The transmembrane topology of presenilins is still the subject of debate despite many experimental topology studies using antibodies or gene fusions. The results from these studies are partly contradictory and consequently several topology models have been proposed. Studies of presenilin-interacting proteins have produced further contradiction, primarily regarding the location of the C-terminus. It is thus impossible to produce a topology model that agrees with all published data on presenilin. We have analyzed the presenilin topology through computational sequence analysis of the presenilin family and the homologous presenilin-like protein family. Members of these families are intramembrane-cleaving aspartyl proteases. Although the overall sequence homology between the two families is low, they share the conserved putative active site residues and the conserved 'PAL' motif. Therefore, the topology model for the presenilin-like proteins can give some clues about the presenilin topology. Here we propose a novel nine-transmembrane topology with the C-terminus in the extracytosolic space. This model has strong support from published data on gamma-secretase function and presenilin topology. Contrary to most presenilin topology models, we show that hydrophobic region X is probably a transmembrane segment. Consequently, the C-terminus would be located in the extracytosolic space. However, the last C-terminal amino acids are relatively hydrophobic and in conjunction with existing experimental data we cannot exclude the possibility that the extreme C-terminus could be buried within the gamma-secretase complex. This might explain the difficulties in obtaining consistent experimental evidence regarding the location of the C-terminal region of presenilin.

BACKGROUND

Triple therapy with proton pump inhibitor, clarithromycin and amoxicillin and, in the event of eradication failure, quadruple therapy with proton pump inhibitor, bismuth, tetracycline and metronidazole have been proposed in Maastricht as the optimal sequential treatment of Helicobacter pylori infection.

OBJECTIVE

To compare two second-line regimens with quadruple therapy.

METHODS

One hundred and eighty patients with a previous failed course of standard therapy were randomly given one of the following 7-day treatments: ranitidine bismuth citrate 400 mg b.d. plus amoxicillin 1 g b.d. and tinidazole 500 mg b.d. (RBCAT), pantoprazole 40 mg b.d. plus amoxicillin 1 g b.d. and levofloxacin 500 mg/day (PAL) and pantoprazole 40 mg b.d., bismuth citrate 240 mg b.d., tetracycline 500 mg q.d.s. and metronidazole 500 mg b.d. (PBTM). The eradication rate was assessed by 13C-urea breath test. Side-effects and compliance were evaluated by a standardized questionnaire and by counting returned medication.

RESULTS

The RBCAT, PAL and PBTM groups achieved mean intention-to-treat eradication rates of 85%, 63% and 83%, respectively (P<0.05 for PAL vs. either RBCAT or PBTM). Compliance was optimal in all patients, although side-effects were more commonly observed in the PBTM group than in the other two patient groups (P<0.0001).

CONCLUSIONS

Both RBCAT and PBTM can be used as second-line therapies. Conversely, PAL did not achieve satisfactory eradication rates.

To better understand the molecular responses of plants to arbuscular mycorrhizal (AM) fungi, we analyzed the differential gene expression patterns of Lotus japonicus, a model legume, with the aid of a large-scale cDNA macroarray. Experiments were carried out considering the effects of contaminating microorganisms in the soil inoculants. When the colonization by AM fungi, i.e. Glomus mosseae and Gigaspora margarita, was well established, four cysteine protease genes were induced. In situ hybridization revealed that these cysteine protease genes were specifically expressed in arbuscule-containing inner cortical cells of AM roots. On the other hand, phenylpropanoid biosynthesis-related genes for phenylalanine ammonia-lyase (PAL), chalcone synthase, etc. were repressed in the later stage, although they were moderately up-regulated on the initial association with the AM fungus. Real-time RT-PCR experiments supported the array experiments. To further confirm the characteristic expression, a PAL promoter was fused with a reporter gene and introduced into L. japonicus, and then the transformants were grown with a commercial inoculum of G. mosseae. The reporter activity was augmented throughout the roots due to the presence of contaminating microorganisms in the inoculum. Interestingly, G. mosseae only colonized where the reporter activity was low. Comparison of the transcriptome profiles of AM roots and nitrogen-fixing root nodules formed with Mesorhizobium loti indicated that the PAL genes and other phenylpropanoid biosynthesis-related genes were similarly repressed in the two organs.

