Intact P1 Research Articles

HpaII-assisted and linear amplification-enhanced isothermal exponential amplification fluorescent strategy for rapid and sensitive detection of DNA methyltransferase activity.

The detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, a HpaII-assisted and linear amplification-enhanced exponential amplification strategy is proposed for sensitive and label-free detection of M.SssI MTase activity. The P1 probe contains self-complementary sequence 5'-CTAGCCGGCTAG-3' at 3'-terminal. After denaturation and annealing, P1 probes hybridize with itself to generate P1 duplexes. M.SssI MTase induces methylation of cytosine at 5'-CG-3' in P1 duplexes, and thus, HpaII fails to cleave at 5'-CCGG-3' due to methylation sensitivity, leaving P1 duplex intact. Then, these intact P1 duplexes are extended along 3'-terminal through Vent (exo-) DNA polymerase to generate dsDNA, which is recognized and nicked at the recognition sites by Nt.BstNBI, releasing two copies of primer X. Primer X hybridizes with X' at the amplification template T1 (X'-Y'-X') and then serves as primers to trigger the exponential amplification reaction (EXPAR). The point of inflection (POI) values of real-time fluorescence curves is linearly correlated with the logarithm of M.SssI MTase concentration in the range of 0.125 [Formula: see text] 8 U mL-1 with a low detection limit of 0.034 U mL-1. In the absence of M.SssI, P1 duplexes are cut by HpaII and separated into ssDNA under the executed temperature of EXPAR and thus unable to trigger the amplification. The strategy provides good selectivity against other types of MTases and protein and is able to detect M.SssI activity in human serum. Furthermore, the analytical method has the generality and can be extended to the analysis of other types of DNA MTases.

What processes are disrupted during the attentional blink? An integrative review of event-related potential research.

Reporting the second of two targets is impaired when these appear in close succession, a phenomenon known as the attentional blink (AB). Despite decades of research, what factors limit our ability to process multiple sequentially presented events remains unclear. Specifically, two central issues remain open: does failure to report the second target (T2) reflect a structural limitation in working memory (WM) encoding or a disruption to attentional processes? And is perceptual processing of the stimulus that we fail to report impaired, or only processes that occur after this stimulus is identified? We address these questions by reviewing event-related potential (ERP) studies of the AB, after providing a brief overview of the theoretical landscape relevant to these debates and clarifying key concepts essential for interpreting ERP studies. We show that failure to report the second target is most often associated with disrupted attentional engagement (associated with a smaller and delayed N2pc component). This disruption occurs after early processing of T2 (associated with an intact P1 component), weakens its semantic processing (typically associated with a smaller N400 component), and prevents its encoding into WM (associated with absent P3b). However, failure to encode T2 in WM can occur despite intact attentional engagement and semantic processing. We conclude that the AB phenomenon, which reflects our limited ability to process sequential events, emerges from the disruption of both attentional engagement and WM encoding.

Characterization of an intermolecular quaternary interaction between discrete segments of the Streptococcusmutans adhesin P1 by NMR spectroscopy.

Cell surface-localized P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcusmutans mediates sucrose-independent adhesion to tooth surfaces. Previous studies showed that P1's C-terminal segment (C123, AgII) is also liberated as a separate polypeptide, contributes to cellular adhesion, interacts specifically with intact P1 on the cell surface, and forms amyloid fibrils. Identifying how C123 specifically interacts with P1 at the atomic level is essential for understanding related virulence properties of S.mutans. However, with sizes of ~51 and ~185kDa, respectively, C123 and full-length P1 are too large to achieve high-resolution data for full structural analysis by NMR. Here, we report on biologically relevant interactions of the individual C3 domain with A3VP1, a polypeptide that represents the apical head of P1 as it is projected on the cell surface. Also evaluated are C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15 N-enriched C3 demonstrate its specific binding to A3VP1. Based on resolved C3 assignments, two binding sites, proximal and distal, are identified. Complementary NMR titration of A3VP1 with a C3/C12 complex suggests that binding of A3VP1 occurs on the distal C3 binding site, while the proximal site is occupied by C12. The MAb 6-8C binding interface to C3 overlaps with that of A3VP1 at the distal site. Together, these results identify a specific C3-A3VP1 interaction that serves as a foundation for understanding the interaction of C123 with P1 on the bacterial surface and the related biological processes that stem from this interaction. DATABASE: BMRB submission code: 27935.

