The esophageal detector device: a rapid and accurate method for assessing tracheal versus esophageal intubation in a porcine model.

To assess time and accuracy of the esophageal detector device (EDD), disposable end-tidal CO2 monitor (ETCO2), and standard clinical methods for detection of endotracheal tube placement. Prospective, randomized, single-blinded, controlled laboratory investigation. Thirty airway managers (physicians, nurse anesthetists, and paramedics) used one pig (Sus scrofa) as the intubated, respiratory depressed/arrest model. Part 1: A standard 7.5-mm endotracheal tube was placed in either the esophagus or the trachea of the anesthetized swine. Anatomic location was verified by bronchoscopy. Airway managers blinded to the endotracheal tube location were assigned randomly to identify tube position by one of three methods (EDD, ETCO2, or clinical methods). Speed and accuracy of the assessment were recorded. Part 2: A second identical tube was placed, so that both the esophagus and the trachea were intubated; then, the esophageal tube was bag-ventilated for one minute. Each blinded airway manager, using only the EDD, determined placement site of both tubes. Part 1: Mean time to determine tube placement for group A (EDD) was 13.8 seconds; group B (ETCO2), 31.5 seconds; and group C (clinical methods), 39 seconds. Comparison by analysis of variance yielded a value of P less than .001. Both groups A and B were 100% accurate, whereas 30% of the subjects from group C mistakenly assessed an esophageal tube as in the trachea. Part 2: The EDD remained 100% sensitive and specific despite prior ventilation of the esophageal tube. In this porcine model, the EDD and ETCO2 were more accurate than clinical methods in determining endotracheal tube placement. The EDD demonstrated a significant time advantage over both ETCO2 and clinical methods. Prior ventilation of the esophageal tube does not interfere with the accuracy of the EDD.


Effects of constriction bands on rattlesnake venom absorption: a pharmacokinetic study.

To determine whether the use of a constriction band alters systemic absorption of rattlesnake venom in pigs and whether constriction band use alters local swelling. Using a crossover design, five pigs were studied with and without the use of a constriction band. 125I-Labeled Western Diamondback rattlesnake (Crotalus atrox) venom was injected subcutaneously into one foreleg. The protocol was repeated using the opposite foreleg six days later. The constriction band was applied at the time of injection and removed four hours later. Plasma radioactivity and leg circumference were measured serially. Maximum plasma venom concentration and area under the venom concentration-time curve were compared in trials with and without constriction band. Within the initial four hours, application of a constriction band decreased maximum plasma venom concentration by 25% and area under the venom concentration-time curve by 33% (P less than .05). After the constriction band removal at four hours, maximum plasma venom concentration and the area under the venom concentration-time curve were not significantly different between groups. Application of a constriction band did not result in a statistically significant increase in maximum leg circumference as compared with trials without a constriction band. The use of a constriction band was effective in reducing venom absorption while it was in place (reduced area under the venom concentration-time curve and maximum plasma venom concentration in the cuffed group), and constriction band removal did not result in a significant increase in maximum plasma venom concentration. Leg swelling was not affected by constriction band use. Because constriction band use delayed venom absorption without causing increased swelling, it may prove to be a useful first aid measure in human beings.


The clinical implications of continuous central venous oxygen saturation during human CPR.

The purpose of this study was to observe, measure, and describe the changes in central venous oxygen saturation during CPR and immediately after return of spontaneous circulation. It also was to examine the clinical utility of continuous central venous oxygen saturation monitoring as a indicator of return of spontaneous circulation during CPR in human beings. Eight-month, prospective, non-outcome, observational, nonrandomized case series in the ED of a large urban hospital. TYPES OF PATIENTS: Adult normothermic, nontraumatic, out-of-hospital cardiopulmonary arrests. All patients were managed according to advanced cardiac life support guidelines. A proximal aortic and double-lumen central venous catheter was placed. Central venous oxygen saturation was measured continuously spectrophotometrically with a fiberoptic catheter in the central venous location. Aortic blood pressure and central venous oxygen saturation were simultaneously measured throughout each resuscitation. Return of spontaneous circulation was defined as a systolic blood pressure of more than 60 mm Hg for more than five minutes. One hundred patients who experienced 68 episodes of cardiac arrest were studied. Patients with return of spontaneous circulation had a higher initial and statistically higher mean and maximal central venous oxygen saturation than those without return of spontaneous circulation (P = .23, .0001, and .0001, respectively; P less than .05 is significant). No patient attained return of spontaneous circulation without reaching a central venous oxygen saturation of at least 30%. Only one of 68 episodes of return of spontaneous circulation was attained without reaching a central venous oxygen saturation of at least 40%. A central venous oxygen saturation of greater than 72% was 100% predictive of return of spontaneous circulation. Continuous central venous oxygen saturation monitoring can serve as a reliable indicator of return of spontaneous circulation during CPR in human beings.


The epidemiology of cardiac arrest in young adults.

To describe the epidemiology of cardiac arrest in young adults and to determine if there are characteristics unique to this group in terms of etiology, rhythm, and outcome. Retrospective, case review. King County, Washington. All out-of-hospital victims of cardiac arrest who received emergency aid. The etiology, cardiac rhythm, and outcome were identified for each case. During the 13-year period from 1976 to 1989, there were 8,054 cardiac arrests; 252 of these were among young adults 18 to 35 years of age. Of those 252 cases, 61 (24%) were caused by ischemic heart disease, and 60 (24%) were caused by overdose. Asystole was the most common rhythm (48%), followed by ventricular fibrillation or tachycardia (31%). Long-term survival following these rhythms was 4% and 28%, respectively. In terms of age, etiology, and rhythm, young adults appear to represent a transitional group between children and older adults. There were no unique characteristics specific to young adults. Long-term survival is dependent more on rhythm than on age. In terms of age, etiology, and rhythm, young adults appear to represent a transitional group between children and older adults.


Improving the time to thrombolytic therapy for myocardial infarction by using a quality assurance audit.

To evaluate a program designed to improve the speed of delivery of thrombolytic therapy for myocardial infarction in an emergency department. Time to administer thrombolytic therapy was evaluated retrospectively in 1988. Then after physician and nurse education, time to therapy was audited in 1989 and 1990, with feedback to physicians. A community hospital ED. Eight board-certified emergency physicians were monitored in their treatment of 58 patients. After the 1988 audit, the results for each physician were made known. Physician and nurse education was undertaken, and it was agreed to continue the audit. Nurses were encouraged to order ECGs rapidly, physicians were encouraged to make the treatment decision before consulting private physicians, and a goal of therapy in less than one hour was established. Further feedback on times was given to the physicians in 1989. Mean time from ED arrival to thrombolytic therapy was 63 minutes in 1988, 47 minutes in 1989, and 38 minutes in 1990. This fall in time was significant (P = .0002). The number of patients treated within one hour rose from 45% in 1988 to 67% in 1989 and to 96% in 1990. Auditing time to thrombolytic therapy, combined with physician and nurse education and increased experience, can significantly improve the time to administration of thrombolytic therapy.


A trial of nebulized magnesium sulfate to reverse bronchospasm in asthmatic patients.

To test the hypothesis that nebulized magnesium sulfate reverses methacholine-induced bronchospasm in asthmatic patients. Randomized, double-blind, crossover clinical trial. Center for Asthma and Allergic Diseases, Children's Hospital of Pittsburgh. Ten patients who were 21 to 37 years old and had stable asthma. Patients withheld asthma medications for 24 hours before each study day. Patients with baseline forced expiratory volume in one second (FEV1) of less than 80% were excluded. All subjects underwent bronchial methacholine challenge to produce bronchospasm. They then received one of three different nebulized treatments: 2.5 mg albuterol in 3 mL saline, 3 mL magnesium sulfate (268 mmol/L, pH 6.4), or 3 mL normal saline. Patients repeated spirometry 15 minutes after completing the study drug and then received albuterol by metered-dose inhaler. Spirometry was repeated after the metered-dose inhaler. Each patient made three separate visits to receive each of the three medications. Methacholine reduced each patient's FEV1 by at least 20% at each testing session. Post-methacholine treatment with nebulized albuterol improved FEV1 by a mean of 56% (SD, 19.6%). Nebulized normal saline led to a mean increase in FEV1 of 29% (SD, 26.5%). Nebulized magnesium sulfate improved FEV1 by a mean of 12% (SD, 12.0%) (P = .054 by paired t-test compared with normal saline). Nebulized magnesium sulfate has a minimal bronchodilatory effect in asthmatic patients with methacholine-induced bronchoconstriction. Responsiveness to magnesium sulfate may be dependent on the mechanism of induction of bronchospasm, and there probably is no role for nebulized magnesium sulfate in the treatment of acute bronchospasm due to cholinergic stimulation.


Ketorolac versus meperidine and hydroxyzine in the treatment of acute migraine headache: a randomized, prospective, double-blind trial.

To compare the effectiveness of IM ketorolac with that of meperidine and hydroxyzine in the treatment of acute migraine headache. Prospective, randomized, double-blind trial. Urban emergency department with an annual census of 42,000 patients. Forty-seven adult patients with migraines enrolled on 50 visits. Patients were randomly assigned to receive a single injection of either 60 mg ketorolac (group 1) or 100 mg meperidine and 50 mg hydroxyzine (group 2). Pain assessment was made using both visual-analog and verbal descriptor scales. At 60 minutes, 15 patients (60%) from group 1 (25) and 14 patients (56%) from group 2 (25) reported a great deal of complete relief (P = .77) Sixty-minute mean pain relief scores (3.35 versus 3.37) were different (P = .76). Nine patients (36%) from group 1 and seven patients (28%) from group 2 required additional analgesia (P = .76). Ketorolac is as effective as meperidine and hydroxyzine for the treatment of acute migraine headache.


