Apoptosis and Its Suppression in Hepatocytes Culture

In order to achieve the goal of developing extracorporeal liver support devices, it is necessary to optimise bioprocess environment such that viability and function are maximised. Optimising culture medium composition and controlling the constitution of the cellular microenvironment within the bioreactor have for many years been considered vital to achieving these aims. Coupled to this is the need to understand apoptosis, the prime suspect in the demise of animal cultures, including those of hepatocytes. Results presented here show that absent nutrients including glucose and amino acids play a substantial part in the induction of apoptosis. The use of chemical apoptosis inhibitors was utilised to investigate key components of hepatic apoptosis where caspases, predominantly caspase 8, were implicated in staurosporine (STS)-induced HepZ apoptosis. Caspase 9 and 3 activation although recorded was of less significance. Interestingly, these results were not consistent with those of mitochondrial membrane depolarisation where inhibition of caspase activation appeared to drive depolarisation. Inhibition of mitochondrial permeability transition and use of anti-oxidants was unsuccessful in reducing apoptosis, caspase activation and mitochondrial membrane depolarisation. In further studies, the anti-apoptotic gene bcl-2 was over-expressed in HepZ, resulting in a cell line that was more robust and resistant to death induced by glucose and cystine deprivation and treatment with STS. Bcl-2 did not however show significant cytoprotectivity where apoptosis was stimulated by deprivation of glutamine and serum. Overall, results indicated that although apoptosis can be curbed by use of chemical inhibitors and genetic manipulation, their success is dependent on apoptotic stimuli.     

Is adaptation to climate change really constrained in niche specialists?

Species with restricted distributions make up the vast majority of biodiversity. Recent evidence suggests that Drosophila species with restricted tropical distributions lack genetic variation in the key trait of desiccation resistance. It has therefore been predicted that tropically restricted species will be limited in their evolutionary response to future climatic changes and will face higher risks of extinction. However, these assessments have been made using extreme levels of desiccation stress (less than 10% relative humidity (RH)) that extend well beyond the changes projected for the wet tropics under climate change scenarios over the next 30 years. Here, we show that significant evolutionary responses to less extreme (35% RH) but more ecologically realistic levels of climatic change and desiccation stress are in fact possible in two species of rainforest restricted Drosophila. Evolution may indeed be an important means by which sensitive rainforest-restricted species are able to mitigate the effects of climate change.     

TRPV1 activation impedes foam cell formation by inducing autophagy in oxLDL-treated vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. The mechanisms underlying VSMC foam cell formation are relatively little known. Activation of transient receptor potential vanilloid subfamily 1 (TRPV1) and autophagy have a potential role in regulating foam cell formation. Our study demonstrated that autophagy protected against foam cell formation in oxidized low-density lipoprotein (oxLDL)-treated VSMCs; activation of TRPV1 by capsaicin rescued the autophagy impaired by oxLDL and activated autophagy–lysosome pathway in VSMCs; activation of TRPV1 by capsaicin impeded foam cell formation of VSMCs through autophagy induction; activation of TRPV1 by capsaicin induced autophagy through AMP-activated protein kinase (AMPK) signaling pathway. This study provides evidence that autophagy plays an important role in VSMC foam cell formation and highlights TRPV1 as a promising therapeutic target in atherosclerosis.     

Inhibition of large-conductance calcium-activated potassium channel by 2-methoxyestradiol in cultured vascular endothelial (HUV-EC-C) cells

2-Methoxyestradiol, an endogenous metabolite of 17β-estradiol, is known to have antitumor and antiangiogenic actions. The effects of 2-methoxyestradiol on ionic currents were investigated in an endothelial cell line (HUV-EC-C) originally derived from human umbilical vein. In the whole-cell patch-clamp configuration, 2-methoxyestradiol (0.3–30 μm) reversibly suppressed the amplitude of K⁺ outward currents. The IC ₅₀ value of the 2-methoxyestradiol-induced decrease in outward current was 3 μm. Evans blue (30 μm) or niflumic acid (30 μm), but not diazoxide (30 μm), reversed the 2-methoxyestradiol-induced decrease in outward current. In the inside-out configuration, application of 2-methoxyestradiol (3 μm) to the bath did not modify the single-channel conductance of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels; however, it did suppress the channel activity. 2-Methoxyestradiol (3 μm) produced a shift in the activation curve of BK_Ca channels to more positive potentials. Kinetic studies showed that the 2-methoxyestradiol-induced inhibition of BK_Ca channels is primarily mediated by a decrease in the number of long-lived openings. 2-Methoxyestradiol-induced inhibition of the channel activity was potentiated by membrane stretch. In contrast, neither 17β-estradiol (10 μm) nor estriol (10 μm) affected BK_Ca channel activity, whereas 2-hydroxyestradiol (10 μm) slightly suppressed it. Under current-clamp condition, 2-methoxyestradiol (10 μm) caused membrane depolarization and Evans blue (30 μm) reversed 2-methoxyestradiol-induced depolarization. The present study provides evidence that 2-methoxyestradiol can suppress the activity of BK_Ca channels in endothelial cells. These effects of 2-methoxyestradiol on ionic currents may contribute to its effects on functional activity of endothelial cells. Received: 27 November 2000/Revised: 13 April 2001     

Epithelial Fluid Transport: Protruding Macromolecules and Space Charges Can Bring about Electro-Osmotic Coupling at the Tight Junctions

The purpose of the present work is to investigate whether the idea of epithelial fluid transport based on electro-osmotic coupling at the level of the leaky tight junction (TJ) can be further supported by a plausible theoretical model. We develop a model for fluid transport across epithelial layers based on electro-osmotic coupling at leaky tight junctions (TJ) possessing protruding macromolecules and fixed electrical charges. The model embodies systems of electro-hydrodynamic equations for the intercellular pathway, namely the Brinkman and the Poisson-Boltzmann differential equations applied to the TJ. We obtain analytical solutions for a system of these two equations, and are able to derive expressions for the fluid velocity profile and the electrostatic potential. We illustrate the model by employing geometrical parameters and experimental data from the corneal endothelium, for which we have previously reported evidence for a central role for electro-osmosis in translayer fluid transport. Our results suggest that electro-osmotic coupling at the TJ can account for fluid transport by the corneal endothelium. We conclude that electro-osmotic coupling at the tight junctions could represent one of the basic mechanisms driving fluid transport across some leaky epithelia, a process that remains unexplained.     

