Enzymatic coupling of phenol vapors onto chitosan.

Phenols are important industrial chemicals, and because they can be volatile, also appear as air pollutants. We examined the potential of tyrosinase to react with the volatile phenol p-cresol. Three lines of evidence support the conclusion that volatile phenols react with tyrosinase and are coupled (i.e., chemisorbed) onto chitosan films. First, phenol-trapping studies indicated that p-cresol can be removed from vapors if the vapors are contacted with tyrosinase-coated chitosan films. Second, the ultraviolet absorbance of tyrosinase-coated chitosan films changes dramatically when they are contacted with cresol-containing vapors, whereas control films are unaffected by contacting with cresol vapors. Third, pressure measurements indicate that tyrosinase-coated chitosan films only react with cresol vapors if the oxygen cosubstrate is present. Additional studies demonstrate the potential of tyrosinase-coated chitosan films/membranes for the detection and removal of phenol vapors.

In vivo and in vitro effects of thiolactomycin on fatty acid biosynthesis in Streptomyces collinus.

A stable-isotope assay was used to analyze the effectiveness of various perdeuterated short-chain acyl coenzyme A (acyl-CoA) compounds as starter units for straight- and branched-chain fatty acid biosynthesis in cell extracts of Streptomyces collinus. In these extracts perdeuterated isobutyryl-CoA was converted to isopalmitate (a branched-chain fatty acid), while butyryl-CoA was converted to palmitate (a straight-chain fatty acid). These observations are consistent with previous in vivo analyses of fatty acid biosynthesis in S. collinus, which suggested that butyryl-CoA and isobutyryl-CoA function as starter units for palmitate and isopalmitate biosynthesis, respectively. Additionally, in vitro analysis demonstrated that acetyl-CoA can function as a starter unit for palmitate biosynthesis. Palmitate biosynthesis and isopalmitate biosynthesis in these cell extracts were both effectively inhibited by thiolactomycin, a known type II fatty acid synthase inhibitor. In vivo experiments demonstrated that concentrations of thiolactomycin ranging from 0.1 to 0.2 mg/ml produced both a dramatic decrease in the cellular levels of branched-chain fatty acids and a surprising three- to fivefold increase in the cellular levels of the straight-chain fatty acids palmitate and myristate. Additional in vivo incorporation studies with perdeuterated butyrate suggested that, in accord with the in vitro studies, the biosynthesis of the palmitate from butyryl-CoA decreases in the presence of thiolactomycin. In contrast, in vivo incorporation studies with perdeuterated acetate demonstrated that the biosynthesis of palmitate from acetyl-CoA increases in the presence of thiolactomycin. These observations clearly demonstrate that isobutyryl-CoA is a starter unit for isopalmitate biosynthesis and that either acetyl-CoA or butyryl-CoA can be a starter unit for palmitate biosynthesis in S. collinus. However, the pathway for palmitate biosynthesis from acetyl-CoA is less sensitive to thiolactomycin, and it is suggested that the basis for this difference is in the initiation step.

Thoracic sonography for diagnosis and intervention.

Many physicians believe that ultrasound has limited usefulness in chest disease. Our clinical experiences and a review of the literature in preparation for this monograph have convinced us that sonography can be a very useful and versatile tool for thoracic diagnosis and intervention. Although there are some limitations caused by interposed ribs and air-containing lung, almost all of the compartments of the chest can be evaluated with ultrasound, which gives unique and clinically useful information. Ultrasound guidance for biopsy and drainage does take some time to learn, but we feel that the effort is very worthwhile. The same advantages ultrasound enjoys for other body regions make it a modality that will see increased use in the chest as well. We hope that this monograph will stimulate our colleagues to explore and expand upon the techniques described.

Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus.

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5' end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

Detection and estimation of the volume of pneumothorax using real-time sonography: efficacy determined by receiver operating characteristic analysis.

OBJECTIVE: The purpose of this study was to determine the efficacy of real-time sonography for the detection and estimation of the volume of pneumothorax using receiver operating characteristic (ROC) analysis in 27 patients evaluated by five radiologists using previously described sonographic findings. MATERIALS AND METHODS: Bilateral chest sonograms were obtained in 27 patients after we performed 26 needle biopsies of the lung and two transcostal biopsies of lesions at the dome of the liver. Thirteen unilateral pneumothoraces were found on radiographs done at the time of the sonograms. The sonograms were recorded on videotape and later viewed by five blinded readers who scored each hemithorax independently as to the likelihood and size of pneumothorax. From these interpretations we were able to calculate ROC curves and standard accuracy statistics for each observer and for pooled data. The results were correlated with the findings on chest radiographs, which were used to verify the diagnoses. RESULTS: The area under the ROC curves ranged from 0.63 to 0.79 in detecting any pneumothorax. The area under the ROC curve derived by pooling readers and hemithoraces using the jackknife method was 0.73. The average sensitivity of the five observers fro pneumothorax was 73%, the specificity was 68%, and the negative and positive predictive values were 89% and 40%, respectively. No significant correlation was found between readers' estimates of pneumothorax size made on the basis of sonographic findings and actual size as ascertained from chest radiographs. CONCLUSION: Real time sonography is useful to localize known pneumothorax but cannot be used to exclude the diagnosis. Moreover, sonography has a significant false-positive rate and is of no use in estimating the volume of a pneumothorax.

Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli.

A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+ trans-2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km = 18 microM for crotonyl-CoA and 15 microM for NADPH. The enzyme was unable to catalyze the reduction of any other enoyl-CoA thioesters or to utilize NADH as an electron donor. A highly effective inhibition by straight-chain fatty acids (Ki = 9.5 microM for palmitoyl-CoA) compared with branched-chain fatty acids (Ki > 400 microM for isopalmitoyl-CoA) was observed. All of these properties are consistent with a proposed role of the enzyme in providing butyryl-CoA as a starter unit for straight-chain fatty acid biosynthesis. The crotonyl-CoA reductase gene was cloned in Escherichia coli. This gene, with a proposed designation of ccr, is encoded in a 1344-bp open reading frame which predicts a primary translation product of 448 amino acids with a calculated molecular mass of 49.4 kDa. Several dispersed regions of highly significant sequence similarity were noted between the deduced amino acid sequence and various alcohol dehydrogenases and fatty acid synthases, including one region that contains a putative NADPH binding site. The ccr gene product was expressed in E. coli and the induced crotonyl-CoA reductase was purified tenfold and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide to the native protein.

Hiring preferences of diagnostic radiology groups: results of a 1994 survey.

RATIONALE AND OBJECTIVES: We surveyed diagnostic radiology group preferences and considerations in hiring radiologists and compared these findings with those of a survey performed in 1990. We sought to identify changes in hiring practices that might have occurred because of socioeconomic changes. We also sought to identify features of job candidates that make them more attractive to hiring groups. METHODS: One hundred surveys were mailed to a stratified random sample of diagnostic radiology groups identified by the American College of Radiology. We solicited information on the importance of various attributes and the level of experience of a candidate, the fellowship training considered most desirable, and the effect of changes in the health care socioeconomic environment. The responses were weighted by group size and geographic location to estimate what results might have been obtained if we had surveyed all groups in the United States. RESULTS: Seventy-five groups returned the survey. The two most important factors in choosing a candidate were motivation and radiologic knowledge. The fellowships that groups that were hiring considered to be the most desirable were body imaging, neuroradiology, and angiography/interventional radiology. Groups overwhelmingly preferred recent training over long experience. CONCLUSION: Fellowship training increases a candidate's marketability, but the two factors that hiring groups consider the most important are motivation and radiologic knowledge.

In vivo analysis of straight-chain and branched-chain fatty acid biosynthesis in three actinomycetes.

The starter units for branched-chain and straight-chain fatty acid biosynthesis was investigated in vivo in three actinomycetes using stable isotopes. Branched-chain fatty acids, which constitute the majority of the fatty acid pool, were confirmed to be biosynthesized using the amino acid degradation products methylbutyrl-CoA and isobutyrl-CoA as starter units. Straight-chain fatty acids were shown to be constructed using butyrl-CoA as a starter unit. Isomerization of the valine catabolite isobutyryl-CoA was shown to be only a minor source of this butyryl-CoA.

Evaluation of a breast biopsy phantom for learning freehand ultrasound-guided biopsy of the liver.

RATIONALE AND OBJECTIVES: We evaluated whether a breast biopsy phantom device would aid in the development of skills in freehand ultrasound liver biopsy. METHODS: Three radiologists who were inexperienced in freehand biopsy of the liver were observed. Each radiologist was timed and scored during attempts to biopsy lesions created in a beef liver. The time required for biopsy and the success of each pass was recorded. A commercially available breast biopsy phantom was then used by each of these radiologists during two 20-min practice sessions. Posttraining testing on the beef liver was performed in the same manner as pretraining testing. RESULTS: Freehand biopsy practice using the breast biopsy phantom resulted in a reduction in the mean time required for biopsy from 32 to 17 sec. Each of the three subjects reduced the mean time required for successful biopsy after training using the breast biopsy phantom. The total number of lesions missed was reduced from 14 of 43 to 0 of 45. CONCLUSION: Practice using the ultrasound breast biopsy phantom improves performance in freehand ultrasound biopsy of the liver.

Ammonium effects on streptonigrin biosynthesis by Streptomyces flocculus.

A defined medium containing glucose and ammonium as the sole carbon and nitrogen sources was developed to support growth and streptonigrin production. In this defined medium, increased initial levels of ammonium resulted in increased growth suggesting that nitrogen is the growth limiting nutrient. In some cases, increased initial ammonium levels resulted in decreased specific streptonigrin productivity, suggesting that nitrogen regulatory mechanisms may adversely affect streptonigrin biosynthesis. This suggestion that nitrogen regulation adversely affects antibiotic biosynthesis is further supported by results from two studies in which the ammonium supply to the cells was controlled. In the first study, streptonigrin productivity and final titer were enhanced by the addition of an ammonium trapping agent. In the second experiment, when ammonium chloride was fed slowly throughout the course of cultivation, the production phase was lengthened and the maximum antibiotic concentration was enhanced compared to the batch controls containing either the same initial or the same total ammonium chloride levels. Although our results indicate streptonigrin production may be subject to nitrogen regulatory mechanisms, the effect of nitrogen on streptonigrin production cannot be strictly correlated to the extracellular ammonium concentration. In fact, we observed that when ammonium was depleted from the medium, streptonigrin production ceased.