CeReS-18, a novel cell surface sialoglycopeptide, induces cell cycle arrest and apoptosis in a calcium-sensitive manner

Very few growth inhibitors have been identified whichcan inhibit the proliferation of a broad spectrumof human breast cancer cell lines. CeReS-18, anovel cell surface sialoglycopeptide growth inhibitor, can reversiblyinhibit the proliferation of both estrogen receptor positive(MCF-7) and negative (BT-20) human breast cancer celllines. In addition, at concentrations above those requiredfor the reversible inhibition of cell proliferation, CeReS-18can also induce cell death in MCF-7 cells.Changes in nuclear and cytoplasmic morphology, characteristic ofapoptosis, were detected in MCF-7 cells treated witha cytotoxic concentration of CeReS-18, and internucleosomal DNAcleavage was also observed. The sensitivity of MCF-7and BT-20 cells to the biological properties ofCeReS-18 could be influenced by altering the calciumconcentration in the extracellular growth medium, such thatwhen the calcium concentration in the environment wasdecreased, an increased sensitivity to CeReS-18-induced growth inhibitionand cytotoxicity were observed. The addition of thecalcium chelating agent EGTA to MCF-7 cells, culturedin a normal calcium environment, could mimic theincreased sensitivity to the biological effects of CeReS-18observed under reduced calcium conditions.     

A biochemical and pharmacological examination of Rhamphiophis oxyrhynchus (Rufous beaked snake) venom.

The venom of R. oxyrhynchus, a member of the psammophiine subfamily of the colubrid assemblage, was examined for biological activity using biochemical and pharmacological techniques. Venom displayed a high protein content, a complex electrophorectic profile and PLA2 activity but no detectable proteolytic or haematological activities. In the chick biventer cervicis nerve muscle preparation, venom (1-10 microg/ml) displayed postsynaptic neurotoxic activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). This inhibitory effect was poorly reversible by washing. Venom (30-50 microg/ml) caused a rapid and readily reversible inhibition of direct (0.1 Hz, 2 ms, supramaximal V) twitches of the chick biventer cervicis nerve muscle preparation without morphological changes to the muscle fibers. Venom (30-100 microg/ml) inhibited electrically-evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibitory effect was not significantly attenuated by 8-phenyltheophylline (8-PT; 20 microM), idazoxan (1 microM), a combination of ranitidine (0.2 microM) and thioperamide (10 microM) or capsazepine (10 microM). Venom (5 mg/kg) induced hypotension with subsequent cardiovascular collapse in the anaesthetised rat. The cardiovascular collapse was prevented by artificial respiration of the animals prior to venom administration. The biological activities demonstrated by R. oxyrhynchus venom may aid in prey envenomation strategies such as prey immobilisation. This study provides further evidence that colubrid venoms are comprised of multiple components which can display a variety of actions, some of which may be novel, therefore reinforcing the largely untapped potential of colubrid venoms.     

Characterization of the in vitro activity of AZD3409, a novel prenyl transferase inhibitor

Purpose

AZD3409 is a novel DPTI that has potent activity against both FTase and GGTase-1. The in vitro inhibition profile of AZD3409 was characterized using three different cell lines: mouse embryogenic fibroblasts, transfected with H-Ras^V12 (MEF), A549 cells (Ki4B-Ras mutation) and MCF-7 cells (no Ras mutation).

Methods

Both cytotoxicity and levels of inhibition of farnesylation and geranylgeranylation were determined in different assays in relation to the concentration of AZD3409. Results were compared with those obtained with the first-generation FTase inhibitor lonafarnib or the GGTase-1 inhibitor GGTI-2147.

Results

The mean IC₅₀ for cytotoxicity of AZD3409 and lonafarnib was 510 and 15,200?nM in MEF cells, 10,600 and 2,740?nM in A549 cells and 6,170 and 9,490?nM in MCF7 cells, respectively. In these cells, the IC₅₀ for FTase activity of AZD3409 ranged from 3.0 to 14.2?nM and of lonafarnib from 0.26 to 31.3?nM. The inhibiting activity of AZD3409 and lonafarnib on general protein farnesylation was comparable with the specific farnesylation levels of HDJ-2. In vitro geranylgeranylation of Rap1a could be inhibited by GGTI-2147 in all three cell lines, but only in MCF-7 cells by AZD3409. These results are in agreement with the IC₅₀ values for GGTase-1 activity as the lowest IC₅₀ for AZD3409 was found in the MCF-7 cell line.

Conclusions

AZD3409 inhibits farnesylation to a higher extent than geranylgeranylation. Both inhibition of farnesylation and geranylgeranylation could not be correlated to the antiproliferative activity of the drug.     

ER-resident STIM1/2 couples Ca2+ entry by NMDA receptors to pannexin-1 activation

Pannexin-1 (Panx1) is a large-pore ion and solute permeable channel highly expressed in the nervous system, where it subserves diverse processes, including neurite outgrowth, dendritic spine formation, and N-methyl D-aspartate (NMDA) receptor (NMDAR)-dependent plasticity. Moreover, Panx1 dysregulation contributes to neurological disorders, including neuropathic pain, epilepsy, and excitotoxicity. Despite progress in understanding physiological and pathological functions of Panx1, the mechanisms that regulate its activity, including its ion and solute permeability, remain poorly understood. In this study, we identify endoplasmic reticulum (ER)-resident stromal interaction molecules (STIM1/2), which are Ca²⁺ sensors that communicate events within the ER to plasma membrane channels, as binding and signaling partners of Panx1. We demonstrate that Panx1 is activated to its large-pore configuration in response to stimuli that recruit STIM1/2 and map the interaction interface to a hydrophobic region within the N terminus of Panx1. We further characterize a Panx1 N terminus–recognizing antibody as a function-blocking tool able to prevent large-pore Panx1 activation by STIM1/2. Using either the function-blocking antibody or re-expression of Panx1 deletion mutants in Panx1 knockout (KO) neurons, we show that STIM recruitment couples Ca²⁺ entry via NMDARs to Panx1 activation, thereby identifying a model of NMDAR-STIM-Panx1 signaling in neurons. Our study highlights a previously unrecognized and important role of the Panx1 N terminus in regulating channel activation and membrane localization. Considering past work demonstrating an intimate functional relation between NMDARs and Panx1, our study opens avenues for understanding activation modality and context-specific functions of Panx1, including functions linked to diverse STIM-regulated cellular responses.

