Ion Regulation of the Kinetics of Potential-Dependent Potassium Channels

We apply a theoretical approach developed earlier. The interaction ofions that permeate a channel with slowly relaxing charged channel-forminggroups (ion-conformational interaction – ICI) is addressed by thisapproach. One can describe the ion concentration influence (ion regulation)on channel functioning in this manner. A patch-clamp method in awhole-cell configuration is used to study the ICI. For this purpose theinfluence of an external concentration of potassium ions on thepotential-dependent potassium current (I_A) in the externalmembrane of GH₃ cells was studied. The increase of[K⁺ _out] from 5 mM to 100 mM causes anon-monotonous shift of current-voltage dependencies. The dependence of bothan activation time constant tgr_n and a steady-state activation(n) on [K⁺]_out have a minimum andmaximum respectively. The analysis of the results suggests that the observedeffects are caused by ICI. A physical model is developed to describe thedependence of the potassium channel kinetics on the external concentrationof the ions and the membrane potential. The deformation of the closedstate of the gate and the corresponding energy shifts cause the observednon-monotonous dependencies due to ICI. Thus, the general theoreticalapproach has an experimental confirmation and is applied to concreteexamples. Formulas for concentrational dependencies of the channel kineticsare given for practical uses.     

Structural, thermodynamic, and cellular characterization of human centrin 2 interaction with xeroderma pigmentosum group C protein

Human centrin 2 (HsCen2), an EF-hand calcium binding protein, plays a regulatory role in the DNA damage recognition during the first steps of the nucleotide excision repair. This biological action is mediated by the binding to a short fragment (N847-R863) from the C-terminal region of xeroderma pigmentosum group C (XPC) protein. This work presents a detailed structural and energetic characterization of the HsCen2/XPC interaction. Using a truncated form of HsCen2 we obtained a high resolution (1.8 A) X-ray structure of the complex with the peptide N847-R863 from XPC. Structural and thermodynamic analysis of the interface revealed the existence of both electrostatic and apolar inter-molecular interactions, but the binding energy is mainly determined by the burial of apolar bulky side-chains into the hydrophobic pocket of the HsCen2 C-terminal domain. Binding studies with various peptide variants showed that XPC residues W848 and L851 constitute the critical anchoring side-chains. This enabled us to define a minimal centrin binding peptide variant of five residues, which accounts for about 75% of the total free energy of interaction between the two proteins. Immunofluorescence imaging in HeLa cells demonstrated that HsCen2 binding to the integral XPC protein may be observed in living cells, and is determined by the same interface residues identified in the X-ray structure of the complex. Overexpression of XPC perturbs the cellular distribution of HsCen2, by inducing a translocation of centrin molecules from the cytoplasm to the nucleus. The present data confirm that the in vitro structural features of the centrin/XPC peptide complex are highly relevant to the cellular context.     

Physical exercise prevents alterations in purinergic system and oxidative status in lipopolysaccharide-induced sepsis in rats

Sepsis is a generalized infection that involves alterations in inflammatory parameters, oxidant status, and purinergic signaling in many tissues. Physical exercise has emerged as a tool to prevent this disease because of its anti-inflammatory and antioxidant properties. Thus, in this study, we investigated the effects of physical exercise on preventing alterations in purinergic system components, oxidative stress, and inflammatory parameters in lipopolysaccharide (LPS)-induced sepsis in rats. Male Wistar rats were divided into four groups: control, exercise (EX), LPS, and EX+LPS. The resisted physical exercise was performed for 12 weeks on a ladder with 1 m height. After 72 hours of the last exercise session, the animals received 2.5 mg/kg of LPS for induction of sepsis, and after 24 hours, lungs and blood samples were collected for analysis. The results showed that the exercise protocol used was able to prevent, in septic animals: (1) the increase in body temperature; (2) the increase of lipid peroxidation and reactive species levels in the lung, (3) the increase in adenosine triphosphate levels in serum; (4) the change in the activity of the enzymes ectonucleotidases in lymphocytes, partially; (5) the change in the density of purinergic enzymes and receptors in the lung, and (6) the increase of IL-6 and IL-1β gene expression. Our results revealed the involvement of purinergic signaling and oxidative damage in the mechanisms by which exercise prevents sepsis aggravations. Therefore, the regular practice of physical exercise is encouraged as a better way to prepare the body against sepsis complications.     

