Modeling the Role of Negative Cooperativity in Metabolic Regulation and Homeostasis

A significant proportion of enzymes display cooperativity in binding ligand molecules, and such effects have an important impact on metabolic regulation. This is easiest to understand in the case of positive cooperativity. Sharp responses to changes in metabolite concentrations can allow organisms to better respond to environmental changes and maintain metabolic homeostasis. However, despite the fact that negative cooperativity is almost as common as positive, it has been harder to imagine what advantages it provides. Here we use computational models to explore the utility of negative cooperativity in one particular context: that of an inhibitor binding to an enzyme. We identify several factors which may contribute, and show that acting together they can make negative cooperativity advantageous.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Complementary strand analysis: a new approach for allelic separation in complex polyallelic genetic systems.

We describe a method, complementary strand analysis (CSA), for separating alleles potentially from any heterozygous genetic locus. Locus specific PCR is performed generating two allelic products. The antisense strands are isolated and hybridised with a sense reference strand to form a chimeric DNA duplex for each allele which is then separated by non-denaturing PAGE. We demonstrate the application of CSA for separation of highly polymorphic HLA-A, -B and -Cw alleles and characterisation of HLA identity in related bone marrow donors and patients. CSA is capable of resolving one nucleotide differences in a DNA fragment nearly as large as a kilobase in length.     

Comparison of point counts and territory mapping for detecting effects of forest management on songbirds

Point counts are commonly used to assess changes in bird abundance, including analytical approaches such as distance sampling that estimate density. Point‐count methods have come under increasing scrutiny because effects of detection probability and field error are difficult to quantify. For seven forest songbirds, we compared fixed‐radii counts (50 m and 100 m) and density estimates obtained from distance sampling to known numbers of birds determined by territory mapping. We applied point‐count analytic approaches to a typical forest management question and compared results to those obtained by territory mapping. We used a before–after control impact (BACI) analysis with a data set collected across seven study areas in the central Appalachians from 2006 to 2010. Using a 50‐m fixed radius, variance in error was at least 1.5 times that of the other methods, whereas a 100‐m fixed radius underestimated actual density by >3 territories per 10 ha for the most abundant species. Distance sampling improved accuracy and precision compared to fixed‐radius counts, although estimates were affected by birds counted outside 10‐ha units. In the BACI analysis, territory mapping detected an overall treatment effect for five of the seven species, and effects were generally consistent each year. In contrast, all point‐count methods failed to detect two treatment effects due to variance and error in annual estimates. Overall, our results highlight the need for adequate sample sizes to reduce variance, and skilled observers to reduce the level of error in point‐count data. Ultimately, the advantages and disadvantages of different survey methods should be considered in the context of overall study design and objectives, allowing for trade‐offs among effort, accuracy, and power to detect treatment effects.     

Primary Cilia Are Lost in Preinvasive and Invasive Prostate Cancer

Prostate cancer is the second most commonly diagnosed cancer in men worldwide. Little is known about the role of primary cilia in preinvasive and invasive prostate cancer. However, reduced cilia expression has been observed in human cancers including pancreatic cancer, renal cell carcinoma, breast cancer, cholangiocarcinoma, and melanoma. The aim of this study was to characterize primary cilia expression in preinvasive and invasive human prostate cancer, and to investigate the correlation between primary cilia and the Wnt signaling pathway. Human prostate tissues representative of stages of prostate cancer formation (normal prostate, prostatic intraepithelial neoplasia (PIN), and invasive prostate cancer (including perineural invasion)) were stained for ciliary proteins. The frequency of primary cilia was determined. A decrease in the percentage of ciliated cells in PIN, invasive cancer and perineural invasion lesions was observed when compared to normal. Cilia lengths were also measured to indirectly test functionality. Cilia were shorter in PIN, cancer, and perineural invasion lesions, suggesting dysfunction. Primary cilia have been shown to suppress the Wnt pathway. Increased Wnt signaling has been implicated in prostate cancer. Therefore, we investigated a correlation between loss of primary cilia and increased Wnt signaling in normal prostate and in preinvasive and invasive prostate cancer. To investigate Wnt signaling in our cohort, serial tissue sections were stained for β-catenin as a measure of Wnt signaling. Nuclear β-catenin was analyzed and Wnt signaling was found to be higher in un-ciliated cells in the normal prostate, PIN, a subset of invasive cancers, and perineural invasion. Our results suggest that cilia normally function to suppress the Wnt signaling pathway in epithelial cells and that cilia loss may play a role in increased Wnt signaling in some prostate cancers. These results suggest that cilia are dysfunctional in human prostate cancer, and increase Wnt signaling occurs in a subset of cancers.     

Epithelial cell population kinetics of isolated ileal loops (thirty-vella fistulae) after 40% small in testinal resection. Autoradiographic studies in the rat.

The influence of 40% small intestinal resection on epithelial cell population kinetics of isolated ileal loops (Thirty-Vella fistulae) was investigated by autoradiography with H3-thymidine in the rat. Sixty days after resection increases were found in the size of the proliferative (0.05 less than P less than 0.1) and villous (P less than 0.01) zones. In isolated ileal loops of animals not subjected to 40% small bowel resection proliferative and villous zones were reduced in size in comparison to those of ileum in continuity in a group subjected to jejunal transection and anastomosis (P less than 0.002). The findings support the hypothesis that villous size is increased after resection by the action of a systemic factor. However, they provide only limited evidence for the influence of a systemic factor on crypt cell proliferation.     

In Vivo Interaction Proteomics Reveal a Novel p38 Mitogen-Activated Protein Kinase/Rack1 Pathway Regulating Proteostasis in Drosophila Muscle

Several recent studies suggest that systemic aging in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila, longevity appears to be tightly linked to myoproteostasis, and the formation of misfolded protein aggregates is a hallmark of senescence in aging muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.g., inclusion body myositis. p38 mitogen-activated protein kinase (MAPK) plays a central role in a conserved signaling pathway activated by a variety of stressful stimuli. Aging p38 MAPK mutant flies display accelerated motor function decline, concomitant with an enhanced accumulation of detergent-insoluble protein aggregates in thoracic muscles. Chemical genetic experiments suggest that p38-mediated regulation of myoproteostasis is not limited to the control of reactive oxygen species production or the protein degradation pathways but also involves upstream turnover pathways, e.g., translation. Using affinity purification and mass spectrometry, we identified Rack1 as a novel substrate of p38 MAPK in aging muscle and showed that the genetic interaction between p38b and Rack1 controls muscle aggregate formation, locomotor function, and longevity. Biochemical analyses of Rack1 in aging and stressed muscle suggest a model whereby p38 MAPK signaling causes a redistribution of Rack1 between a ribosome-bound pool and a putative translational repressor complex.