Structure-based protein engineering coupled with chemical modifications (e.g., pegylation) is a powerful combination to significantly improve the development of proteins as therapeutic agents. As a test case, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was selected for enzyme replacement therapy in phenylketonuria [C.R. Scriver, S. Kaufman, Hyperphenylalaninemia:phenylalanine Hydroxylase Deficiency. The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill, New York, 2001, Chapter 77], an inherited metabolic disorder (OMIM 261600) causing mental retardation due to deficiency of the enzyme l-phenylalanine hydroxylase (EC 1.14.16.1). Previous in vivo studies of recombinant PAL demonstrated a lowering of blood l-phenylalanine levels; yet, the metabolic effect was not sustained due to protein degradation and immunogenicity [C.N. Sarkissian, Z. Shao, F. Blain, R. Peevers, H. Su, R. Heft, T.M. Chang, C.R. Scriver, A different approach to treatment of phenylketonuria:phenylalanine degradation with recombinant phenylalanine ammonia lyase, Proc. Natl. Acad. Sci. USA 96 (1999) 2339; J.A. Hoskins, G. Jack, H.E. Wade, R.J. Peiris, E.C. Wright, D.J. Starr, J. Stern, Enzymatic control of phenylalanine intake in phenylketonuria, Lancet 1 (1980) 392; C.M. Ambrus, S. Anthone, C. Horvath, K. Kalghatgi, A.S. Lele, G. Eapen, J.L. Ambrus, A.J. Ryan, P. Li, Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria, Ann. Intern. Med. 106 (1987) 531]. Here, we report the 1.6A three-dimensional structure of Rhodosporidium toruloides PAL, structure-based molecular engineering, pegylation of PAL, as well as in vitro and in vivo PKU mouse model studies on pegylated PAL formulations. Our results show that pegylation of R. toruloides PAL leads to promising therapeutic efficacy after subcutaneous injection by enhancing the in vivo activity, lowering plasma phenylalanine, and leading to reduced immunogenicity. The three-dimensional structure of PAL provides a basis for understanding the properties of pegylated forms of PAL and strategies for structure-based re-engineering of PAL for PKU treatment.

Antagonists of luteinizing hormone-releasing hormone (LH-RH), unlike the LH-RH agonists, suppress gonadotropins and sex steroid secretion immediately after administration, without initial stimulatory effects. [Ac-D-Nal(2)1,D-Ph(4Cl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-R H (SB-75; Cetrorelix) is a modern, potent antagonistic analog of LH-RH. In this study, the binding characteristics of receptors for LH-RH in membrane fractions from rat anterior pituitaries were investigated after a single injection of Cetrorelix at a dose of 100 microg per rat. To determine whether the treatment with Cetrorelix can affect the concentration of measurable LH-RH binding sites, we applied an in vitro method to desaturate LH-RH receptors by chaotropic agents such as manganous chloride (MnCl2) and ammonium thiocyanate (NH4SCN). Our results show that the percentages of occupied LH-RH receptors at 1, 3, and 6 h after administration of Cetrorelix were approximately 28%, 14%, and 10%, respectively, of total receptors. At later time intervals, we could not detect occupied LH-RH binding sites. Ligand competition assays, following in vitro desaturation, demonstrated that rat pituitary LH-RH receptors were significantly (P < 0.01) down-regulated for at least 72 h after administration of Cetrorelix. The lowest receptor concentration was found 3-6 h after Cetrorelix treatment and a recovery in receptor number began within approximately 24 h. The down-regulation of LH-RH binding sites induced by Cetrorelix was accompanied by serum LH and testosterone suppression. Higher LH-RH receptor concentrations coincided with elevated serum hormone levels at later time intervals. Our results indicate that administration of LH-RH antagonist Cetrorelix produces a marked down-regulation of pituitary receptors for LH-RH and not merely an occupancy of binding sites.

