Tools for assessing health research partnership outcomes and impacts: a systematic review.

OBJECTIVE: To identify and assess the globally available valid, reliable and acceptable tools for assessing health research partnership outcomes and impacts. METHODS: We searched Ovid MEDLINE, Embase, CINAHL Plus and PsycINFO from origin to 2 June 2021, without limits, using an a priori strategy and registered protocol. We screened citations independently and in duplicate, resolving discrepancies by consensus and retaining studies involving health research partnerships, the development, use and/or assessment of tools to evaluate partnership outcomes and impacts, and reporting empirical psychometric evidence. Study, tool, psychometric and pragmatic characteristics were abstracted using a hybrid approach, then synthesized using descriptive statistics and thematic analysis. Study quality was assessed using the quality of survey studies in psychology (Q-SSP) checklist. RESULTS: From 56 123 total citations, we screened 36 027 citations, assessed 2784 full-text papers, abstracted data from 48 studies and one companion report, and identified 58 tools. Most tools comprised surveys, questionnaires and scales. Studies used cross-sectional or mixed-method/embedded survey designs and employed quantitative and mixed methods. Both studies and tools were conceptually well grounded, focusing mainly on outcomes, then process, and less frequently on impact measurement. Multiple forms of empirical validity and reliability evidence was present for most tools; however, psychometric characteristics were inconsistently assessed and reported. We identified a subset of studies (22) and accompanying tools distinguished by their empirical psychometric, pragmatic and study quality characteristics. While our review demonstrated psychometric and pragmatic improvements over previous reviews, challenges related to health research partnership assessment and the nascency of partnership science persist. CONCLUSION: This systematic review identified multiple tools demonstrating empirical psychometric evidence, pragmatic strength and moderate study quality. Increased attention to psychometric and pragmatic requirements in tool development, testing and reporting is key to advancing health research partnership assessment and partnership science. PROSPERO CRD42021137932.

Low-Molecular-Weight Thiols and Thioredoxins Are Important Players in Hg(II) Resistance in Thermus thermophilus HB27.

Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 µM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA.IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.

Contribution of the type III secretion system (TTSS) to virulence of Aeromonas salmonicida subsp. salmonicida.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The DeltaascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or DeltaascC. The experimental infection by immersion with DeltaaopO was indistinguishable from that of the parental strain, that of DeltaaexT was delayed, whilst the virulence of DeltaaopH was reduced significantly but not abolished. By i.p. injection, DeltaaexT, DeltaaopH and DeltaaopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The DeltaascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.

Membrane-associated quinoprotein formaldehyde dehydrogenase from Methylococcus capsulatus Bath.

A membrane-associated, dye-linked formaldehyde dehydrogenase (DL-FalDH) was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The enzyme was the major formaldehyde-oxidizing enzyme in cells cultured in high (above 1 micromol of Cu per mg of cell protein) copper medium and expressing the membrane-associated methane monooxygenase. Soluble NAD(P)(+)-linked formaldehyde oxidation was the major activity in cells cultured in low-copper medium and expressing the soluble methane monooxygenase (Tate and Dalton, Microbiology 145:159-167, 1999; Vorholt et al., J. Bacteriol. 180:5351-5356, 1998). The membrane-associated enzyme is a homotetramer with a subunit molecular mass of 49,500 Da. UV-visible absorption, electron paramagnetic resonance, and electrospray mass spectrometry suggest the redox cofactor of the DL-FalDH is pyrroloquinoline quinone (PQQ), with a PQQ-to-subunit stochiometry of approximately 1:1. The enzyme was specific for formaldehyde, oxidizing formaldehyde to formate, and utilized the cytochrome b(559/569) complex as the physiological electron acceptor.

Topological analysis and role of the transmembrane domain in polar targeting of PilS, a Pseudomonas aeruginosa sensor kinase.

