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.     

Gender-specific effects of prenatal stress on emotional reactivity and stress physiology of goat kids

The aims of this study were to investigate the effects of maternal stress during pregnancy on the emotional reactivity, the hypothalamo-pituitary-adrenocortical (HPA) axis, and the sympatho-adrenomedullary (SAM) system of goat offspring according to their gender, and to investigate the role of maternal cortisol in prenatal stress effects. Goats were exposed to ten transports in isolation or ten ACTH injections (0.125 IU/kg body weight) during the last third of pregnancy. Control goats remained undisturbed. No effect of repeated transport during the last third of pregnancy was found on basal cortisol concentrations of the offspring. However, an increase in phenylethanolamine N-methyl transferase activity in the adrenals was observed in prenatally stressed kids compared to control kids (P = 0.031). In the presence of novelty, prenatally stressed female kids were more active (P = 0.049) than control females; they also showed more signs of arousal (P = 0.039) and tended to explore more of their environment (P = 0.053) in reaction to a startling stimulus. On the contrary, prenatally stressed male kids tended to be less active (P = 0.051) than control male kids but showed more signs of distress (P = 0.047) in the presence of novelty. Intermediate effects were found on the emotional reactivity to novelty of kids born from dams given injections of ACTH. In conclusion, transport stress in pregnant goats affects the sympatho-adrenomedullary system and the emotional reactivity of their offspring in a gender-specific manner. Moreover, the effects of prenatal transport and ACTH injections showed some similarities but differed in some critical details.     

Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase reveal novel features of its pumping mechanism

We have analyzed the Fe2+ -catalyzed oxidative cleavages of Ca2+ -ATPase in the presence of Ca2+, with or without the ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) or in the presence of the inhibitor thapsigargin. To identify the positions of cleavages as precisely as possible, we have used previously identified proteinase K and tryptic fragments as a standard, advanced mass spectrometry techniques, as well as specific antibodies. A number of cleavages are similar to those described for Na+,K+ -ATPase or other P-type pumps and are expected on the basis of the putative Mg2+ binding residues near the phosphorylated Asp351 in E1 or E2P conformations. However, intriguing new features have also been observed. These include a Fe2+ site near M3, which cannot be due to the presence of histidine residues as it was postulated in the case of Na+,K+ -ATPase and H+,K+ -ATPase. This site could represent a Ca2+ binding zone between M1 and M3, preceding Ca2+ occlusion within M4, 5, 6, and 8. In addition, we present evidence that, in the non-crystalline state, the N- and P-domain may approach each other, at least temporarily, in the presence of Ca2+ (E1Ca2 conformation), whereas the presence of Mg.ATP stabilizes the N to P interaction (E1.Mg.ATP conformation).     

Structure based design of nicotinamide phosphoribosyltransferase (NAMPT) inhibitors from a phenotypic screen

Nicotinamide phosphoribosyltransferase is a key metabolic enzyme that is a potential target for oncology. Utilizing publicly available crystal structures of NAMPT and in silico docking of our internal compound library, a NAMPT inhibitor, 1, obtained from a phenotypic screening effort was replaced with a more synthetically tractable scaffold. This compound then provided an excellent foundation for further optimization using crystallography driven structure based drug design. From this approach, two key motifs were identified, the (S,S) cyclopropyl carboxamide and the (S)-1-N-phenylethylamide that endowed compounds with excellent cell based potency. As exemplified by compound 27e such compounds could be useful tools to explore NAMPT biology in vivo.     

SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation

The sarco(endo)plasmic reticulum Ca²⁺‐ATPase (SERCA) couples ATP hydrolysis to transport of Ca²⁺. This directed energy transfer requires cross‐talk between the two Ca²⁺ sites and the phosphorylation site over 50 Å distance. We have addressed the mechano‐structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu³⁰⁹ contributes to Ca²⁺ coordination at site II, and a consensus has been that E309Q only binds Ca²⁺ at site I. The crystal structure of E309Q in the presence of Ca²⁺ and an ATP analogue, however, reveals two occupied Ca²⁺ sites of a non‐catalytic Ca₂E1 state. Ca²⁺ is bound with micromolar affinity by both Ca²⁺ sites in E309Q, but without cooperativity. The Ca²⁺‐bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A‐domain, requiring a shift of transmembrane segment M1 into an ‘up and kinked position’. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca²⁺ site II.     

