Alkaline phosphatase in Amoeba proteus

In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.     

The effect of humoral factors on phagocytic activity of central macrophages in the erythroblast islet culture

A 24-hour cultivation of erythroblast islets (El) in presence of erythropoietin (EP) and macrophage colony stimulating factor (MCSF) led to a concentration-dependent enhancement of phagocytic activity (PA) of the El's "central" macrophages. The latter phenomenon was revealed in "mature" El classes only. The PA of "central" macrophages in "proliferating" El appeared to be affected neither by EP, nor by MCSF.     

Visualization of Flagella during Bacterial Swarming

When cells of Escherichia coli are grown in broth and suspended at low density in a motility medium, they swim independently, exploring a homogeneous, isotropic environment. Cell trajectories and the way in which these trajectories are determined by flagellar dynamics are well understood. When cells are grown in a rich medium on agar instead, they elongate, produce more flagella, and swarm. They move in coordinated packs within a thin film of fluid, in intimate contact with one another and with two fixed surfaces, a surfactant monolayer above and an agar matrix below: they move in an inhomogeneous, anisotropic environment. Here we examine swarm-cell trajectories and ways in which these trajectories are determined by flagellar motion, visualizing the cell bodies by phase-contrast microscopy and the flagellar filaments by fluorescence microscopy. We distinguish four kinds of tracks, defining stalls, reversals, lateral movement, and forward movement. When cells are stalled at the edge of a colony, they extend their flagellar filaments outwards, moving fluid over the virgin agar; when cells reverse, changes in filament chirality play a crucial role; when cells move laterally, they are pushed sideways by adjacent cells; and when cells move forward, they are pushed by flagellar bundles in the same way as when they are swimming in bulk aqueous media. These maneuvers are described in this report.Swarming is a common yet specialized form of surface translocation exhibited by flagellated bacteria and is distinct from swimming (23). When cells are grown on a moist nutrient-rich surface, they differentiate from a vegetative to a swarm state: they elongate, make more flagella, secrete wetting agents, and move across the surface in coordinated packs. Here we focus on the mechanics of bacterial swarming, as exhibited by the model organism Escherichia coli. Others have worked on swarm-cell differentiation in a variety of organisms, including Proteus, Salmonella, Pseudomonas, Serratia, Bacillus, and Vibrio. For example, screens for genes required for swarming in E. coli or Salmonella have been made by Inoue et al. (25) and Wang et al. (40, 41). Vibrio is a special case, because a single polar flagellum enables cells to swim while multiple lateral flagella promote swarming (32). For general reviews, see the work of Allison and Hughes (1), Shapiro (37), Fraser and Hughes (17), and Fraser et al. (16). Also see the work of Eberl et al. (15), Sharma and Anand (38), Harshey (18), Daniels et al. (11), Kaiser (26), O''Toole (33), and Copeland and Weibel (10).Swarming was first observed with Proteus mirabilis by Hauser (22), who named the genus for a sea god able to change his own form. Proteus is distinctive because cells switch periodically from the vegetative to the swarming state, building terraced colonies (36, 42). This is not observed with E. coli under the conditions used here, where swarms expand at a constant rate propelled by cells swimming vigorously in a monolayer behind a smooth outer boundary.Swarming in E. coli was discovered by Harshey, who found that K-12 strains, which lack the lipopolysaccharide O antigen, swarmed on Eiken agar (from Japan) but not on Difco agar (from the United States), presumably because the former is more wettable (19, 20). Chemotaxis is not required: cells lacking the chemotaxis response regulator CheY swarm perfectly well, provided that mutations in the motor protein FliM enable transitions between clockwise (CW) and counterclockwise (CCW) rotational states (31). It was suggested that these reversals promote wetness by causing cells to shed lipopolysaccharide.How do cells in E. coli swarms move across an agar surface? What are their flagella doing? We sought to answer such questions by performing a global analysis of videotaped data (of phase-contrast images) collected from 5 regions of 2 swarms, plotting body lengths, speeds, propulsion angles, local track curvatures, and temporal and spatial correlations, and we found that cells reorient on a time scale of a few tenths of a second, primarily by colliding with one another (13). Our previous report did not describe analyses of individual tracks or visualization of flagella. This aspect of the work is presented here.Most of the time, cells are driven forwards by a flagellar bundle in the usual way. Flagellar filaments from different cells can intertwine and form common bundles, but this is rare. However, cells in swarms do something not ordinarily seen with swimming cells: they back up. They do this without changing the orientation of the cell body by moving back through the middle of the flagellar bundle. This involves changes in filament shape (in polymorphic form), from normal to curly and back to normal. Polymorphic forms were classified by Calladine (7), on the basis of earlier work by Asakura (3), in terms of the relative lengths of 11 protofilaments, longitudinal arrays of protein subunits that comprise the filament. All polymorphic forms are helical, with some being left-handed (e.g., the normal form) and some being right-handed (e.g., the semicoiled and curly forms, which have half the pitch of the normal filament and half the pitch and half the amplitude, respectively). Transformations from one shape to another can be caused in various ways, e.g., by changes in pH, salinity, or temperature (21, 27, 28) or by application of torque (24). The changes observed with swarm cells are driven by the latter mechanism, when motors switch from CCW to CW rotation. When swimming cells tumble, polymorphic transformations also occur, in the order normal to semicoiled to curly and back to normal (14, 39). But we rarely see the semicoiled form with cells in swarms, and when it appears, it is quite transient. We wonder whether polymorphic transformations evolved to enable cells to escape confined environments when the only way out is to back up, keeping the filaments close to the sides of the cell body.     