In Pseudomonas aeruginosa, synthesis of pilin, the major protein subunit of the pili, is regulated by a two-component signal transduction system in which PilS is the sensor kinase. PilS is an inner membrane protein found at the poles of the bacterial cell. It is composed of three domains: an N-terminal hydrophobic domain; a central cytoplasmic linker region; and the C-terminal transmitter region conserved among other sensor kinases. The signal that activates PilS and, consequently, pilin transcription remains unknown. The membrane topology of the hydrophobic domain was determined using the lacZ and phoA gene fusion approach. In this report, we describe a topological model for PilS in which the hydrophobic domain forms six transmembrane helices, whereas the N- and C-termini are cytoplasmic. This topology is very stable, and the cytoplasmic C-terminus cannot cross the inner membrane. We also show that two of the six transmembrane segments are sufficient for membrane anchoring and polar localization of PilS.

Use of the radial forearm fasciocutaneous free flap and montgomery salivary bypass tube for pharyngoesophageal reconstruction.

BACKGROUND: Head and neck reconstructive surgeons involved in pharyngoesophageal reconstruction have several options available to repair the defect after partial or total laryngopharyngectomy. There is no uniform agreement among head and neck surgeons as to which of the most frequently used techniques offers the best results. METHODS: A retrospective study was performed on 20 consecutive patients who had undergone reconstruction of the hypopharynx and cervical esophagus using a radial forearm free flap with Montgomery salivary bypass tube at the Massachusetts Eye and Ear Infirmary in Boston, Massachusetts, and St. Louis University, Department of Otolaryngology-Head and Neck Surgery between 1992 and 1996. This reconstruction was used for primary reconstruction after total or partial laryngopharyngectomy with cervical esophagectomy, partial pharyngectomy sparing the larynx, and for reconstruction of the stenotic neopharynx after laryngectomy. RESULTS: The overall rate of pharyngocutaneous fistula was 20%, and the rate of postoperative stricture was 10%. Of patients reconstructed with this technique, 85% were able to resume oral alimentation, whereas 15% remained G-tube dependent. Of the 18 patients who did not have their larynges remain intact, 6 were able to develop useful tracheoesophageal speech. CONCLUSIONS: The results of this study show that the radial forearm fasciocutaneous free flap in combination with the Montgomery salivary bypass tube is extremely useful for reconstruction of partial and circumferential defects of the hypopharynx and cervical esophagus.

Antepartum detection of macrosomic fetus: clinical versus sonographic, including soft-tissue measurements.

OBJECTIVE: To compare clinical and sonographic estimates of birth weights with five new estimation techniques that involve measurements of soft tissue, for identifying newborns with birth weights of at least 4000 g. METHODS: Over 1 year, each woman at or after 36 weeks' gestation and suspected of having a macrosomic fetus had clinical and sonographic estimates of fetal weight (EFW) based on femur length (FL) and head and abdominal circumference, followed by five additional ways to identify excessive growth: cheek-to-cheek diameter, thigh soft tissue, ratio of thigh soft tissue to FL, upper arm subcutaneous tissue, and EFW derived from it. Areas (+/- standard error) of receiver operating characteristic (ROC) curves were calculated and compared with the area under the nondiagnostic line. P <.05 was considered statistically significant. RESULTS: Among 100 women recruited, 28 newborns weighed 4000 g or more. The areas under the ROC curves with clinical (0.72 +/- 0.06) and sonographic predictions using biometric characteristics (0.73 +/- 0.06) had the highest but similar accuracies (P.05). Three of the five newer methods (upper arm or thigh subcutaneous tissue and ratio of thigh subcutaneous tissue to FL) were poor diagnostic tests (range of areas under ROC 0.52 +/- 0.06 to 0.58 +/- 0.07). Estimated fetal weight based on upper arm soft tissue thickness and cheek-to-cheek diameter (areas 0.70 +/- 0.06 and 0.67 +/- 0.06, respectively) were not significantly better than clinical predictions (P.05) for detecting macrosomic fetuses. About 110 macrosomic and nonmacrosomic infants combined would be needed to have 80% power to detect a difference between ROC curves with areas of 0.58 (thigh subcutaneous tissue) and 0.72 (clinical estimate). CONCLUSION: ROC curves indicated that measurements of soft tissue are not superior to clinical or sonographic predictions in identifying fetuses with weights of at least 4000 g.