Effect of long-term administration of the antidepressant drug milnacipran on serotonergic and noradrenergic neurotransmission in the rat hippocampus

The effect of a long-term administration of the antidepressant milnacipran on the function of the serotonergic (5-HT) and noradrenergic (NE) systems was studied using single cell recording of CA3 hippocampal pyramidal cells in chloral hydrate-anesthetized male Sprague-Dawley rats, and in vitro [3H]5-HT release measurement from hippocampal slices. The sensitivity of neither the extrasynaptic nor that of the postsynaptic 5-HT1A receptors of the pyramidal neurons was altered, as indicated by their unchanged responsiveness to the microiontophoretic application of 5-HT, and by the unchanged effect of the electrical stimulation at low frequency of the ascending 5-HT bundle, respectively. Increasing the frequency of stimulation (from 1 to 5 Hz) decreased its efficacy in control rats; the milnacipran treatment abolished this phenomenon. This cannot be attributed to a desensitisation of the terminal 5-HT1B autoreceptor, since the suppressive effect of 5-HT agonist 5-carboxyamidotryptamine on [3H]5-HT release was enhanced in milnacipran-treated rats. As for the NE system, the unchanged suppressing effect of microiontophoretic applications of NE and that of the 5 Hz stimulation in the locus coeruleus (LC) on the firing activity of pyramidal neurons indicates that the milnacipran treatment not altered the sensitivity of extrasynaptic alpha2- and postsynaptic alpha1-adrenergic receptors on pyramidal cells, as well as that of the presynaptic alpha2-autoreceptor on NE terminals. The decreased inhibitory effect of NE on the [3H]5-HT release in milnacipran-treated rats revealed that this treatment results in a desensitisation of the presynaptic alpha2-heteroreceptor located on serotonergic terminals. Taken together with the decreased suppressive effect of a low frequency of stimulation of the NE tract, the present results suggest that long-term milnacipran treatment enhances the efficacy of the 5-HT and reduces that of the NE neurotransmission.     

Ex vivo expansion of CD4+CD25+FoxP3+ T regulatory cells based on synergy between IL-2 and 4-1BB signaling

Naturally occurring CD4(+)CD25(+)FoxP3(+) T regulatory (Treg) cells require three distinct signals transduced via TCR, CD28, and IL-2R for their development and maintenance. These requirements served as the basis for several recently developed ex vivo expansion protocols that relied on the use of solid support-bound Abs to CD3 and CD28 in the presence of high dose IL-2. We report in this study that Treg cells up-regulate the expression of inducible costimulatory receptor 4-1BB in response to IL-2, and stimulation using this receptor via a novel form of 4-1BB ligand (4-1BBL) fused to a modified form of core streptavidin (SA-4-1BBL) was effective in expanding these cells up to 110-fold within 3 wk. Expanded cells up-regulated CD25, 4-1BB, and membranous TGF-beta, suppressed T cell proliferation, and prevented the rejection of allogeneic islets upon adoptive transfer into graft recipients. Importantly, SA-4-1BBL rendered CD4(+)CD25(-) T effector cells refractive to suppression by Treg cells. This dual function of signaling via 4-1BB, vis-à-vis Treg cell expansion and licensing T effector cells resistant to Treg cell suppression, as well as the up-regulation of 4-1BB by IL-2 may serve as important regulatory mechanisms for immune homeostasis following antigenic challenge. Stimulation using a soluble form of SA-4-1BBL represents a novel approach to expand Treg cells with potential therapeutic applications in autoimmunity and transplantation.     

Inhibitors bound to Ca(2+)-free sarcoplasmic reticulum Ca(2+)-ATPase lock its transmembrane region but not necessarily its cytosolic region, revealing the flexibility of the loops connecting transmembrane and cytosolic domains

Ca2+-free crystals of sarcoplasmic reticulum Ca2+-ATPase have, up until now, been obtained in the presence of inhibitors such as thapsigargin (TG), bound to the transmembrane region of this protein. Here, we examined the consequences of such binding for the protein. We found that, after TG binding, an active site ligand such as beryllium fluoride can still bind to the ATPase and change the conformation or dynamics of the cytosolic domains (as revealed by the protection afforded against proteolysis), but it becomes unable to induce any change in the transmembrane domain (as revealed by the intrinsic fluorescence of the membranous tryptophan residues). TG also obliterates the Trp fluorescence changes normally induced by binding of MgATP or metal-free ATP, as well as those induced by binding of Mg2+ alone. In the nucleotide binding domain, the environment of Lys515 (as revealed by fluorescein isothiocyanate fluorescence after specific labeling of this residue) is significantly different in the ATPase complex with aluminum fluoride and in the ATPase complex with beryllium fluoride, and in the latter case it is modified by TG. All these facts document the flexibility of the loops connecting the transmembrane and cytosolic domains in the ATPase. In the absence of active site ligands, TG protects the ATPase from cleavage by proteinase K at Thr242-Glu243, suggesting TG-induced reduction in the mobility of these loops. 2,5-Di-tert-butyl-1,4-dihydroxybenzene or cyclopiazonic acid, inhibitors which also bind in or near the transmembrane region, also produce similar overall effects on Ca2+-free ATPase.