Evaluation of immunologic parameters in canine glioma patients treated with an oncolytic herpes virus.

Aim: To molecularly characterize the tumor microenvironment and evaluate immunologic parameters in canine glioma patients before and after treatment with oncolytic human IL-12-expressing herpes simplex virus (M032) and in treatment naïve canine gliomas. Methods: We assessed pet dogs with sporadically occurring gliomas enrolled in Stage 1 of a veterinary clinical trial that was designed to establish the safety of intratumoral oncoviral therapy with M032, a genetically modified oncolytic herpes simplex virus. Specimens from dogs in the trial and dogs not enrolled in the trial were evaluated with immunohistochemistry, NanoString, Luminex cytokine profiling, and multi-parameter flow cytometry. Results: Treatment-naive canine glioma microenvironment had enrichment of Iba1 positive macrophages and minimal numbers of T and B cells, consistent with previous studies identifying these tumors as immunologically "cold". NanoString mRNA profiling revealed enrichment for tumor intrinsic pathways consistent with suppression of tumor-specific immunity and support of tumor progression. Oncolytic viral treatment induced an intratumoral mRNA transcription signature of tumor-specific immune responses in 83% (5/6) of canine glioma patients. Changes included mRNA signatures corresponding with interferon signaling, lymphoid and myeloid cell activation, recruitment, and T and B cell immunity. Multiplexed protein analysis identified a subset of oligodendroglioma subjects with increased concentrations of IL-2, IL-7, IL-6, IL-10, IL-15, TNFα, GM-CSF between 14 and 28 days after treatment, with evidence of CD4+ T cell activation and modulation of IL-4 and IFNγ production in CD4+ and CD8+ T cells isolated from peripheral blood. Conclusion: These findings indicate that M032 modulates the tumor-immune microenvironment in the canine glioma model.

Evidence of PKC Binding and Translocation to Explain the Anticancer Mechanism of Chlorogenic Acid in Breast Cancer Cells.

Chlorogenic acid (CGA) exhibits potentials towards liver, breast and skin cancer. Cancer cells stimulated with CGA exhibits differential expression of transcriptional factors and regulatory molecules but the molecular target of the molecule is not known. Superposition of biophoric elements of CGA with Curcumin gives maximum common substructure score of 0.90. Molecular modeling studies further suggest that CGA fits into the C1b domain of PKC with extensive interaction with residues lining binding site. It binds PKC in a concentration dependent manner with dissociation constant KD, 28.84±3.95 µM. PKC-CGA complex is stable with minimal distortion to the 3-D structure and maintains the hydrogen bonding between ligand and receptor during simulation period. Cells stimulated with CGA causes 12.1 ± 0.56% PKC translocation from the cytosol to the plasma membrane. It disturbs the cell cycle and arrest the cancer cell at the G1 phase with a reduction in S-phase. Chlorogenic acid exhibits killing of cancer cells in a dose-dependent manner with an IC50 of 75.88 ± 4.54µg/ml and 52.5 ± 4.72µg/ml towards MDAMB-231 and MCF-7 cells respectively. It induces apoptosis in cancer cells as evident by AO/EtBr staining and degradation of genomic DNA to give a laddering pattern. Apoptosis in cancer cells involves mitochondrial pathway as supported by a reduction in mitochondrial potentials and release of cyt-C into the cytosol. Hence, the current study has established PKC as an important signaling molecule to the observed anti-cancer effects of CGA and provides the impetus to design better CGA analogs for improved anti-cancer potential against the malignant tumor.

3-Amino-4-aminoximidofurazan derivatives: small molecules possessing antimicrobial and antibiofilm activity against Staphylococcus aureus and Pseudomonas aeruginosa.

