Faculty Bibliography

Insulin resistance is often associated with obesity. We tested whether augmentation of triglyceride synthesis in adipose tissue by transgenic overexpression of the diacylglycerol aclytransferase-1 (Dgat1) gene causes obesity and/or alters insulin sensitivity. Male FVB mice expressing the aP2-Dgat1 had threefold more Dgat1 mRNA and twofold greater DGAT activity levels in adipose tissue. After 30 weeks of age, these mice had hyperglycemia, hyperinsulinemia, and glucose intolerance on a high-fat diet but were not more obese than wild-type littermates. Compared with control littermates, Dgat1 transgenic mice were both insulin and leptin resistant and had markedly elevated plasma free fatty acid levels. Adipocytes from Dgat1 transgenic mice displayed increased basal and isoproterenol-stimulated lipolysis rates and decreased gene expression for fatty acid uptake. Muscle triglyceride content was unaffected, but liver mass and triglyceride content were increased by 20 and 300%, respectively. Hepatic insulin signaling was suppressed, as evidenced by decreased phosphorylation of insulin receptor-beta (Tyr(1,131)/Tyr(1,146)) and protein kinase B (Ser473). Gene expression data suggest that the gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, were upregulated. Thus, adipose overexpression of Dgat1 gene in FVB mice leads to diet-inducible insulin resistance, which is secondary to redistribution of fat from adipose tissue to the liver in the absence of obesity.

The discovery of long-lived epithelial stem cells in the bulge region of the hair follicle led to the hypothesis that epidermal renewal and epidermal repair after wounding both depend on these cells. To determine whether bulge cells are necessary for epidermal renewal, here we have ablated these cells by targeting them with a suicide gene encoding herpes simplex virus thymidine kinase (HSV-TK) using a Keratin 1-15 (Krt1-15) promoter. We show that ablation leads to complete loss of hair follicles but survival of the epidermis. Through fate-mapping experiments, we find that stem cells in the hair follicle bulge do not normally contribute cells to the epidermis which is organized into epidermal proliferative units, as previously predicted. After epidermal injury, however, cells from the bulge are recruited into the epidermis and migrate in a linear manner toward the center of the wound, ultimately forming a marked radial pattern. Notably, although the bulge-derived cells acquire an epidermal phenotype, most are eliminated from the epidermis over several weeks, indicating that bulge stem cells respond rapidly to epidermal wounding by generating short-lived 'transient amplifying' cells responsible for acute wound repair. Our findings have implications for both gene therapy and developing treatments for wounds because it will be necessary to consider epidermal and hair follicle stem cells as distinct populations

The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The DeltarasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited approximately 65% survival compared to approximately 10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.

Cardiolipin is a unique mitochondrial phospholipid with an atypical fatty acid profile, but the significance of its acyl specificity has not been understood. We explored the enormous combinatorial diversity among cardiolipin species, which results from the presence of four fatty acids in each molecule, by integrated use of high-performance liquid chromatography, mass spectrometry, diacylglycerol species analysis, fatty acid analysis, and selective cleavage of fatty acids by phospholipase A2. The most abundant cardiolipin species from various organisms and tissues (human heart, human lymphoblasts, rat liver, Drosophila, sea urchin sperm, yeast, mung bean hypocotyls) contained only one or two types of fatty acids, which generated a high degree of structural uniformity and molecular symmetry. However, an exception was found in patients with Barth syndrome, in whom an acyltransferase deficiency led to loss of acyl selectivity and formation of multiple molecular species. These results suggest that restriction of the number of fatty acid species, rather than the selection of a particular kind of fatty acid, is the common theme of eukaryotic cardiolipins. This limits the structural diversity of the cardiolipin species and creates molecular symmetry with implications for the stereochemistry of cardiolipin

