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Background: Abdominal catastrophe in the severely burned patient without abdominal injury has been described. We perceived an alarming recent incidence of this complication in our burn center, both during acute resuscitation and later in the hospital course. We sought to define incidence, outcomes, and associated factors, such as excessive resuscitation volume and treatment issues., Study Design: We examined all severely burned military and civilian patients with abdominal pathology between March 2003 and February 2008. Data included age, gender, total body surface area burn, inhalation injury, Injury Severity Score, disposition, resuscitation volume, time from injury to diagnosis, use of recombinant factor VIIa, vasopressors, and early tube feedings. We assembled a Delphi panel of surgeons experienced in abdominal catastrophes to review these data., Results: Among 1,825 patients admitted to the US Army Institute of Surgical Research Burn Center, 120 (6.6%) were diagnosed with abdominal pathology (burn size 48% +/- 19%), of which 51 (2.8%) had abdominal catastrophe. The majority of these occurred in the first days after injury with associated abdominal compartment syndrome (32 of 51) and increased linearly to burn size. We noted another group of patients who presented primarily with ischemic bowel later in the course, with the same clinical presentation. Resuscitation volume was 6.02 mL/kg/percent total body surface area burned. Vasopressors were used in 71% of patients and tube feedings in 57% before diagnosis., Conclusions: Abdominal catastrophe without abdominal trauma occurs in 2.8% of our population. Associated mortality was 78% without obvious cause. Delphi panel experts recommended more aggressive monitoring of abdominal compartment pressures and earlier operative management to improve outcomes.

An impervious plastic stockinet can facilitate application of a vacuum-assisted closure dressing in complex, traumatic wounds. This article reviews our technique.

Introduction: Acute kidney injury (AKI) is a common and devastating complication in critically ill burn patients with mortality reported to be between 80 and 100%. We aimed to determine the effect on mortality of early application of continuous venovenous hemofiltration (CVVH) in severely burned patients with AKI admitted to our burn intensive care unit (BICU)., Methods: We performed a retrospective cohort study comparing a population of patients managed with early and aggressive CVVH compared with historical controls managed conservatively before the availability of CVVH. Patients with total body surface area (TBSA) burns of more than 40% and AKI were treated with early CVVH and their outcomes compared with a group of historical controls., Results: Overall, the 28-day mortality was significantly lower in the CVVH arm (n = 29) compared with controls (n = 28) (38% vs. 71%, P = 0.011) as was the in-hospital mortality (62% vs. 86%, P = 0.04). In a subgroup of patients in shock, a dramatic reduction in the pressor requirement was seen after 24 and 48 hours of treatment. Compared with controls (n = 19), significantly fewer patients in the CVVH group (n = 21) required vasopressors at 24 hours (100% vs 43%, P < 0.0001) and at 48 hours (94% vs 24%, P < 0.0001). In those with acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), there was a significant increase from baseline in the partial pressure of arterial oxygen (PaO2) to fraction of inspired oxygen (FiO2) ratio at 24 hours in the CVVH group (n = 16, 174 +/- 78 to 327 +/- 122, P = 0.003) but not the control group (n = 20, 186 +/- 64 to 207 +/- 131, P = 0.98)., Conclusions: The application of CVVH in adult patients with severe burns and AKI was associated with a decrease in 28-day and hospital mortality when compared with a historical control group, which largely did not receive any form of renal replacement. Clinical improvements were realized in the subgroups of patients with shock and ALI/ARDS. A randomized controlled trial comparing early CVVH to standard care in this high-risk population is planned.

