Molecular and histochemical characterisation of two distinct poplar Melampsora leaf rust pathosystems.

In this study, we compared interactions of two Melampsora foliar rust species with poplar, which resulted in either limited or abundant pathogen proliferation. In the pathosystem exhibiting limited pathogen growth, a defence response was observed after invasion of poplar leaf tissues by the biotroph, with late and clear production of reactive oxygen species (ROS) and other products. Characterisation of the histological, biochemical and transcriptional events occurring in both pathosystems showed striking similarity with components of plant defence reactions observed during qualitative resistance. Key components associated with development of an active defence response, such as up-regulation of pathogenesis-related (PR) genes, were observed during infection. Moreover, the time course and strength of gene induction appear to be critical determinants for the outcome of the tree-pathogen interaction. This work provides basic biochemical characterisation and expression data for the study of so-called partial resistance in the poplar-rust pathosystem, which is also applicable to other plant-pathogen interactions resulting in quantitative disease resistance.

Unsupervised learning in detection of gene transfer.

The tree representation as a model for organismal evolution has been in use since before Darwin. However, with the recent unprecedented access to biomolecular data, it has been discovered that, especially in the microbial world, individual genes making up the genome of an organism give rise to different and sometimes conflicting evolutionary tree topologies. This discovery calls into question the notion of a single evolutionary tree for an organism and gives rise to the notion of an evolutionary consensus tree based on the evolutionary patterns of the majority of genes in a genome embedded in a network of gene histories. Here, we discuss an approach to the analysis of genomic data of multiple genomes using bipartition spectral analysis and unsupervised learning. An interesting observation is that genes within genomes that have evolutionary tree topologies, which are in substantial conflict with the evolutionary consensus tree of an organism, point to possible horizontal gene transfer events which often delineate significant evolutionary events.

Texture analysis of X-ray radiographs of iliac bone is correlated with bone micro-CT.

INTRODUCTION: Alteration of bone trabecular architecture is a predictor of fracture risk in osteoporosis. Until now, microarchitecture can only be measured on a bone biopsy, thus limiting microarchitecture analysis in routine clinical practice for osteoporosis. Texture analysis on X-ray images has been advocated to be a suitable means to assess two-dimensional (2-D) microarchitecture in the research field. But little is known about the relationships between three-dimensional (3-D) architecture and texture analysis, particularly in clinical practice. The purposes of the study were: (1) to explore the relationship between 3-D histomorphometric parameters and 2-D texture analysis, and (2) to see if cortical assessment may influence results. METHODS: In this study, the anterosuperior part of the iliac bone was removed from 24 cadavers. Large samples were prepared and comprised of the crest and a strip of bone approximately 3 cm wide and 5 cm long. These large specimens were used in order to preserve bone architecture; they also corresponded to the location used by histomorphometrists for the diagnosis of metabolic bone diseases on iliac crest biopsies. Bone samples were examined with a microcomputed tomograph for 3-D microarchitecture [BV/TV, C.BV/C.TV, Tb.P(f), structure model index (SMI), Tb.Th, Tb.N, Tb.Sp]. Texture analysis was done by several methods (skeletonization, run lengths, fractal techniques) from X-ray projection images. No correlation was found between bone mass parameters (BV/TV and C.BV/C.TV, which take into account both cortical and trabecular bone) and texture parameters. RESULTS: However, when specific descriptors of trabecular bone microarchitecture were used, several relationships with texture parameters were found [(Tb.N)/BOUND, r=0.628;/VGLN, r=0.596;/Fractal D, r=0.569]. CONCLUSION: When multiple correlations were used, the correlation coefficients were markedly improved with trabecular characteristics. X-ray texture analysis seemed to be a suitable approach for 2-D bone microarchitecture assessment. Furthermore, there is a good correlation between texture analysis of X-ray radiographs and 3-D bone microarchitecture assessed by microcomputed tomography.

Vascular endothelial growth factor and signaling in the prostate: more than angiogenesis.