Screening of a tobacco (Nicotiana tabacum) cDNA library resulted in the isolation of a clone encoding the bZIP transcription factor BZI-1. With respect to amino acid sequence, conservation of protein domains, genomic exon-intron structure and expression pattern, BZI-1 is closely related to CPRF2, OHP1/2, BLZ1 and REB, a group of bZIP proteins which have been described in a number of dicot and monocot species. BZI-1 exhibits the characteristics of a transcription factor. It binds to G-box and C-box cis-elements in vitro, it is localised in the nucleus, and the N-terminal region of BZI-1 functions as an activation domain in both yeast and plant cells. Since BZI-1-related transcription factors have been isolated from dicots by in vitro binding to G-box elements in the chalcone synthase ( CHS) promoter, it has been suggested that phenylpropanoid pathway genes, such as CHS and PAL (phenylalanine ammonia-lyase), are targets of these proteins in vivo. However, after infection with Pseudomonas syringae or Tobacco Mosaic Virus, no changes in pathogen-induced PAL expression were observed in transgenic plants expressing increased levels of BZI-1 or a dominant negative form of the protein, BZI-1-DeltaN. In contrast to the tissue-specific expression of CHS and PAL, BZI-1 was found to be ubiquitously expressed in tobacco plants. Furthermore, no changes in the tissue-specific expression of PAL or CHS were observed in plants that were transgenic for BZI-1-DeltaN. Expression of a VP16-BZI-1 fusion protein would be expected to result in constitutive activation of the BZI-1 target genes. However, tetracycline-dependent expression of a VP16-BZI-1 protein in tobacco plants did not result in activation of CHS or PAL. On the basis of these data, we conclude that the phenylpropanoid pathway genes analysed are not targets of BZI-1 in vivo. Thus, the pattern of in vitro DNA binding of transcription factors need not always reflect their in vivo function.

The palmitoyl pentapeptide palmitoyl-lysine-threonine-threonine-lysine-serine (pal-KTTKS) is a synthetic material that was designed as a topical agent to stimulate collagen production and thus provide a skin anti-wrinkle benefit. To determine if pal-KTTKS is effective, the clinical study reported here was conducted. Caucasian female subjects (n = 93, aged 35-55) participated in a 12-week, double-blind, placebo-controlled, split-face, left-right randomized clinical study assessing two topical products: moisturizer control product vs. the same moisturizer product containing 3 ppm pal-KTTKS. Pal-KTTKS was well tolerated by the skin and provided significant improvement vs. placebo control for reduction in wrinkles/fine lines by both quantitative technical and expert grader image analysis. In self-assessments, subjects also reported significant fine line/wrinkle improvements and noted directional effects for other facial improvement parameters.

OBJECTIVE

The present study investigated the relationship between parental refractive error and myopia progression in their offspring and the interaction between parental ametropia and the effects of wearing progressive-addition (PALs) or single-vision (SVLs) lenses on the progression of myopia in children enrolled in the Correction of Myopia Evaluation Trial (COMET).

METHODS

The progression of myopia in a subset of COMET children (N= 232; 49% of initial group) was defined as the difference in mean spherical equivalent refraction of both eyes obtained by cycloplegic autorefraction between the baseline and 5-year visit. Parental refractions were obtained by noncycloplegic autorefraction (81%) or from recent eye examination records (19%).

RESULTS

The number of myopic parents (mean spherical equivalent refraction </=-0.75 D) was directly related to myopia progression among children wearing SVLs: myopia in children with no (zero) myopic parents progressed (mean +/- SE) -1.81 +/- 0.18 D and with one myopic parent, -2.04 +/- 0.13) D; these amounts were significantly less than the progression of children with two myopic parents (-2.59 +/- 0.19 D). In the PAL group, progression was not significantly related to the number of myopic parents and was -2.01 D overall. Among children with two myopic parents, progression was -2.00 D in the PAL group, significantly less than the progression of children wearing SVLs (P = 0.03). Among children with zero or one myopic parent, progression did not differ significantly between the lens groups. When the data were adjusted for covariates, the interaction between treatment effect and number of myopic parents was significant (P = 0.01). Over the 5-year study period, axial length increased 0.93 +/- 0.07 mm in children with two myopic parents wearing <em>PALs</em> and 1.18 +/- 0.07 mm in children with two myopic parents wearing SVLs (P = 0.01). The axial length increase in children wearing SVLs and with two myopic parents was significantly more than the 0.89 +/- 0.07 mm increase in children wearing SVLs and with zero myopic parents (P = 0.015).

CONCLUSIONS

Parental refraction was related to myopia progression and changes in axial length. Among COMET children with two myopic parents, myopia progression and increases in axial length were slower in the group wearing PALs than in those wearing SVLs, by a statistically significant but clinically minor amount. Because this study was ancillary to COMET and the present analyses are based on a subset of participants, conclusions must be regarded as suggestive.

