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Targeting the BCL-2 family with BH3-mimetics is proving an effective treatment for several cancers. These drugs initiate apoptosis by neutralising pro-survival BCL-2 members and allowing BH3-only proteins to activate BAK and BAX. However, to date there are no therapeutic agents to date that directly trigger BAK or BAX activation for subsequent apoptosis. Thus, agents that directly target BAK and BAX to trigger apoptosis may augment cancer therapy, and offer alternate means to induce apoptosis in cancer cells that are resistant to other forms of therapy. Although small molecule direct activators of BAX have been described (Lopez et al., 2022), there are none reported for BAK. An antibody, 7D10, that specifically binds and triggers activation of the intracellular pro-apoptotic protein BAK was reported by our group (Iyer et al., 2016), creating the opportunity to directly target the BAK protein to kill cancer cells. This thesis investigates various strategies adopted to obtain proof-of-concept that 7D10, when delivered into cancer cells, can bind, and activate BAK to induce apoptosis. Several approaches were employed to: (1) express 7D10 as "intrabodies" inside the target cells; (2) deliver recombinant 7D10 antibody inside cancer cells; (3) identify alternative "7D10-like" apoptosis inducing antibodies using new antibody discovery platforms; and (4) utilise newly identified antibodies to better understand BAK regulation. Intracellular expression of 7D10 was achieved by grafting complementarity determining regions (CDRs) onto two intrinsically stable scaffolds and by increasing the net negative charge using negatively charged tags. Two out of nine 7D10 intrabodies were able to activate BAK when expressed in cells and release cytochrome c, thereby giving proof-of-concept that 7D10 can indeed activate BAK in cells. Delivery of recombinant BAK inside cancer cells was explored using receptor-mediated endocytosis through the EGF receptor. A 7D10 polyplex was generated wherei

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics.

A treasure trove of intracellular cancer drug targets remains hidden behind cell membranes. However, engineered pathogen-derived toxins such as Shiga toxins can deliver small or macromolecular drugs to specific intracellular organelles. After binding to ganglioglobotriaosylceramide (Gb3, CD77), the non-toxic subunit B (StxB) of the Shiga-holotoxin is endocytosed and delivers its payload by a unique retrograde trafficking pathway via the endoplasmic reticulum to the cytosol. This review provides an overview of biomedical applications of StxB-based drug delivery systems in targeted cancer diagnosis and therapy. Biotechnological production of the Stx-material is discussed from the perspective of developing efficacious and safe therapeutics.

Synthetic polymers, nanoparticles, and carbon-based materials have great potential in applications including drug delivery, gene transfection, in vitro and in vivo imaging, and the alteration of biological function. Nature and humans use different design strategies to create nanomaterials: biological objects have emerged from billions of years of evolution and from adaptation to their environment resulting in high levels of structural complexity; in contrast, synthetic nanomaterials result from minimalistic but controlled design options limited by the authors' current understanding of the biological world. This conceptual mismatch makes it challenging to create synthetic nanomaterials that possess desired functions in biological media. In many biologically relevant applications, nanomaterials must enter the cell interior to perform their functions. An essential transport barrier is the cell-protecting plasma membrane and hence the understanding of its interaction with nanomaterials is a fundamental task in biotechnology. The authors present open questions in the field of nanomaterial interactions with biological membranes, including: how physical mechanisms and molecular forces acting at the nanoscale restrict or inspire design options; which levels of complexity to include next in computational and experimental models to describe how nanomaterials cross barriers via passive or active processes; and how the biological media and protein corona interfere with nanomaterial functionality. In this Perspective, the authors address these questions with the aim of offering guidelines for the development of next-generation nanomaterials that function in biological media.

In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-leucyl-fluorescein isothiocyanate (f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC) binds to and activates formyl peptide receptor1 (FPR1) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, C-terminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10–1000 nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~ 7 nM (microscopy). The nonfluorescent version of the antibody (28 nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly)5-myc, designed to decrease the possible steric hindrance between FPR1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-Nle-Leu-Phe-Nle-Tyr-Lys-myc.

