Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 μM, GE prevents oxidative stress and MPT induced by supraphysiological Ca²⁺ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca²⁺. GE also inhibits Ca²⁺ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca²⁺ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.     

The cardiac ryanodine receptor: Looking for anomalies in permeation properties

Anomalies in the permeation properties of the cardiac RyR channel reconstituted into bilayer lipid membranes were investigated systematically. We tested the presence of the anomalous mole fraction effect (AMFE) for the ion conductance and the reversal potential with varying mole fractions of two permeant ions, while the total ion concentration was lower, as in previous studies, to avoid the masking effect of the channel pore saturation with ions. Mixtures of Ba²⁺ with other divalents (Ca²⁺, Sr²⁺), of Ca²⁺ with monovalents (Li⁺, Cs⁺), and of Na⁺ with other monovalents (Cs⁺, Li⁺) were used. We revealed a clear anomaly only for the ion conductance measured in the Na⁺-Cs⁺ and Ca²⁺-Li⁺ mixtures as computed by a Poisson-Nernst-Planck/density functional theory (PNP/DFT) model. Furthermore, we found a significant minimum in the concentration dependence of the reversal potential determined under Li⁺/Ca²⁺ bi-ionic conditions. Our study led to new observations that may have important implications for understanding the mechanisms involved in ion handling in the RyR channel pore; furthermore our results could be useful for further validation of ion permeation models developed for the RyR channel.     

Single-molecule investigation of the interactions between reconstituted planar lipid membranes and an analogue of the HP(2-20) antimicrobial peptide

In this study, we employed electrophysiology experiments carried out at the single-molecule level to study the mechanism of action of the HPA3 peptide, an analogue of the linear antimicrobial peptide, HP(2–20), isolated from the N-terminal region of the Helicobacter pylori ribosomal protein. Amplitude analysis of currents fluctuations induced by HPA3 peptide at various potentials in zwitterionic lipid membranes reveal the existence of reproducible conductive states in the stochastic behavior of such events, which directly supports the existence of transmembrane pores induced the peptide. From our data recorded both at the single-pore and macroscopic levels, we propose that the HPA3 pore formation is electrophoretically facilitated by trans-negative transmembrane potentials, and HPA3 peptides translocate into the trans monolayers after forming the pores. We present evidence according to which the decrease in the membrane dipole potential of a reconstituted lipid membranes leads to an augmentation of the membrane activity of HPA3 peptides, and propose that a lower electric dipole field of the interfacial region of the membrane caused by phloretin facilitates the surface-bound HPA3 peptides to break free from one leaflet of the membrane, insert into the membrane and contribute to pore formation spanning the entire thickness of the membrane.     

The specific activation of TRPC4 by Gi protein subtype

The classical type of transient receptor potential channel (TRPC) is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Especially, TRPC4 has the similar properties to Ca²⁺-permeable nonselective cation channels (NSCCs) activated by muscarinic stimulation in visceral smooth muscles. In visceral smooth muscles, NSCCs activated by muscarinic stimulation were blocked by anti-Gαi/o antibodies. However, there is still no report which Gα proteins are involved in the activation process of TRPC4. Among Gα proteins, only Gαi protein can activate TRPC4 channel. The activation effect of Gαi was specific for TRPC4 because Gαi has no activation effect on TRPC5, TRPC6 and TRPV6. Coexpression with muscarinic receptor M2 induced TRPC4 current activation by muscarinic stimulation with carbachol, which was inhibited by pertussis toxin. These results suggest that Gαi is involved specifically in the activation of TRPC4.     

Recovery plan for an exploited species, southern bluefin tuna

Southern bluefin tuna (SBT) were heavily depleted in the mid-1980s, and the fishing quota has been restricted since 1985. As a result of this restriction and protection of immature individuals, spawning stock biomass (SSB) recently has shown a slight increase. The Commission for the Conservation of Southern Bluefin Tuna (CCSBT) has the target of recovering SSB to the 1980 level by 2020. We investigated whether SBT populations will recover and reach the target level set by CCSBT. Our projection shows that the SSB of the SBT will temporarily decrease again after 1999. This temporary decrease of SSB does not always mean failure of the recovery plan, because the SSB trend is highly vulnerable to age-composition dynamics. The SBT is an example of this. The number of mature SBT was small during the 1980s because of overfishing. Thus, the number of eggs that were spawned by these mature SBT was small in these years, and when these small numbers of immature fish become mature, the SSB will decrease again. We call this effect the inverse baby-boom effect. The inverse baby-boom effect may be common for managed bioresources that have once been overexploited. We also examine the use of spawning potential (SP) and SSB as an index of stock recovery. Received: August 17, 2000 / Accepted: March 19, 2001     

