Faculty Bibliography

Mice lacking the serotonin 1A receptor (5-HT(1A)R) show increased levels of anxiety-related behavior across multiple tests and background strains. Tissue-specific rescue experiments, lesion studies, and neurophysiological findings all point toward the hippocampus as a potential mediator of the phenotype. Serotonin, acting through 5-HT(1A)Rs, can suppress hippocampal theta-frequency oscillations, suggesting that theta oscillations might be increased in the knock-outs. To test this hypothesis, local field potential recordings were obtained from the hippocampus of awake, behaving knock-outs and wild-type littermates. The magnitude of theta oscillations was increased in the knock-outs, specifically in the anxiety-provoking elevated plus maze and not in a familiar environment or during rapid eye movement sleep. Theta power correlated with the fraction of time spent in the open arms, an anxiety-related behavioral variable. These results suggest a possible role for the hippocampus, and theta oscillations in particular, in the expression of anxiety in 5-HT(1A)R-deficient mice.

Although ovarian estrogen, estradiol-17beta, is a key modulator of normal reproductive functions, natural and synthetic compounds with estrogen-like activities can further influence reproductive functions. Plant-derived phytoestrogens specifically have received much attention because of associated health benefits. However, a comprehensive understanding of the beneficial and/or detrimental impacts of phytoestrogen consumption through commercial rodent diets on uterine biology and early pregnancy at the molecular level remains largely unexplored. Using multiple approaches, we demonstrate here that exposure of adult female mice to a commercial rodent diet with higher phytoestrogen levels facilitates uterine growth in the presence or absence of ovarian estrogen, alters uterine expression of estrogen-responsive genes, and advances the timing of implantation compared with a diet with lower phytoestrogen levels. The finding that variability in phytoestrogen content in commercial rodent diets, both within and between brands, influences experimental results stresses the importance of this investigation and raises caution for investigators using rodents as animal models.

The paradigms for transport along the biosynthetic route have changed dramatically over the past 15 years. Unlike the situation 15 years ago, the current paradigm involves sorting signals practically at every step of the pathway. In particular, at the exit from the Golgi complex, apical, basolateral and lysosomal targeting signals result in the generation of a variety of routes. Furthermore, it is now quite clear that not all sorting in the biosynthetic route occurs in the Golgi complex or the Trans Golgi Network (TGN). Sorting may occur distally to the Golgi, in recycling endosomes or in budded tubulosaccular structures, or it may occur proximally to the Golgi complex, at the exit from the ER. Several adaptors are candidates to sort apical and basolateral proteins but only AP1B and AP4 are currently involved. Progress is fast and future work should elucidate many of the open questions.

We have previously shown that prostatic stem cells are located in the proximal region of mouse prostatic ducts. Here, we show that this region responds differently to transforming growth factor (TGF)-beta than the distal ductal region and that under physiological conditions androgens and TGF-beta are crucial overall regulators of prostatic tissue homeostasis. This conclusion is supported by the observations showing that high levels of TGF-beta signaling are present in the quiescent proximal region of ducts in an androgen-replete animal and that cells in this region overexpress Bcl-2, which protects them from apoptosis. Moreover, androgen ablation reverses the proximal-distal TGF-beta signaling gradient, leading to an increase in TGF-beta signaling in the unprotected distal region (low Bcl-2 expression). This reversal of TGF-beta-mediated signaling accompanies apoptosis of cells in the distal region and gland involution after androgen withdrawal. A physiological TGF-beta signaling gradient (high proximally and low distally) and its functional correlates are restored after androgen replenishment. In addition to highlighting the regulatory role of androgens and TGF-beta, these findings may have important implications for the deregulation of the stem cell compartment in the etiology of proliferative prostatic diseases

The blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1) stimulates cAMP production, promotes Ca2+ influx, and mobilizes an intracellular source of Ca2+ in pancreatic beta cells. Here we provide evidence that these actions of GLP-1 are functionally related: they reflect a process of Ca2+-induced Ca2+ release (CICR) that requires activation of protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). In rat insulin-secreting INS-1 cells or mouse beta cells loaded with caged Ca2+ (NP-EGTA), a GLP-1 receptor agonist (exendin-4) is demonstrated to sensitize intracellular Ca2+ release channels to stimulatory effects of cytosolic Ca2+, thereby allowing CICR to be generated by the uncaging of Ca2+ (UV flash photolysis). This sensitizing action of exendin-4 is diminished by an inhibitor of PKA (H-89) or by overexpression of dominant negative Epac. It is reproduced by cell-permeant cAMP analogues that activate PKA (6-Bnz-cAMP) or Epac (8-pCPT-2'-O-Me-cAMP) selectively. Depletion of Ca2+ stores with thapsigargin abolishes CICR, while inhibitors of Ca2+ release channels (ryanodine and heparin) attenuate CICR in an additive manner. Because the uncaging of Ca2+ fails to stimulate CICR in the absence of cAMP-elevating agents, it is concluded that there exists in beta cells a process of second messenger coincidence detection, whereby intracellular Ca2+ release channels (ryanodine receptors, inositol 1,4,5-trisphosphate (IP3) receptors) monitor a simultaneous increase of cAMP and Ca2+ concentrations. We propose that second messenger coincidence detection of this type may explain how GLP-1 interacts with beta cell glucose metabolism to stimulate insulin secretion.

Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH(2)-terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process.

Metastasis is the primary cause of death from breast cancer. A xenograft model was used to identify genes potentially involved with metastasis, comparing expression in the poorly metastatic GI101A human breast cancer cell line and a highly metastatic variant, GILM2. cDNA microarray analyses of these isogenic variants were done using 16K Operon 70-mer oligonucleotide microarray slides. Differentially expressed genes were identified by ANOVA, and differences of > or =2.5-fold were found for 106 genes. Changes in protein or RNA expression were confirmed for 10 of 12 genes. Three markers, heat shock protein 70 (HSP-70), chemokine (C-X-C motif) ligand 1 (CXCL-1), and secreted leukocyte protease inhibitor (SLPI), were studied further with breast cancer tissue microarrays using a novel method of automated quantitative analysis. This uses cytokeratin to define pixels as breast cancer (tumor mask) within the tissue array spot and then measures intensity of marker expression using a cyanine 5-conjugated antibody within the mask. Scores were correlated with clinicopathologic variables. High HSP-70 expression and high nuclear CXCL-1 expression in primary tumors were both associated with decreased survival (P = 0.05 and 0.027, respectively). Expression of each marker was strongly associated with lymph node involvement (P = 0.0002, 0.008, 0.0012, and 0.012 for HSP-70, nuclear CXCL-1, cytoplasmic CXCL-1, and SLPI, respectively). Identification of genes associated with metastasis in experimental models may have clinical implications for the management of breast cancer, because some of these are associated with lymph node metastasis and survival and might be useful as prognostic markers or molecular targets for novel therapies.

The concept of "three-yin and three-yang" in Shanghan Lun (Treatise on Cold Pathogenic Diseases), a classic written by Zhang Zhongjing in Han Dynasty, has been always the focus of dispute in successive dynasties. The essence of "three-yin and three-yang" has not been fully revealed up till now. Through studying the six divisions of day and night, the six diseases, the combination of syndromes, the complicated diseases, the complete recovery time and the space division of "three-yin and three-yang", the authors draw a conclusion that the "three-yin and three-yang" in Shanghan Lun is a concept of time-sequence, which is associated with the location of disease in space. So it is suggested that the "six diseases" in Shanghan Lun is a categorization for exogenous febrile diseases, and this categorization reveals a sort of inner relationship between the emergence, development, transformation of the febrile diseases and the time.

Fibroblasts represent a highly mechanoresponsive cell type known to play key roles in normal and pathologic processes such as wound healing, joint contracture, and hypertrophic scarring. In this study, we used a novel fibroblast-populated collagen lattice (FPCL) isometric tension model, allowing us to apply graded biaxial loads to dermal fibroblasts in a 3-dimensional matrix. Cell morphology demonstrated dose-dependent transition from round cells lacking stress fibers in nonloaded lattices to a broad, elongated morphology with prominent actin stress fibers in 800-mg-loaded lattices. Using quantitative real-time RT-PCR, a dose dependent induction of both collagen-1 and collagen-3 mRNA up to 2.8- and 3-fold, respectively, as well as a 2.5-fold induction of MMP-1 (collagenase) over unloaded FPCLs was observed. Quantitative expression of the proapoptotic gene Bax was down-regulated over 4-fold in mechanically strained FPCLs. These results suggest that mechanical strain up-regulates matrix remodeling genes and down-regulates normal cellular apoptosis, resulting in more cells, each of which produces more matrix. This 'double burden' may underlie the pathophysiology of hypertrophic scars and other fibrotic processes in vivo

The proliferative responses of cells to mitogens decrease during aging, and this may result from age-related defects in signal transduction in response to mitogens. In this study, we have investigated the age-related alteration of responses to epidermal growth factor (EGF) in cultured human keratinocytes that were senesced in vitro by repeated passage. The stimulation with EGF increased the DNA-binding activity of activator protein 1 (AP-1), an important transcription factor for cell proliferation, in young keratinocytes, whereas the binding activity showed little or slight change in the senescent cells. The induced DNA-binding activity of AP-1 in young cells was inhibited by PD 98059, an inhibitor of MEK, and partially inhibited by GF 109203X, an inhibitor of protein kinase C. Western blot analysis demonstrated that EGF induced dramatic increase in the phosphorylation of EGF receptor (EGFR) and extracellular signal-regulated kinases (ERK) in young cells, while this phosphorylation was much less profound in senescent cells. Finally, the application of EGF to young cells resulted in increased phosphorylation of Fra-2, a Fos protein component of the Jun/Fos heterodimer AP-1 complex. This EGF-induced Fra-2 phosphorylation was attenuated in senescent cells. Taken together, our study suggests that the signal transduction mediated by EGF/ERK pathway is altered in senescent human keratinocytes, and this change may be attributed, in part, to the decreased AP-1 transcription activity observed in senescent keratinocytes