Background: Case-control studies have suggested that air travel may be a risk factor for the development of Venous Thromboembolism (VTE). Burned patients from the current war in Iraq and Afghanistan, are transported across three continents to our Burn Center with total ground and air transport time being approximately 24 hours spread over 3 days to 4 days. We hypothesized global evacuation results in increased VTE rates., Methods: Retrospective review of 1,107 consecutive patients admitted to our burn center from January 2003 to December 2005., Results: In the time period evaluated, no detectible differences were found in incidence of VTE between air-evacuated soldiers and those admitted to our facility from South Texas (1.31% vs. 0.83%, p = ns). The air-evacuated soldiers were younger (26 +/- 7 vs. 41 +/- 19, p < 0.0001) but had a higher incidence of inhalation injury (14.4% vs. 8.0%, p < 0.0001) and higher Injury Severity Score (10.9 +/- 13.0 vs. 6.5 +/- 9.2, p < 0.0001). No difference in average percent total body surface area involvement was found (15.8 +/- 19.4 vs. 15.5 +/- 18.4, p = ns). Overall, 11 of 1,107 (0.99%) burned patients developed VTE., Conclusion: Prolonged global evacuation is not associated with increased risk of VTE.

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.

Background: Acute kidney injury in severely burned patients is associated with high mortality. We wondered whether early use of continuous renal replacement therapy (CRRT) changes outcomes in severely burned military casualties with predetermined criteria for acute kidney injury., Methods: Between November 2005 and June 2007, casualties admitted to our burn intensive care unit after sustaining burns in Iraq and Afghanistan, who subsequently developed acute kidney injury or circulatory shock or both, underwent CRRT. Baseline demographic, laboratory, and hemodynamic parameters were recorded. Both 28-day mortality and in- hospital mortality were evaluated and compared with a consecutive group of burn casualties with greater than 40% total body surface area (TBSA) burns, acute kidney injury, or nephrology consultation in the 2 years before the existence of our CRRT program., Results: One hundred forty-seven severely burned military casualties were admitted to our intensive care unit before CRRT program initiation, and 102 were admitted after CRRT program initiation. Before the CRRT program, 16 patients were identified as having >40% TBSA burns with kidney injury with or without nephrology consultation (control group); 18 were treated with CRRT since (CRRT group). Groups were similar for %TBSA, %full-thickness TBSA, incidence of inhalation injury, blood urea nitrogen, creatinine, and Injury Severity Score. Of the CRRT patients, seven soldiers were treated for isolated acute kidney injury, whereas 11 were treated for a combination of acute kidney injury and shock. The dose of therapy was 50.2 +/- 13 mL/kg/h with a treatment course of 5.2 +/- 3 days. Of the 11 patients in the CRRT group treated for shock, eight were off vasopressors by 24 hours and the remaining three within 48 hours. None of the patients in the control group were placed on renal replacement therapy with nephrology consultation in eight patients. Both 28-day mortality (22% vs. 75%, p = 0.002) and in-hospital mortality (56% vs. 88%, p = 0.04) were lower in the CRRT group compared with that in the control group., Conclusion: Aggressive application of CRRT in severely burned casualties with kidney injury significantly improves survival.

Background: US military burn casualties are evacuated to the US Army Institute of Surgical Research Burn Center in San Antonio, TX. Patients are transported by US Army Institute of Surgical Research Burn Flight Teams, Air Force Critical Care Air Transport Teams, or routine aeromedical evacuation. This study characterizes the military burn casualties transported by each team and reports associated outcomes., Methods: We performed a retrospective review of burn center registry data, identifying all US burn casualties admitted to the Army's burn center between March 2003 and February 2007. Data included total body surface area (TBSA) burn, ventilatory status, inhalational injury, associated injuries, injury severity, disposition, morbidity, and mortality., Results: During 4 years of military operations in Iraq and Afghanistan, 540 casualties were admitted to our burn center for treatment of injuries resulting from war-related operations. Mean burn size was 16.7% total body surface area (range, <1%-95%) with a mean Injury Severity Score of 12.2 +/- 13.7. One hundred eight-one (33.5%) casualties required ventilatory support in flight; inhalation injury was confirmed in 69 (12.7%) patients. Two hundred six (38.1%) were transported by the Burn Flight Team and 174 (32.2%) were transported by Critical Care Air Transport Team, with a mean transit time of 4 days after injury. One hundred sixty (29.6%) patients were routine aeromedical evacuees. There were no in-flight deaths reported; 30 (5.6%) patients died of their wounds at our burn center., Conclusions: Burn casualties represent a group of patients with severe traumatic injuries. Our current system of selectively using specialty medical transport teams for the long-range transport of burn casualties is safe and effective.