In cloning tyrosine kinase genes in dog prostate cells, a fragment of the vascular endothelial growth factor (VEGF) receptor 1 or Flt-1 was sequenced. To test for a functional protein, Flt-1 antibodies were used to probe immunoprecipitated tyrosine phosphorylated proteins. Western blotting revealed a major 170-180 kDa band and a few bands below 116 kDa in dog prostate and human prostatic carcinoma PC-3 cells, with higher levels in PC-3. Similar results were obtained with human placental membranes used as a source of Flt-1. That the major Flt-1 tyrosine phosphorylated protein was likely VEGF-R1 and part of VEGF signaling pathways was shown by enhanced level of only this protein when PC-3 cells were exposed to VEGF. Accordingly specific cell surface receptor complexes, displaced by VEGF but not EGF and compatible with Flt-1 in size, were revealed by chemical cross-linking after 125I-VEGF binding. Similarly to the prostatic neuroproduct, gastrin-releasing peptide/bombesin, VEGF directly triggered the tyrosine phosphorylation of focal adhesion kinase and stimulated PC-3 cell motility. The titration of prostate tissue sections with VEGF-A antibodies revealed a confined staining in chromogranin A and/or serotonin positive neuroendocrine (NE) cells, including in primary tumors and lymph node metastases. Given that NE differentiation is associated with advanced disease, that NE cells are a significant source of VEGF in prostatic tumors, and that VEGF directly act on prostate cancer cells in vitro, VEGF-A may be more than angiogenic in prostate cancer and hence favor progression by affecting tumor cells.

Endoglin expression on human microvascular endothelial cells association with betaglycan and formation of higher order complexes with TGF-beta signalling receptors.

Transforming growth factor-beta (TGF-beta) plays an important role in angiogenesis and vascular function. Endoglin, a transmembrane TGF-beta binding protein, is highly expressed on vascular endothelial cells and is the target gene for the hereditary haemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. The specific function of endoglin responsible for HHT1 is believed to involve alterations in TGF-beta responses. The initial interactions on the cell surface between endoglin and TGF-beta receptors may be an important mechanism by which endoglin modulates TGF-beta signalling, and thereby responses. Here it is shown that on human microvascular endothelial cells, endoglin is co-expressed and is associated with betaglycan, a TGF-beta accessory receptor with which endoglin shares limited amino acid homology. This complex formation may occur in either a ligand-dependent or a ligand-independent manner. In addition, the occurrence of three higher order complexes containing endoglin, type II and/or type I TGF-beta receptors, on these cells is demonstrated. Our findings suggest that endoglin may modify TGF-beta signalling by interacting with both betaglycan and the TGF-beta signalling receptors at physiological receptor concentrations and ratios.

Comparison of urinary desmosine excretion in patients with chronic obstructive pulmonary disease or cystic fibrosis.

Neutrophil elastase is involved in the pathogenesis of several pulmonary diseases; a strategy for monitoring in vivo elastase activity is to measure changes in biochemical markers. The objective of this study was to determine whether differences in the urinary excretion of the elastin crosslinks, desmosine and isodesmosine (which are unique amino acid products of elastase activity), could be discerned between groups of patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF), and non-diseased, age-matched controls. Twenty-four-hour urine collections were analysed to eliminate variations in excretion throughout the day, and urine was collected on four separate days in 29-31 subjects/group to investigate the variability in desmosines excretion among the groups. Both sets of patient populations had significantly more variable desmosines readings (higher standard deviations) relative to their respective age-matched control group. The means for three adult groups (COPD, controls and a COPD-smoker subset) ranged from 28.4 to 35.5 pmol desmosines/mg creatinine and there were no differences among the groups. Values in children were higher: 55 pmol desmosines/mg creatinine in the non-CF children and 77 pmol desmosines/mg creatinine for the CF group (P<0.01 vs. age-matched controls). The results of this study show that urinary desmosines, as a surrogate marker for enhanced elastase activity, are more highly variant in both patient populations relative to age-matched controls, and an overall increase in the mean value is further observed in patients with cystic fibrosis.