Comparison of intravenous and intranasal administration of epinephrine during CPR in a canine model.

Epinephrine improves coronary perfusion pressure during CPR. However, administration of epinephrine during CPR may be delayed or omitted if IV or endotracheal access is not established. Therefore, the objective of this study was to determine if intranasal administration of epinephrine during CPR would provide an alternate route of drug administration that is readily accessible and requires no special technical skills. Randomized blinded study performed in a controlled laboratory environment. Twenty mongrel dogs weighing 19.5 +/- 4.6 kg. All dogs received either IV epinephrine 0.015 mg/kg or intranasal epinephrine 14 mg per nostril. Phentolamine (5 mg per nostril) was administered intranasally one minute before nasal administration of epinephrine to improve absorption. Each dog underwent three minutes of ventricular fibrillation followed by seven minutes of CPR with a pneumatic chest compression device. Epinephrine was administered at two minutes into CPR. Seven dogs were excluded because of inadequate baseline coronary perfusion pressure or compression device displacement, leaving a total of 13 dogs for analysis (six IV epinephrine, seven intranasal epinephrine). Baseline coronary perfusion pressure (mean +/- SD) was similar for IV epinephrine and intranasal epinephrine (16.9 +/- 7.1 mm Hg versus 18.2 +/- 13.8 mm Hg, respectively, P = .84). For IV and intranasal epinephrine, coronary perfusion pressure increased to 21.4 +/- 9.2 mm Hg and 24.4 +/- 18.7 mm Hg one minute after epinephrine, respectively (P = .73). Five minutes after epinephrine coronary perfusion pressure was 18.2 +/- 8.7 mm Hg and 24.3 +/- 13.9 mm Hg for IV epinephrine and intranasal epinephrine, respectively (P = .38). The rate of successful resuscitation was similar for both groups, five of seven dogs for intranasal epinephrine and four of six dogs for IV epinephrine (P = .66). Intranasal epinephrine has similar effects on coronary perfusion pressure and resuscitation compared with standard-dose IV epinephrine. Therefore, the nasal route for administration of epinephrine appears to be an acceptable alternate method of drug delivery during CPR and compares favorably with standard IV therapy in the canine model. Because of the obvious benefits to human patients, these observations suggest further investigation.


Correlation of end-tidal CO2 to cerebral perfusion during CPR.

A number of studies have demonstrated a correlation between end-tidal carbon dioxide (ETCO2), cardiac output, and return of spontaneous circulation in experimental animals and in patients undergoing closed-chest CPR. Our study attempted to correlate ETCO2 to cerebral blood flow during cardiac arrest. Sixteen piglets were anesthetized, intubated, and instrumented for cerebral blood flow studies. An ultrasonic flow probe was placed on both internal carotid arteries for continuous flow measurements. The animal was fibrillated, and closed-chest CPR was begun. Continuous ETCO2 measurements were obtained and compared with simultaneous internal carotid, cardiac output, and cerebral blood flow measurements. Correlations between ETCO2 and carotid and cerebral blood flow were determined using Pearson's method. The correlation between ETCO2 and total internal carotid flow was .58 (P = .01, Bonferroni's adjusted P = .30). Correlation between ETCO2 and cerebral blood flow was .64 (P = .01, Bonferroni's adjusted P = .09). A partial correlation coefficient for ETCO2 versus cardiac output was .70, whereas it was only .30 for ETCO2 versus cerebral blood flow. Partial correlation coefficients suggest that ETCO2 correlates with cerebral blood flow when changes in cerebral blood flow parallel changes in cardiac output.


Evaluation of the Q.E.D. Saliva Alcohol Test: a new, rapid, accurate device for measuring ethanol in saliva.

To evaluate the accuracy of the Q.E.D. A-150 Saliva Alcohol Test, a new device that gives a specific quantitative blood alcohol level by measuring saliva alcohol concentration in the range of 0 to 150 mg/dL. Forty-two healthy volunteers consumed 4.5 to 6 oz of alcohol in the form of beer, wine, or liquor over a 90-minute period. Blood and saliva samples were obtained for alcohol measurement at 30, 60, 90, and 120 minutes after the last drink. Blood samples were analyzed within 24 hours by gas chromatography at a commercial clinical laboratory. Saliva samples were tested immediately using the new Q.E.D. A-150 Saliva Alcohol Test. Excellent correlation was observed between saliva and blood alcohol levels over the range of 0 to 150 mg/dL (slope = 1.0; intercept = 2.4; r = .98). The Q.E.D. Test is an accurate device for specific quantitative measurement of alcohol levels using saliva.


Emergency department presentation and care of heart and heart/lung transplant recipients.

Heart and heart/lung transplants are accepted forms of therapy for patients with end-stage cardiac or pulmonary disease. These patients are likely to present postoperatively to the emergency department. To our knowledge, there have been no previous reports in the medical literature of the ED presentation of these patients. We conducted a retrospective review of the records of all patients who had received a heart or a heart/lung transplant at Stanford University Medical Center from 1988 through 1990 and who had at least one ED visit. Between the time of operation and April 1, 1991, 131 ED visits were recorded. Fever was the single most common presenting complaint, for 48 (37%) of the visits. Difficulty in breathing (13%); gastrointestinal symptoms of nausea, vomiting, or diarrhea (10%); and chest pain (9%) were other common reasons for presentation. The transplant patient, by virtue of requiring chronic immunosuppression, is susceptible to infection with a spectrum of opportunistic organisms. When fever or other symptoms suggest infection, appropriate cultures and aggressive diagnostic procedures (eg, lumbar puncture, bronchoscopy) should be performed.


Loiasis: a case of an unusual ocular foreign body.

Loiasis is a parasitic illness that is endemic in parts of Central and West Africa. In the United States, infection with Loa loa is seen in natives from that region of Africa and in those who have traveled to the area, often in the distant past. There can be significant differences in clinical manifestations between the two groups. We present a case of loiasis in an African native as well as a discussion of Loa loa infection in natives and non-natives and current treatment strategies.


Volar metacarpal phalangeal joint dislocation: a rare and often missed injury.

Volar metacarpal phalangeal joint dislocations are rare injuries. We could find only eight cases reported, with none in the emergency medicine literature. We present a case report and describe the biomechanics and radiographic findings of this injury.


Ruptured splenic abscess secondary to infectious mononucleosis.

A 24-year-old woman with a recent diagnosis of infectious mononucleosis presented with fever and left upper quadrant pain. Abdominal computed tomography revealed a splenic abscess that at laparotomy was found to have ruptured. This case illustrates a rare cause of splenic abscess and a rare complication of infectious mononucleosis.


Comparison of laser trabeculostimulation with laser trabeculoplasty in open-angle glaucoma.

We compared a new method, laser trabeculostimulation (LTS), with laser trabeculoplasty (LTP). The technique LTS stimulates trabecular meshwork tissue and thereby increases the outflow facility. The method LTP causes shrinkage and cicatrization of the meshwork tissue, thus limiting the reduction in postoperative intraocular pressure (IOP). The mean preoperative IOP of 40 eyes was 24.9 +/- 6.8 mmHg (mean +/- standard deviation). The LTS was applied with a 50-microns spot size, a power setting of 50mW, and an exposure time of 1 sec. With LTS, the IOP decreased and then increased afterward. Low power (50mW) was effective if used for a longer duration. The LTS can be applied repeatedly and used in addition to conventional LTP. The overall effect achieved with LTS was less than that with LTP, but it is possible to reduce IOP by selecting more reasonable parameters, e.g., a laser setting of 200mW, a 50 microns spot size and a 0.5 to 1-sec duration.


Efficacy of cryotherapy in vernal catarrh.

Cryotherapy of the palpebral conjunctiva with oral aspirin has been evaluated in mixed-type active vernal keratoconjunctivitis. The symptoms and signs were recorded on a four-point scale. In 15 patients, we used cryotherapy of the palpebral conjunctiva in 30 eyes (-60 degrees C to -80 degrees C for 30 seconds, repeating the freeze-thaw cycle 2-3 times). All patients received oral aspirin 0.5 to 1.5 g in three divided doses daily over six weeks. The relief from symptoms was statistically significant (P less than .001). Objective improvement (palpebral and bulbar signs) also was statistically significant (P less than .001). Follow-up of these cases for one year showed a 3.3% recurrence rate.


Continuous control of intraocular pressure in pseudophakic retinal detachment surgery.

We describe a method for continuous control of intraocular pressure in pseudophakic retinal detachment surgery done shortly after extracapsular cataract extraction and posterior chamber intraocular lens implantation. The system consists of a special needle inserted into the anterior chamber through the corneal limbus. The needle is attached by a regular intravenous administration set to an infusion bag. This allows fluids to exit the eye when the eyeball is compressed and to return to the eye when pressure on the eye is released. The presence of an intact posterior capsule and an intraocular lens do not impose a significant barrier to the passage of fluids between the anterior chamber and the vitreous cavity. Using this technique, intraocular pressure can be stabilized throughout the operation. The implementation of this system is demonstrated by a case report.


Dislocation of the crystalline lens in a patient with Sturge-Weber syndrome.

A moderately retarded institutionalized patient diagnosed with Sturge-Weber syndrome and bilateral retinitis pigmentosa underwent enucleation OD because of intractable pain secondary to glaucoma in 1965. Twenty-three years later, the patient had a dislocated lens OS. Although the lens dislocation might have been caused by trauma, it also might be associated with either Sturge-Weber syndrome or retinitis pigmentosa. The role of protective eye wear in one-eyed institutionalized retarded patients is discussed. To our knowledge, this combination of Sturge-Weber syndrome, bilateral retinitis pigmentosa, and a dislocated lens has not been reported previously.