The plant pathogen Agrobacterium tumefaciens induces the formation of crown gall tumours at wound sites on host plants by directly transforming plant cells. This disease strategy benefits the bacteria as the infected plant tissue produces novel nutrients, called opines, that the colonizing bacteria can use as nutrients. Almost all of the genes that are required for virulence, and all of the opine uptake and utilization genes, are carried on large tumour-inducing (Ti) plasmids. The observation more than 25 years ago that specific opines are required for Ti plasmid conjugal transfer led to the discovery of a cell-cell signalling system on these plasmids that is similar to the LuxR-LuxI system first described in Vibrio fischeri. All Ti plasmids that have been described to date carry a functional LuxI-type N-acylhomoserine lactone synthase (TraI), and a LuxR-type signal receptor and transcriptional regulator called TraR. The traR genes are expressed only in the presence of specific opines called conjugal opines. The TraR-TraI system provides an important model for LuxR-LuxI-type systems, especially those found in the agriculturally important Rhizobiaceae family. In this review, we discuss current advances in the biochemistry and structural biology of the TraR-TraI system.

TraR of Agrobacterium tumefaciens is a member of the LuxR family of transcriptional regulators, and binds to specific DNA sequences (tra boxes) at target promoters of the tumour-inducing (Ti) plasmid. Each tra box has a pronounced dyad symmetry, and each subunit of a TraR dimer binds to one half of a tra box via a helix-turn-helix (HTH) DNA binding motif. Structural analysis has suggested that TraR makes extensive sequence-specific contacts with tra box DNA. In this study, we tested these predictions using synthetic self-complementary oligonucleotides containing variant tra box sequences. Some predictions made from structural analysis were confirmed, while others were shown to be incorrect. Unexpectedly, these experiments also showed that six nucleotides at the centre of the tra box that make no direct contact with TraR are nevertheless critical for high-affinity binding and probably act by facilitating a previously described DNA bend. Variant tra boxes were also tested for transcription activity in vivo. Most transcription assays reflected in vitro binding assays. However, alterations of the outermost nucleotides had little effect on TraR binding but blocked transcription, probably by altering an overlapping -35 promoter motif.

CDC miniature light traps were used to evaluate the general biology of phlebotomine sand flies from April 2003 to November 2004 at Tallil Air Base, Iraq. Factors evaluated include species diversity and temporal (daily and seasonal) and geographic distribution of the sand flies. In addition, the abundance of sand flies inside and outside tents and buildings was observed. In total, 61,630 sand flies were collected during 1,174 trap nights (mean 52 per trap, range 0-1,161), with 90% of traps containing sand flies. Sand fly numbers were low in April, rose through May, were highest from mid-June to early September, and dropped rapidly in late September and October. More than 70% of the sand flies were female, and of these sand flies, 8% contained visible blood. Phlebotomus alexandri Sinton, Phlebotomus papatasi Scopoli, Phlebotomus sergenti Parrot, and Sergentomyia spp. accounted for 30, 24, 1, and 45% of the sand flies that were identified, respectively. P. alexandri was more abundant earlier in the season (April and May) than P. papatasi, whereas P. papatasi predominated later in the season (August and September). Studies on the nocturnal activity of sand flies indicated that they were most active early in the evening during the cooler months, whereas they were more active in the middle of the night during the hotter months. Light traps placed inside tents with and without air conditioners collected 83 and 70% fewer sand flies, respectively, than did light traps placed outside the tents. The implications of these findings to Leishmania transmission in the vicinity of Tallil Air Base are discussed.

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.

