Laboratory studies to assess the risk of development of resistance to zoxamide.

Laboratory studies were conducted to evaluate the risk of developing field resistance to zoxamide, a new Oomycete fungicide which acts on microtubules. Zoxamide, metalaxyl and dimethomorph were compared with respect to the ease with which fungicide-resistant mutants could be isolated and their level of resistance. Attempts to generate mutants of Phytophthora capsici and P infestans with resistance to zoxamide by mycelial adaptation on fungicide-amended medium were unsuccessful. Similarly, changes in sensitivity to zoxamide were small (resistance factors < or = 2.2) in mutants of P capsici isolated by chemical mutagenesis of zoospore cysts. In parallel experiments with metalaxyl, highly resistant mutants were obtained using both adaptation (P capsici or P infestans) and chemical mutagenesis (P capsici). For dimethomorph, chemical mutagenesis (P capsici) yielded moderately resistant mutants (maximum resistance factor = 20.9), and adaptation (P capsici or P infestans) did not induce resistance. It is proposed that failure to isolate mutants resistant to zoxamide results from the diploid nature of Oomycete fungi and the likelihood that target-site mutations would produce a recessive phenotype. Our studies suggest that the risk of a highly resistant pathogen population developing rapidly in the field is much lower for zoxamide than for metalaxyl. However, as with any site-specific fungicide, appropriate precautions against resistance development should be taken.

Structure-activity relationships of phenylcyclohexene and biphenyl antitubulin compounds against plant and mammalian cells.

Phenylcyclohexenes (PCHs) [e.g., trans-4-nitro-5-(2,3,4-trimethoxyphenyl)cyclohexene, 2d] were found to bind weakly to the colchicine site of bovine tubulin, but are the first mimics of colchicine found to have high activity towards plant cells. Structure-activity relationships for PCHs and biphenyl AC-ring analogues of colchicine (e.g., 2,3,4,4'-tetramethoxy-2'-methyl-1,1'-biphenyl, 3e) are discussed.

Covalent binding of the benzamide RH-4032 to tubulin in suspension-cultured tobacco cells and its application in a cell-based competitive-binding assay.

The benzamide, RH-4032, was found to be a potent antimicrotubule agent in tobacco (Nicotiana tabacum) cells. It strongly inhibited root growth and produced swollen club-shaped roots, an accumulation of cells in arrested metaphase, and loss of microtubules. RH-4032 inhibited the in vitro assembly of bovine tubulin into microtubules, with inhibition requiring a relatively long incubation period. Treatment of tobacco suspension-cultured cells or isolated bovine tubulin with [(14)C]RH-4032, and analysis of radiolabeled protein revealed a highly specific covalent attachment to beta-tubulin. Binding of [(3)H]RH-4032 in tobacco suspension-cultured cells was shown to be saturable and to be influenced by pre-incubation of the cells with various antimicrotubule agents: Binding of [(3)H]RH-4032 was inhibited by the benzamides, pronamide and zarilamide, the N-phenylcarbamate, chlorpropham, and the microtubule-stabilizing drug, paclitaxel, whereas trifluralin and amiprophosmethyl were not inhibitory. A common characteristic of agents that cause microtubule disassembly was a slight enhancement of [(3)H]RH-4032 binding at low concentrations, which did not occur with the microtubule-stabilizing agent paclitaxel. For structural analogs of RH-4032 and various N-phenylcarbamates, it was shown that the ability to inhibit binding of [(3)H]RH-4032 was correlated with the ability to inhibit tobacco root elongation. The results suggest a common binding site on beta-tubulin for RH-4032, pronamide, zarilamide, and chlorpropham, which is distinct from the binding site(s) for trifluralin and amiprophosmethyl. RH-4032 provides a unique approach to studying effects of antimicrotubule agents on plant cells by allowing competitive tubulin binding assays to be conducted in whole cells.

Identification of ceramide-phosphorylethanolamine in oomycete plant pathogens: Pythium ultimum, Phytophthora infestans, and Phytophthora capsici.

