A Human Biofilm-Disrupting Monoclonal Antibody Potentiates Antibiotic Efficacy in Rodent Models of both Staphylococcus aureus and Acinetobacter baumannii Infections.

Many serious bacterial infections are antibiotic refractory due to biofilm formation. A key structural component of biofilm is extracellular DNA, which is stabilized by bacterial proteins, including those from the DNABII family. TRL1068 is a high-affinity human monoclonal antibody against a DNABII epitope conserved across both Gram-positive and Gram-negative bacterial species. In the present study, the efficacy of TRL1068 for the disruption of biofilm was demonstrated in vitro in the absence of antibiotics by scanning electron microscopy. The in vivo efficacy of this antibody was investigated in a well-characterized catheter-induced aortic valve infective endocarditis model in rats infected with a methicillin-resistant Staphylococcus aureus (MRSA) strain with the ability to form thick biofilms, obtained from the blood of a patient with persistent clinical infection. Animals were treated with vancomycin alone or in combination with TRL1068. MRSA burdens in cardiac vegetations and within intracardiac catheters, kidneys, spleen, and liver showed significant reductions in the combination arm versus vancomycin alone (P < 0.001). A trend toward mortality reduction was also observed (P = 0.09). In parallel, the in vivo efficacy of TRL1068 against a multidrug-resistant clinical Acinetobacter baumannii isolate was explored by using an established mouse model of skin and soft tissue catheter-related biofilm infection. Catheter segments infected with A. baumannii were implanted subcutaneously into mice; animals were treated with imipenem alone or in combination with TRL1068. The combination showed a significant reduction of catheter-adherent bacteria versus the antibiotic alone (P < 0.001). TRL1068 shows excellent promise as an adjunct to standard-of-care antibiotics for a broad range of difficult-to-treat bacterial infections.

Impact of Exposure of Methicillin-Resistant Staphylococcus aureus to Polyhexanide In Vitro and In Vivo.

Methicillin-resistant Staphylococcus aureus (MRSA) resistant to decolonization agents such as mupirocin and chlorhexidine increases the need for development of alternative decolonization molecules. The absence of reported severe adverse reactions and bacterial resistance to polyhexanide makes it an excellent choice as a topical antiseptic. In the present study, we evaluated the in vitro and in vivo capacity to generate strains with reduced polyhexanide susceptibility and cross-resistance with chlorhexidine and/or antibiotics currently used in clinic. Here we report the in vitro emergence of reduced susceptibility to polyhexanide by prolonged stepwise exposure to low concentrations in broth culture. Reduced susceptibility to polyhexanide was associated with genomic changes in the mprF and purR genes and with concomitant decreased susceptibility to daptomycin and other cell wall-active antibiotics. However, the in vitro emergence of reduced susceptibility to polyhexanide did not result in cross-resistance to chlorhexidine. During in vivo polyhexanide clinical decolonization treatment, neither reduced polyhexanide susceptibility nor chlorhexidine cross-resistance was observed. Together, these observations suggest that polyhexanide could be used safely for decolonization of carriers of chlorhexidine-resistant S. aureus strains; they also highlight the need for careful use of polyhexanide at low antiseptic concentrations.

The shell of the nucleus accumbens has a higher dopamine response compared with the core after non-contingent intravenous ethanol administration.

