Inspection of lead aprons: criteria for rejection.

Lead aprons utilized by personnel performing fluoroscopy are routinely inspected for damage to comply with the requirements of hospital accrediting organizations. Fluoroscopic or radiographic examination of lead aprons may reveal imperfections ranging from small pinholes to large tears. Currently, there are no standards establishing a criteria for acceptance or rejection of lead aprons. As a consequence, many facilities have established arbitrary rejection criteria. Often lead aprons are discarded due to small imperfections, a practice that can become costly to these institutions. We have calculated increases in doses to the whole body for varying sizes of holes, including special consideration of the effects on effective dose equivalent when the hole is over the testes and thyroid. ALARA standards for cost per personsievert averted are used to establish a rational basis for criteria of acceptance or rejection of lead aprons.

Separation of the molecular species of intact phosphatidylethanolamines and their N-monomethyl and N,N-dimethyl derivatives by high-performance liquid chromatography on a C8 column.

We have developed a gradient reversed-phase C8 high-performance liquid chromatography method for the separation of molecular species of phosphatidylethanolamines (PEs) and their N-monomethyl and N,N-dimethyl derivatives. This method uses a 40-min linear gradient of 88-100% methanol, containing ammonium hydroxide as silanol suppressing agent, and is suitable for metabolic studies using both UV detection at 205 nm and radioactivity flow detection. The elution order of a given PE is inversely related to the polarity of its fatty acid constituents. Lipid classes studied here containing the same fatty acyl chains elute in the order: PE-N,N-dimethyl

Metabolism of 1-acyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in castor oil biosynthesis.

We have examined the role of 2-oleoyl-PE (phosphatidylethanolamine) in the biosynthesis of triacylglycerols (TAG) by castor microsomes. In castor microsomal incubation, the label from 14C-oleate of 1-palmitoyl-2-[1-(14)C]oleoyl-sn-glycero-3-phosphoethanolamine is incorporated into TAG containing ricinoleate. The enzyme characteristics, such as optimal pH, and the effect of incubation components of the oleoyl-12-hydroxylase using 2-oleoyl-PE as incubation substrate are similar to those for 2-oleoyl-PC (phosphatidylcholine). However, compared to 2-oleoyl-PC, 2-oleoyl-PE is a less efficient incubation substrate of oleoyl-12-hydroxylase in castor microsomes. Unlike 2-oleoyl-PC, 2-oleoyl-PE is not hydroxylated to 2-ricinoleoyl-PE by oleoyl-12-hydroxylase and is not desaturated to 2-linoleoyl-PE by oleoyl-12-desaturase. We have demonstrated the conversion of 2-oleoyl-PE to 2-oleoyl-PC and vice versa. The incorporation of label from 2-[14C]oleoyl-PE into TAG occurs after its conversion to 2-oleoyl-PC, which can then be hydroxylated or desaturated. We detected neither PE-N-monomethyl nor PE-N,N-dimethyl, the intermediates from PE to PC by N-methylation. The conversion of 2-oleoyl-PE to 2-oleoyl-PC likely occurs via hydrolysis to 1,2-diacyl-sn-glycerol by phospholipase C and then by cholinephosphotransferase. This conversion does not appear to play a key role in driving ricinoleate into TAG.

Biochemical aspects of castor oil biosynthesis.

Castor oil is 90% ricinoleate (12-hydroxy-oleate) and has numerous industrial uses. Components of castor bean (Ricinus communis L.) pose serious problems to processors. We are evaluating two complementary approaches to providing a safe source of castor oil.

Characterization of Neurospora crassa mutants isolated following repeat-induced point mutation of the beta subunit of fatty acid synthase.

