Stem cell isolation

High-dose chemotherapy with autologous hematopoietic progenitor cell support is increasingly used to treat a variety of malignant diseases. A drawback of this technique is the potential for infusing clonogenic tumor cells with the autograft, producing relapse of the disease in the patient. The use of positive selection techniques to isolate stem cells and thus reduce or eliminate tumor cell contamination has been extensively studied over the past few years. Preliminary clinical results have demonstrated that these procedures deplete 2 to 7 logs of tumor cells and do not impair engraftment. It is too early to assess the ultimate clinical benefit of this strategy. Additional applications of CD34-selection include ex vivo expansion of and gene transfer into hematopoietic progenitor cells and T-cell depletion of allogeneic grafts to reduce the incidence of graft-versus-host disease.     

Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma

Peroxisome proliferator-activated receptors (PPARs) alpha and gamma are key regulators of lipid homeostasis and are activated by a structurally diverse group of compounds including fatty acids, eicosanoids, and hypolipidemic drugs such as fibrates and thiazolidinediones. While thiazolidinediones and 15-deoxy-Delta12, 14-prostaglandin J2 have been shown to bind to PPARgamma, it has remained unclear whether other activators mediate their effects through direct interactions with the PPARs or via indirect mechanisms. Here, we describe a novel fibrate, designated GW2331, that is a high-affinity ligand for both PPARalpha and PPARgamma. Using GW2331 as a radioligand in competition binding assays, we show that certain mono- and polyunsaturated fatty acids bind directly to PPARalpha and PPARgamma at physiological concentrations, and that the eicosanoids 8(S)-hydroxyeicosatetraenoic acid and 15-deoxy-Delta12,14-prostaglandin J2 can function as subtype-selective ligands for PPARalpha and PPARgamma, respectively. These data provide evidence that PPARs serve as physiological sensors of lipid levels and suggest a molecular mechanism whereby dietary fatty acids can modulate lipid homeostasis.     

Regulated binding of the protein kinase C substrate GAP-43 to the V0/C2 region of protein kinase C-delta

The interaction between protein kinase C-delta and its neuronal substrate, GAP-43, was studied. Two forms of protein kinase C-delta were isolated from COS cells and characterized by differences in gel mobility, GAP-43 binding, and specific GAP-43 and histone kinase activities. A slow migrating, low specific activity form of protein kinase C-delta bound directly to immobilized GAP-43. Binding was abolished in the presence of EGTA, suggesting Ca2+ dependence of the interaction. The free catalytic domain of protein kinase C-delta did not bind GAP-43, suggesting the existence of a binding site in the regulatory domain. Glutathione S-transferase-protein kinase C-delta regulatory domain fusion proteins were generated and tested for binding to GAP-43. The V0/C2-like amino-terminal domain was defined as the GAP-43-binding site. GAP-43 binding to this region is inhibited by EGTA and regulated at Ca2+ levels between 10(-7) and 10(-6) M. The interaction between protein kinase C-delta and GAP-43 was studied in intact cells by coexpression of the two proteins in human embryonic kidney cells followed by immunoprecipitation. Complex formation occurred only after treatment of the cells with the Ca2+ ionophore ionomycin, indicating that elevation of intracellular Ca2+ is required for interaction in vivo. It is concluded that protein kinase C-delta interacts with GAP-43 through the V0/C2-like domain, outside the catalytic site, and that this interaction is modulated by intracellular Ca2+.     

Serum human chorionic gonadotropin measurement in the diagnosis of ectopic pregnancy when transvaginal sonography is inconclusive

Transition from placental to pulmonary oxygenation at birth depends on a rapid removal of fetal lung fluid from the developing alveoli. Alveolar fluid clearance was examined in ventilated, anesthetized developing guinea pigs of the ages newborn, 2-d-old, 5-d-old, 30-d-old, and 60-d-old (adult). An isosmolar 5% albumin solution was instilled into the lungs of the guinea pigs; the guinea pigs were then studied for 1 h. Alveolar fluid clearance was measured from the increase in alveolar protein concentration as water was reabsorbed. Newborn guinea pigs had a very high alveolar fluid clearance rate that declined rapidly within the first 5 postnatal days towards adult levels. The high alveolar fluid clearance at birth was apparently mediated by the beta-adrenergic system as demonstrated by the elevated plasma epinephrine levels and the increased sensitivity to inhibition by the beta-adrenergic antagonist propranolol immediately after birth. Surprisingly, exogenous addition of epinephrine was not able to stimulate alveolar fluid clearance in the newborn lung, but exogenous epinephrine stimulation increased over time to adult levels. The elevated alveolar fluid clearance at birth was associated with a significantly greater amiloride sensitivity in the newborn guinea pig lung. Northern blot analysis of distal lung tissue as well as isolated alveolar epithelial type II cells showed and confirmed higher levels of the alpha-subunit of the epithelial sodium channel mRNA in the newborn lung that rapidly tapered off toward adult levels. In conclusion, these data demonstrate the importance of the beta-adrenergic system and amiloride-sensitive sodium transporting pathways for clearance of fetal lung fluid at birth.     

Private business: the uptake of confidential HIV testing in remote aboriginal communities on the Anangu Pitjantjatjara Lands

Bacterial populations associated with three sample types from the neck region of poultry carcasses in the dirty area of an abattoir were characterized. Sample types before and after scalding were skin only, feathers only, and a skin and feather combination. The neck skin of carcasses after the defeathering processing stage was also sampled. Bacterial populations associated with water from the scald tank, rubber fingers at the exit of the defeathering machine, and air in the dirty area were also characterized. Bacterial colonies (751) were randomly isolated from yeast extract-supplemented tryptone soya agar plates exhibiting 30 to 300 colonies. Micrococcus spp. were isolated in the highest proportion from pre-and postscalded carcass samples (63.5 to 86.1% of isolates), regardless of the sample type. Conversely, Enterobacteriaceae (40.3%), Acinetobacter (19.4%), and Aeromonas/Vibrio (12.5%) species predominated on neck skin samples taken from mechanically defeathered carcasses. Isolates from the rubber fingers were, however, predominantly Micrococcus spp. (94.4%). Bacterial groups isolated in the highest proportion from scald tank water samples were Micrococcus spp. (38.3%), species of Enterobacteriaceae (29.1%), and lactic acid bacteria (17.0%). Corynebacterium spp., species of Enterobacteriaceae, and Micrococcus spp. were dominant on air settle plates.