Biomedical subjects

Dusica Maysinger

Publications and source records attributed to Dusica Maysinger.

At least 19 recordsLinked to original sources

PubMed2006

Three-dimensional reconstruction of cell nuclei, internalized quantum dots and sites of lipid peroxidation.

BACKGROUND: The purpose of the study was to develop and illustrate three-dimensional (3-D) reconstruction of nuclei and intracellular lipid peroxidation in cells exposed to oxidative stress induced by quantum dots. Programmed cell death is characterized by multiple biochemical and morphological changes in different organelles, including nuclei, mitochondria and lysosomes. It is the dynamics of the spatio-temporal changes in the signalling and morphological adaptations which will ultimately determine the 'shape' and fate of the cell. RESULTS: We present new approaches to the 3-D reconstruction of organelle morphology and biochemical changes in confocal live-cell images. We demonstrate the 3-D shapes of nuclei, the 3-D intracellular distributions of QDs and the accompanying lipid-membrane peroxidation, and provide methods for quantification. CONCLUSION: This study provides an approach to 3-D organelle and nanoparticle visualization in the context of cell death; however, this approach is also applicable more generally to investigating changes in organelle morphology in response to therapeutic interventions, stressful stimuli and internalized nanoparticles. Moreover, the approach provides quantitative data for such changes, which will help us to better integrate compartmentalization of subcellular events and to link morphological and biochemical changes with physiological outcomes.

Journal Article↗

PubMed2006

Fate of micelles and quantum dots in cells.

Micelles and quantum dots have been used as experimental drug delivery systems and imaging tools both in vitro and in vivo. Investigations of their fate at the subcellular level require different surface-core modifications. Among the most common modifications are those with fluorescent probes, dense-core metals or radionucleids. Cellular fate of several fluorescent probes incorporated into poly(caprolactone)-b-copolymer micelles (PCL-b-PEO) was followed by confocal microscopy, and colloidal gold incorporated in poly 4-vinyl pyridine-PEO micelles were developed to explore micelle fate by electron microscopy. More recently, we have examined quantum dots (QDs) as the next-generation-labels for cells and nanoparticulate drug carriers amenable both to confocal and electron microscopic analyses. Effects of QDs at the cellular and subcellular levels and their integrity were studied. Results from different studies suggest that size, charge and surface manipulations of QDs may play a role in their subcellular distribution. Examples of pharmacological agents incorporated into block copolymer micelles, administered or attached to QD surfaces show how the final biological outcome (e.g. cell death, proliferation or differentiation) depends on physical properties of these nanoparticles.

Animals↗

PubMed2006

Assessment of the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles under biological conditions: a fluorogenic-based approach.

The integrity of block copolymer micelles is important for their effectiveness and successful delivery of the incorporated drugs. Here we evaluate the integrity of poly(caprolactone)-b-poly(ethylene oxide) micelles in media of varying chemical complexity and in cells by using fluorogenic micelles. Fluorogenic dye fluorescein-5-carbonyl azide diacetate was covalently attached to the micelle-core-forming part of the block copolymer, poly(caprolactone). The fluorescence was not detectable unless the poly(caprolactone)21-b-poly(ethylene oxide)45 micelles were destroyed and the fluorogenic dye was activated by deesterification. The fluorescence of the activated dye from destroyed micelles was easily detectable in various media and in cells. Micelles were stable in simple media such as phosphate-buffered saline but disassembled to varying extents with increasing chemical complexity of the media and addition of serum. The integrity of the internalized micelles within the cells showed a time-dependent decrease but remained largely preserved (80%) after 20 h of incubation with cells. A proof of principle was also demonstrated in vivo in mice. The fluorogenic approach to micelle integrity assessment presented herein should lend itself to other block copolymer micelles and assessments of their integrity in complex biological systems in vitro and in vivo.

Animals↗

PubMed2006

Interaction of functionalized superparamagnetic iron oxide nanoparticles with brain structures.

