Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on fatty acid availability and neural tube formation in cynomolgus macaque, Macaca fascicularis

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to alter carbohydrate utilization and specific steps in lipid metabolism. TCDD interacts with estradiol in mobilizing specific fatty acids in chickens that may be a cause of cranial/beak malformations in this species. This study was designed to test the hypothesis that TCDD simultaneously alters critical fatty acid mobilization during early pregnancy and determine if those changes correlate to morphological defects of the developing neural tube in the nonhuman primate. Cynomolgus macaques were treated with a single dose of 4 microg/kg body weight (BW) TCDD on gestational day 15 or 20. Pregnancies were terminated by hysterectomy on gestational day 24-26 and embryos were examined to determine morphology of the developing neural tube. Maternal blood samples were used for fatty acid quantification. Embryos exhibited cellular changes, mainly increased cell death, and intercellular spaces in the neural tube, suggestive of an adverse effect on the developing nervous system. Significant decreases on fatty acid composition were found on some of the eight classes of lipids analyzed. Particularly, a decrease was observed in the n-3 (40-60%) and n-6 (47-75%) essential fatty acids in treated pregnancies compared to untreated controls. These data demonstrate the effect of TCDD in decreasing maternal levels of n-3 and n-6 fatty acids that are considered necessary for normal development in mammals. Since neural tube development is dependent, in part, on n-3 and n-6 fatty acids, it is possible that the limitation of these essential fatty acids in plasma resulted in the observed detrimental effects on early brain development.     

In vivo exposure to hyperoxia induces DNA damage in a population of alveolar type II epithelial cells

It is well established that hyperoxia injures and kills alveolar endothelial and type I epithelial cells of the lung. Although type II epithelial cells remain morphologically intact, it remains unclear whether they are also damaged. DNA integrity was investigated in adult mice whose type II cells were identified by their endogenous expression of pro-surfactant protein C or transgenic expression of enhanced green fluorescent protein. In mice exposed to room air, punctate perinuclear 8-oxoguanine staining was detected in approximately 4% of all alveolar cells and in 30% of type II cells. After 48 or 72 h of hyperoxia, 8-oxoguanine was detected in 11% of all alveolar cells and in >60% of type II cells. 8-Oxoguanine colocalized by confocal microscopy with the mitochondrial transmembrane protein cytochrome oxidase subunit 1. Type II cells isolated from hyperoxic lungs exhibited nuclear DNA strand breaks by comet assay even though they were viable and morphologically indistinguishable from cells isolated from lungs exposed to room air. These data reveal that type II cells exposed to in vivo hyperoxia have oxidized and fragmented DNA. Because type II cells are essential for lung remodeling, our findings raise the possibility that they are proficient in DNA repair.     

Evaluating metabolic syndrome in a medical physiology laboratory

The metabolic syndrome, a cluster of factors linked to obesity that contribute to risk for atherosclerosis and Type 2 diabetes, may affect 20-25% of the adults in the United States. We designed a medical physiology laboratory to evaluate and discuss the physiological and nutritional principles involved in the metabolic syndrome. The five criteria used to diagnose this syndrome (fasting blood triglycerides, high-density lipoprotein cholesterol, and glucose, blood pressure, central obesity) were measured by students on each other either previously or during this exercise. In addition, to illustrate nutritional factors involved in causation and treatment of the metabolic syndrome, a meal was provided during the laboratory. Class members were randomized to groups allowed ad libitum meal composition, or constrained to the National Cholesterol Education Program Step I or Step II diets. The composition of the diet (including saturated fat, cholesterol, dietary fiber, and carbohydrate content) was discussed in the context of blood cholesterol, triglyceride, and glucose levels. This laboratory allows a comprehensive analysis of the physiological and nutritional factors involved in the development of the metabolic syndrome.     

