First Report of Leaf Spot of Dracaena reflexa Caused by Burkholderia gladioli Worldwide.

In December 2008 (austral summer), a new disease of Dracaena reflexa Lam. cv. Anita was observed in a postentry quarantine greenhouse near Auckland, New Zealand on plants imported from Costa Rica. Symptoms included rust-colored, water-soaked lesions with chlorotic margins approximately 5 by 10 mm. When the disease was first noticed, incidence approached 80%, but subsequent reduction in greenhouse temperature dramatically reduced symptom expression and lesions were only visible on some leaf tips. Bacteria consistently isolated from the lesions on King's medium B (KB) were cream-colored, shiny, and produced a yellow, diffusible, nonfluorescent pigment. All isolates were able to rot onion slices. On the basis of BIOLOG (Hayward, CA) carbon utilization profiles, isolates were initially identified as Burkholderia gladioli (Severini 1913) Yabuuchi et al. 1993 with a probability index of 100% and a similarity index of 0.85. For molecular identification, a near full-length sequence of the 16S rDNA gene was amplified from all isolates using primers fD2 and rP1 (1), obtaining a PCR product of approximately 1,500 bp. The nucleotide sequences were 100% identical to a number of B. gladioli GenBank entries, including Accession Nos. EF193645 and EF088209. To confirm pathogenicity, three isolates (two isolated prior to greenhouse temperature reduction and one after) were used. Three D. reflexa plants were inoculated per bacterial isolate by wounding three young fully expanded leaves on each plant (four wounds per leaf) and spraying the leaves with a bacterial suspension in sterile distilled water at 108 CFU/ml. At the same time, Gladiolus nanus plants were inoculated in a similar manner. Control plants (D. reflexa and G. nanus) were wounded and sprayed with sterile distilled water. All inoculated plants were covered with plastic bags to maintain humidity and placed in a growth chamber at 25°C. At 3 days, all inoculated plants began to show water soaking and reddish coloration around the inoculation sites, and by 7 days, the lesions had expanded to resemble natural infection. Bacteria isolated on KB from the leading edge of each lesion were morphologically identical to the initial isolates. No bacteria were recovered from the wound sites on the control plants. The 16S rDNA sequences of selected isolates from inoculated plants showed 100% identity to the sequences of the initial isolates, thereby fulfilling Koch's postulates. To our knowledge, this is the first report of B. gladioli causing leaf spot of D. reflexa in the world. Reference: (1) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

Hemostasis biomarkers and risk of sepsis: the REGARDS cohort.

Essentials Few studies have investigated the risk of sepsis by baseline hemostasis biomarkers measures. Baseline hemostasis biomarkers and risk of sepsis was examined using case-control study design. Increased fibrinogen, factor IX, and factor XI levels may be associated with risk of sepsis. Hemostasis biomarkers may provide a target for sepsis mitigation or prevention. SUMMARY: Background Sepsis is a major public health concern, responsible for more than 750 000 hospitalizations and 200 000 annual deaths in the USA. Few studies have investigated the association between baseline measurements of hemostasis biomarkers and the future risk of sepsis. Objective To determine whether hemostasis biomarkers levels measured at baseline in a cohort of community-dwelling participants are associated with the risk of future sepsis events. Methods We performed a nested case-control study within the Reasons for Geographic and Racial Differences in Stroke (REGARDS) cohort. We identified sepsis hospitalizations occurring over a 10-year period. There were 50 incident sepsis cases with baseline measurements of hemostasis (fibrinogen, factor VIII, FIX, FXI, protein C, and D-dimer). Using incidence density sampling, we matched the 50 sepsis cases with 200 controls by age, sex, and race. We used conditional logistic regression to evaluate the association between baseline hemostasis biomarkers and future sepsis events. Results Comparison of 50 sepsis cases with 200 non-sepsis controls showed that sepsis cases had lower education and income, were more likely to live in the stroke belt, had chronic lung disease, and had higher albumin level/creatinine level ratios (ACRs). Individuals with higher baseline fibrinogen levels (adjusted odds ratio [OR] per standard deviation: 1.40, 95% confidence interval [CI] 1.01-1.94), FIX levels ([OR] 1.46, 95% [CI] 1.03-2.07) and FXI levels ([OR]1.52, 95% [CI] 1.04-2.23) were more likely to experience a sepsis event. Conclusion Baseline fibrinogen, FIX and FXI levels are associated with future episodes of sepsis. Hemostasis biomarkers may provide targets for sepsis mitigation or prevention.