Objective: To determine whether treatment with recombinant activated factor VII (rFVIIa) will prevent progression of bleeding in nonsurgical hemorrhagic traumatic brain injury (TBI)., Methods: Chart review from the trauma registry of a level 1 trauma center between January 1, 2002 and December 31, 2004 identified 2 patients who received rFVIIa for progressive hemorrhagic TBI. These patients were given a single dose of rFVIIa (120 mcg/kg) after a repeat head computed tomography (CT) scan showed worsening of intracranial bleeding. Pre-rFVIIa and post-rFVIIa coagulation parameters and postintervention CT scans were performed. A matched convenience sample was drawn from the institution's trauma registry reflecting similar injury patterns., Results: The 2 patients who received rFVIIa were ages 61 and 79 years; the patients in the matched convenience sample were 57 and 63 years. Both sets of patients comprised 1 man and 1 woman who had suffered blunt trauma, including hemorrhagic TBI, and were matched according to age, gender, and injury severity score (ISS). During their hospital course, repeat CT scans documented worsening of intracranial hemorrhage in both cohorts. In the rFVIIa patients, follow-up CT showed overall improvement of head injury compared with the convenience sample. The rFVIIa patients also saw an appreciable decrease in both prothrombin time (PT) and international normalized ratio (INR)., Conclusions: In hemorrhagic TBI, rFVIIa has the potential to limit or even halt the progression of bleeding that would otherwise place growing pressure on the brain. A prospective, randomized multicenter trial is planned to elucidate this hypothesis.

Objective: To assess outcome differences between locally burned civilians and military personnel burned in a distant combat zone treated in the same facility., Summary Background Data: The United States Army Institute of Surgical Research (USAISR) Burn Center serves as a referral center for civilians and is the sole center for significant burns in military personnel. We made the hypothesis that outcomes for military personnel burned in the current conflict in Iraq and Afghanistan would be poorer because of delays to definitive treatment, other associated injury, and distance of evacuation., Methods: We reviewed the civilian and military records of patients treated at the USAISR from the outset of hostilities in Iraq in April 2003 to May 2005. Demographics, injury data, mortality, and clinical outcomes were compared., Results: : We cared for 751 patients during this time period, 273 of whom were military (36%). Military injuries occurred in a younger population (41 +/- 19 vs. 26 +/- 7 years for civilian and military respectively, P < 0.0001) with a longer time from injury to burn center arrival (1 +/- 5 days vs. 6 +/- 5, P < 0.0001), a higher Injury Severity Score (ISS 5 +/- 8 vs. 9 +/- 11, P < 0.0001), and a higher incidence of inhalation injury (8% vs. 13%, P = 0.024). Total burn size did not differ. Mortality was 7.1% in the civilian and 3.8% in the military group (P = 0.076). When civilians outside the age range of the military cohort were excluded, civilian mortality was 5.0%, which did not differ from the military group (P = 0.57). Total body surface area (TBSA) burned, age > or =40 years, presence of inhalation injury, and ventilator days were found to be important predictors of mortality by stepwise regression, and were used in a final predictive model with the area under receiver operator characteristic curve of 0.97 for both populations considered together. No significant effect of either group was identified during development., Conclusions: Mortality does not differ between civilians evacuated locally and military personnel injured in distant austere environments treated at the same center.

This study compared rabbit rectal thermometry with 4 other thermometry techniques: an implantable microchip temperature transponder, an environmental noncontact infrared thermometer, a tympanic infrared thermometer designed for use on humans, and a tympanic infrared thermometer designed for use on animals. The microchip transponder was implanted between the shoulder blades; the environmental noncontact infrared thermometer recorded results from the base of the right pinna and the left inner thigh, and the tympanic infrared thermometer temperatures were taken from the right ear. Results from each technique were compared to determine agreement between the test modality and the rectal temperature. The practicality and reliability of the modalities were reviewed also. According to this study, the implantable microchip transponder measurements agreed most closely with the rectal temperature.

