Calpain mediates a von Hippel-Lindau protein-independent destruction of hypoxia-inducible factor-1alpha

Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1alpha requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1alpha expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2'-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1alpha protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1alpha degradation. Furthermore, chelating intracellular calcium attenuated HIF-1alpha destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1alpha even under normoxia. An active role of calpain in lowering HIF-1alpha amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1alpha that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1alpha destruction involving the calpain system.     

Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression

Mouse p53 is phosphorylated at Ser18 and Ser23 after DNA damage. To determine whether these two phosphorylation events have synergistic functions in activating p53 responses, we simultaneously introduced Ser18/23 to Ala mutations into the endogenous p53 locus in mice. While partial defects in apoptosis are observed in p53S18A and p53S23A thymocytes exposed to IR, p53-dependent apoptosis is essentially abolished in p53S18/23A thymocytes, indicating that these two events have critical and synergistic roles in activating p53-dependent apoptosis. In addition, p53S18/23A, but not p53S18A, could completely rescue embryonic lethality of Xrcc4(-/-) mice that is caused by massive p53-dependent neuronal apoptosis. However, certain p53-dependent functions, including G1/S checkpoint and cellular senescence, are partially retained in p53(S18/23A) cells. While p53(S18A) mice are not cancer prone, p53S18/23A mice developed a spectrum of malignancies distinct from p53S23A and p53(-/-) mice. Interestingly, Xrcc4(-/-)p53S18/23A mice fail to develop tumors like the pro-B cell lymphomas uniformly developed in Xrcc4(-/-) p53(-/-) animals, but exhibit developmental defects typical of accelerated ageing. Therefore, Ser18 and Ser23 phosphorylation is important for p53-dependent suppression of tumorigenesis in certain physiological context.     

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is –98° ± 3° in lily and –124° ± 4° in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.     

Vitrification assays with embryos from a cold tolerant sub-arctic fish species

Pseudopleuronectes americanus is a Northern teleost species that produces antifreeze proteins (AFPs) to protect them from freezing during the winter. These AFPs bind to ice crystals to inhibit their growth, and they also protect cell membranes at low temperatures. In this study, vitrification trials were done with fish embryos at three different developmental stages, using two different protocols for incorporating the vitrifying solutions. Toxicity of the cryoprotectants and permeability to dimethyl sulfoxide were analyzed. Embryos were vitrified in 0.5 ml straws by direct immersion in liquid nitrogen, and their morphology and development analyzed following thaw. The embryos responded well to vitrification as evidenced by the high percentage that exhibited good morphology following thaw. Although none of the embryos hatched, a small percentage (0.92%) of them showed active movements within the chorion and continued to develop for a number of days following thaw. This is the first record of post-thaw development of vitrified fish embryos.     

Vitrification of turbot embryos: preliminary assays

Successful fish embryo cryopreservation is still far from being achieved. Vitrification is considered the most promising option. Many factors are involved in the success of the process. The choice of a proper vitrification solution, the enzymatic permeabilization of embryos to increase cryoprotectant permeability, the adequate container for embryo loading, and the temperature for thawing, were the parameters considered at different developmental stages in the present study. After vitrification, embryo morphology was evaluated under stereoscopic microscopy, establishing the percentage of intact embryos. Two of the studied parameters yielded differences in this percentage, the volume of straw used for embryo loading (1 ml straws were significantly better than 0.5 ml straws, with regard to post-thawed embryo morphologies), and the thawing temperature, achieving 49% of embryos with intact morphology after thawing at 0 degrees C. After thawing, the intact embryos were incubated and periodically observed to detect morphological changes. Changes in the perivitelline space, shrinkage of the yolk and chorion ruptures as well as a progressive whitening of the embryo and yolk were observed. After 8 h all embryos showed clear signs of degradation and during this incubation period no embryo showed any developmental ability.     

A spatial assessment of potential biomass for bioenergy in Australia in 2010, and possible expansion by 2030 and 2050

