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Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

The interaction in multisubunit non-ribosomal peptide synthetases (NRPSs) is mediated by docking domains that ensure the correct subunit-to-subunit interaction. We introduced natural docking domains into the three-module xefoampeptide synthetase (XfpS) to create two to three artificial NRPS XfpS subunits. The enzymatic performance of the split biosynthesis was measured by absolute quantification of the products by HPLC-ESI-MS. The connecting role of the docking domains was probed by deleting integral parts of them. The peptide production data was compared to soluble protein amounts of the NRPS using SDS-PAGE. Reduced peptide synthesis was not a result of reduced soluble NRPS concentration but a consequence of the deletion of vital docking domain parts. Splitting the xefoampeptide biosynthesis polypeptide by introducing docking domains was feasible and resulted in higher amounts of product in one of the two tested split-module cases compared to the full-length wild-type enzyme., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Several peptides in clinical use are derived from non-ribosomal peptide synthetases (NRPS). In these systems multiple NRPS subunits interact with each other in a specific linear order mediated by specific docking domains (DDs), whose structures are not known yet, to synthesize well-defined peptide products. In contrast to classical NRPSs, single-module NRPS subunits responsible for the generation of rhabdopeptide/xenortide-like peptides (RXPs) can act in different order depending on subunit stoichiometry thereby producing peptide libraries. To define the basis for their unusual interaction patterns, we determine the structures of all N-terminal DDs ( N DDs) as well as of an N DD- C DD complex and characterize all putative DD interactions thermodynamically for such a system. Key amino acid residues for DD interactions are identified that upon their exchange change the DD affinity and result in predictable changes in peptide production. Recognition rules for DD interactions are identified that also operate in other megasynthase complexes.

Purpose: For Black women who have sex with women (BWSW), obtaining routine healthcare can be obstructed by a number of psychosocial barriers, including experiences of stigma, related to both sexual orientation and race, and medical mistrust, both race-based and global. Previous research demonstrates that sexual orientation and race-based stigma, as well as global and race-based medical mistrust, each have a negative impact on health outcomes and engagement in care (EIC) independently. This study addresses gaps in the literature by examining the impact of these psychosocial barriers and their interactions among BWSW, an understudied population., Methods: Participants (256 BWSW) were surveyed at a Black Gay Pride festival. Separate generalized linear models assessed the independent and multiplicative effects of participants' self-reported sexual orientation stigma, race-based stigma, race-based medical mistrust, and global medical mistrust related to their engagement in routine physical exams and blood pressure screenings., Results: Prevalence rates of both stigma measures were low, but prevalence rates of global and race-based medical mistrust were high. The results show that experiencing sexual orientation stigma or having race-based medical mistrust predicts significantly lower EIC. Furthermore, the frequencies of obtaining recent physical examinations and blood pressure screenings were significantly related to three- and two-way interactions between stigma and medical mistrust, respectively., Conclusion: There is an urgent need to address the intersectionality of these psychosocial barriers in an effort to increase BWSW's EIC., Competing Interests: Author Disclosure Statement No competing financial interests exist.

Introduction: Antiretroviral therapy (ART) improves the health of people living with HIV and has the potential to reduce HIV infectiousness, thereby preventing HIV transmission. However, the success of ART for HIV prevention hinges on sustained ART adherence and avoiding sexually transmitted infections (STI)., Objectives: To determine the sexual behaviours and HIV transmission risks of individuals with suppressed and unsuppressed HIV replication (i.e., viral load)., Methods: Assessed HIV sexual transmission risks among individuals with clinically determined suppressed and unsuppressed HIV. Participants were 760 men and 280 women living with HIV in Atlanta, GA, USA, who completed behavioural assessments, 28-daily prospective sexual behaviour diaries, one-month prospective unannounced pill counts for ART adherence, urine screening for illicit drug use and medical record chart abstraction for HIV viral load., Results: Individuals with unsuppressed HIV demonstrated a constellation of behavioural risks for transmitting HIV to uninfected sex partners that included symptoms of STI and substance use. In addition, 15% of participants with suppressed HIV had recent STI symptoms/diagnoses, indicating significant risks for sexual infectiousness despite their HIV suppression in blood plasma. Overall, 38% of participants were at risk for elevated sexual infectiousness and just as many engaged in unprotected sexual intercourse with non-HIV-infected partners., Conclusions: Implementation strategies for using HIV treatments as HIV prevention requires enhanced behavioural interventions that extend beyond ART to address substance use and sexual health that will otherwise undermine the potential preventive impact of early ART.

