Faculty Bibliography

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.

Recent work indicates that cartilage oligomeric matrix protein (COMP) plays an important role in extracellular matrix assembly and matrix-matrix protein interactions. In order to identify the proteins in extracellular matrix that interact with COMP, we used an ELISA-based solid-phase binding assay, which revealed a specific, high-affinity interaction between COMP and fibronectin. This interaction is concentration-dependent and saturable, and appears to occur under physiologically relevant conditions. Electron microscopy after negative staining and fragment binding analysis using the solid-phase assay revealed a predominant binding site for the COMP C-terminal globular domain to a molecular domain approximately 14 nm from the N-terminal domain of fibronectin, which can be inhibited by the presence of a polyclonal antibody specific for the C-terminal heptadecapeptide of COMP. This interaction is further demonstrated in vivo by colocalization of both COMP and fibronectin in the chondrocyte pericellular matrix by laser confocal microscopy of chondrocytes grown in agarose culture, and by appositional and colocalization of these proteins in the growth plate of primates by immunohistochemistry

There are tissue specific discrepancies in expression of tryptophan hydroxylase (TPH) between the pineal gland and brainstem. TPH mRNA levels in the pineal are much higher than in the brainstem, however, the two tissues contain comparable protein levels. This discrepancy could result from different translation efficiency of two of the TPH mRNA isoforms. Using PCR-based methods, we analyzed the relative expression, in pineal and brainstem, of two TPH mRNA isoforms differing in the length of their untranslated region (5'UTR). The levels of the TPHalpha were found to be 960-fold more abundant than the 51-nucleotide longer TPHbeta, in the pineal. TPHbeta was also detected for the first time in the brainstem, where TPHbeta/TPHalpha was about five-fold higher than in the pineal. To study the role of the different 5'UTRs, each was cloned in-frame upstream of luciferase, and transfected into PC12 cells. Both 5'UTRs enhanced luciferase activity, with TPHbeta 5'UTR being more effective than TPHalpha 5'UTR, indicating selective regulation of translation efficiency. We also examined whether physiological manipulations alter the distribution of the TPH mRNA isoforms. Repeated stress had no effect in pineal, but led to a marked preferential induction of TPHbeta in brainstem. Modulation of TPH gene expression in serotonergic neurons could result from selective and tissue specific regulation of its mRNA isoforms.

Cochleates containing amphotericin B (CAMB) were administered orally at doses ranging from 0 to 40 mg/kg of body weight/day for 14 days in a murine model of systemic aspergillosis. The administration of oral doses of CAMB (20 and 40 mg/kg/day) resulted in a survival rate of 70% and a reduction in colony counts of more than 2 logs in lungs, livers, and kidneys. Orally administered CAMB shows promise for the treatment of aspergillosis.

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

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism and reverse cholesterol transport. We have recently reported that plasma PLTP concentration correlates positively with plasma HDL cholesterol (HDL-C) but not with PLTP activity in healthy subjects. We have also shown that PLTP exists as active and inactive forms in healthy human plasma. In the present study, we measured plasma PLTP concentration and PLTP activity, and analyzed the distribution of PLTP in normolipidemic subjects (controls), cholesteryl ester transfer protein (CETP) deficiency, and hypo-alphalipoproteinemia (hypo-ALP). Plasma PLTP concentration was significantly lower (0.7 +/- 0.4 mg/l, mean +/- SD, n = 9, P < 0.001) in the hypo-ALP subjects, and significantly higher (19.5 +/- 4.3 mg/l, n = 17, P < 0.001) in CETP deficiency than in the controls (12.4 +/- 2.3 mg/l, n = 63). In contrast, we observed no significant differences in plasma PLTP activity between controls, hypo-ALP subjects, and CETP deficiency (6.2 +/- 1.3, 6.1 +/- 1.8, and 6.8 +/- 1.2 micro mol/ml/h, respectively). There was a positive correlation between PLTP concentration and plasma HDL-C (r = 0.81, n = 89, P < 0.001). By size exclusion chromatography analysis, we found that the larger PLTP containing particles without PLTP activity (inactive form of PLTP) were almost absent in the plasma of hypo-ALP subjects, and accumulated in the plasma of CETP deficiency compared with those of controls. These results indicate that the differences in plasma PLTP concentrations between hypo-ALP subjects, CETP deficiency, and controls are mainly due to the differences in the amount of the inactive form of PLTP

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

We have investigated the role of Bmp signaling in development of the mouse lens using three experimental strategies. First, we have shown that the Bmp ligand inhibitor noggin can suppress the differentiation of primary lens fiber cells in explant culture. Second, we have expressed a dominant-negative form of the type 1 Bmp family receptor Alk6 (Bmpr1b -- Mouse Genome Informatics) in the lens in transgenic mice and shown that an inhibition of primary fiber cell differentiation can be detected at E13.5. Interestingly, the observed inhibition of primary fiber cell development was asymmetrical and appeared only on the nasal side of the lens in the ventral half. Expression of the inhibitory form of Alk6 was driven either by the alpha A-cystallin promoter or the ectoderm enhancer from the Pax6 gene in two different transgenes. These expression units drive transgene expression in distinct patterns that overlap in the equatorial cells of the lens vesicle at E12.5. Despite the distinctions between the transgenes, they caused primary fiber cell differentiation defects that were essentially identical, which implied that the equatorial lens vesicle cells were responding to Bmp signals in permitting primary fiber cells to develop. Importantly, E12.5 equatorial lens vesicle cells showed cell-surface immunoreactivity for bone-morphogenetic protein receptor type 2 and nuclear immunoreactivity for the active, phosphorylated form of the Bmp responsive Smads. This indicated that these cells had the machinery for Bmp signaling and were responding to Bmp signals. We conclude that Bmp signaling is required for primary lens fiber cell differentiation and, given the asymmetry of the differentiation inhibition, that distinct differentiation stimuli may be active in different quadrants of the eye.