Cytochrome oxidase subunit VI of Trypanosoma brucei is imported without a cleaved presequence and is developmentally regulated at both RNA and protein levels

Mitochondrial respiration in the African trypanosome undergoes dramatic developmental stage regulation. This requires co-ordinated control of components encoded by both the nuclear genome and the kinetoplast, the unusual mitochondrial genome of these parasites. As a model for understanding the co-ordination of these genomes, we have examined the regulation and mitochondrial import of a nuclear-encoded component of the cytochrome oxidase complex, cytochrome oxidase subunit VI (COXVI). By generating transgenic trypanosomes expressing intact or mutant forms of this protein, we demonstrate that COXVI is not imported using a conventional cleaved presequence and show that sequences at the N-terminus of the protein are necessary for correct mitochondrial sorting. Analyses of endogenous and transgenic COXVI mRNA and protein expression in parasites undergoing developmental stage differentiation demonstrates a temporal order of control involving regulation in the abundance of, first, mRNA and then protein. This represents the first dissection of the regulation and import of a nuclear-encoded protein into the cytochrome oxidase complex in these organisms, which were among the earliest eukaryotes to possess a mitochondrion

Clinicopathologic Characteristics of 23 Cases of Invasive Low-grade Papillary Urothelial Carcinoma.

OBJECTIVE: To investigate the clinicopathologic and immunohistochemical features associated with invasive low-grade papillary urothelial carcinoma (LGPUC), an uncommon entity not previously described in published studies. METHODS: A multicenter effort originally identified 36 cases diagnosed as invasive LGPUC by urologic pathology subspecialists; after re-review, 23 cases were included. RESULTS: The average patient age was 69 years (range 46-82); 20 patients were men and 3 were women. Stage pT1 disease was present in 19 (83%) of 23 and pT2 disease in 4 patients. Of the 23 cases, 13 (57%) showed a single focus of invasion and 10 multiple foci. The invasive front showed rounded, variably sized nests in 17 cases (74%) and irregular nests with retraction artifact in 6. Additional findings in the noninvasive component included inverted growth in 23, apoptotic debris in 5, focal brisk mitotic activity in 4, dispersed chromatin in individual cells in 4, and a single atypical cell in 2. Immunohistochemical stains showed focal p53 nuclear stain in 23, patchy full-thickness cytokeratin 20 stain in 20, full-thickness CD44 expression in 17, and retention of E-cadherin in 23 cases. Clinical follow-up was available for all patients. The subsequent diagnosis included papilloma in 1 patient (4%), LGPUC in 5 (22%), and high-grade papillary urothelial carcinoma in 8 (35%) of the 23 patients, with 5 demonstrating invasion. Of the latter patients, 2 developed metastatic disease. CONCLUSION: Given the risk of progressive disease in these patients, especially the limited stage pT1 disease in most patients, additional studies investigating the molecular properties and outcomes associated with this uncommon lesion are warranted

Global Transcription Analysis of Krebs Tricarboxylic Acid Cycle Mutants Reveals an Alternating Pattern of Gene Expression and Effects on Hypoxic and Oxidative Genes

To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in α-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth–enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in α-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle

Prognostic markers in invasive bladder cancer: FGFR3 mutation status versus P53 and KI-67 expression: a multi-center, multi-laboratory analysis in 1058 radical cystectomy patients

Objectives: To determine the association between the FGFR3 mutation status and immuno-histochemistry (IHC) markers (p53 and Ki-67) in invasive bladder cancer (BC), and to analyze their prognostic value in a multicenter, multi-laboratory radical cystectomy (RC) cohort. Patients and methods: We included 1058 cN0M0, chemotherapy-naive BC patients who underwent RC with pelvic lymph-node dissection at 8 hospitals. The specimens were reviewed by uro-pathologists. Mutations in the FGFR3 gene were examined using PCR-SNaPshot; p53 and Ki-67 expression were determined by standard IHC. FGFR3 mutation status as well as p53 (cut-off>10%) and Ki-67 (cut-off>20%) expression were correlated to clinicopathological parameters and disease specific survival (DSS). Results: pT-stage was <pT2 in 80, pT2 in 266, pT3 in 513 and pT4 in 199 patients, respectively. Cancer-positive nodes were found in 410 (39%) patients. An FGFR3 mutation was detected in 107 (10%) and aberrant p53 and Ki-67 expression in 718 (68%) and 581(55%) tumors, respectively. The FGFR3 mutation was associated with lower pT-stage (P<0.001), lower grade (P<0.001), pN0 (P=0.001) and prolonged DSS (P<0.001). Aberrant Ki-67 and p53 expression were associated with higher pT-stage and G3-tumors, but not with pN-stage or worse DSS, even if these IHC-biomarkers were combined (P=0.81). Significant predictors for DSS in multivariable analysis were pT-stage (HR1.5, 95%CI:1.3-1.6; P<0.001), lympho-vascular invasion (LVI) (HR1.4, 95%CI:1.2-1.7; P=0.001), pN-stage (HR1.9, 95%CI:1.6-2.4; P<0.001) and FGFR3 mutation status (HR1.6, 95%CI:1.1-2.2; P=0.011). Conclusion: The FGFR3 mutation selectively identified patients with favorable BC at RC while p53 and Ki-67 were only associated with adverse tumor characteristics. Our results suggest that, besides tumor-stage, nodal-status and LVI, the oncogenic FGFR3 mutation may represent a valuable tool to guide adjuvant treatment and follow-up strategies after RC