Frequency domain algorithm for quantifying atrial fibrillation organization to increase defibrillation efficacy.

We hypothesized that frequency domain analysis of an interatrial atrial fibrillation (AF) electrogram would show a correlation of the variance of the signal and the amplitude of harmonic peaks with the periodicity and morphology (organization) of the AF signal and defibrillation efficacy. We sought to develop an algorithm that would provide a high-resolution measurement of the changes in the spatiotemporal organization of AF. AF was initiated with burst atrial pacing in ten dogs. The atrial defibrillation threshold (ADFT50) was determined, and defibrillation was repeated at the ADFT50. Bipolar electrograms from the shocking electrodes were acquired immediately preshock, digitally filtered, and a FFT was performed. The organization index (OI) was calculated as the ratio of the area under the first four harmonic peaks to the total area of the spectrum. For a 4-s window, the mean OI was 0.505 +/- 0.087 for successful shocks, versus 0.352 +/- 0.068 for unsuccessful shocks (p < 0.001). Receiver operator characteristic (ROC) curve analysis was used to determine the optimal sampling window for predicting successful shocks. The area of the ROC curve was 0.8 for a 1-s window, and improved to 0.9 for a 4-s window. We conclude that the spectrum of an AF signal contains information relating to its organization, and can be used in predicting a successful defibrillation.

Early detection of acute myocardial infarction in patients presenting with chest pain and nondiagnostic ECGs: serial CK-MB sampling in the emergency department.

STUDY OBJECTIVES: Patients presenting to the emergency department with chest discomfort are a difficult problem for emergency physicians. Nearly 50% of patients with acute myocardial infarction (AMI) will initially have nondiagnostic ECGs on ED presentation. The purpose of this study was to determine if patients with AMI having nondiagnostic ECGs could be identified using new immunochemical assays for serial CK-MB sampling in the ED. DESIGN: Chest pain patients, more than 30 years old, with pain not caused by trauma or explained by radiographic findings, were eligible for the study. Serial serum samples were drawn on ED presentation (zero hours) and three hours after presentation, then analyzed for CK-MB using four immunochemical methods and electrophoresis. Standard World Health Organization criteria were used to establish the diagnosis of AMI, including new Q-wave formation or elevation of standard in-hospital serum cardiac enzyme markers. SETTING: A tertiary cardiac care community hospital. MEASUREMENTS AND MAIN RESULTS: The serum from 183 patients hospitalized for possible ischemic chest pain was collected and analyzed. Thirty-one of 183 patients (17%) were found to have AMI by standard in-hospital criteria. Sixteen of the 31 patients (52%) with AMI had nondiagnostic ECGs on presentation. Immunochemical determination of serial CK-MB levels provided a sensitive and specific method for detecting AMI in patients within three hours after ED presentation compared with standard electrophoresis. The four immunochemical methods demonstrated a range in sensitivity from 50% to 62.1% on ED presentation versus 92% to 96.7% three hours later. The immunochemical tests demonstrated specificities ranging from 83.0% to 96.4% at three hours, with three of the four tests having specificities of 92% or greater. Electrophoresis had a sensitivity of 34.5% on ED presentation, increasing to 76.9% at three hours, with a specificity of 98.6%. CONCLUSIONS: Immunochemical CK-MB methods allowed rapid, sensitive detection of AMI in the ED. Early detection of AMI offers many potential advantages to the emergency physician. Early detection of AMI, while the patient is in the ED, could direct disposition of this potentially unstable patient to an intensive care setting. Such information may prevent the ED discharge of patients with AMI having nondiagnostic ECGs. The diagnosis of AMI within a six-hour period after symptom onset may allow thrombolytic therapy to be given to patients with AMI not having diagnostic ECGs. This study served as a pilot trial for a multicenter study of the Emergency Medicine Cardiac Research Group, which is currently ongoing.

Purification and properties of polygalacturonic acid trans-eliminase from Bacillus stearothermophilus.

A strain of thermophilic bacteria, Bacillus stearothermophilus, with pectolytic activity has been isolated. It produced an endo-polygalacturonic acid trans-eliminase (endo-PATE, EC 4.2.2.2) extracellularly when grown at 65 degrees C on a pectic acid medium. The PATE was purified 62-fold by the rapid affinity chromatographic method on a Sepharose-polygalacturonamide linked matrix. The absorbed PATE was eluted from the column with a continuous gradient of 0-10(-3)M ethylenediaminetetraacetic acid (EDTA) in phosphate buffer at pH 7.6. The endo-PATE of this organism was much more heat stable than similar enzymes from the mesophilic Bacillus polymyxa and the thermotolerant Bacillus pumilus. The maximum activity of the enzyme occurred at 70 degrees C. With pectic acid as the substrate, the endo-PATE had an optimal pH of 9.0, the highest optimal pH compared with those of similar enzymes from other species of the genus. The molecular weight of the endo-PATE, as determined by chromatography on a Sephadex G-100 gel column, was 24 000.