BACKGROUND

Parenting programs have been shown to work when delivered to motivated ethnic majority parents in demonstration projects, but comparatively little is known about their impact when delivered to high-risk, multi-ethnic populations by routine local services.

METHODS

The Primary Age Learning Skills (PALS) trial was a randomized controlled trial of an evidence-based parenting-group program that targeted the parent-child relationship and child literacy. Parents of 174 children were selected from a population of 672 5- and 6-year-olds attending four primary schools in a high-risk, ethnically diverse, inner-city area. Eighty-eight children were allocated to the Incredible Years preventive program plus a shortened six-week version of the SPOKES literacy program, delivered by local services; 86 to usual community services; 152/174 (87%) of families were successfully followed up. Parent-child relationship quality and child behavior were measured using direct observation and parent interview; child reading was assessed psychometrically.

RESULTS

Two-thirds (58/89) of those offered the parenting program attended at least one session, with similar enrollment rates across the Black African, African-Caribbean, White-British and Other ethnic groups. Mean attendance was four relationship-building sessions and one literacy-development session. Satisfaction questionnaires were completed by 43/58 starters; 93% said they were well or extremely satisfied, with equally high rates across ethnic groups. At follow-up after one year, those allocated to the intervention showed significant improvements in the parent-child relationship on observation and at interview compared to controls; effects were similar across all ethnic groups. However, child behavior problems and reading did not improve. The cost was £1,343 ($2,100) per child.

CONCLUSIONS

Programs can be organized to be engaging and effective in improving parenting among high-risk, multi-ethnic communities, which is of considerable value. To also be cost-effective in achieving child changes may require a set-up that enables parents to attend more sessions and/or an exclusive focus on children with clinically significant behavior problems.

Immunohistochemical studies were performed in thirty skin biopsies from patients with Kaposi's sarcoma, who did and did not have the acquired immune deficiency syndrome (AIDS). Tumour histogenesis was rigorously tested using a battery of endothelial cell markers, which included two new monoclonal antibodies, EN4 and PAL E. These are both specific for endothelial cells and can be visualised in appropriately fixed paraffin embedded tissue. Whereas EN4 labels all endothelial cells, PAL E is negative in endothelium of lymphatic derivation. Lectin binding with Ulex europaeus agglutinin 1 (UEA-1) and the presence of factor VIII related antigen (FVIIIRA) and laminin were also examined. In nodular lesions of Kaposi's sarcoma the spindle cell areas were positive with EN4 and UEA-1, negative with PAL E, and showed focal staining for FVIIIRA and laminin. These results confirm that the tumour is of endothelial cell origin. Six patch stage lesions showed a network of angulated spaces, lined by cells that were positive with EN4 and UEA-1, negative with PAL E and anti-FVIIIRA, and showed only weak staining for laminin. This pattern was observed in both AIDS and non-AIDS related cases and strongly favours a lymphatic derivation for the tumour. This has important implications as it suggests that lymphatic endothelium may have special characteristics that lead to neoplastic transformation in patients with retrovirus infection.

We have shown that the domain V of bacterial 23 S rRNA could fold denatured proteins to their active state. This segment of 23 S rRNA could further be split into two parts. One part containing mainly the central loop of domain V could bind denatured human carbonic anhydrase I stably. This association could be reversed by adding the other part of domain V. The released enzyme was directed in such a way by the central loop of domain V that it could now fold by itself to active form. This agrees with our earlier observation that proteins fold within the cell posttranslationally, a process that is completed after release of the newly synthesized polypeptide from the ribosome (Chattopadhyay, S., Pal, S., Chandra, S., Sarkar, D., and DasGupta, C. (1999) Biochim. Biophys. Acta 1429, 293-298).

