Molecular tagging of a senescence gene by introgression mapping of a stay-green mutation from Festuca pratensis.

* Intergeneric hybrids between Lolium multiflorum and Festuca pratensis (Lm/Fp) and their derivatives exhibit a unique combination of genetic and cytogenetic characteristics: chromosomes undergo a high frequency of homoeologous recombination at meiosis; the chromosomes of the two species can easily be discriminated by genomic in situ hybridization (GISH); recombination occurs along the entire length of homoeologous bivalents; a high frequency of marker polymorphism is observed between the two species. * This combination of characters has been used to transfer and isolate a F. pratensis chromosome segment carrying a mutant 'stay-green' gene conferring a disrupted leaf senescence phenotype into L. multiflorum. * The genetic location within the introgressed F. pratensis segment of the senescence gene has been mapped using amplified fragment length polymorphisms (AFLPs), and F. pratensis-specific AFLP markers closely flanking the green gene have been cloned. * The use of these cloned sequences as markers for the stay-green locus in marker-assisted selection programmes has been tested. The potential application of Lm/Fp introgressions as a tool for the map-based cloning of introgressed Fp genes is discussed.

Interleukin-1 beta rapidly inhibits aortic endothelium-dependent relaxation by a DNA transcription-dependent mechanism.

OBJECTIVES: This study examined the effects of interleukin-1 beta on isometric tension development and relaxation in isolated rat aortic rings in response to the alpha-1 adrenergic agonist phenylephrine, the endothelium-dependent vasodilator acetylcholine, and the endothelium-independent vasodilator sodium nitroprusside. DESIGN: Randomized, controlled, paired design. SETTING: Animal laboratory within a university department of physiology. SUBJECTS Paired aortic thoracic aortic rings from specific pathogen-free Sprague-Dawley rats. INTERVENTIONS: Series I examined the potential for interleukin-1 beta to cause early arterial endothelial dysfunction. Paired aortic rings were incubated for 2 hrs with interleukin-1 beta or vehicle. Series II examined the potential for inhibition of DNA transcription to attenuate interleukin-1 beta-mediated endothelial dysfunction. Paired rings received either dactinomycin or vehicle before interleukin-1 beta incubation. Series III quantified the degree to which inhibition of DNA transcription inhibited early interleukin-1 beta-mediated endothelial dysfunction. Paired rings received either dactinomycin pretreatment followed by interleukin-1 beta incubation, or pretreatment and incubation with inert vehicles. Series IV assessed the effects of interleukin-1 beta on responsiveness to an exogenous nitric oxide donor, sodium nitroprusside, in the presence of the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester. MEASUREMENTS AND MAIN RESULTS: Incubation with interleukin-1 beta for 2 hrs had no effect on contractile response but attenuated endothelium-dependent relaxation significantly relative to control. Dactinomycin pretreatment inhibited early interleukin-1 beta-mediated endothelial dysfunction. The combination of interleukin-1 beta and dactinomycin produced effects on endothelium-dependent relaxation that were not different from that seen in rings not exposed to interleukin-1 beta. Interleukin-1 beta attenuated responsiveness to sodium nitroprusside relative to control. CONCLUSIONS: Interleukin-1 beta causes an early impairment of endothelium-dependent vasorelaxation with an onset that precedes its effects on systemic contractility. This impairment occurs via a mechanism that is wholly or predominantly dependent on DNA transcription. The altered vasorelaxation induced by interleukin-1 beta is at least partly mediated by a reduction in nitric oxide responsiveness.

Abatement of gas-condensate hydrocarbons in a natural wetland.