This paper provides spatial estimates of potentially available biomass for bioenergy in Australia in 2010, 2030 and 2050 (under clearly stated assumptions) for the following biomass sources: crop stubble, native grasses, pulpwood and residues (created either during forest harvesting or wood processing) from plantations and native forests, bagasse, organic municipal solid waste and new short‐rotation tree crops. For each biomass type, we estimated annual potential availability at the finest scale possible with readily accessible data, and then aggregated to make estimates for each of 60 Statistical Divisions (administrative areas) across Australia. The potentially available lignocellulosic biomass is estimated at approximately 80 Mt per year, with the major contributors of crop stubble (27.7 Mt per year), grasses (19.7 Mt per year) and forest plantations (10.9 Mt per year). Over the next 20–40 years, total potentially available biomass could increase to 100–115 Mt per year, with new plantings of short‐rotation trees being the major source of the increase (14.7 Mt per year by 2030 and 29.3 Mt per year by 2050). We exclude oilseeds, algae and ‘regrowth’, that is woody vegetation naturally regenerating on previously cleared land, which may be important in several regions of Australia (Australian Forestry 77 , 2014, 1; Global Change Biology Bioenergy 7 , 2015, 497). We briefly discuss some of the challenges to providing a reliable and sustainable supply of the large amounts of biomass required to build a bioenergy industry of significant scale. More detailed regional analyses, including of the costs of delivered biomass, logistics and economics of harvest, transport and storage, competing markets for biomass and a full assessment of the sustainability of production are needed to underpin investment in specific conversion facilities (e.g. Opportunities for forest bioenergy: An assessment of the environmental and economic opportunities and constraints associated with bioenergy production from biomass resources in two prospective regions of Australia, 2011a).     

Decidual cells in the human ovary at term. I. Incidence, gross anatomy and ultrastructural features of merocrine secretion

Decidual tissue occurring within the human ovarian cortex was examined by light and electron microscopy. Of 21 ovarian specimens obtained at term (36-42 weeks of gestation), decidual cells were confirmed in each. Decidual cells were found within the tunica albuginea as single cells, in nodules, in polyps or in confluent sheets. Decidual cells exhibited several characteristics of cells engaged in secretory activity: abundant rough and smooth endoplasmic reticulum, numerous profiles of the Golgi complex and a large, euchromatic nucleus devoid of heterochromatin and displaying a prominent fibrous lamina. Peduncular protrusions at the periphery of the cell contained numerous dense bodies 0.4-0.9 micron in diameter. These dense bodies were bounded by a single membrane and contained granular subunits 30-60 nm in diameter. These granular subunits were observed in the process of apparent exocytosis, as well as free in the extracellular space. Secretory bodies and their granular content also were observed both in the region of the Golgi complex and partially extruded into peduncular processes. By far the greatest number of secretory bodies occurred within peduncular processes where they may be stored prior to release. Migration of a secretory body into a peduncular process and exocytosis from such a process appears to be an unusual mode of meocrine secretion, perhaps unique to decidual cells.     

Calcium at the Cell Wall-Cytoplast Interface

Attention is given to the role of Ca2+ at the interface between the cell wall and the cytoplast, especially as seen in pollen tubes. While the cytoplasm directs the synthesis and deposition of the wall, it is less well appreciated that the wall exerts considerable self control and influences activities of the cytoplasm. Ca2+ participates as a crucial factor in this two way communication. In the cytoplasm, a [Ca2+] above 0.1 μM, regulates myriad processes, including secretion of cell wall components. In the ...     

RISK OF EXTIRPATION OF STELLER SEA LIONS IN THE GULF OF ALASKA AND ALEUTIAN ISLANDS: A POPULATION VIABILITY ANALYSIS BASED ON ALTERNATIVE HYPOTHESES FOR WHY SEA LIONS DECLINED IN WESTERN ALASKA

We estimated the risk that the Steller sea lion will be extirpated in western Alaska using a population viability analysis (PVA) that combined simulations with statistically fitted models of historical population dynamics. Our analysis considered the roles that density‐dependent and density‐independent factors may have played in the past, and how they might influence future population dynamics. It also established functional relationships between population size, population growth rate and the risk of extinction under alternative hypotheses about population regulation and environmental variability. These functional relationships can be used to develop recovery criteria and guide research and management decisions. Life table parameters (e.g., birth and survival rates) operating during the population decline (1978–2002) were estimated by fitting simple age‐structured models to time‐series of pup and non‐pup counts from 33 rookeries (subpopulations). The PVA was carried out by projecting all 33 subpopulations into the future using these estimated site‐specific life tables (with associated uncertainties) and different assumptions about carrying capacities and the presence or absence of density‐dependent population regulation. Results suggest that the overall predicted risk of extirpation of Steller sea lions as a species in western Alaska was low in the next 100 yr under all scenarios explored. However, most subpopulations of Steller sea lions had high probabilities of going extinct within the next 100 yr if trends observed during the 1990s were to continue. Two clusters of contiguous subpopulations occurring in the Unimak Pass area in the western Gulf of Alaska/eastern Aleutian Islands and the Seguam–Adak region in the central Aleutian Islands had relatively lower risks of extinction. Risks of extinction for a number of subpopulations in the Gulf of Alaska were reduced if the increases observed since the late 1990s continue into the future. The risks of subpopulations going extinct were small when density‐dependent compensation in birth and survival rates was assumed, even when random stochasticity in these vital rates was introduced.