Faculty Bibliography

Disruption of protein synthesis and folding results in ER stress, which is associated with the pathophysiology of diverse diseases affecting secretory and muscle cells. Cells are protected against ER stress by activation of the unfolded protein response (UPR) that is regulated by the protein kinase PERK, which phosphorylates the translation initiation factor 2 eIF2alpha to attenuate protein synthesis. PERK-/- cells are unable to modulate ER protein load and experience high levels of ER stress. In addition to its role in protein synthesis, the ER also orchestrates many signaling events essential for cell survival, prominent among which is Ca2+ signaling. It is not known, however, whether there is a relationship between ER stress and the function of the Ca2+-signaling pathway in muscle and non-muscle cells. To directly address this question we characterized Ca2+ signaling in the secretory pancreatic and parotid acinar cells and in urinary bladder smooth muscle (UBSM) cells obtained from PERK-/- and wild-type mice. Deletion of PERK that results in high levels of ER stress, and distention and fragmentation of the ER slowed the rate of agonist-mediated Ca2+ release from the ER and reduced Ca2+-induced Ca2+ release, although IP3 production, localization of the IP3 receptors, IP3-mediated Ca2+ release, Ca(v)1.2 current and RyRs activity remained unaltered. On the other hand, ER stress disrupted the integrity of the Ca2+-signaling complexes in both secretory and UBSM cells, as revealed by markedly reduced co-immunoprecipitation of plasma membrane- and ER-resident Ca2+-signaling proteins. These findings establish a relationship between the unfolding protein response, ER stress and Ca2+ signaling and highlight the importance of communication within the terminal ER-plasma membrane microdomain for propagation of the Ca2+ signal from the plasma membrane into the cell

This unit describes methods for preparing zymogen granules from rat pancreas. Zymogen granules are storage organelles in pancreatic acinar cells containing digestive enzymes that are released into the pancreatic duct. The protocols in this unit take advantage of the large size (up to 1 microm diameter) and high density (>1.20 g/cm(3) on sucrose gradients) of the granules as compared to other cellular organelles. They use a combination of differential sedimentation and density gradient separation to accomplish the purification. Similar procedures can be used to isolate zymogen granules from mouse pancreas and canine pancreas. A protocol for preparing zymogen granules from dog pancreas is also included

PURPOSE: We report the first case of subconjunctival mucosa-associated lymphoid tissue (MALT) lymphoma arising in Tenon's capsule (fascia bulbi). METHODS: A 75-year-old woman presented with painless swelling of the superior bulbar conjunctiva in her left eye. During the biopsy of the bulbar lymphoid lesion, it was noticed that the conjunctiva was movable and that the lesion was located in the subconjunctiva. The tissues were studied by conventional light microscopy, immunohistochemistry, flow cytometry, and gene rearrangement analysis. RESULTS: Histopathological examination revealed that a diffuse lymphoid infiltrate consisting of small-sized lymphoid cells was present in Tenon's capsule but not in the substantia propria of the conjunctiva. Immunohistochemical and flow cytometric studies documented tumor cells of B-lymphocyte lineage. Molecular analysis demonstrated positive immunoglobulin heavy chain gene rearrangement. The final diagnosis was subconjunctival MALT lymphoma arising in Tenon's capsule. CONCLUSION: Ophthalmologists and pathologists need to distinguish the subconjunctival lymphoma that arises in Tenon's capsule from the conjunctival lymphoma in the substantia propria during diagnosis of epibulbar lymphoid tumors.

Inspired by the usefulness of small molecules to study membrane traffic, we used high-throughput synthesis and phenotypic screening to discover secramine, a molecule that inhibits membrane traffic out of the Golgi apparatus by an unknown mechanism. We report here that secramine inhibits activation of the Rho GTPase Cdc42, a protein involved in membrane traffic, by a mechanism dependent upon the guanine dissociation inhibitor RhoGDI. RhoGDI binds Cdc42 and antagonizes its membrane association, nucleotide exchange and effector binding. In vitro, secramine inhibits Cdc42 binding to membranes, GTP and effectors in a RhoGDI-dependent manner. In cells, secramine mimics the effects of dominant-negative Cdc42 expression on protein export from the Golgi and on Golgi polarization in migrating cells. RhoGDI-dependent Cdc42 inhibition by secramine illustrates a new way to inhibit Rho GTPases with small molecules and provides a new means to study Cdc42, RhoGDI and the cellular processes they mediate.