Results of a five-year research study on natural attenuation processes in a wetland, located downgradient of a sour gas processing plant in central Alberta, Canada, show that natural attenuation may present a favourable remedial solution. Both free-phase and dissolved phase condensate have been discharging to the wetland since 1984. This condensate is primarily composed of C5 to C12 hydrocarbons, including BTEX compounds. The condensate enters the base of the wetland at 1 m below ground surface, resulting in contamination of the wetland peat and underlying clay till. The lateral extent of contamination in the wetland has remained stable, and apparent free product thickness and BTEX concentrations have decreased over time. Sorption, aerobic biodegradation, volatilization, and anaerobic biodegradation were identified as active attenuation processes at this site. Sorption and desorption processes were evaluated by laboratory testing of site soils using 14C-benzene. Linear sorption coefficients (Kd) for the surface and subsurface peat were similar (4.48-4.62 l/kg), while the Kd for the underlying silt was 0.096 l/kg. The significantly higher Kd values for the peat are attributed to the peat's higher organic content (40%), relative to the clayey silt (1%). No significant resistance to desorption was observed, however, indicating that benzene would remain mobile and bioavailable over time. Aerobic biodegradation and volatilization appear to be the main removal processes. They are enhanced by a seasonal drop in the water level from surface down to 1 m depth, resulting in an aerobic unsaturated zone. Respiration testing in the peat indicates a significant aerobic biodegradation rate of 27 mg/kg/day, equating to an estimated hydrocarbon removal rate of 5 kg/day across the 3600 m2 plume area. Surface vapour measurements indicate hydrocarbon volatilization is occurring at a rate of 3 x 10(-4) kg/m2/day, equating to a mass removal of 1 kg/day across the plume. Anaerobic biodegradation is occurring primarily in the clayey silt, based on geochemical indicator parameters, microbial analyses, and soil vapour sampling. Overall, natural attenuation appears to be a feasible remedial solution for this wetland, by providing continued removal and degradation of condensate components before they reach the downgradient surface water receptor.

Airway narrowing associated with inhibition of deep inspiration during methacholine inhalation in asthmatics.

Reduced bronchodilatation in response to deep inspiration (DI) has been demonstrated in asthmatics. We have previously shown that inhibition of DI for 10 min or more during methacholine inhalation increases airway narrowing in normals. We tested the hypothesis that inhibition of DIs during methacholine inhalation in asthmatics would not affect the magnitude of airway narrowing. We administered the PC(15) dose of methacholine to eight asthmatics every 5 min for 5 doses and measured spirometry after each dose. On four separate days, subjects received either 2, 3, 4, or 5 doses selected randomly, but DIs were inhibited during the challenge and spirometry was measured only at the start and after the final dose. Geometric mean PC(15) was 1.6 mg/ml. Mean values for FEV(1) (+/- SEM) after Doses 2 through 5 were 84 +/- 4, 78 +/- 6, 79 +/- 5, and 81 +/- 3% of baseline, respectively, when DIs were allowed. During inhibition of DIs, they were 73 +/- 6, 67 +/- 5, 64 +/- 6, and 61 +/- 7% of baseline values. Decreases in FEV(1) after Doses 4 and 5 were significantly greater when DIs were inhibited (p < 0.05). We conclude that in this group of asthmatics, inhibition of DI for 15 min is associated with increased airway narrowing in response to methacholine inhalation, and therefore, DI may be an important factor limiting induced airway narrowing in asthmatics as well as in normal subjects.

Socioeconomic issues affecting the treatment of obesity in the new millennium.

The prevalence of obesity among the populations of most developed countries has increased to such an extent that the healthcare and social security/disability system will accumulate direct and indirect costs related to obesity that will be more substantial than those for any other primary disease within this generation. For the past decade, the Healthcare Financing Agency, which oversees the Medicare and Medicaid programmes, has required all physicians and healthcare agencies serving beneficiaries of these programmes to include diagnoses using codes established by the ninth revision of the World Health Organization's International Classification of Diseases. This coding system actually distorts data collection and undermines appropriate medical insurance reimbursement for the treatment of obesity. Societal prejudices, inability of governmental agencies to address future concerns and the business community's attempts to control healthcare costs without addressing the underlying issues contributing to these costs have led to confusion on how to confront this emerging epidemic. How will we develop the scientific knowledge and the political willpower to confront this epidemic? First, we need more accurate methods for classifying obesity and for measuring the cost of treatment. We can then determine if it is more cost effective to prevent or treat obesity early in its evolution or pay for its consequences in the form of treatment costs associated with its multiple comorbid diseases, such as hypertension, other cardiovascular disorders, diabetes mellitus, osteoarthritis and cancers, plus the lost productivity from absenteeism, premature retirement and death.

