α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

The effect of some ions on the activity of β-galactosidase and the inhibitory effect of tris (hydroxymethyl) aminomethane

The stimulating effect of phosphate and the inhibitory effect of tris-HCl on the activity of β-galactosidase inEscherichia coli was studied. The phosphate anion antagonizes the inhibitory effect of chloride. Since a similar effect is displayed by sulphate and arsenate no specific “stimulating” effect of phosphate can take place. The tris cation has also an inhibitory effect which is antagonized by univalent cations (K⁺). The resulting β-galactosidase activity reflects the antagonisms between cations and anions present in the reaction medium.     

Cutaneous thermal thresholds—the reproducibility of their measurements and the effect of gender

The present study evaluated the reproducibility of measurements of the forearm thresholds for warm (WT) and cold (CT) sensation, and their dependence on gender. The Middlesex Thermal Testing System was used for this purpose. CT did not differ between the five consecutive trials, whereas WT fell significantly. A minimum of two trial tests are therefore recommended prior to the assessment of WT. Furthermore, CT and WT were induced by significantly smaller skin temperature changes (lower thresholds) in females as compared to males. Should this gender specific difference in thermosensitivity also be observed in other skin regions, then females would be more sensitive to thermal stimulation than males.     

The “survivor effect” and the evolution of parthenogenesis and self-fertilization

Parthenogenetic or self-fertilizing species are often reduced to small numbers of survivors or colonists. These types of reproduction may therefore evolve because they eliminate problems of finding a mate. It is argued here that outbreeding populations which are often reduced to a few survivors will experience heavy offspring losses when population expansion occurs. This ‘survivor effect’ results from the production of many individuals homozygous for deleterious mutations (i.e. inbreeding depression). Parthenogenetic and selfing species do not experience a “survivor effect”. This effect together with mate-finding problems will tend to cause the evolution of parthenogenesis and selfing in weedy or colonizing species.     

Does detritus quality predict the effect of native and non‐native plants on the performance of larval amphibians?

1. The lack of consistent differences between the traits of native and non‐native plant species makes it difficult to make general predictions about the ecological impact of invasive plants; however, the increasing number of non‐native plants in many habitats makes the assessment of the impact of each individual species impracticable. General knowledge about how specific plant traits are linked to their effects on communities or ecosystems may be more useful for predicting the effect of plant invasions. Specifically, we hypothesised that higher carbon‐to‐nitrogen ratio (C:N) and percent lignin in plant detritus would reduce the rate of development and total mass at metamorphosis of tadpoles, resulting in lower metamorph production (total fresh biomass) and amphibian species richness. 2. To test these hypotheses, we raised five species of tadpoles in mesocosms containing senescent leaves of three common native and three common non‐native wetland plants that varied in C:N ratio and % lignin. 3. Leaf mass loss, total metamorph production and the number of species that metamorphosed declined as a function of increasing C:N ratio of plant leaves. Plant lignin content was not related to the production of metamorphs or the number of species that metamorphosed. The percentage of wood frog (Lithobates sylvaticus) and American toad (Anaxyrus americanus) tadpoles reaching metamorphosis declined as a function of increasing plant C:N ratio. Mean time to metamorphosis increased and mean mass at metamorphosis declined as a function of increasing plant C:N ratio. Tadpole performance and metamorph diversity and production (biomass) were similar between native and non‐native plant species with similar C:N ratio in leaves. Percent lignin was not a significant predictor of tadpole performance. 4. Our results show that the impact of a plant invasion on tadpole performance could depend on differences between the quality of the detritus produced by the invading species and that of the native species it replaces. We suggest that plant community changes that lead to dominance by more recalcitrant plant species (those with higher leaf C:N ratio) may negatively affect amphibian populations.     

Recent reports on the effect of low doses of ionizing radiation and its dose–effect relationship

Recently, the risk associated with low doses of ionizing radiation has gained new interest. Here, we analyze and discuss the major differences between two reports recently published on this issue; the report of the French Academy of Sciences and of the French Academy of Medicine published in March 2005, and the BEIR VII—Phase 2 Report of the American National Academy of Sciences published as a preliminary version in July 2005. The conclusion of the French Report is that the linear no-threshold relationship (LNT) may greatly overestimate the carcinogenic effect of low doses (<100 mSv) and even more that of very low doses (<10 mSv), such as those delivered during X-ray examinations. Conversely, the conclusion of the BEIR VII report is that LNT should be used for assessing the detrimental effects of these low and very low doses. The causes of these diverging conclusions should be carefully examined. They seem to be mostly associated with the interpretation of recent biological data. The point of view of the French Report is that these recent data are incompatible with the postulate on which LNT is implicitly based, namely the constancy of the carcinogenic effect per unit dose, irrespective of dose and dose rate.     

