Childhood stress, grown-up brain networks: corticolimbic correlates of threat-related early life stress and adult stress response.

BACKGROUND: Exposure to threat-related early life stress (ELS) has been related to vulnerability for stress-related disorders in adulthood, putatively via disrupted corticolimbic circuits involved in stress response and regulation. However, previous research on ELS has not examined both the intrinsic strength and flexibility of corticolimbic circuits, which may be particularly important for adaptive stress responding, or associations between these dimensions of corticolimbic dysfunction and acute stress response in adulthood. METHODS: Seventy unmedicated women varying in history of threat-related ELS completed a functional magnetic resonance imaging scan to evaluate voxelwise static (overall) and dynamic (variability over a series of sliding windows) resting-state functional connectivity (RSFC) of bilateral amygdala. In a separate session and subset of participants (n = 42), measures of salivary cortisol and affect were collected during a social-evaluative stress challenge. RESULTS: Higher severity of threat-related ELS was related to more strongly negative static RSFC between amygdala and left dorsolateral prefrontal cortex (DLPFC), and elevated dynamic RSFC between amygdala and rostral anterior cingulate cortex (rACC). Static amygdala-DLPFC antagonism mediated the relationship between higher severity of threat-related ELS and blunted cortisol response to stress, but increased dynamic amygdala-rACC connectivity weakened this mediated effect and was related to more positive post-stress mood. CONCLUSIONS: Threat-related ELS was associated with RSFC within lateral corticolimbic circuits, which in turn was related to blunted physiological response to acute stress. Notably, increased flexibility between the amygdala and rACC compensated for this static disruption, suggesting that more dynamic medial corticolimbic circuits might be key to restoring healthy stress response.

Red-shifted fluorescent proteins monitor enzymatic activity in live HT-1080 cells with fluorescence lifetime imaging microscopy (FLIM).

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a membrane-tethered collagenase primarily involved in the mechanical destruction of extracellular matrix proteins. MT1-MMP has also been shown to be upregulated in several types of cancers. Many coordinated functions of MT1-MMP during migration and invasion remain to be determined. In this paper, live cells from the invasive cell line HT-1080 were imaged using an intracellular Förster resonance energy transfer-based biosensor specific for MT1-MMP; a substrate specific for MT1-MMP was hybridized with the mOrange2 and mCherry fluorescent proteins to form the Förster resonance energy transfer-based sensor. The configuration of the biosensor was determined with fluorescence lifetime-resolved imaging microscopy using both a polar plot-based analysis and a rapid data acquisition modality of fluorescence lifetime-resolved imaging microscopy known as phase suppression. Both configurations of the biosensor (with or without cleavage by MT1-MMP) were clearly resolvable in the same cell. Changes in the configuration of the MT1-MMP biosensor were observed primarily along the edge of the cell following the removal of the MMP inhibitor GM6001. The intensities highlighted by phase suppression correlated well with the fractional intensities derived from the polar plot.

Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency.

A spectrograph with continuous wavelength resolution has been integrated into a frequency-domain fluorescence lifetime-resolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross-talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral-FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Förster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral-FLIM data, the spectral dispersion of the acceptor signal can be used to derive the donor lifetime - and thereby the FRET efficiency - without iterative fitting. The lifetime relation between the donor and acceptor, in conjunction with spectral dispersion, is also used to separate the FRET pair signals from the donor alone signal. This method can be applied further to quantify the signals from separate FRET pairs, and provide information on the dynamics of the FRET pair between different states.

Simplification of networks by conserving path diversity and minimisation of the search information.

Alternative paths in a network play an important role in its functionality as they can maintain the information flow under node/link failures. In this paper we explore the navigation of a network taking into account the alternative paths and in particular how can we describe this navigation in a concise way. Our approach is to simplify the network by aggregating into groups the nodes that do not contribute to alternative paths. We refer to these groups as super-nodes, and describe the post-aggregation network with super-nodes as the skeleton network. We present a method to describe with the least amount of information the paths in the super-nodes and skeleton network. Applying our method to several real networks we observed that there is scaling behaviour between the information required to describe all the paths in a network and the minimal information to describe the paths of its skeleton. We show how from this scaling we can evaluate the information of the paths for large networks with less computational cost.