Beliefs about medication necessity and concerns predict treatment adherence in people with a wide-array of medical conditions, including HIV infection. However, medication beliefs have not been examined in people dually treated with psychotropic medications and antiretroviral therapy. In the current study, we used a prospective design to investigate the factors associated with adherence to psychotropic medications and antiretrovirals among 123 dually treated persons living with HIV. We used unannounced phone-based pill counts to monitor adherence to psychiatric and antiretroviral medications over a 6-week period. Hierarchical regression models included demographic, health and psychosocial characteristics as predictors of adherence followed by medication necessity and concerns beliefs. Results showed that medication necessity beliefs predicted both antiretroviral and psychiatric medication adherence over and above established predictors of adherence. Medication concerns also predicted psychotropic adherence, but not antiretroviral adherence. These models accounted for 31 and 22 % of the variance in antiretroviral and psychotropic adherence, respectively. Findings suggest that the necessity-concerns medication beliefs framework has utility in understanding adherence to multiple medications and addressing these beliefs should be integrated into adherence interventions.

HIV infection is concentrated in populations living in poverty. We examined the overlapping and independent effects of multiple poverty indicators on HIV-related health status. Because substance use can create competing survival needs when resources are limited, we also sought to objectively measure expenditures on food relative to alcohol and tobacco products. To achieve these aims, 459 men and 212 women living with HIV infection in Atlanta, GA completed measures of socio-demographic and heath characteristics as well as multiple indicators of poverty including housing stability, transportation, food insecurity, and substance use. Participants were given a $30 grocery gift card for their participation and we collected receipts which were coded for alcohol (beer, wine, liquors) and tobacco purchases. Results showed that participants with unsuppressed HIV replication were significantly more likely to experience multiple indicators of poverty. In addition, one in four participants purchased alcohol or tobacco products with their gift cards, with as much as one-fourth of money spent on these products. A multivariable logistic regression model showed that food insecurity was independently associated with unsuppressed HIV, and purchasing alcohol or tobacco products did not moderate this association. Results confirm previous research to show the primacy of food insecurity in relation to HIV-related health outcomes. Competing survival needs, including addictive substances, should be addressed in programs that aim to alleviate poverty to enhance the health and well-being of people with HIV infection.

Exchange of the native promoter to the arabinose-inducible promoter PBAD was established in entomopathogenic bacteria to silence and/or activate gene clusters involved in natural product biosynthesis. This allowed the "on-demand" production of GameXPeptides, xenoamicins, and the blue pigment indigoidine. The gene clusters for the novel "mevalagmapeptides" and the highly toxic xenorhabdins were identified by this approach., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: Objectives: We assessed how health care-related stigma, global medical mistrust, and personal trust in one's health care provider relate to engaging in medical care among Black men who have sex with men (MSM)., Methods: In 2012, we surveyed 544 Black MSM attending a community event. We completed generalized linear modeling and mediation analyses in 2013., Results: Twenty-nine percent of participants reported experiencing racial and sexual orientation stigma from heath care providers and 48% reported mistrust of medical establishments. We found that, among HIV-negative Black MSM, those who experienced greater stigma and global medical mistrust had longer gaps in time since their last medical exam. Furthermore, global medical mistrust mediated the relationship between stigma and engagement in care. Among HIV-positive Black MSM, experiencing stigma from health care providers was associated with longer gaps in time since last HIV care appointment., Conclusions: Interventions focusing on health care settings that support the development of greater awareness of stigma and mistrust are urgently needed. Failure to address psychosocial deterrents will stymie progress in biomedical prevention and cripple the ability to implement effective prevention and treatment strategies.