Yersinia pseudotuberculosis Thermostable Toxin Dysregulates the Functional Activity of Two Types of Phagocytes in the Holothurian Eupentacta fraudatrix

The effect of a thermostable toxin of Yersinia pseudotuberculosis (in comparison with that of dexamethasone) on the functional activities of two types of phagocytes (P1 and P2) was studied in the holothurian Eupentacta fraudatrix. A high level of NO was shown to be a marker of intact P1 cells, while the high activity of arginase was a marker of P2 cells. The antioxidant defense in the P1 type was more pronounced than that of the P2 type. At the same time, the toxin inhibited the functional activity (generation of reactive oxygen species) of P1 phagocytes after 1 h of incubation and induced primarily the activity of P2 phagocytes, compared to that of P1 cells, after 24 h. In contrast to dexamethasone, which induced the transformation of the P1 phenotype into the P2 phenotype, the toxin promoted the mutual acquisition of the phenotype features by these two types of phagocytes. An analogy between the P1 and P2 phagocytes of the holothurian and M1 and M2 macrophages is discussed.

Specific Interaction of a Naturally Occurring Amyloidogenic Fragment of Streptococcus Mutans Adhesin P1 with Intact P1 on the Cell Surface Measured by Solid State Nuclear Magnetic Resonance Spectroscopy

The P1 adhesin of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 that is covalently attached to the cell wall interacting with non-covalently associated proteolytic fragments thereof, particularly the ∼50-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we have demonstrated that solid state NMR spectroscopy can be used to 1) globally characterize cell walls isolated from a Gram-positive bacterium; 2) quantify P1 localized on the cell surface; and 3) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent adhesion and biofilm formation via NMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy.

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to (1) globally characterize cell walls isolated from a Gram-positive bacterium and (2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

Multiple domains of endopin 2A for serpin cross-class inhibition of papain

The serpin endopin 2A inhibits the cysteine protease papain in cross-class inhibition. This study demonstrates the novel finding that both the non-RSL NH 2-domain and the RSL domain with P1–P1′ residues participate in endopin 2A inhibition. Production of a chimeric mutant of endopin 2A with replacement of its NH 2-domain with that of endopin 1 resulted in less effective inhibition of papain, indicated by its lower k ass association rate constant compared to wild-type endopin 2A. This chimeric mutant formed complexes with papain, but at lower levels compared to that with wild-type endopin 2A. Papain degradation of a portion of the chimeric mutant suggested a role for the NH 2-domain in regulating relative amounts of endopin 2A that enter the substrate pathway compared to the serpin inhibitory pathway. Furthermore, site-directed mutagenesis demonstrated that the RSL domain with intact P1–P1′ residues was necessary for inhibition. These findings indicate that the NH 2-domain and the RSL region both participate in endopin 2A inhibition of papain.

Further characterization of immunomodulation by a monoclonal antibody against Streptococcus mutans antigen P1.

We demonstrated previously that mucosal immunization of mice with Streptococcus mutans coated with the monoclonal antibody (MAb) 6-11A directed against the major surface adhesin protein P1 results in changes in the amount, isotype distribution, and specificity of serum antibodies compared with animals immunized with bacteria only. We now show that the specificity of the mucosal secretory IgA response was also influenced by this MAb. Changes in antibody specificity were associated with changes in biological activity. Serum samples which differed in antibody reactivity with P1 polypeptides generated by partial digestion with N-chlorosuccinimide but not in isotype distribution or overall reactivity with S. mutans or intact P1 demonstrated a statistically significant difference in the ability to inhibit bacterial adherence to salivary-agglutinin-coated hydroxyapatite beads. Serum IgG antibodies against P1 from mice immunized with either S. mutans alone or S. mutans coated with 6-11A were shown to recognize antigenic determinants dependent on the presence of the central proline-rich repeat domain, a segment necessary for the structural integrity of the molecule. However, no statistically significant differences were observed in antibody reactivity with a panel of six partial P1 polypeptides encoded by overlapping spaP subclones, suggesting that the targets of biologically relevant antibodies involve complex epitopes not reconstituted by the recombinant products tested. Lastly, we show that binding of MAb 6-11A to P1 on the surface of S. mutans alters P1's susceptibility to proteolytic digestion. Hence, changes in antigen processing and presentation may contribute to the immunomodulatory effects of this MAb.