Study of the inhibitory effect of fatty acids on the interaction between DNA and polymerase β

The binding of human DNA polymerase β (pol β) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol β inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol β inhibitors. In the interaction between pol β and DNA, pol β could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol β inhibitors prevented the binding of pol β to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol β to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol β-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol β to DNA was mainly due to 7-fold increase in the dissociation rate. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 1000–1006. These authors contributed equally.     

The physical basis of alpha waves in the electroencephalogram and the origin of the “Berger effect”

Synchronised activity, differing in phase in different populations of neurons, plays an important role in existing theories on the function of brain oscillations (e.g., temporal correlation hypothesis). A prerequisite for this synchronisation is that stimuli are capable of affecting (resetting) the phase of brain oscillations. Such a change in the phase of brain waves is also assumed to underlie the Berger effect: when observers open their eyes, the amplitude of EEG oscillations in the alpha band (8–13 Hz) decreases significantly. This finding is usually thought to involve a desynchronisation of activity in different neurons. For functional interpretations of brain oscillations in the visual system, it therefore seems to be crucial to find out whether or not the phase of brain oscillations can be affected by visual stimuli. To answer this question, we investigated whether alpha waves are generated by a linear or a nonlinear mechanism. If the mechanism is linear – in contrast to nonlinear ones – phases cannot be reset by a stimulus. It is shown that alpha-wave activity in the EEG comprises both linear and nonlinear components. The generation of alpha waves basically is a linear process and flash-evoked potentials are superimposed on ongoing alpha waves without resetting their phase. One nonlinear component is due to light adaptation, which contributes to the Berger effect. The results call into question theories about brain-wave function based on temporal correlation or event-related desynchronisation.Electronic Supplementary Material: Supplementary material is available for this article at     

Molecular dynamics simulations of the effect of the G-protein and diffusible ligands on the β2-adrenergic receptor

G-protein-coupled receptors have extraordinary therapeutic potential as targets for a broad spectrum of diseases. Understanding their function at the molecular level is therefore essential. A variety of crystal structures have made the investigation of the inactive receptor state possible. Recently released X-ray structures of opsin and the β₂-adrenergic receptor (β₂AR) have provided insight into the active receptor state. In addition, we have contributed to the crystal structure of an irreversible agonist-β₂ adrenoceptor complex. These extensive studies and biophysical investigations have revealed that agonist binding leads to a low-affinity conformation of the active state that is suggested to facilitate G-protein binding. The high-affinity receptor state, which promotes signal transduction, is only formed in the presence of both agonist and G-protein. Despite numerous crystal structures, it is not yet clear how ligands tune receptor dynamics and G-protein binding. We have now used molecular dynamics simulations to elucidate the distinct impact of agonist and inverse agonist on receptor conformation and G-protein binding by investigating the influence of the ligands on the structure and dynamics of a complex composed of β₂AR and the C-terminal end of the Gα_s subunit (GαCT). The simulations clearly showed that the agonist isoprenaline and the inverse agonist carazolol influence the ligand-binding site and the interaction between β₂AR and GαCT differently. Isoprenaline induced an inward motion of helix 5, whereas carazolol blocked the rearrangement of the extracellular part of the receptor. Moreover, in the presence of isoprenaline, β₂AR and GαCT form a stable interaction that is destabilized by carazolol.     

The effect of environment on plasma cortisol and β-endorphin in the parturient pig and the involvement of endogenous opioids

Previous work has indicated that plasma cortisol increases during farrowing in the pig suggesting increasing physiological stress. The aim of this study was to determine changes in plasma cortisol and β-endorphin over farrowing in the pig to obtain a more detailed profile of pituitary and adrenal release at this time and also to investigate the involvement of endogenous opioids in the mediation of the HPA axis. Indwelling jugular catheters were implanted, under general anaesthesia, in 31 Large White×Landrace gilts approximately 15 days before the expected parturition day (EPD). Gilts were moved into either a farrowing crate, without straw (n=15), or a straw-bedded pen (n=16) 5 days before the EPD. Samples were taken during the pre-farrowing period and then during farrowing itself. At 7.5 min after the birth of the first piglet (BFP), gilts either received naloxone, an opioid antagonist, (1 mg kg⁻¹ body weight, i.v.) or a control dose of saline. Plasma β-endorphin increased following the BFP but remained fairly constant over the third and fourth hour of farrowing. Plasma cortisol continued to increase over the 4 h following the BFP. Changes seen in these hormones were generally insensitive to the environment and there was little evidence of opioid mediation of the HPA axis at parturition. From these results it is suggested that certain aspect(s) of parturition itself stimulate the HPA axis. However it is unknown if the rise in plasma cortisol is a result of some stress-inducing factor of the parturition process or whether it reflects a metabolic function. The study also demonstrates the lack of any inhibitory mediation of the HPA axis by endogenous opioids at parturition.     

