Citations

Eps15 (EGFR pathway substrate clone 15) is well known for its role in clathrin-coated vesicle formation at the plasma membrane through interactions with other clathrin adaptor proteins such as AP-2. Interestingly, we observed that in addition to its plasma membrane localization, Eps15 is also present at the trans-Golgi network (TGN). Therefore, we predicted that Eps15 might associate with clathrin adaptor proteins at the TGN and thereby mediate the formation of Golgi-derived vesicles. Indeed, we have found that Eps15 and the TGN clathrin adaptor AP-1 coimmunoprecipitate from rat liver Golgi fractions. Furthermore, we have identified a 14-amino acid motif near the AP-2–binding domain of Eps15 that is required for binding to AP-1, but not AP-2. Disruption of the Eps15–AP-1 interaction via siRNA knockdown of AP-1 or expression of mutant Eps15 protein, which lacks a 14-amino acid motif representing the AP-1 binding site of Eps15, significantly reduced the exit of secretory proteins from the TGN. Together, these findings indicate that Eps15 plays an important role in clathrin-coated vesicle formation not only at the plasma membrane but also at the TGN during the secretory process.

ObjectiveTo evaluate the image quality of coronal reformatted CT images compared with direct coronal CT images.Study DesignCross‐sectional.Subjects and MethodsPatients who underwent paranasal sinus CT scans using both direct coronal images (DC) and axially acquired images with coronal reconstructions (CR) were consecutively enrolled. Two reconstruction groups were evaluated: group CR1 with 1‐mm axial collimation and group CR2 with 0.625‐mm axial collimation. Scans were presented in a randomized, blinded fashion to two sinus surgeons and a neuroradiologist.ResultsWhen compared with CR1, DC images had higher image quality for each of five key anatomic landmarks (P <. 0001). DC images were more likely to be adequate preoperative imaging (P <. 0001). When compared with CR2, improved DC image quality was noted in the majority of key anatomic landmarks. No difference was noted in the “adequacy rating” between the DC and CR2 (0.625 mm) protocols.ConclusionThe 0.625‐mm reformatted images were judged to be adequate preoperative imaging. The additional information provided by multiple‐image planes make reformatted 0.625‐mm axial images an acceptable approach.

One hundred two patients with Sturge-Weber disease who were seen at the Mayo Clinic between 1942 and 1986 were studied retrospectively to determine the difference in prognosis between unihemispheric (88 patients) and bihemispheric (14 patients) involvement. Seizures occurred in 63 with unihemispheric involvement and 13 with bihemispheric; the mean age at onset of seizures was 24 months in the former and 6 months in the latter. Of the total group, 19% were severely or moderately mentally retarded, 27% were mentally retarded but educable, and 45% had average intelligence. In the bihemispheric involvement group, 46% were severely or moderately mentally retarded, 38% were retarded but educable, and only 8% had average intelligence. Bilateral involvement of the brain by Sturge-Weber disease is associated with earlier onset of seizures and worse prognosis for mental development compared with unilateral involvement. ( J Child Neurol 1988;3:181-184).

The electrophysiologic evaluation of peripheral nerves may provide critically important information, both with respect to diagnosis and prognosis, in the child with a suspected neuromuscular disorder. However, special attention to various technical considerations is necessary to avoid misleading results. Utilizing these techniques, both hereditary and acquired neuropathies may be identified and characterized. The latter has become especially important in view of recent advances in the treatment of acquired demyelinating neuropathies. (J Child Neurol 1986;1:19-26)

