A Novel Approach to Recovery of Function of Mutant Proteins by Slowing Down Translation

Protein homeostasis depends on a balance of translation, folding, and degradation. Here, we demonstrate that mild inhibition of translation results in a dramatic and disproportional reduction in production of misfolded polypeptides in mammalian cells, suggesting an improved folding of newly synthesized proteins. Indeed, inhibition of translation elongation, which slightly attenuated levels of a copepod GFP mutant protein, significantly enhanced its function. In contrast, inhibition of translation initiation had minimal effects on copepod GFP folding. On the other hand, mild suppression of either translation elongation or initiation corrected folding defects of the disease-associated cystic fibrosis transmembrane conductance regulator mutant F508del. We propose that modulation of translation can be used as a novel approach to improve overall proteostasis in mammalian cells, as well as functions of disease-associated mutant proteins with folding deficiencies.     

Electrokinetic properties of isolated cerebral-cortex synaptic vesicles

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.     

Defective Autophagy and mTORC1 Signaling in Myotubularin Null Mice

Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.     

Benthic cyanobacterial bloom impacts the reefs of South Florida (Broward County, USA)

Benthic cyanobacteria of the genus Lyngbya can form prominent mats and blooms in tropical and subtropical coral reef and seagrass habitats worldwide. A Lyngbya bloom on the reef tract offshore of Broward County, Florida, was first noted in 2002, and although it is seasonally variable in its distribution and abundance, it has persisted and spread over the past 3 years. In this study, the most abundant species of Lyngbya found in the blooms have been identified and compared to other species of Lyngbya by morphological and molecular methods. The most common species of Lyngbya is consistent with the properties of Lyngbya confervoides C. Agardh. The 16S ribosomal DNA sequence shares 88–92% identity with other known Lyngbya sequences, suggesting that this bloom consists primarily of a new, previously unsequenced species of Lyngbya. The second most common Lyngbya in the bloom is consistent with Lyngbya polychroa. This persistent bloom is a concern because it smothers octocorals and other invertebrates and negatively impacts these southeastern Florida reefs.     

Progressive induction of β-glucuronidase in individual kidney epithelial cells

The magnitude and kinetics of β-glucuronidase induction in mouse kidney are determined by a cis-acting regulatory gene, Gus-r, that is closely linked to the enzyme structural gene. The accumulation of β-glucuronidase mRNA during induction is much slower than the turnover time of the mRNA, suggesting progressive acquisition of mRNA synthesizing capacity during induction. Counts of the numbers of induced cells present at various times of induction in strains carrying three different alleles of Gus-r show that all potentially responsive cells respond immediately. The level of induction is progressive in individual cells and does not involve continued recruitment of new cells into the induced population. It appears that during induction each chromosome becomes progressively more active in directing the synthesis of β-glucuronidase.     

The Genetic Algorithm and the Conformational Search of Polypeptides and Proteins

Abstract

The genetic algorithm is a technique of function optimization derived from the principles of evolutionary theory. We have adapted it to perform conformational search on polypeptides and proteins. The algorithm was first tested on several small polypeptides and the 46 amino acid protein crambin under the AMBER potential energy function. The probable global minimum conformations of the polypeptides were located 90% of the time and a non-native conformation of crambin was located that was 150kcal/mol lower in potential energy than the minimized crystal structure conformation. Next, we used a knowledge-based potential function to predict the structures of melittin, pancreatic polypeptide, and crambin. A 2.31 Å ΔRMS conformation of melittin and a 5.33 Å ΔRMS conformation of pancreatic polypeptide were located by genetic algorithm-based conformational search under the knowledge-based potential function. Although the ΔRMS of pancreatic polypeptide was somewhat high, most of the secondary structure was correct. The secondary structure of crambin was predicted correctly, but the potential failed to promote packing interactions. Finally, we tested the packing aspects of our potential function by attempting to predict the tertiary structure of cytochrome b ₅₆₂ given correct secondary structure as a constraint. The final predicted conformation of cytochrome b ₅₆₂ was an almost completely extended continuous helix which indicated that the knowledge-based potential was useless for tertiary structure prediction. This work serves as a warning against testing potential functions designed for tertiary structure prediction on small proteins.     

Determinants of Survival in Malignant Pleural Mesothelioma: A Surveillance,Epidemiology, and End Results (SEER) Study of 14,228 Patients

Introduction

Left untreated, malignant pleural mesothelioma (MPM) is associated with uniformly poor prognosis. Better survival has been reported with surgery-based multimodality therapy, but to date, no trial has demonstrated survival benefit of surgery over other therapies. We evaluated whether cancer-directed surgery influenced survival independently from other predictors in a large population-based dataset.

Methods

The SEER database was explored from 1973 to 2009 to identify all cases of pathologically-proven MPM. Age, sex, race, year of diagnosis, histology stage, cancer-directed surgery, radiation, and vital status were analyzed. The association between prognostic factors and survival was estimated using Cox regression and propensity matched analysis.

Results

There were 14,228 patients with pathologic diagnosis of MPM. On multivariable analysis, female gender, younger age, early stage, and treatment with surgery were independent predictors of longer survival. In comparison to no treatment, surgery alone was associated with significant improvement in survival [adjusted hazard ratio (adj HR) 0.64 (0.61–0.67)], but not radiation [adj HR 1.15 (1.08–1.23)]. Surgery and radiation combined had similar survival as surgery alone [adj HR 0.69 (0.64–0.76)]. Results were similar when cases diagnosed between 1973 and 1999 were compared to cases diagnosed between 2000 and 2009.

Conclusions

Despite developments in surgical and radiation techniques, the prognosis for MPM patients has not improved over the past 4 decades. Cancer-directed surgery is independently associated with better survival, suggesting that multimodal surgery-based therapy can benefit these patients. Further research in adjuvant treatment is necessary to improve prognosis in this challenging disease.     

Are rates of species diversification correlated with rates of morphological evolution?

Some major evolutionary theories predict a relationship between rates of proliferation of new species (species diversification) and rates of morphological divergence between them. However, this relationship has not been rigorously tested using phylogeny-based approaches. Here, we test this relationship with morphological and phylogenetic data from 190 species of plethodontid salamanders. Surprisingly, we find that rates of species diversification and morphological evolution are not significantly correlated, such that rapid diversification can occur with little morphological change, and vice versa. We also find that most clades have undergone remarkably similar patterns of morphological evolution (despite extensive sympatry) and that those relatively novel phenotypes are not associated with rapid diversification. Finally, we find a strong relationship between rates of size and shape evolution, which has not been previously tested.