The sequence of the S-adenosyl-L-methionine:trans-caffeoyl-CoA O-methyltransferase (CCoAOMT, EC2.1.1.104) gene, including the 5'-flanking region of 5 kb, was determined from parsley (Petroselinum crispum) plants. The enzyme appears to be encoded by one or two genes, and the ORF is arranged in five exons spaced by introns from 107 to 263 bp in length. The genomic sequence matches the ORF of the cDNA previously reported from elicited parsley cell cultures, showing only three base changes that do not affect the enzyme polypeptide sequence. S1 nuclease protection assays and primer extension analyses with genomic and cDNA templates revealed the transcription start site 67 bp upstream of the translation start codon, indicating a shorter 5'-UTR than reported previously for the transcript. Promoter regulatory consensus elements such as two 'CAAT' boxes and one 'TATA' box were identified at -196, -127 and -31, respectively, relative to the transcription start site, and an SV 40-like enhancer element is located 347 bp upstream. Most notably, three putative cis-regulatory elements were recognized by sequence alignments, which represent motifs recurring in the promoters of several genes of the stress-inducible phenylpropanoid pathway (boxes P, A and L). Transient expression assays with a set of 5'-truncated promoter-GUS fusions show that significant promoter activity is retained in a 354 bp promoter fragment. In vitro DNase 1 footprint experiments and electrophoretic mobilty shift assays (EMSA) identified in this fragment a unique sequence motif with elicitor-inducible trans-factor binding activity, which was unrelated to boxes P, A, or L. This novel cis-regulatory element, designated box E, appears to be conserved in the TATA-proximal regions of other stress-inducible phenylpropanoid genes, and in vitro binding of nuclear protein was confirmed in EMSA assays for such an element from the PAL-1 promoter (-54 to -45). Moreover, the deletion of box E reduced the activity and erased the elicitor-responsiveness of the CCoAOMT promoter in transient expression assays. The results corroborate the proposed physiological function of CCoAOMT in elicited plant cells and may shed new light on the sequential action of trans-active factors in the regulation of phenylpropanoid genes.

Phylogenetic analysis based on the deduced amino acid sequence of phenylalanine ammonia-lyase gene (Sl<em>PAL</em>5) cDNA from tomato (Solanum lycopersicum L.) revealed high sequence similarity to <em>PAL</em> genes in Nicotiana tabacum (92%), Ipomoea nil (87%), Manihot esculenta (84%), and Catharanthus roseus (84%). The Sl<em>PAL</em>5 gene exists as multiple copies in the tomato plant, and its transcription was strongly expressed in old leaves and flowers. From 5 days post-anthesis to the onset of ripening, Sl<em>PAL</em>5 expression decreased gradually but was maintained at a comparatively high level; Sl<em>PAL</em>5 transcript expression was very low at the mature-green stage. Sl<em>PAL</em>5 expression was significantly induced in response to NaCl, mannitol, and cold treatment; Sl<em>PAL</em>5 expression decreased gradually after treatment with abscisic acid and H(2)O(2); Sl<em>PAL</em>5 transcript decreased after exposure to methyl viologen for 3 h and increased after 6 h and maintained a stable expression level until 24 h, suggesting that the Sl<em>PAL</em>5 gene may function in the response to abiotic stress.

Subpopulations of T-cells, B-cells, macrophages and ellipsoid-associated reticular cells (EARC) could be demonstrated by immunohistochemical staining early in the development of chicken spleen. However, the typical structures of the spleen, such as the peri-arteriolar lymphoid sheath (PALS) and the ellipsoids with their surrounding ring of macrophages, were only formed around embryonic day (ED) 20. These structures and especially the B-cell compartment, i.e., the peri-ellipsoid lymphoid sheath (PELS) gradually matured during the first week posthatch. Therefore, we analysed at what age broiler chickens could generate a humoral response against the thymus-dependent antigen bovine serum albumin (BSA). Chickens were immunised in ovo (ED16 and ED18) and at 1, 7 and 12 days of age and subsequent BSA-specific immunoglobulin (Ig) M and IgG responses were measured up to 10 days postimmunisation (DPI). No major differences were observed in the relative growth rates, while hatchability was only slightly reduced. Only in chicks immunised on 12 days of age, IgM and IgG responses were high with a normal kinetic pattern. In chicks immunised on 7 days of age, responses were just detectable, but they were absent in chicks immunised in ovo and on the day of hatching (Day 1). In a subsequent experiment, 1-, 7- and 12-day-old chicks were BSA-immunised and Ig responses were measured for a longer period up to the age of 28 days. The IgG response of chicks immunised at 1 day of age was lower and occurred later (from 28 DPI) than the response of chicks immunised at 7 and 14 days of age (from 14 DPI). It was not increased by a booster immunisation on 29 days of age, in contrast to the response of chicks immunised at 7 and 14 days of age. These findings indicate that vaccination at 1 day of age does not activate the B-cell response resulting in antibody production and support the idea that the immune function of the late embryonic and neonatal chickens is not entirely developed due to the incomplete structural organisation of their secondary immune organs.