AIM: The therapeutic treatment of microbial infections involving biofilm becomes quite challenging because of its increasing antibiotic resistance capacities. Towards this direction, in the present study we have evaluated the antibiofilm property of synthesized 3-amino-4-aminoximidofurazan compounds having polyamine skeleton. These derivatives were synthesized by incorporating furazan and biguanide moieties. METHODS AND RESULTS: Different 3-amino-4-aminoximidofurazan derivatives (PI1-4) were synthesized via protic acid catalysis and subsequently characterized by (1) H NMR and (13) C NMR spectra, recorded at 400 and 100 MHz respectively. We have tested the antimicrobial and antibiofilm activities of these synthetic derivatives (PI1-4) against both Staphylococcus aureus and Pseudomonas aeruginosa. The compounds so tested were also compared with standard antibiotics namely Tobramycin (Ps. aeruginosa) and Azithromycin (Staph. aureus) which were used as a positive control in all experimental sets. All these compounds (PI1-4) exhibited moderate to significant antimicrobial activities against both micro-organisms wherein compound PI3 showed maximum activity. Biofilm inhibition of both micro-organisms was then evaluated by crystal violet and safranin staining, estimation of biofilm total protein and microscopy methods using sub-MIC dose of these compounds. Results showed that all compounds executed anti biofilm activity against both Staph. aureus and Ps. aeruginosa wherein compound PI3 exhibited maximum activity. In relation with microbial biofilm inhibition, we have observed reduction in bacterial motility, proteolytic activity and secreted exo-polysaccharide (EPS) from both Staph. aureus and Ps. aeruginosa when they were grown in presence of these compounds. While addressing the issue of toxicity on host, we have observed that these molecules exhibited minimum level of R.B.C degradation. CONCLUSION: These findings establish the antibacterial and anti biofilm properties of 3-amino-4-aminoximidofurazan derivatives (PI1-4). SIGNIFICANCE AND IMPACT OF THE STUDY: Therefore, our current findings demonstrate that 3-amino-4-aminoximidofurazan derivatives (PI1-4) may hold promise to be effective biofilm and microbial inhibitors that may be clinically significant.

Drosophila KASH-domain protein Klarsicht regulates microtubule stability and integrin receptor localization during collective cell migration.

During collective migration of the Drosophila embryonic salivary gland, cells rearrange to form a tube of a distinct shape and size. Here, we report a novel role for the Drosophila Klarsicht-Anc-Syne Homology (KASH) domain protein Klarsicht (Klar) in the regulation of microtubule (MT) stability and integrin receptor localization during salivary gland migration. In wild-type salivary glands, MTs became progressively stabilized as gland migration progressed. In embryos specifically lacking the KASH domain containing isoforms of Klar, salivary gland cells failed to rearrange and migrate, and these defects were accompanied by decreased MT stability and altered integrin receptor localization. In muscles and photoreceptors, KASH isoforms of Klar work together with Klaroid (Koi), a SUN domain protein, to position nuclei; however, loss of Koi had no effect on salivary gland migration, suggesting that Klar controls gland migration through novel interactors. The disrupted cell rearrangement and integrin localization observed in klar mutants could be mimicked by overexpressing Spastin (Spas), a MT severing protein, in otherwise wild-type salivary glands. In turn, promoting MT stability by reducing spas gene dosage in klar mutant embryos rescued the integrin localization, cell rearrangement and gland migration defects. Klar genetically interacts with the Rho1 small GTPase in salivary gland migration and is required for the subcellular localization of Rho1. We also show that Klar binds tubulin directly in vitro. Our studies provide the first evidence that a KASH-domain protein regulates the MT cytoskeleton and integrin localization during collective cell migration.

Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma.

Combined treatment using adenoviral (Ad)-directed enzyme/prodrug therapy and radiation therapy has the potential to become a powerful method of cancer therapy. We have developed an Ad vector encoding a mutant bacterial cytosine deaminase (bCD) gene (AdbCD-D314A), which has a higher affinity for cytosine than wild-type bCD (bCDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdbCD-D314A with the prodrug 5-fluorocytosine (5-FC) and ionizing radiation against human glioma. The present study demonstrates that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Furthermore, a significant increase in cytotoxicity following AdbCD-D314A and radiation treatment of glioma cells in vitro was demonstrated as compared to AdbCDwt. Animal studies showed significant inhibition of subcutaneous or intracranial tumor growth of D54MG glioma xenografts by the combination of AdbCD-D314A/5-FC with ionizing radiation as compared with either agent alone, and with AdbCDwt/5-FC plus radiation. The results suggest that the combination of AdbCD-D314A/5-FC with radiation produces markedly increased cytotoxic effects in cancer cells in vitro and in vivo. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for cancer therapy.