Tetrahydrobiopterin (BH 4 ) is an essential cofactor required for enzymatic activity of endothelial NO synthase. Recently, it has been shown that vascular protective effects of erythropoietin (EPO) are dependent on activation of endothelial NO synthase. Therefore, our objective was to characterize the effect of EPO on the biosynthesis of BH 4 in the vascular wall. Incubation of isolated C57BL/6J mouse aortas for 18 hours with recombinant human EPO (1 to 50 U/mL) caused a concentration-dependent increase in intracellular BH 4 levels and activity of GTP-cyclohydrolase I. Maximal biosynthesis of BH 4 was detected at therapeutic concentrations of 5 U/mL. Removal of the endothelium abolished EPO-induced biosynthesis of BH 4 demonstrating that the vascular endothelium is a major source of BH 4 . Treatment with a selective phosphatidylinositol 3-kinase inhibitor wortmannin significantly reduced BH 4 biosynthesis stimulated by EPO. The stimulatory effect of EPO on vascular GTP-cyclohydrolase I activity, BH 4 production, and phosphorylation of endothelial NO synthase was also detected in vivo in mice treated with recombinant human EPO. These effects of EPO were abolished in protein kinase Bα/Akt1-deficient mice. In addition, EPO significantly increased systolic blood pressure and the number of circulating platelets in Akt1-deficient mice. Our results demonstrate that EPO stimulates biosynthesis of BH 4 in vascular endothelium and that the increase in BH 4 levels is caused by de novo biosynthesis of BH 4 via the phosphatidylinositol 3-kinase/Akt1 pathway. This effect is most likely designed to provide optimal intracellular concentration of the cofactor necessary for EPO-induced elevation of endothelial NO synthase activity.

Abstract Purpose: The proteasome is a multisubunit cellular organelle that functions as a nonlysosomal threonine protease. Proteasomes play a critical role in the degradation of proteins, regulating a variety of cellular processes, and they are also the target for antineoplastic proteasome inhibitors. Genetic variation in proteasome subunits could influence both proteasome function and response to drug therapy. Experimental Design: We resequenced genes encoding the three active proteasome β subunits using 240 DNA samples from four ethnic groups and the β5 subunit gene in 79 DNA samples from multiple myeloma patients who had been treated with the proteasome inhibitor bortezomib. Resequencing was followed by functional studies of polymorphisms identified in the coding region and 3′-flanking region (3′-FR) of PSMB5, the gene encoding the target for clinically useful proteasome inhibitors. Results: Resequencing of 240 DNA samples identified a series of novel ethnic-specific polymorphisms that are not represented in public databases. The PSMB5 3′-FR 1042 G allele significantly increased transcription during reporter gene studies, observations confirmed by genotype-phenotype correlations between single nucleotide polymorphisms (SNP) in PSMB5 and mRNA expression in the 240 lymphoblastoid cell lines from which the resequenced DNA was obtained. Studies with patient DNA samples identified additional novel PSMB5 polymorphisms, including a SNP and an insertion in the 3′-FR. Reporter-gene studies indicated that these two novel polymorphisms might decrease transcription. Conclusions: These results show that nonsynonymous coding SNPs in the PSMB5 gene did not show significant effects on proteasome activity, but SNPs did influence transcription. Future studies might focus on regulatory region polymorphisms.

Abstract Purpose: Colon tumors with defective DNA mismatch repair (dMMR) have a well-characterized phenotype and accounts for ∼15% to 20% of sporadic colon cancer as well as those colon cancer patients with Lynch syndrome. Although the presence of dMMR seems to be a favorable prognostic marker, data suggest that these patients do not respond as well to adjuvant chemotherapy. Experimental Design: In this study, we examined the prognostic significance of tumor MMR deficiency and the presence of a specific mutation in BRAF (V600E) in a group of patients (n = 533) who participated in a randomized prospective clinical trial through the North Central Cancer Treatment Group. Results: Tumors with dMMR were found to be associated with higher tumor grade (P = 0.001), proximal location (P &lt; 0.0001), and improved overall and disease-free survival (P = 0.05 and 0.04, respectively). Among all cases examined, evaluation of the BRAF V600E mutation status revealed no statistically significant differences in either disease-free or overall survival. Patients were then grouped into four categories for further analysis: dMMR/BRAF(−), dMMR/BRAF(+), pMMR/BRAF(−), and pMMR/BRAF(+). The dMMR/BRAF(−) group had a significantly improved overall survival (5-year overall survival of 100% versus 73%, P = 0.002) compared with all others. The remaining three groups had very similar survival outcomes. An additional cohort of tumors previously classified as having dMMR were also tested for the BRAF V600E alteration. Results remained significant (P = 0.006) when the two groups were combined for analysis. Conclusions: Overall, these data suggest that the underlying molecular etiology of those tumors having dMMR may influence the disease outcome in these patients.