Parallel stranded DNA.

A series of four hairpin deoxyoligonucleotides was synthesized with a four-nucleotide central loop (either C or G) flanked by the complementary sequences d(T)10 and d(A)10. Two of the molecules contain either a 3'-p-3' or 5'-p-5' linkage in the loop, so that the strands in the stem have the same, that is, parallel (ps) polarity. The pair of reference oligonucleotides have normal phosphodiester linkages throughout and antiparallel (aps) stem regions. All the molecules adopt a duplex helical structure in that (i) the electrophoretic mobilities in polyacrylamide gels of the ps and aps oligomers are similar. (ii) The ps hairpins are substrates for T4 polynucleotide kinase, T4 DNA ligase, and Escherichia coli exonuclease III. (iii) Salt-dependent thermal transitions are observed for all hairpins, but the ps molecules denature 10 degrees C lower than the corresponding aps oligomers. (iv) The ultraviolet absorption and circular dichroism spectra are indicative of a base-paired duplex in the stems of the ps hairpins but differ systematically from those of the aps counterparts. (v) The bis-benzimidazole drug Hoechst-33258, which binds in the minor groove of B-DNA, exhibits very little fluorescence in the presence of the ps hairpins but a normal, enhanced emission with the aps oligonucleotides. In contrast, the intercalator ethidium bromide forms a strongly fluorescent complex with all hairpins, the intensity of which is even higher for the ps species. (vi) The pattern of chemical methylation is the same for both the ps and aps hairpins. The combined results are consistent with the prediction from force field analysis of a parallel stranded right-handed helical form of d(A)n.d(T)n with a secondary structure involving reverse Watson-Crick base pairs and a stability not significantly different from that of the B-DNA double helix. Models of the various hairpins optimized with force field calculations are described.

Image analysis for denoising full-field frequency-domain fluorescence lifetime images.

Video-rate fluorescence lifetime-resolved imaging microscopy (FLIM) is a quantitative imaging technique for measuring dynamic processes in biological specimens. FLIM offers valuable information in addition to simple fluorescence intensity imaging; for instance, the fluorescence lifetime is sensitive to the microenvironment of the fluorophore allowing reliable differentiation between concentration differences and dynamic quenching. Homodyne FLIM is a full-field frequency-domain technique for imaging fluorescence lifetimes at every pixel of a fluorescence image simultaneously. If a single modulation frequency is used, video-rate image acquisition is possible. Homodyne FLIM uses a gain-modulated image intensified charge-coupled device (ICCD) detector, which unfortunately is a major contribution to the noise of the measurement. Here we introduce image analysis for denoising homodyne FLIM data. The denoising routine is fast, improves the extraction of the fluorescence lifetime value(s) and increases the sensitivity and fluorescence lifetime resolving power of the FLIM instrument. The spatial resolution (especially the high spatial frequencies not related to noise) of the FLIM image is preserved, because the denoising routine does not blur or smooth the image. By eliminating the random noise known to be specific to photon noise and from the intensifier amplification, the fidelity of the spatial resolution is improved. The polar plot projection, a rapid FLIM analysis method, is used to demonstrate the effectiveness of the denoising routine with exemplary data from both physical and complex biological samples. We also suggest broader impacts of the image analysis for other fluorescence microscopy techniques (e.g. super-resolution imaging).

Short communication: Ability of cultured mammary epithelial cells in a bicameral system to secrete milk fat.