The influence of leaf age on the oviposition preference of Chrysophtharta bimaculata (Olivier) and the establishment of neonates

1 The degree of discrimination shown by a herbivore when selecting oviposition sites has been suggested as a key factor to understanding herbivore population dynamics. Chrysophtharta bimaculata (Coleoptera: Chrysomelidae) is a primary pest of Tasmanian eucalypt forests and can cause severe defoliation. Previous work suggests that females show discrimination when selecting oviposition sites. Our aim was to test the degree of oviposition discrimination exhibited by C. bimaculata with regards to leaf toughness, a character that is critical to neonate survival. 2 We conducted an experiment examining the leaf toughness critical for neonate survival and found that significant larval mortality occurs above a toughness of 46.9 g. We also determined that the maximum toughness of leaves upon which larvae established in the field was 48.2 g, supporting the laboratory result. 3 Field surveys showed that although the majority of eggs were laid on leaves suitable for larval establishment, many eggs were laid on unsuitable, tougher leaves. However, all eggs were normally placed within 20 cm of suitable leaves and glasshouse trials demonstrated the neonates could move this distance without mortality occurring. 4 We conclude that egg batch distribution and larval performance of C. bimaculata will influence the population dynamics of C. bimaculata in two ways. Firstly, the availability of expanding/newly expanding leaves of eucalypt hosts will determine larval carrying capacity. Secondly, at a more localized level, the deposition of large numbers of egg batches on both suitable and unsuitable leaves followed by successful neonate migration increases the risk of resource depletion and poor larval development.     

诱发电位评价脑组织血流量的实验研究

目的：观察实验性大鼠脑损伤后不同时相点大脑皮层体感诱发电位（sensorysomaticevoked potentials,ssep)和局部血流量（regional cerebral blood flow,rCBF)的变化。方法：用流体冲击装置制作中度脑损伤模型SYD4200型神经诱发电位诊断系统监测皮层体感诱发电位,氢清除测定大脑局部血流量。结果：中度脑损伤后rCBF明显低于伤前和正常对照组;大脑皮层体感诱发电位的潜伏期明显延长。结论：SEP的变化与脑血流量有着一定的关系,一定程度上SEP的变化可反映脑损伤后血流量的变化。     

Seasonal dynamics of phytoplankton and phytobenthos,and associated chemical interactions,in a shallow upland lake (Malham Tarn,northern England)

Seasonal changes of phytoplankton were followed over 3 years (1985–87) in a shallow, unstratified and calcareous upland lake.The phytoplankton was of low to moderate abundance and generally dominated by phytoflagellates. Seasonality involved a winter minimum of abundance, a spring maximum of diatoms, and often brief increases in summer that included blue-greens, especially the colonial Gloeotrichia echinulata. Some components were of benthic origin. Seasonal growth of the main component of the phytobenthos, Chara globularisvar. virgata, caused a regular summer depletion in lake water of Ca²⁺ and HCO₃ ^- (alkalinity) by associated CaCO₃ deposition, and a more extreme (and unusual) depletion of K⁺. Chemical analysis of Chara biomass and of underlying sediments indicated a large benthic nutrient stock, much surpassing that represented by the phytoplankton. Growth in this biomass, and the magnitude of water-borne inputs, influenced the removals of Ca²⁺, K⁺ and inorganic N. The phytoplankton was probably limited by a low-P medium, to which co-precipitation of phosphate with CaCO₃ may have contributed. A vernal depletion of Si was probably limiting to diatom growth, and appeared to be mainly induced by benthic rather than planktonic diatoms. Examples of long-term change in composition of the phytoplankton and phytobenthos are noted and discussed in relation to the interaction of these components, nutrient enrichment, and possible alternative stable states.     

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments

The one-electron reduction potentials of the radical cations of five dietary carotenoids (β-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980–1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.