Miriam Shmuel

The activity of antiepileptic drugs as histone deacetylase inhibitors.

Summary:  Purpose: Valproic acid (VPA), one of the widely used antiepileptic drugs (AEDs), was recently found to inhibit histone deacetylases (HDACs). HDAC inhibitors of a wide range of structures, such as hydroxamic acids, carboxylic acids, and cyclic tetrapeptides, have various effects on transformed and nontransformed cells, including neuromodulation and neuroprotection. The aim of this study was to assess comparatively the activity of traditional and newer AEDs as HDAC inhibitors.

ARNO through Its Coiled-coil Domain Regulates Endocytosis at the Apical Surface of Polarized Epithelial Cells

ARNO is a guanine-nucleotide exchange protein for the ARF family of GTPases. Here we show that in polarized epithelial cells, ARNO is localized exclusively to the apical plasma membrane, where it regulates endocytosis. Expression of ARNO stimulates apical endocytosis of the polymeric immunoglobulin receptor, and coexpression of ARF6 with ARNO leads to a synergistic stimulation of apical endocytosis. Expression of a dominant negative ARF6 mutant, ARF6-T27N, antagonizes this stimulatory effect. Deletion of the N-terminal coiled-coil (CC) domain of ARNO causes the mutant ARNO to localize to both the apical and basolateral plasma membranes. Expression of the CC domain alone abolishes ARNO-induced apical endocytosis as well as co-localization of IgA-receptor complexes with ARNO and clathrin. These results suggest that the CC domain contributes to the specificity of apical localization of ARNO through association with components of the apical plasma membrane. We conclude that ARNO acts together with ARF6 to regulate apical endocytosis.

Toxicity Mechanisms of Amphotericin B and Its Neutralization by Conjugation with Arabinogalactan

ABSTRACT Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells.

β-tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure

In epithelial cells, the apical junctional complex (AJC), composed of tight junctions (TJs) and adherens junctions (AJs), maintains cell‐surface polarity by forming a fence that prevents lateral movement and diffusion of proteins and lipids between the apical and basolateral PM and holds the epithelial monolayer intact through cell‐cell contacts. Disassembly of this complex is a prime event in development and cell transformation. Maintenance of the AJC has been shown to involve mainly the actin cytoskeleton. Recent findings also point to the involvement of the microtubule (MT) system. Here we show the first evidence that in polarized epithelial MDCK cells, ARF‐like protein 2 (ARL2) and β‐tubulin cofactor D, known to be involved in MT dynamics, have a role in disassembly of the AJC followed by cell dissociation from the epithelial mono‐layer, which is not dependent on MT depolymerization. In addition, we show that β‐tubulin cofactor D is partially localized to the lateral PM through its 15 C‐terminal amino acids and intact MTs. ARL2 inhibited β ‐tubulin cofactor D‐dependent cell dissociation from the monolayer and AJC disassembly. To our knowledge, this is the first evidence that β‐tubulin cofactor D plays a role in cells independent of its presumed role in folding tubulin heterodimers. We conclude that ARL2 and β ‐tubulin cofactor D participate in AJC disassembly and epithelial depolarization.— Shultz, T., Shmuel, M., Hyman, T., and Altschuler, Y. β‐tubulin cofactor D and ARL2 take part in apical junctional complex disassembly and abrogate epithelial structure. FASEB J. 22, 168–182 (2008)

Multiple Triphenylphosphonium Cations as a Platform for the Delivery of a Pro-Apoptotic Peptide

ABSTRACTPurposeTriphenyl phosphonium cations (TPPs) are delocalized lipophilic cations that accumulate in the mitochondria of cells. We have explore the effect of increasing the number of TPPs on delivery of a cell-impermeable pro-apoptotic peptide to intact cells.MethodsThe pro-apoptotic peptide D-(KLAKLAK)2 (KLA) was extended with 0–3 L-Lysines modified at their ε-amine with TPP. Peptides were studied in HeLa cells to determine their cytotoxic activity and cellular uptake.ResultsIn HeLa cells, the increased cytotoxicity correlates with the number of TPPs; the peptide with 3 TPP molecules (3-KLA) exerts the highest cytotoxic activity. This FITC-labeled peptide is found to accumulate in intact HeLa cells, whereas peptides with 0–2 TPPs are not detected at the same peptide concentration. Mitochondria-dependent apoptosis of HeLa cells in the presence of 3-KLA was followed by propidium iodide, Annexin-V and DiOC fluorescence by FACS.ConclusionA facile synthetic methodology has been presented for the delivery of a biologically active peptide into mitochondria of intact cells by attaching multiple TPP moieties to the peptide. This approach was shown to dramatically increase biological activity of the peptide as a pro-apoptotic agent.

