Fragment P1 Research Articles

Hypoxic Regulation of the KLK4 Gene in two Different Prostate Cancer Cells Treated with TGF- β.

The human kallikrein-related peptidase (KLK) family which consists of 15 members is associated with prostate cancer and other cancers. It has been reported that overexpression of KLK4 in prostate cancer correlates with bone metastasis or advanced stage. Hypoxia occurs in the early stages of prostate cancer due to the accumulation of acidic metabolites or reactive oxygen species (ROS). In our study, KLK4 gene expression in hypoxic conditions in PC-3 and LNCaP cells which are treated with TGF-β was evaluated with mRNA, protein, and promoter activity levels. A chemical hypoxia model was created and confirmed at mRNA and protein level. No statistically significant cytotoxic effect of CoCl2 and TGF-β was observed in PC-3 and LNCaP cells with the MTT test. Four different truncated KLK4 gene promoter constructs were cloned in pmetLuc expression vector and basal activities of all promoter fragments were analyzed. The activities of P1 (-447/ + 657), P2 (-103/ + 657), and P3 (-267/ + 657) promoter fragments increased in hypoxic conditions except P4 (+555/ + 657), which does not contain the SMAD and HRE region. KLK4 mRNA levels in both PC-3 and LNCaP cells increased in the hypoxia and hypoxia/TGF groups compared to the non-treated groups. The stimulating effect of TGF-β is correlated with the increase in SMAD2/3 mRNA levels. KLK4 expression is up-regulated by TGF-β, especially under hypoxic conditions, and its interaction with the SMAD pathway is determined with different inhibitor experiments. HIF-1α and SMAD transcription factors bind to the KLK4 promoter showing the direct interaction of HIF-1α (-80/-52) and SMAD (+163/+194) regions with EMSA.

Functional Analysis of RMA3 in Response to Xanthomonas citri subsp. citri Infection in Citron C-05 (Citrus medica)

Citrus bacterial canker disease, caused by Xanthomonas citri subsp. citri (Xcc), poses a significant global threat to the citrus industry. Lateral organ boundaries 1 (Lob1) is confirmed as a citrus susceptibility gene that induces pathogenesis by interaction with the PthA4 effector of Xcc. Citron C-05 (Citrus medica) is a Citrus genotype resistant to Xcc. However, there is little information available on the regulation of Lob1 in resistant genotypes, which is important for the breeding of citrus cultivars resistant to canker disease. This study aimed to identify upstream regulatory factors of Lob1 in Citron C-05 and to investigate its function in disease resistance. ‘Bingtang’ sweet orange (C. sinensis), a susceptible genotype, was utilized as the control. cDNA yeast libraries of Xcc-induced Citron C-05 and ‘Bingtang’ sweet orange were constructed. The capacities of ‘Bingtang’ and Citron C-05 were 1.896 × 107 and 2.154 × 107 CFU, respectively. The inserted fragments ranged from 500 to 2000 bp with a 100% recombination rate. The promoter of Lob1 was segmented into two pieces and the P1 fragment from both genotypes was used to construct a bait yeast (PAbAi-CsLob1-P1; PAbAi-CmLob1-P1). Through library screening with the bait yeast, upstream regulators interacting with the Lob1-P1 promoter were identified and then validated using Y1H and dual-luciferase tests. The expression analysis of the three transcript factors indicated that RMA3 was upregulated by inoculation with Xcc in the resistant Citron C-05, but not in the susceptible sweet orange. The overexpression of CsRMA3 in ‘Bingtang’ sweet orange led to reduced canker symptoms, with a significantly lower pathogen density in the leaves following Xcc inoculation. When CmRMA3 was silenced by virus-induced gene silencing (VIGS) in Citron C-05, typical canker symptoms appeared on the CmRMA3-silenced leaves at 15 days post-inoculation with Xcc. Further expression analyses revealed that the CmRMA3 transcription factor suppressed the expression of Lob1. These results suggest that RMA3 participates in the resistant reaction of Citron C-05 to Xcc infection, and such a response might be in relation to its suppression of the expression of the pathogenic gene Lob1.

Diastereoselective Synthesis of the HIV Protease Inhibitor Darunavir and Related Derivatives via a Titanium Tetrachloride-Mediated Asymmetric Glycolate Aldol Addition Reaction.

