In vitro susceptibility of equine-obtained isolates of Corynebacterium pseudotuberculosis to gallium maltolate and 20 other antimicrobial agents.

This study's objective was to determine the in vitro antimicrobial activities of gallium maltolate (GaM) and 20 other antimicrobial agents against clinical equine isolates of Corynebacterium pseudotuberculosis. The growth of cultured isolates was not inhibited by any concentration of GaM. MIC data revealed susceptibility to commonly used antimicrobials.

Efficacy of gallium maltolate against Lawsonia intracellularis infection in a rabbit model.

Antimicrobial efficacy against Lawsonia intracellularis is difficult to evaluate in vitro, thus, the effects of gallium maltolate's (GaM) were investigated in a rabbit model for equine proliferative enteropathy (EPE). Juvenile (5-6-week-old) does were infected with 3.0 × 10(8) L. intracellularis/rabbit and allocated into three groups (n = 8). One week postinfection, one group was treated with GaM, 50 mg/kg; one, with doxycycline, 5 mg/kg; and one with a sham-treatment (control). Feces and blood were collected daily and weekly, respectively, to verify presence of L. intracellularis fecal shedding using qPCR, and seroconversion using immunoperoxidase monolayer assay. Rabbits were sacrificed after 1 week of treatment to collect intestinal tissues focusing on EPE-affected sections. Intestinal lesions were confirmed via immunohistochemistry. No difference was noted between treatments regarding EPE-lesions in jejunum (P = 0.51), ileum (P = 0.74), and cecum (P = 0.35), or in L. intracellularis fecal shedding (P = 0.64). GaM and doxycycline appear to have similar efficacy against EPE in infected rabbits.

Pharmacokinetics of gallium maltolate in Lawsonia intracellularis-infected and uninfected rabbits.

Oral gallium maltolate (GaM) pharmacokinetics (PK) and intestinal tissue (IT) concentrations of elemental gallium ([Ga]) and iron ([Fe]) were investigated in a rabbit model of equine proliferative enteropathy (EPE). New Zealand white does (uninfected controls and EPE-infected, n = 6/group) were given a single oral GaM dose (50 mg/kg). Serial blood samples were collected from 0 to 216 h post-treatment (PT) and IT samples after euthanasia. Serology, qPCR, and immunohistochemistry confirmed, or excluded, EPE. Blood and IT [Ga] and [Fe] were determined using inductively coupled plasma-mass spectrometry. PK parameters were estimated through noncompartmental approaches. For all statistical comparisons on [Ga] and [Fe] α = 5%. The Ga log-linear terminal phase rate constant was lower in EPE rabbits vs. uninfected controls [0.0116 ± 0.004 (SD) vs. 0.0171 ± 0.0028 per hour; P = 0.03]; but half-life (59.4 ± 24.0 vs. 39.4 ± 10.8 h; P = 0.12); Cmax (0.50 ± 0.21 vs. 0.59 ± 0.42 µg/mL; P = 0.45); tmax (1.75 ± 0.41 vs. 0.9 ± 0.37 h; P = 0.20); and oral clearance (6.743 ± 1.887 vs. 7.208 ± 2.565 L/h; P = 0.74) were not. IT's [Ga] and [Fe] were higher (P < 0.0001) in controls. In conclusion, although infection reduces IT [Ga] and [Fe], a 48 h GaM dosing interval is appropriate for multidose studies in EPE rabbits.

Evaluation of the effect of gallium maltolate on fecal Salmonella shedding in cattle.

Strategies aimed at reducing fecal shedding of Salmonella and other foodborne pathogens may be effective for limiting transmission of pathogens from food animals to humans. The objective of this study was to determine the effectiveness of gallium maltolate (GaM) against Salmonella in vitro and to determine whether oral administration of GaM would reduce fecal shedding of Salmonella in cattle. Gallium is a semimetal exhibiting antimicrobial properties against some pathogenic bacteria, including Salmonella, by exploiting their need for iron to survive and replicate. In vitro growth studies were performed in pure cultures of Salmonella and in mixed cultures from ruminal fluid. Inclusion of GaM in culture medium or in mixed cultures of ruminal fluid resulted in a significant reduction in growth of Salmonella, suggesting that GaM may be effective for limiting growth and survival in vivo. Therefore, we subsequently administered two doses of GaM to Holstein steers, experimentally infected them with Salmonella, and quantitatively and qualitatively monitored fecal shedding at 12-h intervals. Sixty hours after beginning treatment, cattle were euthanized, and luminal contents and tissue were aseptically harvested from the rumen, jejunum, spiral colon, cecum, and rectum. The luminal contents were processed for quantitative and qualitative analysis of the challenge strains of Salmonella, and tissue samples were enriched and plated for qualitative analysis. We found no significant differences between control and treated animals in quantitative levels of Salmonella in the feces or the luminal contents. Likewise, we observed no pattern between control and treated animals in the frequency of positive or negative results from enriched feces, luminal contents, or tissue samples. These results suggest that GaM was not effective for reducing Salmonella in cattle.