Utilization of oleuropein by microorganisms associated with olive fermentations.

Oleuropein, the bitter glucoside of olives, and its hydrolysis products can possess antibacterial action. However, there is no information on the possible utilization of this polyphenolic compound; therefore studies have been made to assess its utilization as a major source of carbon. Various microorganisms associated with fermentation of olives (both desirable lactic acid bacteria and spoilage organisms) did use oleuropein, many without a significant delay in growth resulting in the appearance of a strong visible turbidity. Although the increase in oleuropein from 0.2 to 0.4% (w/v) had little or no effect on the spoilage organisms, the additional glucoside caused a delay in development of growth with some of the lactic acid bacteria. However, all of the latter cultures tested eventually grew and developed strong visible turbidity.

Preparation, separation and degradation of oligouronides produced by the polygalacturonic acid transeliminase of Bacillus pumilus.

Production of the end products of polygalacturonic acid degradation on a large scale was done by reacting free galacturonic acid with Bacillus pumilus polygalacturonic acid transeliminase (PATE, EC 4.2.2.2) to obtain a mixture of the barium salts of several oligouronides. Small amounts of the unsaturated oligouronides were separated by paper chromatography. Large quantities of unsaturated oligouronides were separated on a AG-1-X8 (formate) column by applying a sample of mixed oligouronides and stepwise elution was carried out with sodium formate buffer (pH 4.7). The unsaturated oligouronides were identified on the basis of chromatographic mobilities, Sephadex gel filtration data, COOH/CHO ratio, thiobarbituric acid-reacting material, bromine uptake, and chemical and enzymatic degradation data as unsaturated tri-, tetra-, and hexagalacturonic acids. The chemical degradation of these unsaturated oligouronides, done with 6 N HCl by heating at 100 degrees for 30 min, gave qualitatively identical products of hydrolysis. These products compared with authentic standards, were identified as galacturonic acid, formic acid, 5-formyl-2-furancarboxylic acid, and 2-furancarboxylic acid. Analysis of the enzymatic breakdown products of the higher unsaturated uronides showed that a minimum of four galacturonic acid units was required for the action of purified endo-PATE from B. pumilus. The unsaturated trimer was not attacked, thus accounting for its accumulation as the major end product of polygalacturonate degradation by this enzyme.

Cellulolytic bacteria associated with sloughing spoilage of California ripe olives.

Sloughing spoilage of California ripe olives during processing is characterized by severe softening, skin rupture, and flesh sloughing. It was assumed that cellulolytic activity was responsible for skin rupture and sloughing of flesh, and so a deliberate search was made for cellulolytic bacteria from olives undergoing sloughing spoilage. A bacterium identified as Cellulomonas flavigena was highly cellulolytic, attacking filter paper, carboxymethyl cellulose (CMC) gel, and olive tissue. Other bacteria attacking CMC, but not filter paper, enhanced the activity of the Cellulomonas strain when grown in mixed culture, although they did not, in pure culture, have any effect on filter paper. These latter cultures (all degraded olive tissue) represented the genera Xanthomonas, Aerobacter, and Escherichia. Other noncellulolytic bacteria belonging to the genera Alcaligenes, Kurthia, and Micrococcus also were used for study of mixed culture fermentation of cellulose by C. flavigena. Cellobiose accumulation at levels of 1.0% (w/v) and above suppressed growth of C. flavigena.

Exosporium formation in sporulating cells of Clostridium botulinum 78A.

In sporulating cells of Clostridium botulinum type A (NCA strain 78A), formation of exosporia was initiated laterally in the sporangia before the synthesis of the spore cortex. Mature spores were enveloped by multilayered exosporia; the layers were uniform in appearance, approximately 3 nm thick, with a center-to-center distance of 7 nm.

Fixation of mature spores of Clostridium botulinum.

A triple fixation method using a sequential application of 15 or 30% formaldehyde, 6% glutaraldehyde, and 1% osmium tetroxide resulted in excellent fixation of mature spores of Clostridium botulinum.

Fermenting yeasts associated with softening and gas-pocket formation in olives.

Fermenting, pectolytic yeasts were isolated from a massive commercial outbreak of softening and gas-pocket formation in olives that had been stored in acidified, low-salt brines in an attempt to reduce the problem of brine disposal. The suspected yeasts represented three different species: Saccharomyces oleaginosus, S. kluyveri, and Hansenula anomala var. anomala. All pectolytic cultures produced pectin esterase and polygalacturonase but no pectic acid trans-eliminase when grown in nutrient glucose broth. Crude, cell-free dialyzed enzyme preparations measured viscosimetrically exhibited optimal activity on sodium polygalacturonate at pH 6.0 and 45 C and were active in the range of pH 4.0 to 9.0 and 10 to 60 C.

