Systematic identification of cellular signals reactivating Kaposi sarcoma-associated herpesvirus

The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.     

Simulation of lipid environment of Ca-dependent ATPase from the sarcoplasmic reticulum of skeletal muscles by non-ionic detergents

To determine the conditions under which the Ca-ATPase from rabbit skeletal sarcoplasmic reticulum can be restored to full activity, a systematic study of reactivation of lipid-depleted enzyme by various non-ionic detergents and surfactants has been carried out. All the non-ionic detergents used were able to reactivate the enzyme. The reactivation potencies of the detergents are closely related to a relative size of their polar head group and hydrophobic hydrocarbon chains. The so-called HLB (hydrophyle/lipophyle balance) numbers were used to estimate the relative hydrophobicities of the detergents studied. A striking correlation between the reactivation potency and the HLB number of any detergent was observed; the inverse correlation coefficient for small samples if equal to -0.95 +/- 0.09. The proper orientation of the ATPase protein in two different phases (lipophyle and hydrophyle) seems to be restored during reactivation. In many cases the protein-bound detergent simulates the lipid environment in the membrane sufficiently well to support the continued activity of the Ca-ATPase.     

SCL/tal-1-dependent process determines a competence to select the definitive hematopoietic lineage prior to endothelial differentiation

Hematopoiesis in most vertebrate species occurs in two distinct phases, primitive and definitive, which diverge from FLK1(+)VE-cadherin(-) mesoderm and FLK1(+)VE-cadherin(+) endothelial cells (EC), respectively. This study aimed at determining the stage at which hematopoietic lineage fate is determined by manipulating the SCL/tal-1 expression that is known to be essential for the early development of the primitive and definitive hematopoietic systems. We established SCL-null ES cell lines in which SCL expression is rescued by tamoxifen-inducible Cre recombinase-loxP site-mediated recombination. While no hematopoietic cells (HPC) were detected in SCL-null ES cell differentiation cultures, SCL gene reactivation from day 2 to day 4 after initiation of differentiation could rescue both primitive and definitive hematopoiesis. SCL reactivation at later phases was ineffective. Moreover, generation of VE-cadherin(+) EC that can give rise to definitive HPC required SCL reactivation prior to VE-cadherin expression. These results indicated that the competence to become HPC is acquired at the mesodermal stage by a SCL-dependent process that takes place independently of determination of endothelial fate.     

Slow inactivation and reactivation of the K+ channel in squid axons. A tail current analysis.

Potassium current inactivation and reactivation in squid axons were measured from tail current amplitudes after voltage clamp prepulses to the potassium equilibrium potential, EK, in seawater containing elevated levels of potassium ion concentration, Ko. Little or no inactivation resulted with prepulses lasting less than 100 ms. Longer pulses caused the current to inactivate in two phases, one between 0.1 and 1 s, and a second phase between 5 and 100 s. Inactivation was incomplete. The time constant of the tail current after a prepulse to EK was independent of pulse duration (0.1-120 s). Inactivation was independent of Ko (10 less than or equal to Ko less than or equal to 300 mM), and it was independent of membrane potential, V, for -40 less than or equal to V less than or equal to 0 mV. Reactivation was measured with a three-pulse protocol. The reactivation time course was sigmoidal with a delay of approximately 100 ms before significant reactivation occurred. These results were described by a model consisting of three inactivated states arranged in a linear sequence. The rate constants of the model are of the form (A + B exp (CV), or 1/(A + B exp (CV], which are required to describe the non-inactivating conductance component.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

Further characterization of the reassembly of creatine kinase and effect of substrate