Acute monocular blindness secondary to fibrous dysplasia of the skull: a case report.

An unusual case of acute monocular visual loss secondary to fibrous dysplasia is presented. Orbital and optic canal decompression restored vision completely. A review of the pertinent literature revealed that operative intervention may be curative in patients with acute monocular visual loss. This finding contrasts with the chronic visual decline found secondary to fibrous dysplasia of the skull.


Eales disease with internuclear ophthalmoplegia.

Eales disease is an idiopathic type of retinal perivasculitis characterized by recurrent retinal and vitreous hemorrhages. Neurologic involvement is rare. We report the case of a patient with Eales disease who had internuclear ophthalmoplegia thought to be a neurologic manifestation of this disease.


Transient fluorescein leakage simulating subretinal membrane: a diagnostic dilemma.

A 68-year-old woman with blurred vision had a foveal spot of fluorescein leakage that later was found to be a transient leakage. The clinical picture resembled central serous chorioretinopathy, a rare diagnosis in older patients. Other possible diagnoses (e.g., subretinal neovascular membrane or epiretinal membrane) are discussed as is the need for laser treatment.


Retinal circulation in primary open-angle glaucoma tested by videodensitometric image analysis.

To investigate the retinal circulation in primary open-angle glaucoma (POAG), we used videodensitometric image analysis of fluorescein angiography to calculate the build-up time, the time constant of the washout rate, and the mean circulation time (MCT) in both the retinal artery and vein. We found the MCT in the POAG group (23 eyes of 20 patients) was significantly longer than in the normal group (3.7 +/- 2.0 sec and 2.8 +/- 1.7 sec, respectively; P less than .05). It correlated significantly with the vertical cup-to-disc ratio (r = .32; P less than .01), the outflow facility (r = -.35, P less than .005), and the glaucomatous visual-field loss. There was no correlation between MCT and intraocular pressure tested at the same time (r = .09, not significant). Therefore, treatment to decrease the intraocular pressure may not be associated directly with improved retinal circulation.


Phonation and swallowing considerations in pediatric laryngotracheal reconstruction.

Experience with laryngotracheal reconstruction (LTR) has resulted in and continues to yield modifications and refinements in approach and technique with the goal to restore and maintain total laryngeal function. In addition to airway obstruction, the laryngeal functions of phonation and swallowing also may be affected by the underlying injury as well as by procedures designed to enlarge the airway. This paper discusses various problems encountered with phonation and swallowing in pediatric patients who underwent LTR and postoperative patients who were seen during the year July 1, 1990, through June 30, 1991. Phonation problems became apparent as long-term difficulties that persisted after tracheotomy decannulation. Swallowing was frequently a short-term perioperative problem while a stent was in place following LTR. The approaches and techniques that have been employed to treat, minimize, and prevent these problems are discussed.


Consumer product aspiration and ingestion in children: analysis of emergency room reports to the National Electronic Injury Surveillance System.

Inadvertent aspiration or ingestion of products in children is a reportable problem. The National Electronic Injury Surveillance System (NEISS) monitors 119 hospitals for product (nonfood) hazards through a network of emergency room physicians. Coins (52%) remain the most frequently ingested object (1988-1989). Pins, including nails and bolts, are second in rank. Spheres (eg, balls) are the most common cause of death. Most deaths (97%) occur in the home. Younger children (13.4 versus 22.7 months) are more at risk for death. This analysis suggests that shape, as well as availability, is important in children's injuries and deaths. Accurate reporting to NEISS of all aspirated objects remains essential so that product safety and the public's health can be improved. Food product injuries, however, are not reported to NEISS.


Pediatric airway manifestations of gastroesophageal reflux.

Gastroesophageal reflux (GER) in children may be classified as physiologic or pathologic, depending on its degree and consequences. There are many head and neck complications of GER in pediatric patients, but most numerous are the airway manifestations, including stridor, recurrent croup, exacerbation of subglottic stenosis, laryngeal irritation with or without laryngospasm, chronic cough, and obstructive apnea. Diagnosis may be difficult unless there is a high index of suspicion for GER and awareness of the concept of "silent" GER. We present the common pediatric airway manifestations of GER, illustrated by case reports, and provide a paradigm to assist in the diagnosis and management of children with airway compromise associated with GER.


Distributions of eustachian tube goblet cells and glands in children with and without otitis media.

The distributions of the goblet cells and submucosal glands in eustachian tubes (ETs) from children with or without otitis media (OM) were studied. From 12 temporal bone-ET specimens obtained from children 1 to 7 years old at death, representative vertically cut histology sections were selected, stained with periodic acid-Schiff-Alcian blue and hematoxylin, and studied by light microscopy. In children without OM, the density of the goblet cells was significantly lower in the roof than in the floor of the ET and was highest in the midcartilaginous portion; glands were most richly distributed near the pharyngeal orifice, and became less densely distributed toward the tympanic portion. In children with OM, the density of the goblet cells was significantly higher both in the roof and floor portions, and the glands were more richly distributed than in the ETs of children without OM. The possibility that the observed increase in ET secretory components in children with OM impairs the passage of air through these tubes is discussed.


Fascia and perichondrium atrophy in tympanoplasty and recurrent middle ear atelectasis.

Fascia and perichondrium grafts to replace and reinforce thin, atrophic tympanic membranes (TMs) are recommended by several authors to correct middle ear atelectasis by forming a fibrous, collapse-resistant TM. This study reviewed the status of connective tissue grafts performed over the 10-year period from 1979 to 1988 to determine if these grafts would maintain sufficient strength and fibrous character to resist recurrent atelectasis. The author used fascia or perichondrium to repair 89 TM defects, and 63 ears were available for follow-up: 54 had cholesteatomas and 9 had perforations. Graft atrophy was judged by microscopic otoscopy and Kodachrome otophotography. Fascia TM grafts atrophied in 35 of 43 ears (80%), and perichondrium atrophied in 8 of 20 ears (40%). Grafts maintained their relatively thick and fibrous character in only 20 of 63 ears (32%). If fascia and perichondrium used to correct atelectasis were to atrophy at the same rate as the grafts in this series, atelectasis would recur after attempts to reinforce atrophic TMs. Atelectasis-prone middle ears require intubation despite surgery.


Clearance function of eustachian tube and negative middle ear pressure.

Two experimental studies were performed using 18 cats in order to elucidate the mechanism of the long-lasting course of otitis media with effusion. First, the middle ear (ME) pressure was monitored for 2.5 to 7 hours after filling the whole ME space with saline. On average, -150 mm H2O of negative ME pressure was induced in 3.1 hours. Second, the residual volume of saline with antibiotics, which was put into the ME space 2 to 7 days before, was compared between the side on which tubal ventilatory function was abolished (resection of tensor veli palatini muscle and hamulus pterygoideus) and the opposite, control side. The percentage of the residual volume to the original volume put into the ME was significantly higher on the experimental side (Wilcoxon's ranking test, t = 27.0, p less than .05), and in one ear on the experimental side, the ME pressure showed-150 mm H2O just before the bulla was opened 2 to 7 days later. These results seem to indicate that tubal ciliary clearance function can induce negative ME pressure when there is fluid in the ME, and that the negative ME pressure induced by clearance of the ME fluid may disturb further clearance of the ME fluid. This condition may cause the long-lasting course of otitis media with effusion.


Chondroblastoma of the temporal bone. Case report and literature review.

Chondroblastoma is a rare tumor of bone that represents approximately 1% of all primary bone tumors. In the temporal bone, only 33 have been reported. We present a case of chondroblastoma of the temporal bone, followed by a literature review and analysis of reported cases. We conclude that the best chance for a prolonged disease-free interval is total excision using a combined otolaryngologic and neurosurgical approach.


Organ-limited laryngeal amyloid deposits: clinical, morphological, and immunohistochemical results of five cases.

Five cases of organ-limited laryngeal amyloid deposits with no evidence of systemic disease are reported in detail and classified immunohistochemically. In four of the five cases the amyloid reacted with anti-A lambda antibodies and in one case with anti-A kappa antibodies. Four of our five female patients had already passed the fifth decade of life. One was 11 years old. Hoarseness was the predominant symptom in four cases, in which we found amyloid deposits in the glottic area. Only one patient, with amyloid deposits in the aryepiglottic fold, complained of pain. The therapy of choice of idiopathic, localized, or organ-limited amyloid deposits without underlying disease may be local excision. In one of the cases reported in this paper, a laryngofissure was performed, and in another a partial laser resection was performed. No therapy was performed in three of our five cases. In the larynx, as in many other locations and only if possible, removal at intervals is more feasible than radical resection, because these amyloid tumors grow slowly.


New device for interdental immobilization.

The immobilization of a simple fractured jaw with arch bars is a time-consuming, laborious procedure. The alternative method of immobilization described here uses a precisely threaded, 22-gauge, malleable stainless steel wire and a threaded nylon nut. It affords the surgeon relative safety from accidental puncture trauma and makes the interdental immobilization a precise and speedy procedure.


Computer recognition of brain stem auditory evoked potential wave V by a neural network.