Introduction: Food insecurity is a well-established predictor of poor health outcomes. Antiretroviral therapies (ARTs) that should be taken with food to increase bioavailability may further challenge food insecure patients. This study examined factors associated with antiretroviral adherence and HIV viral suppression among people living with HIV who are food insecure and prescribed medications that require food., Methods: A community sample of 313 men and 105 women who experienced food insecurity in the previous month and were currently taking ART completed computerized interviews, urine screening for drug use, prospective biweekly unannounced pill count adherence assessments, and obtained their HIV viral load and CD4 cell counts from medical records., Results: Individuals taking ART regimens that should be taken with food were significantly more likely to be unemployed, were living longer with an HIV diagnosis, had lower CD4 cell counts, poorer HIV suppression, and endorsed more beliefs that taking medications was necessary for their health. Multivariable regression models controlling for potential confounding factors showed that receiving ART that requires food was significantly related to poorer ART adherence and unsuppressed HIV in this food insecure sample., Conclusion: People living with HIV who are food insecure likely experience multiple facets of poverty that challenge their medication adherence, but food insecurity is the only such factor that is directly related to the pharmacokinetics of some antiretroviral medications. Achieving optimal treatment outcomes for HIV infection will require routine assessment of access to food when determining patient-tailored ART regimens.

Discovery of new natural products by heterologous expression reaches its limits, especially when specific building blocks are missing in the heterologous host or the production medium. Here, we describe the insect-specific production of the new GameXPeptides E-H (5-8) from Photorhabdus luminescens TTO1, which can be produced heterologously from expression of the GameXPeptide synthetase GxpS only upon supplementation of the production media with the missing building blocks, and thus must be regarded as the true natural products under natural conditions., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Food insecurity is a known barrier to medication adherence among people living with HIV. Antiretroviral therapies (ART) that require food likely pose added challenges to patients who do not have reliable access to food. This study examines the health implications of prescribing ART that requires food to patients who are food insecure., Method: A community sample of 538 men and 221 women currently taking ART to treat their HIV infection completed computerized interviews, biweekly unannounced pill count adherence assessments, and obtained their HIV RNA (viral load) and CD4 cell count from medical records., Results: Sixty-three percent of participants experienced at least 1 indicator of food insecurity during the previous month, of which 274 (57%) were prescribed an ART regimen that requires food. Among participants who were food insecure, individuals taking ART requiring food indicated significantly greater HIV symptoms, had lower CD4 cell counts, and poorer HIV suppression. For participants who were food secure, those taking ART that requires food were significantly less adherent than those whose ART regimen does not require food., Conclusions: People living with HIV who experience food insecurity are significantly more likely to be prescribed ART regimens that require food and experience poorer treatment outcomes. Determination of optimal ART regimens should take patient access to food into account and treatment guidelines should explicitly highlight the importance of food access in selecting ART regimens., (© The Author(s) 2014.)

