Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Macrocyclic piperazinones as potent dual inhibitors of farnesyltransferase and geranylgeranyltransferase-I

A series of macrocyclic piperazinone compounds with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to be potent inhibitors of protein prenylation in cell culture. A hypothesis for the binding mode of compound 3o in FPTase is proposed.     

Suppressive DNA vaccination in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis involves a T1-biased immune response

Vaccination with DNA encoding a myelin basic protein peptide suppresses Lewis rat experimental autoimmune encephalomyelitis (EAE) induced with the same peptide. Additional myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG), may be important in multiple sclerosis. Here we demonstrate that DNA vaccination also suppresses MOG peptide-induced EAE. MOG(91-108) is encephalitogenic in DA rats and MHC-congenic LEW.1AV1 (RT1(av1)) and LEW.1N (RT1(n)) rats. We examined the effects of DNA vaccines encoding MOG(91-108) in tandem, with or without targeting of the hybrid gene product to IgG. In all investigated rat strains DNA vaccination suppressed clinical signs of EAE. There was no requirement for targeting the gene product to IgG, but T1-promoting CpG DNA motifs in the plasmid backbone of the construct were necessary for efficient DNA vaccination, similar to the case in DNA vaccination in myelin basic protein-induced EAE. We failed to detect any effects on ex vivo MOG-peptide-induced IFN-gamma, TNF-alpha, IL-6, IL-4, IL-10, and brain-derived neurotropic factor expression in splenocytes or CNS-derived lymphocytes. In CNS-derived lymphocytes, Fas ligand expression was down-regulated in DNA-vaccinated rats compared with controls. However, MOG-specific IgG2b responses were enhanced after DNA vaccination. The enhanced IgG2b responses together with the requirement for CpG DNA motifs in the vaccine suggest a protective mechanism involving induction of a T1-biased immune response.     

The shifting influence of drought and heat stress for crops in northeast Australia

Characterization of drought environment types (ETs) has proven useful for breeding crops for drought‐prone regions. Here, we consider how changes in climate and atmospheric carbon dioxide (CO₂) concentrations will affect drought ET frequencies in sorghum and wheat systems of northeast Australia. We also modify APSIM (the Agricultural Production Systems Simulator) to incorporate extreme heat effects on grain number and weight, and then evaluate changes in the occurrence of heat‐induced yield losses of more than 10%, as well as the co‐occurrence of drought and heat. More than six million simulations spanning representative locations, soil types, management systems, and 33 climate projections led to three key findings. First, the projected frequency of drought decreased slightly for most climate projections for both sorghum and wheat, but for different reasons. In sorghum, warming exacerbated drought stresses by raising the atmospheric vapor pressure deficit and reducing transpiration efficiency (TE), but an increase in TE due to elevated CO₂ more than offset these effects. In wheat, warming reduced drought stress during spring by hastening development through winter and reducing exposure to terminal drought. Elevated CO₂ increased TE but also raised radiation‐use efficiency and overall growth rates and water use, thereby offsetting much of the drought reduction from warming. Second, adding explicit effects of heat on grain number and grain size often switched projected yield impacts from positive to negative. Finally, although average yield losses associated with drought will remain generally higher than that for heat stress for the next half century, the relative importance of heat is steadily growing. This trend, as well as the likely high degree of genetic variability in heat tolerance, suggests that more emphasis on heat tolerance is warranted in breeding programs. At the same time, work on drought tolerance should continue with an emphasis on drought that co‐occurs with extreme heat.     

The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.     

Pyridyl aminothiazoles as potent inhibitors of Chk1 with slow dissociation rates

Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.     

Increasing drought and diminishing benefits of elevated carbon dioxide for soybean yields across the US Midwest

Elevated atmospheric CO₂ concentrations ([CO₂]) are expected to increase C3 crop yield through the CO₂ fertilization effect (CFE) by stimulating photosynthesis and by reducing stomatal conductance and transpiration. The latter effect is widely believed to lead to greater benefits in dry rather than wet conditions, although some recent experimental evidence challenges this view. Here we used a process‐based crop model, the Agricultural Production Systems sIMulator (APSIM), to quantify the contemporary and future CFE on soybean in one of its primary production area of the US Midwest. APSIM accurately reproduced experimental data from the Soybean Free‐Air CO₂ Enrichment site showing that the CFE declined with increasing drought stress. This resulted from greater radiation use efficiency (RUE) and above‐ground biomass production at elevated [CO₂] that outpaced gains in transpiration efficiency (TE). Using an ensemble of eight climate model projections, we found that drought frequency in the US Midwest is projected to increase from once every 5 years currently to once every other year by 2050. In addition to directly driving yield loss, greater drought also significantly limited the benefit from rising [CO₂]. This study provides a link between localized experiments and regional‐scale modeling to highlight that increased drought frequency and severity pose a formidable challenge to maintaining soybean yield progress that is not offset by rising [CO₂] as previously anticipated. Evaluating the relative sensitivity of RUE and TE to elevated [CO₂] will be an important target for future modeling and experimental studies of climate change impacts and adaptation in C3 crops.     

Presence of CpG DNA and the local cytokine milieu determine the efficacy of suppressive DNA vaccination in experimental autoimmune encephalomyelitis.

We here study the adjuvant properties of immunostimulatory DNA sequences (ISS) and coinjected cytokine-coding cDNA in suppressive vaccination with DNA encoding an autoantigenic peptide, myelin basic protein peptide 68-85, against Lewis rat experimental autoimmune encephalomyelitis (EAE). EAE is an autoaggressive, T1-mediated disease of the CNS. ISS are unmethylated CpG motifs found in bacterial DNA, which can induce production of type 1 cytokines in vertebrates through the innate immune system. Because ISS in the plasmid backbone are necessary for efficient DNA vaccination, we studied the effect of one such ISS, the 5'-AACGTT-3' motif, in our system. Treatment with a DNA vaccine encoding myelin basic protein peptide 68-85 and containing three ISS of 5'-AACGTT-3' sequence suppressed clinical signs of EAE, while a corresponding DNA vaccine without such ISS had no effect. We further observed reduced proliferative T cell responses in rats treated with the ISS-containing DNA vaccine, compared with controls. We also studied the possible impact of coinjection of plasmid DNA encoding rat cytokines IL-4, IL-10, GM-CSF, and TNF-alpha with the ISS-containing DNA vaccine. Coinjection of IL-4-, IL-10-, or TNF-alpha-coding cDNA inhibited the suppressive effect of the DNA vaccine on EAE, whereas GM-CSF-coding cDNA had no effect. Coinjection of cytokine-coding cDNA with the ISS-deficient DNA vaccine failed to alter clinical signs of EAE. We conclude that the presence of ISS and induction of a local T1 cytokine milieu is decisive for specific protective DNA vaccination in EAE.