A neural network simulator was used for the recognition of the presence and location of the peak of wave V of the brain stem auditory evoked potential (BAEP) test. Waveforms selected from BAEPs performed in the last 4 years at the University of Pittsburgh Presbyterian University Hospital were digitized and sampled, and the resulting amplitudes were normalized. A training set was composed of the waveforms resulting from the stimulation of 50 ears. The normalized amplitudes were used as the initial activation values for the networks. The desired outputs (the target locations for wave V) were represented in the output layer by setting the output element, which corresponded to the target location and its immediate neighbors, to high activation levels, and all the remaining output units to zero activity. Two network architectures, differing only in the hidden unit layer, with 40 and 16 hidden units, respectively, were trained by using standard back-propagation. Several trials from different starting points were performed for each architecture. The best network, found after 60 epochs (3,000 presentations), was able to correctly identify 17 of 20 cases (85%) from a set of test cases that were independent from the training set.


Amyloidosis in the upper aerodigestive tracts.

The upper aerodigestive tracts, particularly the larynx, are not uncommon repositories for amyloid. In most instances amyloidosis of the larynx is localized and is not associated with or followed by systemic disease. Oral and nasopharyngeal amyloidoses, on the other hand, are very often manifestations of systemic predisposing disorders. Laryngeal amyloidosis is treated, when indicated, by surgical removal, often repeated because of persistence or multifocal deposits.


[Chronic pancreatitis in Cameroon. Analysis of etiological and clinical aspects].

In a collection of 3,571 patient's files admitted in the University Teaching Hospital and the Yaounde General Hospital, we studied 27 patients suspected of chronic pancreatitis. 20 patients with calcified chronic pancreatitis benefited from a detailed history, physical examination and a complete paraclinical work-up. From the data collected, chronic alcoholism seemed to have been the main aetiology. Industrial beer from barley alone and/or associated with other traditional liquors was most consumed. The majority of patients were heavy alcoholics and daily consumption varied from 75 to 124 g of pure alcohol. The natural history of the disease and physical examination were identical to that observed in the western countries. Associated pathology was observed in 10% of the patients. This included peptic ulcer disease, cirrhosis and bile stones. Complications included diabetics, obstructive jaundice, and malabsorption syndrome. As a conclusion, chronic pancreatitis is a pathology whose prevalence seems to be progressing constantly.


[Difficult diagnosis of a biliary cyst. A case report].

A 26 year-old man presented a hepatic cyst complicated by intracystic bleeding. Clinical and biological features (chronic pancreatitis), ultrasound and computed tomography findings apparent hepatic and splenic location), macroscopic aspects (inflammatory changes, brown-colored fluid) as well as microscopic characteristics (thickened cyst-wall, absence of epithelium) of both cysts suggested the diagnosis of pancreatic pseudocysts. The diagnosis of hepatic cyst was carried out on histological examination showing Meyenburg and neuro-fibrillar complexes. Occurrence of complications (bleeding, size enlargement) may modify typical histological aspect of hepatic cyst.


[A case of acute cardiac insufficiency caused by 5-fluorouracil].

The authors report a case of acute cardiac failure linked to 5 fluoro-uracil. The toxicity seen most commonly involves ischemia. The pathophysiological mechanisms are discussed as well as the role played by pharmacokinetic characteristics in the occurrence of adverse reactions. 5 fluoro-uracil (5 FU) is a compound widely used in the treatment of ENT, breast and gastrointestinal carcinomas. The finding of dose-effect relationship may lead the clinician to use different modes of administration. Continuous administration at high dose 3 g/m2/day from D1 to D5) in combination or not with cisplatin, or continuously at low dose (300 mg/m2/day from D1 to D31) can reduce hematopoietic toxicity but, in contrast, increases gastrointestinal toxicity.


Selective culture of mitotically active human Schwann cells from adult sural nerves.

We devised a simple method to isolate mitotically active human Schwann cells from sural nerve biopsy specimens and expand the population in culture. Nerve fascicles were treated with cholera toxin for 7 days in culture before dissociation, which increased the cell yield at least twenty-five-fold over immediated tissue dissociation. Digesting the tissue completely with enzymes in serum-containing medium resulted in the highest cell viability, and released 2 to 6 x 10(4) cells/mg of tissue. Seeding the cells on a poly-L-lysine substrate in a small volume of serum-free medium optimized the plating efficiency. Although Schwann cells comprised 90% of the initial culture population, their numbers declined over time due to a faster mitotic rate of the fibroblasts in the presence of cholera toxin alone. However, treating the cultures with a combination of cholera toxin and forskolin, which act synergistically to elevate cyclic AMP levels, inhibited fibroblast growth without causing Schwann cell toxicity. Adding glial growth factor to the adenyl cyclase activators maximized Schwann cell proliferation, and the population rapidly and selectively expanded. Therefore, it should be possible to generate large numbers of Schwann cells from diseased nerves to study defects in cell function or from normal nerves to study the effects of Schwann cell grafts on neuronal regeneration.


Recurrent status epilepticus in children.

Status epilepticus is an uncommon but life-threatening seizure. Little is known about the risk of recurrent status epilepticus in patients who present with an initial episode. To determine the risk of recurrent status epilepticus in children, we prospectively followed 95 children, identified at the time of their first episode of status epilepticus, for a mean of 29.0 months (range, 4-60 months). The patients' ages ranged from 1 month to 18 years (mean, 4.6 years). The cause of the status epilepticus was classified as idiopathic (n = 24), remote symptomatic (n = 18), febrile (n = 29), acute symptomatic (n = 18), or progressive neurological disorder (n = 6). Sixteen children (17%) had at least 2 episodes of status epilepticus. The risk of recurrent status was 4% (n = 1) in the idiopathic group, 44% (n = 8) in the remote symptomatic group, 3% (n = 1) in the febrile group, 11% (n = 2) in the acute symptomatic group, and 67% (n = 4) in those with progressive neurological disease. Recurrent status epilepticus occurred primarily in neurologically abnormal children. While neurologically abnormal children accounted for 34% (n = 32) of all children with status epilepticus, they comprised 88% (n = 14) of the children with recurrent status epilepticus (p less than 0.001) and all 5 of the children with multiple (greater than or equal to 3) episodes of status (p less than 0.001). Fifteen of 16 children with recurrent status epilepticus were being treated with antiepileptic drugs at the time of recurrence. The morbidity and mortality of status epilepticus were low.(ABSTRACT TRUNCATED AT 250 WORDS)


[Sigmoid diverticular diseases: surgical treatment].

From 1966 to 1990, 226 consecutive patients were operated on electively for diverticular disease of the sigmoid colon. The indications for surgery were colovesical fistula or suspicion of residual abscess, existence of two or more previous attacks of acute inflammation, existence of chronic symptoms and suspicion of colonic carcinoma. Colonic resection with primary anastomosis was performed in 217 patients with a covering colostomy in 1 case only. The Hartmann procedure was performed in 9 patients with extension of the lesions to the rectum and/or high operative risk. One or more abscesses were found by the surgeon or the pathologist in 50 p. cent of the patients. There were no postoperative deaths, no clinical anastomotic leakages. Long-term results were evaluated for the patients operated on before 1987, with a follow-up from 2 to 22 years. 85 p. cent of the patients had no more symptoms, 11 p. cent complained of persistent symptoms and 3% had recurrent attacks of pain and fever. Colonic barium enema is the best examination for diverticulitis and chronic abscesses. Surgical treatment is easier for abscesses located within the colonic wall and mesentery, than for extracolic abscesses with local peritonitis. Correlations between preoperative symptoms and operative findings are often not good. The good results obtained in 82 p. cent of the patients operated on for chronic symptoms suggest that chronic symptoms should be part of the indications for elective surgery. The low incidence (3 p. cent) of recurrent attacks of pain and fever is in favour of a resection limited to the sigmoid colon even when diffuse colonic diverticula are present.(ABSTRACT TRUNCATED AT 250 WORDS)


Motion-defined letter detection and recognition in patients with multiple sclerosis.

Two important distinctions in visual perception are (1) between the detection and recognition of shape (e.g., letters), and (2) between the recognition of shapes defined by a difference in brightness and the recognition of shapes defined by a difference in motion. We report that 6 of 10 patients with multiple sclerosis showed impaired recognition for motion-defined (MD) letters, although the detection of MD letters was normal as were both detection and recognition of luminance-defined letters. We have shown that this was not a function of acuity loss or the loss of ability to detect motion or a general failure of recognition per se, but was confined to a loss of ability to recognize MD letters. The neurological implications of these findings are discussed, and it is suggested that the MD letter test be used by others interested in the central pathology of visual disorders.


Quantitative magnetic resonance imaging in temporal lobe epilepsy: relationship to neuropathology and neuropsychological function.

Magnetic resonance images (MRIs) were obtained from 25 patients with medically refractory epilepsy of temporal lobe origin (12 on the left, 13 on the right) and 14 right-handed control subjects. The hippocampi and temporal lobes were traced by computer on successive coronal images and the resulting measurements of area were summed for each region. The left and right hippocampi were symmetrical in the control subjects; however, for patients the hippocampus was smaller on the side of the seizure focus. Moreover, the left-right hippocampal ratio significantly differentiated the control subjects from each patient group. The left temporal lobe was significantly smaller than the right in control subjects. The epileptics' temporal lobes were smaller on the side of the seizure focus, compared to the temporal lobes in the control subjects. MRI hippocampal measurements were compared to hippocampal neuronal densities obtained postoperatively. Significant correlations were obtained between the ratio (side ipsilateral to focus/side contralateral to focus) of MRI hippocampal measurements and neuronal densities in all hippocampal subfields except CA2. Prior to surgery, patients were administered the Wechsler Memory Scale and the verbal Selective Reminding Test. Significant correlations existed between MRI measurements of the left hippocampus and the Wechsler logical memory percent retention scores and between the left temporal lobe measurements and the verbal Selective Reminding Test scores for patients with seizure foci in the left temporal lobe.