Apical transport is key in renal function, and the activity of efflux transporters and receptor-mediated endocytosis is pivotal in this process. The conditionally immortalized proximal tubule epithelial cell line (ciPTEC) endogenously expresses these systems. Here, we used ciPTEC to investigate the activity of three major efflux transporters, viz. breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4) and P-glycoprotein (P-gp), as well as protein uptake through receptor-mediated endocytosis using a fluorescence-based setup for transport assays. To this end, cells were exposed to Hoechst33342, chloromethylfluorescein-diacetate (CMFDA) and calcein-AM in presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833) or MRP's (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls and membrane vesicles over-expressing one transporter were used to determine substrate and inhibitor specificities. Receptor-mediated endocytosis was investigated by determining the intracellular accumulation of fluorescently labeled receptor associated protein (RAP-GST). In ciPTEC, BCRP and P-gp showed similar expressions and activities while MRP4 was more abundantly expressed. Hoechst33342, GS-MF and calcein are retained in the presence of KO143, MK571 and PSC833, showing clearly redundancy between the transporters. Noteworthy is the fact that both KO143 and MK571 can block BCRP, P-gp and MRP's, while PSC833 appears a potent inhibitor for BCRP and P-gp, but not the MRP's. Furthermore, ciPTEC accumulate RAP-GST in intracellular vesicles in a dose and time dependent manner, which was reduced in megalin-deficient cells. In conclusion, fluorescent probe-based assays are fast and reproducible in determining apical transport mechanisms, in vitro. We demonstrate that typical substrates and inhibitors are not specific for the designated transporters, reflecting the complex interactions that can take place in vivo. The set of t

Receptor-mediated transcytosis remains a major route for drug delivery across the blood-brain barrier (BBB). PepFect 32 (PF32), a peptide-based vector modified with targeting ligand (Angiopep-2) binding to low-density lipoprotein receptor-related protein-1 (LRP-1), was previously found to be a promising vector for plasmid delivery across an in vitro model of the BBB. Cellular uptake of PF32/plasmid DNA (pDNA) complexes was speculated the internalization via LRP-1 receptor. In this study, we prove that PF32/pDNA nanocomplexes are not only transported into brain endothelial cells via LRP-1 receptor-mediated endocytosis, but also via scavenger receptor class A and B (SCARA3, SCARA5, and SR-BI)-mediated endocytosis. SCARA3, SCARA5, and SR-BI are found to be expressed in the brain endothelial cells. Inhibition of these receptors leads to a reduction of the transfection. In conclusion, this study shows that scavenger receptors also play an essential role in the cellular uptake of the PF32/pDNA nanocomplexes.

In human peripheral blood neutrophils or in myeloid PLB-985 cells differentiated towards a neutrophil-like phenotype, the peptide N-formyl-L-norleucyl-L-leucyl-L-phenylalanyl-L-norleucyl-L-tyrosyl-L-leucyl-fluorescein isothiocyanate (f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC) binds to and activates formyl peptide receptor1 (FPR1) and is submitted to receptor-mediated endocytosis (microscopy, cytofluorometry). This peptide may be considered a C-terminally extended version of f-Met-Leu-Phe which carries a fluorescent cargo into cells. By analogy to other peptide hormones for which we have evaluated epitope-tagged agonists as carriers of antibody cargoes, we have designed and evaluated f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, C-terminally extended with the 10-residue myc tag. This peptide is as potent as f-Met-Leu-Phe to compete for f-Nle-Leu-Phe-Nle-Tyr-Lys-FITC uptake by PLB-985 cells, but did not mediate (10–1000 nM) the internalization of the fluorescent anti-myc monoclonal antibody 4A6 added to the extracellular fluid at ~ 7 nM (microscopy). The nonfluorescent version of the antibody (28 nM) acts as a pre-receptor antagonist of f-Nle-Leu-Phe-Nle-Tyr-Lys-myc, but not of f-Met-Leu-Phe (superoxide release assay in differentiated PLB-985 cells). A further prolonged analog, f-Nle-Leu-Phe-Nle-Tyr-Lys-(Asn-Gly)5-myc, designed to decrease the possible steric hindrance between FPR1 and the bound anti-myc antibody, has little affinity for the receptor, precluding a direct assessment of this issue. Thus, the relatively low-affinity anti-myc antibody used at a high concentration functionally behaves as a selective pre-receptor antagonist of the agonist f-Nle-Leu-Phe-Nle-Tyr-Lys-myc.

