The structure was determined for the capsular polysaccharide (CPS) isolated from a swarming strain of Proteus vulgaris, CP2-96, which was obtained from the spleen of an infected mouse. The CPS was extracted from the cell pellet by hot water, precipitated with ethanol, and further purified by gel-permeation chromatography. The structure was established by glycosyl composition and linkage analyses, and by NMR spectroscopy. The sequence of the glycosyl residues was determined by a NOESY experiment. The CPS is composed of a tetrasaccharide repeating unit with the following structure: OAc [symbol: see text] 4 -->4)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->2)-alpha-D-Glcp-(1-->4)-al pha- D-GlcpA-(1->>.

BACKGROUND

Culture-negative periprosthetic joint infection (PJI) is very intractable when dealing with an infected total knee arthroplasty (TKA) patient. Two-stage revision has been proved to be a reliable solution for PJI patients. Whether it is still credible for culture-negative infected patients remains uncertain.

METHODS

Our group retrospectively reviewed all total knee revision patients from January 2003 to January 2014, 145 PJI patients were diagnosed as infection with the PJI diagnostic criteria and 129 patients were successfully followed. As different treating strategies were utilized, these patients were divided into culture-negative (18 cases, CN) group, culture-positive with one-stage revision group (CP1, 21 cases) and culture-positive with two-stage revision group (CP2, 87 cases) groups. The CN group and CP2 group underwent two-stage revision with antibiotic loaded cement spacers and intravenous antibiotics, CP1 group received one-stage revision. All the culture results and relevant medical records were thoroughly reviewed.

RESULTS

The mean follow-up time was 59.5 ± 32.3 months (range 12-158 months). The culture-negative rate was 14.2%. The overall infection control rate was 92.12%. Infection recurrence was observed in two cases in CP1 group (9.09%), six cases in CP2 group (6.90%) and two cases in CN group (11.1%). The reinfection rate of culture-negative patients and culture-positive patients was 7.34% and 11.1% with no significant difference (p = 0.94). No statistically difference was observed between CP2 group and CN group (p = 0.90). No Spacer fracture or dislocation was observed.

CONCLUSIONS

With combined or broad spectrum antibiotics, two-stage revision showed comparable outcome when treating culture-negative infected TKA patients at five-year follow-up.

Acupuncture can have instant and sustained effects, however, its mechanisms of action are still unclear. Here, we investigated the sustained effect of acupuncture by evaluating centrality changes in resting-state functional magnetic resonance imaging after manually stimulating the acupuncture point ST36 at the lower leg or two control point locations (CP1 same dermatome, CP2 different dermatome). Data from a previously published experiment evaluating instant BOLD effects and S2-seed-based resting state connectivity was re-analyzed using eigenvector centrality mapping and degree centrality mapping. These data-driven methods might add new insights into sustained acupuncture effects on both global and local inter-region connectivity (centrality) by evaluating the summary of connections of every voxel. We found higher centrality in parahippocampal gyrus and middle temporal gyrus after ST36 stimulation in comparison to the two control points. These regions are positively correlated to major hubs of the default mode network, which might be the primary network affected by chronic pain. The stronger integration of both regions within the whole-brain connectome after stimulation of ST36 might be a potential contributor to pain modulation by acupuncture. These findings highlight centrality mapping as a valuable analysis for future imaging studies investigating clinically relevant outcomes associated with physiological response to acupuncture stimulation.

BACKGROUND

NCT01079689, ClinicalTrials.gov.

The aim of this study was to describe and compare findings regarding the prevalence and severity of dental caries when using ICDAS and DMFT/dmft in an epidemiological study with children and their mothers. This cross-sectional study evaluated 150 preschoolers and their mothers. Data were collected with ICDAS and then transformed into DMFT/dmft. ICDAS scores related to caries were analyzed according to three different cut-off-points: CP1 (0-healthy/1-6-caries), CP2 (0-1-healthy/2-6-caries) and CP3 (0-2-healthy/3-6-caries), representing the D/d of DMFT/dmft. ICDAS codes regarding restorations, except sealants, were considered the F/f and the code 97 as the M/m of DMFT/dmft index. Prevalence of caries and its severity with ICDAS were 92%, 84% and 31.3% in children and 97.3%, 96.6% and 80% in adults according to CP1/CP2/CP3, respectively. Admitting CP3 as the standard for data transformation of ICDAS in DMFT/dmft, it was observed that DMFT/dmft index would underestimate 60% of non-cavitated lesions in children and 16.6% in adults. The DMFT/dmft underestimated the presence of disease to disregard non-cavitated lesions for the pediatric population evaluated. The choice of which is the best index for epidemiological surveys will depend on the purpose of the research and the target population: if it is to estimate the needs of the population to determine clinical care in children and adults, the DMFT/dmft may be sufficient. However, if the objective is to have a more comprehensive diagnosis of caries at the population level in order to develop preventive strategies, to halt and reverse the disease, the detection of non-cavitated-lesions becomes important, mainly in young children.