OBJECTIVE

The aim of the study was to evaluate disease progression during supportive periodontal therapy in (i) a group of 225 subjects with "normal" (NG) and (ii) a group with high susceptibility (HSG; n= 109) to periodontal disease (based on their baseline disease status).

METHODS

The following variables were recorded at the baseline examination (1 year after they received non-surgical periodontal therapy) and at the re-examination after 12 years of maintenance: number of teeth, plaque, probing pocket depth, probing attachment level, bone level in full mouth radiographs. All assessments were performed in a standardized manner and by well-trained and calibrated examiners. Supportive periodontal therapy was delivered 3-4 x per year and included repeated oral hygiene instruction and debridement. In addition, sites that bled on probing and had a PPD value of>> or = 5 mm received subgingival instrumentation.

RESULTS

A comparison between the findings at baseline and after 12 years revealed that in the NG, most subjects maintained their periodontal condition unchanged during the maintenance period; only a few subjects experienced tooth loss and the figures describing the mean amount of bone and attachment loss were small (0.5 mm and 0.3 mm respectively). The HSG patients experienced some tooth loss and also lost significant amounts of bone and attachment during the 12 years of SPT. Thus, in this group of subjects, the mean overall PAL loss amounted to 0.8 mm, i.e., 0.06 mm/tooth surface/year. In the NG, the overall attachment loss was significantly smaller: 0.5 mm, i.e. 0.04 mm/tooth surface/year.

CONCLUSIONS

In subjects with a high susceptibility for periodontal disease who had been treated for this condition by non-surgical means, an SPT program including regularly repeated oral hygiene instruction and subgingival debridement, made it possible to maintain bone and attachment levels at a reasonably stable level over a 12-year period. A similar SPT provided to a group of subjects with normal susceptibility to periodontal disease, on the other hand, prevented almost entirely major tooth, bone and attachment loss.

The plant hormone abscisic acid (ABA) is an important regulator in many aspects of plant growth and development, as well as stress resistance. Here, we investigated the effects of exogenous ABA application on the interaction between tomato (Solanum lycopersicon L.) and Alternaria solani (early blight). Foliar spraying of 7.58 μM ABA was effective in reducing disease severity in tomato plants. Previously, increased activities of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) were observed in exogenous ABA-treated tomato leaves. Moreover, these enzyme activities were maintained at higher levels in ABA-pretreated and A. solani challenged tomato plants. Tomato defense genes, such as PR1, β-1, 3-glucanase (GLU), PPO, POD, and superoxide dismutase (SOD), were rapidly and significantly up-regulated by exogenous ABA treatment. Furthermore, a subsequent challenge of ABA-pretreated plants with the pathogen A. solani resulted in higher expression of defense genes, compared to water-treated or A. solani inoculated plants. Therefore, our results suggest that exogenous ABA could enhance disease resistance against A. solani infection in tomato through the activation of defense genes and via the enhancement of defense-related enzymatic activities.

BACKGROUND

Helping young smokers to quit early in life substantially reduces the risk of later morbidity and mortality due to tobacco use. The RealU study demonstrated the efficacy of a smoking-cessation website for college students that incorporated both individually tailored feedback and peer e-mail support. The relationship between peer e-mail support and cessation outcomes among intervention participants is examined here.

METHODS

This study was conducted at the University of Minnesota Twin Cities from Fall 2004 through Spring 2005. During the intervention period, peer-support students (E-pals) wrote weekly e-mails to intervention group participants (n=257) encouraging healthy behaviors including smoking abstinence. Ten survey items assessed perceived E-pal supportiveness. The number of e-mails replies sent by the participants to their E-pal was tracked as a measure of e-mail engagement. The primary outcome was self-reported 30-day abstinence at the end of the intervention period.

RESULTS

Over the course of the intervention, participants sent an average of 4.6 (SD=3.6) e-mails to their E-pals. Perceived E-pal support was significantly correlated with e-mail engagement (p<0.001). At Week 30, 40.5% of individuals in the RealU intervention group (104/257) reported not smoking any cigarettes in the prior 30 days. Bivariate analyses indicated that 30-day abstinence was related to both perceived support from the E-pal (p<0.001) and e-mail engagement (p<0.001). Multivariate analyses indicated that after controlling for age and baseline-level smoking, e-mail engagement remained a significant predictor of 30-day abstinence (p<0.001).