Super Paramagnetic Iron Oxide Nanoparticles (SPIONs) combined with magnetic resonance imaging (MRI) are under clinical evaluation to enhance detection of neurodegenerative diseases. A major improvement would be to link therapeutic drugs to the SPIONs to achieve targeted drug delivery, either at the cell surface or intracellularly, together with active disease detection, without inducing cell reaction. Our objectives were to define the characteristics of SPIONS able to achieve cell-specific interaction with brain-derived structures. Our system consisted in an iron oxide core (9-10 nm diameter) coated either with dextran (Sinerem and Endorem) or various functionalized polyvinyl alcohols (PVAs) (PVA-SPIONs). We investigated the cellular uptake, cytotoxicity, and interaction of these various nanoparticles with brain-derived endothelial cells, microglial cells, and differentiating three-dimensional aggregates. None of the nanoparticles coated with dextran or the various PVAs was cytotoxic or induced the production of the inflammatory mediator NO used as a reporter for cell activation. AminoPVA-SPIONs were taken up by isolated brain-derived endothelial and microglial cells at a much higher level than the other SPIONs, and no inflammatory activation of these cells was observed. AminoPVA-SPIONs did not invade brain cells aggregates lower than the first cell layer and did not induce inflammatory reaction in the aggregates. Fluorescent aminoPVA-SPIONs derivatized with a fluorescent reporter molecule and confocal microscopy demonstrated intracellular uptake by microglial cells. Fluorescent aminoPVA-SPIONs were well tolerated by mice. Therefore, functionalized aminoPVA-SPIONs represent biocompatible potential vector systems for drug delivery to the brain that may be combined with MRI detection of active lesions in neurodegenerative diseases.

Animals↗

PubMed2006

Neurite outgrowth in dorsal root ganglia induced by islet neogenesis-associated protein peptide involves protein kinase A activation.

Islet neogenesis-associated protein (INGAP) peptide is a candidate therapeutic for diabetes and corrects sensory dysfunction in experimental diabetes in mice. In this study, we investigated the mechanism of action by which INGAP peptide promotes neurite outgrowth in sensory neurons of the dorsal root ganglia. Treatment of dorsal root ganglia primary dispersed cultures with INGAP peptide led to the displacement of fluorescently labeled forskolin from adenylate cyclase, the cyclic AMP-generating enzyme that has been implicated in neuritogenesis. The addition of forskolin or dibutyryl cyclic AMP enhanced the effects of INGAP peptide on neurite outgrowth in dorsal root ganglia explant cultures. Furthermore, pharmacological inhibition of adenylate cyclase with SQ22,536 or of protein kinase A with H89 or KT5720 significantly reduced the neurite-promoting effects of INGAP peptide. These results suggest that INGAP peptide-induced neurite outgrowth in the dorsal root ganglia partially involves cyclic AMP-dependent activation of protein kinase A.

Adenylyl Cyclases↗

PubMed2006

Dual-action peptides: a new strategy in the treatment of diabetes-associated neuropathy.

Peripheral neuropathy is one of the most common and debilitating complications of type 1 and type 2 diabetes mellitus. Recent studies have shown that several small, non-neural peptides possess neurotrophic activity and exert beneficial effects on nervous system function in experimental and clinical diabetes. Two of these, C-peptide and islet neogenesis-associated protein peptide, are derived from pancreatic proteins and use related signal transduction mechanisms. Derivatives of erythropoietin possess similar properties in the nervous system. As a group, these peptides are of increasing interest as leads to potential new approaches in the treatment of diabetes-associated neuropathies and other neurodegenerative conditions. This review addresses the recent advances made with these peptides in the context of diabetic neuropathy, and highlights similarities and differences in their mechanisms of action from the perspective of combination therapy.

Animals↗

PubMed2006

Early obesity and age-related mimicry of metabolic syndrome in female mice with sex hormonal imbalances.

