Faculty Bibliography

In the ER, the translocon complex (TC) functions in the translocation and cotranslational modification of proteins made on membrane-bound ribosomes. The oligosaccharyltransferase (OST) complex is associated with the TC, and performs the cotranslational N-glycosylation of nascent polypeptide chains. Here we use a GFP-tagged subunit of the OST complex (GFP-Dad1) that rescues the temperature-sensitive (ts) phenotype of tsBN7 cells, where Dad1 is degraded and N-glycosylation is inhibited, to study the lateral mobility of the TC by FRAP. GFP-Dad1 that is functionally incorporated into TCs diffuses extremely slow, exhibiting an effective diffusion constant (Deff) about seven times lower than that of GFP-tagged ER membrane proteins unhindered in their lateral mobility. Termination of protein synthesis significantly increases the lateral mobility of GFP-Dad1 in the ER membranes, but to a level that is still lower than that of free GFP-Dad1. This suggests that GFP-Dad1 as part of the OST remains associated with inactive TCs. Our findings that TCs assembled into membrane-bound polysomes diffuse slowly within the ER have mechanistic implications for the segregation of the ER into smooth and rough domains

Nicastrin is an integral component of the high molecular weight presenilin complexes that control proteolytic processing of the amyloid precursor protein and Notch. We report here that nicastrin is most probably a type 1 transmembrane glycoprotein that is expressed at moderate levels in the brain and in cultured neurons. Immunofluorescence studies demonstrate that nicastrin is localized in the endoplasmic reticulum, Golgi, and a discrete population of vesicles. Glycosidase analyses reveal that endogenous nicastrin undergoes a conventional, trafficking-dependent maturation process. However, when highly expressed in transfected cells, there is a disproportionate accumulation of the endo-beta-N-acetylglucosaminidase H-sensitive, immature form, with no significant increase in the levels of the fully mature species. Immunoprecipitation revealed that presenilin-1 interacts preferentially with mature nicastrin, suggesting that correct trafficking and co-localization of the presenilin complex components are essential for activity. These findings demonstrate that trafficking and post-translational modifications of nicastrin are tightly regulated processes that accompany the assembly of the active presenilin complexes that execute gamma-secretase cleavage. These results also underscore the caveat that simple overexpression of nicastrin in transfected cells may result in the accumulation of large amounts of the immature protein, which is apparently unable to assemble into the active complexes capable of processing amyloid precursor protein and Notch

Aberrant mossy fiber sprouting and synaptic reorganization are plastic responses in human temporal lobe epilepsy, and in pilocarpine-induced epilepsy in rodents. Although the morphological features of the hippocampal epileptic reaction have been well documented, the molecular mechanisms underlying these structural changes are not understood. The classic cadherins, calcium-dependent cell adhesion molecules, are known to function in development in neurite outgrowth, synapse formation, and stabilization. In pilocarpine-induced status epilepticus, the expression of N-cadherin mRNA was sharply upregulated and reached a maximum level (1- to 2.5-fold) at 1- to 4 weeks postseizure in the granule cell layer and the pyramidal cell layer of CA3. N-cadherin protein was correspondingly increased and became concentrated in the inner molecular layer of the dentate gyrus, consistent with the position of mossy fiber axonal sprouts. Moreover, N-cadherin labeling was punctate; colocalized with definitive synaptic markers, and partially localized on polysialated forms of neural cell adhesion molecule (PSA-NCAM)-positive dendrites of granule cells in the inner molecular layer. Our findings show that N-cadherin is likely to be a key factor in responsive synaptogenesis following status epilepticus, where it functions as a mediator of de novo synapse formation.

The latent TGF-beta binding proteins (LTBP) are believed to control the availability of TGF-beta in the extracellular milieu. To gain insight into the potential roles of LTBP in early development, we isolated the Xenopus LTBP-1 (xLTBP-1) cDNA. The cDNA encodes a protein similar to the mammalian LTBP-1 in both size and domain structure. In addition, we found a novel longer splice isoform of xLTBP. The RNAs for both forms of xLTBP displayed temporal regulation and the shorter transcript is expressed maternally. Both transcripts also display spatial regulation and are found in the dorsal mesoderm of the organizer. In animal cap experiments, LTBP-1 potentiates the activity of activin and nodal. The activity of LTBP-1 did not appear to require covalent association with activin as the addition of medium containing activin and LTBP-1 to animal caps enhanced the activin effect. These results indicate that LTBP-1 may be part of the regulatory system that establishes the threshold of morphogen activity for activins and nodals in the dorsal side of the embryo during gastrulation