Preconditioning of human smooth muscle cells via cyclopentenone prostaglandins protects against toxic effects of oxidized low-density lipoprotein.

Human vascular smooth muscle cells (SMC) exhibit upregulation of inducible heat shock protein 70 (Hsp70), upon exposure to oxidized low-density lipoproteins (LDL(ox)). The presence of Hsp70 is thought to protect the cell against the toxic effects of the modified lipoprotein. In order to test this hypothesis, Hsp70 in SMC was upregulated by exposure to Delta(12) prostaglandin J(2) (Delta(12)PGJ(2)) before cells were exposed to LDL(ox). Hsp70 levels were measured after exposure to Delta(12)PGJ(2) and before exposure to LDL(ox). Cell protection was monitored after LDL(ox) exposure by determination of cell toxicity measured by cell lactate dehydrogenase (LDH) release into the medium. Cells treated with Delta(12)PGJ(2) exhibited a 23-fold increase in Hsp70 levels and 56% lower LDH activity release after exposure to LDL(ox) when compared to cells that were not pretreated with Delta(12)PGJ(2). In addition, cells pretreated with prostaglandins that did not induce Hsp70 did not exhibit increased tolerance against the toxic effects of LDL(ox). The results support a protective role for Hsp70 against the toxic effects of LDL(ox) and hint at the potential for the use of small molecules for the prevention of deleterious effects of LDL(ox) through heat shock protein upregulation.

Induction of heat shock protein 70 by herbimycin A and cyclopentenone prostaglandins in smooth muscle cells.

This study characterizes Hsp70 induction in human smooth muscle cells (SMC) by herbimycin A and cyclopentenone prostaglandins. The magnitude of Hsp70 induction by cyclopentenone prostaglandins was 8- to 10-fold higher than induction by herbimycin A. Hsp70 induction by delta12PGJ2 was first observed at 10 microM, rose to 4000-5000 ng/mL within one log unit and a maximum response was not observed; concentrations of delta12PGJ2 higher than 30 microM were toxic to the cells. A maximum response with herbimycin A (500 ng/mL) was reached at 0.05 microM and maintained to 1 microM without toxicity. Both, delta12PGJ2 and herbimycin A, were inhibited by dithiothreitol (DTT, 100 microM) at lower concentrations and became less sensitive to inhibition at higher concentrations. Hsp70 induction after incubation of SMC with delta12PGJ2 followed by addition of herbimycin A was significantly higher than Hsp70 induction after incubation with herbimycin A followed by addition of delta12PGJ2. When cells were incubated with [3H]-PGJ2, followed by protein denaturation, substantial radioactivity remained protein-bound suggesting that the prostaglandin must be covalently bound. Covalent binding was largely insensitive to DTT. Maximal Hsp70 induction was observed after 5 minutes of exposure of the cells to herbimycin A followed by a 20 hour recovery period in agent-free medium. Cells required 3-4 hours of exposure to delta12PGJ2 followed by a 20 hour recovery period in order to see high Hsp70 induction. Binding of the heat shock factor (HSF) to the heat shock element (HSE) in the presence of herbimycin A or delta12PGJ2, and the effects of DTT, mirrored the results of Hsp70 induction. The results suggest that probable differences between the 2 agents are at the level of the signal transduction prior to HSF activation.

Characterization of endoglin on mouse uterine stromal cells.

