Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells

Although bacterial artificial chromosomes (BACs) provide a well-characterized resource for the analysis of large chromosomal domains, low transfection rates have proven a significant limitation for their use in cell culture models. Using TP53 BAC clones that contain expression cassettes for enhanced green fluorescent protein or red fluorescent protein, we have examined conditions that promote BAC transfection in hamster, human, and mouse cell lines. Atomic force microscopy shows that BAC transfection efficiency correlates with the generation of small, highly condensed but dispersed lipid: BAC DNA transfection complexes. BAC DNA purity and concentration are critical for good transfection; debris from purification columns induces the formation of large aggregates that do not gain entry into the cell, and DNA concentrations must be optimized to promote intramolecular condensation rather than intermolecular linking, which also causes aggregation and diminished transfection efficiency. The expression of both markers and genes within BACs initially occurs at lower levels than observed with plasmids, requiring 3-5 days to evaluate the transfection results. We also show that BACs can be co-transfected with other BACs, which provides for increased experimental flexibility.     

Surfactant protein A and B genetic variants predispose to idiopathic pulmonary fibrosis

Derangement in pulmonary surfactant or its components and alveolar collapse are common findings in idiopathic pulmonary fibrosis (IPF). Surfactant proteins play important roles in innate host defense and normal function of the lung. We examined associations between IPF and genetic polymorphic variants of surfactant proteins, SP-A1, SP-A2, SP-B, SP-C, and SP-D. One SP-A1 (6A⁴) allele and single nucleotide polymorphisms (SNPs) that characterize the 6A⁴ allele, and one SP-B (B1580_C) were found with higher frequency (P0.01) in nonsmoker and smoker IPF (n=84) subgroups, respectively, compared with healthy controls (n=194). To explore whether a tryptophan (present in 6A⁴) or an arginine (present in other SP-A1 alleles and in all SP-A2 alleles) at amino acid 219 alters protein behavior, two truncated proteins that varied only at amino acid 219 were oxidized by exposure to ozone. Differences in the absorption spectra (310–350 nm) between the two truncated recombinant SP-A proteins were observed both before and after protein oxidation, suggesting allele-specific aggregation differences attributable to amino acid 219. The SP-B SNP B1580_C (odds ratio:7.63; confidence interval:1.64–35.4; P0.01), to be a risk factor for IPF smokers, has also been shown to be a risk factor for other pulmonary diseases. The SP-C and SP-D SNPs and SP-B-linked microsatellite markers studied did not associate with IPF. These findings indicate that surfactant protein variants may serve as markers to identify subgroups of patients at risk, and we speculate that these contribute to IPF pathogenesis.     

Drosophila nonmuscle myosin II promotes the asymmetric segregation of cell fate determinants by cortical exclusion rather than active transport

Cell fate diversity can be achieved through the asymmetric segregation of cell fate determinants. In the Drosophila embryo, neuroblasts divide asymmetrically and in a stem cell fashion. The determinants Prospero and Numb localize in a basal crescent and are partitioned from neuroblasts to their daughters (GMCs). Here we show that nonmuscle myosin II regulates asymmetric cell division by an unexpected mechanism, excluding determinants from the apical cortex. Myosin II is activated by Rho kinase and restricted to the apical cortex by the tumor suppressor Lethal (2) giant larvae. During prophase and metaphase, myosin II prevents determinants from localizing apically. At anaphase and telophase, myosin II moves to the cleavage furrow and appears to "push" rather than carry determinants into the GMC. Therefore, the movement of myosin II to the contractile ring not only initiates cytokinesis but also completes the partitioning of cell fate determinants from the neuroblast to its daughter.     

Extinction learning in humans: role of the amygdala and vmPFC

Understanding how fears are acquired is an important step in translating basic research to the treatment of fear-related disorders. However, understanding how learned fears are diminished may be even more valuable. We explored the neural mechanisms of fear extinction in humans. Studies of extinction in nonhuman animals have focused on two interconnected brain regions: the amygdala and the ventral medial prefrontal cortex (vmPFC). Consistent with animal models suggesting that the amygdala is important for both the acquisition and extinction of conditioned fear, amygdala activation was correlated across subjects with the conditioned response in both acquisition and early extinction. Activation in the vmPFC (subgenual anterior cingulate) was primarily linked to the expression of fear learning during a delayed test of extinction, as might have been expected from studies demonstrating this region is critical for the retention of extinction. These results provide evidence that the mechanisms of extinction learning may be preserved across species.     

Detection of caspases activation by fluorochrome-labeled inhibitors: Multiparameter analysis by laser scanning cytometry

BACKGROUND: The fluorochrome-labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308-313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. METHODS: Apoptosis of human MCF-7, U-937, or HL-60 cells was induced by camptothecin (CPT) or tumor necrosis factor-alpha (TNF-alpha) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA-specific fluorochromes was measured by LSC. RESULTS: Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF-7 cells treated with CPT. FLICA binding was largely (> 90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. CONCLUSIONS: The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation.     

NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism

Although microtubule (MT) dynamic instability is thought to depend on the guanine nucleotide (GTP vs GDP) bound to the beta-tubulin of the terminal subunit(s), the MT minus end exhibits dynamic instability even though the terminal beta-tubulin is always crowned by GTP-alpha-tubulin. As an approach toward understanding how dynamic instability occurs at the minus end, we investigated the effects of N-ethylmaleimide-modified tubulin (NTb) on elongation and rapid shortening of individual MTs. NTb preferentially inhibits minus end assembly when combined with unmodified tubulin (PCTb), but the mechanism of inhibition is unknown. Here, video-enhanced differential interference contrast microscopy was used to observe the effects of NTb on MTs assembled from PCTb onto axoneme fragments. MTs were exposed to mixtures of PCTb (25 microM) and NTb (labeled on approximately 1 Cys per monomer) in which the NTb/PCTb ratio varied from 0.025 to 1. The NTb/PCTb mixture had a slight inhibitory effect on the plus end elongation rate, but significantly inhibited or completely arrested minus end elongation. For the majority of mixtures that were assayed (0.1-1 NTb/PCTb ratio), minus end MT length remained constant until the NTb/PCTb mixture was replaced. Replacement with PCTb allowed elongation to proceed, whereas replacement with buffer or NTb caused minus ends to shorten. Taken together, the results indicate that NTb associates with both plus and minus ends and that NTb acts to reversibly cap minus ends only when PCTb is also present. Low-resolution mapping of labeled Cys residues, along with previous experiments with other Cys-reactive compounds, suggests that modification of beta-tubulin Cys(239) may be associated with the capping action of NTb.     

Nigrostriatal reduction of aromatic L-amino acid decarboxylase activity in MPTP-treated squirrel monkeys: in vivo and in vitro investigations

Aromatic L-amino acid decarboxylase (AAAD) activity was examined in vivo with positron emission tomography (PET) using 6-[18F]fluoro-L-DOPA (FDOPA) in squirrel monkeys lesioned with graded doses of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In vitro biochemical determinations of AAAD activity in caudate, putamen, substantia nigra, and nucleus accumbens were performed in the same animals to establish a direct comparison of in vivo and in vitro measurements. In vivo and in vitro AAAD activities in caudate/ putamen were substantially reduced in animals treated with the highest dose of MPTP (2.0 mg/kg). The percent change in the striatal FDOPA uptake (K(i)) and decarboxylation rate constant (k3) values resulting from MPTP treatment showed highly significant correlations with in vitro-determined AAAD activities. However, decarboxylase rates within individual animals presented as approximately 10-fold difference between in vivo and in vitro values. Lower in vivo k3 measurements may be attributed to several possibilities, including transport restrictions limiting substrate availability to AAAD within the neuron. In addition, reductions in AAAD activity in the substantia nigra did not parallel reductions in AAAD activity within the striatum, supporting the notion of a nonlinear relationship between nigrostriatal cell degeneration and terminal losses. This work further explores the role of AAAD in Parkinson's disease, a more important factor than previously thought.     

Regulation of myocardial blood flow response to mental stress in healthy individuals

Mental stress testing has been proposed as a noninvasive tool to evaluate endothelium-dependent coronary vasomotion. In patients with coronary artery disease, mental stress can induce myocardial ischemia. However, even the determinants of the physiological myocardial blood flow (MBF) response to mental stress are poorly understood. Twenty-four individuals (12 males/12 females, mean age 49 +/- 13 yr, range 31-74 yr) with a low likelihood for coronary artery disease were studied. Serum catecholamines, cardiac work, and MBF (measured quantitatively with N-13 ammonia and positron emission tomography) were assessed. During mental stress (arithmetic calculation) MBF increased significantly from 0.70 +/- 0.14 to 0.92 +/- 0.21 ml x min(-1) x g(-1) (P < 0.01). Mental stress caused significant increases (P < 0.01) in serum epinephrine (26 +/- 16 vs. 42 +/- 17 pg/ml), norepinephrine (272 +/- 139 vs. 322 +/- 136 pg/ml), and cardiac work [rate-pressure product (RPP) 8,011 +/- 1,884 vs. 10,416 +/- 2,711]. Stress-induced changes in cardiac work were correlated with changes in MBF (r = 0.72; P < 0.01). Multiple-regression analysis revealed stress-induced changes in the RPP as the only significant (P = 0.0001) predictor for the magnitude of mental stress-induced increases in MBF in healthy individuals. Data from this group of healthy individuals should prove useful to investigate coronary vasomotion in individuals at risk for or with documented coronary artery disease.     

Spatial variations in endothelial barrier function in disturbed flows in vitro

Hindered barrier function has been implicated in the initiation and progression of atherosclerosis, a disease of focal nature associated with altered hemodynamics. In this study, endothelial permeability to macromolecules and endothelial electrical resistance were investigated in vitro in monolayers exposed to disturbed flow fields that model spatial variations in fluid shear stress found at arterial bifurcations. After 5 h of flow, areas of high shear stress gradients showed a 5.5-fold increase in transendothelial transport of dextran (molecular weight 70,000) compared with no-flow controls. Areas of undisturbed fully developed flow, within the same monolayer, showed a 2.9-fold increase. Monolayer electrical resistance decreased with exposure to flow. The resistance measured during flow and the rate of change in monolayer resistance after removal of flow were lowest in the vicinity of flow reattachment (highest shear stress gradients). These results demonstrate that endothelial barrier function and permeability to macromolecules are regulated by spatial variations in shear stress forces in vitro.