Background: Co-infection with human immunodeficiency virus (HIV) and Hepatitis-C virus (HCV) poses a significant threat to personal and public health. Substance use among co-infected persons leads to increased morbidity and mortality. The purpose of this study is to examine the continued substance use of people living with HIV-HCV co-infection and receiving antiretroviral therapy (ART)., Methods: Individuals living with HIV infection in Atlanta, GA and currently receiving ART (N = 678) completed audio-computer-assisted self-interviews for demographic, health, and behavior characteristics; unannounced pill counts to assess ART adherence over one month; finger-stick blood specimens collected for HCV antibody testing and urine specimens for drug use screening; and obtained HIV viral load and CD4 cell counts from their medical provider. We performed cross-sectional analyses for behavioral and biological markers of health, health behaviors, and substance use., Results: Among participants, 131 (19%) were HIV-HCV co-infected; 53% were HIV-mono-infected, and 60% of HIV-HCV co-infected participants tested positive for use of at least one non-alcohol drug: tetrahydrocannabinol (THC) and cocaine were most prevalent. HIV-HCV co-infected individuals were older, with no other significant differences. Within the HIV-HCV co-infected participants, drug users (N = 87) did not differ from non-drug users (N = 53) in terms of ART adherence. However, drug users were significantly more likely to have uncontrolled HIV (17%) compared with those who did not test drug positive (4%)., Conclusions: Substance use is prevalent in persons with HIV-HCV co-infection and may interfere with ART. Research with a larger and more representative sample is needed to replicate and confirm these results.

The Internet is a primary source for health-related information, and Internet search activity is associated with infectious disease outbreaks. The authors hypothesized that Internet search activity for vaccine-related information would predict vaccination coverage. They examined Internet search activity for H1N1 and human papilloma virus (HPV) disease and vaccine information in relation to H1N1 and HPV vaccine uptake. Google Insight for Search was used to assess the volume of Internet search queries for H1N1- and vaccine-related terms in the United States in 2009, the year of the H1N1 pandemic. Vaccine coverage data were also obtained from the Centers for Disease Control and Prevention at the state level for H1N1 vaccinations in 2009. These same measures were collected at the state level for HPV- and vaccine-related search terms in 2010 as well as HPV vaccine uptake in that year. Analyses showed that the search terms H1N1 and vaccine were correlated with H1N1 vaccine uptake; ordinal regression found the H1N1 search term was independently associated with H1N1 vaccine coverage. Similarly, the correlation between vaccine search volume and HPV coverage was significant; ordinal regression showed the search term vaccine independently predicted HPV vaccination coverage. This is among the first studies to show that Internet search activity is associated with vaccination coverage. The Internet should be exploited as an opportunity to dispel vaccine misinformation by providing accurate information to support vaccine decision making.

PoyD is a radical S-adenosyl methionine epimerase that introduces multiple D-configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel post-translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D-amino acid patterns into all-L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide "xenotetrapeptide" from Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D-amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D2 O., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Motile predatory Myxobacteria are producers of multiple secondary metabolites and, on starvation, undergo concerted cellular differentiation to form multicellular fruiting bodies. These abilities demand myxobacterial genomes to encode sophisticated regulatory networks that are not satisfactorily understood. Here, we present two bacterial enhancer binding proteins (bEBPs) encoded in Myxococcus xanthus acting as direct regulators of secondary metabolites intriguingly exhibiting activating and inhibitory effects. Elucidation of a regulon for each bEBP enabled us to unravel their role in myxococcal development, predation, and motility. Interestingly, both bEBPs are able to interact by forming a hetero-oligomeric complex. Our findings represent an alternative mode of operation of bEBPs, which are currently thought to enhance promoter activity by acting as homo-oligomers. Furthermore, a direct link between secondary metabolite gene expression and predation, motility, and cellular development could be shown for the first time., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k(cat) = 153 s(-1), 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although sharing a certain degree of structural uniformity, natural product classes exhibit variable functionalities such as different amino acid or acyl residues. During collision induced dissociation, some natural products exhibit a conserved fragmentation pattern close to the precursor ion. The observed fragments result from a shared set of neutral losses, creating a unique fragmentation pattern, which can be used as a fingerprint for members of these natural product classes. The culture supernatants of 69 strains of the entomopathogenic bacteria Photorhabdus and Xenorhabdus were analyzed by MALDI-MS(2), and a database comprising MS(2) data from each strain was established. This database was scanned for concordant fragmentation patterns of different compounds using a customized software, focusing on relative mass differences of the fragment ions to their precursor ion. A novel group of related natural products comprising 25 different arginine-rich peptides from 16 different strains was identified due to its characteristic neutral loss fragmentation pattern, and the structures of eight compounds were elucidated. Two biosynthesis gene clusters encoding nonribosomal peptide synthetases were identified, emphasizing the possibility to identify a group of structurally and biosynthetically related natural products based on their neutral loss fragmentation pattern.