Mammary epithelial cells from lactating cows were cultured onto inserts coated with type I collagen. Every second day, the rates of fatty acid synthesis and secretion were determined by measuring the amount of [14C]-labeled sodium acetate incorporated into lipids over a 4-h period. The [14C]-containing lipids were identified by thin layer chromatography fractionation. In parallel, the integrity of the cell layer was evaluated by measurement of transepithelial electrical resistance. The integrity increased progressively to reach a maximum after 8 d of culture. Cells incorporated acetate into lipids; 1.34% of acetate was incorporated into lipids produced by freshly isolated cells. This percentage decreased to 0.5% after 2 d of culture. Moreover, this capacity decreased with the duration in culture; on d 8, the rate of incorporation dropped to about 3% of that on d 2. In the cell extracts, the [14C]-labeled lipids were mainly triglycerides, although the proportion of diglycerides and phospholipids progressively increased as a part of total newly synthesized lipids. The proportion of triglycerides decreased 0.66 times between d 2 and 8 when the proportion of diglycerides and phospholipids increased 1.33 and 2.18 times, respectively. About 28% of the newly synthesized lipids were secreted within 4 h of incubation. Around 65 to 85% of these labeled lipids were found in the apical compartment, suggesting a partially vectorial secretion. But 58 to 80% of labeled lipids found in the apical and basolateral medium were free fatty acids. Functional tight junctions and incorporation of labeled fatty acids into triglycerides are not compatible with an inferred status of complete dedifferentiation of the cell layer. Moreover, triglyceride secretion seems compromised, probably due to the lack of an appropriate cell environment and cell shape.

Triacylglycerol hydrolysis by cells isolated from lactating rat mammary gland.

The hydrolysis of exogenous trioleoylglycerol emulsions by suspensions of cells prepared from lactating rat mammary gland has been investigated. Cell integrity remains high throughout short (at least 30 min) incubations, during which extracellular hydrolysis of trioleoylglycerol proceeds at a mean rate (11 preparations) of 1.9 nmol oleate (and 0.6 nmol glycerol) released/min per mg protein. This hydrolysis shows partial dependence upon added serum and partial inhibition by protamine sulphate - both characteristic properties of lipoprotein lipase-catalyzed lipolysis. One of more monoacylglycerol hydrolase enzymes may also contribute to the measured lipolysis. Evidence is presented consistent with the hypothesis that a surface-located lipoprotein lipase is responsible for the observed lipolysis. Very little lipoprotein lipase activity is released from the cell surface by heparin. During trioleoylglycerol hydrolysis, non-esterified oleate does not accumulate in the cells or in the medium in quantities stoicheiometric with glycerol release. Analyses indicate that it passes into the cells without prior equilibration with the extracellular oleate pool(s). Once inside the cells, oleate is rapidly re-esterified into the triacylglycerol fraction. The possible relevance of these findings to the physiological mechanism of fatty acid uptake from triacylglycerol at the capillary endothelium is discussed.

Lipoprotein lipase. Localization on plasma membrane fragments from lactating rat mammary tissue.

Subcellular fractionation studies were done using lactating rat mammary tissue. Lipoprotein lipase (E.C. 3.1.1.34) was found to be associated with plasma membrane-enriched and endoplasmic reticulum-enriched fractions. Considerable amounts of soluble lipoprotein lipase were found after homogenization of this tissue. The membrane-associated lipoprotein lipase activities described here were found to be resistant to release, by heparin, from their membrane sites. Buoyant density gradient fractionation experiments suggest that the lipoprotein lipase activity of lactating mammary tissue is located in part on the secretory epithelial cell plasma membrane. This conclusion is discussed in the context of the known site of action, in vivo, of mammary gland lipoprotein lipase, and of probable routes of its transport to that site.

Transient formation of the phosphoprotein during autophosphorylation of rat mammary gland Golgi vesicles.