Darunavir is a potent HIV protease inhibitor that has been established as an effective tool in the fight against the progression of HIV/AIDS in the global community. The successful application of this drug has spurred the development of derivatives wherein strategic regions (e.g., P1, P1', P2, and P2') of the darunavir framework have been structurally modified. An alternate route for the synthesis of darunavir and three related P1 and P1' derivatives has been developed. This synthetic pathway involves the use of a Crimmins titanium tetrachloride-mediated oxazolidine-2-thione-guided asymmetric glycolate aldol addition reaction. The resultant aldol adduct introduces the P1 fragment of darunavir via an aldehyde. Transamidation with a selected amine (isobutylamine or 2-ethyl-1-butylamine) to cleave the auxiliary yields an amide wherein the P1' component is introduced. From this stage, the amide is reduced to the corresponding β-amino alcohol and the substrate is then bis-nosylated to introduce the requisite p-nitrobenzenesulfonamide component and activate the secondary alcohol for nucleophilic substitution. Treatment with sodium azide yielded the desired azides, and the deprotection of the p-methoxyphenoxy group is achieved with the use of ceric ammonium nitrate. Finally, hydrogenation to reduce both the aniline and azide functionalities with concurrent acylation yields darunavir and its derivatives.

Macrocyclic BACE1 inhibitors with hydrophobic cross-linked structures: Optimization of ring size and ring structure

Based on the X-ray crystallography of recombinant BACE1 and a hydroxyethylamine-type peptidic inhibitor, we introduced a cross-linked structure between the P1 and P3 side chains of the inhibitor to enhance its inhibitory activity. The P1 and P3 fragments bearing terminal alkenes were synthesized, and a ring-closing metathesis of these alkenes was used to construct the cross-linked structure. Evaluation of ring size using P1 and P3 fragments with various side chain lengths revealed that 13-membered rings were optimal, although their activity was reduced compared to that of the parent compound. Furthermore, the optimal ring structure was found to be a macrocycle with a dimethyl branched substituent at the P3 β-position, which was approximately 100-fold more active than the non-substituted macrocycle. In addition, the introduction of a 4-carboxymethylphenyl group at the P1′ position further improved the activity.

Comparison of Lag Versus Nonlag Screw Fixation for Long Oblique Proximal Phalanx Fractures: A Biomechanical Study

To compare lag versus nonlag screw fixation for long oblique proximal phalanx (P1) fractures in a cadaveric model of finger motion via the flexor and extensor tendons. We simulated long oblique P1 fractures with a 45° oblique cut in the index, middle, and ring fingers of 4 matched pairs of cadaveric hands for a total of 24 simulated fractures. Fractures were stabilized using 1 of 3 techniques: two 1.5-mm fully threaded bicortical screws using a lag technique, two 1.5-mm fully threaded bicortical nonlag screws, or 2 crossed 1.14-mm K-wires as a separate control. The fixation method was randomized for each of the 3 fractures per matched-pair hand, with each fixation being used in each hand and 8 total P1 fractures per fixation group. Hands were mounted to a custom frame where a computer-controlled, motor-driven, linear actuator powered movement of the flexor and extensor tendons. All fingers underwent 2,000 full flexion and extension cycles. Maximum interfragmentary displacement was continuously measured using a differential variable reluctance transducer. Our primary outcome was the difference in the mean P1 fragment displacement between lag and nonlag screw fixation at 2,000 cycles. The observed differences in mean displacement between lag and nonlag screw fixation were not statistically significant throughout all time points. A two one-sided test procedure for paired samples confirmed statistical equivalence in the fragment displacement between these fixation methods at all time points, including the primary end point of 2,000 cycles. Nonlag screws provided equivalent biomechanical stability to lag screws for simulated long oblique P1 fractures during cyclic testing in this cadaveric model. Fixation of long oblique P1 fractures with nonlag screws has the potential to simplify treatment without sacrificing fracture stability during immediate postoperative range of motion.

Structure-Activity Relationship Analysis of Cocrystallized Gliptin-like Pyrrolidine, Trifluorophenyl, and Pyrimidine-2,4-Dione Dipeptidyl Peptidase-4 Inhibitors.