Localization of the histidine kinase PilS to the poles of Pseudomonas aeruginosa and identification of a localization domain.

Transcription of the type IV pilus subunit gene of Pseudomonas aeruginosa is controlled by a two-component signal transduction system. PilS, the histidine kinase, is membrane bound and PilR, its cognate response regulator, is cytoplasmic. The signal that activates PilS is unknown. PilS has three domains: (i) The N-terminus, predicted to form six transmembrane (TM) helices; (ii) a central linker domain; and (iii) the C-terminal transmitter domain containing all the conserved residues of sensor kinases. A translational fusion of the gfp gene (green fluorescent protein) to the 3' end of pilS was used to determine the position of PilS in the bacterial cell. Epifluorescence microscopy revealed that PilS is retained to the poles of P. aeruginosa but is distributed evenly about the membrane of Escherichia coli. Deletions of the PilS-GFP fusion revealed that the TM domain was sufficient and necessary to bring GFP to the membrane of P. aeruginosa and E. coli but was not sufficient to confine GFP to the poles. Retention to the poles of P. aeruginosa required both the TM and linker domains. Replacement of the PilS TM domain with an E. coli membrane protein, MalG, still allowed polar localization. Therefore, the PilS TM domain positions the linker domain close to the membrane allowing it to interact with the putative polar anchor which is specific to P. aeruginosa.

BNIP3alpha: a human homolog of mitochondrial proapoptotic protein BNIP3.

Apoptosis is regulated by interaction of viral and cellular BCL-2 family antiapoptotic proteins with various pro-apoptotic proteins, several of which are also members of the BCL-2 family. Cellular protein BNIP3 is a BCL-2 family proapoptotic protein that interacts with viral antiapoptosis proteins such as adenoviruses E1B-19K and EBV-BHRF1 and cellular antiapoptosis proteins such as BCL-2 and BCL-xL. Database searches indicate that the human genome encodes an open reading frame for a protein, BNIP3alpha, that shares substantial homology with BNIP3. The BNIP3alpha open reading frame encodes a protein of 219 amino acids that contains a conserved BH3 domain and a COOH-terminal trans-membrane domain, characteristic of several BCL-2 family proapoptotic proteins. BNIP3alpha interacts with viral antiapoptosis protein E1B-19K and cellular antiapoptosis proteins BCL-2 and BCL-xL. Overexpression of BNIP3alpha in transfected cells results in apoptosis and suppresses the antiapoptosis activity of E1B-19K and BCL-xL. Like BNIP3, BNIP3alpha seems to be predominantly localized in mitochondria. These results suggest that BNIP3alpha is a structural and functional homologue of BNIP3. BNIP3 and BNIP3alpha seem to be the first examples of homologues among the various human proapoptotic proteins. Northern blot analysis reveals that BNIP3alpha is expressed ubiquitously in most human tissues. In contrast, BNIP3 is expressed well in several human tissues and less abundantly in certain tissues such as placenta and lung. These results suggest that although BNIP3 and BNIP3alpha may promote apoptosis simultaneously in most human tissues, BNIP3alpha may play a more universal role.

Interaction between a cellular protein that binds to the C-terminal region of adenovirus E1A (CtBP) and a novel cellular protein is disrupted by E1A through a conserved PLDLS motif.