Combination of cytosine deaminase suicide gene expression with DR5 antibody treatment increases cancer cell cytotoxicity.

Combined treatment using adenoviral-directed enzyme/prodrug therapy and immunotherapy has the potential to become a powerful alternative method of cancer therapy. We have developed adenoviral vectors encoding the cytosine deaminase gene (Ad-CD) and cytosine deaminase:uracil phosphoribosyltransferase fusion gene (Ad-CD:UPRT). A monoclonal antibody, TRA-8, specifically binds to death receptor 5, one of two death receptors bound by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of Ad-CD:UPRT and TRA-8 against human pancreatic cancer and glioma cell lines. The present study demonstrates that Ad-CD:UPRT infection resulted in increased 5-FC-mediated cell killing, compared with Ad-CD. Furthermore, a significant increase of cytotoxicity following Ad-CD:UPRT/5-FC and TRA-8 treatment of cancer cells in vitro was demonstrated. Animal studies showed significant inhibition of tumor growth of MIA PaCa-2 pancreatic and D54MG glioma xenografts by the combination of Ad-CD:UPRT/5-FC plus TRA-8 as compared with either agent alone or no treatment. The results suggest that the combination of Ad-CD:UPRT/5-FC with TRA-8 produces an additive cytotoxic effect in cancer cells in vitro and in vivo. These data indicate that combined treatment with enzyme/prodrug therapy and TRAIL immunotherapy provides a promising approach for cancer therapy.

Prevalence of type 2 diabetes mellitus, other cardiovascular risk factors, and cardiovascular disease in Turkish and Moroccan immigrants in North West Europe: a systematic review.

BACKGROUND: The prevalence of diabetes, other cardiovascular risk factors, and cardiovascular morbidity and mortality varies between immigrant groups in Western societies, but epidemiological data on these topics are scarce for Turks and Moroccan immigrant living in North West Europe. METHODS: Medline and Embase were systematically searched for studies containing data on the prevalence of diabetes, cardiovascular risk factors, and cardiovascular morbidity and mortality in Turkish or Moroccan immigrants living in Northwestern European countries. RESULTS: Eighteen studies were identified. Corresponding findings were a high prevalence of type 2 diabetes in Turkish and Moroccan immigrants, a high prevalence of smoking among Turkish men, and a very low prevalence of smoking in Moroccan women compared to the indigenous population. Because of lack of valid studies, no definite conclusions could be drawn for in particular blood pressure and lipids. One German study showed exceptionally lower cardiovascular mortality rates in Turkish immigrants. CONCLUSION: The reviewed studies yielded insufficient evidence for a good quality comparison of the cardiovascular risk profile between Turkish and Moroccan immigrants and indigenous populations. Diabetes mellitus was more prevalent in Turkish and Moroccan immigrants, smoking more prevalent in Turkish males, and very rare in Moroccan females.

Ethnic specific recommendations in clinical practice guidelines: a first exploratory comparison between guidelines from the USA, Canada, the UK, and the Netherlands.

OBJECTIVES: To investigate whether clinical practice guidelines in different countries take ethnic differences between patients into consideration and to assess the scientific foundation of such ethnic specific recommendations. DESIGN: Analysis of the primary care sections of clinical practice guidelines. SETTING: Primary care practice guidelines for type 2 diabetes mellitus, hypertension, and asthma developed in the USA, Canada, the UK, and the Netherlands. MAIN OUTCOME MEASURES: Enumeration of the ethnic specific information and recommendations in the guidelines, and the scientific basis and strength of this evidence. RESULTS: Different guidelines do address ethnic differences between patients, but to a varying extent. The USA guidelines contained the most ethnic specific statements and the Dutch guidelines the least. Most ethnic specific statements were backed by scientific evidence, usually arising from descriptive studies or narrative reviews. CONCLUSION: The attention given to ethnic differences between patients in clinical guidelines varies between countries. Guideline developers should be aware of the potential problems of ignoring differences in ethnicity.