Grainyhead (Grh)/CP2 transcription factors are highly conserved in multicellular organisms as key regulators of epithelial differentiation, organ development and skin barrier formation. In addition, they have been implicated as being tumor suppressors in a variety of human cancers. Despite their physiological importance, little is known about their structure and DNA binding mode. Here, we report the first structural study of mammalian Grh/CP2 factors. Crystal structures of the DNA-binding domains of grainyhead-like (Grhl) 1 and Grhl2 reveal a closely similar conformation with immunoglobulin-like core. Both share a common fold with the tumor suppressor p53, but differ in important structural features. The Grhl1 DNA-binding domain binds duplex DNA containing the consensus recognition element in a dimeric arrangement, supporting parsimonious target-sequence selection through two conserved arginine residues. We elucidate the molecular basis of a cancer-related mutation in Grhl1 involving one of these arginines, which completely abrogates DNA binding in biochemical assays and transcriptional activation of a reporter gene in a human cell line. Thus, our studies establish the structural basis of DNA target-site recognition by Grh transcription factors and reveal how tumor-associated mutations inactivate Grhl proteins. They may serve as points of departure for the structure-based development of Grh/CP2 inhibitors for therapeutic applications.

In this study, polysaccharides extracted from Cyclocarya paliurus leaves were modified to obtain its three acetylated derivatives, Ac-CP1, Ac-CP2, and Ac-CP3. The physicochemical characteristics and antioxidant activities of acetylated derivatives were investigated. The results of chemical and FT-IR spectrum analysis showed differences between acetylated derivatives and native C. paliurus polysaccharide, which revealed that the acetylation were successful. Relative to unmodified polysaccharide, the protein contents of acetylated derivatives decreased, while carbohydrate values increased. The molecular weight (Mw) of acetylated derivatives were approximately 1.05-1.09×10(6)Da and were mainly composed of Ara, Gal, Glc, Man, GalA. Ac-CP1 with relatively low degree of substitution (0.13±0.01) exhibited excellent antioxidant activity in DPPH radical assay (95.21±0.89%), and also had strong chelating activity on β-carotene-linoleic acid assay (34.64±2.07%) at 0.5mg/ml. In addition, scanning electron microscope (SEM) observations suggested that acetylation could change the morphology and structure of polysaccharides from C. paliurus leaves.

In order to alleviate toxic effects of aflatoxins B₁ (AFB₁) and zearalenone (ZEA) on broiler production performance and gut microbiota, three kinds of compound probiotics (CP) were selected. The optimal ratios of Bacillus subtilis, Lactobacillus casei and Candida utilis in broiler diets were 7, 5 and 6 log CFU/g for ZEA biodegradation (CP1); 6, 7 and 7 log CFU/g for AFB₁ biodegradation (CP2); 7, 6 and 7 log CFU/g for ZEA + AFB₁ biodegradation (CP3). A total of 350 1-day-old Ross broilers were randomly divided into 7 groups. Group A was the basal diet, group B-G contained ZEA, AFB₁, ZEA + AFB₁, ZEA + CP1, AFB₁+CP2, ZEA + AFB₁+CP3, respectively. The experiment showed that AFB₁ or AFB₁+ZEA significantly decreased broiler production performance, damaged liver and jejunum, increased mycotoxin residues in broiler body; however, three kinds of compound probiotics additions could alleviate mycotoxin negative effects on the above parameters (p < 0.05). The gut microbiota analysis indicated that AFB₁+ZEA increased jejunal microbial richness, but which were decreased to almost the same level as the control group by CP3 addition. CP3 addition significantly increased jejunal Firmicutes and Lactobacillus aviarius abundances. The correlative analysis showed that gut Lactobacillus aviarius abundance was positively correlated with average daily gain (ADG) of broilers (p < 0.05), while AFB₁+ZEA addition decreased its relative abundance, indicating that CP3 addition increased broiler growth by increasing Lactobacillus aviarius abundance. AFB₁ and ZEA residues in broiler body were negatively correlated with the gut beneficial bacterial abundances (p < 0.01), but positively correlated with the potentially harmful bacterial abundances (p < 0.05), which inferred that CP3 addition could decrease mycotoxin residues through positively regulating gut relative bacterial abundances. In conclusion, compound probiotics could keep gut microbiota stable, degrade mycotoxins, alleviate histological lesions, increase production performance and reduce mycotoxin toxicity for broilers.