Pulmonary ferritin: differential effects of hyperoxic lung injury on subunit mRNA levels.

Ferritin is an iron storage protein that is regulated at the transcriptional and transcriptional levels, resulting in a complex mixture of tissue- and condition-specific isoforms. The protein shell of ferritin is composed of 24 subunits of two types (heavy or light), which are encoded by two distinct and independently regulated genes. In the present studies, the isoform profile for lung ferritin differed from other tissues (liver, spleen, and heart) as determined by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Lung ferritin was composed of equal amounts of heavy and light subunits. Differences in isoform profiles were the result of tissue-specific differential expression of the ferritin subunit genes as demonstrated by Northern blot analyses. Like heart ferritin, lung ferritin exhibited a low iron content that did not increase extensively in response to iron challenge, which contrasts with ferritins isolated from liver or spleen. When animals were exposed to hyperoxic conditions (95% oxygen for up to 60 h), ferritin heavy subunit mRNA levels did not markedly change at any of the investigated time points. In contrast, ferritin light subunit mRNA increased severalfold in response to hyperoxic exposure. Investigation of the cytoplasmic distribution of ferritin mRNA showed that a substantial portion was associated with the ribonucleoprotein (RNP) fraction of the cytosol, suggesting that a pool of untranslated ferritin mRNA exists in the lung. Upon hyperoxic insult, all ferritin light subunit mRNA pools (RNP, monosomal, polysomal) were elevated, although a specific shift from RNP to polysomal pools was not evident. Therefore, the increase in translatable ferritin mRNA in response to hyperoxia resulted from transcriptional rather than specific translational activation. The observed pattern of light chain-specific transcriptional induction of ferritin is consistent with the hypothesis that hyperoxic lung injury is at least partially iron mediated.

Nucleic acids. 16. Orally active derivatives of ara-cytidine.

Water-soluble derivatives of ara-cytidine (cytarabine, Cytosar) were prepared and tested for antitumor, immunosuppressive, and antiarthritic activities in animals after oral administration. The compounds tested included the 5'-palmitate, 5'-benzoate, and 5'-adamantoate esters of ara-cytidine, made water soluble by use of their hydrochloride salts of peptidyl derivatives, and two basic 5' esters (5'-nicotinoate and 5'-quinuclidinate) as their hydrochloride salts. Five of the compounds had antitumor activity superior to that found with ara-cytidine itself after oral administration in the L1210 leukemic mouse assay. One of these, 5'-adamantoyl-ara-cytidine hydrochloride, had antitumor activity after oral administration approaching that achieved with parenterally administered ara-cytidine.

Attenuation of oxidant-induced lung injury by 21-aminosteroids (lazaroids): correlation with the mRNA expression for E-selectin, P-selectin, ICAM-1, and VCAM-1.