Glutamatergic signaling plays a critical role in diverse processes linked to learning and memory formation. Ca²⁺ signals generated by the N-methyl D-aspartate (NMDA) subtype of glutamate receptors (NMDARs) are indispensable for several forms of synaptic plasticity, including long-term potentiation (LTP), a prototypic form of plasticity linked to memory formation (1–3). NMDAR-initiated Ca²⁺ signals (e.g., time course, amplitude, and spatial spread) are shaped by secondary events, including those engendered via the endoplasmic reticulum (ER) (4, 5). Ca²⁺ entry via NMDARs can promote Ca²⁺-induced Ca²⁺ release from ER stores by stimulating ryanodine (RyRs) (6–8) and/or IP3 receptors (IP3Rs) (9). In turn, NMDAR-initiated Ca²⁺ store depletion recruits ER-resident and Ca²⁺-sensing STIM proteins (10) to negatively regulate L-type voltage-gated Ca²⁺ channels (VGCCs) (13). This establishes the notion that Ca²⁺ entry via NMDARs can stimulate ER- and STIM-dependent cascades that regulate secondary routes of Ca²⁺ entry, thereby sculpting intracellular Ca²⁺ dynamics and in turn the cellular functions influenced by them. As part of a broader search to identify candidate Ca²⁺ channels able to respond to ER signaling dynamics, we found that Pannexin-1 (Panx1) can be activated through ER-based signaling following sarcoendoplasmic reticulum calcium adenosine triphosphatase (ATPase) (SERCA) pump inhibition by thapsigargin. This led us to consider the role of STIM1/2 as a candidate Panx1 activation mechanism.Panx1 is a large-pore nonselective ion and solute permeable channel with prominent central nervous system (CNS) expression (14, 15). Panx1 activation has been linked to pathophysiological disorders, such as excitotoxicity, stroke, migraine, chronic pain, and epilepsy (16–18). However, Panx1 also mediates physiological processes in the CNS, including contributions to neural development (19, 20), spine formation (21, 22), and NMDAR-dependent synaptic plasticity (23, 24). In this context, there remains an important gap in understanding the mechanisms by which Panx1 can mediate such disparate physiological and pathological functions. Intriguingly, evidence suggests that Panx1 ion versus solute permeability may be mediated by distinct channel pore configurations (i.e., small anion vs. large solute permeable) recruited via distinct activation modalities (25). Thus, identifying novel activation mechanisms is fundamental to understanding context- and modality-specific channel function.Here, we uncover a mechanism by which Panx1 is activated in response to ER-initiated signaling, which we demonstrate is dependent on Panx1 interaction with ER-resident STIM1/2. STIM1/2 recruitment and activation stimulates large-pore Panx1 opening, evident on the basis of increased permeability to Ca²⁺ and the large inorganic ion N-methyl-D-glucamine (NMDG). We map the STIM1/2 binding interface to a hydrophobic region in the N terminus of Panx1, a region not previously linked to channel gating. Our detailed structure-function analysis reveals that the Panx1 N-terminal region is necessary for its STIM1/2 responsiveness, but not for its responsiveness to hypotonic stress, demonstrating that this region mediates modality-specific regulation of Panx1 function. Using reverse genetics, ectopic rescue with Panx1 N-terminal deletion mutants, as well as a function inhibiting antibody targeting the critical N-terminal region of Panx1 identified by us, we demonstrate that NMDARs activate Panx1 in hippocampal neurons in a manner contingent upon ER-initiated signaling and reliant upon STIM proteins. Collectively, our data reveal the molecular mechanism by which STIM1/2 activates Panx1 and establishes a previously unrecognized essential role of its N-terminal region in regulating the transition of Panx1 to its large-pore solute permeable state. Our work will benefit studies aimed at understanding diverse functions of Panx1, including those linked to NMDAR-dependent signaling, stimulated in a modality- and context-specific manner by STIM proteins.     

Refeeding Syndrome: A Critical Reality in Patients with Chronic Disease

Malnutrition is one of the most frequent metabolic challenges in the population of chronically ill patients. This results in increased administration of nutritional therapy in inpatient settings, which poses the risk of side effects, in particular, the development of refeeding syndrome. If not managed accordingly, it leads to a significant rise in morbidity and mortality. However, despite its importance, evidence-based recommendations on the management of refeeding syndrome are largely lacking, and only a few randomized controlled trials have been conducted. In light of this, the aim of this review is to raise awareness of refeeding syndrome in chronically ill patients by critically reviewing recent literature and providing a short overview as well as diagnosis and treatment algorithms of this underreported metabolic condition. In summary, recent findings suggest undergoing risk assessment and stratification for every patient receiving nutritional therapy. According to this, adaptation of energy and fluid support during the replenishment phase should be implemented in the nutritional therapy for patients at high risk. Additionally, continuous monitoring should take place, and appropriate actions should be initiated when necessary.     

Insights into pediatric rhabdomyosarcoma research: Challenges and goals

Overall survival rates for pediatric patients with high‐risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.