Cell-penetrating peptides (CPPs) have been used as vehicles to deliver various cargos into cells and are promising as tools to deliver therapeutic biomolecules such as oligonucleotides both in vitro and in vivo. CPPs are positively charged and it is believed that CPPs deliver their cargo in a receptor-independent manner by interactingwith the negatively charged plasmamembrane and thereby inducing endocytosis. In this study we examine the mechanism of uptake of several different, well known, CPPs that form complexes with oligonucleotides.We show that these CPP:oligonucleotide complexes are negatively charged in transfection-media and their uptake is mediated by class A scavenger receptors (SCARA). These receptors are known to promiscuously bind to, and mediate uptake of poly-anionic macromolecules. Uptake of CPP:oligonucleotide complexes was abolished using pharmacological SCARA inhibitors as well as siRNA-mediated knockdown of SCARA. Additionally, uptake of CPP:oligonucleotide was significantly increased by transiently overexpressing SCARA. Furthermore, SCARA inhibitors also blocked internalization of cationic polymer:oligonucleotide complexes.Our results demonstrate that the previous held belief that CPPs act receptor independently does not hold true for CPP:oligonucleotide complexes, as scavenger receptor class A (SCARA) mediates the uptake of all the examined CPP:oligonucleotide complexes in this study.

In this article, we describe a simple method of coating D-(+)-Glucosamine hydrochloride (GlcN) onto gold nanoparticles (AuNPs) by noncovalent binding. The as-prepared AuNPs-GlcN exhibits outstanding properties such as stability, tumor specificity and non-cytoxicity. Tumor cell and tissue-specific targeting by AuNPs-GlcN nanoconjugates were successfully demonstrated in this work. In the colocalization experiment, the AuNPs-GlcN entered tumor cells through receptor-mediated endocytosis. Our strategy provides an efficient method for preparing AuNPs based targeting vectors, which has the potential to be used for tumor specific diagnosis and therapy.

In this article, we describe a simple method of coating D-(+)-Glucosamine hydrochloride (GlcN) onto gold nanoparticles (AuNPs) by noncovalent binding. The as-prepared AuNPs-GlcN exhibits outstanding properties such as stability, tumor specificity and non-cytoxicity. Tumor cell and tissue-specific targeting by AuNPs-GlcN nanoconjugates were successfully demonstrated in this work. In the colocalization experiment, the AuNPs-GlcN entered tumor cells through receptor-mediated endocytosis. Our strategy provides an efficient method for preparing AuNPs based targeting vectors, which has the potential to be used for tumor specific diagnosis and therapy.

We are proposing folate-decorated polymeric nanoparticles as carriers of poorly soluble drug molecules for intracellular and prolonged delivery to retinal pigment epithelium (RPE) cells. RPE is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). In this study, folate-functionalized poly(ethylene glycol)-b-polycaprolactone (folate-PEG-b-PCL) were synthesized for assembling into nanoparticles of ~ 130 nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis, and the cellular uptake was significantly higher than particles without folate modification. Triamcinolone acetonide (TA) was efficiently encapsulated (> 97%) into the folate-decorated nanoparticles and was slowly released over a period of 4 weeks at pH 5.5 and 8 weeks at pH 7.4. The enhanced uptake and controlled release resulted in prolonged anti-angiogenic gene expression of RPE cells. In cell culture, the down-regulation of vascular endothelial growth factor (VEGF) and up-regulation of pigment epithelium derived factor (PEDF) lasted for at least 3 weeks. Unlike benzyl alcohol, the surfactant found in commercial formulation, folate-modified nanoparticles were non-toxic. Furthermore, TA became less cytotoxic by being encapsulated in the nanoparticles. Our findings suggest that folate-PEG-PCL nanoparticles are promising drug carriers for RPE targeting. © 2013 Elsevier B.V.

