A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Acknowledgments

One of the major causes of worldwide amphibian declines is a skin infection caused by a pathogenic chytrid fungus (Batrachochytrium dendrobatidis). This study documents the interactions between this pathogen and a susceptible amphibian host, the boreal toad (Bufo boreas). The amount of time following exposure until death is influenced by the dosage of infectious zoospores, duration of exposure, and body size of the toad. The significant relation between dosage and the number of days survived (dose-response curve) supports the hypothesis that the degree of infection must reach a particular threshold of about 10⁷–10⁸ zoosporangia before death results. Variation in air temperature between 12°C and 23°C had no significant effect on survival time. The infection can be transmitted from infected to healthy animals by contact with water containing zoospores; no physical contact between animals is required. These results are correlated with observations on the population biology of boreal toads in which mortalities associated with B. dendrobatidis have been identified.     

Variation in soil phosphorus, sulfur, and iron pools among south Florida wetlands

To determine relationships between soil nutrient status and known gradients in primary production, we collected and analyzed soils from 17 LTER sampling sites along two transects through south Florida wetland ecosystems. Through upstream freshwater marsh, a middle reach including the oligohaline marsh/mangrove ecotone, and downstream estuarine habitats, we observed systematic variation in soil bulk density, organic content, and pools of phosphorus (P), inorganic sulfur, and extractable iron. Consistent with observed differences in wetland productivity known to be limited by P availability, total P averaged ~200 μg g dw⁻¹ in soils from the eastern Taylor Slough/Panhandle and was on average three times higher in soils from the western Shark River Slough. Along both transects, the largest pool of phosphorus was the inorganic, carbonate-bound fraction, comprising 35–44% of total P. Greater than 90% of the total inorganic sulfur pool in these south Florida wetland soils was extracted as pyrite. Freshwater marsh sites typically were lower in pyrite sulfur (0.2–0.8 mg g dw⁻¹) relative to marsh/mangrove ecotone and downstream estuary sites (0.5–2.9 mg g dw⁻¹). Extractable iron in freshwater marsh soils was significantly higher from the Taylor Slough/Panhandle transect (3.2 mg g dw⁻¹) relative to the western Shark River Slough transect (1.1 mg g dw⁻¹), suggesting spatial variation in sources and/or depositional environments for iron. Further, these soil characteristics represent the collective, integrated signal of ecosystem structure, so any long-term changes in factors like water flow or water quality may be reflected in changes in bulk soil properties. Since the objective of current Everglades restoration initiatives is the enhancement and re-distribution of freshwater flows through the south Florida landscape, the antecedent soil conditions reported here provide a baseline against which future, post-restoration measurements can be compared.     

Capture and Retention of Cryptosporidium parvum Oocysts by Pseudomonas aeruginosa Biofilms

The association of Cryptosporidium oocysts with biofilm communities can influence the propagation of this pathogen through both environmental systems and water treatment systems. We observed the capture and retention of C. parvum oocysts in Pseudomonas aeruginosa biofilms using laboratory flow cells. Biofilms were developed in two different growth media using two different strains of P. aeruginosa, a wild-type strain (PAO1) and a strain that overproduces the exopolysaccharide alginate (PDO300). Confocal laser-scanning microscopy was used in conjunction with image analysis to assess the structure of the biofilms prior to introducing oocysts into the flow cells. More oocysts were captured by the biofilm-coated surfaces than the abiotic glass surface in both media. There was no significant difference in capture across the two strains of P. aeruginosa biofilm, but the fraction of oocysts captured was positively related to biofilm roughness and surface-area-to-volume ratio. Once captured, oocysts were retained in the biofilm for more than 24 h and were not released after a 40-fold increase in the system flow rate. We believe the capture and retention of oocysts by biofilm communities can impact the environmental transmission of C. parvum, and this interaction should be taken into consideration when predicting the migration of pathogens in the environment.     

Artemisinin: The biosynthetic pathway and its regulation in Artemisia annua, a terpenoid-rich species

Summary Artemisinin is a sesquiterpene lactone isolated from the aerial parts of Artemisia annua L. plants. Besides being currently the best therapeutic against both drug-resistant and cerebral malaria-causing strains of Plasmodium falciparum, the drug has also been shown to be effective against other infections diseases including schistosomiasis and hepatitis. More recently, it has also been shown to be effective against numerous types of tumors. Although chemical synthesis of artemisinin is possible, it is not economically feasible. The relatively low yield (0.01–0.8%) of artemisinin in A. annua is a further serious limitation to the commercialization of the drug. Therffore, the enhanced production of artemisinin either in cell/tissue culture or in the whole plant of A. annua is highly desirable. A better understanding of the biochemical pathway leading to the synthesis of artemisinin and its regulation by both exogenous and endogenous factors is essential for facilitating increased yield. Two genes of the artemisinin biosynthetic pathway have now been identified. This critical review covers recent developments related to the biosynthesis of this important compound and related terpenoids, their regulation, and the production of these compounds both in vitro and in whole plants.     

Comparison of Tissue Metal Concentrations in Zucker Lean, Zucker Obese, and Zucker Diabetic Fatty Rats and the Effects of Chromium Supplementation on Tissue Metal Concentrations

Diabetes results in several metabolic changes, including alterations in the transport, distribution, excretion, and accumulation of metals. While changes have been examined in several rat models of insulin resistance and diabetes, the metal ion concentrations in the tissues of Zucker lean, Zucker obese (an insulin resistance and early stage diabetes model), and Zucker diabetic fatty (ZDF, a type 2 diabetes model) have not previously been examined in detail. The concentration of Cu, Zn, Fe, Mg, and Ca were examined in the liver, kidney, heart and spleen, and Cr concentration in the liver and kidney of these rats were examined. Zucker obese rats have a reduction in the concentration of Cu, Zn, Fe, Mg in the liver compared to ZDF and/or lean Zucker rats, presumably as a result of the increased fat content of the liver of the obese rats. ZDF rats have increased concentrations of kidney Cu compared to the lean rats, while kidney Ca concentrations are increased in the Zucker obese rats. Spleen Fe concentrations are decreased in Zucker obese rats compared to the lean rats. No effects on metal concentrations in the heart were observed between the lean, obese, and ZDF rats, and no effects on Cr concentrations were identified. Cr(III) complexes have previously been shown to have beneficial effects on the signs of insulin resistance in Zucker obese and ZDF rats. The effects of daily gavage administration of chromium picolinate ([Cr(pic)₃]) (1 mg?Cr/kg body mass), CrCl₃ (1 mg?Cr/kg body mass), and Cr3 ([Cr₃O(propionate)₆(H₂O)₃]⁺) (33 μg and 1 mg?Cr/kg body mass) on metal concentrations in these tissues were examined. Treatment with CrCl₃ and Cr3, but not [Cr(pic)₃], at 1 mg?Cr/kg resulted in a statistically significant accumulation of Cr in the kidney of lean and obese but not ZDF rats but resulted in lowering the elevated levels of kidney Cu in ZDF rats, suggesting a beneficial effect on this symptom of type 2 diabetes.     

Replication and fine mapping of asthma-associated loci in individuals of African ancestry

Asthma originates from genetic and environmental factors with about half the risk of disease attributable to heritable causes. Genome-wide association studies, mostly in populations of European ancestry, have identified numerous asthma-associated single nucleotide polymorphisms (SNPs). Studies in populations with diverse ancestries allow both for identification of robust associations that replicate across ethnic groups and for improved resolution of associated loci due to different patterns of linkage disequilibrium between ethnic groups. Here we report on an analysis of 745 African-American subjects with asthma and 3,238 African-American control subjects from the Candidate Gene Association Resource (CARe) Consortium, including analysis of SNPs imputed using 1,000 Genomes reference panels and adjustment for local ancestry. We show strong evidence that variation near RAD50/IL13, implicated in studies of European ancestry individuals, replicates in individuals largely of African ancestry. Fine mapping in African ancestry populations also refined the variants of interest for this association. We also provide strong or nominal evidence of replication at loci near ORMDL3/GSDMB, IL1RL1/IL18R1, and 10p14, all previously associated with asthma in European or Japanese populations, but not at the PYHIN1 locus previously reported in studies of African-American samples. These results improve the understanding of asthma genetics and further demonstrate the utility of genetic studies in populations other than those of largely European ancestry.     

Bark beetle infestation impacts on nutrient cycling, water quality and interdependent hydrological effects

Bark beetle populations have drastically increased in magnitude over the last several decades leading to the largest-scale tree mortality ever recorded from an insect infestation on multiple wooded continents. When the trees die, the loss of canopy and changes in water and nutrient uptake lead to observable changes in hydrology and biogeochemical cycling. This review aims to synthesize the current research on the effects of the bark beetle epidemic on nutrient cycling and water quality while integrating recent and relevant hydrological findings, along with suggesting necessary future research avenues. Studies generally agree that snow depth will increase in infested forests, though the magnitude is uncertain. Changes in evapotranspiration are more variable as decreased transpiration from tree death may be offset by increased understory evapotranspiration and ground evaporation. As a result of such competing hydrologic processes that can affect watershed biogeochemistry along with the inherent variability of natural watershed characteristics, water quality changes related to beetle infestation are difficult to predict and may be regionally distinct. However, tree-scale changes to soil–water chemistry (N, P, DOC and base cation concentrations and composition) are being observed in association with beetle outbreaks which ultimately could lead to larger-scale responses. The different temporal and spatial patterns of bark beetle infestations due to different beetle and tree species lead to inconsistent infestation impacts. Climatic variations and large-scale watershed responses provide a further challenge for predictions due to spatial heterogeneities within a single watershed; conflicting reports from different regions suggest that hydrologic and water quality impacts of the beetle on watersheds cannot be generalized. Research regarding the subsurface water and chemical flow-paths and residence times after a bark beetle epidemic is lacking and needs to be rigorously addressed to best predict watershed or regional-scale changes to soil–water, groundwater, and stream water chemistry.