[Less ethnic knowledge in the Dutch College of General Practitioner's practice guidelines on type 2 diabetes mellitus, hypertension and asthma in adults than in the supporting literature]. / Minder etnische kennis in de standaarden van het Nederlands Huisartsen Genootschap over diabetes mellitus type 2, hypertensie en over astma bij volwassenen dan in de onderbouwende literatuur.

OBJECTIVE: To assess whether ethnic differences present in the scientific literature used as the basis for the Dutch College of General Practitioner's (NHG) practice guidelines were reflected in the ethnic-specific information the guidelines contained. DESIGN: Analysis of published information. METHOD: The scientific literature used as the basis for the guidelines about type 2 diabetes mellitus, hypertension and asthma in adults was collected and carefully screened. Relevant ethnic-specific information was compared to the content of the guidelines. RESULTS: Several relevant ethnic differences were stated in the scientific literature used as the basis for the guidelines. Differences in prevalence and clinical progress were stated for type 2 diabetes mellitus, differences in lung-volume were stated for asthma and differences in prevalence, onset, complications, response to pharmacological treatment and dietary salt restriction were stated for hypertension. The type 2 diabetes mellitus guideline stated a higher prevalence of diabetes in Hindustani people and recommended earlier screening in this group. The asthma guideline stated that the lung volume is dependent of ethnicity. The hypertension guideline did not state any ethnic-specific information. CONCLUSION: The guidelines on type 2 diabetes mellitus, hypertension and asthma in adults only adopted a limited number of the ethnic differences contained in the scientific literature on which they were based. Possible explanations are that information was only included if there was a clear scientific basis, and that ethnic distinctions were found to be politically and socially undesirable. However, this lack of information might lead to ineffective or sub-optimal care for ethnic minorities.

Non-invasive gamma camera imaging of gene transfer using an adenoviral vector encoding an epitope-tagged receptor as a reporter.

A model epitope-tagged receptor was constructed by fusing the hemagglutinin (HA) sequence on the extracellular N-terminus of the human somatostatin receptor subtype 2 (hSSTr2) gene. This construct was placed in an adenoviral (Ad-HAhSSTr2) vector. This study evaluated Ad-HAhSSTr2 in vitro and in vivo using FACS, fluorescent microscopy, radioactive binding assays, and gamma camera imaging techniques. Infection of A-427 non-small cell lung cancer cells with Ad-HAhSSTr2 or Ad-hSSTr2 resulted in similar expression of hSSTr2 by FACS analysis and binding assays using a (99m)Tc-labeled somatostatin analogue ((99m)Tc-P2045). HAhSSTr2 expression in A-427 cells was specific for infection with Ad-HAhSSTr2. FITC-labeled anti-HA antibody (FITC-HA) confirmed surface expression in live A-427 cells and the absence of internalization. Gamma camera imaging and gamma counter analysis of normal mice showed significantly greater (P<0.05) liver uptake of (99m)Tc-labeled anti-HA antibody ((99m)Tc-anti-HA) in mice injected i.v. 48 h earlier with Ad-HAhSSTr2 (53.6+/-6.9% ID/g) as compared to mice similarly injected with Ad-hSSTr2 (9.0+/-1.3% ID/g). In a mouse tumor model, imaging detected increased tumor localization of (99m)Tc-anti-HA due to direct intratumor injection Ad-HAhSSTr2. Gamma counter analysis confirmed significantly greater (P<0.05) uptake of (99m)Tc-anti-HA in tumors injected with Ad-HAhSSTr2 (12.5+/-4.1% ID/g) as compared to Ad-hSSTr2-infected tumors (5.1+/-1.5% ID/g). These studies demonstrate the feasibility of using an epitope-tagged reporter receptor for non-invasively imaging gene transfer.

Mu and IS1 transpositions exhibit strong orientation bias at the Escherichia coli bgl locus.