We conducted this study to determine whether heart rate, respiratory rate, and recovery time differed significantly between rats receiving target-controlled infusion (TCI) and those under volatile inhalant anesthesia. TCI rats received intravenous propofol at an average effect site concentration of 11.3 microg/ml or propofol plus ketamine (5 mg/ml of propofol) at an average effect site concentration of 8.7 microg/ml. Inhalant anesthesia rats received isoflurane (average, 1.8%) delivered in medical-grade air. We used a tail-clamp response test to determine when a surgical plane of anesthesia was attained. Anesthesia was continued for 1 h from the first negative tail-clamp test. During this time the test was repeated every 10 min to confirm that a surgical plane of anesthesia was being maintained. Anesthesia then was discontinued, and the animals were monitored continuously until they recovered. Average heart rate was higher for rats during anesthesia with isoflurane compared with TCI propofol-ketamine (P =0.0053). Average respiratory rate was higher for TCI regimens compared with isoflurane anesthesia, with male rats having consistently faster respiratory rates than females (P <0.001). Recovery time was longer for both TCI regimens compared with isoflurane (P <0.001). Once venous access was accomplished, TCI anesthesia with propofol or propofol combined with a low dose of ketamine was comparable to an isoflurane inhalant regimen in ease of administration and control of the anesthetic event when used in rats for procedures of 1-h duration. Respiratory rate was increased and recovery time was longer for rats receiving the TCI regimens.

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.

Burkholderia cenocepacia is an opportunistic human pathogen that can aggressively colonize the cystic fibrosis lung. This organism has a LuxR/LuxI-type quorum sensing system that enables cell-cell communication via exchange of acyl homoserine lactones (AHLs). The CepR and CepI proteins constitute a global regulatory system, controlling expression of at least 40 genes, including those controlling swarming motility and biofilm formation. In this study, we isolated seven lacZ fusions in a clinical isolate of B. cenocepacia that are inducible by octanoyl-HSL. Induction of all of these genes requires CepR. The cepI promoter was tested for induction by a set of 33 synthetic autoinducers and analogues, and was most strongly induced by long-chain AHLs lacking 3-oxo substitutions. Expression of this promoter was inhibited by high concentrations of three different autoinducers, each having six-carbon acyl chains. When CepR protein was overproduced in Escherichia coli, it accumulated in a soluble form in the presence of octanoyl-HSL, but accumulated only as insoluble inclusion bodies in its absence. Purified CepR-OHL complexes bound to specific DNA sequences at the cepI and aidA promoters with high specificity. These binding sites included a 16-nucleotide imperfect dyad symmetry. Both CepR binding sites are centred approximately 44 nucleotides upstream of the respective transcription start sites.

The LuxR-type quorum-sensing transcription factor TraR regulates replication and conjugal transfer of the tumour-inducing (Ti) plasmid in the plant pathogen Agrobacterium tumefaciens. TraR is a two-domain protein with an N-terminal domain that binds to the quorum-sensing signal N-3-oxooctanoyl- l-homoserine lactone (OOHL) and a C-terminal domain that binds to specific DNA sequences called tra boxes. TraR-OOHL complexes form homodimers that activate transcription of at least seven promoters on the Ti plasmid. At five promoters, a tra box overlaps the binding site of core RNA polymerase (class II promoters), while in the other two promoters, this site is located farther upstream (class I promoters). In this study, we performed saturating point mutagenesis of the surface residues of the TraR C-terminal domain. Each mutant was tested for proteolytic stability and transcription activity in vivo, and for DNA binding activity in vitro. Mutants of TraR with single substitutions at positions W184, V187, K189, E193Q, V197 and D217 have wild-type levels of accumulation and DNA binding, but are defective in transcription of both types of promoters. These residues constitute a patch on the surface of the DNA-binding domain. We propose that this patch is an activating region that recruits RNA polymerase to TraR-dependent promoters through direct contact. As residues of this patch are critical for activation at both a class I and a class II promoter, we predict that these residues may contact the C-terminal domain of the RNA polymerase alpha-subunit.

The apical membrane antigen 1 of Plasmodium falciparum is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

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.

In chloroform solution, the syn/anti rotamer ratios for N-(2-pyridyl)carbamates, 3, and N-phenylcarbamates, 4, are close to 0.05. Addition of the double hydrogen bonding acetic acid moderately stabilizes the syn rotamer of 4, but has no measurable effect on the syn/anti ratio for 3. Conversely, the hydrogen bond donor-acceptor-donor triad in 2,6-bis(octylamido)pyridine, 1, strongly stabilizes the syn rotamer of 3, but has no effect on the syn/anti ratio for 4. The K(a) for syn-3:1 is 10(3)-10(4) times higher than the K(a) for anti-3:1. This implies that the alkoxy oxygen in anti-3 is a much poorer hydrogen bond acceptor than the carbonyl oxygen in syn-3, most likely because of a combination of steric and electrostatic factors.

