Interaction between Calpain-1 and HSP90: New Insights into the Regulation of Localization and Activity of the Protease

Here we demonstrate that heat shock protein 90 (HSP90) interacts with calpain-1, but not with calpain-2, and forms a discrete complex in which the protease maintains its catalytic activity, although with a lower affinity for Ca²⁺. Equilibrium gel distribution experiments show that this complex is composed by an equal number of molecules of each protein partner. Moreover, in resting cells, cytosolic calpain-1 is completely associated with HSP90. Since calpain-1, in association with HSP90, retains its proteolytic activity, and the chaperone is displaced by calpastatin also in the absence of Ca²⁺, the catalytic cleft of the protease is not involved in this association. Thus, calpain-1 can form two distinct complexes depending on the availability of calpastatin in the cytosol. The occurrence of a complex between HSP90 and calpain-1, in which the protease is still activable, can prevent the complete inhibition of the protease even in the presence of high calpastatin levels. We also demonstrate that in basal cell conditions HSP90 and calpain-1, but not calpain-2, are inserted in the multi-protein N-Methyl-D-Aspartate receptor (NMDAR) complex. The amount of calpain-1 at the NMDAR cluster is not modified in conditions of increased [Ca²⁺]_i, and this resident protease is involved in the processing of NMDAR components. Finally, the amount of calpain-1 associated with NMDAR cluster is independent from Ca²⁺-mediated translocation. Our findings show that HSP90 plays an important role in maintaining a given and proper amount of calpain-1 at the functional sites.     

Functional Organization of the Action Observation Network in Autism: A Graph Theory Approach

Background

The ability to recognize, understand and interpret other’s actions and emotions has been linked to the mirror system or action-observation-network (AON). Although variations in these abilities are prevalent in the neuro-typical population, persons diagnosed with autism spectrum disorders (ASD) have deficits in the social domain and exhibit alterations in this neural network.

Method

Here, we examined functional network properties of the AON using graph theory measures and region-to-region functional connectivity analyses of resting-state fMRI-data from adolescents and young adults with ASD and typical controls (TC).

Results

Overall, our graph theory analyses provided convergent evidence that the network integrity of the AON is altered in ASD, and that reductions in network efficiency relate to reductions in overall network density (i.e., decreased overall connection strength). Compared to TC, individuals with ASD showed significant reductions in network efficiency and increased shortest path lengths and centrality. Importantly, when adjusting for overall differences in network density between ASD and TC groups, participants with ASD continued to display reductions in network integrity, suggesting that also network-level organizational properties of the AON are altered in ASD.

Conclusion

While differences in empirical connectivity contributed to reductions in network integrity, graph theoretical analyses provided indications that also changes in the high-level network organization reduced integrity of the AON.     

Insulin-Loaded Nanoparticles Based on N-Trimethyl Chitosan: In Vitro (Caco-2 Model) and Ex Vivo (Excised Rat Jejunum, Duodenum, and Ileum) Evaluation of Penetration Enhancement Properties

The aim of this paper was to evaluate the penetration enhancement properties of nanoparticles (NP) based on N-trimethyl chitosan (TMC 35% quaternization degree) loaded with insulin. The permeation performances of TMC NP were compared with those of chitosan (CS) NP and also with TMC and CS solutions. To estimate the mechanism of penetration enhancement, two different approaches have been taken into account: an in vitro study (Caco-2 cells) and an ex vivo study (excised rat duodenum, jejunum, and ileum). Insulin-loaded CS and TMC NP had dimensions of about 250 nm and had high yield and high encapsulation efficiency. The in vitro study evidenced that TMC and CS were able to enhance insulin permeation to the same extent. Penetration enhancement properties of TMC NP seem to be prevalently related to endocytosis while the widening of tight junctions appeared more important as mechanism in the case of CS NP. The ex vivo study put in evidence the role of mucus layer and of its microclimate pH. In duodenum (pH 5–5.5), CS and TMC solutions were more effective than NP while TMC NP were more efficient towards jejunum tissue (pH 6–6.5) for their high mucoadhesive potential. Confocal laser scanning microscopy study supported the hypothesis that penetration enhancement due to TMC NP was mainly due to internalization/endocytosis into duodenum and jejunum epithelial cells. The good penetration enhancement properties (permeation and penetration/internalization) make TMC NP suitable carriers for oral administration of insulin.     

Enhancing therapeutic efficacy by targeting non-oncogene addicted cells with combinations of signal transduction inhibitors and chemotherapy

The effects of inhibition of the Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways and chemotherapeutic drugs on cell cycle progression and drug sensitivity were examined in cytokine-dependent FL5.12 hematopoietic cells. We examined their effects, as these cells resemble normal hematopoietic precursor cells as they do not exhibit “oncogene-addicted” growth, while they do display “cytokine-addicted” proliferation as cytokine removal resulted in apoptosis in greater than 80% of the cells within 48 h. When cytokine-dependent FL5.12 cells were cultured in the presence of IL-3, which stimulated multiple proliferation and anti-apoptotic cascades, MEK, PI3K and mTOR inhibitors transiently suppressed but did not totally inhibit cell cycle progression or induce apoptosis while chemotherapeutic drugs such as doxorubicin and paclitaxel were more effective in inducing cell cycle arrest and apoptosis. Doxorubicin induced a G₁ block, while paclitaxel triggered a G₂/M block. Doxorubicin was more effective in inducing cell death than paclitaxel. Furthermore the effects of doxorubicin could be enhanced by addition of MEK, PI3K or mTOR inhibitors. Cytokine-dependent cells which proliferate in vitro and are not “oncogene-addicted” may represent a pre-malignant stage, more refractory to treatment with targeted therapy. However, these cells are sensitive to chemotherapeutic drugs. It is important to develop methods to inhibit the growth of such cytokine-dependent cells as they may resemble the leukemia stem cell and other cancer initiating cells. These results demonstrate the enhanced effectiveness of targeting early hematopoietic progenitor cells with combinations of chemotherapeutic drugs and signal transduction inhibitors.     