Cellular lipids were extracted from three species of Oomycete plant pathogens (Pythium ultimum, Phytophthora infestans, and Ph. capsici) and analyzed via normal-phase high-performance liquid chromatography with flame-ionization detection. The most abundant polar lipids in each of the three species were the polar membrane lipids, phosphatidylethanolamine (PE), phosphatidylcholine, and a phosphosphingolipid that eluted soon after PE. Structural analysis via mass spectrometry and nuclear magnetic resonance spectrometry revealed that the phosphosphingolipid was ceramide phosphorylethanolamine (Cer-PE). The most abundant molecular species of Cer-PE in P. ultimum had a molecular weight of 670.5, contained an unusual 19-carbon branched triunsaturated sphingoid (C19-delta 4, 8, 10, 9-methyl long-chain base) and palmitic acid as the amide-linked fatty acid. The most abundant molecular species of Cer-PE in Ph. infestans had a molecular weight of 714.5, contained a common 16-carbon 1,3 di-OH sphingoid, and erucic (cis 13-docosenoic, C22-delta 13) acid as the amide-linked fatty acid. The Cer-PE in Ph. capsici comprised a mixture of each of the two molecular species found in P. ultimum and Ph. infestans.

Antifungal properties of taxol and various analogues.

The antimitotic agent taxol was tested for toxicity towards fungi from different taxonomic groups and found to be particularly active against oomycete fungi. In germinating zoospore cysts of the oomycete Phytophthora capsici the mechanism of action of taxol was shown to involve inhibition of mitosis, presumably resulting from an effect on microtubules. Various taxol analogues with deleted A-ring C-13 side chain substituents were tested for toxicity towards P. capsici and Aphanomyces cochlioides to provide insight into structural features required for activity. The importance of the side chain was shown by the much lower activity as compared to taxol of analogues lacking all or part of the side chain. The effect of stereochemistry at the C-2' position on fungitoxicity towards oomycetes was similar to that reported previously on mammalian microtubule assembly.

Female urethral injury secondary to blunt pelvic trauma.

Urethral injury after blunt pelvic trauma is rare in women. We report a case of urethral injury and vaginal laceration secondary to blunt pelvic trauma. Pelvic examination should be performed in any female patient who has sustained blunt pelvic trauma to avoid the morbidity associated with delayed diagnosis of urethral injury.

Effect of nutritional therapy on polyamine metabolism in severely traumatized patients.

The polyamines (PA) spermidine (SD) and spermine and their precursor putrescine (PU) play a leading role in the regulation of protein, RNA and DNA synthesis. We examined the role of PA along with other biomarkers of injury in eight victims of multiple trauma in the early post-traumatic period when they were hypermetabolic and highly catabolic. Intravenous nutritional therapy (TPN) was started 48 to 60h after trauma and continued for 6 days. The basal response to severe trauma was a significant (twofold to threefold) rise in urinary PU (p = 0.05) and SD (p = 0.025) levels compared to normal subjects. Six days of TPN further enhanced the basal excretion of PU (157%) and SD (137%) peaking on the third day. There was a 20% reduction in the excretion of 3-methylhistidine on the first day of TPN, but it was still 40% above normal on the sixth day. The negative nitrogen balance was improved but not reversed. Injury stimulated ribonuclease and catecholamine levels were also enhanced by nutritional therapy, peaking on the first and fourth day of TPN, respectively. This study demonstrated for the first time elevated levels of PA in trauma patients that correlated well with the other known measures of protein metabolic response to injury and changes during nutritional therapy. Extracellular PA levels could be used as markers of both catabolic pathology in trauma and of its response to nutritional therapy.

Obesity and the metabolic response to severe multiple trauma in man.

In the obese state profound metabolic disturbances exist and it is not known how this disrupted metabolism in obese subjects (body mass index greater than 30) may change their ability to respond to the superimposed, injury-induced stress. Understanding the mechanisms that modify the metabolic parameters in traumatized obese patients is essential in their nutritional assessment and further treatment. We have investigated in 7 obese and 10 nonobese multiple trauma patients, on a whole-body level, the energy metabolism, protein kinetics, and lipolysis in the early catabolic "flow phase" of severe injury when they were receiving maintenance fluids without calories or nitrogen. Traumatized obese patients mobilized relatively more protein and less fat compared with nonobese subjects. A relative block both in lipolysis and fat oxidation is experienced by injured obese patients that results in a shift to preferential use of proteins and carbohydrates. Reduced endogenous protein synthetic efficiency observed in obese patients implies increased protein recycling. Thus obese patients could not effectively use their most abundant fat fuel sources and have to depend on other fuel sources. The nutritional management of obese trauma victims should therefore be tailored towards provision of enough glucose calories to spare protein.

