Hyperthermia increases interleukin-6 in mouse skeletal muscle.

Skeletal muscles produce and contribute to circulating levels of IL-6 during exercise. However, when core temperature is reduced, the response is attenuated. Therefore, we hypothesized that hyperthermia may be an important and independent stimulus for muscle IL-6. In cultured C2C12 myotubes, hyperthermia (42°C) increased IL-6 gene expression 14-fold after 1 h and 35-fold after 5 h of 37°C recovery; whereas exposure to 41°C resulted in a 2.6-fold elevation at 1 h. IL-6 protein was secreted and significantly elevated in the cell supernatant. Similar but reduced responses to heat were seen in C2C12 myoblasts. Isolated soleus muscles from mice, exposed ex vivo to 41°C for 1 h, yielded similar IL-6 gene responses (>3-fold) but without a significant effect on protein release. When whole animals were exposed to passive hyperthermia, such that core temperature increased to 42.4°C, IL-6 mRNA in soleus increased 5.4-fold compared with time matched controls. Interestingly, TNF-α gene expression was routinely suppressed at all levels of hyperthermia (40.5-42°C) in the isolated models, but TNF-α was elevated (4.2-fold) in the soleus taken from intact mice exposed, in vivo, to hyperthermia. Muscle HSP72 mRNA increased as a function of the level of hyperthermia, and IL-6 mRNA responses increased proportionally with HSP72. In cultured C2C12 myotubes, when heat shock factor was pharmacologically blocked with KNK437, both HSP72 and IL-6 mRNA elevations, induced by heat, were suppressed. These findings implicate skeletal muscle as a "heat stress sensor" at physiologically relevant hyperthermia, responding with a programmed cytokine expression pattern characterized by elevated IL-6.

Use of magnetic nanoparticles to monitor alginate-encapsulated betaTC-tet cells.

Noninvasive monitoring of tissue-engineered constructs is an important component in optimizing construct design and assessing therapeutic efficacy. In recent years, cellular and molecular imaging initiatives have spurred the use of iron oxide-based contrast agents in the field of NMR imaging. Although their use in medical research has been widespread, their application in tissue engineering has been limited. In this study, the utility of monocrystalline iron oxide nanoparticles (MIONs) as an NMR contrast agent was evaluated for betaTC-tet cells encapsulated within alginate/poly-L-lysine/alginate (APA) microbeads. The constructs were labeled with MIONs in two different ways: 1) MION-labeled betaTC-tet cells were encapsulated in APA beads (i.e., intracellular compartment), and 2) MION particles were suspended in the alginate solution prior to encapsulation so that the alginate matrix was labeled with MIONs instead of the cells (i.e., extracellular compartment). The data show that although the location of cells can be identified within APA beads, cell growth or rearrangement within these constructs cannot be effectively monitored, regardless of the location of MION compartmentalization. The advantages and disadvantages of these techniques and their potential use in tissue engineering are discussed.

Non-invasive evaluation of alginate/poly-l-lysine/alginate microcapsules by magnetic resonance microscopy.

In this report, we present data to demonstrate the utility of (1)H MR microscopy to non-invasively examine alginate/poly-l-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly-l-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6+/-6.2 microm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 min). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T(2) relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T(2) over the duration of the experiment, while ADC was unaffected. This decrease in T(2) is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T(2) was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T(2) or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions.

Cytochrome c association with the inner mitochondrial membrane is impaired in the CNS of G93A-SOD1 mice.

A "gain-of-function" toxic property of mutant Cu-Zn superoxide dismutase 1 (SOD1) is involved in the pathogenesis of some familial cases of amyotrophic lateral sclerosis (ALS). Expression of a mutant form of the human SOD1 gene in mice causes a degeneration of motor neurons, leading to progressive muscle weakness and hindlimb paralysis. Transgenic mice overexpressing a mutant human SOD1 gene (G93A-SOD1) were used to examine the mitochondrial involvement in familial ALS. We observed a decrease in mitochondrial respiration in brain and spinal cord of the G93A-SOD1 mice. This decrease was significant only at the last step of the respiratory chain (complex IV), and it was not observed in transgenic wild-type SOD1 and nontransgenic mice. Interestingly, this decrease was evident even at a very early age in mice, long before any clinical symptoms arose. The effect seemed to be CNS specific, because no decrease was observed in liver mitochondria. Differences in complex IV respiration between brain mitochondria of G93A-SOD1 and control mice were abolished when reduced cytochrome c was used as an electron donor, pinpointing the defect to cytochrome c. Submitochondrial studies showed that cytochrome c in the brain of G93A-SOD1 mice had a reduced association with the inner mitochondrial membrane (IMM). Brain mitochondrial lipids, including cardiolipin, had increased peroxidation in G93A-SOD1 mice. These results suggest a mechanism by which mutant SOD1 can disrupt the association of cytochrome c with the IMM, thereby priming an apoptotic program.

Limitations of allotopic expression of mitochondrial genes in mammalian cells.