Neurospora crassa cel-2 mutants were isolated following repeat-induced point mutation using part of the gene encoding beta-fatty acid synthase. These mutants are phenotypically less leaky than cel-1, which has a defective alpha-fatty acid synthase. The cel-2 mutant had a strict fatty acid (16:0) requirement for growth, and synthesized less fatty acid de novo than cel-1. Unlike cel-1, cel-2 has impaired fertility, and homozygous crosses are infertile, suggesting a low but strict requirement for fatty acid synthesis during sexual development. Like cel-1, cel-2 synthesized unusually high levels of the polyunsaturate 18:3(Delta9,12,15), and elongated 18:2(Delta9,12 )and 18:3(Delta9,12,15 )to 20:2(Delta11,14) and 20:3(Delta11,14,17), respectively. These fatty acids are not synthesized by wild-type, except following treatment with cerulenin (a fatty acid synthase inhibitor), demonstrating that inhibition of fatty acid biosynthesis results in a relative increase in both fatty acid desaturation and elongation activity.

Biosynthesis of ricinoleate in castor oil.

Castor oil is 90% ricinoleate (12-hydroxyoleate) and has numerous industrial uses. Components of castor bean (Ricinus communis L.) pose serious problems to processors. Other researchers have cloned the gene for the oleoyl hydroxylase, but transgenic plants produce only about 20% hydroxy fatty acid. To improve such transgenic substitutes for castor, we are using HPLC analysis of castor bean microsomal suspensions to follow the hydroxylase reaction and the movement of 14C-ricinoleate through phospholipid into triacylglycerol. Most labeled ricinoleate is rapidly removed from the phospholipid fraction as free fatty acid and incorporated into triacylglycerol, with triricinolein predominating. Elucidation of the basis for high incorporation of ricinoleate and exclusion of oleate from triacylglycerols will identify genes that can be used to engineer high ricinoleate production in transgenic plants.

Conversion of palmitate to unsaturated fatty acids differs in a Neurospora crassa mutant with impaired fatty acid synthase activity.

The Neurospora crassa cel (fatty acid chain elongation) mutant has impaired fatty acid synthase activity. The cel mutant requires exogenous 16:0 for growth and converts 16:0 to other fatty acids. In contrast to wild-type N. crassa, which converted only 42% of the exogenous [7,7,8,8-(2)H4]16:0 that was incorporated into cell lipids to unsaturated fatty acids, cel converted 72%. In addition, cel contains higher levels of 18:3(delta 9,12,15) than wild-type, and synthesizes two fatty acids, 20:2(delta 11,14 and 20:3(delta 11,14,17, found at only trace levels in wild-type. Thus, the delta 15-desaturase activity and elongation activity on 18-carbon polyunsaturated fatty acids are higher for cel than wild-type. This altered metabolism of exogenous 16:0 may be directly due to impaired flux through the endogenous fatty acid biosynthetic pathway, or may result from altered regulation of the synthesis of unsaturated fatty acids in the mutant.

Biosynthesis of triacylglycerols containing ricinoleate in castor microsomes using 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine as the substrate of oleoyl-12-hydroxylase.