Pharmacokinetics of an orally administered methylcellulose formulation of gallium maltolate in neonatal foals.

Gallium is a trivalent semi-metal with anti-microbial effects because of its incorporation into crucial iron-dependent reproductive enzyme systems. Gallium maltolate (GaM) provides significant gallium bioavailability to people and mice following oral administration and to neonatal foals following intragastric administration. To study the prophylactic and therapeutic effects of GaM against Rhodococcus equi pneumonia in foals, we developed a methylcellulose formulation of GaM (GaM-MCF) for oral administration to neonatal foals. Normal neonatal foals were studied. Six foals received 20 mg/kg and another six foals received 40 mg/kg of GaM-MCF orally. Serial serum samples were collected and serum gallium concentrations were determined using inductively coupled plasma mass spectroscopy. Gallium was rapidly absorbed (T(max) of 4 h), and a mean C(max) of 0.90 or 1.8 microg/mL was achieved in foals receiving 20 or 40 mg/kg respectively. Marked variability existed in C(max) among foals: only half of the foals receiving 20 mg/kg attained serum concentrations of >0.7 microg/mL, a level suggested to be therapeutic against R. equi by previous studies. Mean elimination half-life was 32.8 or 32.4 h for foals receiving 20 or 40 mg/kg respectively. The results of this study suggest that at least 30 mg/kg orally every 24 h should be considered in future pharmacodynamic and efficacy studies.

Antimicrobial activity of gallium against virulent Rhodococcus equiin vitro and in vivo.

Rhodococcus equi, a facultative intracellular bacterium, causes severe pneumonia in foals. Evidence suggests that most foals become infected very early in life, when they have immature or ineffective innate immune responses. This study evaluated the antimicrobial activity of gallium against R. equi, as a potential chemoprophylactic and therapeutic agent. Rhodococcus equi was grown in media with various concentrations of gallium nitrate (GN), with and without excess iron. GN significantly inhibited growth and killed R. equi, and these effects were abolished with excess iron. Antimicrobial effects of Ga appear to be related to its interference with iron metabolism. Mice were treated orally with gallium maltolate (GaM), 10 or 50 mg/kg BW, or distilled H2O prior to and after experimental infection with R. equi. Six days post-infection, organs were harvested and R. equi concentrations assessed, and serum gallium concentrations determined. GaM was absorbed in a dose-dependent manner, and R. equi tissue burdens were greater in control mice than in all GaM-treated mice. GaM may aid in the control of disease by preventing development of overwhelming R. equi tissue burdens prior to the establishment of requisite innate and adaptive immune responses.

A new analytical scale DNA affinity binding assay for analyses of specific protein-DNA interactions.

We describe a rapid analytical assay for identification of proteins binding to specific DNA sequences. The DAPSTER assay (DNA affinity preincubation specificity test of recognition assay) is a DNA affinity chromatography-based microassay that can discriminate between specific and nonspecific protein-DNA interactions. The assay is sensitive and can detect protein-DNA interactions and larger multicomponent complexes that can be missed by other analytical methods. Here we describe in detail the optimization and utilization of the DAPSTER assay to isolate AP-1 complexes and associated proteins in multimeric complexes bound to the AP-1 DNA element.

Auditory processing of interaural timing information: new insights.

