Faculty Bibliography

Cotranslational translocation of apoB100 across the endoplasmic reticulum (ER) membrane is inefficient, resulting in exposure of nascent apoB on the cytosolic surface of the ER. This predisposes apoB100 to ubiquitinylation and targeting for proteasomal degradation. It has been suggested that pause transfer sequences (PTS) present throughout apoB cause inefficient translocation. On the other hand, our previous study demonstrated that the translocation efficiency of apoB100 is dependent on the presence of a beta-sheet domain between 29 and 34% of full-length apoB100 (Liang, J.-S., Wu, X., Jiang, H., Zhou, M., Yang, H., Angkeow, P., Huang, L.-S., Sturley, S. L., and Ginsberg, H. N. (1998) J. Biol. Chem. 273, 35216-35221); this region of apoB has no PTS. However, the effects of the beta-sheet domain may require the presence of PTS elsewhere in the N-terminal region of apoB100. To further investigate the roles of PTS and beta-sheet domains in the translocation of apoB100 across the ER, we transfected McArdle RH7777, HepG2, or Chinese hamster ovary cells with human albumin (ALB)/human apoB chimeric cDNA constructs: ALB/B12-17 (two PTS but no beta-sheet), ALB/B29-34 (beta-sheet but no PTS), ALB/B36-41 (two PTS and a beta-sheet), and ALB/B49-54 (neither PTS nor a beta-sheet). ALB/ALB1-40 served as a control. Compared with ALB/ALB1-40, secretion rates of ALB/B12-17, ALB/B29-34, and ALB/B36-41 were reduced. Secretion of ALB/B49-54 was similar to that of ALB/ALB1-40. However, only ALB/B29-34 and ALB/B36-41 had increased proteinase K sensitivity, ubiquitinylation, and increased physical interaction with Sec61alpha. These results indicate that the translocation efficiency of apoB100 is determined mainly by the presence of beta-sheet domains. PTS do not appear to affect translocation, but may affect secretion by other mechanisms.

CopA from the extreme thermophile Archaeoglobus fulgidus is a P-type ATPase that transports Cu(+) and Ag(+) and has individual metal-binding domains (MBDs) at both N- and C-termini. We expressed and purified full-length CopA as well as constructs with MBDs deleted either individually or collectively. Cu(+) and Ag(+)-dependent ATPase assays showed that full-length CopA had submicromolar affinity for both ions, but was inhibited by concentrations above 1muM. Deletion of both MBDs had no effect on affinity but resulted in loss of this inhibition. Individual deletions implicated the N-terminal MBD in causing the inhibition at concentrations >1muM. Rates of phosphoenzyme decay indicated that neither the dephosphorylation step, nor the E1P-E2P equilibrium accounted for this inhibition, suggesting the involvement of a different catalytic step. Alternative hypotheses are discussed by which the N-terminal MBD could influence the catalytic activity of CopA

OBJECTIVE: To investigate the barrier function of esophagogastric junction (EGJ) in the patients with gastroesophageal reflux diseases (GERD). METHODS: Ninety-six GERD patients and 18 healthy subjects (HSs) underwent esophageal manometry. Forty-two of the 96 patients were with reflux esophagitis (RE) and were divided into 2 subgroups according to the LA typing: 31 subgroup of LA-A or B (n = 31), and subgroup of LA-C or D (n = 11). Fifty-four of the 96 patients suffered from non-erosive reflux disease (NERD) and were divided into 2 subgroups based on 24 h esophageal pH monitoring: 31 patients with normal acid exposure (pH < 14.72 according to DeMeester scoring) and 23 with excessive acid exposure (pH > 14.72). The EGJ functions, including lower esophageal sphincter pressure (LESP), crura of diaphragm pressure (CDP), esophagogastric junction pressure (EGJP, sum of LESP and CDP), and post-lower esophageal sphincter relaxation pressure (post-LESRP), and the contractive pressure of esophageal body, including proximal esophageal contractive pressure (PECP) and distal esophageal contractive pressure (DECP), were measured. RESULTS: The levels of LESP, CDP, EGJP, and post-LESRP of the RE patients were (8 +/- 3), (13 +/- 7), (20 +/- 9), and (31 +/- 13) mm Hg respectively, all significantly lower than those of the NERD patients [(9 +/- 3), (18 +/- 6), (28 +/- 8), and (39 +/- 15) mm Hg, all P < 0.05]. The levels of LESP and post-LESRP of the NERD patients were significantly lower than those of the HSs [(32 +/- 7) and (50 +/- 15) mm Hg, both P < 0.05], however, the levels of CDP and EGJP of the NERD patients were both not significantly different from those of the HSs [(16 +/- 6) and (32 +/- 7) mm Hg, both P > 0.05]. The PECP and DECP of the RE and NERD patients were all significantly lower than those of the HSs (all P < 0.05), however, there were not significant differences in DECP and PECP between the RE and NERD patients (both P > 0.05). Among the NERD patients there were not significant differences in the PECP and DECP between the LA-A and B subgroup and RE LA-C and D subgroup, and among the NERD patients there were not significant differences in PECP and DECP between the subgroups with and without excessive esophageal acid exposure (all P > 0.05). CONCLUSION: The EGF anti-reflux function of the GERD patients is impaired. The function of crura of diaphragm of the NERD patients is almost normal, however, that of the RE patients is impaired. NERD and RE have different path physiological basis.