We compared the effects of treatment with methylprednisolone or the 21-aminosteroids, U-74389 and U-74006F (Tirilizad mesylate), on hyperoxic lung injury and the associated expression of mRNA for several adhesion molecules in rats. Inhalation of > 95% oxygen for up to 72 hr in Sprague-Dawley rats produced a marked increase in lung weight and an accumulation of fluid in the thorax when compared with air-breathing controls. Hyperoxia also induced a marked neutrophil-rich influx of inflammatory cells into the bronchial lumen as measured by bronchoalveolar lavage. Neutrophil numbers in bronchoalveolar lavage fluid peaked after 60 hr of exposure to s 95% oxygen; this was associated with a marked upregulation of mRNA for the adhesion molecules P-selectin and E-selectin but not VCAM-1. mRNA for ICAM-1 was constitutively expressed at high levels in both air-breathing controls and in the lungs of rats exposed to high concentrations of oxygen. Pretreatment with the 21-aminosteroids reduced hyperoxic lung damage and improved survival times in animals exposed to > 95% oxygen. However, treatment with methylprednisolone significantly decreased survival times. Treatment with U-74389 did not significantly (p > 0.05) inhibit the BAL neutrophilia and did not significantly (p > 0.05) reduce hyperoxia-induced increases in mRNA expression for P-selectin and E-selectin. The inhibition of hyperoxic lung damage coupled with improved survival seen in treated animals suggests that 21-aminosteroids may provide valuable treatments for pulmonary disorders in which oxidant damage has been implicated.

Contribution of leukotriene B4 to airway inflammation and the effect of antagonists.

Inhalation of aerosols of ovalbumin in sensitized guinea pigs produced a marked, bronchoalveolar eosinophilia 24 hr after challenge. The lung eosinophilia was not prevented by the cyclooxygenase inhibitors, indomethacin or PAF antagonists (WEB-2086 and L-652731) but was inhibited by methylprednisolone, the 5-LO inhibitor, U-66858 and a series of structural analogs of LTB4, U-75302, U-77692, U-75485 and U-78489. The effectiveness of LTB4 antagonists but not PAF antagonists in vivo was consistent with in vitro studies in which LTB4 was shown to be far more chemotactic than PAF for guinea pig eosinophils. LTB4 elicited maximal directional migration of guinea pig eosinophils at concentrations from 10(-7) M to 10(-9) M while PAF showed no effect over the same concentration range. The structural analogs of LTB4 were shown to inhibit LTB4 induced chemotaxis of guinea pig eosinophils and produced a dose-related inhibition of binding of LTB4 to guinea pig eosinophil membranes. To add further proof to the hypothesis that LTB4 contributed to the antigen-induced lung eosinophilia we attempted to measure LTB4 release into BAL fluid immediately after and at various time points up to 24 hr after antigen inhalation. However, using a sensitive radioimmunoassay (detection limit 10 pg/ml) very low levels of LTB4 (24.9-67.9 pg/ml) or its metabolite, 20-OH LTB4 (24.9-98.2 pg/ml) were detected in BAL fluid and these levels did not increase significantly following antigen provocation. Inhalation of LTB4 aerosols in unsensitized Brown-Norway rats or inhalation of aerosols of ovalbumin in sensitized Brown-Norway rats also produced a marked "late-phase" eosinophil-rich influx of inflammatory cells into the lungs. The lung eosinophilia in the rat was prevented by two structurally unrelated leukotriene B4 (LTB4) antagonists, U-75302 and Ly255283. These data implicate LTB4 as a mediator of allergen-induced bronchopulmonary eosinophilia. Leukotriene B4 antagonists may provide leads for the development of compounds which inhibit the chronic airway inflammation associated with asthma in man.

Distinction of dicarboxylic aciduria due to medium-chain triglyceride feeding from that due to abnormal fatty acid oxidation and fasting in children.

Increased amounts of dicarboxylic acids are excreted in human urine under conditions of medium-chain triglyceride (MCT) feeding, abnormal fatty acid oxidation (FAO) and fasting. Criteria to distinguish dicarboxylic aciduria originating from MCT feeding and other conditions are needed in urinary organic acid profiling for detecting inborn errors of metabolism. Patterns of dicarboxylic aciduria in children under various conditions were compared. The relative amounts of medium-chain saturated dicarboxylic acids in urine are not reliable for identifying MCT-induced dicarboxylic aciduria. On the other hand, low ratios of unsaturated to saturated dicarboxylic acids (<0.1) and 3- hydroxydecenedioic to 3-hydroxydecanedioic acids were found to be useful in identifying dicarboxylic aciduria due to MCT ingestion. Additional unique features of dicarboxylic aciduria from MCT are low ratios of 3-hydroxydodecanedioic to 3-hydroxydecanedioic acid (<0.14) and 3-hydroxyadipic to adipic acid (<0.02).