Approved and potent reported dipeptidyl peptidase-4 (DPP-4) inhibitors with gliptin-like structures are classified here according to their structures and mechanisms of the inhibition in three groups: (i) those with pyrrolidine or analogs as P1 fragment with α-aminoacyl linker, (ii) structures with trifluorophenyl moiety or analogs as P1 fragment with β-aminobutanoyl linker, and (iii) DPP-4 inhibitors with pyrimidine-2,4-dione or analogs as P1' fragment. The structure-activity relationship analysis was performed for those whose cocrystallized structures with the enzyme were published. While inhibitors with pyrrolidine and trifluorophenyl moiety or analogs as P1 fragment bind in a similar way in S1, S2 and S2 extensive domains of the enzyme, the binding mode of pyrimidine-2,4-dione derivatives/analogs differs with additional interactions in S1' and S2' pockets. Three general schemes of fragmented gliptins and gliptin-like structures with the enzyme and protein-ligand interaction fingerprints were made, which might be useful in the creation of DPP-4 inhibitor's design strategies.

Design, synthesis and biological evaluation of novel FXIa inhibitors with 2-phenyl-1H-imidazole-5-carboxamide moiety as P1 fragment.

Factor XIa, as a blood coagulation enzyme, amplifies the generation of the last enzyme thrombin in the blood coagulation cascade. It was proved that direct inhibition of factor XIa could reduce pathologic thrombus formation without an enhanced risk of bleeding. WSJ-557, a nonpurine imidazole-based xanthine oxidase inhibitor in our previous reports, could delay blood coagulation during its animal experiments, which prompted us to investigate its action mechanism. Subsequently, during the exploration of the action mechanism, it was found that WSJ-557 exhibited weak invitro factor XIa binding affinity. Under the guide of molecular modeling, we adopted molecular hybridization strategy to develop novel factor XIa inhibitors with WSJ-557 as an initial compound. This led to the identification of the most potent compound 44g with a Ki value of 0.009μM, which was close to that of BMS-724296 (Ki=0.0015μM). Additionally, serine protease selectivity study indicated that compound 44g display a desired selectivity, more 400-fold than those of thrombin, factor VIIa and factor Xa in coagulation cascade. Moreover, enzyme kinetics studies suggested that the representative compound 44g acted as a competitive-type inhibitor for FXIa, and molecular modeling revealed that it could tightly bind to the S1, S1' and S2' pockets of factor XIa. Furthermore, invivo efficacy in the rabbit arteriovenous shunt model suggested that compound 44g demonstrated dose-dependent antithrombotic efficacy. Therefore, these results supported that compound 44g could be a potential and efficacious agent for the treatment of thrombotic diseases.

Coordination pattern and reactivity of two model peptides from porin protein P1

It has been reported that numerous of Fusobacterium nucleatum outer membrane proteins take part in cancerogenesis. Therefore, it is very interesting to study their interactions with metal ions and the ability to produce reactive oxygen species, which may be involved in cancer progression. Since investigations of metal binding to proteins are often based on fragments that contain the metal-binding domains, designing model peptides should be very mindful. As was shown in this paper, very similar protein fragments may behave differentially. Herein, combined potentiometric, spectroscopic, and computational studies were performed to determine metal ion binding by ligands constituting fragments of porin protein P1. Two studied tetrapeptides (Ac–KEHK–NH2 and Ac–EHKA–NH2) that have common EHK motif have different coordination properties and reactivity. Therefore, we should be cautious when transferring the behavior of small peptide fragments to whole protein.

Elevated CO2 alters transgene methylation not only in promoterregion but also in codingregion of Bt rice under different N-fertilizer levels