Differences in the time-of-arrival of sounds at the two ears, or interaural temporal disparities (ITDs), constitute one of the major binaural cues that underlie our ability to localize sounds in space. In addition, ITDs contribute to our ability to detect and to discriminate sounds, such as speech, in noisy environments. For low-frequency signals, ITDs are conveyed primarily by "cycle-by-cycle" disparities present in the fine-structure of the waveform. For high-frequency signals, ITDs are conveyed by disparities within the time-varying amplitude, or envelope, of the waveform. The results of laboratory studies conducted over the past few decades indicate that ITDs within the envelopes of high-frequency are less potent than those within the fine-structure of low-frequency stimuli. This is true for both measures of sensitivity to changes in ITD and for measures of the extent of the perceived lateral displacement of sounds containing ITDs. Colburn and Esquissaud (1976) hypothesized that it is differences in the specific aspects of the waveform that are coded neurally within each monaural (single ear) channel that account for the greater potency of ITDs at low frequencies rather than any differences in the more central binaural mechanisms that serve these different frequency regions. In this review, the results of new studies are reported that employed special high-frequency "transposed" stimuli that were designed to provide the high-frequency channels of the binaural processor with envelope-based information that mimics waveform-based information normally available only in low-frequency channels. The results demonstrate that these high-frequency transposed stimuli (1) yield sensitivity to ITDs that approaches, or is equivalent to, that obtained with "conventional" low-frequency stimuli and (2) yield large extents of laterality that are similar to those measured with conventional low-frequency stimuli. These findings suggest that by providing the high-frequency channels of the binaural processor with information that mimics that normally available only at low frequencies, the potency of ITDs in the two frequency regions can be made to be similar, if not identical. These outcomes provide strong support for Colburn and Esquissaud's (1976) hypothesis. The use of high-frequency transposed stimuli, in both behavioral and physiological investigations offers the promise of new and important insights into the nature of binaural processing.

Ten ERK-related proteins in three distinct classes associate with AP-1 proteins and/or AP-1 DNA.

We have identified seven ERK-related proteins ("ERPs"), including ERK2, that are stably associated in vivo with AP-1 dimers composed of diverse Jun and Fos family proteins. These complexes have kinase activity. We designate them as "class I ERPs." We originally hypothesized that these ERPs associate with DNA along with AP-1 proteins. We devised a DNA affinity chromatography-based analytical assay for DNA binding, the "nucleotide affinity preincubation specificity test recognition" (NAPSTER) assay. In this assay, class I ERPs do not associate with AP-1 DNA. However, several new "class II" ERPs do associate with DNA. p41 and p44 are ERK1/2-related ERPs that lack kinase activity and associate along with AP-1 proteins with AP-1 DNA. Class I ERPs and their associated kinase activity thus appear to bind AP-1 dimers when they are not bound to DNA and then disengage and are replaced by class II ERPs to form higher order complexes when AP-1 dimers bind DNA. p97 is a class III ERP, related to ERK3, that associates with AP-1 DNA without AP-1 proteins. With the exception of ERK2, none of the 10 ERPs appear to be known mitogen-activated protein kinase superfamily members.

Sensitivity to brief changes of interaural time and interaural intensity.

The purpose of this study was to measure listeners' abilities to detect brief changes in interaural temporal disparities (ITDs) or interaural intensitive disparities (IIDs) conveyed by bursts of noise (probes) temporally and symmetrically flanked by segments of diotic or uncorrelated noise. Thresholds were measured using a four-interval, two-alternative, forced-choice adaptive task and the total duration of the bursts of noise was either 20, 40, or 100 ms. Probes were temporally centered within each burst and the durations of the probes ranged from 2 to 100 ms, depending upon the duration of the (longer) total burst of noise within which they were embedded. The results indicate that, for a given total duration of noise, there is a rapid decrease in threshold ITD or threshold IID as the duration of the probe is increased so that it occupies a larger portion of the total burst of noise. Mathematical analyses revealed that both threshold ITDs and threshold IIDs could be well accounted for by assuming that the listener processes both types of binaural cues via a single, symmetric, double-exponential temporal window. Interestingly, the shapes of the temporal windows that fit the data obtained from the human listeners resemble the shapes of the temporal windows derived by Wagner [H. Wagner, J. Comp. Physiol. A 169, 281-289 (1991)], who studied the barn owl. The time constants and relative weightings yielded temporal window functions that heavily emphasize information occurring within the very temporal center of the window. This temporal window function was found to be generalizable in the sense that it also accounts for classic data reported by Grantham and Wightman [D.W. Gratham and F.L. Wightman, J. Acoust. Soc. Am. 63, 511-523 (1978)] concerning sensitivity to dynamically changing interaural disparities.

A consideration of the normalization that is typically included in correlation-based models of binaural detection.