During the oestrous cycle and early pregnancy, the uterus undergoes a variety of morphological and physiological modifications involving uterine cell proliferation and differentiation as well as extensive tissue remodelling. Transforming growth factor beta (TGF-beta) has powerful effects on these events and thus is thought to have a critical role in uterine physiology. Endoglin is a transmembrane glycoprotein that binds TGF-beta 1 and -beta 3 and interacts with TGF-beta signalling receptors to modulate many effects of this growth factor in different types of cell. Studies in mice revealed the highest concentrations of endoglin in the reproductive tract, notably on stromal cells of cyclic and pregnant uteri. The objective of the present study was to investigate the role of endoglin expressed on uterine stromal cells in binding TGF-beta and in the cellular responses induced by this growth factor. Highly purified populations of uterine stromal cells were isolated by cell affinity to the monoclonal antibody MJ7/18, which is specific to mouse endoglin. Affinity labelling of these cells with 125I-labelled TGF-beta followed by immunoprecipitation with endoglin-specific polyclonal 1256:4b antiserum indicated that endoglin expressed at the surface of uterine stromal cells binds TGF-beta 1 and interacts with TGF-beta signalling receptors. Treatment of uterine stromal cells with different concentrations of TGF-beta 1 induced a biphasic proliferative response and addition of MJ7/18 as well as neutralizing TGF-beta antibodies showed endoglin to be a modulator of TGF-beta-induced stromal cell proliferation. Given the importance of TGF-beta in the regulation of uterine physiology, these results indicate a role for endoglin during uterine tissue remodelling and decidualization.

Direct demonstration of a humorally-mediated inhibition of renal phosphate transport in the Hyp mouse.

The murine Hyp model reproduces the characteristics of human X-linked hypophosphatemia (XLH), an inherited disease causing renal loss of phosphate (Pi), severe rickets and osteomalacia. A current hypothesis considers that a humoral factor may be responsible for the renal Pi loss, although in vitro experiments with renal cell models have failed to demonstrate the presence of such a factor in XLH or in the Hyp mouse model. To test this hypothesis directly, we prepared primary mouse proximal tubule cell cultures (MPTC), expressing normal features of proximal tubule cells. These cells possess high alkaline phosphatase activity, and respond to human parathyroid hormone fragment 1-34 (PTH) with a four- to sixfold increase in cAMP production but do not respond to either arginine vasopressin (AVP) or to salmon calcitonin (sCT). They also show sodium-dependent phosphate, glucose and amino acid uptake. The presence of 10% Hyp mouse serum in HAMF12/DMEM media (1 mM Pi) for the last 48 hours of culture of MPTC reduced Pi uptake (0.1 mM 32P-Pi in the presence of 140 mM NaCl) by 45.7 +/- 3.9% (P < 0.01) as compared to normal mouse serum. This effect of Hyp mouse serum was dose-dependent between 5 to 20% (final concentration) in culture media for the last 48 hours of culture (P < 0.01 by analysis of variance). This effect of Hyp mouse serum was also time-dependent, with a lag time of at least 12 hours. Indeed, no significant inhibition of Pi uptake could be detected with incubations less than 12 hours in the presence of 10% Hyp mouse serum, whereas a maximal effect was obtained after 24 hours of incubation and remained unchanged after 36 and 48 hours. The inhibition of phosphate uptake by Hyp mouse serum was specific, since neither sodium-dependent glucose nor alpha-aminobutyric acid uptake was modified under these conditions. MPTC cells showed a very nice adaptation to Pi concentration in the media; low Pi (0.4 mM final concentration in the presence of 10% serum) stimulated Pi uptake, whereas high Pi concentration (3 mM) reduced Pi uptake by these cells as compared to regular HAMF12/DMEM media containing 1 mM Pi. Normal and Hyp mouse serum both inhibited Pi uptake by MPTC following adaptation in low or normal Pi media, however, Hyp mouse serum always showed a stronger inhibition than normal serum. In contrast, adaptation of MPTC in high Pi media resulted in no inhibition of phosphate uptake either in the presence of normal or Hyp mouse serum. We next questioned whether conditioned media from confluent Hyp mouse primary osteoblast-like cell cultures could affect Pi uptake by MPTC. These osteoblast-like cells expressed high alkaline phosphatase and produced the bone specific protein, osteocalcin. When MPTC were treated for 48 hours with Hyp mouse bone cell media conditioned for the last 48 hours of cultures, Pi uptake was specifically inhibited by 30.5 +/- 4.1% (P < 0.025) as compared to normal mouse bone cell-conditioned media. This effect of primary Hyp mouse bone cell-conditioned media is specific for these cells since it was not observed with CHO cell-conditioned media, nor with either mouse fibroblast (NCTC), normal mouse Kupffer cell- or Hyp mouse Kupffer cell-conditioned media. This effect also persisted through a number of passages of Hyp mouse bone cells, since conditioned-media from cells at their third passage still resulted in a 32 +/- 9.4% inhibition (P < 0.02). These results are the first to show an effect of Hyp mouse serum on Pi uptake by primary renal cell cultures in vitro. This effect is dose- and time-dependent, requiring 24 hours for maximum response, and is blocked in Pi rich media. These results also suggest that a specific intrinsic cellular defect, present in Hyp mouse osteoblasts, is responsible for the release of and/or the modification of a factor that can reach the circulation and which inhibits renal phosphate reabsorption. The molecular nature of this factor and its mode of action remains to be determined.