[New research techniques in human nutrition].

Accurate determination of energy requirement and nutrients metabolism is essential to improve physiological knowledge and for physiopathological purpose in human nutrition. This evaluation is an absolute necessity for food industry. Energy expenditure could be precisely determined by indirect calorimetry or doubly labeled water technic. Nutrients metabolism and substrates turn-over studies are now accessible without health hazard using tracers labelled with stable isotopes. These three methods are the basic tools for the new Research Nutrition Center which are now in progress.


3-O-methyldopa administration does not alter fluorodopa transport into the brain.

To determine if 3-O-methyldopa (3OMD) significantly inhibits the transport of 6-[18F]fluorodopa (6-FD) into the brain at the concentration normally encountered during L-dopa administration, we performed 6-FD studies with positron emission tomography in cynomolgus monkeys in the presence and absence of 3OMD. Infusion of 3OMD was designed to produce plasma concentrations equivalent to those seen in patients on chronic L-dopa therapy. Plasma 3OMD levels of 39 +/- 4 microM did not alter the blood-brain transfer rate of 6-FD. 6-FD positron emission tomographic studies in parkinsonian patients will therefore not be altered by 3OMD present in the blood in those patients taking L-dopa preparations. These results do not support the hypothesis that transport of L-dopa into the brain is inhibited by 3OMD to cause the declining response seen in patients with advanced Parkinson's disease.


Neurological complications following liver transplantation.

Neurological complications occurred in 4 (19%) of 21 consecutive patients (Group II) undergoing orthotopic liver transplantation, compared with a 47% (9/19) incidence in our initial series (Group I). In Group II, the neurological problems included new recurrent headaches and delayed intracerebral hemorrhage (1 patient), partial third nerve palsy and brachial plexopathy (1 patient), and ataxic dysarthria with encephalopathy (2 patients). Seizures, noted in 8 of 9 neurologically affected Group I patients, were not encountered in Group II. Of the 4 patients in the entire series with the cerebrocerebellar syndrome, 2 had partial recovery after stopping treatment with cyclosporine, 1 stabilized when cyclosporine was discontinued but later worsened when rechallenged, and 1 had full recovery but died following a second transplantation. Brain magnetic resonance images appeared normal in 3 of the 4 patients. Complications affecting vision included cortical blindness in 2 patients and occipital lobe hemorrhage in 1. All completely recovered. Survival was comparable for patients with and those without neurological complications (69% and 63%, respectively). Immediate withdrawal of cyclosporine at the onset of a change in mental status or dysarthria and improvement in intra- and postoperative management may have contributed to the decreased incidence of neurological complications.


Functional neuroimaging strategy in temporal lobe epilepsy: a comparative study of 18FDG-PET and 99mTc-HMPAO-SPECT.

We performed 99mTc-hexamethylpropyleneamineoxime-single-photon emission computed tomography (SPECT) and 18F-fluorodeoxyglucose-positron emission tomography (PET) in 20 epileptic patients with well-lateralized temporal electroencephalographic focus, normal computed tomographic scan, and brain magnetic resonance imaging (MRI) either normal (n = 10) or showing nonspecific changes in the epileptogenic temporal lobe (n = 10). In patients with a normal MRI, PET exhibited focal hypometabolism in 80%, whereas SPECT showed corresponding hypoperfusion in only 20%. In patients with an abnormal MRI, PET and SPECT yielded 100% and 90% sensitivity, respectively. The metabolic and regional cerebral blood flow disturbances were topographically concordant with electroencephalographic and MRI findings in all these patients. Only patients with a large and pronounced hypometabolism on PET images exhibited hypoperfusion on SPECT. Spatial resolution appeared to be the critical factor responsible for the higher sensitivity of PET. However, this superiority of PET did not prove clinically useful in patients whose SPECT was abnormal, particularly when brain MRI showed nonspecific changes in the epileptogenic temporal lobe.


New horizons in neurology. The twentieth century.

Neurology in the twentieth century has undergone extraordinary change, particularly in the past 40 years. We trace the major trends, including the growth of the field, the technological advances, the revolution in neuroscience, the emergence of neurology as a free-standing specialty, the rise of child neurology, and the development of neurological subspecialization. Subsequent articles in this series will expand on these subjects.


A novel point mutation in the mitochondrial tRNA(Leu)(UUR) gene in a family with mitochondrial myopathy.

A T-to-C transition mutation at nucleotide position 3,250 in the mitochondrial tRNA(Leu)(UUR) gene was present in a family with mitochondrial myopathy. Two of three muscle biopsies examined had complex I (NADH-ubiquinone oxidoreductase) deficiency. Heteroplasmy of wild and mutant mitochondrial DNA was detected by Nae I digestion of the polymerase chain reaction products with a modified primer. This was found in blood or muscle samples or both from all seven members examined. Similar to the 3,243 mutation in most patients with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), the new mutation site was located in the dihydrouridine loop and embedded in the binding region of mitochondrial transcription termination factor. Elucidation of the effects of this mutation may help clarify the role of mitochondrial tRNAs and transcription termination.


Idiopathic childhood stroke is associated with human leukocyte antigen (HLA)-B51.

We report four children with idiopathic stroke syndromes who were assayed for human leukocyte antigen (HLA) class I markers and found to have HLA-B51 in common. This finding suggests that there may be a genetic predisposition for "idiopathic" childhood stroke, and host factors, possibly in concert with environmental factors or viruses, may play a major role in the mechanism of unexplained occlusion of the cerebral vasculature in children.


Serum IgG antibody to ganglioside GQ1b is a possible marker of Miller Fisher syndrome.

We studied serum anti-glycolipid antibodies by enzyme-linked immunosorbent assay and thin-layer chromatography-enzyme immunoassay in six consecutive patients with typical Miller Fisher syndrome. In all six, increased activity of IgG antibody against ganglioside GQ1b was present in the early phase and reduced with time, whereas such activity was not detected in normal control subjects and disease control subjects including those with Guillain-Barré syndrome. Anti-GQ1b IgG antibody is a new possible diagnostic marker of Miller Fisher syndrome and could well be related to the disease process itself.


Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.


Cloning, expression, and nucleotide sequence of genes involved in production of pediocin PA-1, and bacteriocin from Pediococcus acidilactici PAC1.0.

The production of pediocin PA-1, a small heat-stable bacteriocin, is associated with the presence of the 9.4-kbp plasmid pSRQ11 in Pediococcus acidilactici PAC1.0. It was shown by subcloning of pSRQ11 in Escherichia coli cloning vectors that pediocin PA-1 is produced and, most probably, secreted by E. coli cells. Deletion analysis showed that a 5.6-kbp SalI-EcoRI fragment derived from pSRQ11 is required for pediocin PA-1 production. Nucleotide sequence analysis of this 5.6-kbp fragment indicated the presence of four clustered open reading frames (pedA, pedB, pedC, and pedD). The pedA gene encodes a 62-amino-acid precursor of pediocin PA-1, as the predicted amino acid residues 19 to 62 correspond entirely to the amino acid sequence of the purified pediocin PA-1. Introduction of a mutation in pedA resulted in a complete loss of pediocin production. The pedB and pedC genes, encoding proteins of 112 and 174 amino acid residues, respectively, are located directly downstream of the pediocin structural gene. Functions could not be assigned to their gene products; mutation analysis showed that the PedB protein is not involved in pediocin PA-1 production. The mutation analysis further revealed that the fourth gene, pedD, specifying a relatively large protein of 724 amino acids, is required for pediocin PA-1 production in E. coli. The predicted pedD protein shows strong similarities to several ATP-dependent transport proteins, including the E. coli hemolysin secretion protein HlyB and the ComA protein, which is required for competence induction for genetic transformation in Streptococcus pneumoniae.(ABSTRACT TRUNCATED AT 250 WORDS)


Nitrate and nitrite microgradients in barley rhizosphere as detected by a highly sensitive denitrification bioassay.

A highly sensitive denitrification bioassay was developed for detection of NO3- and NO2- in rhizosphere soil samples. Denitrifying Pseudomonas aeruginosa ON12 was grown anaerobically in citrate (30 mM) minimal medium with KClO3 (10 mM) and NaNO2 (3 mM), which gave cells capable of NO2- reduction to N2O but incapable of NO3- reduction to NO2-. Growth on citrate minimal medium further resulted in the absence of N2O reduction. When added to small soil samples in O2-free vials, such cells could be used to convert the indigenous NO2- pool to N2O, which was subsequently quantified by gas chromatography. Cells grown in KClO3-free citrate medium with 10 mM NaNO3 as the electron acceptor were capable of reducing both NO3- and NO2-, and these cells could subsequently be added to the sample to convert the indigenous NO3- pool to N2O. Concentrations of both NO3- and NO2- were thus determined as N2O, with a detection limit of approximately 10 pmol of N. The bioassay could be used to determine NO3- and NO2- pools in 10-mg soil samples taken along a microgradient in the rhizosphere of field-grown barley plants. At both low (10%, wt/wt) and high (18%, wt/wt) water content, relatively high levels of NO2- were found in the rhizosphere compared with bulk soil. Under dry conditions, NO3- was also more abundant in the rhizosphere than in the bulk soil, whereas such a difference was not observed at the high water content. The roles of plant metabolism and bacterial nitrification and denitrification processes for NO3- and NO2- availability in the rhizosphere are discussed.


Characterization of bacteriocins from two strains of Bacillus thermoleovorans, a thermophilic hydrocarbon-utilizing species.