We are proposing folate-decorated polymeric nanoparticles as carriers of poorly soluble drug molecules for intracellular and prolonged delivery to retinal pigment epithelium (RPE) cells. RPE is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). In this study, folate-functionalized poly(ethylene glycol)-b-polycaprolactone (folate-PEG-b-PCL) were synthesized for assembling into nanoparticles of ~ 130 nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis, and the cellular uptake was significantly higher than particles without folate modification. Triamcinolone acetonide (TA) was efficiently encapsulated (> 97%) into the folate-decorated nanoparticles and was slowly released over a period of 4 weeks at pH 5.5 and 8 weeks at pH 7.4. The enhanced uptake and controlled release resulted in prolonged anti-angiogenic gene expression of RPE cells. In cell culture, the down-regulation of vascular endothelial growth factor (VEGF) and up-regulation of pigment epithelium derived factor (PEDF) lasted for at least 3 weeks. Unlike benzyl alcohol, the surfactant found in commercial formulation, folate-modified nanoparticles were non-toxic. Furthermore, TA became less cytotoxic by being encapsulated in the nanoparticles. Our findings suggest that folate-PEG-PCL nanoparticles are promising drug carriers for RPE targeting. © 2013 Elsevier B.V.

We are proposing folate-decorated polymeric nanoparticles as carriers of poorly soluble drug molecules for intracellular and prolonged delivery to retinal pigment epithelium (RPE) cells. RPE is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). In this study, folate-functionalized poly(ethylene glycol)-b-polycaprolactone (folate-PEG-b-PCL) were synthesized for assembling into nanoparticles of ~ 130 nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis, and the cellular uptake was significantly higher than particles without folate modification. Triamcinolone acetonide (TA) was efficiently encapsulated (> 97%) into the folate-decorated nanoparticles and was slowly released over a period of 4 weeks at pH 5.5 and 8 weeks at pH 7.4. The enhanced uptake and controlled release resulted in prolonged anti-angiogenic gene expression of RPE cells. In cell culture, the down-regulation of vascular endothelial growth factor (VEGF) and up-regulation of pigment epithelium derived factor (PEDF) lasted for at least 3 weeks. Unlike benzyl alcohol, the surfactant found in commercial formulation, folate-modified nanoparticles were non-toxic. Furthermore, TA became less cytotoxic by being encapsulated in the nanoparticles. Our findings suggest that folate-PEG-PCL nanoparticles are promising drug carriers for RPE targeting. © 2013 Elsevier B.V.

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.

Extracellular protein misfolding and aggregation underlie many of the most serious amyloidoses including Alzheimer's disease, spongiform encephalopathies and type II diabetes. Despite this, protein homeostasis (proteostasis) research has largely focussed on characterising systems that function to monitor protein conformation and concentration within cells. We are now starting to identify elements of corresponding systems, including an expanding family of secreted chaperones, which exist in the extracellular space. Like their intracellular counterparts, extracellular chaperones are likely to play a central role in systems that maintain proteostasis; however, the precise details of how they participate are only just emerging. It is proposed that extracellular chaperones patrol biological fluids for misfolded proteins and facilitate their clearance via endocytic receptors. Importantly, many amyloidoses are associated with dysfunction in rates of protein clearance. This is consistent with a model in which disruption to, or overwhelming of, the systems responsible for extracellular proteostasis results in the accumulation of pathological protein aggregates and disease. Further characterisation of mechanisms that maintain extracellular proteostasis will shed light on why many serious diseases occur and provide us with much needed strategies to combat them.

Iron is a transition element which has two biologically important oxidation states, +2 and +3. It is an essential trace element and, as such, takes part in a large number of varied biological processes, many of which utilize the interconversion of ferrous and ferric iron. Ferrous iron is relatively soluble but is readily oxidized to the ferric form which is sparingly soluble at physiological pH and will precipitate if not solubilized. It is also possible for the electron released from the oxidation reaction to participate in the formation of reactive oxygen species which can cause oxidative damage if present in high enough concentrations. Consequently, many elegant processes have evolved which render iron both soluble and non-toxic, allowing organisms to accumulate iron in forms which are both safe and functional.This chapter will provide an overview of the normal processes of iron transport in mammals, including a summary of the transport molecules and their mechanisms of iron capture and release. We will also outline the mechanisms of iron acquisition by tissues and the regulation of these processes.