Ritual behaviors associated with spermatophore transfer in Deuterosminthurus bicinctus (Collembola: Bourletiellidae)

The springtail Deuterosminthurus bicinctus, similarly to other members of Bourletiellidae, use their antennae, legs or heads to monopolize, stimulate, and direct female partners to spermatophores. The mating behavior of this species was examined by analysis of video recordings made on the leaves of its host plant. The characteristic stages of the behavioral sequence leading to sperm transfer were (1) preliminary courtship, highly variable in time and intensity—a male (rarely a female) endeavors to recruit a partner for further courtship, (2) push-and-retreat ritual—a rigid and rhythmical head-to-head dance of both partners, composed of some 180 phases and interspersed by several (3) free turns of a male and ended by (4) spermatophore deposition in front of a female, followed by stimulation of a female by a male to walk over the spermatophore, (5) spermatophore pick-up by a female with her gonopore, or alternately, (6) total spermatophore consumption by a female, as happened in 29% of the observed pairs. The mating ended always with (7) competition by both partners for the consumption of spermatophore residuals, usually won by the female. Comparison of mating elements and morphological features of partners engaged in mating sessions that ended either with spermatophore pick-up or consumption did not reveal any substantial differences. This suggests that changes in female motivation (hunger, state of impregnation) may be crucial for the male success in sperm transfer to a female. A digital video image is available at An erratum to this article can be found at     

NAD(P)H Quinone-Oxydoreductase 1 Protects Eukaryotic Translation Initiation Factor 4GI from Degradation by the Proteasome

The eukaryotic translation initiation factor 4GI (eIF4GI) serves as a central adapter in cap-binding complex assembly. Although eIF4GI has been shown to be sensitive to proteasomal degradation, how the eIF4GI steady-state level is controlled remains unknown. Here, we show that eIF4GI exists in a complex with NAD(P)H quinone-oxydoreductase 1 (NQO1) in cell extracts. Treatment of cells with dicumarol (dicoumarol), a pharmacological inhibitor of NQO1 known to preclude NQO1 binding to its protein partners, provokes eIF4GI degradation by the proteasome. Consistently, the eIF4GI steady-state level also diminishes upon the silencing of NQO1 (by transfection with small interfering RNA), while eIF4GI accumulates upon the overexpression of NQO1 (by transfection with cDNA). We further reveal that treatment of cells with dicumarol frees eIF4GI from mRNA translation initiation complexes due to strong activation of its natural competitor, the translational repressor 4E-BP1. As a consequence of cap-binding complex dissociation and eIF4GI degradation, protein synthesis is dramatically inhibited. Finally, we show that the regulation of eIF4GI stability by the proteasome may be prominent under oxidative stress. Our findings assign NQO1 an original role in the regulation of mRNA translation via the control of eIF4GI stability by the proteasome.In eukaryotes, eukaryotic translation initiation factor 4G (eIF4G) plays a central role in the recruitment of ribosomes to the mRNA 5′ end and is therefore critical for the regulation of protein synthesis (14). Two homologues of eIF4G, eIF4GI and eIF4GII, have been cloned (15). Although they differ in various respects, both homologues clearly function in translation initiation. The most thoroughly studied of these is eIF4GI, which serves as a scaffolding protein for the assembly of eIF4F, a protein complex composed of eIF4E (the mRNA cap-binding factor) and eIF4A (an ATP-dependent RNA helicase). Thus, via its association with the mRNA cap-binding protein eIF4E and with another translation initiation factor (eIF3) which is bound to the 40S ribosomal subunit, eIF4GI creates a physical link between the mRNA cap structure and the ribosome, thus facilitating cap-dependent translation initiation (25). eIF4GI functions also in cap-independent, internal ribosome entry site (IRES)-mediated translation initiation. For instance, upon picornavirus infection, eIF4G is rapidly attacked by viral proteases. The resulting eIF4GI cleavage products serve to reprogram the cell''s translational machinery, as the N-terminal cleavage product inhibits cap-dependent translation of host cell mRNAs by sequestering eIF4E while the C-terminal cleavage product stimulates IRES-mediated translation of viral mRNAs (23). Similarly, apoptotic caspases cleave eIF4G into an N-terminal fragment that blocks cap-dependent translation and a C-terminal fragment that is utilized for IRES-mediated translation of mRNAs encoding proapoptotic proteins (22).The regulation of eIF4GI cleavage by viral proteases or apoptotic caspases has been extensively studied. Little is known, however, about the regulation of eIF4GI steady-state levels. Yet the eIF4GI amount that exists at a given moment results from the sum of the effects of de novo synthesis and ongoing degradation. Many cellular proteins are physiologically degraded by the proteasome. This has been shown to be true for eIF4GI, as the factor can be degraded by the proteasome in vitro (5) and in living cells (6). However, how eIF4GI targeting for or protection from destruction by the proteasome is regulated remains unknown.There are two major routes to degradation by the proteasome. In the more conventional route, polyubiquitinated proteins are targeted to the 26S proteasome. Alternatively, a few proteins can be degraded by the 20S proteasome (and sometimes by the 26S proteasome) in a ubiquitin-independent manner (16). Interestingly, it has been shown recently that a few of these proteins (1, 2, 13) can be protected from degradation by the 20S proteasome by binding to the NAD(P)H quinone-oxydoreductase 1 (NQO1). It has been proposed that NQO1 may interact with the 20S proteasome and may consequently block access of target proteins to the 20S degradation core. Because eIF4GI can be degraded in vitro by the 20S proteasome (5) and since it appears that proteasomes can degrade eIF4GI in living cells independently of ubiquitination (6), we asked whether NQO1 could protect eIF4GI from degradation by the proteasome.     