Dopamine increases in the nucleus accumbens after ethanol administration in rats, but the contributions of the core and shell subregions to this response are unclear. The goal of this study was to determine the effect of various doses of i.v. ethanol infusions on dopamine in these two subregions of the nucleus accumbens. Male Long-Evans rats were infused with either acute i.v. ethanol (0.5, 1.0, 1.5 g/kg), repeated i.v. ethanol (four 1.0 g/kg infusions resulting in a cumulative dose of 4.0 g/kg), or saline as a control for each condition. Dopamine and ethanol were measured in dialysate samples from each experiment. The in vivo extraction fraction for ethanol of probes was determined using i.v. 4-methylpyrazole, and was used to estimate peak brain ethanol concentrations after the infusions. The peak brain ethanol concentrations after the 0.5, 1.0 and 1.5 g/kg ethanol infusions were estimated to be 20, 49 and 57 mM, respectively. A significant dopamine increase was observed for the 0.5 g/kg ethanol group when collapsed across subregions. However, both the 1.0 g/kg and 1.5 g/kg ethanol infusions produced significant increases in dopamine levels in the shell that were significantly higher than those in the core. An ethanol dose-response effect on dopamine in the shell was observed when saline controls, 0.5, 1.0, and 1.5 g/kg groups were compared. For the cumulative-dosing study, the first, second, and fourth infusions resulted in significant increases in dopamine in the shell. However, these responses were not significantly different from one another. The results of this study show that the shell has a stronger response than the core to i.v. ethanol, that dopamine in the shell increases in a dose-dependent manner between 0.5-1.0 g/kg doses, but that the response to higher ethanol doses reaches a plateau.

City tuberculosis control coordinators' perspectives of patient adherence to DOT in São Paulo State, Brazil, 2005.

SETTING: Thirty-six priority cities in São Paulo State, Brazil, with a high incidence of tuberculosis (TB) cases, deaths and treatment default. OBJECTIVE: To identify the perspectives of city TB control coordinators regarding the most important components of adherence strategies adopted by health care teams to ensure patient adherence in 36 priority cities in the State of São Paulo, Brazil. DESIGN: Qualitative research with semi-structured interviews conducted with the coordinators of the National TB Control Programme involved in the management of TB treatment services in the public sector. RESULTS: The main issues thought to influence adherence to directly observed treatment (DOT) by coordinators include incentives and benefits delivered to patients, patient-health care worker bonding and comprehensive care, the encouragement given by others to follow treatment (family, neighbours and health professionals), and help provided by health professionals for patients to recover their self-esteem. CONCLUSION: The main aspects mentioned by city TB control coordinators regarding patient adherence to treatment and to DOT in São Paulo are improvements in communications, relationships based on trust, a humane approach and including the patients in the decision-making process concerning their health.

DARPP-32 and regulation of the ethanol sensitivity of NMDA receptors in the nucleus accumbens.

The medium spiny neurons of the nucleus accumbens receive both an excitatory glutamatergic input from forebrain and a dopaminergic input from the ventral tegmental area. This integration point may constitute a locus whereby the N-methyl-D-aspartate (NMDA)-subtype of glutamate receptors promotes drug reinforcement. Here we investigate how dopaminergic inputs alter the ethanol sensitivity of NMDA receptors in rats and mice and report that previous dopamine receptor-1 (D1) activation, culminating in dopamine and cAMP-regulated phosphoprotein-32 kD (DARPP-32) and NMDA receptor subunit-1 (NR1)-NMDA receptor phosphorylation, strongly decreases ethanol inhibition of NMDA responses. The regulation of ethanol sensitivity of NMDA receptors by D1 receptors was absent in DARPP-32 knockout mice. We propose that DARPP-32 mediated blunting of the response to ethanol subsequent to activation of ventral tegmental area dopaminergic neurons initiates molecular alterations that influence synaptic plasticity in this circuit, thereby promoting the development of ethanol reinforcement.

Short pulse laser train for laser plasma interaction experiments.

A multiframe, high-time resolution pump-probe diagnostic consisting of a consecutive train of ultrashort laser pulses (approximately ps) has been developed for use with a chirped pulse amplification (CPA) system. A system of high quality windows is used to create a series of 1054 nm picosecond-laser pulses which are injected into the CPA system before the pulse stretcher and amplifiers. By adding or removing windows in the pulse train forming optics, the number of pulses can be varied. By varying the distance and thickness of the respective optical elements, the time in between the pulses, i.e., the time in between frames, can be set. In our example application, the CPA pulse train is converted to 527 nm using a KDP crystal and focused into a preformed plasma and the reflected laser light due to stimulated Raman scattering is measured. Each pulse samples different plasma conditions as the plasma evolves in time, producing more data on each laser shot than with a single short pulse probe. This novel technique could potentially be implemented to obtain multiple high-time resolution measurements of the dynamics of physical processes over hundreds of picoseconds or even nanoseconds with picosecond resolution on a single shot.