The region upstream of the Escherichia coli bgl operon is an insertion hot spot for several transposons. Elements as distantly related as Tn1, Tn5, and phage Mu home in on this location. To see what characteristics result in a high-affinity site for transposition, we compared in vivo and in vitro Mu transposition patterns near the bgl promoter. In vivo, Mu insertions were focused in two narrow zones of DNA near bgl, and both zones exhibited a striking orientation bias. Five hot spots upstream of the bgl cyclic AMP binding protein (CAP) binding site had Mu insertions exclusively with the phage oriented left to right relative to the direction of bgl transcription. One hot spot within the CAP binding domain had the opposite (right-to-left) orientation of phage insertion. The DNA segment lying between these two Mu hot-spot clusters is extremely A/T rich (80%) and is an efficient target for insertion sequences during stationary phase. IS1 insertions that activate the bgl operon resulted in a decrease in Mu insertions near the CAP binding site. Mu transposition in vitro differed significantly from the in vivo transposition pattern, having a new hot-spot cluster at the border of the A/T-rich segment. Transposon hot-spot behavior and orientation bias may relate to an asymmetry of transposon DNA-protein complexes and to interactions with proteins that produce transcriptionally silenced chromatin.

[Relation between ethnicity and reason for referral of new patients to the Internal Medicine outpatient clinic of the Academic Hospital Rotterdam-Dijkzigt, 1997]. / Samenhang tussen etniciteit en verwijsreden bij nieuwe patiënten op de polikliniek Inwendige Geneeskunde van het Academisch Ziekenhuis Rotterdam-Dijkzigt, 1997.

OBJECTIVE: To inventory the ethnic composition of the patients referred to an internal medicine outpatient clinic of a Dutch academic hospital and to determine to what extent ethnic minorities differ from Dutch patients in terms of referral reasons, taking relevant background characteristics into account. DESIGN: Cross-sectional analysis. METHOD: Data were collected on all new patients referred in 1997 for the first time to the internal medicine outpatient clinic of the Academic Hospital Dijkzigt, Rotterdam, the Netherlands, using the hospital information system (n = 3205). Patients were categorised into ethnic groups based on country of birth or name. Ethnic differences in referral reasons were tested for the 4 largest ethnic groups by means of logistic regression analysis with adjustment for age, sex, mean income of the zipcode area of the patients' residence and type of health insurance. RESULTS: The percentage of ethnic minorities amongst all referred patients was 22% (696/3205). The percentage of ethnic minorities among the patients referred from the catchment area of the outpatient clinic was 48% (209/440). Compared with Dutch patients Turkish patients were referred more often with stomach ache (odds ratio (OR): 4.26) and joint problems (OR: 7.16) as reasons. Moroccans were more often referred with stomach ache (OR: 4.10) and diabetes (OR: 4.51). Ethnic minorities were referred less often with dyslipidemia (Turks: OR: 0.11; Surinamese: OR: 0.17; Moroccans: 0 patients).

Phage Mu transposition immunity reflects supercoil domain structure of the chromosome.

Transposition immunity is the negative influence that the presence of one transposon sequence has on the probability of a second identical element inserting in the same site or in sites nearby. A transposition-defective Mu derivative (MudJr1) produced transposition immunity in both directions from one insertion point in the Salmonella typhimurium chromosome. To control for the sequence preference of Mu transposition proteins, Tn10 elements were introduced as targets at various distances from an immunity-conferring MudJr1 element. Mu transposition into a Tn10 target was not detectable when the distance of separation from MudJr1 was 5 kb, and transposition was unencumbered when the separation was 25 kb. Between 5 kb and 25 kb, immunity decayed gradually with distance. Immunity decayed more sharply in a gyrase mutant than in a wild-type strain. We propose that Mu transposition immunity senses the domain structure of bacterial chromosomes.

In vivo localization of [(111)In]-DTPA-D-Phe1-octreotide to human ovarian tumor xenografts induced to express the somatostatin receptor subtype 2 using an adenoviral vector.

