Citations

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca 2+ -binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (∼300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.

Abstract An abnormal serum immunoglobulin free light chain (FLC) ratio at diagnosis may identify risk of progression to myeloma in patients with solitary bone plasmacytoma (SBP). In the cohort of 116 patients, 43 have progressed to myeloma, with a median time to progression of 1.8 years. The FLC ratio was determined in all 116 patients on serum collected at time of diagnosis and was abnormal in 54 patients (47%). An abnormal FLC ratio was associated with a higher risk of progression to myeloma (P = .039). The risk of progression at 5 years was 44% in patients with an abnormal serum FLC ratio at diagnosis compared with 26% in those with a normal FLC ratio. One to 2 years following diagnosis, a persistent serum M protein level of 5 g/L (0.5 g/dL) or higher was an additional risk factor for progression. A risk stratification model was constructed using the 2 variables of FLC ratio and M protein level: patients with a normal FLC ratio at baseline and M protein level less than 5 g/L (0.5 g/dL) at 1 to 2 years following diagnosis (low risk, n = 31); with either risk factor abnormal (intermediate risk, n = 26); and with both an abnormal FLC ratio and M protein level of 5 g/L (0.5 g/dL) or higher (high risk, n = 18). The corresponding progression rates at 5 years were significantly different in the low, intermediate, and high groups: 13%, 26%, and 62%, respectively (P < .001).

The electron transport system of muscle mitochondria was examined in a familial syndrome of lactacidemia, mitochondrial myopathy, and encephalopathy. The propositus, a 14-year-old female, and her 12-year-old sister had suffered from progressive muscle weakness, abnormal fatigability, and central nervous system dysfunction since early childhood. In the propositus, the state 3 respiratory rate of muscle mitochondria with NADH-linked substrates and with succinate was markedly reduced. The levels of cytochromes a + a3, b, and c + c1 were normal. The activities of complexes I, II, III, and IV of the electron transport chain were normal or increased. By contrast, the activities of complex I-III and of complex II-III, both of which need coenzyme Q10 (CoQ10), were abnormally low. On direct measurement, the mitochondrial CoQ10 content was 3.7% of the mean value observed in 10 controls. Serum and cultured fibroblasts of the propositus had normal CoQ10 contents. In the younger sister, the respiratory activities and CoQ10 level of muscle mitochondria were similar to those observed in the propositus. The findings establish CoQ10 deficiency as a cause of a familial mitochondrial cytopathy and suggest that the disease results from a tissue-specific defect of CoQ10 biosynthesis.

The potential of nonterminal cellular differentiation to stably repress the expression of the neoplastic phenotype of transformed cells is established. Nonterminal differentiation induces spontaneously transformed 3T3 T cells to revert to a nontransformed state and induces the revertant cell clones to become resistant to retransformation by UV irradiation or 4-nitroquinoline oxide treatment. Nonterminal differentiation also induces simian virus 40-transformed 3T3 T cells to repress expression of the large tumor antigen and to revert to a nontransformed state. Although the molecular mechanisms that mediate these and other forms of anticancer activity have not been definitively established, data are presented which suggest that differentiation-induced repression of large tumor antigen expression can be regulated at the level of transcription and/or RNA processing.

In a congenital myasthenic syndrome with a severe endplate myopathy, patch-clamp studies revealed markedly prolonged acetylcholine receptor (AChR) channel openings. Molecular genetic analysis of AChR subunit genes demonstrated a heterozygous adenosine-to-cytosine transversion at nucleotide 790 in exon 8 of the epsilon-subunit gene, predicting substitution of proline for threonine at codon 264 and no other mutations in the entire coding sequences of genes encoding the alpha, beta, delta, and epsilon subunits. Genetically engineered mutant AChR expressed in a human embryonic kidney fibroblast cell line also exhibited markedly prolonged openings in the presence of agonist and even opened in its absence. The Thr-264-->Pro mutation in the epsilon subunit involves a highly conserved residue in the M2 domain lining the channel pore and is likely to disrupt the putative M2 alpha-helix. Our findings indicate that a single mutation at a critical site can greatly alter AChR channel kinetics, leading to a congenital myasthenic syndrome. This observation raises the possibility that mutations involving subunits of other ligand-gated channels may also exist and be the basis of various other neurologic or psychiatric disorders.