An analysis of binaural detection and new data that elucidate the nature and precision of normalization that must be assumed if binaural detection is accomplished via mechanisms that effectively compute the coefficient of cross correlation is presented. Based on that analysis, it is argued that the precision of normalization required to remove deleterious effects resulting from variations in the levels of the stimuli is so great that it is highly unlikely that normalization, per se, actually occurs as part of binarual processing. Instead, it appears more likely that binaural processing is accomplished via "subtractive" mechanisms, such as the one originally described by Durlach [J. Acoust. Soc. Am. 35, 1206-1218 (1963)]. Within that framework, deleterious effects that could result from variations in the levels of the stimuli simply do not arise.

Manipulating the "straightness" and "curvature" of patterns of interaural cross correlation affects listeners' sensitivity to changes in interaural delay.

The purpose of this study was to test the hypothesis that stimuli characterized by "straight" trajectories of their patterns of cross correlation foster greater sensitivity to changes in interaural temporal disparities (ITDs) than do stimuli characterized by more "curved" trajectories of their patterns of cross correlation. To do so, sensitivity to changes in ITD was measured, as a function of duration, using a set of "reference" stimuli that yielded differing relative amounts of straightness within their patterns of cross correlation while keeping the dominant trajectory at or near midline. The relative amounts of straightness were manipulated by employing specific combinations of bandwidth, ITD, and interaural phase disparity (IPD) of Gaussian noises centered at 500 Hz. The results were consistent with expectations in that the patterning of the threshold ITDs revealed increasingly poorer sensitivity as greater and greater curvature was imposed on the dominant, "midline," trajectory. The variations in threshold ITD across the stimulus conditions can be accounted for quite well quantitatively by assuming either that the listeners based their judgments on changes in the position of the most central peak of the cross-correlation function or that they based their judgments on changes in the centroid of a second-level cross-correlation function. In a second experiment, binaural detection was measured using a subset of the reference stimuli as maskers. As expected, sensitivity was poorest with the maskers characterized by the greatest curvature, which were also those having the lowest interaural correlation.

The variation across time of sensitivity to interaural disparities: behavioral measurements and quantitative analyses.

Zurek (1980) measured listeners' sensitivities to interaural disparities conveyed by a 5-ms "probe" segment embedded within a 50-ms burst of otherwise diotic broadband noise [P. M. Zurek, J. Acoust. Soc. Am. 67, 952-964 (1980)]. He found that thresholds for interaural time delay (ITD) and interaural intensitive difference (IID) were markedly elevated when the onset of the probe segment occurred between 1 and 5 ms after the onset of the burst. Zurek postulated that this occurred because the leading portion of the noise briefly inhibited sensitivity to subsequent binaural information. If such inhibition were the primary factor responsible for the elevation in thresholds, then the omission of the portion of the noise trailing the probe segment would be expected to have little, if any, influence on performance. In order to test this hypothesis, listeners' sensitivities to ITD and IID were measured using a paradigm similar to that employed by Zurek. The results revealed that the omission of either the leading or the trailing portions of the diotic noise led to substantial reductions in threshold ITDs and IIDs. The data were successfully accounted for by a model based upon a combination of a temporal window with an equivalent rectangular duration of approximately 10 ms and a weighting function representing a brief loss of binaural sensitivity just after the onset of a sound.

Chemistry and pharmacokinetics of gallium maltolate, a compound with high oral gallium bioavailability.

Gallium maltolate, tris(3-hydroxy-2-methyl-4H-pyran-4-onato)gallium (GaM), is an orally active gallium compound for therapeutic use. It is moderately soluble in water (10.7 +/- 0.9 mg/mL at 25 composite functionC) with an octanol partition coefficient of 0.41+/-0.08. The molecule is electrically neutral in aqueous solution at neutral pH; a dilute aqueous solution (2.5 x10-(-5) M) showed little dissociation at pH 5.5-8.0. Single crystal X-ray diffraction analysis found the GaM molecule to consist of three maltolate ligands bidentately bound to a central gallium atom in a propeller-like arrangement, with one of the ligands disordered in two possible orientations. The compound is orthorhombic, space group Pbca, unit cell a = 16.675(3), b = 12.034(2), c = 18.435(2) A at 158K. GaM was administered to healthy human volunteers at single doses of 100, 200, 300, and 500 mg (three subjects per dose). GaM was very well tolerated. Oral absorption of Ga into plasma was fairly rapid (absorption half life = 0.8-2.0h), with a central compartment excretion half life of 17-21h. Absorption appeared dose proportional over the dosage range studied. Estimated oral gallium bioavailability was approximately 25-57%, based on comparison with published data on intravenous gallium nitrate. Urinary Ga excretion following oral GaM administration was approximately 2% of the administered dose over 72h, in contrast to 49-94% urinary Ga excretion over 24h following i.v. gallium nitrate administration. We suggest that oral administration of GaM results in nearly all plasma gallium being bound to transferrin, whereas i.v. administration of gallium nitrate results in formation of considerable plasma gallate [Ga(OH)(4) (-)], which is rapidly excreted in the urine. These data support the continued investigation of GaM as an orally active therapeutic gallium compound.