A 7-year-old girl presented with asymptomatic bruise-like hairy nodules on her right lower leg since 8 months of age. Histopathology demonstrated an increased number of blood vessels and eccrine glands, thicker collagen bundles, and a terminal hair follicle in catagen phase. The patient was diagnosed with multiple eccrine pilar angiomatous nevi, an unusual variant of eccrine angiomatous hamartomas. The natural course of eccrine angiomatous hamartomas is typically slow growth and benign behavior. Simple excision is usually curative and is reserved for painful or cosmetically disfiguring lesions. Our patient's nevus is large and multifocal, making excision more challenging

The ability of organs such as the liver or the lymphoid system to maintain their original size or regain it after injury is well documented. However, little is known about how these organs sense that equilibrium is breached, and how they cease changing when homeostasis is reached. Similarly, it remains unclear how, during normal development, different cell types within an organ coordinate their growth. Here we show that during gonad development in the fruitfly Drosophila melanogaster the proliferation of primordial germ cells (PGCs) and survival of the somatic intermingled cells (ICs) that contact them are coordinated by means of a feedback mechanism composed of a positive signal and a negative signal. PGCs express the EGF receptor (EGFR) ligand Spitz, which is required for IC survival. In turn, ICs inhibit PGC proliferation. Thus, homeostasis and coordination of growth between soma and germ line in the larval ovary is achieved by using a sensor of PGC numbers (EGFR-mediated survival of ICs) coupled to a correction mechanism inhibiting PGC proliferation. This feedback loop ensures that sufficient numbers of PGCs exist to fill all the stem-cell niches that form at the end of larval development. We propose that similar feedback mechanisms might be generally used for coordinated growth, regeneration and homeostasis

Orthologous gene structures in eight vertebrate species were compared on a genomic scale to detect the birth and maturation of new internal exons during the course of evolution. We found that 40% of new human exons are alternatively spliced, and most of these are cassette exons (exons that are either included or skipped in their entirety) with low inclusion rates. This proportion decreases steadily as older and older exons are examined, even as splicing efficiency increases. Remarkably, the great majority of new cassette exons are composed of highly repeated sequences, especially Alu. Many new cassette exons are 5' untranslated exons; the proportion that code for protein increases steadily with age. New protein-coding exons evolve at a high rate, as evidenced by the initially high substitution rates (K(s) and K(a)), as well as the SNP density compared with older exons. This dynamic picture suggests that de novo recruitment rather than shuffling is the major route by which exons are added to genes, and that species-specific repeats could play a significant role in recent evolution.