Early deletion of neuromeres in Wnt-1-/- mutant mice: evaluation by morphological and molecular markers.

The Wnt-1 gene is required for the development of midbrain and cerebellum; previous work showed that knockout of Wnt-1 causes the loss of most molecular markers of these structures in early embryos and deletion of these structures by birth. However, neither the extent of early neuronal defects nor any possible alterations in structures adjacent to presumptive midbrain and cerebellum were examined. By using a neuron-specific antibody and fluorescent axon tracers, we show that central and peripheral neuronal development are altered in mutants during initial axonogenesis on embryonic day 9.5. The absence of neuronal landmarks, including oculomotor and trochlear nerves and cerebellar plate, suggests that both mesencephalon and rhombomere 1 (r1) are delected, with the remaining neural tube fused to form a new border between the caudalmost portion of the prosencephalon (prosomere 1, or p1) and r2. Central axons accurately traverse this novel border by forming normal longitudinal tracts into the rhombencephalon, implying that the cues that direct these axons are aligned across neuromeres and are not affected by the delection. The presence of intact p1 and r2 is further supported by the retention of markers for these two neuromers, including a marker of p1, the Sim-2 gene, and an r2-specific lacZ transgene in mutant embryos. In addition, alterations in the Sim-2 expression domain in ventral prosencephalon, rostral to p1, provide novel evidence for Wnt-1 function in this region.

Participation of the lytic replicon in bacteriophage P1 plasmid maintenance.

P1 bacteriophage carries at least two replicons: a plasmid replicon and a viral lytic replicon. Since the isolated plasmid replicon can maintain itself stably at the low copy number characteristic of intact P1 prophage, it has been assumed that this replicon is responsible for driving prophage replication. We provide evidence that when replication from the plasmid replicon is prevented, prophage replication continues, albeit at a reduced rate. The residual plasmid replication is due to incomplete repression of the lytic replicon by the c1 immunity repressor. Incomplete repression was particularly evident in lysogens of the thermoinducible P1 c1.100 prophage, whose replication at 32 degrees C remained almost unaffected when use of the plasmid replicon was prevented. Moreover, the average plasmid copy number of P1 in a P1 c1.100 lysogen was elevated with respect to the copy number of P1 c1+. The capacity of the lytic replicon to act as an auxiliary in plasmid maintenance may contribute to the extraordinary stability of P1 plasmid prophage.

Participation of Escherichia coli integration host factor in the P1 plasmid partition system.

Stable maintenance of the plasmid prophage of bacteriophage P1 requires the P1 ParB protein, which acts on a DNA site termed parS. Fractionation of extracts from Escherichia coli cells overproducing ParB revealed that a host factor, in addition to ParB, is required to observe maximal binding to parS, as detected by a nitrocellulose filter retention assay. Two observations indicated that this factor is E. coli integration host factor (IHF): purified IHF substituted specifically for host factor from a crude lysate, and lysates prepared from cells deficient in the beta subunit of IHF (E. coli hip mutants; also called himD) contained no host factor activity. Binding studies in vitro and competition experiments in vivo suggest that two types of ParB-parS DNA complexes can exist that differ in (i) the presence of IHF, (ii) the amount of parS sequence with which the proteins interact, and (iii) the specificity of their participation in partition. Under normal conditions, with the intact P1 partition region and wild-type bacteria, P1 plasmids apparently use IHF to assist ParB in the assembly of a functional partition complex at parS.

A novel role for site-specific recombination in maintenance of bacterial replicons

If daughter copies of unit-copy replicons recombine with each other, a replicon dimer results that cannot be partitioned equally to daughter cells at cell division. We present evidence that dimer formation interferes with plasmid equipartition in the case of a miniplasmid derived from the unit-copy plasmid prophage of bacteriophage P1. Asymmetric partition occurs, leading to a relatively high rate of loss of the plasmid from the growing population. In contrast, the wild-type P1 plasmid is maintained very efficiently in host cells. We show that this efficient maintenance is due to the presence of the loxP-cre site-specific recombination system present on the intact P1 plasmid. This system promotes rapid recombination between two IoxP sites on dimer molecules, resolving them into monomeric substrates for proper partition. We suggest that bacterial replicons that are maintained with great accuracy in recombination-proficient cells might also encode high-efficiency recombination systems.