The Medicago Genome Initiative: a model legume database.

The Medicago Genome Initiative (MGI) is a database of EST sequences of the model legume MEDICAGO: truncatula. The database is available to the public and has resulted from a collaborative research effort between the Samuel Roberts Noble Foundation and the National Center for Genome Resources to investigate the genome of M.truncatula. MGI is part of the greater integrated MEDICAGO: functional genomics program at the Noble Foundation (http://www.noble.org ), which is taking a global approach in studying the genetic and biochemical events associated with the growth, development and environmental interactions of this model legume. Our approach will include: large-scale EST sequencing, gene expression profiling, the generation of M.truncatula activation-tagged and promoter trap insertion mutants, high-throughput metabolic profiling, and proteome studies. These multidisciplinary information pools will be interfaced with one another to provide scientists with an integrated, holistic set of tools to address fundamental questions pertaining to legume biology. The public interface to the MGI database can be accessed at http://www.ncgr.org/research/mgi.

Superoxide dismutase, catalase, and glutathione peroxidase in red blood cells from patients with malignant diseases.

Superoxide dismutase (SOD), catalase, and glutathione peroxidase activities have been determined in red blood cells isolated from patients with acute myelogenous leukemia, chronic lymphocytic leukemia, Hodgkin's disease, lymphosarcoma, and various visceral cancers. In all investigated cases, both catalase and glutathione peroxidase were found to be in normal ranges of activity. In the group of patients with visceral cancers, SOD activity was found to be normal as well. In contrast, SOD activity was found to be significantly increased in red blood cells from patients with acute myelogenous leukemia and lymphoproliferative syndromes. This increase in superoxide level was not related to either reticulocytosis or hypochromic anemia. No relationship was found between the SOD level and the stage, the extension of the disease, or the presence of an inflammatory syndrome. The highest SOD levels were observed in untreated patients or during the early time period of the treatment. SOD levels further decrease as a function of the increase in the duration of the treatment. These results suggest an abnormality in the regulation of the expression of the SOD gene in the pluripotent stem cells.

Organization and structure of the human tissue transglutaminase gene.

Analysis of the genomic organization of tissue transglutaminase shows that the gene is 32.5 kilobases, contains 13 exons and 12 introns. Our results show that the sites for the two alternative splicing forms of tissue transglutaminase we reported earlier are located within exons 6 and 10 respectively. The 5'-upstream region of the gene has several potential regulatory promoter elements, and the 3'-exon contains about 50% of the total cDNA size and codes for the C-terminus of the protein. Alignment of deduced tissue transglutaminase amino acid sequence with other transglutaminases showed very similar intron splice positions.

A third human tissue transglutaminase homologue as a result of alternative gene transcripts.

A 2.4 kilobase (kb) cDNA encoding a new form of human tissue transglutaminase homologue (TGH2) was isolated from retinoic acid-induced human erythroleukemia cell (HEL) library. Full-length cDNA analysis gives an open reading frame coding for a polypeptide of 349 amino acid residues with a molecular mass of 38,700 Da. This variant differs from the previously reported homologue TGH in that it is 199 amino acids shorter and has an alternative, 63 amino acid COOH-terminal peptide. The 3'-untranslated region of the cDNA also differs from the previously reported sequences for both TGH and human tissue transglutaminase. The region coding for the first 286 amino acids of TGH2, which contains the active site is identical to TGH. Immunoprecipitation of the in vitro translation product from a synthetic TGH2 mRNA and immunoprecipitation of total protein of human heart, liver, kidney and cultured erythroleukemia HEL cell, revealed a protein with a molecular mass of 37,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the cDNA sequence for the previously known tissue transglutaminases with genomic DNA and the TGH2 cDNA described here indicate that the sequence divergence points correlate with known intron-exon boundaries. The smaller RNA species encode for truncated proteins with novel carboxyl termini. The TGH cDNA and the TGH2 cDNA both produce transcripts which start with the regular coding sequence for TGase and then fail to splice at specific donor sites, resulting in the use of an alternative exon that contains a stop codon.

Acid-labilization of sterols for extraction from yeast.