Using suppressive subtractive hybridization, we have identified a novel gene, which we named early epithelial differentiation associated (EEDA), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA, and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine, and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca(2+) conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the 'normal' differentiation pathway in a wide range of stratified epithelia. (c) 2005 Wiley-Liss, Inc

Identification of tyrosine phosphorylation by MS is challenging due to its low abundance in biological samples. Therefore, specific enrichment of tyrosine phosphorylated peptides prior to their analysis is highly desirable. The application of immunopurification of phosphotyrosine (pY) peptides using pY antibodies has been greatly limited by poor selectivity. In the present study, we have shown that the selectivity of pY peptide immunopurification can be dramatically improved by adding detergents to immunoprecipitation buffers. Optimum selectivity and sensitivity were achieved using an immunoprecipitation buffer containing n-octyl glucoside with a concentration above its critical micelle concentration (0.7%). The optimized method was used to identify in vivo tyrosine phosphorylation on proteins isolated from cell extract by anti-pY protein immunoprecipitation. After immunopurification, non-pY-containing peptides from protein digests were readily removed and pY peptides became the dominant peaks in MALDI quadrupole-TOF mass spectra. In addition, the signal intensities from pY-containing peptides were enhanced significantly after enrichment, allowing characterization of tyrosine phosphorylation sites with greater sensitivity

A sensitive liquid chromatographic-tandem mass spectrometry(LC-MS/MS) method was developed for the determination of rizatriptan in human plasma. The analytes were extracted from plasma samples by liquid-liquid extraction, separated on a Zorbax XDB C8 column (150 x 4.6 mm i.d.) and detected by tandem mass spectrometry with an electrospray ionization interface. Zomitriptan was used as the internal standard. The method had a lower limit of quantitation of 50 pg/mL for rizatriptan, which showed more sensitivity and speed of analysis compared with reported methods. The within- and between-day precision was measured to be below 11.71% and accuracy between -5.87 and 0.86% for all quality control samples. This quantitation method was successfully applied to the evaluation of the pharmacokinetic profiles of rizatriptan after single oral administration of 5, 10 and 15 mg rizatriptan tablets to 10 healthy volunteers (five males and five females).

Keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, necessary for wound closure and restoration of barrier function. They solely express the beta2-adrenergic receptor (beta2-AR) subtype of beta-ARs and can also synthesize beta-AR agonists generating a hormonal mediator network in the skin. Emerging studies from our laboratory demonstrate that beta-AR agonists decrease keratinocyte migration via a protein phosphatase (PP) 2A-dependent mechanism. Here we have extended our investigations to observe the effects of beta2-AR activation on keratinocyte polarization, migration, and ERK phosphorylation at the wound edge, cytoskeletal organization, phospho-ERK intracellular localization, proliferation, human skin wound re-epithelialization, wound-induced ERK phosphorylation, and murine skin wound healing. We demonstrate that in keratinocytes, beta2-AR activation is anti-motogenic and anti-mitogenic with both mechanisms being PP2A dependent. beta2-AR activation dramatically alters the organization of the actin cytoskeleton and prevents localization of phospho-ERK to the lamellipodial edge and its colocalization with vinculin. Finally, we demonstrate a beta2-AR-mediated delay in re-epithelialization and decrease in wound-induced epidermal ERK phosphorylation in human skin wounds and a delay in re-epithelialization in murine tail-clip wounds. Our work uncovers novel keratinocyte biology and a previously unrecognized role for the adrenergic hormonal mediator network in the wound repair process

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model