The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

Effects of growth regulation on conditionally-transformed alginate-entrapped insulin secreting cell lines in vitro.

The ability to control cell growth is an issue of critical importance for the use of transformed beta-cell lines within a bioartificial pancreas. Such control can be achieved either by entrapping the cells in a biomaterial that can inhibit cell proliferation or by genetically modifying the cells to regulate growth. Integrating tetracycline-off or -on operon systems into murine insulinoma cell lines (betaTC-tet and R7T1, respectively) allows cell growth regulation upon exposure to tetracycline (TC) or its derivative doxycycline (Dox), respectively. However, the effects of this regulatory approach on the long-term phenotypic metabolic and secretory stability of alginate-entrapped cells have yet to be thoroughly investigated. In this study, cultures of betaTC-tet and R7T1 cells entrapped in alginate beads were allowed to grow freely, or were growth-regulated, either at the onset, or after 20 days of growth. The data show that growth regulation of alginate-entrapped cells is achievable with chronic administration of the regulatory compound in a concentration-dependent manner. However, as these cultures age, the amount of insulin released does not always reflect the metabolic and histological characteristics of the cultures. This change, coupled with a loss of glucose stimulated insulin secretion in the Dox treated R7T1 cell line, indicate a phenotypic shift of cells with an activated tet-operon. These observations have implications on the selection and long-term function of three-dimensional bioartificial pancreatic constructs that include conditionally transformed beta-cell lines.

Repurposed biological scaffolds: kidney to pancreas.

Advances in organ regeneration have been facilitated by gentle decellularization protocols that maintain distinct tissue compartments, and thereby allow seeding of blood vessels with endothelial lineages separate from populations of the parenchyma with tissue-specific cells. We hypothesized that a reconstituted vasculature could serve as a novel platform for perfusing cells derived from a different organ: thus discordance of origin between the vascular and functional cells, leading to a hybrid repurposed organ. The need for a highly vascular bed is highlighted by tissue engineering approaches that involve transplantation of just cells, as attempted for insulin production to treat human diabetes. Those pancreatic islet cells present unique challenges since large numbers are needed to allow the cell-to-cell signaling required for viability and proper function; however, increasing their number is limited by inadequate perfusion and hypoxia. As proof of principle of the repurposed organ methodology we harnessed the vasculature of a kidney scaffold while seeding the collecting system with insulin-producing cells. Pig kidneys were decellularized by sequential detergent, enzymatic and rinsing steps. Maintenance of distinct vascular and collecting system compartments was demonstrated by both fluorescent 10 micron polystyrene microspheres and cell distributions in tissue sections. Sterilized acellular scaffolds underwent seeding separately via the artery (fibroblasts or endothelioma cells) and retrograde (murine ßTC-tet cells) up the ureter. After three-day bioreactor incubation, histology confirmed separation of cells in the vasculature from those in the collecting system. ßTC-tet clusters survived in tubules, glomerular Bowman's space, demonstrated insulin immunolabeling, and thereby supported the feasibility of kidney-to-pancreas repurposing.

Techniques and pitfalls in the detection of pathogenic mitochondrial DNA mutations.

Mutations in the mitochondrial DNA (mtDNA) are now recognized as major contributors to human pathologies and possibly to normal aging. A large number of rearrangements and point mutations in protein coding and tRNA genes have been identified in patients with mitochondrial disorders. In this review, we discuss genotype-phenotype correlations in mitochondrial diseases and common techniques used to identify pathogenic mtDNA mutations in human tissues. Although most of these approaches employ standard molecular biology tools, the co-existence of wild-type and mutated mtDNA (mtDNA heteroplasmy) in diseased tissues complicates both the detection and accurate determination of the size of the mutated fractions. To address these problems, novel approaches were developed and are discussed in this review.

Reactive oxygen species scavenging properties of ZrO2-CeO2 solid solution nanoparticles.

AIMS: The hypothesis that an increase in defects in cerium oxide (CeO(2)) nanoparticles induced by solid solutions with differences in valency and ionic radius of the solute will yield superior reactive oxygen species (ROS) scavengers at room temperature will be tested. METHODS: Solid solutions of zirconium in CeO(2), that is, Ce(x)Zr(1-x)O(2) nanoparticles, were synthesized by a reverse micelle method. Their crystal structures, particle sizes and level of agglomeration were characterized. The nanoparticles' activities to scavenge ROS were tested in response to hydrogen peroxide at physiological levels and room temperature using an enzyme peroxidase-based assay. RESULTS: Solid solutions of Zr in CeO(2) nanoparticles enhanced ROS scavenging fourfold. The hypothesis is confirmed that more defects are formed and that the scavenging activities of Ce(x)Zr(1-x)O(2) correlate to the nanoparticles' oxygen-storage capacity. CONCLUSIONS: The antioxidant efficacy of CeO(2) nanoparticles can be enhanced by dissolving zirconium in the CeO(2) lattice. The Ce(x)Zr(1-x)O(2) nanoparticles act as an enhanced catalyst at room temperature that scavenges ROS. Increased efficacy will enable lower nanoparticle dosages to protect cells from ROS, thus increasing the therapeutic width of these compounds.