Inhibition of Cell Expansion by Rapid ABP1-Mediated Auxin Effect on Microtubules? A Critical Comment

Critical analysis of a recent article raises questions regarding the inhibition of cell expansion by rapid ABP1-mediated auxin effect on microtubules.How do cortical microtubules shape plant cells? This has been an important question ever since the microtubular cytoskeleton was found to orientate the deposition of cellulose microfibrils in the primary cell wall and control long-term anisotropic cell expansion under isotropic turgor pressure (Green, 1962). In axial plant organs, longitudinal microtubule/microfibril arrays hamper expansion in length and favor expansion in girth, while transverse microtubule/microfibril arrays have the opposite effect (Baskin, 2001). By generating mechanical anisotropy in the cell wall, microtubule orientation controls the ratio of longitudinal versus circumferential cell expansion (the allometric ratio). A recent study by Chen et al. (2014) concludes that auxin inhibits cell growth by causing a rapid reorientation of microtubules from a transverse to a longitudinal orientation in cells of the submeristematic root zone and the elongation zone of the hypocotyl of Arabidopsis (Arabidopsis thaliana) seedlings. This conclusion warrants attention because the cell expansion that drives auxin-mediated organ elongation is generally thought to be controlled by the regulated breaking of bonds between existing cell wall polymers by chemical means (wall loosening; Cosgrove, 2005).It has been clear for more than 25 years that auxin does induce rapid changes in the orientation of microtubules in growing cells, either during straight growth (Bergfeld et al., 1988) or tropic bending (Nick et al., 1990). These studies form part of a group of investigations that show that numerous growth-affecting endogenous, environmental, and even artificial physical factors have very similar effects on microtubule orientation: during active cell expansion or related mechanical strains, microtubules are aligned against the direction of expansion, and they are aligned with it during the inhibition of expansion (Fischer and Schopfer, 1997). An important insight that emerges from this extensive evidence is that the type of reorientation elicited by a particular factor depends on its physiological context, thereby allowing auxin to induce either transverse or longitudinal microtubule orientations depending on whether elongation growth is promoted (as in shoot organs) or inhibited (as in roots). Clearly, ordered microtubule reorientations require the input of directional information (Williamson, 1990). Auxin signaling as such cannot provide this information, but the directional growth responses produced by auxin can. This brings us to the crucial question: is the orientation of microtubules determined by the effector signal directly or by changes in growth? The answer given by Chen et al. (2014) comes as a surprise: the inhibition of cell expansion is mediated by the rapid AUXIN-BINDING PROTEIN1 (ABP1)-dependent action of auxin on microtubules. The authors imply that it is microtubular reorientation per se that is responsible for the sudden growth inhibition caused by auxin in roots and hypocotyls rather than any changes in cell wall structure. Related microtubule reorientations at the concave side of gravitropically curving roots are interpreted in a similar way.Coming as an even bigger surprise, Chen and collaborators do not provide any evidence to support the claim made in their title, nor do they touch the obvious question of how microtubule reorientation from transverse to longitudinal might produce the growth inhibition elicited by auxin in the Arabidopsis root so quickly (Evans et al., 1994). Modification of the allometric ratio by changing the orientation of newly deposited cellulose microfibrils happens over hours and, therefore, is much too slow to account directly for the inhibition of cell expansion by auxin. Accompanying data on the growth changes produced by auxin in their experiments are not included in the report, nor are specifications of the investigated cell layers, despite the fact that the different cell layers show different responses to hormones (Ubeda-Tomás et al., 2012). Hence, critical questions regarding the quantitative relationship between microtubule reorientation and cell elongation remain unanswered. The gap between the experimental data presented and the far-reaching conclusions derived from them has been pointed out by Baskin (2015).There is, to our knowledge, no evidence for any fast growth-controlling mechanisms involving microtubules. There is, on the