Changes in the levels of pituitary and steroid hormones in ovine and human ovarian follicular fluid

Changes in the protein and steroid hormones of follicular fluid, aspirated from different follicles of sheep and human ovaries, have been measured and correlated with the size of the follicles. As the fluid contains a number of proteins, steroids have been measured directly and after ether extraction. The follicular fluid concentrations of progesterone and 17 beta-oestradiol measured directly in the fluid increased with the size of the follicles. The levels of free testosterone remained constant in all sizes of follicles, while those of bound hormone showed a 10- to 15-fold increase over the free testosterone concentrations in both the sheep and human follicular fluid. A decrease in the levels of bound testosterone in the fluid of large follicles (LFFL) coincided with the increase in bound 17 beta-oestradiol, suggesting the possible conversion of bound testosterone to oestrogen as the follicle attained maturity. The ratio of follicle-stimulating hormone (FSH) to luteinizing hormone (LH) varied in the fluid obtained from different size follicles, being 1:7 in small (SFFL), 1.3.5 in medium (MFFL) and 1:2.3 in large (LFFL) follicles of sheep ovaries. The LH content of follicular fluid of different size follicles appeared to be the same, with LFFL showing a minor increase over SFFL. In the human, the fluid from medium follicles contained very little LH compared to LFFL. These differences in the pattern of LH levels present in the fluid from different size follicles between human and sheep ovaries presumably reflect species variations in the entry of LH into the follicles.     

Development and characterization of novel microsatellite markers in the Asiatic lion (Panthera leo persica)

All cat species, except domestic cats, are either threatened or endangered. The Asiatic lion (Panthera leo persica), a critically endangered species, is driven to a single habitat zone in Gir forests in India for its survival. The present status of this big cat compels us to acquire deeper knowledge of its genetic variability and phylogenetic relationships. Here we report the isolation of highly polymorphic microsatellite loci from a partial genomic library of the Asiatic lion. These loci were characterized and have shown high levels of variation ranging from six to 11 alleles per locus in the population studied.     

Fyn Tyrosine Kinase Increases Apolipoprotein E Receptor 2 Levels and Phosphorylation

Apolipoprotein E Receptor 2 (ApoER2) and the tyrosine kinase Fyn are both members of the Reelin pathway, a signaling pathway essential for the laminar formation of the cortex during development and proper dendritic spine density and long-term potential (LTP) in the adult brain. In the presence of extracellular Reelin, ApoER2 binds the intracellular protein Dab1, an adaptor protein that is phosphorylated by Fyn. However, direct interactions between ApoER2 and Fyn are not well defined. Here, we show that total levels of ApoER2 and surface levels of ApoER2 are increased by active Fyn. Via a separate mechanism, ApoER2 is also phosphorylated by Fyn, an event that peaks in the postnatal cortex at day 5 and can occur at multiple ApoER2 tyrosine residues. Dab1 is also involved in this phosphorylation, promoting the phosphorylation of ApoER2 by Fyn when it is itself phosphorylated. These results elucidate some of the intracellular mechanisms that give rise to a functional Reelin pathway.     

Spontaneous Genomic Alterations in a Chimeric Model of Colorectal Cancer Enable Metastasis and Guide Effective Combinatorial Therapy

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and β-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.     

Suppression of reactive oxygen species production enhances neuronal survival in vitro and in vivo in the anoxia-tolerant turtle Trachemys scripta

Hypoxia-ischemia with reperfusion is known to cause reactive oxygen species-related damage in mammalian systems, yet, the anoxia tolerant freshwater turtle is able to survive repeated bouts of anoxia/reoxygenation without apparent damage. Although the physiology of anoxia tolerance has been much studied, the adaptations that permit survival of reoxygenation stress have been largely ignored. In this study, we examine ROS production in the turtle striatum and in primary neuronal cultures, and examine the effects of adenosine (AD) on cell survival and ROS. Hydroxyl radical formation was measured by the conversion of salicylate to 2,3-dihydroxybenzoic acid (2,3-DHBA) using microdialysis; reoxygenation after 1 or 4 h anoxia did not result in increased ROS production compared with basal normoxic levels, nor did H₂O₂ increase after anoxia/reoxygenation in neuronally enriched cell cultures. Blockade of AD receptors increased both ROS production and cell death in vitro , while AD agonists decreased cell death and ROS. As turtle neurons proved surprisingly susceptible to externally imposed ROS stress (H₂O₂), we propose that the suppression of ROS formation, coupled to high antioxidant levels, is necessary for reoxygenation survival. As an evolutionarily selected adaptation, the ability to suppress ROS formation could prove an interesting path to investigate new therapeutic targets in mammals.     