Tracking inflammation in the epileptic rat brain by bi-functional fluorescent and magnetic nanoparticles

Correct localization of epileptic foci can improve surgical outcome in patients with drug-resistant seizures. Our aim was to demonstrate that systemically injected nanoparticles identify activated immune cells, which have been reported to accumulate in epileptogenic brain tissue. Fluorescent and magnetite-labeled nanoparticles were injected intravenously to rats with lithium-pilocarpine-induced chronic epilepsy. Cerebral uptake was studied ex vivo by confocal microscopy and MRI. Cellular uptake and biological effects were characterized in vitro in murine monocytes and microglia cell lines. Microscopy confirmed that the nanoparticles selectively accumulate within myeloid cells in the hippocampus, in association with inflammation. The nanoparticle signal was also detectable by MRI. The in vitro studies demonstrate rapid nanoparticle uptake and good cellular tolerability. We show that nanoparticles can target myeloid cells in epileptogenic brain tissue. This system can contribute to pre-surgical and intra-surgical localization of epileptic foci, and assist in detecting immune system involvement in epilepsy.

Indocyanine Green Liposomes for Diagnosis and Therapeutic Monitoring of Cerebral Malaria.

Cerebral malaria (CM) is a major cause of death of Plasmodium falciparum infection. Misdiagnosis of CM often leads to treatment delay and mortality. Conventional brain imaging technologies are rarely applicable in endemic areas. Here we address the unmet need for a simple, non-invasive imaging methodology for early diagnosis of CM. This study presents the diagnostic and therapeutic monitoring using liposomes containing the FDA-approved fluorescent dye indocyanine green (ICG) in a CM murine model. Increased emission intensity of liposomal ICG was demonstrated in comparison with free ICG. The Liposomal ICGs emission was greater in the brains of the infected mice compared to naïve mice and drug treated mice (where CM was prevented). Histological analyses suggest that the accumulation of liposomal ICG in the cerebral vasculature is due to extensive uptake mediated by activated phagocytes. Overall, liposomal ICG offers a valuable diagnostic tool and a biomarker for effectiveness of CM treatment, as well as other diseases that involve inflammation and blood vessel occlusion.

Imaging the urinary pathways in mice by liposomal indocyanine green

UNLABELLED Intraoperative ureter identification can assist in the prevention of ureteral injury and consequently improve surgery outcomes. Our aim was to take advantage of the altered pharmacokinetics of liposomal indocyanine green (ICG), the only FDA-approved near-infrared (NIR) dye, for imaging of ureters during surgeries. ICG was passively adsorbed to liposomes. NIR whole mice body and isolated tissue imaging were used to study liposomal ICG properties vs. free ICG. In vivo, the urinary bladder could be clearly observed in most of the liposome-treated mice. Liposomal encapsulation of ICG enhanced ureteral emission up to 1.9 fold compared to free ICG (P<0.01). Increase in liposomal micropolarity and microviscosity and differential scanning calorimetry supported ICG localization within the liposomal bilayer. Our findings suggest that liposomal ICG could be utilized for ureteral imaging intra-operatively, thus potentially improving surgical outcomes. FROM THE CLINICAL EDITOR Iatrogenic ureteral injury is a serious complication of abdominal surgery and intra-operative recognition of the ureters is usually the best method of injury prevention. In this article, the authors developed liposomal indocyanine green, which could be excreted via the urinary system and investigated its in-vivo use in mice.

Maternal-fetal transfer of indocyanine green across the perfused human placenta.

Indocyanine green (ICG) is an FDA-approved near-infrared imaging probe, given also to pregnant women. We aimed to characterize ICGs transplacental transfer using the ex-vivo perfusion model. Placentas were obtained from caesarean deliveries. Cotyledons were cannulated and dually perfused. ICG, 9.6μg/mL and antipyrine (50μg/mL) were added to the maternal circulation in the absence (n=4) or the presence of the organic anion transporting polypeptide (OATPs) inhibitor rifampin (10μg/mL; n=5) or the P-glycoprotein inhibitor valspodar (2μg/mL; n=3). ICGs maternal-to-fetal transfer was evaluated over 180min. The cumulative percent of ICG in the fetal reservoir was minor. When ICG transfer was normalized to that of antipyrine, it was lower in the presence of rifampin (a 41% decrease; p<0.05). Valspodar did not appear to modify the kinetics of ICG. ICGs transplacental transfer is minimal and is probably OATP-mediated. The placenta is an effective protective barrier to ICGs distribution into the fetus.

Adverse placental effects of valproic acid: Studies in perfused human placentas

In utero exposure to valproic acid (VPA) has been associated with worse pregnancy outcomes compared to all other antiepileptic drugs. We have previously shown that VPA alters the expression of placental transporters for hormones and nutrients in vitro and in pregnant mice. Here, our aim was to characterize the effects of short exposure to VPA on the expression of carriers for compounds essential for fetal development in human placentas ex vivo, under controlled conditions.