Photosynthetic pigment concentrations, gas exchange and vegetative growth for selected monocots and dicots treated with two contrasting coal fly ashes.

There is uncertainty as to the rates of coal fly ash needed for optimum physiological processes and growth. In the current study we tested the hypothesis that photosynthetic pigments concentrations and CO(2) assimilation (A) are more sensitive than dry weights in plants grown on media amended with coal fly ash. We applied the Terrestrial Plant Growth Test (Guideline 208) protocols of the Organization for Economic Cooperation and Development (OECD) to monocots [barley (Hordeum vulgare) and ryegrass (Secale cereale)] and dicots [canola (Brasica napus), radish (Raphanus sativus), field peas (Pisum sativum), and lucerne (Medicago sativa)] on media amended with fly ashes derived from semi-bituminous (gray ash) or lignite (red ash) coals at rates of 0, 2.5, 5.0, 10, or 20 Mg ha(-1). The red ash had higher elemental concentrations and salinity than the gray ash. Fly ash addition had no significant effect on germination by any of the six species. At moderate rates (

Growth and elemental accumulation by canola on soil amended with coal fly ash.

To explore the agronomic potential of an Australian coal fly ash, we conducted two glasshouse experiments in which we measured chlorophyll fluorescence, CO2 assimilation (A), transpiration, stomatal conductance, biomass accumulation, seed yield, and elemental uptake for canola (Brassica napus) grown on soil amended with an alkaline fly ash. In Experiment 1, application of up to 25 Mg/ha of fly ash increased A and plant weight early in the season before flowering and seed yield by up to 21%. However, at larger rates of ash application A, plant growth, chlorophyll concentration, and yield were all reduced. Increases in early vigor and seed yield were associated with enhanced uptake of phosphorus (P) by the plants treated with fly ash. Fly ash application did not influence accumulation of B, Cu, Mo, or Zn in the stems at any stage of plant growth or in the seed at harvest, except Mo concentration, which was elevated in the seed. Accumulation of these elements was mostly in the leaves, where concentrations of Cu and Mo increased with any amount of ash applied while that of B occurred only with ash applied at 625 Mg/ha. In Experiment 2, fly ash applied at 500 Mg/ha and mixed into the whole 30 cm soil core was detrimental to growth and yield of canola, compared with restricting mixing to 5 or 15 cm depth. In contrast, application of ash at 250 Mg/ha with increasing depth of mixing increased A and seed yield. We concluded that fly ash applied at not more than 25 Mg/ha and mixed into the top 10 to 15 cm of soil is sufficient to obtain yield benefits.

Association analysis of endothelial nitric oxide synthase gene polymorphism with primary hypertension in a Singapore population.

Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Endothelial nitric oxide synthase (eNOS) gene polymorphisms are associated with coronary artery disease, but their linkage with primary hypertension is controversial. A total of 103 individuals with primary hypertension and 104 normotensive control subjects were studied in Singapore. The specific genotypes for G894T missense variant in exon 7, variable number tandem repeats (VNTR) in intron 4 (eNOS 4A/B/C) and T-786C in the promoter were isolated using allele-specific gene amplification and restriction fragment length polymorphism to examine the association of genotype and allelic frequency in both groups. Logistic regression analysis was also used to detect the association between genotypes and hypertension. Five genotypes of intron 4 VNTR (AA, AB, BB, AC and BC) were observed. Intron 4 B/B genotype was significantly associated with the hypertension group (P = 0.035), but disequilibrium of G894T and T-786C was absent between the two groups (P = 0.419 and P = 0.227), respectively. The overall distribution of allelic frequency differed significantly between the two groups, with four-repeat allele (4A) of intron 4 more frequent in the normotensive group than the hypertensive group (P = 0.019). Logistic regression analysis showed that intron 4 B/B genotype was significantly associated with systolic blood pressure of individuals with body mass index greater than 25 kg/m2 (P = 0.04). In conclusion, the eNOS 4 B/B genotype is a genetic susceptibility factor for primary hypertension in a Singapore population.

Does nonsense-mediated mRNA decay explain the ovarian cancer cluster region of the BRCA2 gene?

BRCA2 (BReast CAncer susceptibility gene 2) germline mutation carriers are at increased risk for breast and ovarian cancers. Mutations occurring in the ovarian cancer cluster region (OCCR) are linked to higher ovarian cancer and/or lower breast cancer risk(s) than mutations occurring elsewhere in BRCA2. Most BRCA2 germline mutations introduce premature termination codons (PTCs), making their mRNAs likely targets of nonsense-mediated mRNA decay (NMD), a mechanism that eliminates PTC-bearing transcripts to prevent expression of truncated proteins. Contradictory evidence exists regarding whether NMD can be triggered by PTCs located far upstream of the nearest exon-exon junction (EEJ). Since the OCCR comprises a major portion of the 4.9 kb exon 11 of BRCA2, we investigated if transcripts bearing PTCs in this large exon are unable to trigger NMD, and if this might contribute to the phenotypic difference associated with the OCCR. We examined cDNA from 18 carriers of PTC-introducing germline mutations located throughout BRCA2, and found that PTC-bearing transcripts were 1.4-3.3-fold less prevalent than their nonmutated counterparts irregardless of PTC position. We conclude that NMD can recognize PTCs up to 4.5 kb upstream of the nearest EEJ, demonstrating that a general inability of NMD to recognize PTCs in exon 11 is unlikely to explain the genotype-phenotype correlation associated with the OCCR.