CONCLUSIONS

Greater peer engagement via e-mail was associated with increased smoking abstinence and reduced frequency of smoking. These findings suggest that online peer support may be an important strategy when delivering Internet-assisted cessation programs to young adults.

G protein-coupled receptor kinases (GRKs) utilize diverse mechanisms to associate with the plasma membrane and mediate phosphorylation of agonist-occupied receptors. For example, two members of this family, GRK4 and GRK6, contain C-terminal cysteine residues that are palmitoylated. To address whether the activity and membrane association of GRK6 is regulated by palmitoylation, we overexpressed and characterized wild-type GRK6 and two GRK6 mutants, one with the palmitoylation sites mutated to serines (GRK6-pal-) and one containing a C-terminal CAAX motif to promote geranylgeranylation (GRK6-GG). Compared with wild-type GRK6, GRK6-pal- had a approximately 5-fold higher Km and approximately 2-fold lower Vmax for phosphorylating rhodopsin, whereas GRK6-GG exhibited a approximately 2-fold lower Km and approximately 14-fold higher Vmax for rhodopsin. In contrast, wild-type GRK6 and GRK6-pal- displayed similar activity toward the nonreceptor substrate phosvitin, indicating that nonpalmitoylated GRK6 is catalytically active. Wild-type GRK6 and GRK6-GG, but not GRK6-pal-, also bound significantly to phosphatidylcholine vesicles (36 +/- 3, 79 +/- 4, and 4 +/- 2%, respectively) suggesting that GRK6 activity is dependent upon its ability to interact with the plasma membrane. When assayed in COS-1 cells GRK6-pal- promoted minimal agonist-dependent sequestration of the beta2-adrenergic receptor, while sequestration was significantly increased in cells expressing either wild-type GRK6 or GRK6-GG. These data demonstrate an important functional link between the ability of GRK6 to bind to the plasma membrane, a process that appears to be regulated by palmitoylation, and its activity toward receptor substrates.

BACKGROUND

Elevated levels of plasminogen activator inhibitor 1 (PAI-1) have been associated with the occurrence of thrombotic disease, and inhibition of PAI-1 activity in vivo resulted in enhanced thrombolysis and a reduction in reocclusion. Besides monoclonal antibodies and peptides, no suitable agents that are able to block PAI-1 activity are available to date. The present study was designed to test the interaction between a nonantibody, nonpeptide, diketopiperazine-based inhibitor of PAI-1, XR5118, and PAI-1 and to assess the effect of XR5118 on PAI-1 activity in vitro and on in vivo thrombolysis and thrombus growth in an experimental thrombosis model in rabbits.

RESULTS

The binding site of XR5118 on the PAI-1 molecule was studied by competitive binding experiments with mapped anti-PAI-1 monoclonal antibodies by use of surface plasmon resonance experiments. XR5118 selectively and competitively inhibited binding of the PAl-1-inhibiting monoclonal antibody CLB-2C8, indicating that binding of XR5118 to PAI-1 takes place at the area between amino acids 110 and 145 of the PAI-1 molecule, which is known to be involved with the binding of PAI-1 to tissue plasminogen activator (TPA). Incubation of plasma or platelet releasate with XR5118 resulted in a dose-dependent inhibition of PAI-1 activity. Systemic infusion of XR5118 induced a significant reduction in plasma PAI-1 activity levels from 23.7+/-4.9 to 10.9+/-3.4 IU/mL. Administration of XR5118 resulted in a significant, twofold increase in endogenous thrombolysis compared with the control. Thrombus growth in rabbits receiving both XR5118 and rTPA was significantly attenuated compared with rabbits receiving rTPA alone (13.5+/-2.7% versus 19.9+/-3.8%, respectively).

CONCLUSIONS

XR5118 binds to PAI-1 and reduces plasma PAI-1 activity levels. Furthermore, XR5118 promotes endogenous thrombolysis and inhibits thrombus accretion and is the first nonpeptide compound with significant anti-PAI-1 activity in vivo in these models.