The effects of nifedipine and verapamil on antigen-induced bronchoconstriction in dogs.

The effects of calcium channel blockers, nifedipine and verapamil (i.v. and aerosol), were investigated in beagle dogs natively allergic to Ascaris suum antigen. Control exposures to an aerosol of Ascaris antigen provoked significant bronchopulmonary changes, i.e., increases in pulmonary resistance (RL) and decreases in dynamic lung compliance (CDYN). Pretreatment with either nifedipine or verapamil (200 micrograms/kg, i.v.) provided significant inhibition in the RL responses to Ascaris antigen (P less than 0.015) while neither agent significantly affected CDYN changes. When administered as an aerosol, verapamil (1.0%; 10 breaths) significantly inhibited both the RL and CDYN responses to Ascaris antigen (P less than 0.05), whereas a similar concentration of nifedipine was without effect. Resting basal levels of RL and CDYN were unaffected by either the i.v. route or by aerosols of either nifedipine or verapamil. These results suggest that calcium channel blockers may have beneficial effects against allergen-provoked bronchoconstriction; however, differences appear to exist in the choice of agent, route of administration and site of action.

Bronchodilator effects of prostacyclin (PGI2) in dogs and guinea pigs.

prostacyclin (PGI2), a recently discovered unstable product in the biosynthetic conversion of prostaglandin endoperoxides, was examined for bronchopulmonary actions. in anesthetized dogs, PGI2 given i.v. (0.3-30.0 microgram/kg) and by aerosol (0.002-0.2%) inhibited significantly PGF2 alpha-induced increases in pulmonary resistance and decreases in dynamic lung compliance in a dose-related fashion. Intrinsically, PGI2 affected resting bronchopulmonary and cardiac functions minimally, but decreased peripheral and pulmonary vascular pressures. PGI2 (0.1-10 mg/kg, i.p.) afforded protection against histamine-induced asphyxial collapse in normal guinea pigs and ovalbumin-induced anaphylaxis in sensitized animals. Cumulative concentrations of PGI2 (1.0 x 10(-9)--3.0 x 10(-4) M) relaxed contractions of the isolated guine pig trachea produced by carbachol. These bronchodilator and hemodynamic effects could not be ascribed to the stable metabolic product of PGI2, because 6-keto-PGF1 alpha was inactive or markedly less active than PGI2 in these test systems. The results of this investigation suggest that PGI2 possesses considerable bronchodilator and vasodilator activity in experimental animal systems.

The effects of hydrocephalus upon the developing brain. Histological and quantitative studies of the ependyma and subependyma in hydrocephalic rats.

A suspension of kaolin was injected into the cisterna magna of 44 rats at 2 weeks of age. Animals killed at intervals from 5--19 weeks of age showed varying degrees of hydrocephalus. Light microscopy, scanning and transmission electron microscopy revealed stretching and flattening of the ependymal cells but no significant loss of cilia. Histological evidence of periventricular tissue damage in these chronically hydrocephalic animals was only present when the ventricular dilatation was extensive. A quantitative assessment was made of the ependymal and subependymal cell reactions around the lateral ventricles of the hydrocephalic animals. Although the ependymal cells were clearly stretched around the ventricles, there was no apparent proliferation of these cells. An increase in the total number of subependymal cells was observed in hydrocephalic animals when compared with a series of 39 aged-matched controls. The greatest proliferation was in the dorsal and lateral walls of the ventricles which were the regions most severely stretched by the ventricular dilatation. There is evidence that subependymal cells differentiate into astrocytes and microglia so that proliferation of these cells may be interpreted as a response to continuing and progressive brain damage in chronic hydrocephalus. Such progressive tissue damage could adversely affect the developing brain.

Lectin from Codium fragile ssp. tomentosoides conjugated to colloidal gold: a new histochemical reagent.