other hand, ample evidence for a causal relationship between wall-relaxing processes (such as the secretion or activation of wall-loosening enzymes or the generation of reactive oxygen species) and the rapid regulation of cell growth by auxin (Cosgrove, 2005; Perrot-Rechenmann, 2010). Modification of the allometric ratio by changing the orientation of newly deposited cellulose microfibrils appears much too slow (happening over hours) to account for the inhibition of cell expansion within minutes (Evans et al., 1994). Gravitropic bending of maize (Zea mays) roots has been shown to proceed normally even after the disassembly or immobilization of microtubules, and the inhibition of bending prohibits unilateral microtubule reorientation (Baluška et al., 1996). Chen et al. (2014) do not consider any of this evidence.So, can the data presented by Chen and collaborators be explained without coming into conflict with previously published results? The answer to this question is straightforward and has long been accessible in the pertinent literature: the observed changes in microtubule pattern are trivial consequences of either growth inhibition or the auxin insensitivity of growth in abp1 (Tromas et al., 2009). Consider the following points.First, mechanical forces can reorientate microtubules. There is a large body of experimental evidence that indicates that microtubule reorientations in single cells or tissues can be induced by oriented mechanical forces causing oriented stresses and strains in the affected cell walls (Landrein and Hamant, 2013). For example, Fisher and Cyr (2000) subjected protoplasts, embedded in an elastic agarose matrix, to mechanically induced stretching. The originally randomly oriented microtubules responded to this treatment by aligning at right angles to the major tensive force vector. Similarly, growing coleoptile segments respond to mechanical bending by reorientating the microtubules of epidermal cells at right angles to the direction of tension (extended side) and parallel to the direction of compression (compressed side; Fischer and Schopfer, 1997).Second, anisotropic cell growth mirrors patterns of stress and strain within the cell wall (Hamant and Traas, 2010). In biophysical terms, turgid plant cells can be regarded as pressurized vessels surrounded by an elastically stretched wall. Auxin-driven cell enlargement is brought about by changing the yielding properties of the wall and the resulting expansion in the direction of growth (Cosgrove, 2005).Third, experiments with maize coleoptiles have shown that auxin, in addition to effecting growth-related microtubule reorientations, strongly promotes their responsivity to mechanical forces. The epidermal microtubules of auxin-deprived coleoptile segments barely respond to bending stresses but reorientate rapidly after a 1-h treatment with auxin (Fischer and Schopfer, 1997). Hence, cell wall strains generated by growth or applied stresses interact in orientating microtubules in a synergistic manner, pointing to a common signaling mechanism activated by changes in strain rate.Summing up, there is well-founded evidence for the conclusion that the microtubule reorientations observed by Chen and collaborators occur as a result of changes in the physical strain pattern that underlies the auxin-induced changes in cell expansion. In agreement with established knowledge, the primary effect of auxin may be a rapid inhibition of cell wall loosening, mediated by the production of hydrogen peroxide (Ivanchenko et al., 2013). Based on these arguments and the weight of published evidence, we conclude that Chen and collaborators have inversed cause and effect.Their observation that the inactivation of ABP1 (and downstream components of the ABP1 pathway) causes microtubules to become unresponsive to auxin and lose their transverse pattern in roots may be explained as trivial consequences of growth inhibition (Tromas et al., 2009). Serious questions now hang over the roles for ABP1 in auxin signaling and auxin-controlled development (Gao et al., 2015; Liu, 2015). However, this does not come as a complete surprise. Hayashi et al. (2008) previously showed that the auxin-induced growth inhibition of root and hypocotyl in Arabidopsis can be suppressed by α[2,4-dimethylphenly-ethyl-2-oxo]-IAA (auxinole). This antagonist specifically competes with auxin at the TIR1-AUX/IAA-type receptor complexes and does not bind to ABP1, thereby suggesting that ABP1 does not act as a receptor in these pathways.There are lessons here, not the least that literature from the pre-Arabidopsis era remains a relevant and valuable source of information.     

