An approximately 64% increase in the ADP ribosylation of actin was observed at 2 h following exposure to UV light. When MIBG or novobiocin was present, the ADP ribosylation of actin was only 14-18% above the levels observed in control nonirradiated cells. The current study is the first to demonstrate a relationship between ADP-ribosylation of actin and the formation of apoptotic bodies.     

Indian hedgehog signaling from endothelial cells is required for sclera and retinal pigment epithelium development in the mouse eye

The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.