The effect of the use of copper carbonate and copper nanoparticles in the diet of rats on the level of β-amyloid and acetylcholinesterase in selected organs

BackgroundCopper has an important role in nervous system function, as a cofactor of many enzymes and in the synthesis of neurotransmitters. Both the dose and the chemical form of copper can determine the impact of this element on metabolism, the neurological system and the immune system.AimsThe aim of the study was to determine whether and in what form the addition of copper changes the level of amyloid beta and acetylcholinesterase level in selected rat tissues.MethodsThirty, healthy, male, albino Wistar rats aged 7 weeks were randomly divided into 3 groups. Three experimental treatments were used to evaluate the effects of different levels and sources of Cu (6.5 mg kg of diet) in the diet: Cu₀ – rats fed a diet without Cu supplementation; Cu_salt – rats fed a diet with CuCO₃ (6.5 mg kg of diet) during two months of feeding; Cu_NPs - rats fed a diet with Cu nanoparticles (6.5 mg kg of diet) during two months of feeding. In blood serum and tissue homogenates there rated the indicators proving the potential neurodegenerative effect and epigenetic DNA damage induced by chemical form of copper or lack of additional copper supplementation in diet were determined. There were analysed: level of acetylcholinesterase, β-amyloid, low-density lipoprotein receptor-related protein 1, apyrimidinic endonuclease, thymidine glycosidase, alkylpurine-DNA-N-glycosylase and glycosylated acetylcholinesterase.ResultsIrrespective of the form of copper added, it was found to increase acetylcholinesterase level in the brain, spleen and liver, as well as in the blood plasma of the rats. Copper in the form of CuCO₃ was found to increase acetylcholinesterase level in the kidneys. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu. The addition of both forms of copper caused a marked increase in the plasma concentration of β-amyloid in comparison with the diet with no added Cu.ConclusionsA lack of added Cu in the diet of rats reduces the concentration of amyloid-β in the blood, whereas administration of copper, in the form of either CuNPs or CuCO₃, increases the level of this peptide in the blood. The use of copper in the form of CuNPs in the diet of rats does not increase the level of β-amyloid more than the use of the carbonate form of this element. The use of CuNPs or CuCO₃ in the diet of rats increases acetylcholinesterase level in the brain, spleen, liver, and blood. CuNPs in the diet of rats were not found to increase acetylcholinesterase level to a greater extent than Cu⁺² carbonate.     

Model of synthesis and release of insulin from rat islet β-cells and the effect of pretreatment with tolbutamide

A kinetic model involving synthesis of proinsulin in the rough endoplasmic reticulum, maturation through the Golgi apparatus and granules, with conversion to insulin, is proposed to account for data on the amount of insulin and of proinsulin both secreted during various time intervals and remaining in islets. Introducing three compartments for granules makes it possible to account for the measurement of both hot (pulse labeled with tritiated leucine) and cold proinsulin and insulin over a period of 21/2 hr under constant glucose. Data from islets from animals pretreated with tolbutamide are also presented and modeled. The model is then expanded so that it can be successfully applied to available data on the effects of a period of glucose deprivation on secretion of both hot and cold hormone. Parameters have essentially the same values, where they overlap, as were obtained (Landahl and Grodsky, 1982Bull. math. Biol. 44, 399–410) from insulin secretion by perfused rat pancreas stimulated by a variety of temporal patterns of glucose concentration.     

The effect of NBD-Cl in nucleotide-binding of the major subunit α and B of the motor proteins F<Subscript>1</Subscript>F<Subscript>O</Subscript> ATP synthase and A<Subscript>1</Subscript>A<Subscript>O</Subscript> ATP synthase

Subunit α of the Escherichia coli F₁F_O ATP synthase has been produced, and its low-resolution structure has been determined. The monodispersity of α allowed the studies of nucleotide-binding and inhibitory effect of 4-Chloro-7-nitrobenzofurazan (NBD-Cl) to ATP/ADP-binding. Binding constants (K _d ) of 1.6 μM of bound MgATP-ATTO-647N and 2.9 μM of MgADP-ATTO-647N have been determined from fluorescence correlation spectroscopy data. A concentration of 51 μM and 55 μM of NBD-Cl dropped the MgATP-ATTO-647N and MgADP-ATTO-647N binding capacity to 50% (IC₅₀), respectively. In contrast, no effect was observed in the presence of N,N′-dicyclohexylcarbodiimide. As subunit α is the homologue of subunit B of the A₁A_O ATP synthase, the interaction of NBD-Cl with B of the A-ATP synthase from Methanosarcina mazei Gö1 has also been shown. The data reveal a reduction of nucleotide-binding of B due to NBD-Cl, resulting in IC₅₀ values of 41 μM and 42 μM for MgATP-ATTO-647N and MgADP-ATTO-647N, respectively.     

