Limited retention of micro-organisms using commercialized needle filters.

A study was undertaken to compare a commercialized needle filter with a 0.2-µm filtered epidural set and a non-filtered standard needle. No culturable bacteria were detected following filtration through the 0.2-µm filter. Bacterial breakthrough was observed with the filtered needle (pore size 5 µm) and the non-filtered needle. Filtered systems (0.2 µm) should be employed to achieve total bacterial retention. This highlights that filtration systems with different pore sizes will have varying ability to retain bacteria. Healthcare professionals need to know what type/capability of filter is implied on labels used by manufacturers, and to assess whether the specification has the desired functionality to prevent bacterial translocation through needles.

Determination of thirteen common elements in food samples by inductively coupled plasma atomic emission spectrometry: comparison of five digestion methods.

Five sample digestion procedures were evaluated for the determination of Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Sr, and Zn in food samples by inductively coupled plasma atomic emission spectrometry. The 5 procedures include dry ashing at 500 degrees C, wet digestion with HNO3-HClO4, microwave digestion with HNO3, microwave digestion with HNO3-H2O2, and microwave digestion with HNO3-H2O2-HF. For microwave digestion with HNO3-H2O2-HF, silicon (IV) oxide was used to eliminate the excess HF, making it possible to determine total Al, B, and other common elements accurately and simultaneously. Seven National Institute of Standards and Technology standard reference materials (SRMs) were analyzed to compare the recovery of 13 elements with above digestion procedures. The results demonstrated that the microwave digestion procedure with HNO3-H2O2-HF yielded the best recoveries for all 13 elements in the selected SRMs. The determined concentrations of most elements were close for all 3 microwave digestion procedures with the exception of Al in oyster tissue, bovine liver, and spinach. Notably, the wet digestion with HNO3-HClO4 is the simplest and the most effective procedure for the selected elements except Al and B. Although there are several concerns with the dry ashing procedure, it might be a preferable procedure for those analyses where only nonvolatile elements are to be determined and the concentrations of the elements are low.

Alanine, not ammonia, is excreted from N2-fixing soybean nodule bacteroids.

Symbiotic nitrogen fixation, the process whereby nitrogen-fixing bacteria enter into associations with plants, provides the major source of nitrogen for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobia-leguminous plant symbioses, the current model of nitrogen transfer from the symbiotic form of the bacteria, called a bacteroid, to the plant is that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids outside of the bacteroid. We purified soybean nodule bacteroids by a procedure that removed contaminating plant proteins and found that alanine was the major nitrogen-containing compound excreted. Bacteroids incubated in the presence of 15N2 excreted alanine highly enriched in 15N. The ammonium in these assays neither accumulated significantly nor was enriched in 15N. The results demonstrate that a transport mechanism rather than diffusion functions at this critical step of nitrogen transfer from the bacteroids to the plant host. Alanine may serve only as a transport species, but this would permit physiological separation of the transport of fixed nitrogen from other nitrogen metabolic functions commonly mediated through glutamate.

Gas-liquid chromatography-mass spectrometry of hydroxy fatty acids as their methyl esters tert.-butyldimethylsilyl ethers.

tert.-Butyldimethylsilyl ethers of secondary hydroxy fatty acid methyl esters (tBDMS-O-FAMEs) produce stable derivatives amenable to gas-liquid chromatography (GLC) and mass spectrometry (MS). Derivatives produce prominent molecular mass minus 57 [M-57]+ fragment ions and unique marker fragment ions indicating the location of the secondary hydroxyl groups along the aliphatic chain from the omega-2 carbon to carbon numbers 5 from the carboxylic terminus, in addition to yielding information regarding carbon chain length, and degree of unsaturation. The tBDMS-derivatives of C-2, C-3 hydroxy fatty acids and the unique GLC-MS data of gamma- and delta-lactones are also presented. Though several tBDMS-O-FAMEs with centrally located hydroxyl groups were not chromatographically resolved, the combination of GLC retention times and monitoring of key diagnostic fragment ions of each tBDMS-derivative, when applied to mixtures containing all hydroxy isomers of palmitic through arachidic acid methyl esters, and to several monounsaturated, monohydroxylated fatty acid methyl esters, allowed for their unambiguous identification. Coupled with derivative stability, permitting their purification and concentration, this method was applied to the identification of trace lipids isolated from bovine skim milk which contained a complex mixture of hydroxy fatty acids of which 19 were newly identified.

Disulfated oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis.

Twenty novel disulfated oligosaccharides were purified in nanomolar quantities from tracheo-bronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, followed by anion-exchange chromatography, size-exclusion chromatography, and high-performance liquid chromatography (HPLC). In addition to positive ion fast-atombombardment mass spectrometry (FABMS), proposed structures for the resulting purified disulfated oligosaccharides were also based on carbohydrate permethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, and parallel analysis of desulfated products. Sulfate esters were found to reside on C-3 or C-6 of terminal D-galactose and on C-6 of internal D-galactose or 2-acetamido-2-deoxy-D-glucose residues. For this group of oligosaccharides, ranging in size from tri- to undeca-saccharides and possessing linear, di- and tri-antennary forms, it was also observed that sulfate esters could be located on the same or on different branches and that branched oligosaccharides can possess sulfate esters on C-3 and C-6 of different terminal galactose residues within the same structure.

Characterization of Gallus domesticus alpha-N-acetyl-galactosaminidase blood group A2 activity.

Soluble A antigens and an enzyme-linked immunosorbent assay (ELISA) using type A2 erythrocyte membranes were used to study the activity of an alpha-N-acetyl-galactosaminidase from Gallus domesticus (domestic chicken). The enzyme readily hydrolyzed the terminal N-acetyl-alpha-D-galactosamine of the A antigen under a variety of conditions, converting it to H antigen. Conversion of the A antigen to H antigen produces blood type O, which is universally transfusable. These preliminary studies are important in determining optimal conditions for enzymatic conversion of blood type A to O if efficient large scale production of enzymatically converted, universally transfusable red blood cells is to be achieved.

Sulfated sialyl-oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis.

Thirteen novel oligosaccharides, each possessing both a sulfate ester and a sialic acid residue, were isolated from tracheobronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, and by employing immobilized Limulus polyphemus lectin affinity chromatography, SynChroprep AX300 anion-exchange chromatography, Bio-Gel P-2 size-exclusion chromatography, and Hypersil 120A APS-2 high-performance liquid chromatography (HPLC). Proposed structures for the resulting purified sulfated sialyl-oligosaccharides were based on carbohydrate/permethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, analysis of desulfated products and, analysis by positive-ion fast-atom-bombardment mass spectrometry (FABMS). Sulfate esters on these sialyl-oligosaccharides resided on C-6 of a terminal or an internal D-galactose or 2-acetamido-2-deoxy-D-glucose residue or C-4 of a terminal D-galactose residue. The sialic acid residues were found to be either bound (2-->6)-alpha to 2-acetamido-2-deoxy-D-galactitol or (2-->3)-alpha or (2-->6)-alpha to a D-galactose residue occupying a nonreducing terminus. For this group of oligosaccharides, ranging in size from tri- to hepta-saccharides, it was also observed that a sialic acid residue and a sulfate ester did not residue on the same oligosaccharide branch when more than one branch existed. On linear unbranched sulfated sialyl-oligosaccharides, the sialic acid residue was bound to a D-galactose residue occupying a nonreducing terminus with the sulfate ester residing on an internal D-galactose or a 2-acetamido-2-deoxy-D-glucose residue. These results demonstrate that it is possible for sialic acid and a sulfate ester to exist on the same oligosaccharide and that this oligosaccharide can be as small as a trisaccharide.

Clinical observations of nebulized flunisolide in infants and young children with asthma and bronchopulmonary dysplasia.

Severe bronchopulmonary dysplasia (BPD) is frequently associated with asthma. The combination is often severe enough to necessitate corticosteroid therapy. There are no commercially available nebulizer solutions of corticosteroids for use in infants and young children. Seven infants and small children with very severe BPD and asthma aged 6-24 months, were treated with flunisolide, 187-250 micrograms q.i.d. in the form of nasal spray delivered by nebulizer. After treatment for 2.5-20 months, four patients showed clinical improvement, one initially improved but later deteriorated and died of cardiac failure, and two patients showed no improvement and died within 3 months. The number of days of hospitalization was significantly reduced from 8.4/month to 2.5/month (P less than 0.05). No side-effects were detected and it was felt that the three patients who died, did so as a consequence of very severe BPD and its cardiac consequences. The suspension remained stable for 80 min when mixed with normal saline, cromolyn sodium, albuterol, or acetylcysteine. It is concluded that nebulized flunisolide is a potentially useful treatment for infants and young children with asthma and BPD.

Structural analysis of monosulfated side-chain oligosaccharides isolated from human tracheobronchial mucous glycoproteins.

To determine the location of some sulfate esters on respiratory mucins, an unambiguous sequencing strategy was developed for a crude, monosulfated oligosaccharide fraction derived from tracheobronchial mucous glycoproteins, isolated from sputum from a patient with cystic fibrosis, and which possessed Ricinus communis-I lectin affinity. Employing fractionation by Bio-Gel P-2 chromatography and high-voltage paper electrophoresis of the pool, eighteen branched and four straight-chained monosulfated oligosaccharides, each possessing at least one neutral D-galactose residue at a nonreducing terminus, were purified. Desulfated analogs of each sulfated oligosaccharide were then produced. Elucidation of their structures and sulfate ester locations was accomplished through a parallel comparative sequencing approach for the sulfated oligosaccharide and its desulfated analog. The method was based on their carbohydrate composition and parallel analysis by sequential exoglycosidase degradations, endoglycosidase digestion, permethylation analyses, and specific lectin affinities. Key to this approach was the inability for specific exoglycosidases and lectins to cleave or bind to, respectively, carbohydrates of their specificity which occupied nonreducing termini and possessed a sulfate ester. Herein we report the structures of twenty-two novel sulfated oligosaccharides. Oligosaccharides ranged from trisaccharides to heptasaccharides, were branched and unbranched, and each possessed a single sulfate ester on either C-6 of a terminal or an internal D-galactose residue or on C-6 of an internal residue of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine).

Superior-subordinate dyads: Dependence of leader effectiveness on mutual reinforcement contingencies.

Task contingencies were modeled from bureaucratic organizations in which vague job descriptions provide incomplete contingency specifications. Response rates within dyads were examined using two nonsocial, two social, and two control contingencies. In the first social contingency, responses by the superior produced monetary reinforcement for a subordinate while the superior received no reinforcement from his subordinate. A second social contingency was identical to the first except that the subordinate's rate of responding determined the rate of reinforcement delivered to his superior. Within this contingency, mutual reinforcement occurred whenever rates of superior and subordinate responding were correlated. Two control contingencies were identical to the second social contingency except that either the superior or the subordinate received a rate of response-independent reinforcement virtually identical to the rate received during the second social contingency. Leadership, in this context, was the difference between rates of subordinate responding produced by a nonsocial contingency and rates produced by each of the two social contingencies. The two nonsocial contingencies supported almost no responding among subjects. The first social contingency produced minimal levels of leadership within every dyad. The second social contingency produced high levels of leadership. Response-independent reinforcement generally reduced or eliminated responding.

Perilymphatic fistula: analysis of free amino acids in middle ear microaspirates.

High-performance liquid chromatography was used to determine 19 free amino acid concentrations in perilymph, serum/plasma, and red blood cell intracellular fluid. Significant differences were found between perilymph and these fluids. Free amino acid analysis was then used to quantitatively analyze middle ear microaspirates in order to test the hypothesis that perilymph is a potential source of clear fluid in perilymphatic fistulas (PLF). Fourteen unknown samples from patients with visually identified PLF, including patients with no identifiable otic capsule defect, were studied. Six samples on amino acid pattern analysis were correlated most similarly with perilymph (rrho greater than 0.95). Four of these six samples were scored on the basis of quantitative amino acid values as similar to perilymph. However, three samples of clear fluid were more similar to serum/plasma than to perilymph on both amino acid pattern and quantitative amino acid score analysis. These results objectively suggest perilymph as a potential source of clear fluid in some patients with a diagnosis of PLF. Not all clear fluid observed in the middle ear, however, is potentially perilymph.

Free amino acid analysis of guinea pig perilymph: a possible clinical assay for the PLF enigma?

Controversy prevails regarding the accuracy of the clinical diagnosis of perilymphatic fistula (PLF). The diagnosis of PLF has been based on the subjective evaluation of vestibular function tests and the intraoperative macroscopic visualization of "clear fluid" from the oval/round windows at the time of exploratory tympanotomy. However, the subjective visual characterization of PLF varies among observing surgeons. Furthermore, perilymph can be "contaminated" with serum, blood, cerebrospinal fluid (CSF), and local anesthesia. This article presents a scientific biochemical microassay for the free amino acid profile of perilymph. Microaliquots of uncontaminated perilymph were sampled from the bilateral round windows (scala tympani) of 20 guinea pigs and analyzed for 19 free amino acid concentrations (FAAC) by high-performance liquid chromatography (HPLC). These samples were compared with the FAAC of guinea pig serum samples. Perfect predictor value ranges were nonoverlapping for 12 of 19 free amino acids in perilymph vs. plasma. Amino acid microassay of middle ear fluid for verification of "true" perilymph vs. nonperilymph fluids by the identification of nonoverlapping FAA markers may allow scientific verification of the existence of PLF in "suspected" patients.

Identification of an antigenic marker of slime production for Staphylococcus epidermidis.

The pathogenic Staphylococcus epidermidis strain RP62A (ATCC 35984) adheres to smooth surfaces by forming a tenacious bacterial film known as slime. The mechanism of slime production is not known; however, workers in the laboratory of G. Pier (Harvard Medical School, Boston, Mass.) have isolated from RP62A a galactose-rich capsular polysaccharide adhesin (CPA) which mediates the attachment of the organism to smooth surfaces. We have obtained two daughter strains from RP62A that no longer produce slime. One daughter strain, H4A, was obtained by selection for a spontaneous variant; the other strain, HAM892, was obtained by treating growing cultures of RP62A with acriflavin. Using an antiserum generated against whole cells of RP62A, we have examined lysozyme-lysostaphin digests of RP62A, H4A, and HAM892 by double immunodiffusion. The two strains that no longer produced slime no longer produced a particular antigen, which we refer to as the slime-associated antigen (SAA). SAA was also produced by unrelated strains of slime-producing S. epidermidis. SAA was heat and protease stable, had a molecular weight of greater than 50,000, and could be partially purified by chromatographing trypsin-digested material over a Sephadex G-200 column. Chemical analysis of partially purified SAA by gas-liquid chromatography found SAA to be glucose rich (59%) and galactose poor (1.4%). This analysis chemically distinguished SAA from CPA. When tested together by double immunodiffusion with anti-RP62A and anti-CPA antisera, partially purified SAA did not cross-react with CPA. Kinetic studies suggested that SAA is a marker for surface accumulation whereas CPA mediates initial adherence.

Anaphylaxis due to cornstarch surgical glove powder.