The normalized interaural correlation: accounting for NoS pi thresholds obtained with Gaussian and "low-noise" masking noise.

Recently, Eddins and Barber [J. Acoust. Soc. Am. 103, 2578-2589 (1998)] and Hall et al. [J. Acoust. Soc. Am. 103, 2573-2577 (1998)] independently reported that greater masking of interaurally phase-reversed (S pi) tones was produced by diotic low-noise noise than by diotic Gaussian noise. Based on quantitative analyses, Eddins and Barber suggested that their results could not be accounted for by assuming that listeners' judgments were based on constant-criterion changes in the normalized interaural correlation produced by adding the S pi signal to the diotic masker. In particular, they showed that a model like the one previously employed by Bernstein and Trahiotis [J. Acoust. Soc. Am. 100, 3774-3784 (1996)] predicted an ordering of thresholds between the conditions of interest that was opposite to that observed. Bernstein and Trahiotis computed the normalized interaural correlation subsequent to half-wave, square-law rectification and low-pass filtering, the parameters of which were chosen to mimic peripheral auditory processing. In this report, it is demonstrated that augmenting the model by adding a physiologically valid stage of "envelope compression" prior to rectification and low-pass filtering provides a remedy. The new model not only accounts for the data obtained by Eddins and Barber (and the similar data obtained by Hall et al.), but also does not diminish the highly successful account of the comprehensive set of data that gave rise to the original form of the model. Therefore, models based on the computation of the normalized interaural correlation appear to remain valid because they can account, both quantitatively and qualitatively, for a wide variety of binaural detection and discrimination data.

The effects of signal duration on NoSo and NoS pi thresholds at 500 Hz and 4 kHz.

A two-interval, two-alternative temporal forced-choice procedure was used to measure NoSo and NoS pi masked thresholds with 500-Hz and 4-kHz tonal signals. The duration of the signal was either 10, 20, 40, or 320 ms. The maskers were 200-Hz-wide bands of Gaussian noise centered at the frequency of the signal and presented continuously. Decreasing the duration of the 500-Hz tonal signal resulted in a modest increase (1.5 dB or so) in the masking-level difference (MLD) measured between NoSo and NoS pi conditions. In contrast, decreasing the duration of the 4-kHz tonal signal resulted in a substantial decrease (4.5 dB or so) in the MLD. Comparisons of the data with thresholds predicted from analyses based on "windows of temporal integration" provided quantitatively acceptable accounts of the data. The data obtained in the NoS pi condition at 4 kHz, which are novel and were of primary interest, were well-accounted for in a statistical sense. However, there were small, but systematic, discrepancies between the predictions and the data. Those discrepancies, although small in magnitude, suggest that binaural temporal integration at high frequencies, where the envelopes of the stimuli convey the information, may be inherently different from both monaural temporal integration and binaural temporal integration at low frequencies.

Tumor promotion resistant cells are deficient in AP-1 DNA binding, JunD DNA binding and JunD expression and form different AP-1-DNA complexes than promotion sensitive cells.

The JB6 cell culture model is used to identify molecular determinants of susceptibility to the promotion of neoplastic transformation. Clonal variants susceptible to transformation ('P+' cells) form numerous anchorage-independent colonies in soft agar upon treatment with the phorbol ester tumor promoter TPA, whereas resistant variants ('P-' cells) do not. We now report that there is significantly less binding of activator protein-1 (AP-1) to its DNA binding site in P- cells than in P+ cells. Gel supershift assays were performed to detect association of all seven AP-1 family members with their DNA binding site in TPA-treated and -untreated P+ and P- cells. Significantly lower DNA binding and protein expression of JunD were detected in P- cells than in P+ cells. c-Jun was detected in P+, but not P-, AP-1-DNA complexes, and c-Fos was detected in P-, but not P+, AP-1-DNA complexes. These and other phenotype-specific differences in abundance and composition of AP-1-DNA complexes may play a role in the resistance of P- cells to tumor promoter-induced transformation.