Srs2 Plays a Critical Role in Reversible G2 Arrest upon Chronic and Low Doses of UV Irradiation via Two Distinct Homologous Recombination-Dependent Mechanisms in Postreplication Repair-Deficient Cells

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G₂ arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G₂ arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G₂ arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes and exogenous DNA-damaging agents. In nature, the steady-state level of DNA damage is usually very low. However, chronic low-level DNA damage can lead to age-related genome instability as a consequence of the accumulation of DNA damage (12, 27). Increasing evidence implicates DNA damage-related replication stress in genome instability (7, 21). Replication stress occurs when an active fork encounters DNA lesions or proteins tightly bound to DNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source of genome instability, which can contribute to tumorigenesis and aging in humans (4, 11). Confronted with this risk, cells have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA replication (8).In budding yeast Saccharomyces cerevisiae, the Rad6-dependent postreplication repair (PRR) pathway is subdivided into three subpathways, which allow replication to resume by bypassing the lesion without repairing the damage (3, 22, 33). Translesion synthesis (TLS) pathways dependent on the DNA polymerases eta and zeta promote error-free or mutagenic bypass depending on the DNA lesion and are activated upon monoubiquitination of proliferating cell nuclear antigen (PCNA) at Lys164 (K164) (5, 16, 37). The Rad5 (E3) and Ubc13 (E2)/Mms2 (E2 variant)-dependent pathway promotes error-free bypass by template switching and is activated by polyubiquitination of PCNA via a Lys63-linked ubiquitin chain (16, 38, 41). It remains mechanistically unclear how polyubiquitinated PCNA promotes template switching at the molecular level. In addition to its ubiquitin E3 activity, Rad5 also has a helicase domain and was recently shown to unwind and reanneal fork structures in vitro (6). This led to the proposal that Rad5 helicase activity is required at replication forks to promote fork regression and subsequent template switching. It is possible that PCNA polyubiquitination acts to facilitate Rad5-dependent template switching by inhibiting monoubiquitination-dependent TLS activity and/or by recruiting alternative proteins to the fork.In addition to modification by ubiquitin, PCNA can also be sumoylated on Lys164 by the SUMO E3 ligase Siz1 (16). A second sumoylation site, Lys127, is also targeted by an alternative SUMO E3 ligase, Siz2, albeit with lower efficiency (16, 30). PCNA SUMO modification results in recruitment of the Srs2 helicase and subsequent inhibition of Rad51-dependent recombination events (29, 32). The modification can therefore allow the replicative bypass of lesions by promoting the RAD6 pathway. Srs2 is known to act as an antirecombinase by eliminating recombination intermediates. This can occur independently of PCNA sumoylation, and when srs2Δ cells are UV irradiated or other antirecombinases, such as Sgs1, are concomitantly deleted, toxic recombination structures accumulate (1, 10). Such genetic data are consistent with the ability of Srs2 to disassemble the Rad51 nucleoprotein filaments formed on single-stranded DNA (ssDNA) in vitro (20, 40). In addition to directly inhibiting homologous recombination (HR), Srs2 is also involved in regulating HR outcomes to not produce crossover recombinants in the mitotic cell cycle (18, 34, 35).The UV spectrum present in sunlight is a primary environmental cause of exogenous DNA damage. Sunlight is a potent and ubiquitous carcinogen responsible for much of the skin cancer in humans (17). In the natural environment, organisms are exposed to chronic low-dose UV light (CLUV), as opposed to the acute high doses commonly used in laboratory experiments. Hence, understanding the cellular response to CLUV exposure is an important approach complementary to the more traditional laboratory approaches for clarifying the biological significance of specific DNA damage response pathways. A recently developed experimental assay for the analysis of CLUV-induced DNA damage responses was used to show that the PCNA polyubiquitination-dependent error-free PRR pathway plays a critical role in tolerance of CLUV exposure by preventing the generation of excessive ssDNA when replication forks arrest, thus suppressing counterproductive checkpoint activation (13).Mutants of SRS2 were first isolated by their ability to suppress the radiation sensitivity of rad6 and rad18 mutants (defective in PRR) by a mechanism that requires a functional HR pathway (23, 36). In this study, we analyzed the function of Srs2 in CLUV-exposed PRR-deficient cells. We established that Srs2 acts in conjunction with SUMO-modified PCNA to lower the threshold for checkpoint activation and maintenance by suppressing the function of HR in rad18Δ cells exposed to CLUV. We also showed that Srs2 is separately involved in an HR-dependent recovery process following cessation of CLUV exposure and that this second role for Srs2, unlike its primary role in checkpoint activation and maintenance, is regulated by CDK1-dependent phosphorylation. Thus, Srs2 is involved in both CLUV-induced checkpoint-mediated arrest and recovery from CLUV exposure in PRR-deficient cells, and these two functions, while both involving HR, are separable and thus independent.     