Bacillus thermoleovorans S-II and B. thermoleovorans NR-9 produce bacteriocins, and these bacteriocins are designated thermoleovorin-S2 and thermoleovorin-N9, respectively. The bacteriocins are effective against all but the producing strain of B. thermoleovorans, as well as being effective against Salmonella typhimurium, Branhamella catarrhalis, Streptococcus faecalis, and Thermus aquaticus. Thermoleovorins are produced during log-phase growth and are inhibitory to actively growing cells. The bacteriocins are proteinaceous in nature, being sensitive to selected proteases (protease type XI and pepsin). They are stable at pHs of 3 to 10. Thermoleovorin-S2 was more thermostable than thermoleovorin-N9 at 70 and 80 degrees C. Thermoleovorins-S2 and -N9 apparently act by binding to the susceptible organisms, resulting in lysis of the cell. Thermoleovorins-S2 has an estimated M(r) of 42,000, while thermoleovorin-N9 has a M(r) of 36,000.


Degradation of azo dyes by the lignin-degrading fungus Phanerochaete chrysosporium.

Under nitrogen-limiting, secondary metabolic conditions, the white rot basidiomycete Phanerochaete chrysosporium extensively mineralized the specifically 14C-ring-labeled azo dyes 4-phenylazophenol, 4-phenylazo-2-methoxyphenol, Disperse Yellow 3 [2-(4'-acetamidophenylazo)-4-methylphenol], 4-phenylazoaniline, N,N-dimethyl-4-phenylazoaniline, Disperse Orange 3 [4-(4'-nitrophenylazo)-aniline], and Solvent Yellow 14 (1-phenylazo-2-naphthol). Twelve days after addition to cultures, the dyes had been mineralized 23.1 to 48.1%. Aromatic rings with substituents such as hydroxyl, amino, acetamido, or nitro functions were mineralized to a greater extent than unsubstituted rings. Most of the dyes were degraded extensively only under nitrogen-limiting, ligninolytic conditions. However, 4-phenylazo-[U-14C]phenol and 4-phenylazo-[U-14C]2-methoxyphenol were mineralized to a lesser extent under nitrogen-sufficient, nonligninolytic conditions as well. These results suggest that P. chrysosporium has potential applications for the cleanup of textile mill effluents and for the bioremediation of dye-contaminated soil.


Roles of manganese and organic acid chelators in regulating lignin degradation and biosynthesis of peroxidases by Phanerochaete chrysosporium.

We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic 14C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.


Moderation of ruminal fermentation by ciliated protozoa in cattle fed a high-grain diet.

The objective of this study was to assess the influence of ciliated protozoa on ruminal fermentation in cattle fed high-grain diets. Six ruminally cannulated steers fed a corn-based grain diet (85% concentrate plus 15% alfalfa hay) at 12-h intervals were assigned randomly to two groups, ciliate free and faunated, in a crossover design. Defaunation was by ruminal emptying, omasal flushing, and treatment with sodium sulfosuccinate. Two to 3 weeks after defaunation, the ruminal contents of all steers were sampled before the morning feeding (0 h) and at 1, 2, 4, 6, 8, and 12 h after feeding to measure pH, analyze fermentation products, and monitor counts of ciliated protozoa and lactic acid-producing and -fermenting bacterial groups. Total numbers of ciliated protozoa in the faunated steers averaged 4.3 x 10(5)/g, and the protozoa consisted of nine genera. Ciliate-free steers had lower (P less than 0.01) ruminal pHs (pH 5.97) than faunated cattle (pH 6.45); however, the treatment-time interaction was not significant. Ruminal lactate and ammonia concentrations were similar in both groups. The total volatile fatty acid concentration was higher (P less than 0.05) in the ciliate-free steers than in the faunated steers and exhibited a treatment-time interaction (P less than 0.05). The acetate-to-propionate ratio was higher (P less than 0.05) in the faunated group than in the ciliate-free group and showed a treatment-time interaction (P less than 0.05). Total anaerobic bacterial counts were about fourfold higher in the ciliate-free group than in the faunated group.(ABSTRACT TRUNCATED AT 250 WORDS)


Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation.

Survival studies were conducted on Legionella pneumophila cells that had been grown intracellularly in Acanthamoeba polyphaga and then exposed to polyhexamethylene biguanide (PHMB), benzisothiazolone (BIT), and 5-chloro-N-methylisothiazolone (CMIT). Susceptibilities were also determined for L. pneumophila grown under iron-sufficient and iron-depleted conditions. BIT was relatively ineffective against cells grown under iron depletion; in contrast, iron-depleted conditions increased the susceptibilities of cells to PHMB and CMIT. The activities of all three biocides were greatly reduced against L. pneumophila grown in amoebae. PHMB (1 x MIC) gave 99.99% reductions in viability for cultures grown in broth within 6 h and no detectable survivors at 24 h but only 90 and 99.9% killing at 6 h and 24 h, respectively, for cells grown in amoebae. The antimicrobial properties of the three biocides against A. polyphaga were also determined. The majority of amoebae recovered from BIT treatment, but few, if any, survived CMIT treatment or exposure to PHMB. This study not only shows the profound effect that intra-amoebal growth has on the physiological status and antimicrobial susceptibility of L. pneumophila but also reveals PHMB to be a potential biocide for effective water treatment. In this respect, PHMB has significant activity, below its recommended use concentrations, against both the host amoeba and L. pneumophila.


Sequence variation in the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Our previous molecular epidemiologic study with gene probes (H. Shirai, H. Ito, T. Hirayama, Y. Nakamoto, N. Nakabayashi, K. Kumagai, Y. Takeda, and M. Nishibuchi, Infect. Immun. 58:3568-3573, 1990) demonstrated that the gene (trh) encoding a thermostable direct hemolysin-related hemolysin was strongly associated with clinical strains of Vibrio parahaemolyticus. Strain-to-strain variation in the intensities of the hybridization signals observed in the above study also suggested that the trh genes in different strains may have significantly divergent nucleotide sequences. To assess the public health significance of the rare environmental strains which exhibited very weak hybridization signals with the trh gene-specific DNA probe, the trh-like sequence was cloned from one of the environmental strains and the nucleotide sequence was determined in this study. A hemolysin gene (trh2) which was 84% homologous to the trh gene (newly named trh1) and 54.8 to 68.8% homologous to the genes (tdh) encoding thermostable direct hemolysins was detected in the cloned sequence. The trh2 gene product showed a profile of hemolytic activities against various animal erythrocytes different from that of the trh1 gene product. The trh2 gene product was antigenically related (partially identical) to the trh1 and tdh gene products. DNA colony blot and Southern blot hybridization analyses with trh1- and trh2-specific DNA probes showed that the trh1 probe-positive strains exhibiting hybridization signals with varying intensities could be clustered into trh1 and trh2 subgroups. In addition, hybridization analysis with oligonucleotide probes demonstrated significant strain-to-strain variation in the trh1 and trh2 gene sequences.(ABSTRACT TRUNCATED AT 250 WORDS)


Multiple periplasmic catalases in phytopathogenic strains of Pseudomonas syringae.

Phytopathogenic strains of Pseudomonas syringae are exposed to plant-produced, detrimental levels of hydrogen peroxide during invasion and colonization of host plant tissue. When P. syringae strains were investigated for their capacity to resist H2O2, they were found to contain 10- to 100-fold-higher levels of total catalase activity than selected strains belonging to nonpathogenic related taxa (Pseudomonas fluorescens and Pseudomonas putida) or Escherichia coli. Multiple catalase activities were identified in both periplasmic and cytoplasmic fluids of exponential- and stationary-phase P. syringae cells. Two of these activities were unique to the periplasm of P. syringae pv. glycinea. During the stationary growth phase, the specific activity of cytoplasmic catalases increased four- to eightfold. The specific activities of catalases in both fluids from exponential-phase cells increased in response to treatment with 0.25 to 10 mM H2O2 but decreased when higher H2O2 concentrations were used. In stationary-growth phase cultures, the specific activities of cytoplasmic catalases increased remarkably after treatment with 0.25 to 50 mM H2O2. The growth of P. syringae into stationary phase and H2O2 treatment did not induce synthesis of additional catalase isozymes. Only the stationary-phase cultures of all of the P. syringae strains which we tested were capable of surviving high H2O2 stress at concentrations up to 50 mM. Our results are consistent with the involvement of multiple catalase isozymes in the reduction of oxidative stress during plant pathogenesis by these bacteria.


Two types of bacterial alginate lyases.

The extracellular alginate lyases were purified from Vibrio harveyi AL-128 and V. alginolyticus ATCC 17749. The former enzyme appears to be specific for alpha-1,4 bonds involving L-guluronate units in alginate, whereas the latter exhibits specificity for beta-1,4 bonds involving D-mannuronate units. The molecular weights of the enzymes were estimated to be 57,000 and 47,000, and they had isoelectric points of 4.3 and 4.6, respectively. The enzyme from strain AL-128 was most active at NaCl concentrations of 0.3 to 1.0 M. Optimum activity of the enzyme from strain ATCC 17749 was found in the presence of 5 to 10 mM CaCl2.


Characterization of Lactococcus lactis phage antigens.

Phage phi 197 is representative of a widespread lactococcal phage group characterized by a particular morphology (prolate head with a noncontractile tail). In order to develop an immunoenzymatic phage detection test, fusion proteins containing beta-galactosidase fused to epitopes of phage phi 197 structural proteins were constructed by cloning random DNA fragments from the phage genome upstream of a lacZ gene on a plasmid vector. Recombinant plasmids containing certain fragments encoded the synthesis of fusion proteins which react with polyclonal antibodies against the phage and confer a Lac+ phenotype on Escherichia coli. Three different epitopes were represented; phage-specific DNA fragments encoding these epitopes were mapped at three locations on the phage genome, and their nucleotide sequences were determined. Two fused phage antigens were conformational epitopes, whereas the phage epitope of protein encoded by the recombinant plasmid designated pOA17 was a denaturation-resistant epitope. This epitope was very immunogenic. Protein encoded by plasmid pOA17 was synthesized in large amounts from a strong promoter. Antibodies raised against this hybrid protein were used to identify the 46-kDa minor phage protein which provides the epitope. Antibody cross-reactivity of phages related to phi 197 showed that this epitope is well conserved in this genetic group.


