Nerve growth factor (NGF) stimulation of cholinergic telencephalic neurons in aggregating cell cultures.

The addition of nerve growth factor (2.5S NGF) to serum-free aggregating cell cultures of fetal rat telencephalon greatly stimulated the developmental increase in choline acetyltransferase activity. Two other neuronal enzymes, acetylcholinesterase and glutamic acid decarboxylase, showed only slightly increased activities after NGF treatment whereas the total protein content of the cultures and the activity of 2',3'- cyclic nucleotide phosphodiesterase remained unchanged. The stimulation of choline acetyltransferase was dependent on the NGF media concentrations, showing a 50% maximum effect (120% increase) at approximately 3 ng/ml (10-10 M 2.5S NGF). NGF treatments during different culture periods showed that the cholinergic neurons remained responsive for at least 19 days. The continued treatment was the most effective; however, an initial treatment for only 5 days still caused a significant stimulation of choline acetyltransferase on day 19. The observed stimulation appeared to be specific to NGF. Univalent antibody fragments (Fab) against 2.5S NGF completely abolished the NGF-dependent increase in choline acetyltransferase activity, whereas Fab fragments of control IgG were ineffective. Furthermore, angiotensin II, added in high amounts to the cultures, showed no stimulatory effect. The present results suggest that certain populations of rat brain neurons are responsive to nerve growth factor.

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways.

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione.

Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent.

Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum B90A.

Soil pollution with hexachlorocyclohexane (HCH) has caused serious environmental problems. Here we describe the targeted degradation of all HCH isomers by applying the aerobic bacterium Sphingobium indicum B90A. In particular, we examined possibilities for large-scale cultivation of strain B90A, tested immobilization, storage and inoculation procedures, and determined the survival and HCH-degradation activity of inoculated cells in soil. Optimal growth of strain B90A was achieved in glucose-containing mineral medium and up to 65% culturability could be maintained after 60 days storage at 30 degrees C by mixing cells with sterile dry corncob powder. B90A biomass produced in water supplemented with sugarcane molasses and immobilized on corncob powder retained 15-20% culturability after 30 days storage at 30 degrees C, whereas full culturability was maintained when cells were stored frozen at -20 degrees C. On the contrary, cells stored on corncob degraded gamma-HCH faster than those that had been stored frozen, with between 15 and 85% of gamma-HCH disappearance in microcosms within 20 h at 30 degrees C. Soil microcosm tests at 25 degrees C confirmed complete mineralization of [(14)C]-gamma-HCH by corncob-immobilized strain B90A. Experiments conducted in small pits and at an HCH-contaminated agricultural site resulted in between 85 and 95% HCH degradation by strain B90A applied via corncob, depending on the type of HCH isomer and even at residual HCH concentrations. Up to 20% of the inoculated B90A cells survived under field conditions after 8 days and could be traced among other soil microorganisms by a combination of natural antibiotic resistance properties, unique pigmentation and PCR amplification of the linA genes. Neither the addition of corncob nor of corncob immobilized B90A did measurably change the microbial community structure as determined by T-RFLP analysis. Overall, these results indicate that on-site aerobic bioremediation of HCH exploiting the biodegradation activity of S. indicum B90A cells stored on corncob powder is a promising technology.

Laboratory Evaluation of Asphalt-Rubber in Asphalt Paving Mixture, MLR-89-15, 1990

Interest in the use of ground rubber from used tires as a hot asphalt mix binder has been increasing due to the magnitude of the disposal problem posed by the annual addition of millions of waste tires to the refuse stream. This study evaluates, through laboratory means, the performance of asphalt-rubber as a hot mix binder as compared to conventional asphalt. The results indicate that asphalt-rubber outperforms its base asphalt in mixes of identical gradation and comparable void content on tests that are heavily dependent on binder characteristics (resilient modulus and indirect tension). An appreciable increase in rut resistance due to the use of asphalt-rubber is not indicated.