Mitochondrial DNA Toxicity in Forebrain Neurons Causes Apoptosis,Neurodegeneration, and Impaired Behavior

Mitochondrial dysfunction underlying changes in neurodegenerative diseases is often associated with apoptosis and a progressive loss of neurons, and damage to the mitochondrial genome is proposed to be involved in such pathologies. In the present study we designed a mouse model that allows us to specifically induce mitochondrial DNA toxicity in the forebrain neurons of adult mice. This is achieved by CaMKIIα-regulated inducible expression of a mutated version of the mitochondrial UNG DNA repair enzyme (mutUNG1). This enzyme is capable of removing thymine from the mitochondrial genome. We demonstrate that a continual generation of apyrimidinic sites causes apoptosis and neuronal death. These defects are associated with behavioral alterations characterized by increased locomotor activity, impaired cognitive abilities, and lack of anxietylike responses. In summary, whereas mitochondrial base substitution and deletions previously have been shown to correlate with premature and natural aging, respectively, we show that a high level of apyrimidinic sites lead to mitochondrial DNA cytotoxicity, which causes apoptosis, followed by neurodegeneration.A variety of both exogenous and endogenous reactive compounds present a constant threat to the integrity of DNA in living cells. DNA damage introduced by such compounds can lead to high and deleterious mutation rates as well as DNA cytotoxicity, both to the nuclear and the mitochondrial genome. This has triggered the evolution of several different DNA repair pathways (28). One is the base excision repair (BER) pathway, which repairs small base alterations that do not distort the DNA helix. Repair of such highly abundant lesions by BER is performed by a multistep process that is initiated by a damage-specific DNA glycosylase, which removes the damaged base. One of these glycosylases is uracil-DNA glycosylase (UDG), which acts to preserve the genome by removing mutagenic uracil residues from the DNA. This glycosylase, as well as the OGG1 glycosylase that is specialized for the removal of oxidized bases, exists in a nuclear and mitochondrial splice form (1, 11, 37, 45). Accordingly, BER of a variety of lesions has been observed in mitochondria (26, 31).Damage to the mitochondrial DNA (mtDNA) can cause respiratory chain deficiency and lead to disorders that have varied phenotypes (35, 41). Many involve neurological features that are often associated with cell loss within specific brain regions. These pathologies, along with the increasing evidence of a decline in mitochondrial function with aging, have raised speculation that key changes in mitochondrial DNA sequences and functions could have a vital role in age-related neurodegenerative diseases (41). This has also been studied in several model organisms. Mouse models with respiratory chain deficient dopamine neurons have demonstrated adult onset Parkinsonism phenotype (16), and cell death induced by mitochondrial toxicity is likely to underlie Alzheimer disease (32). Mitochondrial oxidative stress and accumulation of mtDNA damage are believed to be particularly devastating to postmitotic differentiated tissue, including neurons (30). The mtDNA contains genetic information for 13 polypeptides that are a part of the electron transport chain and for rRNAs and tRNAs that are necessary for mitochondrial protein synthesis. Thus, damage to the mtDNA genome will affect the energetic capacities of the mitochondria and also influence the level of reactive oxygen species (ROS) and ultimately the susceptibility to apoptosis (30, 35).Some recent influential studies have assessed the effect of mtDNA mutagenesis, including small base-pair substitutions and larger mtDNA deletions, on the life span of mice. It was concluded that a massive increase in the frequency of mtDNA base-pair substitutions are required for inducing premature aging, whereas the number of mtDNA deletions coincides better with natural aging (25, 47-49).In the present study, we have combined two novel transgenic mouse models, which allow the induction of a high number of apyrimidinic (AP) sites specifically to the mitochondrial genome in adults simply by the addition of doxycycline to the diet. Such AP sites are created by the expression of a mutated version of mitochondrion-targeted human UDG (abbreviated here as mutUNG1), whereby an amino acid substitution results in an enzyme that removes thymine, in addition to uracil, from DNA (23). The CaMKIIα promoter restricts expression of the mutUNG1 to forebrain neurons (34). We demonstrate that a continuous generation of AP sites leads to apoptosis, accelerated neurodegeneration, and impaired behavior.     