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 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.

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 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.

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.

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.

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.

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.

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.

Eight fetal pigs, in utero, were injected ip with 20 microCi/fetus [U14C]-fructose between d 55 and 65 pregnancy. The isotope was allowed to equilibrate between blood and tissues within injected fetuses for a period of 240 min. Fetal pigs were then sacrificed and nucleic acids were extracted from cold tissue homogenates of skeletal muscle and liver. Nuclide disintegrations per minute recovered in extracted DNA and RNA were used to calculate incorporation of labeled C from fructose. The recovery of labeled C per mmol of nucleic acids from skeletal muscle was greater (P less than .05) than that from liver. Relative incorporation of labeled C into skeletal muscle RNA (395.9 pmol/mmol) was greater (P less than .05) than for DNA (189.5 pmol/mmol). The same trend was observed for liver RNA (78.0 pmol/mmol) and DNA (55.6 pmol/mmol), but differences were nonsignificant. These data suggest that at least part of the high concentration of endogenous fructose measured in fetal pigs in utero is involved in synthesis of nucleic acids, thereby providing substrate for anabolic functions necessary for fetal growth and development.

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.

Twenty-two Hampshire-Yorkshire X Large White sows of second and third parity were allotted randomly to one of three dietary treatments. Five sows were fed 6.0 kg/day of a corn-soybean meal lactation diet (control diet). Twelve sows were fed the control diet in which 24% of the composition was supplied by corn syrup containing 72% fructose on a dry matter basis (fructose diet) and five sows were fed the control diet in which 24% of the composition was supplied by powdered dextrose (glucose diet). All diets were fed from days 1 through 21 of lactation. Blood was collected from all sows immediately prior to feeding and hourly for 6 hours postprandial via jugular vein cannulae following a single feeding on seven separate but nonconsecutive days during the 21-day period. Fructose was absorbed from the digestive tract of sows as evidenced by elevated (P less than 0.01) conventions of fructose in plasma. Sows fed the fructose diet also had higher (P less than 0.01) plasma glucose concentrations than did those fed the glucose and control diets. The concomitant elevated glucose concentration following ingestion of the fructose diet was not associated (P greater than 0.10) with increased insulin concentration. Fructose in plasma was associated with a slight but significant increase in insulin although the mean concentration of insulin in plasma was only one-third that measured in sows fed the glucose and control diets. These data suggest that fructose in vivo has a glucose-sparing effect presumably mediated through a physiological mechanism that lowers insulin concentration.

Yield and composition of milk and growth of nursing pigs in response to dietary treatment were estimated from 25 lactating sows during a 22-d period. Eight sows were fed 6 kg/d of a corn-soybean control diet (C sows). Nine were fed the control diet in which approximately 6.5 g X kg body weight (BW-1) X d-1 of carbohydrate was supplied by fructose corn syrup (F sows) and eight were fed the control diet containing equivalent carbohydrate supplied by powdered dextrose (D sows). Blood samples collected via jugular cannulae were analyzed for plasma concentrations of fructose, glucose and insulin. Concentrations of fructose and glucose from F sows were significantly higher throughout the study than that from D and C sows, while insulin concentration was approximately 2.5-fold lower. Milk yield from F sows on d 14 and 21 was significantly higher and pigs weaned on d 21 were heavier than those from D and C sows. Sows fed the diet containing fructose experienced significant BW loss during lactation. Coefficients of gross correlation across treatments showed milk yield and litter weight gain to be negatively associated with percentages of protein, lipids and total solids in milk, but positively associated with concentrations of lactose and gross energy. Nursing pig weight gain at weaning was more responsive to total yields of milk and milk nutrients than to composition. These data support the hypothesis that source of metabolizable energy (ME) affects milk yield, composition and efficiency at which the sow converts dietary nutrients into milk.