Cardiovascular risk screening for women.

Cardiovascular disease is the leading cause of death of both men and women in Canada and the United States. The medical and societal emphasis on the occurrence of cardiovascular disease in men has resulted in an inclination to minimize its existence and severity in women. The purpose of this article is to assist clinical nurse specialists in cardiovascular risk-screening of women by providing a review of cardiovascular risk factors specific to women. Current knowledge about lipids, hypertension, diabetes, smoking, menopause, obesity, sedentary lifestyle, stress, and multiple roles are discussed. The clinical presentation for women and the clinical implications are presented. Lastly, implications for future research are described.

Novel and potent adenosine 3',5'-cyclic phosphate phosphodiesterase III inhibitors: thiazolo[4,5-b][1,6]naphthyridin-2-ones.

The transformation of 3-bromo-1,6-naphthyridin-2(1H)-ones 8 to thiazolo[4,5-b][1,6]naphthyridin-2(1H)-ones 12 resulted in a 2-9-fold increase in cAMP phosphodiesterase (PDE) III inhibitory potency. Unlike the secondary binding sites on the cAMP PDE III isozyme which interact with the methyl group of milrinone (2) and CI-930 (4), the site which interacts with the 5-substituents of 1,6-naphthyridin-2(1H)-ones and the 8-substituents of thiazolo[4,5-b][1,6]naphthyridin-2(1H)-ones 12 is able to accommodate a diverse group of substituents which have different steric and electronic requirements.

In vitro and in vivo evaluation of a once-daily controlled-release pseudoephedrine product.

The functionality of a once-daily, osmotic dosage form--gastrointestinal therapeutic system (pseudoephedrine HCl) or GITS (PeHCl)--was studied in vitro and in vivo. The in vitro release profiles were close to identical from pH 1 to 7.5 and between USP apparatus 2 and 7, independent of paddle speeds from 50 to 200 rpm; GITS also released drug at the normal rate in aqueous media after incubation in bile salts or fatty media. Both strengths of GITS (PeHCl)--240 and 120 mg--were then compared with a commercially available pseudoephedrine solution given every 6 hours and a timed-release 12-hour pseudoephedrine capsule given every 12 hours in a randomized 4-way crossover study in 24 healthy men. All four formulations were equivalent in total drug absorbed. Both GITS treatments had AUCinf values equivalent to those of PeHCl solution and capsules, and Cmax values equivalent to PeHCl capsules. Cmax for GITS and capsule treatments were each significantly lower than for solution, but the differences were small (14-17%). A one-to-one correlation was shown between rate of absorption and in vitro release profiles for the GITS products, indicating that drug release from GITS controls absorption. Insensitivity to conditions of in vivo release accounts for the close in vitro/in vivo correlation of release rates. In a second randomized crossover trial (12 men), the effect of a high-fat breakfast on GITS performance was evaluated. Mean pseudoephedrine concentrations in plasma were close to identical with or without the breakfast, and the treatments were bioequivalent.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclic GMP potentiation by WIN 58237, a novel cyclic nucleotide phosphodiesterase inhibitor.

