Twelve-hour duration testing of cream formulations of three repellents against Amblyomma americanum.

The repellent efficacies of the U.S. military repellent 33% N,N-diethyl-3-methylbenzamide (deet), 10% and 20% (1S, 2'S) 2-methylpiperidinyl-3-cyclohexene-1-carboxamide (SS220) and 10% and 20% 1-methyl-propyl-2-(hydroxyethyl)-1-piperidinecarboxylate (Bayrepel) cream formulations on human volunteers against the lone star tick Amblyomma americanum (L.) were evaluated in a simulated forest floor environment over a 12-h testing period. At 2-h intervals, volunteers, with repellent applied in a 5-cm-wide band around each ankle, stood for 5 min in plastic tubs containing leaf litter and 100 host-seeking A. americanum nymphs. Ticks were allowed to remain on a volunteer's feet and ankles for an additional 5 min after the volunteer exited the tub. All repellent formulations provided high levels of protection for the entire 12 h. No ticks crossed 5-cm-wide bands of 20% SS220 and Bayrepel during any challenge, and thus 100% protection was afforded throughout the test. These formulations showed a long-lasting efficacy hitherto unknown in tick repellents intended for use on human skin.

A comparison of non-contact, subcutaneous, and rectal temperatures in captive owl monkeys (Aotus sp.).

BACKGROUND: Three methods of body temperature measurement were compared in owl monkeys: non-contact infrared thermometry, subcutaneous transponder, and rectal probe. METHODS: Forty owl monkeys (Aotus sp.) were used. Animals were implanted with subcutaneous microchip transponders. Infrared thermometry was used at three different sites. All measurements were taken and repeated randomly. RESULTS: Infrared and subcutaneous measurements did not agree well with rectal thermometry. Subcutaneous and rectal temperatures were highly repeatable, and subcutaneous temperatures were most agreeable to rectal temperatures. CONCLUSIONS: Further investigation is needed into subcutaneous implant sites and use of different infrared thermometers in this species.

Evaluation of a timed and repeated perianal tape test for the detection of pinworms (Trypanoxyuris microon) in owl monkeys (Aotus nancymae).

The purpose of this project was to determine if the detection of pinworm infections in owl monkeys (Aotus nancymae) could be improved by performing perianal tape testing at specific times of the day and/or by performing repeated sampling. Eight Aotus known to be infected with pinworms were sampled at four selected time points (06:00, 12:00, 18:00 and 24:00 hours) over the course of a 3-week period. Samples were examined microscopically and oxyurid eggs were quantified. Results revealed no significant differences in time points, but did indicate that repeated sampling significantly improved pinworm egg detection. Results also determined that Aotus housed with an infected cage mate are at an approximately 14-times greater risk of being infected than animals housed without an infected cage mate. Lastly, results indicated no significant difference between peripheral eosinophil and basophil numbers from infected and clean animals.

Two-dimensional restoration of motion-degraded intensified CCD imagery.

A Wiener filter-based deconvolution algorithm is developed to restore vibration-degraded video imagery from an intensified CCD camera. The method is based on the use of azimuth and elevation angular optical line-of-sight data recorded from external sensors to estimate a two-dimensional vibration-blur impulse response on a per frame basis. Flight conditions are reproduced in the laboratory by use of prerecorded in-flight vibration data. The performance of the algorithm varies from frame to frame, following the time-varying characteristics of the vibration-blur impulse response. However, real-time display of the restored video minimizes these effects because of eye integration, and near-full restoration of the original uncorrupted imagery is observed for both high-light- and low-light-level conditions with minimal amplification of noise.

Energetics of thrombin-thrombomodulin interaction.