Whereas in vision a large amount of information may in theory be extracted from instantaneous images, sound exists only in its temporal extent, and most of its information is contained in the pattern of changes over time. The "echoic memory" is a pre-attentive auditory sensory store in which sounds are apparently retained in full temporal detail for a period of a few seconds. From the long-latency auditory evoked potentials to spectro-temporal modulation of complex harmonic tones, at least two automatic sound analysis processes can be identified whose time constants suggest participation of the echoic memory. When a steady tone changes its pitch or timbre, "change-type" CP1, CN1 and CP2 potentials are maximally recorded near the vertex. These potentials appear to reflect a process concerned with the distribution of sound energy across the frequency spectrum. When, on the other hand, changes occur in the temporal pattern of tones (in which individual pitch changes are occurring at a rate sufficiently rapid for the C-potentials to be refractory), a large mismatch negativity (or MN1) and following positivity (MP2) are generated. The amplitude of these potentials is influenced by the degree of regularity of the pattern, larger responses being generated to a "deviant" tone when the pitch and time of occurrence of the "standards" are fully specified by the preceding pattern. At the sudden cessation of changes, on resumption of a steady pitch, a mismatch response is generated whose latency is determined with high precision (in the order of a few milliseconds) by the anticipated time of the next change, which did not in fact occur. The mismatch process, therefore, functions as spectro-temporal auditory pattern analyser, whose consequences are manifested each time the pattern changes. Since calibration of the passage of time is essential for all conscious and subconscious behaviour, is it possible that some states of unconsciousness may be directly due to disruption of internal "clocks"? Abnormal mismatch potentials may provide a manifestation of a disordered auditory time-sense, sometimes being abolished in comatose patients while the C-potentials and similar responses to the onset of tones are preserved. Both C- and M-potentials were usually found to be preserved, however, in patients who had emerged from coma and were capable of discriminating sounds. Substantially intact responses were also recorded from three patients who were functionally in a "vegetative" state. The C- and M-potentials were once again dissociated in a group of patients with multiple sclerosis, only the mismatch potentials being found to be significantly delayed. This subclinical impairment of a memory-based process responsible for the detection of change in temporal sound patterns may be related to defects in other memory domains such as working memory.

During Dictyostelium development, the differentiation inducing factor (DIF) triggers expression of the prestalk gene ecmB and induces stalk cell differentiation, a form of programmed cell death. The effects of DIF are mediated by a sustained increase in cytosolic Ca2+ levels. The Ca2+ ATPase inhibitor BHQ causes a similar rise in Ca2+ levels and also induces prestalk gene expression. We show here that Ca2+ is a specific intermediate for prestalk gene induction, since BHQ represses transcription of the cAMP-inducible aggregative gene PDE, the postaggregative gene CP2, and the prespore gene D19. The prestalk gene ecmA is also induced by DIF, but induction appears to occur in two steps, which occur within 1 h and after 2 h, respectively. The slow step shows the same kinetics as ecmB induction and similar to ecmB induction, this step is BHQ inducible and requires an initial round of protein synthesis. The fast step does not require protein synthesis and cannot be induced by BHQ. This indicates that in addition to the slow Ca2+-mediated pathway, there is probably a second fast Ca2+-independent signal transduction pathway for DIF.