Adenovirus E1A proteins immortalize primary animal cells and cooperate with several other oncogenes in oncogenic transformation. These activities are primarily determined by the N-terminal half (exon 1) of E1A. Although the C-terminal half (exon 2) is also essential for some of these activities, it is dispensable for cooperative transformation with the activated T24 ras oncogene. Exon 2 negatively modulates in vitro cooperative transformation with T24 ras as well as the tumorigenic and metastatic potentials of transformed cells. A short C-terminal sequence of E1A governs the oncogenesis-restraining activity of exon 2. This region of E1A binds with a cellular phosphoprotein, CtBP, through a 5-amino acid motif, PLDLS, conserved among the E1A proteins of human adenoviruses. To understand the mechanism by which interaction between E1A and CtBP results in tumorigenesis-restraining activity, we searched for cellular proteins that complex with CtBP. Here, we report the cloning and characterization of a 125-kDa protein, CtIP, that binds with CtBP through the PLDLS motif. E1A exon 2 peptides that contain the PLDLS motif disrupt the CtBP-CtIP complex. Our results suggest that the tumorigenesis-restraining activity of E1A exon 2 may be related to the disruption of the CtBP-CtIP complex through the PLDLS motif.

Managed Care in Rural Health: The Stroke Survivor on Horseback.

Managed care has been slow to come to underserved rural areas, where profits may not be as large as in metropolitan areas. In the last decade, increased threat of takeover by out-of-state managed care organizations (MCOs) has prompted rural community hospitals and providers to develop some new models of managed care that fit both their economic and health care needs. Stroke survivors in rural areas have the same need for coordinated, ongoing care during their lifetime, but it is more difficult to deliver it in rural areas. Rehabilitation providers are urged to be proactive in creating innovative managed care approaches that will improve both the health and recovery outcomes of rural stroke survivors.

Differential gene expression by Pseudomonas aeruginosa during interaction with respiratory mucus.

Pseudomonas aeruginosa is a common respiratory tract pathogen that causes serious infections in patients with cystic fibrosis. A number of putative virulence factors have been characterized in several laboratories, and some have been implicated in human infections, based on criteria such as the phenotype of isolates from infected patients, an immune response to a particular antigenic factor, and the effect of a virulence factor on infectivity in an animal model. We have developed a series of genetic tools to study the selective regulation of expression of P. aeruginosa genes during interactions of the pathogen with host tissues. These tools are based on direct enrichment of bacteria, when a particular promoter is induced or repressed. We have found that interaction of bacteria with mucus from patients with cystic fibrosis results in marked induction of expression of several genes, including one that encodes a lipopolysaccharide biosynthetic enzyme, a gene for a protein responsible for uptake of the ferric pyochelin siderophore, and a new gene homologous with a class of iron-responsive repressors. The tools described here are useful for identification of induced or repressed genes in various animal models of infection or in controlled laboratory conditions that mimic natural infections of humans. Such genes might not be detectable when bacteria are cultured in laboratory conditions, and these tools are therefore useful for general probing of a bacterial genome for genes regulated during different stages of infection.

Dual function of PilS during transcriptional activation of the Pseudomonas aeruginosa pilin subunit gene.

The polar pili of Pseudomonas aeruginosa are composed of subunits encoded by the pilA gene. Expression of pilA requires the alternative sigma factor RpoN and a pair of regulatory elements, PilS and PilR. These two proteins are members of the two-component regulatory family, in which PilS is the sensory component and PilR is the response regulator. By using expression and localization analyses, in this work we show that PilS is synthesized as a 59-kDa polypeptide located in the P. aeruginosa cytoplasmic membrane. When the pilS gene is expressed in Escherichia coli, aberrant translational initiation results in a smaller, 40-kDa polypeptide. Unexpectedly, overexpression of pilS in P. aeruginosa results in decreased transcription of the pilA gene. Moreover, fully functional PilS was not required for this inhibitory effect. A mutation in the histidine residue essential for kinase activity resulted in a protein unable to activate transcription, yet when overexpressed in the presence of the wild-type PilS protein, this protein still repressed pilin synthesis. A shorter form of PilS, lacking its transmembrane segments, was active and fully capable of stimulating pilA transcription but when overexpressed did not show the inhibitory effect on pilin expression seen with full-length PilS. We also show that overexpression of pilR can activate transcription of pilA even in the absence of PilS. On the basis of our studies, we propose a complex mechanism of regulation of PilS function, involving other cellular factors that control PilS and its activities during the phosphorelay mechanism of signal transduction.