We have examined the biosynthetic pathway of triacylglycerols containing ricinoleate to determine the steps in the pathway that lead to the high levels of ricinoleate incorporation in castor oil. The biosynthetic pathway was studied by analysis of products resulting from castor microsomal incubation of 1-palmitoyl-2-[14C]oleoyl-sn-glycero-3-phosphocholine, the substrate of oleoyl-12-hydroxylase, using high-performance liquid chromatography, gas chromatography, mass spectrometry, and/or thin-layer chromatography. In addition to formation of the immediate and major metabolite, 1-palmitoyl-2-[14C]ricinoleoyl-sn-glycero-3-phosphocholine, 14C-labeled 2-linoleoyl-phosphatidylcholine (PC), and 14C-labeled phosphatidylethanolamine were also identified as the metabolites. In addition, the four triacylglycerols that constitute castor oil, triricinolein, 1,2-diricinoleoyl-3-oleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linoleoyl-sn-glycerol, 1,2-diricinoleoyl-3-linolenoyl-sn-glycerol, were also identified as labeled metabolites in the incubation along with labeled fatty acids: ricinoleate, oleate, and linoleate. The conversion of PC to free fatty acids by phospholipase A2 strongly favored ricinoleate among the fatty acids on the sn-2 position of PC. A major metabolite, 1-palmitoyl-2-oleoyl-sn-glycerol, was identified as the phospholipase C hydrolyte of the substrate; however, its conversion to triacylglycerols was blocked. In the separate incubations of 2-[14C]ricinoleoyl-PC and [14C]ricinoleate plus CoA, the metabolites were free ricinoleate and the same triacylglycerols that result from incubation with 2-oleoyl-PC. Our results demonstrate the proposed pathway: 2-oleoyl-PC-->2-ricinoleoyl-PC-->ricinoleate-->triacylglycerols. The first two steps as well as the step of diacylglycerol acyltransferase show preference for producing ricinoleate and incorporating it in triacylglycerols over oleate and linoleate. Thus, the productions of these triacylglycerols in this relatively short incubation (30 min), as well as the availability of 2-oleoyl-PC in vivo, reflect the in vivo drive to produce triricinolein in castor bean.

Trichomonas gallinae in budgerigars and columbid birds in Perth, Western Australia.

OBJECTIVE: To estimate the prevalence of infection with Trichomonas gallinae and other parasites of the alimentary tract in psittacine and columbid birds in Perth and to determine in vitro the effectiveness of drugs commonly recommended for treating trichomoniasis. DESIGN AND PROCEDURES: Samples of crop contents were collected from aviary flocks of budgerigars (Melopsittacus undulatus) and other psittacine and columbid birds in both private and commercial collections in Perth. Similar samples from wild Senegal doves (Streptopelia senegalensis) also were collected. Crop contents were examined and cultured for Trichomonas gallinae and in vitro studies were conducted on the susceptibility of isolates to several drugs used commonly. Other parasites also were detected by faecal examination and/or necropsy. RESULTS: T gallinae was recovered from birds in 1 of 13 private collections of budgerigars (2/289 birds in total). Direct wet-mount examination of crop fluid identified 36.4% of samples at four commercial bird dealers which were later determined by culture to contain T gallinae. The prevalence of T gallinae infection range from 0 to 11.4% in budgerigars. The prevalence of T gallinae infection of wild Senegal doves was 46% and from one flock of racing pigeons was 59%. The in vitro minimum lethal concentrations of metronidazole, dimetridazole and ronidazole ranged from 40 to 96, 30 to 80 and 40 to 92 micrograms/mL respectively for six isolates of T gallinae. Other alimentary parasites detected during the survey included Spironucleus sp (syn. Hexamita sp), coccidia, Ascaridia platycerci and Raillietina sp. CONCLUSIONS: Thirteen budgerigar flocks belonging to members of avicultural societies in Perth had a low prevalence of trichomoniasis and other parasitic infections. The dose rate currently recommended for ronidazole may not result in complete protozoacidal activity against T gallinae infection.

Pathways for fatty acid elongation and desaturation in Neurospora crassa.

Neurospora crassa incorporated exogenous deuterated palmitate (16:0) and 14C-labeled oleate (18:1 delta 9) into cell lipids. Of the exogenous 18:1 delta 9 incorporated, 59% was desaturated to 18:2 delta 9,12 and 18:3 delta 9,12,15. Of the exogenous 16:0 incorporated, 20% was elongated to 18:0, while 37% was elongated and desaturated into 18:1 delta 9, 18:2 delta 9,12, and 18:3 delta 9,12,15. The mass of unsaturated fatty acids in phospholipid and triacylglycerol is 12 times greater than the mass of 18:0. Deuterium label incorporation in unsaturated fatty acids is only twofold greater than in 18:0, indicating a sixfold preferential use of 16:0 for saturated fatty acid synthesis. These results indicate that the release of 16:0 from fatty acid synthase is a key control point that influences fatty acid composition in Neurospora.