Trend toward nonoperative management of splenic injuries.

Treatment of splenic injuries has evolved over the past decade to reflect more effort to conserve function of the spleen. Records of 169 patients admitted over a 6-year period were identified as documenting the treatment of splenic injuries. We collected data regarding patient age, gender, degree of hemodynamic stability, number of units of blood required, severity of splenic injury, Injury Severity Score, and results of treatment. There were 143 adults (age greater than 16 years) and 26 pediatric patients (age less than 17 years), with mean age in the 2 groups of 31.6 and 11.4 years, respectively. Males comprised 72% of the group, and blunt injury occurred in 154 of the 169 patients. In the adults, splenectomy, splenorrhaphy, laparotomy without operative treatment of the spleen, and nonoperative management were observed 48%, 30%, 14%, and 8% of the time and in the pediatric group 31%, 27%, 19%, and 23% of the time, respectively. By using operative splenic repair techniques and increased use of nonoperative management, the splenic salvage rate has increased in the last 6 years from 41% to 61% without an increase in morbidity and mortality. Incidence of spleen salvage correlated with severity of spleen and overall injury and cardiovascular stability.

Effect of major trauma on plasma free amino acid concentrations in geriatric patients.

Age-associated decrease in lean body mass may lead to varied responses to severe trauma. Hypoaminoacidemia is generally common among trauma victims. We measured the plasma free amino acids in the early "flow," ie, catabolic, phase of injury in 9 elderly (aged 61-81 y) and 13 young (aged 20-38 y) traumatized patients. Postabsorptive control samples were obtained from 8 elderly and 10 young volunteers. The results were analyzed for age-related responses to major trauma. Older healthy control subjects showed a decrease in total amino acids, essential amino acids, proline, histidine, taurine, and cystine. The hypoaminoacidemia was less pronounced in geriatric trauma, mainly because of a larger decrease in nonessential amino acids in young trauma victims. Significant decreases in arginine and methionine and increases in ornithine and citrulline concentrations were seen in geriatric trauma. These results suggest a sluggish protein metabolic response to trauma in elderly individuals, which should be considered in their nutritional management.

Glucose turnover, oxidation, and indices of recycling in severely traumatized patients.

Hyperglycemia is often seen in trauma patients and its etiology is not clearly understood. We have determined parameters of glucose metabolism by using simultaneous primed-constant intravenous infusion of both [6-3H] glucose and [U-14C] glucose in ten severely traumatized hypermetabolic subjects during the early "flow phase" of injury and in six post-absorptive normal volunteers. The mean rate of glucose production (determined by means of [6-3H] glucose) was 3.96 +/- 0.40 mg/kg/min in trauma patients, which was significantly (p = 0.025) higher than the value of 2.75 +/- 0.13 observed in normal volunteers. Glucose turnover rates determined with [U-14C] glucose as tracer were lower in all subjects. The difference between the turnover rates determined by the two tracers represents an index of recycling of glucose through three-carbon fragments. This recycling index was similar in both groups of subjects in amount (0.24 +/- 0.07 vs. 0.26 +/- 0.08 mg glucose/kg/min) but different when expressed as percentage of total glucose turnover (5.6 +/- 1.4% vs. 9.8 +/- 1.7%; p = 0.05). The absolute rates of glucose clearance, oxidation, and recycling were similar in stressed trauma patients and unstressed controls although the rate of production was increased by 44% due to injury. Post-trauma hyperglycemia was mainly due to an increased hepatic output of glucose and not due to a decreased ability of the tissue to extract glucose from the plasma. Hyperglycemia may be the driving force in the metabolic effects of injury.

Influence of parenteral nutrition on rates of net substrate oxidation in severe trauma patients.

Optimal nutritional support should use a patient's energy expenditure as a guide for administering sufficient but not excessive caloric intake. Eight patients requiring parenteral nutrition were evaluated, using indirect calorimetry measurements, to determine the nutritional influence on the rates of substrate utilization in the critical period of catabolic illness due to accidental trauma. Five days of total parenteral nutrition, providing calories to match the measured basal resting energy expenditure and N to replace the initial urinary losses a) shifted the RQ from 0.74 +/- 0.03 to 0.81 +/- 0.03, b) improved but could not reverse negative N balance, c) decreased net fat oxidation, d) increased carbohydrate and protein oxidation, e) elevated daily norepinephrine and epinephrine excretion rates, and f) attained positive energy balance. The results suggest that positive energy balance could be achieved in trauma patients by providing total energy intake matching their basal measured energy expenditure plus 7% to 10% for activity energy expenditure. To prevent further loss of lean body mass, an N intake of 350 mg/kg.day was needed in these catabolic ICU patients.