Adenoviral vectors, encoding genes for cell surface antigens or receptors, have been used to induce their high level expression on tumor cells in vitro and in vivo. These induced antigens and receptors can then be targeted with radiolabeled antibodies or peptides for potential radiotherapeutic applications. The purpose of this study was to determine a dosing schema of an adenoviral vector encoding the human somatostatin receptor subtype 2 (AdCMVhSSTr2) for achieving the highest tumor localization of [(111)In]-DTPA-D-Phe1-octreotide, which binds to this receptor, in a human ovarian cancer model as a prelude to future therapy studies. AdCMVhSSTr2 was produced and used to induce hSSTr2 on A427 human nonsmall cell lung cancer cells and on SKOV3.ipl human ovarian cancer cells in vitro, as demonstrated by competitive binding assays using [125I]-Tyr1-somatostatin and [(111)In]-DTPA-D-Phe1-octreotide. Mice bearing i.p. SKOV3.ip1 tumors administered 1 x 10(9) plaque-forming units of AdCMVhSSTr2 i.p. 5 days after tumor cell inoculation, followed by an i.p. injection of [(111)In]-DTPA-D-Phe1-octreotide 2 days later, showed a range of 15.3-60.4% median injected dose/gram (ID/g) in tumor at 4 h after injection compared with 3.5% ID/g when [125I]-Tyr1-somatostatin was administered and 0.3% ID/g when the negative control peptide [125I]-mIP-bombesin was administered. Mice administered a control adenoviral vector encoding the gastrin-releasing peptide receptor did not have tumor localization of [(111)In]-DTPA-D-Phe1-octreotide (<1.6% ID/g), demonstrating specificity of [(111)In]-DTPA-D-Phe1-octreotide for the AdCMVhSSTr2 induced tumor cells. In another set of experiments, the tumor localization of [(111)In]-DTPA-D-Phe1-octreotide was not different 1, 2, or 4 days after AdCMVhSSTr2 injection (31.8, 37.7, and 40.7% ID/g, respectively; P = 0.88), indicating that multiple injections of radiolabeled peptide can be administered with equivalent uptake over a 4-day period. [(111)In]-DTPA-D-Phe1-octreotide tumor localization in animals administered AdCMVhSSTr2 on consecutive days or 2 days apart was 22.4% ID/g and 53.2% ID/g, respectively (P = 0.009) when [(111)In]-DTPA-D-Phe1-octreotide was given 1 day after the second AdCMVhSSTr2 injection. There was no difference in [(111)In]-DTPA-D-Phe1-octreotide localization after a single AdCMVhSSTr2 injection (40.7% ID/g) or two injections of AdCMVhSSTr2 given 1 (45.9% ID/g) or 2 (53.2% ID/g) days apart, where [(111)In]-DTPA-D-Phe1-octreotide was given in each case 4 days after the first AdCMVhSSTr2 injection (P = 0.65). Therefore, two AdCMVhSSTr2 injections did not increase [(111)In]-DTPA-D-Phe1-octreotide tumor localization compared with one injection, which eliminates concerns about an immune response to a second dose of AdCMVhSSTr2. This will be the basis for a therapeutic protocol with multiple administrations of an octreotide analogue labeled with a therapeutic radioisotope.

How is osmotic regulation of transcription of the Escherichia coli proU operon achieved? A review and a model.

The proU operon in enterobacteria encodes a binding-protein-dependent transporter for the active uptake of glycine betaine and L-proline, and serves an adaptive role during growth of cells in hyperosmolar environments. Transcription of proU is induced 400-fold under these conditions, but the underlying signal transduction mechanisms are incompletely understood. Increased DNA supercoiling and activation by potassium glutamate have each been proposed in alternative models as mediators of proU osmoresponsivity. We review here the available experimental data on proU regulation, and in particular the roles for DNA supercoiling, potassium glutamate, histone-like proteins of the bacterial nucleoid, and alternative sigma factors of RNA polymerase in such regulation. We also propose a new unifying model, in which the pronounced osmotic regulation of proU expression is achieved through the additive effects of at least three separate mechanisms, each comprised of a cis element [two promoters P1 and P2, and negative-regulatory-element (NRE) downstream of both promoters] and distinct trans-acting factors that interact with it: stationary-phase sigma factor RpoS with P1, nucleoid proteins HU and IHF with P2, and nucleoid protein H-NS with the NRE. In this model, potassium glutamate may activate proU expression through each of the three mechanisms whereas DNA supercoiling has a very limited role, if any, in the osmotic induction of proU transcription. We also suggest that proU may be a virulence gene in the pathogenic enterobacteria.