Reassessment of the prevalence of heat-stable enterotoxin (NAG-ST) among environmental Vibrio cholerae non-O1 strains isolated from Calcutta, India, by using a NAG-ST DNA probe.

A collection of 521 environmental isolates of Vibrio cholerae which were previously examined by the suckling mouse assay and found to be negative for the heat-stable enterotoxin NAG-ST were reassessed by a recently developed DNA probe for NAG-ST. A total of 12 (2.3%) of the isolates hybridized with the NAG-ST probe. By using a cholera toxin (CT) DNA probe, the CT gene was detected in six of the strains in the collection, although none of the isolates of V. cholerae non-O1 hybridized with both of the toxin probes. All of the NAG-ST and CT probe-positive strains were hemolysin positive. Thirty-fold-concentrated supernatants of the three representative NAG-ST DNA probe-positive V. cholerae non-O1 strains gave positive fluid accumulation ratios in the suckling mouse assay even after heating (100 degrees C for 5 min) and also inhibited the binding of a NAG-ST monoclonal antibody to the bound NAG-ST in a competitive enzyme-linked immunosorbent assay (ELISA). Likewise, all six CT probe-positive V. cholerae non-O1 strains produced in vitro CT when examined by the CT bead ELISA. HindIII digest patterns of chromosomal DNA from the representative NAG-ST gene-positive strains were visually indistinguishable. Between the groups of NAG-ST probe-positive strains examined, there was a variation in the hybridizable fragments, with one group of strains exhibiting a hybridizable fragment similar to that of the NRT 36 reference strain; a smaller HindIII fragment hybridized with the NAG-ST probe in the other group of strains.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation and characterization of a variety of microcystins from seven strains of the cyanobacterial genus Anabaena.

Hepatotoxins (microcystins) from seven freshwater Anabaena strains originating from three different Finnish lakes and one lake in Norway were isolated by high-performance liquid chromatography and characterized by amino acid analysis and fast atom bombardment mass spectrometry. All strains produced three to seven different microcystins. A total of 17 different compounds were isolated, of which 8 were known microcystins. The known compounds identified from six strains were MCYST (microcystin)-LR, [D-Asp3]MCYST-LR, [Dha7]MCYST-LR, [D-Asp3,Dha7]MCYST-LR, MCYST-RR, [D-Asp3]MCYST-RR, [Dha7]MCYST-RR, and [D-Asp3,Dha7]MCYST-RR. With the exception of MCYST-LR and [D-Asp3]MCYST-LR, this is the first time that isolation of these toxins from Anabaena strains has been reported. Three of the strains produced one to three toxins as minor components which could not be identified. Anabaena sp. strain 66 produced four unidentified toxins. The other Anabaena strains always contained both MCYST-LR and MCYST-RR and/or their demethyl variants. Quantitative differences between toxins within and between strains were detected; at times MCYST-LR and at other times MCYST-RR or demethyl derivatives thereof were the most abundant toxins found in a strain.


Lipoxygenase inhibitors shift the yeast/mycelium dimorphism in Ceratocystis ulmi.

The yeast-mycelium dimorphism in Ceratocystis ulmi, the causative agent of Dutch elm disease, was switched by gossypol, nordihydroguaiaretic acid, and propylgallate. In each case the mycelial form was converted to the yeast form. These compounds are recognized lipoxygenase inhibitors. Inhibitors of cyclooxygenase and thromboxane synthetase did not cause mycelia to shift to the yeast form. We suggest the following two-part hypothesis: (i) that lipoxygenase is a target for antifungal antibiotics and (ii) that many phytoalexins (antimicrobial compounds of plant origin) are targeted toward fungal lipoxygenases. In addition, in a study to determine potential lipoxygenase substrates, a fatty acid analysis indicated that C. ulmi conidiospores contained high levels of oleic, linoleic, and linolenic acids but no arachidonic acid.


Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.


Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.


Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae.

A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described. The crystal proteins are produced as bipyramidal crystals. These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa. Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested. In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae. The crystal protein-encoding gene was cloned and sequenced. The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively. Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins. Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins. CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae. Natural CryIIIC crystals are not toxic. Toxicity is revealed only after an in vitro solubilization and activation step.


Comparison of bioaerosol sampling methods in barns housing swine.

The air in livestock buildings contains bioaerosol levels that are sufficiently high to cause adverse health effects in animals and workers. These bioaerosols are complex mixtures of live and dead microorganisms and their products as well as other aeroallergens. The effectiveness of sampling methods used for quantifying the very high concentrations of microorganisms in these environments has not been well studied. To facilitate an accurate assessment of respiratory hazards from viable organisms in agricultural environments, three bioaerosol sampling methods were investigated: the Andersen microbial sampler method (AMS), the all-glass impinger method (AGI), and the Nuclepore filtration-elution method (NFE). These methods were studied in a parallel fashion in 24 swine confinement buildings. Measurements were taken in two seasons with three types of culture media in duplicate to assess total bacteria, gram-negative enteric bacteria, and total fungi. Methods were analyzed for the proportion of samples yielding data within the limits of detection, intraclass reliability, and correlation between methods. For sampling viable bacteria, the AMS had a poor data yield because of overloading and demonstrated weak correlation with the AGI. Conversely, the AGI and NFE gave sufficient numbers of valid data points (90%), yielded high intraclass reliabilities (alpha greater than or equal to 0.92), and were highly correlated with each other (r = 0.86). The AGI and the NFE were suitable methods for assessing bacteria in this environment, but the AMS was not. The AMS was the only method that consistently recovered enteric bacteria (73% data yield). For sampling fungi, the AGI and AMS both yielded sufficient data and all three methods demonstrated high intraclass reliability.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of antibacterial agents on in vitro ovine ruminal biotransformation of the hepatotoxic pyrrolizidine alkaloid jacobine.

Ingestion of pyrrolizidine alkaloids, naturally occurring plant toxins, causes illness and death in a number of animal species. Senecio jacobaea pyrrolizidine alkaloids cause significant economic losses due to livestock poisoning, particularly in the Pacific Northwest. Some sheep are resistant to pyrrolizidine alkaloid poisoning, because ovine ruminal biotransformation detoxifies free pyrrolizidine alkaloids in digesta. Antibacterial agents modify ruminal fermentation. Pretreatment with antibacterial agents may account for some animal variability in resistance to pyrrolizidine alkaloid toxicosis, and antibacterial agents can also be used for characterizing ruminal pyrrolizidine alkaloid-biotransforming microflora. The objective of this study was to evaluate the effects of antibacterial agents on biotransformation of a predominant S. jacobaea pyrrolizidine alkaloid, jacobine, in ovine ruminal contents. Ovine ruminal jacobine biotransformation was tested in vitro with 20 independent antibacterial agents. Low amounts of rifampin and erythromycin prevented jacobine biotransformation. Chlortetracycline, lasalocid, monensin, penicillin G, and tetracycline were slightly less effective at inhibiting jacobine biotransformation. Bacitracin, crystal violet, kanamycin, and neomycin were moderately inhibitory against jacobine biotransformation. Brilliant green, chloramphenicol, gramicidin, nalidixic acid, polymyxin B SO4, sodium azide, streptomycin, sulfisoxazole, and vancomycin had little to no effect on jacobine biotransformation. The antibiotics that were most effective at inhibiting biotransformation were those that are active against gram-positive bacteria. Therefore, gram-positive bacteria are most likely critical members of the jacobine-biotransforming consortia.


Contribution of anaerobic protozoa and methanogens to hindgut metabolic activities of the American cockroach, Periplaneta americana.

The ciliate Nyctotherus ovalis occurs in high numbers in the hindgut of the American cockroach (Periplaneta americana) and harbors methanogenic bacteria as endosymbionts. The contribution of these hindgut microorganisms to metabolic and developmental processes of P. americana was studied by comparing cultures of cockroaches in which the composition of the hindgut microbial population was altered in various ways. Rearing the insects protozoan free resulted in increased insect generation time, decreased adult body weight, and absence of methane production. After feeding of protozoan-free adult cockroaches with a hindgut suspension containing N. ovalis and methanogens, methane increased to normal values and insect body weight was restored during the development of the second generation of insects. Feeding the protozoan-free cockroaches a hindgut suspension which was made free of N. ovalis resulted in an increase in methane production to only about 20% of the normal methane production level. This suggests that the methanogenic endosymbionts of N. ovalis are the major source of methane production in the hindgut. Inhibition of methanogens by addition of bromoethanesulfonic acid to the drinking water of a normal cockroach culture resulted in a reduction of methane production to about 2% of the normal level. No effects on insect body weight or the number of N. ovalis organisms were observed, but the fermentation pattern in the hindgut was shifted towards a relative increase in propionate levels. Similar results were obtained for in vitro cultures of hindgut microorganisms treated with bromoethanesulfonic acid. The results suggest a major role for hindgut protozoa in cockroach metabolic activities, especially during the insect growth period. The relatively large amounts of methane produced by cockroaches and by other methane-producing xylophagous insects suggest a major contribution by insects to global methane production.