OBJECTIVE: To investigate the relationship of early obesity to metabolic syndrome during sex hormonal imbalances in mutant female mice at different ages. RESEARCH METHODS AND PROCEDURES: Hormonal imbalances, accumulation and nature of adipose tissue, food intake, glucose tolerance, and expression of candidate genes and markers of inflammation were studied by comparing wild-type, null, and haploinsufficient follitropin receptor knockout female mice at different ages. RESULTS: Follitropin receptor deletion in mice produced null females that are infertile and haploinsufficient mice that undergo accelerated biological aging. Both types of mutants with sex hormonal imbalances have central obesity without hyperphagia, but circulating leptin is elevated. Adipocyte hyperplasia and hypertrophy is attributed to elevated peroxisome proliferator-activated receptor gamma expression. Adiponectin protein levels increase in fat tissue and plasma. Only mutants but not controls acquire age-dependent decline in glucose tolerance with high insulin and altered pancreatic beta cells. Changes in inflammation markers, decreased muscle insulin receptor phosphorylation, and increase of the enzyme protein tyrosine phosphatase 1B indicate insulin resistance. DISCUSSION: In this animal model, the chronological appearance of early obesity induced by hormonal imbalances culminates in characteristics that are attributable to metabolic syndrome, including cardiovascular abnormalities. Dissection of the depot-specific alterations and defining molecular interrelationships could help in developing targeted remedies and resolving complications and controversies related to health benefits and adversities of current hormone replacement therapy.

Adiponectin↗

PubMed2006

Block copolymer micelles as delivery vehicles of hydrophobic drugs: micelle-cell interactions.

One-third of drugs in development are water insoluble and one-half fail in trials because of poor pharmacokinetics. Block copolymer micelles are nanosized particles that can solubilize hydrophobic drugs and alter their kinetics in vitro and in vivo. However, block copolymer micelles are not solely passive drug containers that simply solubilize hydrophobic drugs; cells internalize micelles. To facilitate the development of advanced, controlled, micellar drug delivery vehicles, we have to understand the fate of micelles and micelle-incorporated drugs in cells and in vivo. With micelle-based drug formulations recently reaching clinical trials, the impetus for answers is ever so strong and detailed studies of interactions of micelles and cells are starting to emerge. Most notably, the question arises: Is the internalization of block copolymer micelles carrying small molecular weight drugs an undesired side effect or a useful means of improving the effectiveness of the incorporated drugs?

Drug Delivery Systems↗

PubMed2005

Block copolymer micelles: preparation, characterization and application in drug delivery.

Block copolymer micelles are generally formed by the self-assembly of either amphiphilic or oppositely charged copolymers in aqueous medium. The hydrophilic and hydrophobic blocks form the corona and the core of the micelles, respectively. The presence of a nonionic water-soluble shell as well as the scale (10-100 nm) of polymeric micelles are expected to restrict their uptake by the mononuclear phagocyte system and allow for passive targeting of cancerous or inflamed tissues through the enhanced permeation and retention effect. Research in the field has been increasingly focused on achieving enhanced stability of the micellar assembly, prolonged circulation times and controlled release of the drug for optimal targeting. With that in mind, our group has developed a range of block copolymers for various applications, including amphiphilic micelles for passive targeting of chemotherapeutic agents and environment-sensitive micelles for the oral delivery of poorly bioavailable compounds. Here, we propose to review the innovations in block copolymer synthesis, polymeric micelle preparation and characterization, as well as the relevance of these developments to the field of biomedical research.

Animals↗

PubMed2005

Differences in subcellular distribution and toxicity of green and red emitting CdTe quantum dots.

Quantum dots (QDs) are emerging as alternative or complementary tools to the organic fluorescent dyes currently used in bioimaging. QDs hold several advantages over conventional fluorescent dyes including greater photostability and a wider range of excitation/emission wavelengths. However, recent work suggests that QDs exert deleterious effects on cellular processes. This study examined the subcellular localization and toxicity of cadmium telluride (CdTe) QDs and pharmacological means of preventing QD-induced cell death. The localization of CdTe QDs was found to depend upon QD size. CdTe QDs exhibited marked cytotoxicity in PC12 and N9 cells at concentrations as low as 10 microg/ml in chronic treatment paradigms. QD-induced cell death was characterized by chromatin condensation and membrane blebbing and was more pronounced with small (2r=2.2+/-0.1 nm), green emitting positively charged QDs than large (2r=5.2+/-0.1 nm), equally charged red emitting QDs. Pretreatment of cells with the antioxidant N-acetylcysteine and with bovine serum albumin, but not Trolox, significantly reduced the QD-induced cell death. These findings suggest that the size of QDs contributes to their subcellular distribution and that drugs can alter QD-induced cytotoxicity.