Estrogen-binding properties of rat serum alpha 1-fetoprotein and its isoforms. Investigation of the apparent non-integrality of sites on the unfractionated protein.

Rat fetal serum alpha 1-fetoprotein (AFP), a heterogeneous glycoprotein, binds estrogens with high affinity but at a fractional number of sites even after treatment with charcoal (n = 0.6), which may mean 60% of the protein has 1 site and the remainder none. To investigate the origin of this fractional number of sites the "native" protein (purified by negative affinity chromatography) was further purified (step 1) and fractionated (step 2) into its two main charge variants (electrophoretically "slow" and "fast") by a two-step fast-protein liquid chromatography method. The binding parameters for estrone and estradiol-17 beta of the "native" and "repurified" proteins and of each charge variant were determined by equilibrium microdialysis. The molar extinction coefficient at 278 nm of each sample was also determined. (1) The "repurified" AFP and each charge variant had a number of binding sites for estrogens close to unity. This increase in the number of sites could neither be explained by the loss of a non-binding isoform (corresponding to 40% of the protein) during chromatography, nor by the existence of complex negative modulatory interactions between isoforms. (2) The affinities for estrogens of the "repurified" protein and the two charge variants were slightly decreased compared to that of "native" AFP, except that the "fast" form had the "native" protein's high affinity for estrone--but not for estradiol-17 beta. (3) The molar extinction coefficients at 278 nm of the "repurified" AFP and the isoforms were much lower than that of the "native" protein. These results suggest that the presence of (an) inhibitor(s) of estrogen binding on the "native" protein which is/are removed by the ion-exchange fast protein liquid chromatography (FPLC) column. A ligand absorbing at 278 nm, which may or may not be the inhibitor, is also removed. The isoform heterogeneity with respect to estrone binding is discussed.     

Efficient Class II Major Histocompatibility Complex Presentation of Endogenously Synthesized Hepatitis C Virus Core Protein by Epstein-Barr Virus-Transformed B-Lymphoblastoid Cell Lines to CD4+ T Cells

The induction of an efficient CD4⁺ T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4⁺ T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10⁴ transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.Hepatitis C virus (HCV) has been known as a major etiologic agent of posttransfusion and sporadic community-acquired non-A, non-B hepatitis. Like the other members in the family Flaviviridae, HCV contains a single, positive-strand RNA genome with a single long open reading frame (ORF) coding for a polyprotein precursor of about 3,000 amino acids (aa) (12). HCV infection is frequently persistent in the majority of patients and is closely associated with the later development of liver cirrhosis and hepatocellular carcinoma (3, 12, 16, 32). The effective control of HCV infection has been limited by the high frequency of viral genetic heterogeneity (7), the low rate of response to alpha interferon (46), and inadequate production of protective immunity (44, 45). These features strongly suggest that there is a great need to establish a new, highly effective therapy.CD4⁺ T cells are considered to play a central role in the generation of protective immunity against infections, because they can provide help to B cells for antibody production (42) and to cytotoxic precursor T cells for their maturation to effectors (21). Some CD4⁺ T cells may also act as cytotoxic effectors (30). It has been recognized that CD4⁺ T-cell response to HCV antigens is important for determining the clinical course of HCV infection (17, 37). Generally, T-cell proliferation is more frequent and stronger in patients with a benign course (6, 17, 20, 33, 37) that is accompanied by the normalization of serum alanine aminotransferase and, in some cases, the clearance of viral RNA (17, 37). In contrast, patients who have a poor T-cell response tend to develop persistent infection (17, 37). These findings support the hypothesis that a sufficient CD4⁺ T-lymphocyte response is critical for limiting HCV infection.Activation of T lymphocytes depends on the recognition of processed viral peptides, but not native antigens, in the context of major histocompatibility complex (MHC) molecules that are presented by antigen-presenting cells (APCs) (56). The B cell is an important professional APC, and its role in mediating antigen-specific immune response has been described extensively (11). Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells are frequently used as APCs in in vitro analyses for antigen processing and presentation to T cells. These cells are characterized by high-level expression of class I and class II MHC molecules, along with accessory molecules such as ICAM-1, B7/BB1, and LFA-3, known to be important costimulatory molecules for T-cell activation (9, 15, 24). Importantly, transfected EBV-immortalized B cells expressing a tumor antigen have been shown to be capable of eliciting both T-helper and cytotoxic-T-lymphocyte (CTL) responses following in vivo inoculation (40). Nevertheless, dendritic cells have been shown to be critical for initiating responses by naive T cells (53), and in some situations presentation by B cells has been suggested to be toleragenic (35). To date, the role of B cells in processing and presenting HCV antigens has not been studied in detail and the mechanisms underlying T-cell–B-cell interaction are still being worked out.In the present study, EBV-transformed B-lymphoblastoid cell lines (B-LCLs) established from HCV-infected patients were transfected with an expression vector coding for the whole structural region and part of the NS2 region of the HCV genome. The capacity of transfected B-LCL APCs for presenting intracellularly synthesized peptides was assessed by in vitro induction of the HCV-specific lymphoproliferative response of autologous T-cell lines. Our results indicated that core protein was properly expressed and efficiently presented by B-LCL APCs to CD4⁺ T cells. We demonstrated that the endogenous core peptides were presented through the class II MHC pathway and that they need B7/BB1 for providing costimulatory signals.     