The RNA polymerase complex of human parainfluenza virus type 3 (HPIV 3), a member of the family Paramyxoviridae, is composed of two virally encoded polypeptides: a multifunctional large protein (L, 255 kDa) and a phosphoprotein (P, 90 kDa). From extensive deduced amino acid sequence analyses of the cDNA clones of a number of L proteins of nonsegmented negative-strand RNA viruses, a cluster of high-homology sequence segments have been identified within the body of the L proteins. Here, we have focused on the NH(2)-terminal domain of HPIV 3 L protein that is also highly conserved. Following mutational analyses within this domain, we examined the ability of the mutant L proteins to (i) transcribe an HPIV 3 minireplicon, (ii) transcribe the viral RNA in vitro using the HPIV 3 nucleocapsid RNA template, and (iii) interact with HPIV 3 P protein. Our results demonstrate that the first 15 amino acids of the NH(2)-terminal domain spanning a highly conserved motif is directly involved in transcription of the genome RNA and in forming a functional complex with the P protein. Substitution of eight nonconserved amino acids within this domain by the corresponding Sendai virus L protein residues yielded mutants with variable transcriptional activities. However, one mutant in which all eight amino acids were replaced with the corresponding residues of Sendai virus L protein failed to both transcribe the minireplicon and interact with HPIV 3 P and the Sendai virus P protein. The possible functional significance of the NH(2)-terminal domain of paramyxovirus L protein is discussed.

To perform vectorial secretory and transport functions that are critical for the survival of the organism, epithelial cells sort plasma membrane proteins into polarized apical and basolateral domains. Sorting occurs post-synthetically, in the trans Golgi network (TGN) or after internalization from the cell surface in recycling endosomes, and is mediated by apical and basolateral sorting signals embedded in the protein structure. Basolateral sorting signals include tyrosine motifs in the cytoplasmic domain that are structurally similar to signals involved in receptor internalization by clathrin-coated pits. Recently, an epithelial-specific adaptor protein complex, AP1B, was identified. AP-1B recognizes a subset of basolateral tyrosine motifs through its mu 1B subunit. Here, we characterized the post-synthetic and post-endocytic sorting of the fast recycling low density lipoprotein receptor (LDLR) and transferrin receptor (TfR) in LLC-PK1 cells, which lack mu 1B and mis-sort both receptors to the apical surface. Targeting and recycling assays in LLC-PK1 cells, before and after transfection with mu 1B, and in MDCK cells, which express mu 1B constitutively, suggest that AP1B sorts basolateral proteins post-endocytically.

Course materials for a Human Anatomy and Development Course were placed on the World Wide Web (WWW). The materials included a lab manual, lecture notes and slides, faculty-generated atlases, Web links, and examinations. The lab manual, lecture notes, and atlases were also provided as black-and-white hardcopy. The Office of Education assigned students a code name that allowed them to use the Web site and take exams anonymously. Student Web use was tracked and correlated with their performance on the final examination. Overall use patterns revealed that most students used the Web site to prepare for examinations, but not for daily studying. Old examinations were the most accessed documents; lecture notes were the least accessed. The access patterns of the students with top 20, middle 20 (closest to the mean), and bottom 20 scores on the final examination were compared. In general, there was little difference between the middle and top groups. Students in the bottom group used computer resources significantly less than the other groups. In a second analysis, the 10 students who used the Web site most frequently scored below the mean. The study suggests that interactive exercises will be heavily used, but that the preparation of all course materials for the WWW may not be an efficient use of institutional resources.

OBJECTIVES: To determine the frequency of readmission for early postoperative small-bowel obstruction (SBO), to highlight factors that may predispose to this condition, to define the risks of strangulation and to compare the immediate and long-term risks and benefits of operative versus nonoperative treatment. DESIGN: A chart review. SETTING: The Sir Mortimer B. Davis-Jewish General Hospital, a university-affiliated teaching hospital in Montreal. PATIENTS: Out of a total of 1001 cases of SBO in 552 patients, 30 patients were readmitted within 50 days of a previous laparotomy with the diagnosis of SBO. INTERVENTION: Selective nonoperative management and exploratory laparotomy. MAIN OUTCOME MEASURES: The value of nonoperative management and need for operation. RESULTS: Adhesions were the cause of the obstruction in most cases (24); other causes were Crohn's disease (2), hernia (1), malignant neoplasm (1) and a combination of adhesions and malignant disease (2). Thirteen (43%) of the procedures preceding the obstruction were primary small-bowel operations. There was only 1 episode of strangulated bowel. Of the patients readmitted for SBO, 7 (23%) were treated operatively with a long-term recurrence rate of 57% compared with 63% for those treated nonoperatively for the SBO. The median time to recurrence was 0.1 years (range from 0.02-6 yr) for those whose SBO was managed operatively, compared with 0.7 years (range from 0.08-5 yr) for those managed nonoperatively for the SBO. The median length of stay for patients managed operatively for SBO was 12 days (range from 9-17 d) compared with 6 days (range from 2-33 d) for those managed nonoperatively. CONCLUSIONS: Readmission for SBO within 50 days of a previous laparotomy represents a small percentage of all cases of SBO. They frequently follow small-bowel operations. Cases of strangulation are no more common than in general cases of SBO. Patients treated nonoperatively for SBO did not experience less favourable outcomes with respect to resolution of symptoms, length of stay, risk of recurrence and reoperation. Thus, operative intervention is not necessary in an otherwise stable patient

Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii