Elevated expression of adenosine A1 receptor in bronchial biopsy specimens from asthmatic subjects.

Asthmatics, unlike healthy subjects, experience bronchoconstriction in response to inhaled adenosine, and extracellular adenosine concentrations are elevated in the bronchoalveolar lavage fluid and exhaled breath condensate of asthmatic subjects. However, little is known about the location and expression of adenosine receptors in asthmatic airways. The aim of the present study was to investigate the distribution of adenosine A(1) receptors in bronchial biopsy specimens from mildly asthmatic steroid-naïve subjects and then compare the degree of expression with that of healthy subjects. Biopsy sections were immunostained using an adenosine A(1) receptor antibody, the selectivity of which was validated in specific experiments. Image analysis was then performed in order to determine differences in immunostaining intensity. Immunostaining of biopsy sections from the asthmatic subjects revealed strong expression of the A(1) receptor, located predominantly in the bronchial epithelium and bronchial smooth muscle. In comparison, very weak immunostaining was observed in biopsy specimens obtained from healthy subjects. Image analysis revealed that the intensity of positive staining of the asthmatic bronchial epithelium and smooth muscle regions was significantly greater than that observed for the healthy epithelium and smooth muscle. In conclusion, the sensitivity of asthmatics to inhaled adenosine coupled with increased adenosine A(1) receptor expression implies that these receptors play a role in the pathophysiology of this disease.

Airway subepithelial fibrosis in a murine model of atopic asthma: suppression by dexamethasone or anti-interleukin-5 antibody.

Fibrosis in the reticular layer beneath the epithelial basement membrane is a feature of airway remodeling in human asthma. We previously reported the presence of subepithelial fibrosis (SEF) in a disease model of atopic asthma in which mice were sensitized and intratracheally challenged with ovalbumin (OVA) (Blyth and colleagues, Am. J. Respir. Cell Mol. Biol. 1996;14:425-438). Here, we describe further studies to quantify the degree of SEF after its induction by repeated exposure of the airways to allergen. The amount of subepithelial reticulin in the airways of animals challenged three times with 80 microg OVA was typically increased 1. 4-fold. The increased amount of reticulin showed no reduction after a 50-d period after the third allergen challenge. A reduction in SEF was achieved by daily treatment with dexamethasone (DEX) for 8 d during the allergen challenge period, or by treatment with anti-interleukin-5 antibody (TRFK5) at the time of allergen challenge. Postchallenge treatment with DEX for 15 d resulted in significant resolution of previously established SEF. Severe nonallergic inflammation during repeated exposure of airways to lipopolysaccharide did not induce SEF. The results indicate that development of SEF is associated with eosinophil infiltration into airways, and may occur only when the inflammatory stimulus is allergic in nature.

Monoterpene synthases of loblolly pine (Pinus taeda) produce pinene isomers and enantiomers.

The turpentine fraction of conifer oleoresin is a complex mixture of monoterpene olefins and plays important roles in defense and in the mediation of chemical communication between conifer hosts and insect predators. The stereochemistry of the turpentine monoterpenes is critical in these interactions, influencing host recognition, toxicity, and potency of derived pheromones, and the stereochemical composition of these compounds lends insight into their biogenetic origin, with implications for the numbers and types of enzymes responsible and their corresponding genes. Analysis of the oleoresin from several tissues of loblolly pine (Pinus taeda) showed the derived turpentine to consist mainly of (+)-(3R:5R)-alpha-pinene and (-)-(3S:5S)-beta-pinene. Cell-free extracts from xylem tissue yielded three monoterpene synthases which together account for the monoterpene isomer and enantiomer content of the turpentine of this tissue. The major products of these enzymes, produced from the universal precursor of monoterpenes, geranyl diphosphate, were shown to be (+)-alpha-pinene, (-)-alpha-pinene, and (-)-beta-pinene, respectively. In most properties (molecular mass of approximately 60 kDa, K(m) for geranyl diphosphate of 3 microM, requirement for monovalent and divalent cations), these enzymes resemble other monoterpene synthases from conifer species.

Induction, duration, and resolution of airway goblet cell hyperplasia in a murine model of atopic asthma: effect of concurrent infection with respiratory syncytial virus and response to dexamethasone.

We recently described a murine model of atopic asthma in which a marked, extensive hyperplasia of airway goblet cells is induced by repeated challenge of ovalbumin (OA)-sensitized mice with intratracheally administered allergen (Am. J. Respir. Cell Mol. Biol. 1996;14:425-438). We report here the time course of the duration of this feature and of its spontaneous resolution in the absence of further allergen exposure. Induction of severe neutrophilic inflammation in the airways by repeated intratracheal administration of lipopolysaccharide failed to induce goblet cell hyperplasia (GCH) to as great a degree as that induced by allergen, suggesting that nonallergic inflammation is a relatively poor inducer of this phenotype change in mice. When a "subclinical" infection of the lungs with the human A2 strain of respiratory syncytial virus was superimposed on the model of atopic asthma, recruitment of monocytes and lymphocytes to the airways was enhanced and a discharge of goblet cell mucin contents was observed. This may partly explain the respiratory difficulty that typifies virally induced exacerbations of asthma in humans. Daily systemic treatment of sensitized mice with dexamethasone during the period of allergen challenge produced a dose-related suppression of developing GCH, while similar treatment during the period following the establishment of extensive hyperplasia induced an accelerated resolution toward a normal epithelial phenotype. These results confirm and extend the relevance of this model as a representation of the human disease.

Monoterpene synthases from common sage (Salvia officinalis). cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase.

Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of the p-menthane (1,8-cineole), pinane (alpha- and beta-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli. The heterologously expressed synthases produce (+)-bornyl diphosphate, 1, 8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-alpha-pinene, (+)-camphene, and (+/-)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (-)-alpha-pinene, (+)- and (-)-beta-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of gamma-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.

Evidence for an Elongation/Reduction/C1-Elimination Pathway in the Biosynthesis of n-Heptane in Xylem of Jeffrey Pine.

The biosynthetic pathway to n-heptane was investigated by examining the effect of the [beta]-keto acyl-acyl carrier protein synthase inhibitor (2R,3S)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide (cerulenin), a thiol reagent ([beta]-mercaptoethanol), and an aldehydetrapping reagent (hydroxylamine) on the biosynthesis of n-[14C]heptane and putative intermediates in xylem sections of Jeffrey pine (Pinus jeffreyi Grev.& Balf.) incubated with [14C]acetate. Cerulenin inhibited C18 fatty acid biosynthesis but had relatively little effect on radiolabel incorporation into C8 fatty acyl groups and n-heptane. [beta]-Mercaptoethanol inhibited n-heptane biosynthesis, with a corresponding accumulation of radiolabel into both octanal and 1-octanol, whereas hydroxylamine inhibited both n-heptane and 1-octanol biosynthesis, with radiolabel accumulation in octyl oximes. [14C]Octanal was converted to both n-heptane and 1-octanol when incubated with xylem sections, whereas [14C]1-octanol was converted to octanal and n-heptane in a hydroxylamine-sensitive reaction. These results suggest a pathway for the biosynthesis of n-heptane whereby acetate is polymerized via a typical fatty acid synthase reaction sequence to yield a C8 thioester, which subsequently undergoes a two-electron reduction to generate a free thiol and octanal, the latter of which alternately undergoes an additional, reversible reduction to form 1-octanol or loss of C1 to generate n-heptane.

Lung inflammation and epithelial changes in a murine model of atopic asthma.

A murine model of allergen-induced airway inflammation and epithelial phenotypic change, and the time-courses of these events, are described. Mice were sensitized to ovalbumin using an adjuvant-free protocol, and challenged by multiple intratracheal instillations of ovalbumin by a non-surgical technique. Many of the characteristic features of human atopic asthma were seen in the mice. A marked eosinophilic infiltration of lung tissue and airways followed allergen challenge, and its severity increased with each challenge, as did the number of eosinophils in the blood. Lymphocytes, neutrophils, and monocytes also invaded the lungs. Airway macrophages showed signs of activation, their appearance resembling those recovered from antigen-challenged human asthmatic airways. The airway epithelium was thickened and displayed a marked goblet cell hyperplasia in terminal bronchioles and larger airways. After repeated challenges, the reticular layer beneath the basement membrane of the airway epithelium showed fibrosis, reproducing a commonly observed histologic feature of human asthma. Goblet cell hyperplasia began to appear before eosinophils or lymphocytes had migrated across the airway epithelium, and persisted for at least 11 days after the third intratracheal challenge with ovalbumin, despite the number of inflammatory cells in the lungs and airways having decreased to near-normal levels by 4 days. Plugs of mucus occluded some of the airways. These results indicate that some of the phenotypic changes in airway epithelium that follow an allergic response in the lung can be initiated before the migration of eosinophils or lymphocytes across the epithelial layer.

Biochemistry of Short-Chain Alkanes (Tissue-Specific Biosynthesis of n-Heptane in Pinus jeffreyi).

Short-chain (C7-C11) alkanes accumulate as the volatile component of oleoresin (pitch) in several pine species native to western North America. To establish the tissue most amenable for use in detailed studies of short-chain alkane biosynthesis, we examined the tissue specificity of alkane accumulation and biosynthesis in Pinus jeffreyi Grev. & Balf. Short-chain alkane accumulation was highly tissue specific in both 2-year-old saplings and mature trees; heart-wood xylem accumulated alkanes up to 7.1 mg g-1 dry weight, whereas needles and other young green tissue contained oleoresin with monoterpenoid, rather than paraffinic, volatiles. These tissue-specific differences in oleoresin composition appear to be a result of tissue-specific rates of alkane and monoterpene biosynthesis; incubation of xylem tissue with [14C]sucrose resulted in accumulation of radiolabel in alkanes but not monoterpenes, whereas incubation of foliar tissue with 14CO2 resulted in the accumulation of radiolabel in monoterpenes but not alkanes. Furthermore, incubation of xylem sections with [14C]acetate resulted in incorporation of radiolabel into alkanes at rates up to 1.7 nmol h-1 g-1 fresh weight, a rate that exceeds most biosynthetic rates reported with other plant systems for the incorporation of this basic precursor into natural products. This suggests that P. jeffreyi may provide a suitable model for elucidating the enzymology and molecular biology of short-chain alkane biosynthesis.

Internal fixation of distal fibula fractures: a case presentation demonstrating a unique technique for a severely comminuted fibula.

The laterally comminuted fracture-dislocation of the ankle can be associated with devastating consequences. Previously described surgical as well as nonsurgical-treatment results have been disappointing. Accurate anatomical reduction and rigid fracture stabilization of a comminuted fibula can be extremely difficult. This manuscript presents some of the more common methods of comminuted fibular fracture fixation described in the literature. A case report demonstrates successful anatomical stabilization of a comminuted fibula, utilizing a method for internal fibular fixation which has been previously employed, but has not been advocated, in the literature. Clinical and radiographic results at 12 and 20 months post-injury are promising.

The internal fixation of ankle fracture repair.

A descriptive overview of the type of internal fixation, the biomechanical principles of this fixation technique, and the methods of application are outlined. Clinical illustrations demonstrate some of the more commonly used internal fixation techniques.

Monoterpene synthases from gymnosperms and angiosperms: stereospecificity and inactivation by cysteinyl- and arginyl-directed modifying reagents.

To further define specific structural and mechanistic differences among monoterpene synthases from divergent plant sources, the stereospecificity of the enzyme-catalyzed isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization to monoterpene olefins (which have been well established for monoterpene synthases from herbaceous angiosperms) were examined for monoterpene synthases from a conifer, lodgepole pine (Pinus contorta). The chiral monoterpenes isolated from lodgepole pine oleoresin and the major chiral products from cell-free assays of each of the four lodgepole pine monoterpene synthases belonged to the stereochemical family related by the biosynthetic intermediacy of 3S-linalyl pyrophosphate. Furthermore, both the putative intermediate, 3S-linalyl pyrophosphate, and the natural substrate, geranyl pyrophosphate, were enzymatically converted to the same monoterpene enantiomers. Thus, like monoterpene synthases from herbaceous angiosperms, monoterpene synthases from lodgepole pine appear to catalyze both the stereospecific isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound intermediate to multiple, stereochemically related monoterpene olefin isomers. The susceptibility of monoterpene synthases to inactivation by cysteinyl- and arginyl-directed chemical modification reagents was also examined to identify specific structural differences between enzymes from conifers and angiosperms. Like monoterpene synthases from peppermint (Mentha x piperita) and culinary sage (Salvia officinalis), monoterpene synthases from lodgepole pine were inactivated by thiol-directed reagents; however, unlike monoterpene synthases from these herbaceous angiosperms, monoterpene synthases from lodgepole pine were not protected against inactivation by coincubation with substrate and metal ion cofactor. Lodgepole pine monoterpene synthases were also inactivated by the arginyl-directed reagent phenylglyoxal, and coincubation with substrate and cofactor, to effect active-site protection, reduced the rate of inactivation 10-fold. (+)-Pinene synthase and (-)-pinene synthase from sage were also inactivated by phenylglyoxal, but no protection was afforded by coincubation with substrate and cofactor. Thus, monoterpene synthases of conifers appear to have catalytically important arginyl residues specifically located at or near the active site and have at least some catalytically important thiol residues at a non-substrate-protectable region of the enzyme, in contrast to monoterpene synthases from angiosperms which appear to have catalytically important cysteinyl residues at the active site and have catalytically important arginyl residues located at a non-substrate-protectable region of the enzyme.

Monoterpene synthases of Pinus contorta and related conifers. A new class of terpenoid cyclase.

A cell-free extract from the xylem of lodgepole pine (Pinus contorta) catalyzes the conversion of [1-3H1]geranyl pyrophosphate to a variety of monoterpene olefins found in lodgepole pine oleoresin. This monoterpene synthase activity is similar to previously described terpenoid cyclases from grand fir (Abies grandis) and other higher plants in molecular mass (67 +/- 2 kDa as estimated by size-exclusion chromatography), Km for geranyl pyrophosphate (7.8 +/- 1.9 microM), and isoelectric point (4.75 +/- 0.2 as determined by isoelectric focusing), but the cyclases from both lodgepole pine and grand fir are unlike previously characterized terpenoid cyclases from angiosperms and fungi, in that they have an alkaline pH optimum (pH 7.8), are activated by K+, Rb+, Cs+, or NH+4 (Li+ and Na+ are not effective), require either Mn2+ or Fe2+ as divalent metal ion cofactors (Mg2+ is not effective), and are not protected by the substrate-metal ion complex against inhibition by the histidine-directed reagent diethyl pyrocarbonate. Chromatography of the pine xylem extracts on a quaternary amino anion-exchange resin results in the separation of four similar, but distinct, multiple product monoterpene synthases that produce sabinene, beta-phellandrene, 3-carene, and beta-pinene as the principal components, respectively. The major cyclase (phellandrene synthase) was subsequently purified by hydroxyapatite chromatography and electrophoresis. V8 proteolysis provided a peptide map significantly different from that obtained with limonene synthase from spearmint (Mentha spicata), and limited NH2-terminal sequencing of the phellandrene synthase fragments revealed no significant similarity to the deduced amino acid sequence of the angiosperm limonene synthase, the only monoterpene cyclase to be cloned and sequenced thus far. Furthermore, polyclonal antibodies raised against the angiosperm limonene synthase did not detectably cross-react with any proteins in extracts from either lodgepole pine or grand fir by immunoblotting analysis. In addition to these structural differences between cyclases from conifers and herbaceous angiosperms, the unusual pH optimum, mono- and divalent metal ion requirement, and reactivity toward histidine carbethoxylation indicate that monoterpene cyclases isolated from conifers may also have a different complement of active-site amino acid residues involved in substrate binding and catalysis than those of terpenoid cyclases previously isolated from angiosperms.

Biosynthesis of monoterpenes: regio- and stereochemistry of (+)-3-carene biosynthesis.

Incubation of [1-3H1]geraniol with stem disks of Douglas fir (Pseudotsuga menziesii) and incubation of [1-3H1]geranyl pyrophosphate with both a soluble enzyme extract from Douglas fir and a partially purified preparation of (+)-3-carene synthase from lodgepole pine (Pinus contorta) resulted in the production of (+)-3-[3H] carene. Subsequent conversion of the product to car-3-en-5-one and to 4-isocaranone followed by base-catalyzed exchange of the alpha-hydrogens established that the 3H located at C1 in the geranyl substrate resided at C5 of (+)-3-carene. Incubation of the (+)-3-carene synthase preparation with (S)-[5-3H1, 4-14C]geranyl pyrophosphate resulted in the production of (+)-3-carene without loss of tritium, indicating that the 5-proR hydrogen is eliminated during cyclopropyl ring closure. Analysis of the conformational requirements for this 1,3 elimination involving the 5-proR hydrogen suggested that cyclopropyl ring formation occurs via a (4S)-alpha-terpinyl cation derived from the anti-endo cyclization of a (3S)-linalyl pyrophosphate intermediate. Kinetic analyses of the conversion of (1Z,3R)-[1-3H1]linalyl pyrophosphate, (1Z, 3S)-[1-3H1]linalyl pyrophosphate and [1-3H1]geranyl pyrophosphate by (+)-3-carene synthase revealed that the velocity of the reaction with the (3S)-linalyl enantiomer was 25-fold greater than the velocity with the (3R)-enantiomer and twice that of the natural substrate, geranyl pyrophosphate, thereby confirming this stereochemical prediction and also indicating that the cyclization of the linalyl intermediate is faster than the coupled isomerization and cyclization of the geranyl substrate. From these results, a model that details the regio- and stereochemistry of the enzymatic conversion of geranyl pyrophosphate to (+)-3-carene is proposed.

Defense mechanisms of conifers : relationship of monoterpene cyclase activity to anatomical specialization and oleoresin monoterpene content.

Cell-free extracts from Pinus ponderosa Lawson (ponderosa pine) and Pinus sylvestris L. (Scotch pine) wood exhibited high levels of monoterpene synthase (cyclase) activity, whereas bark extracts of these species contained no detectable activity, and they inhibited cyclase activity when added to extracts from wood, unless polyvinylpyrrolidone was included in the preparation. The molecular mass of the polyvinylpyrrolidone added was of little consequence; however, polyvinylpolypyrrolidone (a cross-linked insoluble form of the polymer) was ineffective in protecting enzyme activity. Based on these observations, methods were developed for the efficient extraction and assay of monoterpene cyclase activity from conifer stem (wood and bark) tissue. The level of monoterpene cyclase activity for a given conifer species was shown to correlate closely with the monoterpene content of the oleoresin and with the degree of anatomical complexity of the specialized resin-secreting structures. Cyclase activity and monoterpene content were lowest in the stems of species containing only isolated resin cells, such as western red cedar (Thuja plicata D. Don). Increasing levels of cyclase activity and oleoresin monoterpenes were observed in advancing from species with multicellular resin blisters (true firs [Abies]) to those with organized resin passages, such as western larch (Larix occidentalis Nutt.), Colorado blue spruce (Picea pungens Engelm.) and Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco). The highest levels of cyclase activity and oleoresin monoterpenes were noted in Pinus species that contain the most highly developed resin duct systems. The relationship between biosynthetic capacity, as measured by cyclase activity, monoterpene content, and the degree of organization of the secretory structures for a given species, may reflect the total number of specialized resin-producing cells per unit mass of stem tissue.

Navigating the system governance maze.

Health care system governance today is a complex maze of concerns that assume a unique character in Catholic-sponsored multi-institutional systems. Most Catholic health care systems began with a common sponsor or mission and several shared services and gradually moved from separately incorporated entities to a system with few centralized operating functions but a governing body between the local facilities and the sponsoring religious institute. The next step was development of a managed system with consolidated services and centralized decision making. Now, many systems are attempting the most important and difficult effort--systemwide strategic planning. The phases described have required a rethinking of governance structures, and conflicts often arise during restructuring. Such turmoil requires many Catholic health care systems to develop a clearer sense of direction and purpose. To achieve their objectives, system leaders can use a governance compass that has five key points: Information. Boards must determine what they need to know, where to secure this information, and what form the information should take. Agenda. Boards must make reflective and intentional use of their agenda by reviewing and categorizing agenda items discussed in the past 12 months and establishing an agenda plan for the next 12 months. Structural mechanisms. Boards must decide structural issues such as relationships between system board and local boards, sizes of boards, and kinds of committees needed. Culture. Boards should reflect on their culture--values and traditions that have characterized them in the past--to assess whether changes are needed to strengthen or improve the culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Ketoconazole in experimental vaginal candidosis in rats.

Rats in pseudoestrous were treated subcutaneously with ketoconazole at 25 mg/kg twice a day. In both uninfected and infected rats ketoconazole inhibited the cornification of the vaginal epithelium. Thus, ketoconazole, in addition to having an antifungal effect, may aid in the removal of candida by inhibiting the epithelial conditions suitable for hyphal invasion.

A morphological study of experimental rabbit staphylococcal endocarditis and aortitis. I. Formation and effect of infected and uninfected vegetations on the aorta.

In this study the development of sterile thrombic vegetations on the aorta resulting from catheterization and the effect of subsequent infection with Staphylococcus aureus were examined by light and electron microscopy. Thrombi of various sizes, comprising fibrin, platelets and a few leucocytes and erythrocytes, develop on the damaged surface of the aorta with minimal changes in the underlying aortic wall. After intravenous inoculation of Staph. aureus most vegetations become infected, as shown by the presence of bacterial colonies, and the underlying aortic wall is markedly inflamed. The inflammatory cells invade the wall from the base of the aorta and cause swelling plus disruption of the elastic laminae with ulceration of the luminal surface in some cases. This structural damage appears to be a direct result of the bacterial infection of the lesions on the luminal surface.

Trusteeship: salvaging the myth of the governance role.

A myth of trusteeship--that governance is the central role of board members--has been difficult for trustees to put into practice. The process of initiating new trustees implicitly communicates a set of largely unspoken practices that do not fulfill the myth. In actual practice, trustees find themselves involved in management, rather than governance. Although regulatory bodies, the courts, or a constituent can clarify the job of governance, trustees must call one another to action to reshape their practice and refurbish the myth with credibility. Trustees generally are expected to ensure quality of service and performance standards through governance, although many question their competence to do so. Since a direct link exists between a board's ability to evaluate itself and its ability to evaluate others, members first must learn to evaluate themselves in an open, verifiable manner. Trustees also must take account of the cyclical emphasis society places on private interests versus public issues, noting shifts in the cycle to identify and define board responsibilities. Members should act not as representatives of a single group but keep in touch with as many constituencies as possible. Though models are being developed to evaluate board performance and to chart management/governance functions, the most effective change in practice will come from within boards--through members' study and reflection. A board retreat that allows trustees to share information and examine principles of practice may facilitate this role definition.