Renal tubular reabsorption is important for extra-cellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterised by low-molecular-weight-proteinuria, hypercalciuria, nephrolithiasis and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here we report that CLC-5 interacts with Kinesin Family Member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, Glutathione-S-Transferase (GST) pull-down and co-immunoprecipitation assays, the C-terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous co-immunoprecipitation of CLC-5 and KIF3B and co-distribution with endosomal markers in mouse kidney fractions. Confocal live-cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5 containing vesicles along KIF3B microtubules. KIF3B over-expression and under-expression, using siRNA, had reciprocal effects on whole-cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarised cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression, and for facilitating endocytosis and microtubular transport in the kidney. Key words: Chloride/proton antiporter, Kinesin family, proximal tubular reabsorption, hypercalciuric nephrolithiasis.

To better understand the mechanism by which Merlin functions as a tumor suppressor, we have confirmed that Drosophila Merlin plays important roles in the control of mitosis exit and in the determination of dorsal/ventral compartment border during wing imaginal disc development. We show that the Merlin protein is dynamically redistributed during meiosis and demonstrate, for the first time, Merlin immunoreactivity in mitochondria. Also, we have found that Merlin colocalizes with Wingless morphogen in the cells at the dorsal/ventral compartment border of the wing imaginal disc. Merlin inactivation may alter the determination/maintenance of Wingless stripe expression. Cells lacking Merlin possess greater ability to overcome vein restriction. In addition, we provide evidence for potential genetic interactions between Merlin and the proteins involved in vesicular trafficking, including Porcupine, Shibire, and Lap. By analyzing the evolution, diversity, and overall distribution of Merlin among different taxa, we demonstrate a monophyletic origin of the Merlin proteins with their root in the early metazoa. The overall similarity among the primary and secondary structures of all merlin proteins and the conservation of several functionally important residues suggest a universal role for merlin in a wide range of metazoa. Furthermore, we show that the AKT pathway is frequently activated in NF2- tumor cells. We have tested two novel compounds, OSU03012 and (S)-HDAC-42, which inhibit AKT phosphorylation, and found that these drugs effectively inhibit the growth of vestibular schwannoma cells. These findings set the stage for a phase I clinical trial on VS in the future.

To better understand the mechanism by which Merlin functions as a tumor suppressor we have shown that mutations in the Drosophila Merlin gene lead to increased mitosis and alter the duration of the G2 phase of the cell cycle. We have also found that the Merlin protein is dynamically redistributed during meiosis and discovered for the first time Merlin immunoreactivity in the mitochondria. In support of the finding of a genetic interaction between Merlin and lap which encodes an adapter protein involved in vesicular trafficking we show that both the Merlin and Lap proteins colocalize at the cellular cortex of the wing imaginal disc cell. In addition we demonstrate that the distribution of the Merlin protein in the wing imaginal disc is not affected by other tumor suppressor mutations. We also show that the Drosophila Merlin protein is regulated by phosphorylation; while the non-phospho-Merlin protein appears mostly in the cytoplasm the phospho-Merlin protein can be seen in the membrane region. Furthermore we have found that the AKT pathway is frequently activated in NF2- tumor cells. We have tested two novel compounds OSU03012 and (S)-HDAC-42, which inhibit AKT phosphorylation. We show that these drugs effectively inhibit the growth of vestibular schwannoma cells. These findings set the stage for a phase 1 clinical trial on VS in the future., The original document contains color images.

A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immunoblotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and gamma-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by Na(+) similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures.

Although the oral route of drug administration is the most acceptable way of self-medication with a high degree of patient compliance, the intestinal absorption of many drugs is severely hampered by different biological barriers. These barriers comprise of biochemical and physical components. The biochemical barrier includes enzymatic degradation in the gastrointestinal lumen, brush border and in the cytoplasm of the epithelial cells as well as efflux transporters that pump drug molecules from inside the epithelial cell back to the gastrointestinal lumen. The physical barrier consists of the epithelial cell membranes, tight junctions and mucus layer. Different strategies have been applied to improve the absorption of drugs after oral administration, which range from chemical modification of drug molecules and formulation technologies to the targeting of receptors, transporters and specialized cells such as the gut-associated lymphoid tissues. This review focuses specifically on the targeting of receptor-mediated endocytosis, transporters and the absorption-site as methods of optimizing intestinal drug absorption. Intestinal epithelial cells express several nutrient transporters that can be targeted by modifying the drug molecule in such a way that it is recognized as a substrate. Receptor-mediated endocytosis is a transport mechanism that can be targeted for instance by linking a receptor substrate to the drug molecule of interest. Many formulation strategies exist for enhancing drug absorption of which one is to deliver drugs at a specific site in the gastrointestinal tract where optimum drug absorption takes place., non disponibile