Incubation with [gamma-32P] ATP of Golgi vesicles, prepared from the mammary tissue of lactating rats, resulted in the phosphorylation of four of the proteins in the preparation which were resolvable by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Three of these had electrophoretic properties identical to the three major caseins of rat milk: their phosphorylation was approximately linear with respect to time during the course of the short (1 min) incubations analyzed. The fourth component (Mr,app. 70,000) behaved differently. It was very rapidly phosphorylated to a maximum level within 5 S at 0 degree C; its 32 P-content declined thereafter, with a t 1/2 for dephosphorylation of approx. 20 s. The extent of 32P incorporation into this component, measured after incubation for 20 s at 0 degree C with [gamma-32P] ATP, was sensitive to the concentration of Ca2+ in the incubation medium, being enhanced at low concentrations (less than 10-8 M) of Ca2+ and depressed at high (10-4 M) ones. Inclusion of ADP (100 microM) in such incubation also depressed 32P incorporation into the 70 kDa component. This phosphoprotein was further distinguished from the other three by virtue of the lability of its incorporated phosphorus to treatment with hot trichloroacetic acid. The properties and possible function of this phosphoprotein are discussed in relation to the ATP-dependent Ca2+ transport that occurs in this Golgi vesicle preparation.

Cholesterol ester hydrolysis and hormone-sensitive lipase in lactating rat mammary tissue.

Neutral cholesterol esterase activity is expressed in extracts of mammary epithelial cells. The identity of the enzyme catalyzing this hydrolysis was investigated. Anti-hormone-sensitive lipase immunoglobulin elicited the total inhibition of this activity and also immunoprecipitated a single phosphoprotein of Mr 84 kDa from mammary cell extracts previously phosphorylated in vitro with [gamma-32P]ATP and cyclic AMP-dependent protein kinase. It is concluded that mammary cell cholesterol esterase activity results from the presence of hormone-sensitive lipase.

Salt-resistant (hepatic) lipase. Evidence for its presence in bovine liver and adrenal cortex.

Alkaline lipolytic activities in bovine liver and adrenal cortex have been investigated; each tissue has a salt-resistant, hepatic-type lipase activity of which we describe a partial purification. Properties of the partially purified enzymes have been compared directly with those of authentic hepatic lipase prepared from rat liver. Furthermore, a similar activity has been detected in bovine post-heparin plasma. These findings contrast with a previous report that bovine post-heparin plasma and liver extracts lack hepatic salt-resistant lipase.

The effects of cerulenin on lipid metabolism in vitro in cellular preparations from the rat.

The effects of the fatty acid-like antibiotic, cerulenin, on fatty acid biosynthesis in preparations of rat adipocytes and mammary cells in vitro have been investigated. Synthesis of palmitic acid was most strongly inhibited, although the magnitude of the effect was dependent on the nature of the tissue, and was especially diminished in the larger adipocytes from older rats. Cerulenin had no effect on the chain-elongation of preformed fatty acids in any of the tissues studied. Some inhibition of the esterification of preformed palmitic acid was also observed, but this appeared to be due to disruption of the cells rather than direct inhibition of the acyltransferases. It is concluded that cerulenin is a valuable experimental tool in studies of lipogenesis in preparations of intact mammalian cells in vitro.

Mitochondrial DNA of Tetrahymena pyriformis strain ST contains a long terminal duplication-inversion.

1. We have studied denatured Tetrahymena mtDNA by electron microscopy using the formamide technique. 2. After denaturation all DNA is single stranded, but within a few minutes single-stranded circles with a duplex tail are formed. 3. The duplex tail is 1.3 mum long, i.e. 8 percent of the length of native mtDNA, and it often contains a small single-stranded eye. 4. Digestion of the duplex DNA with exonuclease III of Escherichia coli abolishes its ability to form circles and duplex tails after denaturation. 5. Renaturation of denatured mtDNA leads to the formation of duplex circles with single-stranded section and/or duplex tails. In addition, a minority of duplex circles without apparent tails is formed, but these circles contain a small ambiguous section. 6. We conclude that this mtDNA contains a long terminal duplication-inversion, that could be involved in the replication of this linear mtDNA.

Heterogeneity of E. coli RNA polymerase revealed by high pressure.