A new histochemical reagent has been developed utilising a lectin from a marine alga for the first time. Colloidal gold was coupled to the N-acetyl-alpha-D-galactosamine specific lectin from the green alga Codium fragile ssp. tomentosoides. The lectin--gold conjugate bound to the membranes of blood-group A1 human erythrocytes which were used as a model system. The bound complex could be detected, readily, by transmission electron microscopy. This novel reagent incorporating a lectin of low molecular weight (15 kDa) has potential value for studies of cell-surface topography of a variety of tissues.

On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field.

Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities.

Cerebral ventriculitis in the hydrocephalic mouse: a histological and scanning electron microscope study.

Staphylococci are the commonest organisms found in infected cerebrospinal fluid shunts in hydrocephalic patients. In the present study, staphylococcus albus was injected (1200-500,000 organisms in 10 microliters saline) into the ventricles of the hydrocephalic mouse SUMS - hy - 3/+ (temporary strain name). The bacteria appeared to have little direct effect upon the ependymal cells lining the ventricles and were not found as free organisms in the subependymal tissue. Staphylococci were phagocytosed by polymorphonuclear leukocytes which entered the ventricle from the choroid plexus and through the ependyma from subependymal vessels. Macrophages on the lining of the hydrocephalic ventricles also played a significant role in the phagocytosis of bacteria in the early stages of infection.

Potent bronchoconstrictor effects of aerosolized prostaglandin D2 in dogs.

Previously, we demonstrated that prostaglandin D2 (PGD2), a natural product of the endoperoxide PGH2, evoked bronchoconstriction when given I.V. to dogs (PROSTAGLANDINS 13:255-269, 1977). The present investigation in anesthetized dogs demonstrated that aerosols of PGD2 (0.001-0.1%) produced concentration-dependent increases in pulmonary resistance (RL) and decreases in dynamic lung compliance(CDYN) which were short-lived and equipotent to PGF2 alpha. These alterations in pulmonary mechanics were partially, yet significantly, inhibited by atropine, thereby suggesting that at least a portion of the bronchoconstriction may be cholinergically mediated. Concomitant cardiovascular depressant effects of both PGD2 and PFG2 alpha aerosols were much less and more variable than their bronchopulmonary effects. These results demonstrate a potent bronchoconstrictor effect of aerosolized PGD2 in dogs. PGD2 warrants further attention as a possible mediator of the bronchospasm seen in acute, reversible airways obstruction.

Inhibition of bronchoconstriction by aerosols of prostaglandins E1 and E2.

The proposal that some naturally occurring prostaglandins (PGs) or their by-products may be implicated in the pathogenesis of the asthmatic bronchospasm has been suggested. Other PGs may be potentially useful in the treatment of this lung disease. The present investigation compared the bronchodilator effects of PGE1 and PGE2 in pharmacologically constricted experimental animals. In pentobarbital-anesthetized, spontaneously breathing dogs, aerosols of PGE1 and PGE2, 0.0002 to 0.2%, effectively inhibited the increases in pulmonary resistance (RL) and decreases in dynamic lung compliance (CDYN) produced by PGF2 alpha (3.0 micrograms/kg i.v.). PGE2 was found to be more effective than PGE1 in preventing RL responses to PGF2 alpha; however, both bronchodilators were equally effective vs. CDYN changes. These agents inhibited central airway constriction more than peripheral. Transient decreases in systemic arterial pressure and increases in heart rate occurred especially at the higher concentrations. In a group of trained conscious dogs, effective concentrations did not evoke adverse subjective discomfort or irritation. Higher concentrations, i.e., 1.0%, did produce coughing, breathholding, restlessness and altered patterns of breathing. In normal or sensitized guinea pigs, PGE aerosols were effective in reducing the bronchopulmonary provocation produced by histamine or specific antigen. These in vivo results suggest that aerosols of the classical PGEs are effective bronchospasmolytics in laboratory animals and that irritation may be related to concentration.