BACKGROUND

Some non-pathogenic rhizobacteria called Plant Growth Promoting Rhizobacteria (PGPR) possess the capacity to induce in plant defense mechanisms effective against pathogens. Precedent studies showed the ability of Pseudomonas putida BTP1 to induce PGPR-mediated resistance, termed ISR (Induced Systemic Resistance), in different plant species. Despite extensive works, molecular defense mechanisms involved in ISR are less well understood that in the case of pathogen induced systemic acquired resistance.

RESULTS

We analyzed the activities of phenylalanine ammonia-lyase (PAL) and lipoxygenase (LOX), key enzymes of the phenylpropanoid and oxylipin pathways respectively, in tomato treated or not with P. putida BTP1. The bacterial treatment did not stimulate PAL activity and linoleate-consuming LOX activities. Linolenate-consuming LOX activity, on the contrary, was significantly stimulated in P. putida BTP1-inoculated plants before and two days after infection by B. cinerea. This stimulation is due to the increase of transcription level of two isoforms of LOX: TomLoxD and TomLoxF, a newly identified LOX gene. We showed that recombinant TomLOXF preferentially consumes linolenic acid and produces 13-derivative of fatty acids. After challenging with B. cinerea, the increase of transcription of these two LOX genes and higher linolenic acid-consuming LOX activity were associated with a more rapid accumulation of free 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids, two antifungal oxylipins, in bacterized plants.

CONCLUSIONS

In addition to the discovery of a new LOX gene in tomato, this work is the first to show differential induction of LOX isozymes and a more rapid accumulation of 13-hydroperoxy-octadecatrienoic and 13-hydroxy-octadecatrienoic acids in rhizobacteria mediated-induced systemic resistance.

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

In grass, the evolutionary relationship between lemma and <em>pal</em>ea, and their relationship to the flower organs in dicots have been variously interpreted and wildely debated. In the present study, we carried out morphological and genetic analysis of a <em>pal</em>ealess mutant (<em>pal</em>) from rice (Oryza sativa L.), and fine mapping the gene responsible for the mutated trait. Together, our findings indicate that the <em>pal</em>ea is replaced by two leaf-like structures in the <em>pal</em> flowers, and this trait is controlled by one recessive gene, termed <em>pal</em>ealess1 (<em>pal</em>1). With a large F2 segregating population, the <em>pal</em>1 gene was finally mapped into a physical region of 35 kb. Our results also suggest that the lemma and <em>pal</em>ea of rice are not homologous organs, <em>pal</em>ea is likely evolutionarily equivalent to the eudicot se<em>pal</em>, and the <em>pal</em>1 should be an A function gene for rice floral organ identity.

Target identification of biologically active small-molecules is often the rate-determining step in forward chemical genetics. Photo-affinity labeling (PAL) represents a useful biochemical strategy for target identification in complex protein mixtures. This unit describes the use of alkyl diazirine-based photo-affinity probes and Cu(I)-catalyzed click chemistry to covalently label and visualize the targets of biologically active small-molecules. A general method for affinity purification of probe-modified proteins, useful for identification of protein targets, is also described.

OBJECTIVE

Physical activity (PA) is associated with a reduced risk of colorectal cancer. Thus, we examined the colon transit time (CTT) according to the physical activity level (PAL) in Korean adults.

METHODS

The study subjects were 49 adults: 24 males and 25 females. The subjects used an accelerometer for 7 consecutive days to measure the 1-week PAL. The subjects took a capsule containing 20 radio-opaque markers for 3 days. On the fourth day, a supine abdominal radiography was performed. According to the total activity count of all study subjects, the upper 25%, middle 50% and lower 25% were classified into the high (H), moderate (M) and low (L) physical activity (PA) groups, respectively.

RESULTS

The total CTT was significantly longer in the female (25.8 hours) than in the male subjects (7.4 hours) (P = 0.002). In regard to difference on PAL, although there was no significant difference among the male subjects, the right CTT in the female subjects was significantly shorter in H group than in M group (P = 0.048), and the recto-sigmoid CTT was significantly shorter in H group than in L group (P = 0.023). Furthermore, there were significant differences in total CTT between L and M groups (P = 0.022), M and H groups (P = 0.026) and between L and H groups (P = 0.002).

CONCLUSIONS

The female, but not male, subjects showed that moderate and high PAL assisted colon transit.