Novel synthesis of cerium oxide nanoparticles for free radical scavenging.

AIMS: The aim of this article is to present a novel synthetic route to form CeO(2) nanoparticles that protects against the detrimental influence of oxidative stress in mammalian cells. METHODS: The noncytotoxic surfactant lecithin was used to synthesize CeO(2) nanoparticles and the products were colloidally stabilized in a biocompatible tri-sodium citrate buffer. These nanoparticles were delivered into murine insulinoma betaTC-tet cells, and intracellular free radical concentrations responding to exposure to hydroquinone were measured in a variety of extracellular CeO(2) concentrations. RESULTS: Well-dispersed, highly crystallized CeO(2) nanoparticles of 3.7 nm in size were achieved that are chemically and colloidally stable in Dulbecco's modified Eagle's medium for extended periods of time. Treating betaTC-tet cells with these nanoparticles alleviated detrimental intracellular free radical levels down to the primary level. CONCLUSION: CeO(2) nanoparticles synthesized from this route are demonstrated to be effective free radical scavengers within betaTC-tet cells. Furthermore, it is shown that CeO(2) nanoparticles provide an effective means to improve cellular survival in settings wherein cell loss due to oxidative stress limits native function.

Magnetic resonance spectroscopic investigation of mitochondrial fuel metabolism and energetics in cultured human fibroblasts: effects of pyruvate dehydrogenase complex deficiency and dichloroacetate.

The pyruvate dehydrogenase complex (PDC) is integral to metabolism and energetics. Congenital PDC deficiency leads to lactic acidosis, neurological degeneration and early death. An investigational compound for such defects is dichloroacetate (DCA), which activates the PDC (inhibiting reversible phosphorylation of the E1alpha subunit) and decreases its turnover. Here, primary human fibroblast cultures from five healthy subjects and six patients with mutations in the PDC-E1 component were grown in media+/-DCA, exposed to media containing (13)C-labeled glucose, and studied (as cell extracts) by nuclear magnetic resonance (NMR) spectroscopy. Computer modeling of NMR-derived (13)C-glutamate isotopomeric patterns estimated relative carbon flow through TCA cycle-associated pathways and characterized effects of PDC deficiency on metabolism and energetics. Rates of glucose consumption (GCR) and lactate production (LPR) were measured. With the exception of one patient cell line expressing an unusual splicing mutation, PDC-deficient cells had significantly higher GCR, LPR and label-derived acetyl-CoA, indicative of increased glycolysis vs. controls. In all cells, DCA caused a major shift (40% decrease) from anaplerotic-related pathways (e.g., pyruvate carboxylase) toward flux through PDC. Ignoring the patient with the splicing mutation, DCA decreased average glycolysis (29%) in patient cells, but had no significant effect on control cells, and did not change LPR or the nucleoside triphosphate to diphosphate ratio (NTP/NDP) in either cell type. Maintenance of NTP despite reduced glycolysis indicates that DCA improves metabolic efficiency by increasing glucose oxidation. This study demonstrates that NMR spectroscopy provides insight into biochemical consequences of PDC deficiency and the mechanism of putative therapeutic agents.

BCL-2 improves oxidative phosphorylation and modulates adenine nucleotide translocation in mitochondria of cells harboring mutant mtDNA.

Members of the BCL-2-related antiapoptotic family of proteins have been shown previously to regulate ATP/ADP exchange across the mitochondrial membranes and to prevent the loss of coupled mitochondrial respiration during apoptosis. We have found that BCL-2/BCL-x(L) can also improve mitochondrial oxidative phosphorylation in cells harboring pathogenic mutations in mitochondrial tRNA genes. The effect of BCL-2 overexpression in mutated cells was independent from apoptosis and was presumably associated with a modulation of adenine nucleotide exchange between mitochondria and cytosol. These results suggest that BCL-2 can regulate respiratory functions in response to mitochondrial distress by regulating the levels of adenine nucleotides.

Magnetically labeled insulin-secreting cells.

Iron oxide nanoparticles have been shown to magnetically label cells in order to visualize them in vivo via MR imaging. This technology has yet to be implemented in insulin secreting cells, thus it is not known whether the presence of these nanoparticles in the cytoplasm of the cells affects insulin secretion. This study investigates the effectiveness and consequence of labeling mouse insulinoma betaTC3 and betaTC-tet cells with monocrystalline iron oxide nanoparticles (MION). Our data show that MION can be internalized in both betaTC3 and betaTC-tet cells following a 24h exposure to 0.02mg/ml MION solution. The metabolic and secretory activities of both MION-labeled cell lines were statistically indistinguishable from sham treatment. Furthermore, cell viability and apoptosis remained constant throughout the cell's exposure to MION. Finally, MR images demonstrated significant contrast between labeled and sham-treated cells. Thus, labeling murine insulinoma cell lines with magnetic iron oxide nanoparticles does not hinder their insulin secretion, while it provides MR imaging contrast.