CP2b activates alpha-globin expression in an erythroid cell-specific manner, through interaction with CP2c and PIAS1. Although CP2a is identical to CP2b except for lacking an exon encoding additional 36 amino acids and has the intrinsic DNA binding and transactivation properties, it does not exert any role in alpha-globin expression. Investigation of subcellular localization of exogenous CP2 proteins revealed that CP2a and CP2b were exclusively localized in the cytosol and nucleus, respectively. The CP2b-specific exon was in charge of the nuclear localization of CP2b. Interestingly, subcellular localization of CP2c was either in the nucleus or cytosol depending on the relative level of CP2a and CP2b although CP2c intrinsically localized in the cytosol in the absence of CP2a/CP2b. Finally, dramatic increment of hemoglobin expression was correlated with nuclear translocation of CP2c during MEL cell differentiation. Our data suggest that CP2b potentiate erythroid cell-specific alpha-globin expression by recruiting CP2c into the nucleus.

(1) GARS-AIRS-GART is an important candidate gene in studies of Down syndrome (DS)-related Alzheimer's disease (AD), due to its chromosomal localization (21q22.1) in the Down syndrome critical region, involvement in de novo purine biosynthesis, and over-expression in DS brain. The aim of this study was to identify factor(s) likely to enhance transcription of GARS-AIRS-GART in DS-related AD. (2) Based on a bio-informatics approach, the PromoterInspector, Promoter Scan II, and EBI toolbox CpG plot software programs were used to identify GARS-AIRS-GART sequences important for gene transcription. Transcription factor binding motifs within these regions were mapped with the help of the MatInspector and TFSEARCH programs. Factors implicated in neurodevelopment or neurodegeneration were the focus of attention, and mining of human (T1Dbase) and murine (GNF) expression databases revealed information on the regional distribution of these factors and their relative abundance vis-a-vis GARS-AIRS-GART. (3) The Leader-binding protein 1-c (LBP-1c/CP2/LSF) emerged as a promising candidate from these studies, as MatInspector and TFSEARCH analyses revealed a total of four CP2 binding sites with potential for functional interaction(s) within the promoter and CpG islands of GARS-AIRS-GART. Furthermore, two of these sites harbor sequences for methylation-sensitive restriction enzymes, which suggest that methylation status may, in part, regulate CP2-mediated transcription of GARS-AIRS-GART. A search of T1Dbase and GNF expression databases reveals co-expression of CP2 and GARS-AIRS-GART in brain regions relevant to DS-related AD. (4) The virtual screen identified CP2/LBP-1c/LSF as a factor that likely mediates enhanced transcription of GARS-AIRS-GART in DS-related AD.

Early gastric carcinoma (GC) is considered to be a curable cancer, as it progresses to the advanced stage following varying durations. Understanding the early stage of GC may provide an insight into its pathogenesis and contribute to reducing the mortality rate of this disease. To investigate the genomic aberrations associated with 22 cases of early GC, high-density microarray comparative genomic hybridization was performed in the present study. The most notable finding was copy number gains (log2 ratio >0.25) on the long arm of chromosome 8, which occurred in 77.3% (17/22) of GC cases, and the delineated minimal common region was 8q22.1-q24.3. More specifically, two amplified (log2 ratio >1) loci in the 8q22.1-q24.3 region were detected in 18.2% (4/22) of GC cases. The first loci covered a region of 102.4-107.9 kb, mapping on 8q22.3-q23.1, and comprised the transcription factor CP2-like 3 gene. The second loci, spanning 128.7-145.7 kb on 8q24.21-q24.3, comprised the representative oncogene of myelocytomatosis. Furthermore, the following possible target genes that were not previously considered to play a pathogenic role in GC were identified: Plasmacytoma variant translocation 1, cysteine/histidine rich 1, kinesin family member C2, forkhead box H1, protein phosphatase 1 regulatory subunit 16A, glutamic-pyruvate transaminase, LOC113655 and RecQ protein-like 4. In the present study, previous findings showing that 8q mutations accumulate early during the multistage pathogenesis of GC were confirmed and expanded upon. The confirmation of previously reported 8q gains and the identification of novel target genes at 8q22.1-q24.3 amplified chromosomal sites should aid in improving our understanding of the molecular mechanisms underlying the tumorigenesis of early GC.