Coactivator binding promotes the specific interaction between ligand and the pregnane X receptor

The pregnane X receptor (PXR) detects the presence of a wide variety of endogenous and xenobiotic compounds, and is a master regulator of the expression of genes central to drug metabolism and excretion. We present the 2.0A crystal structure of the human PXR ligand-binding domain (LBD) in complex with the cholesterol-lowering compound SR12813 and a 25 amino acid residue fragment of the human steroid receptor coactivator-1 (SRC-1) containing one LXXLL motif. PXR crystallizes as a homodimer in the asymmetric unit in this structure and possesses a novel alpha2 helix adjacent to its ligand-binding cavity. The SRC-1 peptide forms two distinct helices and binds adjacent to the ligand-dependent transactivation AF-2 helix on the surface of PXR. In contrast with previous PXR structures, in which SR12813 bound in multiple orientations, the small SR12813 agonist in this structure binds in a single, unique orientation within the receptor's ligand-binding pocket and contacts the AF-2 helix. Thermal denaturation studies reveal that the SR12813 ligand and SRC-1 coactivator peptide each stabilize the LBD of PXR, and that together they exert an additive effect on the stability of the receptor. These results indicate that the binding of coactivator to the surface of PXR limits the ability of this promiscuous receptor to "breathe" and helps to trap a single, active conformation of SR12813. They further reveal that specificity is required for PXR activation.     

Adenovirus-transduced dendritic cells injected into skin or lymph node prime potent simian immunodeficiency virus-specific T cell immunity in monkeys

Adenoviral vectors can be used to deliver complex Ag to dendritic cells (DC), and thus may be ideal for stimulating broad T cell responses to viral pathogens and tumors. To test this hypothesis in a relevant primate model, we used recombinant adenovirus serotype 5 vectors expressing SIV Gag Ag to transduce monocyte-derived DC from rhesus macaques, and then immunized donor animals either by intradermal or intranodal injections. T cell responses were evaluated by ELISPOT assay using previously frozen PBMC pulsed with pools of 15-mer peptides representing the Gag sequence. Immunization resulted in rapid and potent induction of T cell responses to multiple regions of Gag, with frequencies approaching 1 Gag-specific T cell per 500 uncultured PBMC. Surprisingly, intradermal and intranodal injections generated a similar intensity and breadth of response, indicating that administration of Ag-expressing DC by either route may be equally effective at inducing immune responses. Detailed analysis of two monkeys revealed CD8(+) T cell responses to several peptide epitopes of Gag not previously described, at least two of which are restricted by MHC class I alleles not currently identified. Repeated vaccination did not induce T cell responses to the adenoviral vector and did not prevent Ag-expressing DC injected under the capsule of the lymph node from migrating to the paracortex and interposing between T cells. However, boost injections of adenovirus-transduced DC were generally limited in efficacy. These findings support the use of adenovirus-transduced DC in the therapy of HIV infection and cancer.     

Genetics and population history of Caucasus populations

We describe aspects of genetic diversity in several ethnic populations of the Caucasus Mountains of Daghestan using mitochondrial DNA sequences and a sample of 100 polymorphic Alu insertion loci. The mitochondrial DNA (mtDNA) sequences are like those of Europe. Principal coordinates and nearest neighbor statistics show that there is little detectable structure in the distances among populations computed from mtDNA. The Alu frequencies of the Caucasus populations suggest that they have undergone more genetic drift than most other groups since the dispersal of modern humans. Genetic differences among these populations are not large; instead, they are of the same order as distances among populations of Europe. We compare two methods of inference about the demography of ancient colonizing populations from Africa, one based on conventional FST statistics and one based on mean Alu insertion frequencies. The two approaches agree reasonably well if we assume that there was demographic growth in Africa before the diaspora of ancestors of contemporary regional human groups outside Africa.     

A novel conus peptide ligand for K+ channels

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.     

Dynamic changes in p27kip1 variant expression in activated lymphocytes

The p27Kip1 cell cycle inhibitor (p27) has emerged as a critical mediator of normal cellular growth control. We report the expression of a 24 kD C-terminal variant of p27 in normal peripheral blood lymphocytes. This variant is rapidly degraded in a proteasome-dependent manner when lymphocytes are activated by interleukin-2 or by superantigen. Whereas p24 degradation is complete within 16 h of mitogen addition, full-length p27 is decreased only modestly over 72 h of mitogen exposure and is present in activated and cycling lymphocytes. Persistent p27 is present in a complex with cyclin D3 in activated lymphocytes, and is localized both in the nucleus and cytoplasm. These results indicate that lymphocytes exiting from quiescence use several mechanisms to overcome the p27Kip1-enforced cell cycle checkpoint, and that elimination of p27 is not required for cell cycle entry.