Electroporation has been recently adapted for the transfer of macromolecules into cells of tissues in vivo. Although mature adipocytes constitute Ͻ20% of cells residing in adipose tissue, we hypothesized that fat cells might be susceptible to selective electrotransfer of plasmid DNA owing to their large size relative to other cells in the tissue. Results demonstrate the feasibility of electroporating DNA into mature fat cells with Ͼ99% selectivity over other cells in the tissue. Further experiments used the "adiporation" technique to image the subcellular targeting of fluorescent bioreporter molecules to the nucleus, mitochondria, and lipid droplets of adipocytes within intact adipose tissue. Finally, we utilized fluorescent bioreporters to examine the effects of constitutive activation of the -adrenergic signaling pathway in adipocytes. These results demonstrate that overexpression of rat 1-adrenergic receptors alters the cellular morphology of white adipocytes in a fashion that mimics the effects of systemic infusion of 3-adrenergic receptor agonists. Hallmarks of the altered morphology include pronounced fragmentation of the single lipid droplet, repositioning of the nucleus, and induction of mitochondrial biogenesis. These results indicate that activation of -adrenergic signaling within adipocytes is sufficient to induce a phenotype that resembles typical brown adipocytes and suggest that in vivo electroporation will allow molecular dissection of the mechanisms involved. lipid droplet; morphology; perilipin; subcellular targeting; plasticity; mitochondrial biogenesis; gene transfer; biological imaging WORK OVER THE LAST DECADE has established the significance of adipocytes in the regulation of body energy homeostasis (7). Not only are adipocytes the principal energy reservoir in mammals, but these cells also regulate various physiological functions through the secretion of numerous adipokines, like leptin and adiponectin. Analysis of fat cell function by acute genetic manipulation has relied on use of primary cell culture and established cell lines. Although use of cultured cells has provided invaluable information regarding fat cell differentiation and metabolism, these in vitro models have several limitations compared with adipocytes in intact tissue, including low phenotypic gene expression of continuous cell lines and rapid dedifferentiation of freshly isolated cells (4,12,15). These and other data indicate that full phenotypic expression and morphology require important interactions with the extracellular matrix of the intact tissue that are difficult, if not impossible, to replicate in vitro and point to the need for developing techniques for adipocyte-specific gene transfer in vivo.Adipose tissue is a complex tissue that contains several cell types in addition to lipid-laden adipocytes, including those that constitute the vasculature (endothelial and vascular smooth muscle) and adipocyte progenitors. Of the cells residing in adipose tissue, mature adipocytes are by far the largest, with diameters ...
Background: Open heart surgery with cardiopulmonary bypass is recognized as a common cause of acute kidney injury (AKI). The conventional biomarker creatinine is not sensitive enough to detect AKI until a significant decline in renal filtration has occurred. Urine neutrophil gelatinase-associated lipocalin (NGAL), part of an acute response to the release of tissue iron from cells, is an early biomarker and a predictor of AKI in a variety of clinical settings. We sought to evaluate the relationship between urine catalytic iron (unbound iron) and NGAL over the course of AKI due to cardiac surgery. Methods: Fourteen patients who underwent open heart surgery had the following measured: serum creatinine (0, 12, 24, 48 and 72 h postoperatively), urine NGAL and urine catalytic iron (0, 8, 24 and 48 h postoperatively). Urine NGAL and urine catalytic iron were quantified by immunoassay and bleomycin-detectable iron assay, respectively. AKI was defined by the Acute Kidney Injury Network (AKIN) criteria. Results: Urine catalytic iron increased significantly (p < 0.05) within 8 h and peaked at 24 h postoperatively in patients who developed AKI (n = 8, baseline 101.96 ± 177.48, peak 226.35 ± 238.23 nmol/l, p = 0.006), but not in non-AKI patients (n = 6, baseline 131.08 ± 116.21, peak 163.99 ± 109.62 nmol/l, p = 0.380). Urine NGAL levels also peaked at 24 h with significant increase observed only in AKI patients: AKI – baseline 34.88 ± 26.47, peak 65.50 ± 27.03 ng/ml, p = 0.043; non-AKI – baseline 59.33 ± 31.72, peak 71.00 ± 31.76 ng/ml, p = 0.100. The correlation between baseline levels of urine catalytic iron and NGAL and peak levels of urine catalytic iron and NGAL was r = 0.86, p < 0.0001. Conclusion: Urine catalytic iron appears to rise and fall in concert with NGAL in patients undergoing cardiac surgery and may be indicative of early AKI. Future research into the role that catalytic iron plays in acute organ injury syndromes and its potential diagnostic and therapeutic implications is warranted.
The illicit use of cocaine has increased dramatically over the last 10-12 years. There has been a corresponding increase in cocaine abuse among obstetric patients and in the number of "cocaine babies." According to some estimates, these children make up more than half of the drug-associated births. This problem is therefore a major public health concern. Consequently, our laboratory investigated the effects of prenatal cocaine exposure on hearing, vision, growth, and exploratory/stress behavior. This chapter summarizes the literature on animals and humans on these topics and presents new observations from our laboratory. In terms of maternal toxicity, prenatal cocaine exposure causes hypertension, placental abruption, spontaneous abortion, poor pregnancy weight gain, and undernutrition secondary to appetite suppression. Some offspring effects include in utero growth retardation, cephalic hemorrhage, fetal edema, altered body composition, congenital malformations, and even pre- and postnatal death. The offspring can also exhibit a variety of behavioral, visual, hearing, and language disorders. Differential effects of animal strain and late gestational cocaine exposure are discussed. Comparisons are made between prenatal cocaine, the fetal alcohol syndrome, and the effects of prenatal undernutrition. Recommendations for clinical assessment and intervention are made.
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