The activity and subunit association of Escherichia coli RNA polymerase has been investigated by high pressure techniques (up to 2000 atm). The extent of subunit dissociation in the presence and absence of DNA was monitored by carrying out electrophoresis directly at elevated pressure. The degree of inactivation brought about by high pressure was determined by measuring the enzyme activity following decompression. The loss of activity if the enzyme molecules are not actively involved in transcription is correlated with the extent of association of the polymerase subunits. At any particular pressure only a fraction of polymerase molecules becomes inactivated; the remaining fraction retains its original activity characteristics. If the enzyme molecules are actively involved in transcription when the high pressure is applied, the RNA synthesis can be completely halted, but the elongation activity is fully recovered on decompression. The experimental results are consistent with the existence of a broad distribution of protein conformers that are differentially sensitive to the level of pressure. RNA polymerase molecules that display similar catalytic properties have large differences in their free energy of subunit association in the absence of substrates.

Fluorescence energy transfer analysis of DNA structures containing several bulges and their interaction with CAP.

DNA molecules with three bulges separated by double-stranded helical sections of B-DNA were constructed to be used as substrates for DNA-protein binding assays. Fluorescence resonance energy transfer (FRET) between dye molecules attached to the 5'-ends of the DNA molecules is used to monitor the protein binding. The A5 bulge, which consists of five unpaired adenine nucleotides, alters the direction of the helical axis by approximately 80 to 90 at every bulge site. Computer molecular modeling facilitated a pre-selection of suitable helix lengths that bring the labeled ends of the three-bulge DNA molecules (60 to 70 base-pairs long) into close proximity. The FRET experiments verified that the labeled ends of the helices of these long molecules were indeed close. A series of FRET experiments was carried out with two A5 and two A7 bulge molecules. The relative positions of the bulges were varied along the central helical DNA sequence (between the bulges) in order to determine the relative angular juxtapositions of the outlying helical arms flanking the central helical region. The global structural features of the DNA molecules are manifested in the FRET data. The FRET experiments, especially those of the two-bulge series, could be interpreted remarkably well with molecular models based on the NMR structure of the A5 bulge. These models assume that the DNA molecules do not undergo large torsional conformational fluctuations at the bulge sites. The magnitude of the FRET efficiency attests to a relatively rigid structure for many of the long 5'-end-labeled molecules. The changes in the FRET efficiency of three-bulge structures containing the specific binding sequence of the catabolite activator protein (CAP) demonstrated significant deformation of the DNA upon binding of CAP. No direct interaction of CAP with the dyes was observed.

Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics.

The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH

Cyclic AMP-dependent protein kinase in mammary epithelial cells; activity and subcellular distribution are acutely modulated by isoprenaline.

Brief incubation of a mammary epithelial cellular preparation from lactating rats with isoprenaline is shown to result in major re-distribution of the activity of cyclic AMP-dependent protein kinase (measured in the presence of saturating cyclic AMP) within the cell. Activity in the soluble fraction was halved and a corresponding increase in the sedimentable activity occurred. Similar effects were observed when cell-free extracts were treated with cyclic AMP in the presence of inhibitors of phosphodiesterase and subsequently fractionated by a simple one-step centrifugation procedure. The concentration of the catalytic subunit of cyclic AMP-dependent protein kinase, assessed by quantitative Western blot analysis, did not reflect these activity changes. Quantitation of the regulatory subunits (R-I plus R-II) of A-kinase enabled independent assessment of the possible total A-kinase holoenzyme in mammary epithelial cells and was in reasonable agreement with the measured total A-kinase activity. Isoprenaline selectively increased the apparent mean specific catalytic activity of the C-subunit in the particulate fraction.

Fluorescence resonance energy transfer.

In the past year, a number of studies have demonstrated the utility of fluorescence resonance energy transfer as a technique for probing complex intermolecular interactions and for determining the spatial extension and geometrical characteristics of multicomponent structures composed of diverse molecular constituents, such as proteins, lipids, carbohydrates, nucleic acids, and even cells with viruses. The benefits of fluorescence resonance energy transfer are becoming increasingly evident to researchers who require measurements with high sensitivity, specificity, non-invasiveness, rapidity, and relative simplicity.