Sustainable management of leakage from wastewater pipelines.

Wastewater pipeline leakage is an emerging concern in Europe, especially with regards to the potential effect of leaking effluent on groundwater contamination and the effects infiltration has on the management of sewer reticulation systems. This paper describes efforts by Australia, in association with several European partners, towards the development of decision support tools to prioritize proactive rehabilitation of wastewater pipe networks to account for leakage. In the fundamental models for the decision support system, leakage is viewed as a function of pipeline system deterioration. The models rely on soil type identification across the service area to determine the aggressiveness of the pipe environment and for division of the area into zones based on pipe properties and operational conditions. By understanding the interaction between pipe materials, operating conditions, and the pipe environment in the mechanisms leading to pipe deterioration, the models allow the prediction of leakage rates in different zones across a network. The decision support system utilizes these models to predict the condition of pipes in individual zones, and to optimize the utilization of rehabilitation resources by targeting the areas with the highest leakage rates.

Aldehyde oxidase is coamplified with the World's most common Culex mosquito insecticide resistance-associated esterases.

The evolution and spread of insecticide resistance is an important factor in human disease prevention and crop protection. The mosquito Culex quinquefasciatus is the main vector of the disease filariasis and a member of a species complex which is a common biting nuisance worldwide. The common insecticide resistance mechanism in this species involves germline amplification of the esterases estalpha21 and estbeta21. This amplification has arisen once and rapidly spread worldwide. Less common and more variable resistance phenotypes involve coamplification of estalpha3 and estbeta1, or individual amplification of a single estbeta1, different alleles of the same estalpha and estbeta gene loci. Estalpha21 and estbeta21 are on the same large fragment of amplified DNA (amplicon) 2.7 kb apart. We have now shown that this amplicon contains another full-length gene immediately 5' of estalpha21 which codes for a molybdenum-containing hydroxylase, with highest homology to aldehyde oxidase (AO) from other organisms. The full-length putative AO gene is not present on the estalpha3/estbeta1 or estbeta1 amplicons, but multiple truncated 5' ends of this gene are present around the presumed estalpha3/estbeta1 amplicon breakpoint. Polymerase chain reaction (PCR) analysis of insecticide-susceptible genomic DNA demonstrated that a different allele of the putative AO gene in its non-amplified form is immediately 5' of estalpha. The 'AO' gene on the estalpha21/estbeta21 amplicon is expressed and resistant insects have greater AO activity. This AO activity is sensitive to inhibition by an aldehyde-containing herbicide and pesticide. This enzyme may confer a selective advantage to these insects in the presence of insecticide, as AO in mammals is believed to be important in the detoxification process of several environmental pollutants.

A study involving venlafaxine overdoses: comparison of fatal and therapeutic concentrations in postmortem specimens.

The distribution and redistribution of venlafaxine were investigated in two overdoses and several cases involving the therapeutic use of venlafaxine. Blood, liver, bile vitreous humor, urine and gastric contents were analyzed using high performance liquid chromatography with ultraviolet detection. Blood concentrations of venlafaxine in the two overdose cases were 53 mg/L and 78 mg/L. Comparison of venlafaxine concentrations in blood samples taken at different times after death revealed increases in concentrations over time, suggesting the possible postmortem redistribution of venlafaxine.

Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy.

Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.

Resistance to insecticides in insect vectors of disease: est alpha 3, a novel amplified esterase associated with amplified est beta 1 from insecticide resistant strains of the mosquito Culex quinquesfasciatus.

Vector control programmes in many countries face the dual problems of parasite drug resistance and insecticide resistance in the insect vectors of the disease. Here we report for the first time a new esterase-based insecticide resistance mechanism in the filariasis vector Culex quinquefasciatus. The field collected COL strain of C. quinquefasciatus from Columbia was heterogeneous for organophosphorus insecticide resistance. On native polyacrylamide gels it had an elevated beta-naphthyl acetate specific esterase with the same Rf as that for the Est beta 1s involved in insecticide resistance in other strains of this mosquito species. After five generations of temephos insecticide selection, both the esterase specific activity with p-nitrophenyl acetate and the temephos LC50 values were increased, suggesting that elevation of esterase activity was the underlying mechanism of resistance. Western blots with antisera raised to Est alpha 2(1) and Est beta 2(1) from C. quinquefasciatus indicated that the COL strain had an elevated Est alpha 3 enzyme which co-migrated on native gels with Est beta 1. Southern blots indicated that an est alpha 3 gene was amplified in the COL strain and a Cuban mosquito strain (MRes), although the restriction digest patterns of the est beta 1 genes in these two strains are different. In contrast, the Californian TEMR strain, with the amplified est beta 1(1) gene, had no associated elevated Est alpha. Restriction digest patterns for COL and TEMR DNA suggest that they contain an identical est beta 1(1) gene, but our data suggest that the est alpha 3 gene occurs on the same amplicon as an est beta 1 gene although the genes are probably > 10 kb apart. Hence, either the COL strain has two est beta 1 genes or the est beta 1(1) amplicon in TEMR has been disrupted at some stage during the long colonisation of this strain and the amplified est alpha has been lost.