Geochemistry of the thermal springs and fumaroles of Basse-Terre Island, Guadeloupe, Lesser Antilles

The purpose of this work was to study jointly the volcanic-hydrothermal system of the high-risk volcano La Soufriere, in the southern part of Basse-Terre, and the geothermal area of Bouillante, on its western coast, to derive an all-embracing and coherent conceptual geochemical model that provides the necessary basis for adequate volcanic surveillance and further geothermal exploration. The active andesitic dome of La Soufriere has erupted eight times since 1660, most recently in 1976-1977. All these historic eruptions have been phreatic. High-salinity, Na-CI geothermal liquids circulate in the Bouillante geothermal reservoir, at temperatures close to 250 degrees C. These Na-CI solutions rise toward the surface, undergo boiling and mixing with groundwater and/or seawater, and feed most Na-CI thermal springs in the central Bouillante area. The Na-Cl thermal springs are surrounded by Na-HCO3 thermal springs and by the Na-Cl thermal spring of Anse a la Barque (a groundwater slightly mixed with seawater), which are all heated through conductive transfer. The two main fumarolic fields of La Soufriere area discharge vapors formed through boiling of hydrothermal aqueous solutions at temperatures of 190-215 degrees C below the ``Ty'' fault area and close to 260 degrees C below the dome summit. The boiling liquid producing the vapors of the Ty fault area has SD and delta(18)O values relatively similar to those of the Na-CI liquids of the Bouillante geothermal reservoir, whereas the liquid originating the vapors of the summit fumaroles is strongly enriched in O-18, due to input of magmatic fluids from below. This process is also responsible for the paucity of CH;I in the fumaroles. The thermal features around La Soufriere dome include: (a) Ca-SO4 springs, produced through absorption of hydrothermal vapors in shallow groundwaters; (b) conductively heated, Ca-Na-HCO3 springs; and (c) two Ca-Na-Cl springs produced through mixing of shallow Ca-SO4 waters and deep Na-Cl hydrothermal liquids. The geographical distribution of the different thermal features of La Soufriere area indicates the presence of: (a) a central zone dominated by the ascent of steam, which either discharges at the surface in the fumarolic fields or is absorbed in shallow groundwaters; and (b) an outer zone, where the shallow groundwaters are heated through conduction or addition of Na-Cl liquids coming from hydrothermal aquifer(s).

Improvement of cardiovascular risk prediction: time to review current knowledge, debates, and fundamentals on how to assess test characteristics.

Cardiovascular risk assessment might be improved with the addition of emerging, new tests derived from atherosclerosis imaging, laboratory tests or functional tests. This article reviews relative risk, odds ratios, receiver-operating curves, posttest risk calculations based on likelihood ratios, the net reclassification improvement and integrated discrimination. This serves to determine whether a new test has an added clinical value on top of conventional risk testing and how this can be verified statistically. Two clinically meaningful examples serve to illustrate novel approaches. This work serves as a review and basic work for the development of new guidelines on cardiovascular risk prediction, taking into account emerging tests, to be proposed by members of the 'Taskforce on Vascular Risk Prediction' under the auspices of the Working Group 'Swiss Atherosclerosis' of the Swiss Society of Cardiology in the future.

Fungicidal synergism of fluconazole and cyclosporine in Candida albicans is not dependent on multidrug efflux transporters encoded by the CDR1, CDR2, CaMDR1, and FLU1 genes.