Enumeration of phenanthrene-degrading bacteria by an overlayer technique and its use in evaluation of petroleum-contaminated sites.

Bacteria that are capable of degrading polycyclic aromatic hydrocarbons were enumerated by incorporating soil and water dilutions together with fine particles of phenanthrene, a polycyclic aromatic hydrocarbon, into an agarose overlayer and pouring the mixture over a mineral salts underlayer. The phenanthrene-degrading bacteria embedded in the overlayer were recognized by a halo of clearing in the opaque phenanthrene layer. Diesel fuel- or creosote-contaminated soil and water that were undergoing bioremediation contained 6 x 10(6) to 100 x 10(6) phenanthrene-degrading bacteria per g and ca. 5 x 10(5) phenanthrene-degrading bacteria per ml, respectively, whereas samples from untreated polluted sites contained substantially lower numbers. Unpolluted soil and water contained no detectable phenanthrene degraders (desert soil) or only very modest numbers of these organisms (garden soil, municipal reservoir water).


Response surface analysis of the effects of pH and dilution rate on Ruminococcus flavefaciens FD-1 in cellulose-fed continuous culture.

The ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 was grown in cellulose-fed continuous culture with 20 different combinations of pH and dilution rate (D); the combinations were selected according to the physiological pH range of the organism (6.0 to 7.1) and growth rate of the organism on cellulose (0.017 to 0.10 h-1). A response surface analysis was used to characterize the effects of pH and D on the extent of cellulose consumption, growth yield, soluble sugar concentration, and yields of fermentation products. The response surfaces indicate that pH and D coordinately affect cellulose digestion and growth yield in this organism. As expected, the net cellulose consumption increased with increasing D while the fraction of added cellulose that was utilized decreased with increasing D. The effect of changes in pH within the physiological range on cellulose consumption was smaller than that of changes in D. Cellulose degradation was less sensitive to low pH than to high pH. At low Ds (longer retention times), cellulose degradation did not follow first-order kinetics. This decreased rate of cellulose digestion was not due to poor mixing, limitation by other medium components, or preferential utilization of the more amorphous fraction of the cellulose. The cell yield increased from 0.13 to 0.18 mg of cells per mg of cellulose with increasing Ds from 0.02 to 0.06 h-1 and decreased when the pH was shifted from the optimum of 6.5 to 6.8. The effect of pH on cell yield increased with increasing D. The reduced cell yield at low pH appears to be due to both an increase in maintenance energy requirements and a decrease in true growth yield.


Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase.

The bifunctional enzyme chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which is encoded by the pheA gene of Escherichia coli K-12, is subject to strong feedback inhibition by L-phenylalanine. Inhibition of the prephenate dehydratase activity is almost complete at concentrations of L-phenylalanine greater than 1 mM. The pheA gene was cloned, and the promoter region was modified to enable constitutive expression of the gene on plasmid pJN302. As a preliminary to sequence analysis, a small DNA insertion at codon 338 of the pheA gene unexpectedly resulted in a partial loss of prephenate dehydratase feedback inhibition. Four other mutations in the pheA gene were identified following nitrous acid treatment of pJN302 and selection of E. coli transformants that were resistant to the toxic phenylalanine analog beta-2-thienylalanine. Each of the four mutations was located within codons 304 to 310 of the pheA gene and generated either a substitution or an in-frame deletion. The mutations led to activation of both enzymatic activities at low phenylalanine concentrations, and three of the resulting enzyme variants displayed almost complete resistance to feedback inhibition of prephenate dehydratase by phenylalanine concentrations up to 200 mM. In all four cases the mutations mapped in a region of the enzyme that has not been implicated previously in feedback inhibition sensitivity of the enzyme.


Volatile metabolites produced by six fungal species compared with other indicators of fungal growth on cereal grains.

Six fungal species, Penicillium brevicompactum, P. glabrum, P. roqueforti, Aspergillus flavus, A. versicolor, and A. candidus, were inoculated on moistened and autoclaved wheat and oat grains. They were cultivated in glass vessels provided with an inlet and outlet for air. Air was passed through the vessels to collect volatile fungal metabolites on porous polymer adsorbents attached to the outlet. Samples were collected at two fungal growth stages. Adsorbed compounds were thermally desorbed, separated by gas chromatography, and identified by mass spectrometry. Differences in the production of volatile metabolites depended more on the fungal species than on the grain type. The fungal growth stage was not an important factor determining the composition of volatiles produced. 3-Methylfuran was produced in similar amounts regardless of the fungal species and substrate (oat versus wheat). The production of volatile metabolites was compared with the production of ergosterol and CO2 and the number of CFU. The production of volatile metabolites was more strongly correlated with accumulated CO2 production than with actual CO2 production and more strongly correlated with ergosterol contents of the grain than with numbers of CFU.


Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.


The homologous and heterologous regions within the iap gene allow genus- and species-specific identification of Listeria spp. by polymerase chain reaction.

The iap gene of Listeria species encodes protein p60. The comparison of iap-related genes from different Listeria species indicated common and variable regions within these genes which appeared to be specific for each Listeria species. On the basis of the iap gene sequences, pairs of polymerase chain reaction (PCR) primers which allowed the unambiguous identification of all members of the genus Listeria, of groups of related Listeria species, and of L. monocytogenes, exclusively, were selected. The PCR primers specific for L. monocytogenes yielded PCR products which represented essentially the repeat region of the iap gene. The size of these PCR products allowed an estimate of the number of the TN repeat units within the repeat region of the p60 protein of an L. monocytogenes strain. The data indicated that the number of repeat units differed among L. monocytogenes isolates.


Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.


Impact of growth conditions on resistance of Klebsiella pneumoniae to chloramines.

The resistance of Klebsiella pneumoniae to inorganic monochloramine (1.5 mg/liter; 3:1 Cl2:N ratio, pH 8.0) was examined in relation to growth phase, temperature of growth, and growth under decreased nutrient conditions. Growth phase did not impact resistance to chloramines. Mid-exponential and stationary-phase cells, grown in a yeast extract-based medium, had CT99 values and standard deviations of 4.8 +/- 0.1 and 4.6 +/- 0.2 mg.min/liter, respectively. Growth temperature did not alter chloramine resistance at short contact times. CT99 values of cells grown at 15 and 23 degrees C were 4.5 +/- 0.2 and 4.6 +/- 0.2 mg.min/liter, respectively. However, at longer contact times, CT99.99 values of cells grown at 15 and 23 degrees C were 14 and 8 mg.min/liter, respectively, suggesting a small resistant subpopulation for cells grown at the lower temperature. Growth under decreased nutrient conditions resulted in a concomitant increase in resistance to chloramines. When K. pneumoniae was grown in undiluted Ristroph medium and Ristroph medium diluted by 1:100 and 1:1,000, the CT99 values were 4.6 +/- 0.2, 9.6 +/- 0.4, and 24 +/- 7.0 mg.min/liter, respectively. These results indicate that nutrient availability has a greater impact than growth phase or growth temperature in promoting the resistance of K. pneumoniae to inorganic monochloramine.


Growth-promoting effect of thermophilic fungi on the mycelium of the edible mushroom Agaricus bisporus.

The growth-promoting effect of the thermophilic fungus Scytalidium thermophilum in mushroom compost on the mycelium of the edible mushroom Agaricus bisporus was investigated. Results obtained by others were confirmed by showing that S. thermophilum leads to an increased hyphal extension rate of the mushroom mycelium. However, it was demonstrated that hyphal extension rates were not clearly related to mushroom biomass increase rates. A number of experiments pointed strongly towards CO2 as the determinant of hyphal extension rates. In compost, CO2 is produced mainly by thermophilic fungi. Several experiments did not reveal any other specific compound produced by S. thermophilum that increases the hyphal extension rate of the mushroom mycelium.


Complete mineralization of benzene by aquifer microorganisms under strictly anaerobic conditions.

Benzene was mineralized to CO2 by aquifer-derived microorganisms under strictly anaerobic conditions. The degradation occurred in microcosms containing gasoline-contaminated subsurface sediment from Seal Beach, California, and anaerobic, sulfide-reduced defined mineral medium supplemented with 20 mM sulfate. Benzene, at initial concentrations ranging from 40 to 200 microM, was depleted in all microcosms and more than 90% of 14C-labeled benzene was mineralized to 14CO2.


Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.


Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in sewage.

A new medium has been adapted for the growth of Bacteroides fragilis so that its phages can be recovered from environmental samples, and its efficiency has been assessed. Polyvinylidene difluoride membranes allow significantly higher recoveries among different membrane filters used to decontaminate the samples. In all cases, a number of phages remain in the filters and a percentage of them can be recovered by treatment with an eluant.


Use of degenerate primers for polymerase chain reaction cloning and sequencing of the Lactococcus lactis subsp. lactis recA gene.

Two particularly well-conserved stretches in the RecA protein sequences were chosen as templates to synthesize degenerate oligonucleotides, which were used in polymerase chain reaction to amplify an internal recA DNA fragment of Lactococcus lactis subsp. lactis ML3. Using this fragment, we recovered and sequenced the entire lactococcal recA gene. The end of an open reading frame present upstream of the recA gene shows strong homology with formamidopyrimidine-DNA-glycosylase, a protein involved in DNA repair.


Development of the immunomagnetic enrichment method selective for Vibrio parahaemolyticus serotype K and its application to food poisoning study.

A method using immunomagnetic separation was developed to isolate the specified K serotype of Vibrio parahaemolyticus from a mixture of a large number of bacteria with other K serotypes. This method was applied to food poisoning studies and could recover the V. parahaemolyticus serotype found in the patient from the incriminated foods.