Temperature and salt dependence studies of thrombin interaction with thrombomodulin, with and without chondroitin sulfate, and two fragments containing the EGF-like domains 4-5 and 4-5-6 reveal the energetic signatures and the mechanism of recognition of this physiologically important cofactor. Binding of thrombomodulin is affected drastically by the particular salt present in solution and is positively linked to Na+ binding to thrombin and the conversion of the enzyme from the slow to the fast form, but is opposed by Cl- binding to the fibrinogen recognition site and especially to the heparin binding site. Binding of thrombomodulin has an unusually large salt dependence (gamma(salt) = -4.8) contributed mostly by the polyelectrolyte-like nature of the chondroitin sulfate moiety that binds to the heparin binding site and increases the affinity of the cofactor by almost 10-fold. On the other hand, the chondroitin sulfate has no effect on the deltaCp of binding, which is determined predominantly by contacts made by the EGF-like domains 5 and 6 with the fibrinogen recognition site. The modest heat capacity change (-0.2 kcal mol(-1) K(-1)) observed when thrombomodulin binds to the fast form suggests a rigid-body association of the cofactor with the enzyme. In the slow form, however, the heat capacity change is significantly more pronounced (-0.5 kcal mol(-1) K(-1)) and signals the presence of a conformational transition of the enzyme linked to binding of the cofactor that mimics the slow-->fast conversion. These results demonstrate that recognition of thrombomodulin by thrombin is steered electrostatically by the highly charged regions of the fibrinogen recognition site and the heparin binding site, to which the chondroitin sulfate moiety binds and enhances the affinity of the interaction. The recognition event also involves conformational changes of the enzyme in the slow form mediated by binding of the EGF-like domains 5-6 to the fibrinogen recognition site. Consistent with this model, binding of thrombomodulin to the fast form has only a small effect on the hydrolysis of nine chromogenic substrates carrying substitutions at P1, P2, and P3 aimed at probing the environment of the specificity sites S1, S2, and S3 of the enzyme. Binding to the slow form, on the other hand, enhances the specificity toward all substrates up to 15-fold. For substrates carrying a Gly at P2, binding of thrombomodulin changes the relative specificity of the slow and fast forms and makes the slow form more specific. Interestingly, these effects are not specific of thrombomodulin and depend solely on binding to the fibrinogen recognition site of the enzyme. In fact, they are also observed with the hirudin C-terminal fragment 55-65. The characterization of the mechanism of thrombin-thrombomodulin interaction and the effects of the cofactor on the hydrolysis of chromogenic substrates probing the interior of the catalytic pocket bear on the thrombomodulin-induced enhancement of protein C cleavage by thrombin. We propose that this enhancement is due predominantly to an effect of thrombomodulin on the bound protein C in the ternary complex. Therefore, thrombomodulin would carry out its physiological function by making protein C a better substrate for thrombin, rather than making thrombin a better enzyme for protein C.

The fifth epidermal growth factor-like domain of thrombomodulin does not have an epidermal growth factor-like disulfide bonding pattern.

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thrombomodulin have been determined. In previous work, this fragment was expressed and purified to homogeneity, and its cofactor activity, as measured by Kcat for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single methionine in the connecting region between the domains and subsequent deglycosylation yielded the individual EGF-like domains. The disulfide bonds were mapped by partial reduction with tris(2-carboxyethyl)phosphine according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is the same as that found previously in EGF and in a synthetic version of the fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found in EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bound to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulfide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.

Large-scale expression, purification and characterization of small fragments of thrombomodulin: the roles of the sixth domain and of methionine 388.

Fragments of human thrombomodulin (TM) have been expressed in large quantities in the Pichia pastoris yeast expression system and purified to homogeneity. Fermentation of P. pastoris resulted in yields of 170 mg/l TM. Purification to homogeneity resulted in an overall 10% yield, so that quantities of approximately 20 mg purified fragments can be readily obtained. Smaller fragments of TM, such as the individual fourth or fifth domains, were not active, nor were equimolar mixtures of the two domains. These results demonstrate that the fourth and fifth epidermal growth factor (EGF)-like domains together comprise the smallest active fragment of TM. The fragment containing the fourth and fifth EGF-like domains [TMEGF(4-5)] had 10% the specific activity of rabbit TM. Comparison of the M388L mutant TMEGF(4-5) fragment with the same mutant TMEGF(4-5-6) fragment showed that the fragment with the sixth domain had a 10-fold better Km value for thrombin than the fragment that did not contain the sixth domain; this factor completely accounts for the higher specific activity of the fragments containing the sixth domain. Comparison of the wild-type and M388L mutants showed that the M388L mutation resulted in a 2-fold increase in kcat for the activation of protein C by the thrombin-TM fragment complex, completely accounting for the 2-fold increase in specific activity of these mutant fragments.