The combination of fluconazole (FLC) and cyclosporine (CY) is fungicidal in FLC-susceptible C. albicans (O. Marchetti, P. Moreillon, M. P. Glauser, J. Bille, and D. Sanglard, Antimicrob. Agents Chemother. 44:2373-2381, 2000). The mechanism of this synergism is unknown. CY has several cellular targets including multidrug efflux transporters. The hypothesis that CY might inhibit FLC efflux was investigated by comparing the effect of FLC-CY in FLC-susceptible parent CAF2-1 (FLC MIC, 0.25 mg/liter) and in FLC-hypersusceptible mutant DSY1024 (FLC MIC, 0.03 mg/liter), in which the CDR1, CDR2, CaMDR1, and FLU1 transporter genes have been selectively deleted. We postulated that a loss of the fungicidal effect of FLC-CY in DSY1024 would confirm the roles of these efflux pumps. Time-kill curve studies showed a more potent fungistatic effect of FLC (P = 0.05 at 48 h with an inoculum of 10(3) CFU/ml) and a more rapid fungicidal effect of FLC-CY (P = 0.05 at 24 h with an inoculum of 10(3) CFU/ml) in the FLC-hypersusceptible mutant compared to those in the parent. Rats with experimental endocarditis were treated for 2 or 5 days with high-dose FLC, high-dose CY, or both drugs combined. FLC monotherapy for 5 days was more effective against the hypersusceptible mutant than against the parent. However, the addition of CY to FLC still conferred a therapeutic advantage in animals infected with mutant DSY1024, as indicated by better survival (P = 0.04 versus the results obtained with FLC) and sterilization of valves and kidneys after a very short (2-day) treatment (P = 0.009 and 0.002, respectively, versus the results obtained with FLC). Both in vitro and in vivo experiments consistently showed that the deletion of the four membrane transporters in DSY1024 did not result in loss of the fungicidal effect of FLC-CY. Yet, the accelerated killing in the mutant suggested a "dual-hit" mechanism involving FLC hypersusceptibility due to the efflux pump elimination and fungicidal activity conferred by CY. Thus, inhibition of multidrug efflux transporters encoded by CDR1, CDR2, CaMDR1, and FLU1 genes is not responsible for the fungicidal synergism of FLC-CY. Other cellular targets must be considered.

A LC-tandem MS assay for the simultaneous measurement of new antiretroviral agents: Raltegravir, maraviroc, darunavir, and etravirine.

Raltegravir (RAL), maraviroc (MVC), darunavir (DRV), and etravirine (ETV) are new antiretroviral agents with significant potential for drug interactions. This work describes a sensitive and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of plasma drug levels. Single-step extraction of RAL, MVC, DRV, ETV and RTV from plasma (100 microl) is performed by protein precipitation using 600 microl of acetonitrile, after the addition of 100 microl darunavir-d(9) (DRV-d(9)) at 1000 ng/ml in MeOH/H(2)O 50/50 as internal standard (I.S.). The mixture is vortexed, sonicated for 10 min, vortex-mixed again and centrifuged. An aliquot of supernatant (150 microl) is diluted 1:1 with a mixture of 20 mM ammonium acetate/MeOH 40/60 and 10 microl is injected onto a 2.1 x 50 mm Waters Atlantis-dC18 3 microm analytical column. Chromatographic separations are performed using a gradient program with 2 mM ammonium acetate containing 0.1% formic acid and acetonitrile with 0.1% formic acid. Analytes quantification is performed by electrospray ionisation-triple quadrupole mass spectrometry using the selected reaction monitoring detection in the positive mode. The method has been validated over the clinically relevant concentrations ranging from 12.5 to 5000 ng/ml, 2.5 to 1000 ng/ml, 25 to 10,000 ng/ml, 10 to 4000 ng/ml, and 5 to 2000 ng/ml for RAL, MRV, DRV, ETV and RTV, respectively. The extraction recovery for all antiretroviral drugs is always above 91%. The method is precise, with mean inter-day CV% within 5.1-9.8%, and accurate (range of inter-day deviation from nominal values -3.3 to +5.1%). In addition our method enables the simultaneous assessment of raltegravir-glucuronide. This is the first analytical method allowing the simultaneous assay of antiretroviral agents targeted to four different steps of HIV replication. The proposed method is suitable for the Therapeutic Drug Monitoring Service of these new regimen combinations administered as salvage therapy to patients having experienced treatment failure, and for whom exposure, tolerance and adherence assessments are critical.

Evaluation of Limestone Filler in Gravel Mixtures "Asphaltic Concrete", R-228, 1968

This study was designed to provide background information on asphaltic concrete mixtures peculiar to northwest Iowa. This background is necessary to provide the basis for future specifications. There were several projects let in 1967 involving l", 3/4" and 3/8" mixes of Type "B'' asphaltic concrete which specified in part, II Not less than 40% of the material passing the No. 200 sieve shall be pulverized limestone or mineral filler, but in no case shall the per cent of pulverized limestone or mineral filler passing the No. 200 sieve be less than 2%. No credit will be allowed for limestone in gravel - II Northwest Iowa has no suitable limestone or mineral filler locally available. As a result, this material has to be imported, raising the cost of the mix approximately twenty-five cents per ton. The purpose of this study, therefore, was designed to compare some original job mix samples with alternate mixes from the same local material, but without the addition of pulverized limestone or mineral filler. Since the filler from the crushed gravel does not have the same crushing characteristics or sieve analysis as the pulverized limestone or mineral filler, they could not be compared on an equal percentage basis. Therefore, the alternate mixes were made to conform to the following proposed specification, "No less than 40% of the material passing No. 200 sieve shall be pulverized limestone or mineral filler or a 100% crushed gravel, but in no case shall the per cent of pulverized limestone or mineral filler or a 100% crushed gravel passing the No. 200 sieve be less than 2%."