Energy cost of fat-fuel mobilization in geriatric trauma.

Age-related changes in body composition may result in varied responses to acute accidental injury. Gaining fat as age advances is common and therefore the mobilization of fat fuel resources in traumatized geriatric patients needs closer examination. We have measured in six elderly (age, 60 to 74 years) and seven young (age, 18 to 46) traumatized, hypermetabolic, and highly catabolic patients, in the "flow phase" of the metabolic response to injury, the rates of whole-body lipolysis and net fat oxidation. This enabled us to calculate the rate of triglyceride/free fatty acid (TG/FFA) cycling in the whole body and to assess its contribution to energy expenditure. Energy metabolism in general and the fat metabolism in particular were found to be somewhat slowed in elderly trauma patients compared with equally injured young individuals, although the aged patients had more total body fat. The energy cost of TG/FFA cycling is significantly (P less than .025) lower in elderly trauma victims (0.28 +/- 0.06 kcal/kg/d) compared with young patients (0.63 +/- 0.1 kcal/kg/d). This can account for approximately 3% to 4% of the elevation in metabolic rate over that predicted in the uninjured state.

Nutritional impact on the energy cost of fat fuel mobilization in polytrauma victims.

In the "flow" phase of severe injury, mobilization of body fat and protein sources are accelerated to meet increased demands. Fat mobilization usually exceeds the need for oxidative substrates and the leftover fatty acids are re-esterified, resulting in a "futile" TG/FFA cycle. This contributes to increased energy expenditure and provision of nutrients may alter the activity of this cycle. We measured in nine adult (age 40 +/- 7 years, weight, 80 +/- 4 kg) severely traumatized (ISS 32 +/- 4) patients, the resting energy expenditure (REE) and net fat oxidation rate (NFO) by indirect calorimetry and whole body lipolysis rate (WBLR) by a two-stage, primed-constant infusion of glycerol. Fasting postinjury kinetic studies were performed within 48-96 hours after admission when the patients were receiving saline without calories or nitrogen. Glucose-based nutritional therapy was then started and continued for 5 to 7 days. The kinetic measurements were repeated after the nutritive solutions were replaced by normal saline for 12 to 15 hours to achieve a postabsorptive state. Trauma elicits an accelerated rate of fat mobilization with increased TG/FFA cycle activity. Adequate nutritional support for 5 to 7 days tends to reduce but cannot normalize the lipid metabolism. In acute trauma 47 +/- 7% of the mobilized fat was recycled to triglyceride and this is increased to 54 +/- 9% after providing nutrition. The energy cost of this cycling was 38 kcal/day, which is five times that seen in normals and corresponds to 1.34% REE.

Endogenous protein-synthesis efficiency in trauma victims.

Hyperglycemia despite increased glucose oxidation and hypoaminoacidemia despite increased rate of protein breakdown are frequently seen in hypermetabolic and highly catabolic patients with major trauma. The extent of reutilization of endogeneously produced amino acids in the synthesis of new proteins (protein recycling) by the traumatized human is poorly understood. The protein synthesis efficiency was measured in ten multiple trauma patients during the early "flow" phase of injury, and these data were compared with normal and depleted patients. In acute trauma the synthetic efficiency of protein is significantly (P = .01) low (61% +/- 4%) compared with other pathologic states (78% +/- 1%). This suggests that the priority is preferentially set to meet the increased demands of glucose production, and a major portion of the amino acids are utilized for purposes other than synthesis of body proteins. This observation should be taken into account in the early management of acute trauma patients before the body adapts to accept the exogenous supply of amino acids.

Preinjury psychopathology in trauma patients.

