An ER clamp for endosome fission

Endosomes are known to undergo budding and fission reactions that separate regions destined for lysosomal degradation from carriers to be recycled to the plasma membrane. A recent paper (Rowland et al, 2014 ) shows that contact sites between endosomes and the endoplasmic reticulum (ER) define the position and timing for fission. This uncovers an unanticipated role for the ER in controlling endosomal sorting and maturation.     

Production of phosphatidylinositol 5‐phosphate via PIKfyve and MTMR3 regulates cell migration

Although phosphatidylinositol 5‐phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3‐kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P‐binding motifs, we identified the phosphoinositide 5‐kinase PIKfyve and the phosphoinositide 3‐phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P₂. The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P‐producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.     

Stereotactic Body Radiation Therapy for Primary and Metastatic Liver Tumors

OBJECTIVES: The full potential of stereotactic body radiation therapy (SBRT), in the treatment of unresectable intrahepatic malignancies, has yet to be realized as our experience is still limited. Thus, we evaluated SBRT outcomes for primary and metastatic liver tumors, with the goal of identifying factors that may aid in optimization of therapy. METHODS: From2005 to 2010, 62 patients with 106 primary and metastatic liver tumors were treated with SBRT to a median biologic effective dose (BED) of 100 Gy (42.6-180). The majority of patients received either three (47%) or five fractions (48%). Median gross tumor volume (GTV) was 8.8 cm³ (0.2-222.4). RESULTS: With a median followup of 18 months (0.46-46.8), freedom from local progression (FFLP) was observed in 97 of 106 treated tumors, with 1- and 2-year FFLP rates of 93% and 82%. Median overall survival (OS) for all patients was 25.2 months, with 1- and 2-year OS of 81%and 52%. Neither BED nor GTV significantly predicted for FFLP. Local failure was associated with a higher risk of death [hazard ratio (HR) = 5.1, P = .0007]. One Child-Pugh Class B patient developed radiationinduced liver disease. There were no other significant toxicities. CONCLUSIONS: SBRT provides excellent local control for both primary and metastatic liver lesions with minimal toxicity. Future studies should focus on appropriate selection of patients and on careful assessment of liver function to maximize both the safety and efficacy of treatment.     

Phosphorylation of Hrs downstream of the epidermal growth factor receptor.

The hepatocyte growth factor-regulated tyrosine kinase substrate Hrs is an early endosomal protein that is thought to play a regulatory role in the trafficking of growth factor/receptor complexes through early endosomes. Stimulation of cells with epidermal growth factor (EGF) rapidly leads to phosphorylation of Hrs, raising the question whether the receptor tyrosine kinase phosphorylates Hrs directly. Here, we present evidence that a downstream kinase, rather than the active receptor kinase is responsible. We show that the nonreceptor tyrosine kinase Src is able to phosphorylate Hrs in vitro and in vivo, but that Hrs is nevertheless phosphorylated in Src-, Yes- and Fyn-negative cells. Moreover, we show that only 10-20% of Hrs is phosphorylated following EGF stimulation, and that phosphorylation occurs at multiple tyrosines located in different parts of Hrs. These results suggest that Hrs is a substrate for several kinases downstream of the EGF receptor.     

Production of Diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011

Background  

Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP.     

Modelling the transition from simple to complex Ca2+oscillations in pancreatic acinar cells

A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP₃) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca²⁺ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP₃ is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.     

Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants.

We have developed a system to study the interactions of diphtheria toxin with the cell surface using non-toxic mutant proteins synthesized in vitro. Proteins obtained by N-terminal deletions containing the whole B fragment bound strongly to cells. Deletions extending into the B fragment did not yield an autonomous binding domain. Loss of only the N-terminal 3 kd of the B fragment significantly impaired the ability to recognize the receptor. This, together with previous reports that the C-terminal end of the B fragment is required for binding, suggests that both ends of the B fragment are necessary for receptor recognition. Receptor bound diphtheria toxin undergoes a conformational change at pH less than 5.3 that results in translocation of the A fragment to the cytosol and the appearance of a B fragment-derived 25 kd polypeptide (P25) resistant to externally applied protease. Only the B fragment was required for generation of P25. N-terminal deletions of 130 amino acids or more resulted in proteins that gave rise to P25 at higher pH than full length toxin. Furthermore, a second protease-inaccessible polypeptide of 18 kd (P18) was observed.     

Permeabilization of the plasma membrane by deletion mutants of diphtheria toxin.

Diphtheria toxin B-fragment binds to cell-surface receptors and facilitates translocation of the enzymatically active A-fragment to the cytosol. In this process the B-fragment inserts into the plasma membrane and induces formation of cation-selective channels. We examined the ability of a number of diphtheria toxin-derived molecules translated in vitro to permeabilize cells. Two proteins consisting of the whole B-fragment and small parts of the A-fragment, and one protein comprising most of the B-fragment alone, were more efficient than full-length toxin in permeabilizing the plasma membrane to monovalent cations. Two shorter B-fragment-derived proteins, with 3 and 10 kd N-terminal deletions, permeabilized the cells to sulfate and sucrose in addition to monovalent cations. The relationship between channel formation and toxin translocation is discussed.