Upon exposure to 8 M urea, creatine kinase from rabbit muscle exhibited a rapid increase in intrinsic fluorescence and a rapid decrease in fluorescence polarization. Polarization changes were complete after 5 min, while fluorescence changes continued for at least 15 min. Fluorescence polarization changes accompanying reassembly were complex, and appeared to involve a concentration dependent reaction. Enzyme sampled at intervals during denaturation exhibited refolding kinetics displaying two first-order rate constants, the first dependent and the second independent of the duration of exposure to urea. There was evidence for an additional renaturation step, occurring within the mixing phase of the denatured protein with solvent. Reactivation kinetics and yield of reactivated enzyme exhibited a dependency upon length of exposure to denaturant. The exposure of renaturing creatine kinase to trypsin was shown to prevent further reactivation, and provided use of a method to determine reactivation rates at discrete intervals after initiation of reassembly. The presence of 2 mM MgADP during reactivation enhanced the rate of reactivation immediately after initiation of reactivation. Reactivation was not accelerated if nucleotide substrate was added after reactivation was initiated nor did nucleotide substrate increase the overall reactivation yield. The presence of MgADP also enhanced the rate of refolding at an early stage as judged by changes in intrinsic fluorescence and resistance to tryptic hydrolysis. While in addition to MgADP, creatine phosphate accelerated resistance by refolding creatine kinase to trypsin, according to the other criteria measured, the phosphagen substrates did not promote reactivation or renaturation. The unfolding-refolding studies and role of substrate in reassembly were consistent with a mechanism involving at least two steps, possibly involving cis-trans isomerization of proline. These data also supported the suggestion that the formation of the nucleotide binding region is an early event in the refolding of creatine kinase in vitro.     

Reactivation of M. tuberculosis infection in trans-membrane tumour necrosis factor mice

Of those individuals who are infected with M. tuberculosis, 90% do not develop active disease and represents a large reservoir of M. tuberculosis with the potential for reactivation of infection. Sustained TNF expression is required for containment of persistent infection and TNF neutralization leads to tuberculosis reactivation. In this study, we investigated the contribution of soluble TNF (solTNF) and transmembrane TNF (Tm-TNF) in immune responses generated against reactivating tuberculosis. In a chemotherapy induced tuberculosis reactivation model, mice were challenged by aerosol inhalation infection with low dose M. tuberculosis for three weeks to establish infection followed chemotherapeutic treatment for six weeks, after which therapy was terminated and tuberculosis reactivation investigated. We demonstrate that complete absence of TNF results in host susceptibility to M. tuberculosis reactivation in the presence of established mycobacteria-specific adaptive immunity with mice displaying unrestricted bacilli growth and diffused granuloma structures compared to WT control mice. Interestingly, bacterial re-emergence is contained in Tm-TNF mice during the initial phases of tuberculosis reactivation, indicating that Tm-TNF sustains immune pressure as in WT mice. However, Tm-TNF mice show susceptibility to long term M. tuberculosis reactivation associated with uncontrolled influx of leukocytes in the lungs and reduced IL-12p70, IFNγ and IL-10, enlarged granuloma structures, and failure to contain mycobacterial replication relative to WT mice. In conclusion, we demonstrate that both solTNF and Tm-TNF are required for maintaining immune pressure to contain reactivating M. tuberculosis bacilli even after mycobacteria-specific immunity has been established.     

Reactivation processes of three inward current systems involved in the rising phase of the action potentials in embryonic chick hearts

In embryonic chick hearts during development, there are three inward current systems which are involved in the rising phases of the action potentials (APs): fast INa, slow ICa, and tetrodotoxin-insensitive slow INa. To assess reactivation processes for these three types of inward current channels (fast Na+, slow Ca2+, and slow Na+ channels), diastolic recovery of Vmax was examined in embryonic chick hearts using a paired-pulse protocol. In all cases, the diastolic recoveries were approximated by single exponential functions. The time constants of recovery (tau(V)) and T90% (the diastolic interval which allows 90% recovery of Vmax of the premature AP) were, respectively, 53.1 +/- 5.2 and 61.5 +/- 8.6 ms for Na+-dependent fast AP (n = 10), 376.9 +/- 49.3 and 659.2 +/- 113.1 ms for the Ca2+-dependent slow AP (n = 10), and 40.7 +/- 5.3 and 45.6 +/- 12.0 ms for the Na+-dependent slow AP (n = 10). In the presence of lidocaine, the recovery kinetics also appeared to be single exponentials for diastolic intervals up to 500 ms (fast APs) or 250 ms (slow APs). The reactivation processes for the Na+-dependent fast and slow channels were significantly slowed by 100 microM lidocaine. In addition, in the presence of 100 microM lidocaine, Vmax was depressed in a frequency-dependent manner; the higher the stimulation frequency, the greater the depression. Hence, the fast Na+ channels and the slow Na+ channels had the following similarities: rapid reactivation, reactivation slowed by lidocaine, and frequency-dependent depression in the presence of lidocaine.     