OBJECTIVE

To evaluate the diagnostic performance of novel latex-protein complexes obtained from different antigens of Trypanosoma cruzi through immunoagglutination test using a panel of T. cruzi-positive sera, leishmaniasis-positive sera and negative sera for both parasites.

METHODS

Complexes' behaviour using total parasite homogenate (TPH), two simple recombinant proteins (RP1 and RP5) and two chimeric recombinant proteins (CP1 and CP2) was comparatively evaluated. The area under ROC curves was used as an index of accuracy. Sensitivity, specificity and discrimination efficiency were assessed.

RESULTS

All recombinant antigens showed higher specificity than TPH. The lower specificity of TPH was mainly due to cross-reacting peptides between T. cruzi and Leishmania spp. In turn, all performance indicators were higher for CP1 and CP2 than for RP1 and RP5. The carboxylated latex-CP2 (C2-CP2) complex was able to detect antibodies against T. cruzi. The values of area under ROC curve (0.96), sensitivity (92.3%, 95% CI: 79.4-100.0%) and specificity (84.0%, 95% CI: 67.6-100.0%) indicate that the assay could be used as a screening test.

CONCLUSIONS

The C2-CP2 complex could be an important tool to carry out sero-epidemiological studies.

BACKGROUND

Although metal ceramic fixed restorations are commonly preferred by clinicians, there remain a limited number of studies on how opaque porcelain color is affected by fabrication procedures, such as the number of firings and types of metal alloys.

OBJECTIVE

The purpose of this study was to determine the effects of various types of metal alloys on the color of opaque porcelain after repeated firings.

METHODS

Seven different types of metal ceramic alloys (3 base metals: Metalloy CC, chromium cobalt (B-MCC); Heraenium NA, nickel chromium (B-HNA); Argeloy NP, nickel chromium beryllium (B-ANP); 3 noble metals: Ceradelta, palladium silver (N-CD); Cerapall 2, palladium (N-CP2); V-Delta SF, gold palladium (N-VDSF); and 1 high noble metal: V-Gnathos Plus, gold platinum (HN-GP)) were used to support a 0.1-mm-thick layer of opaque porcelain (IPS d.SIGN Opaquer, shade B1) to determine the metal alloys' effect on the opaque porcelain color after repeated porcelain firings. Opaque porcelain was applied on specimens (16 mm x 1 mm) prepared from each type of alloy. The specimens (n=21) were subjected to 1 opaque firing, 4 consecutive dentin firing cycles, and 1 glaze firing cycle. Delta E values were calculated for all metal alloy groups from opaque firing (control group) to each subsequent firing stage within each tested alloy group. One-way ANOVA and Fisher's least significant difference tests were performed to determine the differences between alloys. In addition, DeltaE values calculated after repeated firings were analyzed by 1-way ANOVA and paired t test, to determine whether repeated dentin firing stages affected the color of opaque porcelain (alpha=.05).

RESULTS

After the first and second dentin firings, the color shift in opaque porcelain was significant for all tested alloy groups (P<.001). The color of opaque porcelain changed significantly after the third dentin firing for all groups except for B-HNA and N-VDSF (P<.001). After the fourth dentin firing, the color of opaque porcelain changed significantly for all tested alloy groups (P=.022 for B-ANP, P=.042 for N-VDSF, and P<.001 for remaining alloys). After glaze firing, the color change in opaque porcelain was significant in all but the N-CP2 group (P=.002 for N-VDSF, P=.014 for HN-GP, and P<.001 for remaining alloys). Delta E values showed that B-MCC after the first dentin firing, N-CD after the second dentin firing and glaze firing, and B-ANP after the third and fourth dentin firings showed significantly different DeltaE values than all remaining test alloys (P<.001).

CONCLUSIONS

Subsequent porcelain firings significantly affected the color of a 0.1-mm-thick layer of opaque porcelain for all alloys tested. After the third and fourth firings, 1 base metal alloy (B-ANP) showed significantly greater color change than the remaining dental alloys when the color difference was compared to baseline. In addition, the color change in a noble alloy (N-CD) was significantly less than that of the other alloys after glaze firing. However, color shifts after repeated dentin firings were imperceptible (DeltaE<2.6) and clinically acceptable (DeltaE<5.5) for each type of alloy.