Use of epicardial pacing wires after coronary artery bypass surgery.

OBJECTIVE: To replicate a previous study that described the incidence and characteristics of patients after coronary artery bypass graft surgery who required the use of epicardial pacing wires and to explore the reasons for epicardial pacing wire use in this patient population. DESIGN: Ex post facto descriptive correlational. SETTING: Cardiothoracic intensive care and step down units of a 500-bed medical center. SUBJECTS: Convenience sample of 196 patients after coronary artery bypass graft surgery, 165 who did not use the epicardial pacing wires and 31 who used the epicardial pacing wires to augment cardiac output, diagnose dysrhythmias, suppress dysrhythmias, or treat heart block. Patients receiving other surgical techniques in combination with coronary artery bypass graft surgery were not included. PROCEDURE: Recording of demographic and clinical data for all of the sample population, with additional data collected when the epicardial pacing wires were used. DATA ANALYSIS: Independent t test and chi-square analysis were used to determine significance between the means and frequencies in the variables of the patients who used the epicardial pacing wires and those who did not. The significance level was set at 0.05. RESULTS: There were no statistically significant differences between the groups in terms of age or previous or recent myocardial infarction, which was opposite of the replicated study's findings. A statistically significant difference (p < 0.001) was found between the groups for the use of inotropic support, which was also opposite of the findings of that study. The group requiring epicardial pacing wire utilization demonstrated a greater need for diuretics in the preoperative phase than those who did not (p < 0.01), as well as a higher use of digitalis therapy before surgery (p < 0.01). Additionally, those who were paced experienced a greater cardiac output (p = 0.013) and cardiac index (p = 0.018) after pacing was initiated. CONCLUSIONS: The variation in findings between this study and the one replicated may be the result of variations in the patient populations, treatment practices, or preoperative condition. Replication of this study at a future date may reveal variables not identified here.

Evidence for involvement of proteins HU and RpoS in transcription of the osmoresponsive proU operon in Escherichia coli.

Transcription of the proU operon of Escherichia coli is induced several hundred-fold upon growth at elevated osmolarity, but the underlying mechanisms are incompletely understood. Three cis elements appear to act additively to mediate proU osmoresponsivity: (i) sequences around a promoter, P1, which is situated 250 bp upstream of the first structural gene proV; (ii) sequences around another (sigma 70-dependent) promoter, P2, which is situated 60 bp upstream of proV; and (iii) a negative regulatory element present within the proV coding region. These three cis elements are designated, respectively, P1R, P2R, and NRE. trans-acting mutants with partially derepressed proU expression have been obtained earlier, and a vast majority of the mutations affect the gene encoding the nucleoid protein HNS. In this study we employed a selection for trans-acting mutants with reduced proU+ expression, and we obtained a derivative that had suffered mutations in two separate loci designated dpeA and dpeB. The dpeB mutation caused a marked reduction in promoter P1 expression and was allelic to rpoS, the structural gene for the stationary-phase-specific sigma factor of RNA polymerase. Expression from P1 was markedly induced, in an RpoS-dependent manner, in stationary-phase cultures. In contrast to the behavior of the isolated P1 promoter, transcription from a construct carrying the entire proU cis-regulatory region (P1R plus P2R plus NRE) was not significantly affected by either growth phase or RpoS. The dpeA locus was allelic to hupB, which along with hupA encodes the nucleoid protein HU. hupA hupB double mutants exhibited a pronounced reduction in proU osmotic inducibility. HU appears to affect proU regulation through the P2R mechanism, whereas the effect of HNS is mediated through the NRE.