Structure elucidation of natural products including the absolute configuration is a complex task that involves different analytical methods like mass spectrometry, NMR spectroscopy, and chemical derivation, which are usually performed after the isolation of the compound of interest. Here, a combination of stable isotope labeling of Photorhabdus and Xenorhabdus strains and their transaminase mutants followed by detailed MS analysis enabled the structure elucidation of novel cyclopeptides named GameXPeptides including their absolute configuration in crude extracts without their actual isolation., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The production of the blue pigment indigoidine has been achieved in the entomopathogenic bacterium Photorhabdus luminescens by a promoter exchange and in Escherichia coli following heterologous expression of the biosynthesis gene indC. Moreover, genes involved in the regulation of this previously "silent" biosynthesis gene cluster have been identified in P. luminescens., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Isovaleryl-CoA (IV-CoA) is usually derived from the degradation of leucine by using the Bkd (branched-chain keto acid dehydrogenase) complex. We have previously identified an alternative pathway for IV-CoA formation in myxobacteria that branches from the well-known mevalonate-dependent isoprenoid biosynthesis pathway. We identified 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (MvaS) to be involved in this pathway in Myxococcus xanthus, which is induced in mutants with impaired leucine degradation (e.g., bkd(-)) or during myxobacterial fruiting-body formation. Here, we show that the proteins required for leucine degradation are also involved in the alternative IV-CoA biosynthesis pathway through the efficient catalysis of the reverse reactions. Moreover, we conducted a global gene-expression experiment and compared vegetative wild-type cells with bkd mutants, and identified a five-gene operon that is highly up-regulated in bkd mutants and contains mvaS and other genes that are directly involved in the alternative pathway. Based on our experiments, we assigned roles to the genes required for the formation of IV-CoA from HMG-CoA. Additionally, several genes involved in outer-membrane biosynthesis and a plethora of genes encoding regulatory proteins were decreased in expression levels in the bkd(-) mutant; this explains the complex phenotype of bkd mutants including a lack of adhesion in developmental submerse culture.

The genus Sorangium synthesizes approximately half of the secondary metabolites isolated from myxobacteria, including the anti-cancer metabolite epothilone. We report the complete genome sequence of the model Sorangium strain S. cellulosum So ce56, which produces several natural products and has morphological and physiological properties typical of the genus. The circular genome, comprising 13,033,779 base pairs, is the largest bacterial genome sequenced to date. No global synteny with the genome of Myxococcus xanthus is apparent, revealing an unanticipated level of divergence between these myxobacteria. A large percentage of the genome is devoted to regulation, particularly post-translational phosphorylation, which probably supports the strain's complex, social lifestyle. This regulatory network includes the highest number of eukaryotic protein kinase-like kinases discovered in any organism. Seventeen secondary metabolite loci are encoded in the genome, as well as many enzymes with potential utility in industry.

In the attempt to establish a reliable real-time PCR protocol for transcriptional analysis of secondary metabolism in Sorangium cellulosum strain So ce56, a RNA extraction method and a reverse transcription protocol was developed. In order to validate chivosazol or etnangien gene cluster transcripts as good candidates to develop the real-time PCR protocol, stability measurements of the transcripts were performed proving both transcripts to be very stable. The chivosazol biosynthetic gene cluster was taken as the test case to evaluate the special problems arising from the large size of the transcripts and the high G/C-content of the encoding DNA. A set of primer pairs targeting the presumed 90 kbp chivosazol transcript at different positions was employed. The production rate of chivosazol was compared to the transcription of the operon in time course experiments revealing that during the logarithmic growth phase transcription is maximally induced and levels out during the stationary phase. Some deviations in transcript numbers could be measured depending on the primer pair used, but cross-evaluation strengthened the notion that the measured numbers reflect the whole transcript quantities and the in vivo level. Finally, a putative promoter located between chiA and chiB was examined by using the developed real-time PCR protocol.