The combined effect of acetylation and glycation on the chaperone and anti-apoptotic functions of human α-crystallin

N^ε-acetylation occurs on select lysine residues in α-crystallin of the human lens and alters its chaperone function. In this study, we investigated the effect of N^ε-acetylation on advanced glycation end product (AGE) formation and consequences of the combined N^ε-acetylation and AGE formation on the function of α-crystallin. Immunoprecipitation experiments revealed that N^ε-acetylation of lysine residues and AGE formation co-occurs in both αA- and αB-crystallin of the human lens. Prior acetylation of αA- and αB-crystallin with acetic anhydride (Ac₂O) before glycation with methylglyoxal (MGO) resulted in significant inhibition of the synthesis of two AGEs, hydroimidazolone (HI) and argpyrimidine. Similarly, synthesis of ascorbate-derived AGEs, pentosidine and N^ε-carboxymethyl lysine (CML), was inhibited in both proteins by prior acetylation. In all cases, inhibition of AGE synthesis was positively related to the degree of acetylation. While prior acetylation further increased the chaperone activity of MGO-glycated αA-crystallin, it inhibited the loss of chaperone activity by ascorbate-glycation in both proteins. BioPORTER-mediated transfer of αA- and αB-crystallin into CHO cells resulted in significant protection against hyperthermia-induced apoptosis. This effect was enhanced in acetylated and MGO-modified αA- and αB-crystallin. Caspase-3 activity was reduced in α-crystallin transferred cells. Glycation of acetylated proteins with either MGO or ascorbate produced no significant change in the anti-apoptotic function. Collectively, these data demonstrate that lysine acetylation and AGE formation can occur concurrently in α-crystallin of human lens, and that lysine acetylation improves anti-apoptotic function of α-crystallin and prevents ascorbate-mediated loss of chaperone function.     

The influence of the acyl chain composition of cardiolipin on the stability of mitochondrial complexes; An unexpected effect of cardiolipin in α-ketoglutarate dehydrogenase and prohibitin complexes

The role of cardiolipin acyl chain composition in assembly/stabilization of mitochondrial complexes was investigated using three yeast deletion mutants (acb1Δ strain; taz1Δ strain; and acb1Δtaz1Δ strain). Deletion of the TAZ1 gene, involved in cardiolipin acyl chain remodeling, is known to increase the content of monolyso-cardiolipin (MLCL) at the expense of CL, and to decrease the unsaturation of the remaining CL. Deletion of the ACB1 gene encoding the acyl-CoA-binding protein, involved in fatty acid elongation, decreases the average length of the CL acyl chains. Furthermore, a TAZ1ACB1 double deletion mutant strain was used in this study which has both a decrease in the length of the CL acyl chains and an increase in MLCL. BN/SDS PAGE analysis revealed that cardiolipin is important for the prohibitin–m-AAA protease complex, the α-ketoglutarate dehydrogenase complex and respiratory chain supercomplexes. The results indicate that the decreased level of complexes in taz1Δ and acb1Δtaz1Δ mitochondria is due to a decreased content of CL or the presence of MLCL.     

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1α on the distribution of these collagens

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion.     

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid β-spectrin on the properties of natural membranes and skeletal structures

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na⁺K⁺ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.     

Modulation of aggregation of silk fibroin by synergistic effect of the complex of curcumin and β-cyclodextrin

Amyloid aggregation has been associated with numerous human pathological diseases. A recent study has demonstrated that silk fibroin intermittently endorses amyloidogenesis in vivo. In the current study, we explored the propensity of silk fibroin to undergo amyloid-like aggregation and its prevention using an optimized concoction of curcumin with β-cyclodextrin. Aggregation of silk fibroin resulted in the formation of fibrils with a diameter of ~3.2 nm. However, addition of the optimized concentration of curcumin and β-cyclodextrin to silk fibroin inhibited aggregation and preserved the random coil conformation even under aggregation inducing conditions, as demonstrated by CD and FTIR spectroscopy. Benzene rings of curcumin interact with the aromatic residues of fibroin via hydrophobic interactions. However, β-cyclodextrin preferentially interacts with the non-polar residues, which are the core components for nucleation dependent protein aggregation. The present study demonstrates the ability of the concoction of curcumin and β-cyclodextrin in tuning the self assembly process of fibroin. It also provides a platform to explore the assembly process of nano-fibril and hierarchical structures in vitro along with a novel insight for designing clinically relevant silk-based functional biomaterials.