Multiple subsets of the bone marrow contain T cell precursors, but it remains unclear which is most likely to replenish the adult thymus. Therefore, RAG-1+ early lymphoid progenitors (RAG-1+ ELP), and CD62L/L-selectin+ progenitors (LSP), as well as common lymphoid progenitors from C57BL6-Thy1.1-RAG-1/GFP mouse bone marrow were directly compared in transplantation assays. The two c-Kit(high) populations vigorously regenerated the thymus and were superior to common lymphoid progenitors in magnitude and frequency of thymic reconstitution. Regeneration was much faster than the 22 days described for transplanted stem cells, and RAG-1+ ELP produced small numbers of lymphocytes within 13 days. As previously reported, LSP were biased to a T cell fate, but this was not the case for RAG-1+ ELP. Although RAG-1+ ELP and LSP had reduced myeloid potential, they were both effective progenitors for T lymphocytes and NK cells. The LSP subset overlapped with and included most RAG-1+ ELP and many RAG-1- TdT+ ELP. LSP and RAG-1+ ELP were both present in the peripheral circulation, but RAG-1+ ELP had no exact counterpart among immature thymocytes. The most primitive of thymocytes were similar to Lin- c-Kit(high) L-selectin+ TdT+ RAG-1- progenitors present in the marrow, suggesting that this population is normally important for sustaining the adult thymus.

Transcription factor NF-E2-related factor 2 (Nrf2) regulates antioxidant response element (ARE)-mediated expression and coordinated induction of chemoprotective proteins in response to chemical stress. In this report, we investigated Nrf2 response to low and high dose UVB irradiation. Low dose (7.5 J/m(2)) UVB exposure of mouse hepatoma, mouse keratinocyte, and human skin fibroblast cells led to the nuclear accumulation of Nrf2 and up-regulation of ARE-mediated gene expression. On the contrary, and intriguingly, high dose (20 J/m(2)) UVB exposure of cells led to the nuclear exclusion of Nrf2 and down-regulation of chemoprotective gene expression with possible implications in UVB carcinogenesis. We investigated the mechanism by which high dose UVB induced the nuclear exclusion of Nrf2. Prior treatment with nuclear export inhibitor, leptomycin B, abrogated the UVB-induced nuclear exclusion of Nrf2, indicating that the decrease of Nrf2 in the nucleus was due to the nuclear export of Nrf2. High dose UVB increased the phosphorylation of Nrf2Y568 which stimulated the nuclear export of Nrf2. Mutation of Nrf2Y568 to phenylalanine and src kinase inhibitor PP2 abrogated/reduced the UVB-induced phosphorylation of Nrf2Y568 and nuclear exclusion of Nrf2. Transfection with src family member Fyn small interfering RNA resulted in the nuclear accumulation of Nrf2 and an increase in the expression and UVB induction of ARE-mediated gene expression. UVB exposure also induced the nuclear localization of Fyn. These results suggest that high dose UVB induced the activation/nuclear localization of Fyn which led to increased phosphorylation of Nrf2Y568 and enhanced nuclear export of Nrf2. This resulted in nuclear exclusion of Nrf2 and down-regulation of ARE-mediated chemoprotective gene expression.

PURPOSE: Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where expression levels correlate with decreased survival. Therapies that block EGFR have shown limited efficacy in clinical trials and primarily when combined with standard therapy. The most common form of mutant EGFR (EGFRvIII) has been described in several cancers, chiefly glioblastoma. The present study was undertaken to determine the incidence of EGFRvIII expression in HNSCC and the biological consequences of EGFRvIII on tumor growth in response to EGFR targeting. EXPERIMENTAL DESIGN: Thirty-three HNSCC tumors were evaluated by immunostaining and reverse transcription-PCR for EGFRvIII expression. A representative HNSCC cell line was stably transfected with an EGFRvIII expression construct. EGFRvIII-expressing cells and vector-transfected controls were compared for growth rates in vitro and in vivo as well as chemotherapy-induced apoptosis and the consequences of EGFR inhibition using the chimeric monoclonal antibody C225/cetuximab/Erbitux. RESULTS: EGFRvIII expression was detected in 42% of HNSCC tumors where EGFRvIII was always found in conjunction with wild-type EGFR. HNSCC cells expressing EGFRvIII showed increased proliferation in vitro and increased tumor volumes in vivo compared with vector-transfected controls. Furthermore, EGFRvIII-transfected HNSCC cells showed decreased apoptosis in response to cisplatin and decreased growth inhibition following treatment with C225 compared with vector-transfected control cells. CONCLUSIONS: EGFRvIII is expressed in HNSCC where it contributes to enhanced growth and resistance to targeting wild-type EGFR. The antitumor efficacy of EGFR targeting strategies may be enhanced by the addition of EGFRvIII-specific blockade.