Nicotonic and muscarinic components in acetylcholine stimulation of porcine adrenal medullary cells

Adrenal medullary chromaffin cells secrete catecholamines (CA) in response to cholinergic receptor activation by acetylcholine (ACh) released from splacnic nerve terminals. In cultured bovine chromaffin cells nicotinic receptors play a preponderant (> 90%) role in the control of CA release. By contrast, we found and report here that up to 40% of the ACh-evoked CA secretion from cultured porcine chromaffin cells can be associated with muscarinic receptor activation. The following results support our belief that in porcine adrenal medullary cells ACh (100 M) evoked CA secretion is mediated by both nicotinic and muscarinic cholinergic receptors. 1) Hexamethonium (100 M), a nicotinic receptor antagonist, inhibited ACh-induced CA secretion to ca. 40% of the control release and atropine (1 M), a muscarinic receptor antagonist, inhibited to ca. 60% of the control value. 2) We also found that ACh (100 M) evoked intracellular Ca2+ concentration ([Ca2+]i) rise was inhibited by these receptor antagonists to a different extent, and reversibly reduced by lowering the concentration of Ca2+ in the external medium ([Ca2+]o). This last maneuver ([Ca2+]o < 0.1 M) per se caused a marked reduction in the peak phase of the [Ca2+]i rise evoked by ACh (40% of the control response). Switching the external medium back to physiologic [Ca2+]o in the continued presence of ACh caused a partial recovery of the elevated [Ca2+]i. This [Ca2+]o-dependent [Ca2+]i rise was blocked by hexamethonium (100 M) but not by atropine (1 M). Conversely, the ACh-evoked [Ca2+]i rise in low external [Ca2+]o was blocked by atropine but not by hexamethonium. From these data we conclude that in porcine adrenal medullary cells an important fraction (ca. 0.4) of both ACh-induced CA secretion and peak [Ca2+]i rise is due to muscarinic receptor activation.     

Enhanced proliferation of murine T cell lines following interaction of Poly(Glu60, Phe40) (GPhe) and antigen-presenting accessory cells. III. Possible mechanisms responsible for activity of GPhe.

The random copolymers (Glu80, Phe20)n (GPhe20), (Glu60, Phe40)n (GPhe), and (Glu50, Phe50)n (GPhe50) were compared for the capacity to augment proliferation of antigen-reactive murine T cell lines. GPhe20, GPhe, and GPhe50 showed "augmenting" activity in order of increasing potency. Phenylalanyl residues constituted a significant portion of the "active" determinant(s) in the GPhe polymers tested. High titer murine anti-GPhe (ascites fluid) inhibited augmentation by GPhe of exogenous (IL-1 + rat-conditioned media (RCM] driven T cell proliferation, indicating that (a) the antibodies by binding to specific active determinant(s) in GPhe may have prevented critical GPhe-APC membrane interaction, and/or (b) "GPhe-anti-GPhe" complexes interfered with necessary "processing" of GPhe by APCs. Time course studies demonstrated that the appearance of increased T cell proliferation after GPhe addition occurred after proliferation to (a) nominal antigen or (b) exogenous (IL-1 + RCM) had reached peak [3H]thymidine incorporation ([3HT]). This suggested that more than GPhe-APC membrane interaction was necessary for GPhe activity. Leupeptin, a lysosomal protease inhibitor, inhibited the augmentation of T cell proliferation by GPhe, which led to the conclusion that GPhe must be "processed" by APCs to exhibit activity.