The p38 mitogen-activated protein kinase pathway in activated and anergic Th1 cells.

Stimulation of T cells through the TCR leads to activation of the mitogen-activated protein kinase (MAPK) family members ERK (extracellular signal-regulated kinase) and JNK (jun NH2-terminal kinase). These kinases act in synergy to increase the activity of the transcription factor AP-1 which is involved in the transcriptional upregulation of IL-2. Recently a third MAPK member, p38, has been identified. The effects of T cell activation on this pathway have not yet been elucidated. Using two murine Th1 clones, we demonstrate that the p38 pathway is induced upon anti-CD3 plus anti-CD28 crosslinking or PMA plus ionomycin stimulation. p38 activity was induced fully by anti-CD3 or PMA alone and is not enhanced by costimulation even at low levels of TCR signaling. p38 activity peaked at 20 min and was significantly decreased by 2 hr. Anergic (tolerant) Th1 cells showed decreased p38 activity as well as decreased ERK and JNK activities even though levels of these proteins remained unchanged.

Defective transcription of the IL-2 gene is associated with impaired expression of c-Fos, FosB, and JunB in anergic T helper 1 cells.

Anergic CD4+ Th cells do not produce IL-2 when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical IL-2 DNA enhancer elements: NF-AT (nuclear factor of activated T cells; a sequence that binds a heterotrimeric NFATp, Fos, and Jun protein complex) and Activator Protein-1 (AP-1) (that binds Fos and Jun heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible AP-1 family members c-Fos, FosB, and JunB. However, the reduced expression of these proteins was not the result of a global TCR/CD3-signaling defect because CD3 cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as NFATp dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells. Thus, defective IL-2 gene transcription appears to be due, at least in part, to a selective block in the expression of the AP-1 Fos and Jun family members in anergic T cells.

Anergic T cells are defective in both jun NH2-terminal kinase and mitogen-activated protein kinase signaling pathways.

T helper type 1 cells (Th1) become anergic when stimulated through the antigen receptor in the absence of costimulation. They do not produce IL-2 or proliferate in response to subsequent stimulation. Previous studies have indicated that anergic T cells are defective in the trnsactivational activity of the transcription factor, AP-1, which is required for optimal IL-2 transcription. Using two murine Th1 cell clones, we demonstrate that anergic Th1 cells have defects in both jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activities. These kinases have been shown to be important for the upregulation of AP-1 activity. Furthermore, our data show that ERK and JNK activities are restored when anergy is induced in the presence of the protein synthesis inhibitor cycloheximide, or when anergic T cells are allowed to proliferate in response to exogenous IL-2. These treatments have previously been shown to prevent or reverse the anergic state. Our results suggest that defects in both JNK and ERK may result in the decreased AP-1 activity and the reduced IL-2 transcription observed in anergic T cells.

Clonal anergy is induced in vitro by T cell receptor occupancy in the absence of proliferation.

Murine Th1 clones that receive signals through their TCR in the absence of APC-derived co-stimulatory signals do not produce IL-2 and instead become anergic, i.e., they are subsequently unable to produce IL-2 in response to Ag and normal APC. The critical cellular event required to prevent the induction of this anergic state appears to be T cell proliferation. Anergy was induced when T cell clones were stimulated under conditions where both TCR occupancy and costimulatory signals were provided but where proliferation in response to the IL-2 produced was prevented. Once induced, anergy could be reversed if the T cells were allowed to undergo multiple rounds of cell division. These results show that anergy is induced as a consequence of TCR occupancy in the absence of cell division; this can be achieved either by limiting IL-2 production because of deficient provision of co-stimulatory signals or by preventing response to IL-2.

Effects of glycyl-histidyl-lysyl chelated Cu(II) on ferritin dependent lipid peroxidation.

The copper binding tripeptide, glycyl-L-histidyl-L-lysine [GHK:Cu(II)] has a plethora of biological effects related to the wound healing process. The presence of iron complexes in damaged tissues is detrimental to wound healing, due to local inflammation, as well as microbial infection mediated by iron. To test if the wound healing properties of GHK:Cu(II) are due to an affect on iron metabolism, we examined the effects of GHK:Cu(II) on iron catalyzed lipid peroxidation. GHK:Cu(II) inhibited lipid peroxidation only if the iron source was ferritin. Whereas GHK:Cu(II) inhibited ferritin iron release it did not exhibit significant superoxide dismutase-like or ceruloplasmin-like activity. We propose that GHK:Cu(II) binds to the channels of ferritin involved in iron release and physically prevents the release of Fe(II). Thus, a biological effect of GHK:Cu(II), possibly related to wound healing, may be the inhibition of ferritin iron release in damaged tissues, preventing inflammation and microbial infections.