OBJECTIVETo review the recent innovations in hearing aid technology.STUDY DESIGNReview of the literature.SETTINGPrivate tertiary referral otology/neurotology practice.RESULTSRecent innovations including digital signal processing (DSP), directional microphone technology, and open mold hearing aid fitting have resulted in improved satisfaction with current hearing aids.CONCLUSIONCurrent hearing aid technology with its innovations better meet needs of individuals who are hard of hearing.SIGNIFICANCEThe image commonly held of hearing aids by the general public is a negative one that can be improved with greater education.

ABSTRACTSeveral studies have demonstrated roles for eosinophils during innate and adaptive immune responses to helminth infections. However, evidence that eosinophils are capable of initiating an immune response to parasite antigens is lacking. The goal of the present in vitro study was to investigate the potential of eosinophils to serve as antigen-presenting cells (APC) and initiate an immune response to parasite antigens. Purified eosinophils were exposed to solubleStrongyloides stercoralisantigens, and the expression of various surface markers involved in cell activation was examined. Antigen-exposed eosinophils showed a sixfold increase in expression levels of CD69 and major histocompatibility complex (MHC) class II, a fourfold increase in levels of T-cell costimulatory molecule CD86, and a twofold decrease in levels of CD62L compared to eosinophils cultured in medium containing granulocyte-macrophage colony-stimulating factor. The ability of eosinophils to present antigen to T cells was determined by culturing them with T cells in vitro. Eosinophils pulsed with antigen stimulated antigen-specific primed T cells and CD4+T cells to increase interleukin-5 (IL-5) production. The blocking of MHC class II expression on eosinophils inhibited their ability to induce IL-5 production by CD4+T cells in culture. Antigen-pulsed eosinophils were able to prime naïve T cells and CD4+T cells in culture and polarized them into Th2 cells producing IL-5 similar to that induced by antigen-loaded dendritic cells. These results demonstrate that eosinophils are capable of activating antigen-specific Th2 cells inducing the release of cytokines and assist in the priming of naïve T cells to initiate Th2 responses against infection. This study highlights the potential of eosinophils to actively induce immune responses against infection by amplifying antigen-specific Th2-cell responses.

Background— The importance of completeness of revascularization by percutaneous coronary intervention in patients with multivessel disease is unclear in that there is little information on the impact of incomplete revascularization outside of randomized trials. The objective of this study is to compare long-term mortality and subsequent revascularization for percutaneous coronary intervention patients receiving stents who were completely revascularized (CR) with those who were incompletely revascularized (IR). Methods and Results— Patients from New York State’s Percutaneous Coronary Interventions Reporting System were subdivided into patients who were CR and IR. Then subsets of IR patients were contrasted with CR patients. Differences in long-term survival and subsequent revascularization for CR and IR patients were compared after adjustment for differences in preprocedural risk. A total of 68.9% of all stent patients with multivessel disease who were studied were IR, and 30.1% of all patients had total occlusions and/or ≥2 IR vessels. At baseline, the following patients were at higher risk: those who were older and those with more comorbid conditions, worse ejection fraction, and more renal disease and stroke. After adjustment for these baseline differences, IR patients were significantly more likely to die at any time (adjusted hazard ratio=1.15; 95% confidence interval, 1.01 to 1.30) than CR patients. IR patients with total occlusions and a total of ≥2 IR vessels were at the highest risk compared with CR patients (hazard ratio=1.36; 95% confidence interval, 1.12 to 1.66). Conclusions— IR with stenting is associated with an adverse impact on long-term mortality, and consideration should be given to either achieving CR, opting for surgery, or monitoring percutaneous coronary intervention patients with IR more closely after discharge.

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.