A membrane potential threshold for phage T4 DNA injection

DNA penetration from T4 phage adsorbed to $\text{Escherichia coli}$ was measured at different membrane potentials. There was a precipitous reduction in DNA penetration between 110 mV and 60 mV. This threshold of membrane potential for DNA penetration is independent of ΔpH and rather insensitive to external pH between 6 and 8.     

红壤茶树根层土壤基础呼吸作用和酶活性

对不同树龄茶树根层土壤的呼吸作用(包括代谢熵qCO2)和土壤酶(脲酶、转化酶和酸性磷酸单酯酶)活性进行了研究、不同树龄茶树根层土壤日基础呼吸作用强度(36．23—58．52mg·kg^-1·d^-1)和日代谢墒(0．30一0．68)都以40和90年茶树较为接近,分别显著大于和小于10年树龄茶树根层土壤;服酶活性(41．48—47、72mg·kg^-1·d^-1)则三者间差异不大,虽然随树龄增长而下降;转化酶活性(189．29—363．40mg·kg^-1·d^-1)也随树龄增长而下降,并且10年茶树根层土壤显著大于40和90年树龄茶树;而酸性磷酸单酯酶活性(444．22—828．32mg·kg^-1·d^-1)相反,随树龄增长而增强．结果表明,土壤基础呼吸作用、代谢熵和3种土壤酶活性都与茶树树龄、土壤pH、土壤有机碳、土壤全氮、土壤可活性酚总量、及土壤微生物生物量密切相关．     

Interspecific Differences of Leaf Gas Exchange and Water Relations of Three Evergreen Mediterranean Shrub Species

Leaf gas exchange and plant water relations of three co-occurring evergreen Mediterranean shrubs species, Quercus ilex L. and Phillyrea latifolia L. (typical evergreen sclerophyllous shrubs) and Cistus incanus L. (a drought semi-deciduous shrub), were investigated in order to evaluate possible differences in their adaptive strategies, in particular with respect to drought stress. C. incanus showed the highest annual rate of net photosynthetic rate (P _N) and stomatal conductance (g _s) decreasing by 67 and 69 %, respectively, in summer. P. latifolia and Q. ilex showed lower annual maximum P _N and g _s, although P _N was less lowered in summer (40 and 37 %, respectively). P. latifolia reached the lowest midday leaf water potential (₁) during the drought period (–3.54±0.36 MPa), 11 % lower than in C. incanus and 19 % lower than in Q. ilex. Leaf relative water content (RWC) showed the same trend as ₁. C. incanus showed the lowest RWC values during the drought period (60 %) while they were never below 76 % in P. latifolia and Q. ilex; moreover C. incanus showed the lowest recovery of ₁ at sunset. Hence the studied species are well adapted to the prevailing environment in Mediterranean climate areas, but they show different adaptive strategies that may be useful for their co-occurrence in the same habitat. However, Q. ilex and P. latifolia by their water use strategy seem to be less sensitive to drought stress than C. incanus.     

The ecosystem wilting point defines drought response and recovery of a Quercus-Carya forest

Soil and atmospheric droughts increasingly threaten plant survival and productivity around the world. Yet, conceptual gaps constrain our ability to predict ecosystem-scale drought impacts under climate change. Here, we introduce the ecosystem wilting point (Ψ_EWP), a property that integrates the drought response of an ecosystem's plant community across the soil–plant–atmosphere continuum. Specifically, Ψ_EWP defines a threshold below which the capacity of the root system to extract soil water and the ability of the leaves to maintain stomatal function are strongly diminished. We combined ecosystem flux and leaf water potential measurements to derive the Ψ_EWP of a Quercus-Carya forest from an “ecosystem pressure–volume (PV) curve,” which is analogous to the tissue-level technique. When community predawn leaf water potential (Ψ_pd) was above Ψ_EWP (=−2.0 MPa), the forest was highly responsive to environmental dynamics. When Ψ_pd fell below Ψ_EWP, the forest became insensitive to environmental variation and was a net source of carbon dioxide for nearly 2 months. Thus, Ψ_EWP is a threshold defining marked shifts in ecosystem functional state. Though there was rainfall-induced recovery of ecosystem gas exchange following soaking rains, a legacy of structural and physiological damage inhibited canopy photosynthetic capacity. Although over 16 growing seasons, only 10% of Ψ_pd observations fell below Ψ_EWP, the forest is commonly only 2–4 weeks of intense drought away from reaching Ψ_EWP, and thus highly reliant on frequent rainfall to replenish the soil water supply. We propose, based on a bottom-up analysis of root density profiles and soil moisture characteristic curves, that soil water acquisition capacity is the major determinant of Ψ_EWP, and species in an ecosystem require compatible leaf-level traits such as turgor loss point so that leaf wilting is coordinated with the inability to extract further water from the soil.