A wild type strain of yeast, Saccharomyces cerevisiae, pretreated with a mild acid hydrolysis, exhibited a 4-fold increase in sterol yield upon saponification and extraction. This increased yield is reflected in both major and minor sterols (ergosterol; zymosterol) and sterol esters.

New algorithm for the localization of accessory atrioventricular connections using a baseline electrocardiogram.

OBJECTIVES: In this study, we propose a new algorithm for accessory atrioventricular pathway localization using a 12-lead electrocardiogram (ECG). BACKGROUND: Radiofrequency catheter ablation produces a very discrete lesion, and ECG localization based on surgical dissection is obsolete. METHODS: Stepwise discriminant analysis was used to assess the relation of 18 pre-excited ECG (QRS duration > 100 ms) variables to the site of successful ablation in 93 patients. The most discriminating variables were combined to form rules for each location. The ECGs were retested by these rules to determine predictive accuracy. RESULTS: If the precordial QRS transition was at or before lead V1, the pathway had been ablated on the left side. If it was after lead V2, the pathway had been ablated on the right side. If the QRS transition was between leads V1 and V2 or at lead V2, then if the R wave amplitude in lead I was greater than the S wave by > or = 1.0 mV, it was right-sided; otherwise, it was left-sided (p < 0.0001, sensitivity 100%, specificity 97%). Right-side pathways. If the QRS transition was between leads V2 and V3, the pathway was right septal; if after lead V4, it was right lateral. If it was between leads V3 and V4, then if the delta wave amplitude in lead II was > or = 1.0 mV, it was right septal; otherwise, it was right lateral (p < 0.0001, sensitivity 97%, specificity 95%). In right lateral locations, if the delta wave frontal axis was > or = 0 degrees, or if it was < 0 degrees but the R wave amplitude in lead III was > or = 0 mV, it was anterolateral; otherwise, it was posterolateral (p < 0.0001, sensitivity 100%, specificity 87.3%). Anteroseptal pathways had a sum of delta wave polarities in leads II, III and aVF > or = +2(p < 0.0001, sensitivity 100%, specificity 100%). Posteroseptal pathways (inferior delta wave sum < or = -2) were less well discriminated from right midseptal pathways (inferior delta wave sum < or = 1 > or = -1) (p < 0.0001, sensitivity 76.5%, specificity 71%) [corrected]. Left-sided pathways. Two or more positive delta waves in the inferior leads or the presence of an S wave amplitude in lead aVL greater than the R wave, or both, discriminated left anterolateral pathways from posterior pathways (p < 0.001, sensitivity and specificity 100%). If the R wave in lead I was greater than the S wave by > or = 0.8 mV, and the sum of inferior delta wave polarities was negative, the location was posteroseptal; otherwise, it was posterolateral (p < 0.05, sensitivity 71.4%, specificity 100%). CONCLUSIONS: Using the algorithm derived, a right-sided accessory pathway can be reliably distinguished from one that is left-sided, right free wall from right septal, right anterolateral from posterolateral and anteroseptal from other right septal pathways. Left anterolateral pathways can be distinguished from left posterior pathways and left posterolateral pathways from left posteroseptal pathways.

N-methyl-D-aspartate mediated responses decrease with age in Fischer 344 rat brain.

N-Methyl-D-aspartate (NMDA) receptor-mediated responses were studied in hippocampus, cortex, and striatum of Fischer 344 rats of various ages (3-5, 12-14, or 24-28 months old; young, middle-aged, and senescent or old, respectively) to determine whether aging alters the function of NMDA receptors. NMDA-induced inhibition of muscarinic-stimulated phosphoinositide hydrolysis in hippocampus, and NMDA-stimulated release of [3H]norepinephrine (NE) or [3H]dopamine (DA) were used as indices of NMDA receptor function. The muscarinic agonist carbachol (1 mM) stimulated PI hydrolysis in hippocampi from all three age groups with no significant differences between the groups. NMDA inhibited the carbachol-evoked PI response in a concentration-dependent manner (10-100 microM) in all age groups. However, the NMDA-induced (100 microM) inhibition of the carbachol-stimulated response was markedly reduced in an age-dependent manner with losses of 25% and 53% in middle-aged and senescent rats compared to young. Concentration-effect curves for NMDA-stimulated [3H]NE release were determined using hippocampal and cortical slices from rats of the three age groups. In the hippocampus the maximal response for NMDA was significantly decreased from 6.55 fractional [3H]NE release in young to 4.51 and 4.18 in middle-aged and old rats, respectively, with no age-related changes in the potency of NMDA or slope of the curves. In cortical slices the maximal response was significantly reduced in an age-dependent manner by 23% in the senescent rats compared to the young rats. NMDA-stimulated [3H]DA release from striatal slices was significantly lower in the senescent rats at concentrations of NMDA from 500-2000 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