Acetylcysteine↗

PubMed2005

Unmodified cadmium telluride quantum dots induce reactive oxygen species formation leading to multiple organelle damage and cell death.

Quantum dots (QDs) are luminescent nanoparticles with unique optical properties that have been exploited for single-cell and whole-animal imaging. When coated with proteins or biocompatible polymers, QDs are not deleterious to cells and organisms. However, when QDs are retained in cells or accumulated in the body for a long period of time, their coatings may be degraded, yielding "naked" QDs. Here, we show that "naked" QDs induce damage to the plasma membrane, mitochondrion, and nucleus, leading to cell death. Reactive oxygen species (ROS) are important players in mediating QD-induced cellular damage. QD-induced cytotoxicity can be reduced or even eliminated without covalent binding of protective agents to the QD surface. Results from these studies suggest the critical role of several subcellular compartments in QD-induced cytotoxicity and point toward multiple molecular targets in nonclassical apoptosis.

Acetylcysteine↗

PubMed2004

INGAP peptide improves nerve function and enhances regeneration in streptozotocin-induced diabetic C57BL/6 mice.

INGAP peptide comprises the core active sequence of Islet Neogenesis Associated Protein (INGAP), a pancreatic cytokine that can induce new islet formation and restore euglycemia in diabetic rodents. The ability of INGAP peptide in vitro to enhance nerve growth from sensory ganglia suggests its potential utility in peripheral nerve disorders. In this study, INGAP peptide was administered alone or in combination with insulin to streptozotocin-induced diabetic mice exhibiting signs of peripheral neuropathy. Following a 2-wk treatment period, thermal hypoalgesia in diabetic mice was significantly improved in groups that received INGAP peptide, without development of hyperalgesia. Explanted dorsal root ganglia (DRG) from these groups showed enhanced nerve outgrowth and evidence of increased mitochondrial activity. Western blotting experiments revealed attenuation of neurofilament hyperphosphorylation, up-regulation of beta-tubulin and actin, and increased phosphorylation of the transcription factor STAT3 in DRG. These findings suggest that INGAP peptide can activate some of the signaling pathways implicated in nerve regeneration in sensory ganglia, thereby providing a means of improvement of nociceptive dysfunction in the peripheral nervous system.

Animals↗

PubMed2004

Cross-talk between phosphatidylinositol 3-kinase/AKT and c-jun NH2-terminal kinase mediates survival of isolated human islets.

Therapeutic strategies aimed at the inhibition of specific cell death mechanisms may increase islet yield and improve cell viability and function after routine isolation. The aim of the current study was to explore the possibility of AKT-JNK cross-talk in islets after isolation and the relevance of c-jun NH2-terminal kinases (JNK) suppression on islet survival. After routine isolation, increased AKT activity correlated with suppression of JNK activation, suggesting that they may be related events. Indeed, the increase in AKT activation after isolation correlated with suppression of apoptosis signal-regulating kinase 1 (ASK1), a kinase acting upstream of JNK, by phosphorylation at Ser83. We therefore examined whether modulators of phosphatidylinositol 3-kinase (PI3K)/AKT signaling affected JNK activation. PI3K inhibition led to increased JNK phosphorylation and islet cell death, which could be reversed by the specific JNK inhibitor SP600125. In addition, IGF-I suppressed cytokine-mediated JNK activation in a PI3K-dependent manner. We also demonstrate that inhibition of PI3K rendered islets more susceptible to cytokine-mediated cell death. SP600125 transiently protected islets from cytokine-mediated cell death, suggesting that JNK may not be necessary for cytokine-induced cell death. When administered immediately after isolation, SP600125 improved islet survival and function, even 48 h after removal of SP600125, suggesting that JNK inhibition by SP600125 may be a viable strategy for improving isolated islet survival. Taken together, these results demonstrate that PI3K/AKT suppresses the JNK pathway in islets, and this cross-talk represents an important antiapoptotic consequence of PI3K/AKT activation.