One hundred patients admitted to a Level I trauma center were interviewed using the Langner 22-item index of psychiatric symptoms to evaluate pre-existing psychological pathology. The medical chart was also examined for any type of note or evidence of psychiatric consultation efforts by the surgical attending staff. There were 74 males and 26 females in the sample, with a mean age of 33 years. Types of injuries included blunt trauma in 71% and penetrating trauma in 29% of the group, respectively. Eight per cent of these injuries were self inflicted. The mean Injury Severity Score was 17. Alcoholic intoxication was documented in 49%. We found that severe psychopathology as defined by the Langner Index was present in 88% of those admitted with penetrating trauma, in 47% of those admitted with blunt trauma, and in 75% of the intoxicated group. In only 14% of the sample was psychopathology documented in the chart by the attending staff. Preinjury psychopathology in trauma patients was commonly present and seemed to be most highly associated with penetrating trauma and alcohol use.

Polyamine levels as biomarkers of injury response in polytrauma victims.

Enhanced protein mobilization and synthesis are common responses to severe trauma. The hypothesis that extracellular polyamine levels could be valid biomarkers for these responses has been investigated. The three polyamines, spermidine, spermine and their precursor putrescine are directly involved in cell growth/death kinetics through regulation of protein metabolism. The lack of tissue uptake of extracellular polyamines and their rapid conjugation and excretion make them excellent biomarkers of variations in cellular kinetics. The polyamine levels in plasma and urine of severely traumatized patients were measured during the early "flow" phase of injury and compared with unstressed normals. Significantly elevated urinary levels of free and total putrescine and spermidine indicate the increase in the protein synthesis and breakdown rates, respectively, in polytrauma patients. Urinary spermidine level correlates well with other known parameters of protein catabolism, such as isotopically measured whole body protein breakdown rate in the basal state and 3-methylhistidine excretion and nitrogen loss in the basal condition and during nutritional therapy. Whole-body protein synthesis rate positively correlates with putrescine levels in urine. Based on these observations, urinary levels of the polyamines spermidine and putrescine may be applied as valid biomarkers of protein breakdown and synthesis rates, respectively, both for the existing pathology of severe trauma and for the response to nutritional therapy.

Aminoaciduria of severe trauma.

Plasma and urine levels of free amino acids were measured in 15 severely traumatized adult patients while they were receiving fluids free of calories and nitrogen. Endogenous plasma clearance and the relative rates of reabsorption of free amino acids from renal tubules were calculated. These data were compared with similar studies of eight control subjects. Multiple injury provoked distinct patterns of free amino acids in plasma and urine. Hypoaminoacidemia and hyperaminoaciduria were seen in severe trauma. There was a marked depletion of nonessential amino acids in plasma of trauma victims. In contrast, the urinary loss of all amino acids was increased 5-10 times. This enhanced loss in patients, however, represented only 2.1% of total N excreted compared with 0.7% in control subjects. Considerable variations were seen in the selectivity with which various amino acids were reabsorbed by renal tubules. This may partly be due to the abnormal pattern of amino acids presented to renal tubules.

Fungitoxicity of m-fluorophenylalanine-containing peptides towards Pythium ultimum.

The tripeptide L-m-fluorophenylalanyl-L-alanyl-L-alanine was much more fungitoxic towards Pythium ultimum than the dipeptide L-m-fluorophenylalanyl-L-alanine or m-fluorophenylalanine. The fungitoxicity of the tripeptide was reduced by L-alanyl peptides and phenylalanine, but not by other amino acids. In contrast, the fungitoxicity of m-fluorophenylalanine was unaffected by peptides, and was antagonized by several amino acids. These results suggest the effective delivery of m-fluorophenylalanine into the cell by a tripeptide carrier.

Effect of helicopter transport of trauma victims on survival in an urban trauma center.

This paper reports a retrospective analysis of patients with serious yet substantially survivable injuries represented by ISS scores from 20 to 39 and whether or not survival was influenced by the use of helicopters. A review of 606 of these patients with blunt trauma was performed for the period from 1983 through 1986. When the group was evaluated there were 451 patients in the ISS cohort of 20-29 and 155 in the 30-39 group. The mean age was 30.5 years and 76% were males. A total of 259 patients were transported by ambulance and 347 by helicopter. Characteristics of the two groups were similar. The mean TS was 12.7 for ambulance and 12.1 for helicopter patients. Mean GCS was 10.4 in the ambulance group and 9.6 for helicopter patients. Overall the mortality for ambulance transported patients was 13% compared to 18% for the helicopter group. We conclude that there is no survival advantage in the helicopter transported group in an urban area with a sophisticated prehospital care system. Patients of rural origin deserve further study.