Sucrose Phosphate Synthase, Sucrose Synthase, and Invertase Activities in Developing Fruit of Lycopersicon esculentum Mill. and the Sucrose Accumulating Lycopersicon hirsutum Humb. and Bonpl

The green-fruited Lycopersicon hirsutum Humb. and Bonpl. accumulated sucrose to concentrations of about 118 micromoles per gram fresh weight during the final stages of development. In comparison, Lycopersicon esculentum Mill. cultivars contained less than 15 micromoles per gram fresh weight of sucrose at the ripe stage. Glucose and fructose levels remained relatively constant throughout development in L. hirsutum at 22 to 50 micromoles per gram fresh weight each. Starch content was low even at early stages of development, and declined further with development. Soluble acid invertase (EC 3.2. 1.26) activity declined concomitant with the rise in sucrose content. Acid invertase activity, which was solubilized in 1 molar NaCl (presumably cell-wall bound), remained constant throughout development (about 3 micromoles of reducing sugars (per gram fresh weight) per hour. Sucrose phosphate synthase (EC 2.4.1.14) activity was present at about 5 micromoles of sucrose (per gram fresh weight) per hour even at early stages of development, and increased sharply to about 40 micromoles of sucrose (per gram fresh weight) per hour at the final stages of development studied, parallel to the rise in sucrose content. In comparison, sucrose phosphate synthase activity in L. esculentum remained low throughout development. The possible roles of the sucrose metabolizing enzymes in determining sucrose accumulation are discussed.     

Self-organization effects induced by ion-conformational interaction in biomembrane channels

It is shown that phenomena of molecular self-organization can take place in biomembrane channels. A strong interaction between charged groups of channel-forming protein and ion flux passing through the channel causes bistable or steady limit cycle regimes. These regimes are possible only far from the equilibrium state when the ion flux exceeds a certain threshold value. A number of such synergetic models is reviewed. Each time, only the simplest (monostable) regime takes place near the zero value of the ion flux. Some experimental data may be explained by this approach.     

Effects of bud phenology and foliage chemistry of balsam fir and white spruce trees on the efficacy of Bacillus thuringiensis against the spruce budworm, Choristoneura fumiferana

Abstract 1 Efficacy of commercial formulations of Bacillus thuringiensis ssp. kurstaki (Btk) against spruce budworm Choristoneura fumiferana was investigated in mixed balsam fir‐white spruce stands. Btk treatments were scheduled to coincide with early flaring of balsam fir shoots, and later with flaring of white spruce shoots. Btk efficacy on the two host trees was compared and examined according to the foliar content of nutrients and allelochemicals and the insect developmental stage at the time of spray. 2 Larvae fed white spruce foliage were less vulnerable to Btk ingestion than larvae fed balsam fir foliage. Higher larval survival on white spruce, observed 10 days after spray, was related to higher foliage content in tannins and a lower N/tannins ratio, which might have induced inactivation of Btk toxins. 3 Larval mortality due to Btk did not depend on spruce budworm larval age. 4 Foliage protection of both host trees was similar in plots treated with Btk: larval mortality due to Btk treatment reduced insect grazing pressure on balsam fir trees; meanwhile, suitability of white spruce foliage seemed to decrease very rapidly, which induced high larval mortality among spruce budworm fed on white spruce trees. Nevertheless, following Btk sprays, 50% more foliage remained on white spruce than on balsam fir trees, because of the higher white spruce foliage production. 5 Both spray timings achieved similar protection of white spruce trees, but Btk treatments had to be applied as early as possible (i.e. during the flaring of balsam fir shoots to optimally protect balsam fir trees in mixed balsam fir‐white spruce stands).     

Acute effects of glyphosate herbicide on metabolic and enzymatic parameters of silver catfish (Rhamdia quelen)

Silver catfish (Rhamdia quelen; Teleostei) were exposed to commercial formulation Roundup, a glyphosate herbicide: 0 (control), 0.2 or 0.4 mg/L for 96 h. Fish exposed to glyphosate showed an increase in hepatic glycogen, but a reduction in muscle glycogen at both concentrations tested. Glucose decreased in liver and increased in muscle of fish at both herbicide concentrations. Glyphosate exposure increased lactate levels in liver and white muscle at both concentrations. Protein levels increased in liver and decreased in white muscle while levels of ammonia in both tissues increased in fish at both glyphosate concentrations. Specific AChE activity was reduced in brain after treatments, no changes were observed in muscle tissue. Catalase activity in liver did not change during of exposure. Fish exposed to glyphosate demonstrated increased TBARS production in muscle tissue at both concentrations tested. For both glyphosate concentrations tested brain showed a reduction of TBARS after 96 h of exposure. The present results showed that in 96 h, glyphosate changed AChE activity, metabolic parameters and TBARS production. The parameters measured can be used as herbicide toxicity indicators considering environmentally relevant concentration.