Androstadienes↗

PubMed2004

Influence of metalation on the morphologies of poly(ethylene oxide)-block-poly(4-vinylpyridine) block copolymer micelles.

Micellization of a poly(ethylene oxide)-block-poly(4-vinylpyridine) (PEO45-b-P4VP28) copolymer in water during metalation (incorporation of gold compounds and gold nanoparticle formation) with three types of gold compounds, NaAuCl4, HAuCl4, and AuCl3, was studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The transformations of the PEO45-b-P4VP28 block copolymer micelles in water were found to depend on a number of parameters including the thermal history of the as-prepared block copolymer, the type of the metal compound, and the metal loading. For the HAuCl4-filled PE045-b-P4VP28 micelles, the subsequent reduction with hydrazine hydrate results in a significant fraction of rodlike micelles, suggesting that slow nucleation (confirmed by the formation of the large gold nanoparticles) and facilitated migration of gold ions yields the ideal conditions for sphere-to-rod micellar transition.

Journal Article↗

PubMed2004

Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans.

The current procedure for isolation of islet cells from the pancreas for transplantation by enzymatic digestion is accompanied by significant islet cell loss. Therapeutic strategies aimed at the inhibition of islet cell damage could be expected to increase islet yield and improve cell viability, thereby making more efficient use of available donor tissue. The aim of the present work was to examine the effects of caspase and PARP-1 inhibition on islet survival. We demonstrate that following isolation, islets become increasingly necrotic and display a PARP-1 cleavage pattern typical of necrotic cells, characterized by the appearance of a 50 kDa cleavage product. Caspase inhibition using Z-VAD-fmk resulted in increased necrosis in both human and canine islets by a nicotinamide-sensitive mechanism. Necrosis was also induced by DEVD-fmk, but not by YVAD-cmk, indicating that only inhibitors of caspase-3 were able to cause necrosis. Moreover, increased mitochondrial depolarization was observed in islets following 72 h in culture, which correlated with increased expression of Bax. Mitochondrial depolarization was also visible in islets treated with both Z-VAD-fmk and nicotinamide, indicating that mitochondrial dysfunction may account for the necrotic-like death observed in the absence of PARP-1 and caspase activity. Our results demonstrate that inhibition of PARP-1 cleavage results in increased levels of PARP-1-mediated necrotic cell death, highlighting the importance of PARP-1 cleavage in assuring the execution of the apoptotic program. Taken together, these findings reveal the interdependence of necrosis and apoptosis in isolated islets, suggesting therapeutic strategies which target early events in cell death signaling in order to prevent multiple forms of islet cell death.

Amino Acid Chloromethyl Ketones↗

PubMed2003

The menopausal mouse: a new neural paradigm of a distressing human condition.

Progressive and long-term sex hormone imbalance in the FSH-R haploinsufficient menopausal mouse leads to degenerative changes in the CNS associated with increased anxiety. The brain region most affected by aging in these mice is the hippocampus. Choline acetyltransferase (ChAT) enzymatic activity and synapsin immunoreactivity are reduced at 20 months of age. Neurons in the dentate gyrus show signs of progressive degenerative changes, hypertrophy and glyosis, and subsequent cell shrinkage and death. These results suggest that the menopausal mouse mimics degenerative changes in the hippocampus of hormonally imbalanced aging humans. We propose using this animal model to test the effectiveness of potential therapeutics in paradigms of accelerated aging.

Aging↗

PubMed2003

Micellar nanocontainers distribute to defined cytoplasmic organelles.

Block copolymer micelles are water-soluble biocompatible nanocontainers with great potential for delivering hydrophobic drugs. An understanding of their cellular distribution is essential to achieving selective delivery of drugs at the subcellular level. Triple-labeling confocal microscopy in live cells revealed the localization of micelles in several cytoplasmic organelles, including mitochondria, but not in the nucleus. Moreover, micelles change the cellular distribution of and increase the amount of the agent delivered to the cells. These micelles may thus be worth exploring for their potential to selectively deliver drugs to specified subcellular targets.

Animals↗

PubMed2003

BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1.

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.

Animals↗