Metabolism of ricinoleate by Neurospora crassa.

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate 14 microM-3mM [1-14C]ricinoleate. The fate of the [14C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.

Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine.

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5 degrees C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual substrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.

Performance measurement: integrating quality management and activity-based cost management.

The development of an activity-based management system provides a framework for developing performance measures integral to quality and cost management. Performance measures that cross operational boundaries and embrace core processes provide a mechanism to evaluate operational results related to strategic intention and internal and external customers. The author discusses this measurement process that allows managers to evaluate where they are and where they want to be, and to set a course of action that closes the gap between the two.

Benchmarks and performance indicators: two tools for evaluating organizational results and continuous quality improvement efforts.

Benchmarks are tools that can be compared across companies and industries to measure process output. The key to benchmarking is understanding the composition of the benchmark and whether the benchmarks consist of homogeneous groupings. Performance measures expand the concept of benchmarking and cross organizational boundaries to include factors that are strategically important to organizational success. Incorporating performance measures into a balanced score card will provide a comprehensive tool to evaluate organizational results.

Activity-based management: a tool to complement and quantify continuous quality improvement efforts.

An activity-based cost management system provides a framework to integrate the disciplines of quality and cost management. The integration occurs through the development of performance measures that collectively measure operations with respect to internal and external customers. It is the measurement process that allows management staff to evaluate where they are, determine where they want to be, and set a course of action that closes the gap between the two.

Fatty acid biosynthesis in novel ufa mutants of Neurospora crassa.

New mutants of Neurospora crassa having the ufa phenotype have been isolated. Two of these mutants, like previously identified ufa mutants, require an unsaturated fatty acid for growth and are almost completely blocked in the de novo synthesis of unsaturated fatty acids. The new mutations map to a different chromosomal location than previously characterized ufa mutations. This implies that at least one additional genetic locus controls the synthesis of unsaturated fatty acids in Neurospora.

Vasoactive intestinal peptide and secretin produce long-term increases in tyrosine hydroxylase activity in the rat superior cervical ganglion.

Electrical stimulation of the preganglionic fibers innervating the rat superior cervical ganglion (SCG) produces both short-term and long-term increases in tyrosine hydroxylase (TH) activity that are not completely blocked by nicotinic antagonists. Vasoactive intestinal peptide (VIP) and secretin, two neuropeptides known to produce short-term increases in TH activity, were examined for their ability to produce long-term increases in this enzyme activity. Culturing the SCG in the presence of either peptide produced a 30-50% increase in TH activity measured 2 days later. The results raise the possibility that one of these peptides or a related molecule participates in the transsynaptic induction of ganglionic TH.

Phenotypic plasticity in adult sympathetic neurons: changes in neuropeptide expression in organ culture.

Vasoactive intestinal peptide (VIP)-like immunoreactivity is present at low levels in the superior cervical ganglion of the adult rat, where immunostained neural processes, but only an occasional immunostained cell body, are found. However, when ganglia are maintained for 24 or 48 hr in organ culture, their content of VIP-like immunoreactivity increases 6- or 31-fold, respectively. When examined at 24 hr, the increase in VIP-like immunoreactivity is totally blocked by an inhibitor of RNA or protein synthesis. Many neuronal cell bodies and processes with immunoreactivity for VIP and the related peptide histidine isoleucine amide (PHI) are seen in cultured ganglia. In addition, VIP/PHI mRNA is abundant in cultured ganglia but only barely detectable in ganglia prior to culture. Under the same culture conditions, neuropeptide Y-like immunoreactivity increases to a small extent, and tyrosine hydroxylase activity and total ganglion protein remain unchanged. These results support the idea that adult sympathetic neurons exhibit plasticity in neuropeptide expression and that this plasticity, in the case of VIP, depends on changes in gene expression.