Time course of increased airway narrowing caused by inhibition of deep inspiration during methacholine challenge.

Inhibition of deep inspiration (DI) enhances methacholine-induced airway narrowing in normal subjects. However, the time course over which excessive airway narrowing develops during inhibition of DI is not known. We hypothesized that the development of enhanced airway narrowing when DI is inhibited is time dependent. Ten normal volunteers (five males and five females) inhaled five doses of methacholine (16 mg/ml for 2 min) at 5-min intervals during an initial methacholine challenge. FEV(1) was measured at baseline and after each dose. On four subsequent days, the subjects again inhaled two, three, four, or five doses, in random order, without DIs during the challenge. FEV(1) was measured only at baseline and after the last dose. Baseline FEV(1) was normal in all subjects. The maximal mean percent decrease in FEV(1) after the initial challenge was 10 +/- 1.5%, but was 28 +/- 6.0% when DIs were inhibited throughout the five inhalations (p < 0.01). The difference in decrease in FEV(1) between days with and without DI became significant after 10 min (three doses), and remained stable thereafter when the response plateaued. The reversal of airway narrowing after three DIs was incomplete after 15 min (four doses). In conclusion, the increased airway narrowing associated with inhibition of DI during airway smooth-muscle contraction occurs after 10 min in normal subjects, at which time the response plateaus. However, the ability of DI to reverse airway narrowing appears to diminish progressively.

Mechanism of methacholine dose-response plateaus in normal subjects.

Normal subjects develop plateaus on dose-response curves produced from inhalation challenge tests with bronchoconstricting agonists. These plateaus occur after only mild degrees of airway narrowing despite the fact that, if unloaded, maximally activated airway smooth muscle (ASM) should be able to cause airway closure. Plateaus may develop because, despite maximal activation, the muscle load provided by lung parenchymal recoil and tidal swings in airway transmural pressure are sufficient to prevent further ASM shortening. Alternatively, progressive ASM activation may occur throughout the plateau, but progressive hyperinflation and/or parenchymal stiffening could increase parenchymal load and attenuate further airway narrowing. In the first case, maximal ASM activation causes the plateau and in the second case the plateau is caused by progressive activation balanced by progressive loading. To test which of these mechanisms is responsible for the plateau, we measured pulmonary resistance (RL) and the maximal, minimal, and mean pulmonary elastic recoil pressure (PELmax, PELmin, and PELmean) during tidal breathing throughout methacholine challenge in 10 normal subjects. PELmean served as our measure of ASM afterload. Subjects swallowed an esophageal balloon and inhaled doubling concentrations of methacholine (1 to 256 mg/ml). RL was measured after each dose, as was PEL. All subjects developed a plateau on the dose-response curve defined by < 25% change in RL over three successive doses. During the RL plateau, there was no significant further increase in PELmean, i.e., PELmean also plateaued. These data are consistent with the hypothesis that maximal activation of ASM is balanced by an equal afterload at the maximal dose-response plateau. Airway hyperresponsiveness could result from a failure of afterload to attenuate muscle shortening after maximal activation.

Ferrets (Mustela putoius furo) inefficiently convert beta-carotene to vitamin A.

The ferret has recently been used as a model to evaluate the absorption and metabolism of several carotenoids; however, little is known about the vitamin A (VA) requirements of this species or the ability of ferrets to convert dietary beta-carotene (betaC) to VA. Three studies were conducted to estimate the daily utilization of VA in ferrets and to determine the effect of prior VA status on the ability of ferrets to utilize betaC as a source of VA. Weanling male ferrets were fed a pelleted, low carotenoid, semipurified diet either with (+VA) or without VA (-VA) for 21- to 35-d prefeeding periods. Upon initiation of the experiments, several ferrets were killed to determine base-line VA status. The remaining ferrets were fed VA, betaC, or VA and betaC in pelleted feed (Studies 1-3) or liquid carrier (Study 3) for 16-21 additional days. Hepatic VA and betaC concentrations were used as the primary indicators of VA status, although serum and adrenal VA and betaC also were measured. The results showed the following: 1) provision of betaC at up to a 15:1 weight ratio of betaC to VA in pelleted feed or liquid carrier was not sufficient to maintain hepatic VA stores after a -VA prefeeding period; 2) the daily utilization rate of VA by ferrets ranged from 80 to 171 microg in the three studies; 3) the ferret was confirmed to be a species that has the majority of its serum VA in ester form; and 4) feeding -VA diets significantly reduced serum retinyl esters but had less effect on serum retinol. We conclude that although ferrets can convert betaC to VA, the process is inefficient. The ferret model can be most appropriately used when studying the biological effect of tissue betaC stores on VA status and is less appropriate for the evaluation of dietary betaC conversion to VA.

The effect of deep inspiration on methacholine dose-response curves in normal subjects.

Normal subjects develop exaggerated airway narrowing when deep inspiration (DI) is voluntarily suppressed during methacholine challenge. Failure of periodic inflation may interfere with the bronchodilating effect of DI, and this may be fundamental to the difference in bronchodilation caused by DI in asthmatics and normal subjects. To determine whether repeated exhalations to residual volume (RV) and/or incomplete inspiration to baseline total lung capacity (TLC) could contribute to exaggerated narrowing during challenge, we tested 10 subjects on three separate days using modified methacholine challenge protocols. On Day 1, partial and complete flow volume curves were obtained after each dose. On Day 2, DI was prohibited, but partial curves were performed. On Day 3, DI and exhalation to RV were prohibited. TLC was measured pre- and post-challenge on each day. After comparable doses of methacholine, there was a greater change in FEV1 on Day 2 (27+/-15) and Day 3 (38+/-17) than on Day 1 (14+/-8) (p < 0.05). There were no differences in changes in FEV1 and FVC between Days 2 and 3, or in TLC between all 3 d. We conclude that exaggerated airway narrowing occurs in normal subjects when DI is prohibited and that this effect is not due to repeated expiration to RV, nor due to an artifact caused by a failure to inhale to TLC.

Integration of 101 DNA markers across human Xp11 using a panel of somatic cell hybrids.

One hundred and one DNA markers previously assigned to the short arm of the human X chromosome were localized on a hybrid mapping panel consisting of ten radiation-reduced, and four classical somatic cell hybrids. Of the 101 DNA markers, 16 are genes, two are pseudogenes, 13 are expressed sequence tags, 32 are simple tandem repeats (STRs), four are restriction fragment length polymorphisms, one is a variable number of tandem repeats, and 33 are sequence tagged sites (STSs). Three of these markers, two STSs and one STR, were generated from the products of an inter-Alu PCR library of a radiation-reduced hybrid containing Xp11.4-->p11.22 as its only human DNA content. A second STR was isolated from a region-specific cosmid containing the gene ZNF21. The 101 DNA markers fell into 22 bins based on their retention on the hybrids of this panel, which, in combination with YAC contig data, could be further resolved into 24 bins. This hybrid map of Xp11 has an average resolution of approximately 0.8 Mb.

cis-trans lycopene isomers, carotenoids, and retinol in the human prostate.