Functional substitution identifies a cell survival promoting domain common to adenovirus E1B 19 kDa and Bcl-2 proteins.

The adenovirus E1B 19 kDa protein provides a cell survival function during adenovirus infection and facilitates efficient virus replication by preventing premature cell death. These functions resemble those performed by the protein coded by the proto-oncogene Bcl-1. The Bcl-1 protein, which provides a survival function in cells exposed to a number of cell death-inducing stimuli, can substitute for the 19 kDa protein during adenovirus infection. Although these two survival- promoting proteins are not overtly related by primary amino acid sequence, they appear to share short homologous sequences. In order to determine if these sequence motifs constitute common functional domains, we carried out domain exchanges between the 19 kDa and Bcl-2 proteins. Our results indicate that a seven amino acid region of the Bcl-2 protein (108-YRRDFAE-114) can efficiently substitute for the corresponding 19 kDa domain (47-YKWEFEE-53). Mutagenizing this domain in Bcl-2 abolishes the survival promoting activity of Bcl-2. Substitution of the kDa sequences into Bcl-2 restores the survival promoting activity, albeit at reduced levels. Our results suggest that this domain (designated NH1) may constitute a common functional domain for the 19 kDa protein and survival promoting members of the Bcl-2 family of proteins.

Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins.

The survival-promoting activity of the Bcl-2 family of proteins appears to be modulated by interactions between various cellular proteins. We have identified a novel cellular protein, Bik, that interacts with the cellular survival-promoting proteins, Bcl-2 and Bcl-xL, as well as the viral survival-promoting proteins, Epstein Barr virus-BHRF1 and adenovirus E1B-19 kDa. In transient transfection assays, Bik promotes cell death in a manner similar to the death-promoting members of the Bcl-2 family, Bax and Bak. This death-promoting activity of Bik can be suppressed by coexpression of Bcl-2, Bcl-XL, EBV-BHRF1 and E1B-19 kDa proteins suggesting that Bik may be a common target for both cellular and viral anti-apoptotic proteins. While Bik does not show overt homology to the BH1 and BH2 conserved domains characteristic of the Bcl-2 family, it does share a 9 amino acid domain (BH3) with Bax and Bak which may be a critical determinant for the death-promoting activity of these proteins.

Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation.

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Intermolecular interactions between protein C inhibitor and coagulation proteases.

Protein C inhibitor (PCI) inhibits multiple plasma serine proteases. To determine which residues contribute to its specificity of inhibition, 19 mutations in the reactive site loop of PCI (from Thr352 to Arg357) were generated and assayed with thrombin, activated protein C (APC), and factor Xa. To identify the intermolecular interactions responsible for these kinetics, a molecular model of PCI was generated using alpha 1-protease inhibitor and ovalbumin as templates. This model of PCI was docked with thrombin, followed by extensive energy minimization, to determine a lowest energy complex. The resulting docked complex was used as a template to form molecular models of PCI-APC and PCI-factor Xa complexes. The best inhibitors of thrombin contained Pro or Gly at the P2 position in place of Phe353, with 2- and 7-fold increases in activity, respectively. These substitutions reduced steric interactions with the 60-insertion loop unique to thrombin. The best inhibitors of APC and factor Xa contained Arg at the P3 position in place of Thr352, with 2- and 5-fold increases in inhibition rates, respectively. The molecular model predicts that Arg in this position could form a salt bridge with Glu217 of each protease. Changing Arg357 at the P3' position had little effect on protease inhibition, consistent with the observation in the model that this residue points toward the body of PCI, forming a salt bridge with Glu220. Given its broad specificity of inhibition, PCI has proven very useful in understanding the nature of serpin-protease interactions using multiple mutations in a serpin assayed with multiple proteases.