Respiratory dysfunction during sepsis is common. However, although lung function can often be adequately supported, death frequently results from cardiovascular collapse. Despite intense investigation, the mechanism underlying the myocardial dysfunction of sepsis remains unclear. Macrophage migration inhibitory factor (MIF), an important cytokine released in sepsis and the acute respiratory distress syndrome, is a known cardiac depressant. We hypothesized that MIF released from the lung results in myocardial dysfunction during sepsis. In murine models of polymicrobial sepsis, we demonstrate a significant increase in the lungs of total and lavagable MIF between 20 and 30 h post induction of sepsis. At 30 h post sepsis, the lungs released MIF into the pulmonary circulation, increasing the plasma concentration by up to 51% in a single pass. Exogenous MIF, instilled into the lungs, increased alveolar keratinocyte-derived chemokine (KC), Macrophage inflammatory protein-2 (MIP2), and tumor necrosis factor alpha (TNFalpha) at 3 h, and plasma KC and MIP2 at 6 h postinstillation. This was associated with an increase in p38 mitogen-activated protein kinase and c-Jun N-terminal kinase phosphorylation. Because changes in mitogen-activated protein kinase activation can lead to myocardial depression, these data suggest that MIF released from the lungs may be responsible, at least in part, for the cardiac dysfunction seen in the late stages of sepsis

Molecular motors are enzymatic proteins that couple the consumption of chemical energy to mechanical displacement. In order to elucidate the translocation mechanisms of these enzymes, it is of fundamental importance to measure the physical step size. The step size can, in certain instances, be directly measured with single-molecule techniques; however, in the majority of cases individual steps are masked by noise. The step size can nevertheless be obtained from noisy single-molecule records through statistical methods. This analysis is analogous to determining the charge of the electron from current shot noise. We review methods for obtaining the step size based on analysing, in both the time and frequency domains, the variance in position from noisy single-molecule records of motor displacement. Additionally, we demonstrate how similar methods may be applied to measure the step size in bulk kinetic experiments.

Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and metastasis. Currently, most of the available information on the function of Rho proteins in malignant transformation is based on the use of dominant-negative mutants of these GTPases. The specificity of these dominant-negative mutants is limited however. In this study, we used small interfering RNA directed against either Rac1 or Rac3 to reduce their expression specifically. In line with observations using dominant-negative Rac1 in other cell types, we show that RNA interference-mediated depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and invasion in SNB19 glioblastoma cells. Surprisingly however, Rac1 depletion has a much smaller inhibitory effect on SNB19 cell proliferation and survival. Interestingly, whereas depletion of Rac3 strongly inhibits SNB19 cell invasion, it does not affect lamellipodia formation and has only minor effects on cell migration and proliferation. Similar results were obtained in BT549 breast carcinoma cells. Thus, functional analysis of Rac1 and Rac3 using RNA interference reveals a critical role for these GTPases in the invasive behavior of glioma and breast carcinoma cells

Interneurons of the cerebral cortex represent a heterogeneous population of cells with important roles in network function. At present, little is known about how these neurons are specified in the developing telencephalon. To explore whether this diversity is established in the early progenitor populations, we conducted in utero fate-mapping of the mouse medial and caudal ganglionic eminences (MGE and CGE, respectively), from which most cortical interneurons arise. Mature interneuron subtypes were assessed by electrophysiological and immunological analysis, as well as by morphological reconstruction. At E13.5, the MGE gives rise to fast-spiking (FS) interneurons, whereas the CGE generates predominantly regular-spiking interneurons (RSNP). Later at E15.5, the CGE produces RSNP classes distinct from those generated from the E13.5 CGE. Thus, we provide evidence that the spatial and temporal origin of interneuron precursors in the developing telencephalic eminences predicts the intrinsic physiological properties of mature interneurons

In most organisms, primordial germ cells (PGCs) arise far from the region where somatic gonadal precursors (SGPs) are specified. Although PGCs in general originate as a single cluster of cells, the somatic parts of the gonad form on each site of the embryo. Thus, to reach the gonad, PGCs not only migrate from their site of origin but also split into two groups. Taking advantage of high-resolution real-time imaging, we show that in Drosophila melanogaster PGCs are polarized and migrate directionally toward the SGPs, avoiding the midline. Unexpectedly, neither PGC attractants synthesized in the SGPs nor known midline repellents for axon guidance were required to sort PGCs bilaterally. Repellent activity provided by wunen (wun) and wunen-2 (wun-2) expressed in the central nervous system, however, is essential in this migration process and controls PGC survival. Our results suggest that expression of wun/wun-2 repellents along the migratory paths provides faithful control over the sorting of PGCs into two gonads and eliminates PGCs left in the middle of the embryo