An evaluation of the Health Professionals Follow-Up Study has detected a lower prostate cancer risk associated with the greater consumption of tomatoes and related food products. Tomatoes are the primary dietary source of lycopene, a non-provitamin A carotenoid with potent antioxidant activity. Our goal was to define the concentrations of lycopene, other carotenoids, and retinol in paired benign and malignant prostate tissue from 25 men, ages 53 to 74, undergoing prostatectomy for localized prostate cancer. The concentrations of specific carotenoids in the benign and malignant prostate tissue from the same subject are highly correlated. Lycopene and all-trans beta-carotene are the predominant carotenoids observed, with means +/- SE of 0.80 +/- 0.08 nmol/g and 0.54 +/- 0.09, respectively. Lycopene concentrations range from 0 to 2.58 nmol/g, and all-trans beta-carotene concentrations range from 0.09 to 1.70 nmol/g. The 9-cis beta-carotene isomer, alpha-carotene, lutein, alpha-cryptoxanthin, zeaxanthin, and beta-cryptoxanthin are consistently detectable in prostate tissue. No significant correlations between the concentration of lycopene and the concentrations of any other carotenoid are observed. In contrast, strong correlations between prostate beta-carotene and alpha-carotene are noted (correlation coefficient, 0.88; P < 0.0001), as are correlations between several other carotenoid pairs, which reflects their similar dietary origins. Mean vitamin A concentration in the prostate is 1.52 nmol/g, with a range of 0.71 to 3.30 nmol/g. We further evaluated tomato-based food products, serum, and prostate tissue for the presence of geometric lycopene isomers using high-performance liquid chromatography with a polymeric C30 reversed phase column. All-trans lycopene accounts for 79 to 91% and cis lycopene isomers for 9 to 21% of total lycopene in tomatoes, tomato paste, and tomato soup. Lycopene concentrations in the serum of men range between 0.60 and 1.9 nmol/ml, with 27 to 42% all-trans lycopene and 58 to 73% cis-isomers distributed among 12 to 13 peaks, depending upon their chromatographic resolution. In striking contrast with foods, all-trans lycopene accounts for only 12 to 21% and cis isomers for 79 to 88% of total lycopene in benign or malignant prostate tissues. cis Isomers of lycopene within the prostate are distributed among 14 to 18 peaks. We conclude that a diverse array of carotenoids are found in the human prostate with significant intra-individual variation. The presence of lycopene in the prostate at concentrations that are biologically active in laboratory studies supports the hypothesis that lycopene may have direct effects within the prostate and contribute to the reduced prostate cancer risk associated with the reduced prostate cancer risk associated with the consumption of tomato-based foods. The future identification and characterization of geometric lycopene isomers may lead to the development of novel agents for chemoprevention studies.

Shape and position of the complete dose-response curve for inhaled methacholine in normal subjects.

Plateaus on the inhalation concentration-response curve have been described in normal subjects and patients with mild asthma. To determine the prevalence of plateaus on inhalation concentration-response curves, and the position of the curves in normal subjects, we measured complete dose-response curves for methacholine (1 mg/ml to 256 mg/ml) in 73 nonatopic, nonsmoking, nonasthmatic normal subjects between the ages of 20 and 76 yr. Measurements included FEV1, maximal expiratory flow at 50% and 30% of vital capacity on partial and complete forced expiratory flow-volume curves (Vmax50p, Vmax50c, Vmax30p, Vmax30c) and pulmonary resistance (RL). Plateau responses, EC50 values and slopes were measured. Plateaus were present in 25, 27, 24, 34, 35, and 16 subjects for FEV1, Vmax50c, Vmax30c, Vmax50p, Vmax30p, and RL, respectively. In those who achieved a plateau, the mean maximal decrease in FEV1 (+/- SD) was 21 +/- 8%, in Vmax50c it was 46 +/- 16%, in Vmax50p it was 67 +/- 12%, in Vmax30c it was 58 +/- 21%, and in Vmax30p it was 75 +/- 15%, and the increase in RL was 213 +/- 89%. In summary, the results of this study showed that easily identifiable plateaus develop on the inhalation concentration-response curves of approximately 40% of normal subjects after only moderate decreases in maximum flow and increases in RL. Maximal response at the plateau was greater on partial flow-volume curves and at lower lung volumes (30% versus 50% of VC). Comparison of these data with data from patients at risk for airway hyperresponsiveness will allow definition of the mechanisms leading to airway hyperresponsiveness.

Measurement of lung expansion with computed tomography and comparison with quantitative histology.