Adenovirus E1B 19 kDa and Bcl-2 proteins interact with a common set of cellular proteins.

Adenovirus E1B 19 kDa protein protects against cell death induced by viral infection and certain external stimuli. The Bcl-2 protein can functionally substitute for the E1B 19 kDa protein. To identify cellular targets for the 19 kDa protein, we used the two-hybrid screen in yeast. We have isolated cDNAs for three different proteins, designated Nip1, Nip2, and Nip3, that interact with the 19 kDa protein. Mutational analysis indicates that these proteins do not associate with 19 kDa mutants defective in suppression of cell death, suggesting a correlation between interaction of these proteins and suppression of cell death. These proteins also associate with discrete sequence motifs in the Bcl-2 protein that are homologous to motifs of the 19 kDa protein. Our results suggest that two diverse proteins, the E1B 19 kDa and the Bcl-2 proteins, promote cell survival through interaction with a common set of cellular proteins.

Identification and characterization of PilS, an essential regulator of pilin expression in Pseudomonas aeruginosa.

Expression of the pilin gene, pilA, of Pseudomonas aeruginosa requires the alternative sigma factor, sigma 54, and also two other transcriptional regulators encoded by the pilS and pilR genes. These two linked genes, which have been identified by transposon insertion mutagenesis, share significant amino acid sequence homology with members of the two-component family of regulators. The transcriptional regulator, PilR, has been described previously. PilS, a 37,285 Dalton protein, shares significant homology with the protein kinase sensors of the two-component regulatory family. PilS, however, has no hydrophobic domains which might be membrane-spanning alpha-helices, suggesting that PilS is a cytoplasmic protein. Characterization of the pilS gene revealed that when overexpressed in Escherichia coli by the bacteriophage T7 promoter it specifies a protein of approximately 40,000 daltons, corresponding to the molecular weight of PilS predicted from the deduced amino acid sequence. Deletion analysis of the pilS promoter fused to a promoterless lacZ gene further showed that a significant region upstream of pilS is essential for expression of pilS and pilR, suggesting a need for transcriptional activation. The pilA promoter can be activated in E. coli but only when PilR and sigma 54 are present. This work suggests that the PilS activation signal is received in the bacterial cytoplasm, and that the mechanism of PilS/PilR-mediated signal transduction resulting in activation of the pilin gene promoter is likely to be similar to that of other two-component systems.

Mutational analysis of the transforming and apoptosis suppression activities of the adenovirus E1B 175R protein.

The role of the adenovirus-2 E1B 19-kDa (175R) T antigen in E1a-cooperative transformation was determined by cotransfection of plasmids expressing E1A or E1B 175R T antigens into primary rat kidney (BRK) cells. Transformed cells were selected by virtue of their resistance to the antibiotic Geneticin (G418) conferred by neo gene co-expression from plasmids coding for 175R. 175R cooperated efficiently with genomic E1a and specifically with the 289R protein coded by the 13S mRNA in the transformation of primary BRK cells. Mutational analysis of the 175R protein revealed that the N terminus and the C-terminal 30 amino acids are not essential for E1a-cooperative transformation. Several conserved sequences located in the middle of the 175R protein are essential for transformation. The effect of various mutants to suppress apoptosis (programmed cell death) induced by an anti-cancer agent, cisplatin, was examined in cells producing the E1A and E1B 175R proteins. Apoptosis was measured by flow cytometric analysis and indicates that the 175R protein efficiently prevents cisplatin-induced apoptosis. This suggests that the 175R function involved in transformation segregates with its ability to suppress cisplatin-induced apoptosis.