Two nurses suffered anaphylaxis to cornstarch glove powder. Both exhibited (1) positive prick skin tests to cornstarch powder in water, with resultant anaphylaxis in one and (2) negative prick skin tests and RAST to corn. Analysis of the powder revealed only glucose and inorganic salts. We were unable to detect in vitro histamine release or cornstarch powder specific IgE. Because of the positive skin tests and the resultant anaphylaxis, we suspect that cornstarch is the responsible allergen.

Gas-liquid chromatography-mass spectrometry of mono- and dithiols as their tert.-butyldimethylsilyl derivatives.

The use of gas-liquid chromatography and mass spectrometry with derivatizing agents that give stable derivatives and consistent fragmentation patterns allows for accurate identification of a variety of compounds. In this study either N-methyl-N-tert.-butyldimethylsilyltrifluoroacetamide or N-tert.-butyldimethylsilylimidazole were employed to derivatize a range of mono- and dithiols: from ethanethiol to 1-hexadecanethiol and 1,2-ethanedithiol to 1,9-nonanedithiol. When analyzed in this way, the resulting tert.-butyldimethylsilyl derivatives of the 24 thiols tested were readily distinguishable. Complete baseline separation of each derivative by capillary gas-liquid chromatography was achieved, and each produced a prominent mass minus 57 [M+. - 57] fragment ion. The tert.-butyldimethylsilyl-thioethers were colorless and were stable at room temperature for over 3 months. This method may provide a convenient approach to the analysis of thiol compounds from a wide variety of sources.

Boron fertilization and carbohydrate relations in mycorrhizal and nonmycorrhizal shortleaf pine.

Eight-week-old shortleaf pine seedlings (Pinus echinata Mill.) with and without ectomycorrhizae formed by Pisolithus tinctorius were treated for two to eight weeks with 25 microg borate ml(-1) solution applied either to the soil, or as a foliar spray, or in both ways. Control seedlings were fertilized only with modified Hoagland's solution containing 0.03 microg ml(-1) borate. Five sugars (pinitol, fructose, glucose, myoinositol and sucrose) were quantitated in both mycorrhizal and nonmycorrhizal roots by gas-liquid chromatography. Fertilization with boron increased the total carbohydrate content of mycorrhizal roots except in seedlings receiving foliar applications of boron. Foliar + soil fertilization yielded a 24% increase in total carbohydrates in mycorrhizal roots, whereas foliar fertilization alone decreased the total carbohydrate content. Carbohydrate content of nonmycorrhizal roots was significantly increased only by soil fertilization with boron. Individual sugars were affected less by boron fertilization in nonmycorrhizal roots than in ectomycorrhizal roots. However, significant increases in sugars in response to boron fertilization were observed in both ectomycorrhizal and nonmycorrhizal plants.

Inositol is a required nutrient for keratinocyte growth.

Bovine hypothalamus is known to contain a growth-promoting activity for human epidermal keratinocytes. By sequential purification, the substance was isolated and found to be myo-inositol. The identity of the substance as myo-inositol was confirmed by ion modified partition, gas liquid, thin layer chromatography, by mass spectrometry, and quantitative bioassay. The inositol content of the crude hypothalamic extract and of an active acetone precipitate (the first step in the purification) was determined to be sufficient to account for their observed bioactivity. At an optimal concentration of 55 microM (10 micrograms/ml), myo-inositol approximately tripled keratinocyte yield compared to paired cultures in basal medium containing 0.3 microM, although this yield was only half that produced by a crude saline extract of hypothalamus, suggesting that there are additional growth-promoting activities in the tissue extract. No other skin-derived cell type tested was stimulated by supplemental inositol. These results establish that the inositol requirement for cultured human keratinocytes is markedly higher than for any other normal or malignant cell type investigated to date, and expand the list of brain-derived phospholipid precursors known to stimulate epithelial proliferation in vitro. These data suggest that inositol may subserve quantitatively or qualitatively different functions in the keratinocyte than in other cell types.