Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome

The cell-cycle regulator p21(Cip1) is degraded by proteasomes independently of ubiquitination. We now show that degradation of p21 in vivo does not require the 19S proteasome lid, which contains the ubiquitin-binding subunit. Instead, the major proteasomal pathway for p21 degradation involves an alternative proteasome lid, the REGgamma complex. REGgamma binds to p21 in vivo, and deletion of p21's REGgamma-binding site greatly extends its half-life. Knockdown of REGgamma by RNA interference stabilizes p21, p21 has a significantly extended half-life in REGgamma(-/-) murine embryonic fibroblasts, and the p21 abundance is elevated in REGgamma(-/-) mice. The role of REGgamma in cell-cycle regulation may extend beyond p21 regulation, because p16(INK4A) and p19(Arf) also bind to REGgamma and are stabilized in REGgamma-deficient cells.     

Classification and prediction of clinical Alzheimer's diagnosis based on plasma signaling proteins

A molecular test for Alzheimer's disease could lead to better treatment and therapies. We found 18 signaling proteins in blood plasma that can be used to classify blinded samples from Alzheimer's and control subjects with close to 90% accuracy and to identify patients who had mild cognitive impairment that progressed to Alzheimer's disease 2-6 years later. Biological analysis of the 18 proteins points to systemic dysregulation of hematopoiesis, immune responses, apoptosis and neuronal support in presymptomatic Alzheimer's disease.     

Computational design of antibody-affinity improvement beyond in vivo maturation

Antibodies are used extensively in diagnostics and as therapeutic agents. Achieving high-affinity binding is important for expanding detection limits, extending dissociation half-times, decreasing drug dosages and increasing drug efficacy. However, antibody-affinity maturation in vivo often fails to produce antibody drugs of the targeted potency, making further affinity maturation in vitro by directed evolution or computational design necessary. Here we present an iterative computational design procedure that focuses on electrostatic binding contributions and single mutants. By combining multiple designed mutations, a tenfold affinity improvement to 52 pM was engineered into the anti-epidermal growth factor receptor drug cetuximab (Erbitux), and a 140-fold improvement in affinity to 30 pM was obtained for the anti-lysozyme model antibody D44.1. The generality of the methods was further demonstrated through identification of known affinity-enhancing mutations in the therapeutic antibody bevacizumab (Avastin) and the model anti-fluorescein antibody 4-4-20. These results demonstrate computational capabilities for enhancing and accelerating the development of protein reagents and therapeutics.     

Concordant evolution of mitochondrial and nuclear genomes in the wheat pathogen Phaeosphaeria nodorum

We compared patterns of mitochondrial restriction fragment length polymorphism (RFLP) diversity with patterns of nuclear RFLP diversity to investigate the effects of selection, gene flow, and sexual reproduction on the population genetic structure and evolutionary history of the wheat pathogen Phaeosphaeria nodorum. A total of 315 fungal isolates from Texas, Oregon, and Switzerland were analyzed using seven nuclear RFLP probes that hybridized to discrete loci and purified mitochondrial DNA that hybridized to the entire mtDNA genome. Forty-two different mitochondrial haplotypes and 298 different nuclear haplotypes were detected. The two most frequent mtDNA haplotypes were present in every population and represented 32% of all isolates. High levels of gene flow, low levels of population subdivision, no evidence for either host specificity or cyto-nuclear disequilibrium were inferred from the analysis of both genomes. The concordance in estimates of these population genetic parameters from both genomes suggests that the two genomes experienced similar degrees of migration, genetic drift and selection.     

Anthropology and the Dialectic of Enlightenment: A Discourse on the Definition and Ideals of a Threatened Discipline

The knowledge practices of social and cultural anthropology can be conceived as undergoing constant methodological reconsideration or reformulation as a consequence of internal critique and of institutional change effected in the larger educational and political environment. Neo‐liberal shifts affecting the institutional context may have influenced a deepening of the crisis in anthropology where the nature of its project has become less certain or has threatened a reconfiguration of such proportion that anthropology may be losing sight of its direction. This essay explores some of the Enlightenment roots of social and cultural anthropology. It is presented as very much an idea that embodies and reflects what Adorno and Horkheimer discussed as the dialectic of Enlightenment. The argument presented is less pessimistic claiming that the distinction of anthropology is in its pursuit of Enlightenment ideals that it has maintained and rehoned as a consequence of its own routine internal critique. The vital implication of the discussion is that anthropology is in a situation of serious threat in largely a post‐Enlightenment world. In such a context, the methodological ideals that emerged as integral to the spirit of anthropology are well worth maintaining rather than abandoning. The ideals that are addressed are conceived to be integral to the importance of anthropology as critique and as a knowledge practice capable of sustaining a profound contribution to the understanding of the potential that is human being.     