Although the oral route of drug administration is the most acceptable way of self-medication with a high degree of patient compliance, the intestinal absorption of many drugs is severely hampered by different biological barriers. These barriers comprise of biochemical and physical components. The biochemical barrier includes enzymatic degradation in the gastrointestinal lumen, brush border and in the cytoplasm of the epithelial cells as well as efflux transporters that pump drug molecules from inside the epithelial cell back to the gastrointestinal lumen. The physical barrier consists of the epithelial cell membranes, tight junctions and mucus layer. Different strategies have been applied to improve the absorption of drugs after oral administration, which range from chemical modification of drug molecules and formulation technologies to the targeting of receptors, transporters and specialized cells such as the gut-associated lymphoid tissues. This review focuses specifically on the targeting of receptor-mediated endocytosis, transporters and the absorption-site as methods of optimizing intestinal drug absorption. Intestinal epithelial cells express several nutrient transporters that can be targeted by modifying the drug molecule in such a way that it is recognized as a substrate. Receptor-mediated endocytosis is a transport mechanism that can be targeted for instance by linking a receptor substrate to the drug molecule of interest. Many formulation strategies exist for enhancing drug absorption of which one is to deliver drugs at a specific site in the gastrointestinal tract where optimum drug absorption takes place., non disponibile

Although the oral route of drug administration is the most acceptable way of self-medication with a high degree of patient compliance, the intestinal absorption of many drugs is severely hampered by different biological barriers. These barriers comprise of biochemical and physical components. The biochemical barrier includes enzymatic degradation in the gastrointestinal lumen, brush border and in the cytoplasm of the epithelial cells as well as efflux transporters that pump drug molecules from inside the epithelial cell back to the gastrointestinal lumen. The physical barrier consists of the epithelial cell membranes, tight junctions and mucus layer. Different strategies have been applied to improve the absorption of drugs after oral administration, which range from chemical modification of drug molecules and formulation technologies to the targeting of receptors, transporters and specialized cells such as the gut-associated lymphoid tissues. This review focuses specifically on the targeting of receptor-mediated endocytosis, transporters and the absorption-site as methods of optimizing intestinal drug absorption. Intestinal epithelial cells express several nutrient transporters that can be targeted by modifying the drug molecule in such a way that it is recognized as a substrate. Receptor-mediated endocytosis is a transport mechanism that can be targeted for instance by linking a receptor substrate to the drug molecule of interest. Many formulation strategies exist for enhancing drug absorption of which one is to deliver drugs at a specific site in the gastrointestinal tract where optimum drug absorption takes place., non disponibile

Improper regulation of the level and duration of activated erbB family growth factor receptors at the cell surface can lead to uncontrolled cell proliferation and transformation via over-stimulation of mitogenic signaling cascades. The large GTPase dynamin is a key regulator both of transport of receptors to the plasma membrane after receptor biosynthesis and down-regulation of receptors via receptor-mediated endocytosis (RME), during which it is involved in the scisson of endocytic vesicles. Disruption of RME has been shown to render the epidermal growth factor receptor (erbBl) oncogenic (1), illustrating the importance of proper attenuation of signaling by down-regulation. This proposal addresses the mechanistic role of the pleckstrin homology (PH) domain in dynamin function, which may provide a pharmacologic target for modulating dynamin activity. The PH domain binds% phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane (PM), but the role of this binding is not yet understood. The experiments detailed below address whether PI(4,5)P2 binding is involved in targeting of dynamin to the PM, or whether phosphoinositide binding instead plays a more physical role in the scission of endocytic vesicles, and therefore receptor downregulation., The original document contains color images. All DTIC reproductions will be in black and white.

We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/µg DNA produced the highest luciferase gene expression, exceeding 1 × 109 relative light units/s/mg protein (>3 µg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.

We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/µg DNA produced the highest luciferase gene expression, exceeding 1 × 109 relative light units/s/mg protein (>3 µg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.