Multiconfigurational, intermediate valent ground states are established in several methyl-substituted bipyridine complexes of bis(pentamethylcyclopentadienyl)ytterbium, Cp2*Yb (Me(x)-bipy). In contrast to Cp2*Yb(bipy) and other substituted-bipy complexes, the nature of both the ground state and the first excited state are altered by changing the position of the methyl or dimethyl substitutions on the bipyridine rings. In particular, certain substitutions result in multiconfigurational, intermediate valent open-shell singlet states in both the ground state and the first excited state. These conclusions are reached after consideration of single-crystal X-ray diffraction (XRD), the temperature dependence of X-ray absorption near-edge structure (XANES), extended X-ray absorption fine-structure (EXAFS), and magnetic susceptibility data, and are supported by CASSCF-MP2 calculations. These results place the various Cp2*Yb(bipy) complexes in a new tautomeric class, that is, intermediate-valence tautomers.

CRTR-1 is a member of the CP2 family of transcription factors. Unlike other members of the family which are widely expressed, CRTR-1 expression shows specific spatio-temporal regulation. Gene targeting demonstrates that CRTR-1 plays a central role in the maturation and function of the salivary glands and the kidney. CRTR-1 has also recently been identified as a component of the complex transcriptional network that maintains pluripotency in embryonic stem (ES) cells. CRTR-1 was previously shown to be a repressor of transcription. We examine the activity of CRTR-1 in ES and other cells and show that CRTR-1 is generally an activator of transcription and that it modulates the activity of other family members, CP2, NF2d9 and altNF2d9, in a cell specific manner. We also demonstrate that CRTR-1 activity is regulated by sumoylation at a single major site, residue K30. These findings imply that functional redundancy with other family members may mask important roles for CRTR-1 in other tissues, including the blastocyst stage embryo and embryonic stem cells.

Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation.

The activity of peptidyl-tRNALys-CpCp2'dA was measured in an in vitro poly(A)-dependent polypeptide synthesizing system derived from Escherichia coli. It has already been shown that Lys-tRNALys-CpCp2'dA is active as an acceptor and Ac2-Lys-tRNALys-Cp2'dA can donate its peptidyl residue but that the overall poly(A)-dependent synthesis of polylysine does not take place with Lys-tRNALys-CpCp2'dA [Wagner, T., Cramer, F., & Sprinzl, M. (1982) Biochemistry 21, 1521-1529]. This is due to the efficient inhibition of the EF-G-dependent translocation of the peptidyl-tRNA CpCp2'dA from the ribosomal A to the ribosomal P site. In addition, the EF-G-dependent release of the deacylated tRNALys-CpCp2'dA from the ribosomes is also inhibited. The action of the elongation factor G or some other ribosomal component participating in the translocation process requires the presence of the 2'-hydroxyl group on the terminal adenosine of tRNA. If this hydroxyl group is not present on the tRNA, the ribosomes remain locked in their pretranslocational state.

Although ubiquitously expressed, the transcriptional factor CP2 also exhibits some tissue- or stage-specific activation toward certain genes such as globin in red blood cells and interleukin-4 in T helper cells. Because this specificity may be achieved by interaction with other proteins, we screened a peptide display library and identified four consensus motifs in numerous CP2-binding peptides: HXPR, PHL, ASR and PXHXH. Protein-database searching revealed that RE-1 silencing factor (REST), Yin-Yang1 (YY1) and five other proteins have one or two of these CP2-binding motifs. Glutathione S-transferase pull-down and coimmunoprecipitation assays showed that two HXPR motif-containing proteins REST and YY1 indeed were able to bind CP2. Importantly, this binding to CP2 was almost abolished when a double amino acid substitution was made on the HXPR sequence of REST and YY1 proteins. The suppressing effect of YY1 on CP2's transcriptional activity was lost by this point mutation on the HXPR sequence of YY1 and reduced by an HXPR-containing peptide, further supporting the interaction between CP2 and YY1 via the HXPR sequence. Mapping the sites on CP2 for interaction with the four distinct CP2-binding motifs revealed at least three different regions on CP2. This suggests that CP2 recognizes several distinct binding motifs by virtue of employing different regions, thus being able to interact with and regulate many cellular partners.