Phase variations inBifidobacterium animalis

Strains isolated from rabbit, chicken, and rat feces and from sewage and fermented milk products, all identified asBifidobacterium animalis, were found to show phase variations in colony appearance and in cellular morphology. The rate of transition in a switching system from opaque to transparent colonies and vice versa was determined. Differences in protein components and in penicillin-binding proteins (PBPs) of the cells from different colony types are shown.     

Immune surveillance properties of human NK cell-derived exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.     

The surface roughness of lactose particles can be modulated by wet-smoothing using a high-shear mixer

The purpose of this research was to encapsulate superoxide dismutase (SOD) and catalase (CAT) in biodegradable microspheres (MS) to obtain suitable sustained protein delivery. A modified water/oil/water double emulsion method was used for poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) PLA MS preparation co-encapsulating mannitol, trehalose, and PEG400 for protein stabilization. Size, morphology, porosity, mass loss, mass balance, in vitro release and in vitro activity were assessed by using BCA protein assay, scanning electron microscopy, BET surface area, and particle-sizing techniques. In vitro activity retention within MS was evaluated by nicotinammide adenine dinucleotide oxidation and H₂O₂ consumption assays. SOD encapsulation efficiency resulted in 30% to 34% for PLAMS and up to 51% for PLGA MS, whereas CAT encapsulation was 34% and 45% for PLGA and PLAMS, respectively. All MS were spherical with a smooth surface and low porosity. Particle mean diameters ranged from 10 to 17 μm. CAT release was prolonged, but the results were incomplete for both PLA and PLGA MS, whereas SOD was completely released from PLGA MS in a sustained manner after 2 months. CAT results were less stable and showed a stronger interaction than SOD with the polymers. Mass loss and mass balance correlated well with the release profiles. SOD and CAT in vitro activity was preserved in all the preparations, and SOD was better stabilized in PLGA MS. PLGA MS can be useful for SOD delivery in its native form and is promising as a new depot system.     

The key to enabling biosurveillance is cooperative technology development

The world population will continue to face biological threats, whether they are naturally occurring or intentional events. The speed with which diseases can emerge and spread presents serious challenges, because the impact on public health, the economy, and development can be huge. The U.S. government recognizes that global public health can also have an impact on national security. This global perspective manifests itself in U.S. policy documents that clearly articulate the importance of biosurveillance in providing early warning, detection, and situational awareness of infectious disease threats in order to mount a rapid response and save lives. In this commentary, we suggest that early recognition of infectious disease threats, whether naturally occurring or man-made, requires a globally distributed array of interoperable hardware and software fielded in sufficient numbers to create a network of linked collection nodes. We argue that achievement of this end state will require a degree of cooperation that does not exist at this time-either across the U.S. federal government or among our global partners. Successful fielding of a family of interoperable technologies will require interagency research, development, and purchase ("acquisition") of biosurveillance systems through cooperative ventures that likely will involve our strategic allies and public-private partnerships. To this end, we propose leveraging an existing federal interagency group to integrate the acquisition of technologies to enable global biosurveillance.     

Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance

Growth factors and mitogens use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their receptors to regulate gene expression and prevent apoptosis. Some components of these pathways are mutated or aberrantly expressed in human cancer (e.g., Ras, B-Raf). Mutations also occur at genes encoding upstream receptors (e.g., EGFR and Flt-3) and chimeric chromosomal translocations (e.g., BCR-ABL) which transmit their signals through these cascades. Even in the absence of obvious genetic mutations, this pathway has been reported to be activated in over 50% of acute myelogenous leukemia and acute lymphocytic leukemia and is also frequently activated in other cancer types (e.g., breast and prostate cancers). Importantly, this increased expression is associated with a poor prognosis. The Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways interact with each other to regulate growth and in some cases tumorigenesis. For example, in some cells, PTEN mutation may contribute to suppression of the Raf/MEK/ERK cascade due to the ability of activated Akt to phosphorylate and inactivate different Rafs. Although both of these pathways are commonly thought to have anti-apoptotic and drug resistance effects on cells, they display different cell lineage specific effects. For example, Raf/MEK/ERK is usually associated with proliferation and drug resistance of hematopoietic cells, while activation of the Raf/MEK/ERK cascade is suppressed in some prostate cancer cell lines which have mutations at PTEN and express high levels of activated Akt. Furthermore the Ras/Raf/MEK/ERK and Ras/PI3K/PTEN/Akt pathways also interact with the p53 pathway. Some of these interactions can result in controlling the activity and subcellular localization of Bim, Bak, Bax, Puma and Noxa. Raf/MEK/ERK may promote cell cycle arrest in prostate cells and this may be regulated by p53 as restoration of wild-type p53 in p53 deficient prostate cancer cells results in their enhanced sensitivity to chemotherapeutic drugs and increased expression of Raf/MEK/ERK pathway. Thus in advanced prostate cancer, it may be advantageous to induce Raf/MEK/ERK expression to promote cell cycle arrest, while in hematopoietic cancers it may be beneficial to inhibit Raf/MEK/ERK induced proliferation and drug resistance. Thus the Raf/MEK/ERK pathway has different effects on growth, prevention of apoptosis, cell cycle arrest and induction of drug resistance in cells of various lineages which may be due to the presence of functional p53 and PTEN and the expression of lineage specific factors.