Secretory phospholipases A2 stimulate mucus secretion, induce airway inflammation, and produce secretory hyperresponsiveness to neutrophil elastase in ferret trachea

Secretory phospholipases A(2) (sPLA(2)) are increased in the bronchoalveolar lavage fluid of patients with asthma and acute respiratory distress syndrome. Intratracheal sPLA(2) instillation induces acute lung injury in the rat and guinea pig. We hypothesized that sPLA(2) would stimulate mucus secretion in vitro and that intratracheal sPLA(2) exposure would induce mucus hypersecretion and airway inflammation in the ferret trachea in vivo. In vitro, porcine pancreatic sPLA(2) at a concentration of 0.5 or 5 U/ml significantly increased mucous glycoconjugate (MG) secretion from the excised ferret trachea. P-bromophenacylbromide (a sPLA(2) inhibitor), quercetin (a lipoxygenase inhibitor), or MK-886 (a 5-lipoxygenase inhibitor), each at 10(-4) M, significantly reduced sPLA(2)-induced MG secretion. sPLA(2)-stimulated MG secretion was decreased in Ca(2+)-free medium. In vivo, ferrets were intubated for 30 min once per day for 3 days using an ETT coated with 20 units of porcine pancreatic sPLA(2) mixed in water-soluble jelly. Constitutive MG secretion increased 1 day after sPLA(2) exposure and returned to control 5 days later. Human neutrophil elastase (HNE) at 10(-8) M increased MG secretion in the sPLA(2)-exposed trachea compared with that in the control trachea, but methacholine at 10(-7) M did not. sPLA(2)-induced secretory hyperresponsiveness continued for at least 5 days after sPLA(2) exposure ended. sPLA(2) increased tracheal inflammation, MG secretion, and secretory hyperresponsiveness to HNE probably through enzymatic action rather than by activation of its receptor.     

Sustained biological effects of porcine interleukin 5 delivered to pigs as recombinant protein or via a DNA vector

The ability of cytokines to act as natural immunotherapeutics to enhance the health and the disease resistance of animals is of particular interest to the intensive livestock industries. Antibiotics have been used for such purposes over a long period of time, however, there is growing concern that this practice will enhance the development of antibiotic resistance in a range of bacterial pathogens. In several species, interleukin 5 (IL-5) is known to enhance B cell activity and to increase the numbers of eosinophils in blood and tissues. In this report, IL-5 was delivered to pigs, either as a recombinant protein or via a DNA delivery vector and was shown to elevate eosinophils in blood over a sustained period. Interleukin 3, a potent haemopoietic factor, did not synergise with IL-5 when both cytokines were given together, but did prime the pigs for a stronger response to IL-5. These results demonstrate that IL-5 can readily be delivered to commercial pigs to elicit a significant biological effect.     

Six months of supervised high-intensity low-volume resistance training improves strength independent of changes in muscle mass in young overweight men

To determine the effects of a 6-month supervised low-volume resistance training (RT) program (1 set, 85-90%, one repetition maximum, 1RM, 3 d x wk(-1)) on muscular strength (1RM) and skeletal muscle mass (SMM) in previously sedentary, overweight men on an ad libitum diet. Nineteen men were randomly assigned to a control (CON, n = 8) or RT (n = 11) group. The exercise protocol consisted of 5 upper- and 4 lower-body exercises using weight machines. CON maintained their sedentary lifestyle. One RM for upper body (chest press [CP] + lat pull-down [LPD]) and lower body (leg press [LP]) and SMM were assessed at baseline, and at 3 and 6 months. Adherence was 96 +/- 2% with an average time to complete each exercise session of 15 +/- 2 minutes. Volume completed per exercise session significantly increased from baseline (2,812 +/- 670 kg) to 6 months (6,411 +/- 2,128 kg). There was a group by time interaction in 1RM for CP, LPD, and LP. Upper-body strength increased significantly (p < 0.001) (31.3 +/- 9.3%) from baseline to 3 months and from 3 to 6 months (17.9 +/- 8.7%). Lower-body strength also increased significantly from baseline to 3 months (17.8 +/- 16.6%) and from 3 to 6 months (32.0 +/- 33.7%). No changes in upper- or lower-body strength occurred in the CON group. There was no group by time interaction for SMM (CON, 34.5 +/- 2.9 kg vs. RT, 34.2 +/- 2.9 kg; p > 0.05) or for energy intake (p > 0.05). In conclusion, a single set resistance training program at 85% of 1RM, 3 d x wk(-1) resulted in continued increases in muscular strength and a very high adherence rate over a 6-month period in sedentary, overweight men independent of significant changes in SMM. This training protocol may increase adherence and produce long-term increases in muscular fitness as part of an adult fitness program.