The cDNA sequence and infectious transcripts of peanut stripe virus.

A full-length cDNA clone of the blotch isolate of the peanut stripe potyvirus (PStV) RNA genome was constructed downstream from the bacteriophage SP6 RNA polymerase promoter. The full-length PStV cDNA clone (PStVSF9) was sequenced and compared to the previously published sequence of PStV-B. In vitro-synthesized PStV transcripts capped with m7GpppG were infectious in Nicotiana benthamiana plants, and the progeny virus was aphid transmissible. To confirm the origin of infection, a mutant PStV (PStVDAE), with a Gly14 to Glu mutation in the coat protein-encoding gene, was constructed. Transcripts from PStVDAE produced symptoms indistinguishable from native or PStVSF9 virus, but was not transmitted by aphids.

Genomic structure of rat liver aryl sulfotransferase IV-encoding gene.

This report contains the first description of the genomic structure for a sulfotransferase (ST). The gene (ASTIV) encodes rat hepatic aryl ST IV, also known as tyrosine-ester ST (EC 2.8.2.9). A phage genomic clone containing 70% of the 3' AST gene coding sequence was isolated after screening a rat genomic library with an ASTIV cDNA. The remaining 5' sequence was determined from a PCR product obtained from rat genomic DNA and ASTIV cDNA-specific primers. ASTIV spans 3.5 kb and contains eight exons and seven introns. The fourth intron of this gene contains sequences homologous to rodent B1 repetitive elements and an Alu repeat found in rat. An alignment of the primary structures of ten different ST revealed several conserved regions, as well as a putative binding site for the cofactor for enzymatic sulfation reactions, 3'-phosphoadenosine-5'-phosphosulfate.

Distribution of thorny excrescences on CA3 pyramidal neurons in the rat hippocampus.

Thorny excrescences are the postsynaptic components of synapses between mossy fibers of granule cells and dendrites of CA3 pyramidal neurons in the hippocampal formation. Very little quantitative data on the number and distribution of excrescences in adult rats are available because, first, the vast majority are grouped into clusters and it is not possible to identify single excrescences within these clusters at the light microscope level. Second, clusters are of varying lengths and are distributed over hundreds of micrometers, making ultrastructural analysis prohibitively time-consuming. Here, by using three-dimensional analysis techniques at the light microscope level, we quantified the number, length, and distribution of excrescence clusters on proximal and midfield pyramidal neurons in the rat. Results indicated that proximal neurons had similar numbers of clusters on their apical and basal trees, and that cluster length was also similar. In contrast, midfield neurons had more apical than basal clusters, and apical clusters were longer. For neurons in both regions, basal clusters were located about 50% closer to somata. Overall, proximal neurons had more clusters than did midfield neurons, but the clusters were shorter; thus, proximal and midfield neurons had about the same total cluster length, and presumably the same number of single excrescences. Based on these data and on published ultrastructural measurements of single excrescences, we estimated an average of 41 excrescences/neuron, and suggest that a pyramidal neuron can be contacted by a maximum of 41 mossy fiber boutons, each from a different granule cell.

Alcohol-induced inhibition of N-methyl-D-aspartate-evoked release of [3H]norepinephrine from brain is related to lipophilicity.