The effects of three models of airway disease on tidal breathing flow-volume loops of thoroughbred horses.

The effects of histamine and methacholine aerosols and of a fixed inspiratory resistance on tidal breathing flow-volume loops (TBFVL) were investigated using 18 unsedated, standing, healthy thoroughbred horses. The data were first analysed using traditional flow-volume loop indices and then reduced using standardized factor scoring coefficients obtained in a previous study in this laboratory using similar experimental techniques. On the basis of resting TBFVL analysis, the degree of pulmonary dysfunction caused by inhalation of histamine and methacholine aerosols with concentrations of 10 and 2 mg/ml, respectively, was similar. The fixed resistance also caused significant changes in the resting spirogram and TBFVL indices, suggesting that this model may prove valuable for further studies involving upper respiratory tract (URT) conditions. Administration of histamine and methacholine aerosols resulted in significant changes in all factor scores, although most of the observed changes were due to the effects of these aerosols on the respiratory rate. These findings re-emphasize the importance of the effects of respiratory rate on pulmonary mechanics. Application of the resistance resulted in significant changes in factor score 3, the 'inspiratory' factor, which lends support to the validity of this model for URT conditions. The close agreement between the factor scores obtained under controlled conditions in this study and in a previous study in this laboratory confirms that the factor analysis used for both of these studies provides an adequate means of reducing TBFVL data obtained from thoroughbred horses. The large intra- and inter-individual variation observed both with the indices of TBFVL and with the factor scores limits the potential of these variables for detecting individual animals with obstructive airway disease. Re-evaluation of these indices under the stress of exercise may reduce the variability observed in these data and may increase the magnitude of differences between different animals, providing a means of detecting individual animals with subclinical obstructive airway conditions.

Characterization of normal tidal breathing flow-volume loops for thoroughbred horses.

The purpose of this study was to characterize the normal equine tidal breathing flow-volume loop (TBFVL). The study was performed using 18 healthy Thoroughbred horses. TBFVLs constructed from data collected from resting horses had a typical biphasic inspiratory and expiratory phase. The interindividual variability of the indices used to describe TBFVLs was in the range 16-32%, which is comparable to the variability of other measures of equine pulmonary mechanics. The large variability of these data probably limits the value of resting TBFVL indices for detecting subclinical respiratory conditions in individual horses. Factor analysis of these data revealed that in excess of 90% of the variance of the initial response variables could be explained in terms of three common factors. Varimax rotation of these three common factors provided three subsequent factors that were readily identifiable as (1) a factor describing the time-volume relationships of TBFVLs, responsible for 81% of the total variance, (2) a factor explaining the expiratory portion of the TBFVL, explaining 12% of the variance, and (3) a factor describing the inspiratory portion of the loops, responsible for the remaining 7% of the variance. The analysis also provided standardized factor scoring coefficients for use in subsequent studies using similar experimental techniques.

The effect of hyperbaric oxygen treatment on a compromised axial pattern flap in the cat.

A pedicle skin flap based on the caudal superficial epigastric artery was created in 12 cats. The artery was ligated and the vein left intact. Six cats were treated with hyperbaric oxygen at 2 atm absolute for 90 minutes daily for 14 days, starting the day after surgery. Skin flap color was significantly better in the treated cats on days 1, 2, and 3. The amount of exudate was significantly less in the treated cats on day 3. There was no difference in the amount of swelling between treated and nontreated control cats. Although five of six of the treated cats had 100% skin flap survival, there was no significant difference in total flap survival between treated and nontreated control cats.