Carbonic anhydrase activity in primary sensory neurons. II. Influence of environmental factors on the phenotypic expression of the enzyme in dissociated cultures of chicken dorsal root ganglion cells.

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.

Laboratory Testing of SHRP SPS-2 P.C.C. Mixes, MLR-94-5, 1995

This research evaluated the concrete strength of two mixes which were used in the Polk County project NHS-500-1(3)--10-77 and were developed to meet a contract requirement of 900 psi third-point 28-day flexural strength. Two concrete mixes, the Proposed Mix and the Enhanced Mix, were tested for strength. Based on the experimental results, it was found that the addition of 50 lb of cementitious materials did not significantly increase concrete strength. The requirement of 900 psi 28-day third-point flexural strength (MOR-TPL) was not achieved by this amount of addition of cementitious materials.

Plant regeneration from protoplast of Brazilian citrus cultivars

A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

Association of major and minor ECG abnormalities with coronary heart disease events.

CONTEXT: In populations of older adults, prediction of coronary heart disease (CHD) events through traditional risk factors is less accurate than in middle-aged adults. Electrocardiographic (ECG) abnormalities are common in older adults and might be of value for CHD prediction. OBJECTIVE: To determine whether baseline ECG abnormalities or development of new and persistent ECG abnormalities are associated with increased CHD events. DESIGN, SETTING, AND PARTICIPANTS: A population-based study of 2192 white and black older adults aged 70 to 79 years from the Health, Aging, and Body Composition Study (Health ABC Study) without known cardiovascular disease. Adjudicated CHD events were collected over 8 years between 1997-1998 and 2006-2007. Baseline and 4-year ECG abnormalities were classified according to the Minnesota Code as major and minor. Using Cox proportional hazards regression models, the addition of ECG abnormalities to traditional risk factors were examined to predict CHD events. MAIN OUTCOME MEASURE: Adjudicated CHD events (acute myocardial infarction [MI], CHD death, and hospitalization for angina or coronary revascularization). RESULTS: At baseline, 276 participants (13%) had minor and 506 (23%) had major ECG abnormalities. During follow-up, 351 participants had CHD events (96 CHD deaths, 101 acute MIs, and 154 hospitalizations for angina or coronary revascularizations). Both baseline minor and major ECG abnormalities were associated with an increased risk of CHD after adjustment for traditional risk factors (17.2 per 1000 person-years among those with no abnormalities; 29.3 per 1000 person-years; hazard ratio [HR], 1.35; 95% CI, 1.02-1.81; for minor abnormalities; and 31.6 per 1000 person-years; HR, 1.51; 95% CI, 1.20-1.90; for major abnormalities). When ECG abnormalities were added to a model containing traditional risk factors alone, 13.6% of intermediate-risk participants with both major and minor ECG abnormalities were correctly reclassified (overall net reclassification improvement [NRI], 7.4%; 95% CI, 3.1%-19.0%; integrated discrimination improvement, 0.99%; 95% CI, 0.32%-2.15%). After 4 years, 208 participants had new and 416 had persistent abnormalities. Both new and persistent ECG abnormalities were associated with an increased risk of subsequent CHD events (HR, 2.01; 95% CI, 1.33-3.02; and HR, 1.66; 95% CI, 1.18-2.34; respectively). When added to the Framingham Risk Score, the NRI was not significant (5.7%; 95% CI, -0.4% to 11.8%). CONCLUSIONS: Major and minor ECG abnormalities among older adults were associated with an increased risk of CHD events. Depending on the model, adding ECG abnormalities was associated with improved risk prediction beyond traditional risk factors.