Kinetic aspects of the role of phospholipids in D-beta-hydroxybutyrate dehydrogenase activity

Interactions of phospholipids with D-beta-hydroxybutyrate dehydrogenase (BDH), a lecithin-requiring enzyme, have been studied by a kinetic approach. The process of reactivation of BDH by phospholipids, which follows a second-order mechanism, reveals that (1) at least 2 mol of lecithins is essential for the reactivation of the enzyme, and (2) the enzyme contains two dependent binding sites for lecithins. The graphic representation of the time course of reactivation shows a latent phase which decreases when there is an increase in the amount of phospholipids. A Scatchard plot treatment of the reactivation kinetic data reveals the presence of two classes of phospholipid binding sites, which exhibit high and low affinities related to the binding of four and two lecithin molecules, respectively. The effect of temperature on BDH activity and on the inactivation of the apoenzyme with N,N'-dicyclohexylcarbodiimide (a specific carboxyl reagent) or with phenylglyoxal (a specific arginine reagent) shows a break at 22-24 degrees C, indicating a slight structural change in the enzyme-active site around this temperature. In addition, the variations in enzyme kinetic parameters, according to the nature of phospholipids, are in agreement with conformational changes related to the nature and to the fluidity state of phospholipids. However, the apparent NAD+ binding constant does not depend on the phospholipid's fluidity.     

The dendrogeomorphic spatio-temporal reconstruction of flow-like landslides activity in one of the most susceptible region of Central Europe (the Vsetínské vrchy Mts.)

Flow-like landslides are a dangerous landslide type. They often express gradual movement or seeming dormancy, but occasional reactivation can, in extreme cases, result in catastrophic events. To predict their future behaviour, knowledge of past spatio-temporal development and relationships with hydrometeorological triggers is crucial. Moreover, regional data are more robust than case studies. Dendrogeomorphic (tree-ring-based) methods are a very precise approach for reconstructing past landslide behaviour. Nevertheless, regional reconstructions are very rare, which is probably due to their time-consuming procedures. This paper presents the results of a regional tree-ring-based reconstruction of the spatio-temporal development of flow-like landslides in a selected region in the Outer Western Carpathians. Six selected landslides were studied via analysis of 614 increment cores that came from 307 disturbed trees. The reconstruction provided data for approximately 70 individual landslide reactivation phases that were distributed in 44 event years. Events with regional extension (at least half of the studied landslides were active) were detected in six years (1940, 1941, 1953, 1961, 1985, and 1997). Periods of increased (1950s, 1990s) as well as decreased (1940s, 1970s, 2010s) landslide activity were reconstructed. The use of tree-ring data enabled the construction of landslide probability maps. Based on this analysis, all studied landslides exhibit extremely high probabilities of reactivation during a temporal horizon of 100 years, but even over shorter periods (5 and 20 years), their probability of reactivation is very high. Finally, analysis of meteorological triggers suggests the positive effect of precipitation in May (and possibly in September) to activate landslides with regional extent. Extreme short-duration (1-day) precipitation events probably do not play a role in landslide triggering. Moreover, gradual increases in precipitation totals during periods of at least one-half year preceding the event years were detected.     