The total and regional lung volumes were estimated from computed tomography (CT), and the pleural pressure gradient was determined by using the milliliters of gas per gram of tissue estimated from the X-ray attenuation values and the pressure-volume curve of the lung. The data show that CT accurately estimated the volume of the resected lobe but overestimated its weight by 24 +/- 19%. The volume of gas per gram of tissue was less in the gravity-dependent regions due to a pleural pressure gradient of 0.24 +/- 0.08 cmH2O/cm of descent in the thorax. The proportion of tissue to air obtained with CT was similar to that obtained by quantitative histology. We conclude that the CT scan can be used to estimate total and regional lung volumes and that measurements of the proportions of tissue and air within the thorax by CT can be used in conjunction with quantitative histology to evaluate lung structure.

Donor selection limits use of living-related liver transplantation.

The purpose of this investigation was to assess the applicability of living-related liver transplantation in an established regional transplant program by determining the frequency of acceptable living donors from an unselected population of pediatric transplant candidates and identify specific factors limiting application of this technique. During the period May 1992 to May 1994, all children accepted as transplant candidates at the University of California-San Francisco were evaluated for potential living-related liver transplantation. Indications for transplantation and patient demographics represented the spectrum anticipated at a regional center. Donor evaluation was performed using a three-phase evaluation process we have previously reported. Retrospective analysis identified 75 potential donors for 38 pediatric candidates (age range, 17 days to 14.5 years; mean, 5.1 years). Twenty-three percent of potential donors declined evaluation. Of the 75 potential donors, only 10 (13%) were found to be acceptable for donation. The leading causes for donor declination were significant medical history (23%), ABO blood group incompatibility (23%), and psychosocial history (20%). Of the 38 recipient candidates, 9 (23%) were offered living-donor transplantation. Five patients have received living-donor transplantation, and 4 patients await the procedure when medical indications exist. Seventy-seven percent of recipient candidates received or are awaiting cadaveric transplantation. These results suggest that current donor criteria markedly limit the application of living-related liver transplantation.

Developmental changes in diaphragm contractile properties.

The contractile properties of pre- and early postnatal respiratory muscles are incompletely understood. We examined the effects of development on isometric contractile properties, with an emphasis on properties at 37 degrees C. One-day-old (n = 10), 3-wk-old (n = 10), and adult (n = 10) rabbits were studied. Isometric contractile properties of costal diaphragm strips were measured in vitro by using direct stimulation. Twitch and maximal, i.e., fused, tetanic force production increased with strip dimension and with age. Maximal tetanic force developed per unit cross-sectional area (stress) was significantly decreased in muscle from 1-day olds, whereas it was greatest in muscle from 3-wk olds. Twitch stress was similar in all three groups. Only when the stimulus duration was prolonged did twitch and fused tetanic force achieve maximal values values for the 1-day-old and 3-wk-old strips, suggesting less effective excitation-contraction coupling in those muscles. We conclude that immature rabbit diaphragm has unique isometric contractile properties and stimulus parameter requirements that cannot be deduced from studies using mature diaphragm.

Epithelial reorganization and irregular growth following carbendazim-induced injury of the efferent ductules of the rat testis.

Occlusions of the rat efferent ductules were induced by an oral gavage of carbendazim and the capacity for regeneration or recanalization of the ductules was histologically evaluated between 2 and 70 days post-treatment. At 2 days post-treatment, the efferent ductules were occluded by sloughed materials and showed various degrees of inflammation. Severely damaged epithelium showed few regenerative features at later intervals. On the other hand, epithelia with medium inflammation often exhibited irregular epithelial growth along the luminal contents or the formation of multiple abnormal ductules. These abnormal ductules were formed by migrated and original epithelia at the periphery of the occluded lumen at 16 days post-treatment, indicating attempted recanalization. At later time periods, 32 and 70 days post-treatment, the occluded original lumen was filled in by fibrotic connective tissue and surrounded by a series of abnormal ductules. These abnormal ductules were characterized by cuboidal epithelia, a small luminal diameter, fewer cilitated cells than normal, less developed organelles in the epithelial cells, and basal laminae of irregular thickness. However, there was no evidence that occluded ductules formed patent re-connections via abnormal ductules. The results suggest that occluded efferent ductules have the ability to initiate epithelial regrowth and to form new ductules, but the newly formed ductules are abnormal and are not adequate to recover from azoospermia at least at 70 days post-treatment.