Discovery and Characterization of a Small Molecule Inhibitor of the PDZ Domain of Dishevelled

Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.The Wnt signaling pathways are regulated by a family of secreted Wnt glycoproteins. The canonical Wnt pathway, which is highly conserved, is best understood. In this pathway, Wnt molecules interact with the seven-transmembrane Frizzled (Fz)² proteins (1) by binding to an N-terminal cysteine-rich-domain (2). The signal is then transduced into the cell through an internal sequence of Fz, C-terminal to the seventh transmembrane domain, which binds directly to the PDZ (postsynaptic density-95/discs large/zonula occludens-1) domain of the cytoplasmic protein Dishevelled (Dvl) (3). Dvl then transduces the Wnt signals to downstream components (4). Three Dvl homologs (Dvl-1, -2, and -3) have been identified in humans; all are expressed in both embryonic and adult tissues, including brain, heart, lung, kidney, skeletal muscle, and others (4). Up-regulation and overexpression of Dvl proteins have been reported in many cancers, including those of breast, colon, prostate, mesothelium, and lung (non-small cell) (5–8).The Dvl protein is made up of three conserved domains: an N-terminal DIX domain, a central PDZ domain, and a C-terminal DEP domain (9). The central PDZ domain is of particular interest because of its interaction with Fz and other Wnt pathway proteins (3, 10). The direct interaction between the PDZ domain and Fz peptides is relatively weak, and other factors may play a role to ensure the communication between the two molecules (3). For example, several studies suggest that the DEP domain of Dvl has a membrane-targeting function that may facilitate PDZ-Fz interaction (11–14). However, the weak PDZ-Fz interaction provides an opportunity to block Wnt signaling at the Dvl level by using a small molecule inhibitor. An earlier study in our laboratories used an NMR-assisted virtual ligand screening approach to identify a peptide mimic that can bind to the Dvl PDZ domain (15). We have now used an improved algorithm to conduct an additional structure-based virtual screen of the PDZ domain of Dvl and have discovered a group of drug-like compounds that bind to the PDZ domain with moderate to low micromolar affinity. One of these compounds effectively blocked Wnt signaling in vivo and reduced the growth rate of a prostate cancer cell line.     

Rac1 targeting suppresses human non-small cell lung adenocarcinoma cancer stem cell activity

The cancer stem cell (CSC) theory predicts that a small fraction of cancer cells possess unique self-renewal activity and mediate tumor initiation and propagation. However, the molecular mechanisms involved in CSC regulation remains unclear, impinging on effective targeting of CSCs in cancer therapy. Here we have investigated the hypothesis that Rac1, a Rho GTPase implicated in cancer cell proliferation and invasion, is critical for tumor initiation and metastasis of human non-small cell lung adenocarcinoma (NSCLA). Rac1 knockdown by shRNA suppressed the tumorigenic activities of human NSCLA cell lines and primary patient NSCLA specimens, including effects on invasion, proliferation, anchorage-independent growth, sphere formation and lung colonization. Isolated side population (SP) cells representing putative CSCs from human NSCLA cells contained elevated levels of Rac1-GTP, enhanced in vitro migration, invasion, increased in vivo tumor initiating and lung colonizing activities in xenografted mice. However, CSC activity was also detected within the non-SP population, suggesting the importance of therapeutic targeting of all cells within a tumor. Further, pharmacological or shRNA targeting of Rac1 inhibited the tumorigenic activities of both SP and non-SP NSCLA cells. These studies indicate that Rac1 represents a useful target in NSCLA, and its blockade may have therapeutic value in suppressing CSC proliferation and metastasis.