Expression of the Dictyostelium discoideum pst-cath (CP2) gene is transcriptionally regulated during multicellular development, and the gene is inducible in competent single cells following administration of exogenous cAMP. The 5' flanking region of pst-cath (CP2) that extends from -313 to the Cap site (+1) has previously been shown to contain sufficient cis-acting regulatory elements for proper developmental and cAMP-inducible expression of a foreign gene [Datta and Firtel, 1987, Mol Cell Biol 7:149-159]. The -283 to -201 region includes two exceptional "G-boxes" centered at -233 and -217 respectively, and this approximately 80 bp region is essential for basal as well as regulated expression of the pst-cath (CP2) gene. Here we summarize results obtained from a detailed analysis of a series of linker-scanner mutants and mutants that carry small internal deletions within the essential 80-bp region. Insertion of a synthetic oligonucleotide that includes the downstream G-box is demonstrated to rescue a low level of cAMP-inducible expression following insertion into cassette mutants. The effect of introducing a change in the relative spacing between regulatory elements has also been investigated. We have analyzed nuclear extracts for the presence of DNA-binding proteins that interact specifically with the pst-cath (CP2) regulatory region and identified two such putative trans-acting factors: 1) the AT-factor that is observed within a few hours following the onset of starvation and that binds tightly to stretches of alternating adenine-thymine residues (poly(dA-dT]; and 2) the AG-factor that is present in nuclear extracts of aggregated cells. Competition studies have demonstrated significant differences in the affinity that characterizes the binding of the two factors to G-box-containing sequences. The binding specificities of these DNA-binding proteins have been analyzed using gel mobility-shift and DNaseI footprinting assays.

Accumulation of β-amyloid protein (Aβ) is one of the most important pathological features of Alzheimer's disease. Although Aβ induces neurodegeneration in the cortex and hippocampus through several molecular mechanisms, few studies have evaluated the modulation of transcription factors during Aβ-induced neurotoxicity. Therefore, in this study, we investigated the transcriptional activity of transcription factor CP2 in neuronal damage mediated by Aβ (Aβ(1-42) and Aβ(25-35) ). An unbiased motif search of the transferrin promoter region showed that CP2 binds to the transferrin promoter, an iron-regulating protein, and regulates transferrin transcription. Ectopic expression of CP2 led to increased transferrin expression at both the mRNA and protein levels, whereas knockdown of CP2 down-regulated transferrin mRNA and protein expression. Moreover, CP2 trans-activated transcription of a transferrin reporter gene. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay showed that CP2 binds to the transferrin promoter region. Furthermore, the binding affinity of CP2 to the transferrin promoter was regulated by Aβ, as Aβ (Aβ(1-42) and Aβ(25-35) ) markedly increased the binding affinity of CP2 for the transferrin promoter. Taken together, these results suggest that CP2 contributes to the pathogenesis of Alzheimer's disease by inducing transferrin expression via up-regulating its transcription.

The phytochrome-encoding gene Cerpu;PHY;2 (CP2) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P phi B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [Pr]/far-red-absorbing phytochrome [Pfr]) for the PCB adduct, and 659/724 nm for the P phi B-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that P phi B is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophytochrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the Pr->>Pfr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I700), decaying with a time constant of approximately 110 microseconds. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the Pfr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.

The leaves of Artocarpus tonkinensis are used in Vietnamese traditional medicine for treatment of arthritis, and the compound maesopsin 4-O-β-D-glucoside (TAT-2), isolated from them, inhibits the proliferation of activated T cells. Our goal was to test the anti-proliferative activity of TAT-2 on the T-cell leukemia, Jurkat, and on the acute myeloid leukemia, OCI-AML. TAT-2 inhibited the growth of OCI-AML (and additional acute myeloid leukemia cells) but not Jurkat cells. Growth inhibition was shown to be due to inhibition of proliferation rather than increase in cell death. Analysis of cytokine release showed that TAT-2 stimulated the release of TGF-β, yet TGF-β neutralization did not reverse the maesopsin-dependent effect. Gene expression profiling determined that maesopsin modulated 19 identifiable genes. Transcription factor CP2 was the gene most significantly modulated. Real-time PCR validated that up-regulation of sulphiredoxin 1 homolog (SRXN1), hemeoxygenase 1 (HMOX1), and breast carcinoma amplified sequence 3 (BCAS3) were consistently modulated.