Activation sequence-1 (as-1)-like regulatory cis elements mediate transcriptional activation in response to increased levels of plant signalling molecules auxin and salicylic acid (SA). Our earlier work has shown that tobacco cellular as-1-binding complex SARP (salicylic acid responsive protein) is primarily comprised of bZIP protein TGA2.2 and of minor amounts of a protein that cross-reacts with an antibody directed against related bZIP factor TGA2.1. As this protein was significantly smaller than recombinant TGA2.1, the origin of this protein had remained unresolved. Here we demonstrate that it corresponds to a distinct cleavage product of TGA2.1 generated during extract preparation. Overexpression of TGA2.1 led to increased levels of the TGA2.1/TGA2.2 heterodimer which was as effective with regard to enhancing the SA-inducibility of as-1 containing target gene Nt103 as corresponding amounts of the TGA2.2 homodimer. Thus, the TGA2.1 specific N-terminal domain, which had revealed transcriptional activation potential in yeast, did not show enhanced transcriptional activation in planta. TGA2.1 even had a negative effect on the SA-induced expression of the truncated CaMV 35S (-90) promoter that contains an isolated as-1-element upstream of the TATA-box. Plants expressing a TGA mutant deficient in DNA binding (TGA2.1trd) showed reduced levels of SA-inducible Nt103 expression, thus resembling plants expressing the analogous TGA2.2 derivative TGA2.2trd. In contrast to TGA2.2trd, TGA2.1trd did not reduce auxin-induced expression of Nt103 and SA-induced expression of pathogenesis related protein PR-1a, indicating that TGA2.1trd and TGA2.2trd differ in their capacity to outcompete regulatory factors involved in these regulatory pathways.

In higher plants, activating sequence-1 (as-1) of the cauliflower mosaic virus 35 S promoter mediates both salicylic acid (SA)- and auxin-inducible transcriptional activation. Originally found in promoters of several viral and bacterial plant pathogens, as-1-like elements are also functional elements of plant promoters activated in the course of a defense response upon pathogen attack. Nuclear as-1-binding factor (ASF-1) and cellular salicylic acid response protein (SARP) bind specifically to as-1. Four different tobacco bZIP transcription factors (TGA1a, PG13, TGA2.1, and TGA2.2) are potential components of either ASF-1 or SARP. Here we show that ASF-1 and SARP are very similar in their composition. TGA2.2 is a major component of either complex, as shown by supershift analysis and Western blot analysis of DNA affinity-purified SARP. Minor amounts of a protein immunologically related to TGA2.1 were detected, whereas TGA1a was not detectable. Overexpression of either TGA2.2 or a dominant negative TGA2.2 mutant affected both SA and auxin (2, 4D) inducibility of various target promoters encoding as-1-like elements, albeit to different extents. This indicates that TGA2.2 is a component of the enhancosome assembling on these target promoters, both under elevated SA and 2,4D concentrations. However, the effect of altered TGA2.2 levels on gene expression was more pronounced upon SA treatment than upon 2,4D treatment.

Patients with gastroenterological or cardiovascular functional syndromes (n = 109) were examined by means of an extensive psychological standardized diagnostic programme and psychological strain analysis was carried out under psychogenic stress in laboratory. The results were compared with healthy probands (n = 51) likewise examined. Patients with functional syndromes significantly discriminated of healthy probands by more psychosocial risk constellation under strain caused by more distress and in parameters of performance and in psychophysiological field in some strain variables (heart rate, blood pressure, acral vasomotricity) and their dynamics.