The earth has been undergoing climate change, especially in recent years, driven by increasing concentration of atmospheric carbon dioxide (CO2) and rising earth-surface temperature, which could reduce N allocation to Bt toxin for transgenic Bt crops (Bt crops), but the N fertilization is considered to be an effective method to enhance the C–N balance in Bt crops in the case of elevated CO2 in future. DNA methylation not only in promoterregion but also in codingregion of transgene plays a critical role in transgene expression regulation and silencing of transgenic crops. Recent research has emphasized the risks of increased transgene silencing of Bacillus thuringiensis (Bt) rice under elevated CO2. In this study, the effects of elevated CO2 (vs. ambient CO2) on exogenous Bt toxins and transgene expression in promoterregion and codingregion of Bt rice during tillering stage (cv. HH1 expressing fused Cry1Ab/Cry1Ac) were evaluated under three nitrogen (N) fertilizer rate (1/4, 1 and 2 N levels). The aboveground and belowground biomass, and foliar Bt protein content of Bt rice were all significantly increased with the augmentation of N-fertilizer. And elevated CO2 significantly increased belowground biomass, total soluble protein content, transgene methylation levels in promoterregion (P1), and in total of promoterregion(P1) and codingregion (P2 + P3) (i.e., P1 + P2 + P3) at 1 N level, and it also increased transgene methylation levels in codingregion (P2), and in total of promoterregion and codingregion (P1 + P2 + P3) at 2 N level. In addition, elevated CO2 decreased foliar Bt protein content at 1 N level. The transgene methylation levels in promoterregion and codingregion were negatively correlated with Bt-transgene expression level. The methylation level of cytosines located at CG sites was higher than those at CHG and CHH sites in P1, P2 and P3 fragments regardless of the CO2 or N-fertilizer level. The correlation of transgene mehtylation in promoterregion with transgene expression is even stronger than that in codingregion. These data indicate that N fertilization supply will increase the Bt toxin content in transgenic Bt rice, especially under elevated CO2.

Synthetic fragment (60–76) of RAGE improves brain mitochondria function in olfactory bulbectomized mice

The receptor for advanced glycation end products (RAGE) is considered to contribute to the pathogenesis of Alzheimer's disease (AD), mediating amyloid beta (Aβ) accumulation, mitochondrial damage, and neuroinflammation. Previously, we have synthesized small peptides corresponding to the fragments (60–76) (P1) and (60–62) (P2) of the RAGE extracellular domain, and have shown that administration of P1 fragment but not P2 results in restoration of the spatial memory and decreases the brain Aβ (1–40) level in olfactory bulbectomized (OBX) mice demonstrating main features of Alzheimer's type neurodegeneration. In the present study, we have investigated the supposed mechanism of the therapeutic efficacy of P1 RAGE fragment and compared it to P2 short fragment. We have found that P1 restored activities of the respiratory chain in the Complexes I and IV in both cortical and hippocampal mitochondria of the OBX mice while P2 had no effect. Besides, fluorescein-labeled analog Flu-P1 bound to Aβ (1–40) and Aβ (1–42) with high affinity (Kd in the nanomolar range) whereas Flu-P2 revealed low affinity with tenfold higher Kd value for Aβ (1–40) and did not bind to Aβ (1–42). However, neither of the peptides had a notable impact on inflammation, estimated as mRNA expression of proinflammatory cytokines in the brain tissues of OBX mice. Taken together, our results suggest that direct Aβ-P1 interaction is one of the molecular events mediating the protection of the mitochondria in OBX animals from Aβ toxic effect. The RAGE fragment P1 would be the soluble decoy for Aβs and serve as a promising therapeutic agent against neurodegeneration accompanied by mitochondrial dysfunction.

Enhanced purification coupled with biophysical analyses shows cross-\u03b2 structure as a core building block for Streptococcus mutans functional amyloids

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-β amyloid structure.

MYC regulates coelomocytes apoptosis by targeting Bax expression in sea cucumber Apostichopus japonicus

Myelocytomatosis viral oncogene (MYC), a multifunctional transcription factor, (TF) exerts various physiological and pathological effects on animals. AjMYC could induce coelomocyte apoptosis in Apostichopus japonicus, but the underlying molecular mechanism remains poorly understood. In this study, the promoter sequence of apoptosis regulator Bcl-2-associated X (Bax) was cloned by genomic walking. The AjBax promoter region spaning 1189 bp, containing several transcription factor binding sites, included four potential E-boxes (−1030 bp to −1019 bp, −785 bp to −774 bp, −570 bp to −559 bp, −100 bp to −89 bp), two P53 binding sites (−439 bp to −430 bp, −845 bp to −836 bp), and one NF-κB site (−191 bp to −182 bp). Transient transfection of EPC cells with 5′–deletion constructs linked to luciferase reporter revealed that the region −1189/+454 contributed importantly to the expression of the AjBax. In addition, the AjBax promoter was induced by LPS, PGN or MAN. The four potential MYC binding sites were cotransfected with AjMYC in EPC cell whether AjMYC could activate AjBax expression as a transcriptional factor. Only P1 (−1189/+454) fragment containing the first MYC binding site transfection increased the luciferase activity by 2.08-fold (p < 0.01) compared with the control. The first MYC binding site −1030/−1019 was essential to induce AjBax transcription. Further functional assay indicated that AjBax was significantly induced by 3.54-fold increase (p < 0.01) after AjMYC overexpression in sea cucumber coelomocytes. All our findings supported that AjMYC could regulate coelomocyte apoptosis by directly targeting AjBax expression in A. japonicus.

Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease.

Basement membranes provide structural support to epithelium, endothelium, muscles, fat cells, Schwann cells, and axons. Basement membranes are multifunctional: they modulate cellular behavior, regulate organogenesis, promote tissue repair, form a barrier to filtration and tumor metastasis, bind growth factors, and mediate angiogenesis. All basement membranes contain type IV collagen (Col IV), laminin, nidogen, and perlecan. Col IV and laminin self-assemble into two independent supramolecular networks that are linked to nidogen and perlecan to form a morphological discernable basement membrane/basal lamina. The triple helical region, 7S domain and NCI domain of Col IV, laminin and laminin fragment P1 have been evaluated as noninvasive fibrosis biomarkers of alcoholic liver disease, viral hepatitis, and nonalcoholic fatty liver disease. Elevated serum Col IV and laminin are related to degrees of fibrosis and severity of hepatitis, and may reflect hepatic basement membrane metabolism. But the serum assays have not been linked to disclosing the anatomical sites and lobular distribution of perisinusoidal basement membrane formation in the liver. Hepatic sinusoids normally lack a basement membrane, although Col IV is a normal matrix component of the space of Disse. In liver disease, laminin deposits in the space of Disse and codistributes with Col IV, forming a perisinusoidal basement membrane. Concomitantly, the sinusoidal endothelium loses its fenestrae and is transformed into vascular type endothelium. These changes lead to capillarization of hepatic sinusoids, a significant pathology that impairs hepatic function. Accordingly, codistribution of Col IV and laminin serves as histochemical marker of perisinusoidal basement membrane formation in liver disease. Anat Rec, 300:1371-1390, 2017. © 2017 Wiley Periodicals, Inc.

Selective [3+1] Fragmentations of P4 by "P" Transfer from a Lewis Acid Stabilized [RP4 ]- Butterfly Anion.

Two [3+1] fragmentations of the Lewis acid stabilized bicyclo[1.1.0]tetraphosphabutanide Li[Mes*P4 ⋅ BPh3 ] (Mes*=2,4,6-tBu3 C6 H2 ) are reported. The reactions proceed by extrusion of a P1 fragment, induced by either an imidazolium salt or phenylisocyanate, with release of the transient triphosphirene Mes*P3 , which was isolated as a dimer and trapped by 1,3-cyclohexadiene as a Diels-Alder adduct. DFT quantum chemical computations were used to delineate the reaction mechanisms. These unprecedented pathways grant access to both P1 - and P3 -containing organophosphorus compounds in two simple steps from white phosphorus.

Selective [3+1] Fragmentations of P4 by “P” Transfer from a Lewis Acid Stabilized [RP4]− Butterfly Anion

AbstractTwo [3+1] fragmentations of the Lewis acid stabilized bicyclo[1.1.0]tetraphosphabutanide Li[Mes*P4⋅ BPh3] (Mes*=2,4,6‐tBu3C6H2) are reported. The reactions proceed by extrusion of a P1 fragment, induced by either an imidazolium salt or phenylisocyanate, with release of the transient triphosphirene Mes*P3, which was isolated as a dimer and trapped by 1,3‐cyclohexadiene as a Diels–Alder adduct. DFT quantum chemical computations were used to delineate the reaction mechanisms. These unprecedented pathways grant access to both P1‐ and P3‐containing organophosphorus compounds in two simple steps from white phosphorus.

Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface

P1 (antigen I/II) is a sucrose-independent adhesin of Streptococcus mutans whose functional architecture on the cell surface is not fully understood. S. mutans cells subjected to mechanical extraction were significantly diminished in adherence to immobilized salivary agglutinin but remained immunoreactive and were readily aggregated by fluid-phase salivary agglutinin. Bacterial adherence was restored by incubation of postextracted cells with P1 fragments that contain each of the two known adhesive domains. In contrast to untreated cells, glutaraldehyde-treated bacteria gained reactivity with anti-C-terminal monoclonal antibodies (mAbs), whereas epitopes recognized by mAbs against other portions of the molecule were masked. Surface plasmon resonance experiments demonstrated the ability of apical and C-terminal fragments of P1 to interact. Binding of several different anti-P1 mAbs to unfixed cells triggered release of a C-terminal fragment from the bacterial surface, suggesting a novel mechanism of action of certain adherence-inhibiting antibodies. We also used atomic force microscopy-based single molecule force spectroscopy with tips bearing various mAbs to elucidate the spatial organization and orientation of P1 on living bacteria. The similar rupture lengths detected using mAbs against the head and C-terminal regions, which are widely separated in the tertiary structure, suggest a higher order architecture in which these domains are in close proximity on the cell surface. Taken together, our results suggest a supramolecular organization in which additional P1 polypeptides, including the C-terminal segment originally identified as antigen II, associate with covalently attached P1 to form the functional adhesive layer.

Creating novel activated factor XI inhibitors through fragment based lead generation and structure aided drug design.

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.

Characterization of anti-P monoclonal antibodies directed against the ribosomal protein-RNA complex antigen and produced using Murphy Roths large autoimmune-prone mice.

Autoantibodies, including anti-ribosomal P proteins (anti-P), are thought to be produced by an antigen-driven immune response in systemic lupus erythematosus (SLE). To test this hypothesis, we reconstituted the ribosomal antigenic complex in vitro using human P0, phosphorylated P1 and P2 and a 28S rRNA fragment covering the P0 binding site, and immunized Murphy Roths large (MRL)/lrp lupus mice with this complex without any added adjuvant to generate anti-P antibodies. Using hybridoma technology, we subsequently obtained 34 clones, each producing an anti-P monoclonal antibody (mAb) that recognized the conserved C-terminal tail sequence common to all three P proteins. We also obtained two P0-specific monoclonal antibodies, but no antibody specific to P1, P2 or rRNA fragment. Two types of mAbs were found among these anti-P antibodies: one type (e.g. 9D5) reacted more strongly with the phosphorylated P1 and P2 than that with their non-phosphorylated forms, whereas the other type (e.g. 4H11) reacted equally with both phosphorylated and non-phosphorylated forms of P1/P2. Both 9D5 and 4H11 inhibited the ribosome/eukaryotic elongation factor-2 (eEF-2)-coupled guanosine triphosphate (GTP)ase activity. However, preincubation with a synthetic peptide corresponding to the C-terminal sequence common to all three P proteins, but not the peptide that lacked the last three C-terminal amino acids, mostly prevented the mAb-induced inhibition of GTPase activity. Thus, at least two types of anti-P were produced preferentially following the immunization of MRL mice with the reconstituted antigenic complex. Presence of multiple copies of the C-termini, particularly that of the last three C-terminal amino acid residues, in the antigenic complex appears to contribute to the immunogenic stimulus.

Immunogenicity and in vitro and in vivo protective effects of antibodies targeting a recombinant form of the Streptococcus mutans P1 surface protein.

Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1(39-512)), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1(39-512) in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1(39-512) preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1(39-512)-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1(39-512) antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1(39-512) as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.

Isolation and identification of age-related DNA methylation markers for forensic age-prediction.

Age-prediction is an important part of forensic science. There is no available method of individual age-prediction for general forensic biological samples at crime scenes. Accumulating evidence indicates that aging resembles a developmentally regulated process tightly controlled by specific age-associated methylation exists in human genome. This study isolated and identified eight gene fragments in which the degree of cytosine methylation is significantly correlated with age in blood of 40 donors. Furthermore, we validated two CpG sites of each gene fragment and replicated our results in a general population sample of 40 males and 25 females with a wide age-range (11-72 years). The methylation of these fragments is linear with age over a range of six decades (Fragment P1 (r=-0.64), P2 (r=-0.58), P3 (r=-0.79), R1 (r=0.82), R2 (r=0.63), R3 (r=0.59), R4 (r=0.63) and R5 (r=0.62)). Using average methylation of two CpG sites from each fragment, we built a regression model that explained 95% of the variance in age and is able to predict the age of an individual with great accuracy (R(2)=0.918). The predicted values are highly correlated with the observed age in the sample (r=0.91). This study implicates that DNA methylation will be an available biological marker of age-prediction. Furthermore, measurement of relevant sites in the genome could be a tool in routine forensic screening to predict age of biological samples.