The objectives of this study were to determine the potency and selectivity of the structurally novel cyclic nucleotide phosphodiesterase (PDE) inhibitor, WIN 58237 (1-cyclopentyl-3-methyl-6-(4- pyridyl)pyrazolo[3,4-d]pyrimidin-4-(5H)-one), and to determine if this compound possesses cyclic GMP (cGMP) PDE inhibitory activity in vitro and in vivo. WIN 58237 is a competitive inhibitor of cGMP PDE V from canine aorta, with a Ki value of 170 nM. It is a relatively less potent inhibitor of calmodulin-sensitive PDE I and cGMP-inhibitable cyclic AMP PDE III; but does inhibit cyclic AMP PDE IV with an IC50 value of approximately 300 nM. In vitro, WIN 58237 is a functional cGMP PDE inhibitor at submicromolar concentrations as evident by potentiation of both sodium nitroprusside- and atrial natriuretic factor-mediated vasorelaxation of contracted, endothelial-denuded rat aortic rings. Moreover, WIN 58237 possesses vasorelaxant activity in the presence of an intact endothelium or nitric oxide. Similar results are evident in vivo, as WIN 58237 (0.3-3.0 mg/kg i.v.) decreases mean arterial pressure in conscious spontaneously hypertensive rats with an associated increase in vascular (aortic) cGMP content in vivo. Both the decrease in mean arterial blood pressure and increase in aortic cGMP content are attenuated by the nitric oxide synthase inhibitor, N omega-nitro-l-arginine. However, WIN 58237 may possess an additional depressor mechanism of action. WIN 58237 restores vasorelaxation responsiveness to nitroglycerin in vitro and in vivo in models of vascular tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct profiles of phosphodiesterase isozymes in cultured cells derived from nonpigmented and pigmented ocular ciliary epithelium.

Alterations in either cyclic AMP (cAMP) or cyclic GMP (cGMP) may modulate the production of aqueous humor by the ciliary epithelium of the eye, thereby affecting intraocular pressure. We have found distinct profiles of phosphodiesterase (PDE) isozyme activity in cultured cells derived from bovine pigmented ciliary epithelium (PE) and cells derived from human nonpigmented ciliary epithelium (NPE), as well as corresponding differences in the effects of selective PDE inhibitors on the accumulation of cAMP and cGMP. In NPE cells, but not in PE cells, the major peak of PDE activity was stimulated by Ca++/calmodulin-stimulated (PDE I), and hydrolyzed both cAMP and cGMP. In contrast, PE cells contained a cGMP-specific PDE V not found in NPE cells. Rolipram, a selective inhibitor of PDE IV, was more potent and effective than the selective PDE III inhibitor CI-930 at potentiating intracellular cAMP accumulation in both cell types. Zaprinast, a selective inhibitor of PDE V, potentiated cGMP accumulation in PE but not in NPE cells. The results suggest that selective PDE inhibitors may modulate aqueous humor production by pigmented and nonpigmented ciliary epithelium, the two cell types may have different functional roles, and selective modulation of their functions may be possible. Furthermore, there may be distinct roles for intracellular calcium in regulating cGMP and cAMP in pigmented vs. nonpigmented ciliary epithelial cells.

Corona destruction: an innovative control technology for VOCs and air toxics.

This paper discusses the work and results to date leading to the demonstration of the corona destruction process at pilot scale. The research effort in corona destruction of volatile organic compounds (VOCs) and air toxics has shown significant promise for providing a valuable contribution to critical U.S. Environmental Protection Agency and national goals of reducing the health effects associated with exposures to hazardous air pollutants. The corona destruction technology could be especially useful in future years in helping industry meet the residual risk requirements of the Clean Air Act Amendments of 1990. Since 1988, EPA has conducted research in the area of corona destruction of VOCs and air toxics. EPA's interest in corona destruction of molecular species started with modeling of a point-plane reactor for destroying toxic organic compounds. EPA's goal is to develop a technology capable of controlling low concentration streams at low capital and operating costs. The purpose of this work is to develop an industrial scale corona reactor capable of efficiently and cost-effectively destroying VOCs and air toxics at ambient temperature and pressure. Results show that corona destruction is a promising control technology for many VOC-contaminated air streams, especially at low concentrations. Cost comparisons are presented for corona destruction and conventional control devices, carbon adsorption, catalytic incineration and thermal incineration.