Purification and properties of an polygalacturonic acid trans-eliminase produced by Bacillus pumilus.

A strain of Bacillus pumilus produced an extracellular pectic enzyme with polygalacturonic acid as the substrate. This enzyme, with optimal activity at pH 8.0 to 8.5, produced reaction products that strongly absorbed light at 232 nm, indicating the presence of a pectic acid trans-eliminase (PATE). Neither pectin esterase nor polygalacturonase was detected in the cell-free culture fluid. Chromatographic examination of the end products revealed the presence of large quantities of unsaturated oligouronides unlike those found with B. polymyxa. It was found that the PATE was produced extracellularly during the negative logarithmic death phase of the organism. The filtrate from sonically treated cells did not show any activity for PATE or hydrolases for lower oligogalacturonides at any time during the growth cycle. The enzyme was inducible. Pectin, National Formulary (NF) was the best inducer, followed by polygalacturonic acid and galacturonic acid. Enzyme activity was markedly stimulated by calcium and other divalent ions. Copper and cobalt ions were inhibitory. The partially purified enzyme showed no significant activity on pectin containing a high methoxyl content (96% esterified). However, pectin NF with a lower methoxyl content (68% esterified) was attacked to a degree by the partially purified and crude enzyme preparations. The initial rate of PATE activity increased up to 60 C, about 16-fold higher than that observed at room temperature. The activation energy was calculated as 12,183 cal/mole. A protective action of calcium chloride against heat inactivation of the PATE was observed. Degradation of polygalacturonic acid by this enzyme produced several unsaturated oligouronides soon after its addition to the substrate. The major endproduct was thought to be different from that of other known PATE enzymes. Paper chromatographic studies and viscosity measurements disclosed the random cleaving nature of the enzyme an endo-PATE.

Some pink yeasts associated with softening of olives.

Pink yeasts identified as Rhodotorula glutinis var. glutinis, R. minuta var. minuta, and R. rubra produce polygalacturonases which cause a slow softening of olive tissue. Both pectin methyl esterase and polygalacturonase are produced when cultures are grown in appropriate media. Crude, cell-free dialyzed enzyme preparations measured viscosimetrically exhibited optimal activity on sodium polygalacturonate at pH 6.0 and 40 C, and were active in the range of pH 4.0 to 9.0 and 10 to 50 C. Cultures grown in sterilized olives and brine at pH 4.0 with sterile glucose added aseptically caused a slow softening of tissue as measured with a Christel texturometer. Similar results were obtained when crude, cell-free enzyme preparations were added to olives in buffer solution at pH 6.0 with Merthiolate. Commercial control of these yeasts is easy if anaerobic conditions can be provided. Otherwise, the industry has to resort to manual removal of the film from the brine surface, either by skimming or by flagellation.

Incidence of Escherichia coli in black walnut meats.

Examination of commercially shelled black walnut meats showed inconsistent numbers of total aerobic bacteria, coliforms, and Escherichia coli; variation occurred among different meat sizes and within each meat size. The incidence of E. coli on meats of commercially hulled black walnuts depended on the physical condition of the nuts. Apparently tightly sealed ones contained only a few or none, whereas those with visibly separated sutures and spoiled meats yielded the most. This contamination was in part correlated to a hulling operation. Large numbers of E. coli on the husk of the walnuts contaminated the hulling water, subsequently also contaminating the meats by way of separated sutures. Chlorination of the hulling wash water was ineffective. Attempts were made to decontaminate the walnut meats without subsequent deleterious changes in flavor or texture. A treatment in coconut oil at 100 C followed by removal of excess surface oil by centrifugation was best.

Death of Salmonella typhimurium and Escherichia coli in the presence of freshly reconstituted dehydrated garlic and onion.

The kinetics of the decline of populations of Salmonella typhimurium inoculated into freshly reconstituted dehydrated onion and garlic powders was studied. Measurable bactericidal activity was observed for onion and garlic concentrations of 1 and 5% (w/v), respectively, with maximal death rates occurring for concentrations of 5 and 10%. At these concentrations, the decimal reduction times were 1.1 and 1.2 hr, respectively, for resting cell cultures and 1.8 and 2.1 hr, respectively, for growing cultures. Of the major volatile aliphatic disulfide compounds of onions, n-propyl allyl and di-n-propyl, at concentrations of 0.1%, showed a comparable activity against resting cells but only a bacteriostatic effect toward actively growing cultures, which overcame this effect in 2 to 6 hr. At comparable concentrations, growing cultures of Escherichia coli were as susceptible to garlic, but apparently more resistant to onion, than were those of S. typhimurium.

Production and catabolite repression of the constitutive polygalacturonic acid trans-eliminase of Aeromonas liquefaciens.

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.