Temporal effects of inhaled histamine and methacholine aerosols on the pulmonary mechanics of thoroughbred horses.

This paper presents a method for on-line determination of pulmonary mechanics in standing, non-sedated horses during and following inhalation of aerosolized drug solutions. This method was used to evaluate the temporal effects of inhaled histamine and methacholine aerosols on pulmonary mechanics in 18 Thoroughbred horses. The following were concluded from this study. The extremely large between-breath variation, for all variables used to evaluate pulmonary mechanics in the horse, limits the usefulness of these variables for modeling the non-specific pulmonary responses to inhaled stimulants on a breath-by-breath basis. Following the implementation of averaging techniques to control the variation of these response variables, respiratory rate appears to be affected most predictably by inhalation of non-specific bronchoconstrictors. In the 18 subjects studied, the response of respiratory rate to inhaled histamine and methacholine aerosols was well described by a complex function consisting of exponential treatment and post-treatment phases in 10 and six of the subjects, respectively. Large intra-individual variation of the non-specific response to these stimulants in repeated studies of some subjects, suggests that differences in responses on a particular day may be due to inter-day variations in minute ventilation or baseline airway caliber.

The management of mid-face fractures with intracranial injury.

Recent advances have radically changed the management of facial fractures. CT scanning, extensive exposure, and rigid plate fixation in the setting of the trauma center have permitted early operation with improved results. A subset of patients with facial fractures will also have intracranial injuries (ICI). We sought to identify parameters associated with an increased risk for ICI. We also sought to examine the safety and limits of early craniofacial repair in patients with intracranial injuries. Of 114 mid-face fractures treated over a 1-year period, 43 (38%) had a concomitant ICI. The majority, 36 (84%), were from motor vehicle accidents (MVA). Frontal sinus and orbitoethmoid fractures were at the highest risk for ICI, although orbitozygomatic fractures caused by MVAs also had a surprisingly high incidence of ICI. Our results show that early craniofacial repair can be performed safely with appropriate general surgical and neurosurgical support.

Effect of dietary fructose on broiler chick performance.

The effect of feeding various levels of dietary fructose, furnished by high fructose corn syrup (HFCS), to broiler-type chicks housed in Petersime batteries was studied in two experiments. In experiment 1, HFCS was added to a corn-soybean meal basal diet at levels of 0, 5, 10, and 15%. In Experiment 2, HFCS was added to the diet at levels of 0, 2, 4, 6, and 15%. In both experiments diets were isocaloric and isonitrogenous. In Experiment 1, chicks fed HFCS consumed more feed (P less than or equal to .05) and grew more rapidly (P less than or equal to .05) than chicks fed the control diet. No differences in the feed to gain ratio were observed among treatments. Feeding HFCS resulted in non-significantly higher liver weight and percent liver lipid. Values for kidney weights and plasma uric acid were higher (P less than or equal to .05) in chicks fed 15% HFCS. In Experiment 2, adding 4, 6, and 15% HFCS resulted in more rapid growth (P less than or equal to .05). No significant differences were observed in feed intake and kidney weights. Chicks fed the 15% HFCS diet had heavier (P less than or equal to .05) livers.

Relationship between estrone sulfate in plasma and litter size at farrowing for sows and gilts.

A positive association (P < 0.01) was detected between estrone sulfate (ES) concentrations in maternal plasma at Day 30 of pregnancy and litter size at parturition in swine. This relationship was best described by a fifth order regression equation (R(2) = 0.5) which indicated that as ES increased from 1 to 7.5 ng/ml on Day 30, litter size increased from 0 (nonpregnant) to 18 piglets farrowed. Day of sampling (P < 0.02), month (P < 0.04) and parity (P < 0.08) were major sources of variation in the model. This indicated that effects of environmental factors such as heat stress, which influence conception rate and embryonic survival, are reflected in changes in maternal ES. Also, larger litter size associated with parous sows is reflected in increased ES in maternal plasma. We conclude that measurement of ES early in gestation may be useful in reproductive management to identify nonpregnant gilts and sows as well as those with small litters.