A series of short chain alkanols were studied for their effects on N-methyl-D-aspartate (NMDA)-evoked release [3H]norepinephrine (NE) from slices of cortex of the rat. Methanol, ethanol, isopropanol, n-propanol, n-butanol and isoamyl alcohol inhibited NMDA-stimulated release of [3H]NE in a concentration-dependent manner, while having no effect on non-stimulated release of [3H]NE. The inhibitory potencies of the alkanols varied with the shorter chain alkanols, such as methanol, being less potent than the longer chain alkanols, such as isoamyl alcohol. Direct comparison of the effects of 100 mM methanol, ethanol, isopropanol, n-propanol and n-butanol indicated that increasing the chain length led to a greater efficacy for producing inhibition of NMDA-stimulated release of [3H]NE. A plot of the log IC50 values, versus the log of the membrane/buffer partition coefficients for the various alkanols was linear, indicating that lipophilicity played some role in the inhibitory effect. The alkanols did not significantly depress the release of [3H]NE stimulated by 25 mM KCl, in the presence of 300 microM 2-amino-5-phosphonopentanoic acid (AP-5), suggesting that the alkanols have a selective effect at the NMDA receptor, as opposed to altering the release of neurotransmitter at the nerve terminal. The inhibitory effects of the alcohols were reversible, which suggests that the alcohols were not causing non-specific toxic effects on the slices. It is concluded that the inhibitory effect of ethanol and related short chain alkanols on NMDA-stimulated release of [3H]NE involves an interaction with a lipophilic target, at or near the NMDA receptor-ionophore complex.

Electrophysiologic testing in the management of patients with the Wolff-Parkinson-White syndrome and atrial fibrillation.

Twenty patients with the Wolff-Parkinson-White (WPW) syndrome and 1 or more episodes of symptomatic atrial fibrillation (AF) due to rapid anterograde bypass tract conduction underwent electrophysiologic testing. The mean ventricular rate during spontaneous AF was 242 +/- 56 beats/min (+/- standard deviation) and the shortest preexcited R-R interval was 194 +/- 40 ms. Six patients underwent surgical bypass tract ablation and 14 were treated medically, based on the results of electropharmacologic testing. Over a mean follow-up period of 35 +/- 19 months (+/- standard deviation), only 1 patient treated medically had a recurrence of minimally symptomatic AF. The successful chemoprophylaxis of symptomatic AF was associated with the inability to induce AF and atrioventricular reciprocating tachycardia during drug testing (7 patients) or with the induction of AF with a ventricular rate less than 200 beats/min and a shortest preexcited R-R interval of greater than 250 ms (7 patients). Electrophysiologic testing can identify a subgroup of patients with WPW and AF in whom medical therapy is a suitable alternative to bypass tract ablation.

A salivary vasodilator in the blood-sucking bug, Rhodnius prolixus.

1. Salivary gland homogenates of the blood-sucking bug, Rhodnius prolixus induced transient, dose-dependent relaxation of rabbit aortic preparations pre-constricted with 200 ng ml-1 noradrenaline, 1 microgram ml-1 histamine or 20 ng ml-1 angiotensin II. Such relaxations were less marked when the aorta was constricted by 60 mM KC1. These effects were observed with as little as 0.2 microgram ml-1 of crude salivary gland protein. 2. The vasodilator effect was endothelium-independent, abolished by 50 microM hydroquinone or 50 microM methylene blue, and potentiated by 30 mu ml-1 superoxide dismutase. 3. Salivary homogenates generated a coloured compound when reacted with sulfanilic acid in the presence of N-(1-naphthyl)-ethylediamine, indicating the presence of reactive nitrogen groups, equivalent to 35 +/- 3 ng of sodium nitrite per pair of glands. 4. Molecular sieving high performance liquid chromatography of salivary gland homogenates generated a single peak of vasorelaxant activity which coincided with the presence of platelet antiaggregating and spasmolytic (guinea-pig ileum contracted with histamine) activities, as well as with reactive nitrogen groups. 5. It is concluded that a protein of molecular weight 16.500 daltons in the salivary glands of R. prolixus contains reactive nitrogen groups which assist the bug during a blood meal. It is suggested that saliva of blood sucking anthropods is a natural resource of novel pharmacological activities.