Comparison of activity and conformation changes during refolding of urea-denatured creatine kinase

The course of the recovery of the enzymatic activity and the native conformation during the renaturation of urea-denatured creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) has been studied. Under suitable conditions, an activity recovery of 95% can be obtained and the reactivation follows a triphasic course. The initial two phases are relatively fast, whereas the slow phase takes some 24 h to reach completion. The recovery of the native conformation has been followed by changes in fluorescence, ultraviolet absorption and in exposed SH groups and has been shown to be a biphasic process. Both the reactivation and the refolding processes are independent of protein concentrations within a certain range, showing that the dimerization of the enzyme molecule is not rate-limiting. A comparison of the rate constants for the refolding of the molecule with those for the recovery of its catalytic activity shows that these are not synchronized and the activity recovery approaches completion after the refolding and dimerization of the subunits so far as can be detected by the methods employed. The final stage of refolding with complete activity recovery probably involves subtle conformational changes of the dimeric enzyme molecule not detectable by the physiochemical methods used in the present study.     

Reactivation of Latent Tuberculosis in Cynomolgus Macaques Infected with SIV Is Associated with Early Peripheral T Cell Depletion and Not Virus Load

HIV-infected individuals with latent Mycobacterium tuberculosis (Mtb) infection are at significantly greater risk of reactivation tuberculosis (TB) than HIV-negative individuals with latent TB, even while CD4 T cell numbers are well preserved. Factors underlying high rates of reactivation are poorly understood and investigative tools are limited. We used cynomolgus macaques with latent TB co-infected with SIVmac251 to develop the first animal model of reactivated TB in HIV-infected humans to better explore these factors. All latent animals developed reactivated TB following SIV infection, with a variable time to reactivation (up to 11 months post-SIV). Reactivation was independent of virus load but correlated with depletion of peripheral T cells during acute SIV infection. Animals experiencing reactivation early after SIV infection (<17 weeks) had fewer CD4 T cells in the periphery and airways than animals reactivating in later phases of SIV infection. Co-infected animals had fewer T cells in involved lungs than SIV-negative animals with active TB despite similar T cell numbers in draining lymph nodes. Granulomas from these animals demonstrated histopathologic characteristics consistent with a chronically active disease process. These results suggest initial T cell depletion may strongly influence outcomes of HIV-Mtb co-infection.     

Mechanism for reactivation of N-cyclopropylbenzylamine-inactivated monoamine oxidase by amines

The effect of 18 different amines, two mercaptans, and two alcohols on the reactivation of N-cyclopropylbenzylamine- (N-CBA-) inactivated bovine liver monoamine oxidase (MAO) is described. All of the compounds that reactivate the enzyme produce a time-dependent pseudo-first-order return of enzyme activity and exhibit saturation kinetics. There is no direct correlation between the ability of a compound to serve as a substrate for native MAO and its ability to reactivate N-CBA-inactivated MAO. Amines containing an aromatic moiety, in general, are better reactivators than the aliphatic amines. The amine must be primary or secondary in order for reactivation to occur. The distance between the aromatic portion and the amino group is critical to the reactivation properties of the compound. The mercaptans and alcohols do not reactivate N-CBA-inactivated MAO, nor do they interfere with the reactivation reaction by benzylamine. Three mechanisms for the reactivation reaction are considered. One involves initial Schiff base formation with the active site adduct produced by N-CBA inactivation of MAO followed by base-catalyzed beta-elimination to the imine of acrolein. The second mechanism is the same as the first except no prior Schiff base formation is invoked. The third mechanism is an SN2 displacement by the amine of the active site amino acid residue attached to the adduct. Experiments are carried out to exclude the SN2 mechanism. The results of the reactivation experiments favor the Shiff base mechanism.     

The sulfhydryl groups responsible for bilitranslocase transport activity respond to the interaction of the carrier with bilirubin and functional analogues

Both inactivation of sulfobromophthalein transport in rat liver plasma membrane vesicles by sulfhydryl group reagents and subsequent reactivation by 2-mercaptoethanol are shown to be modulated by ligands to bilitranslocase. In particular, bilirubin, sulfobromophthalein and Thymol blue behave as negative effectors in the inactivation reaction and as positive effectors in the reactivation reaction. Kinetic data provide further evidence of the existence of two classes of sulfhydryl groups involved in transport activity. The effect brought about by remarkably low concentrations of bilirubin is in line with the physiological function of bilitranslocase as a bilirubin carrier.