Effects of non-ionogenic surface active compounds on yeast protoplasts]

The lytic effect of non-ionogenic surface active compounds (SAC), based on polyoxyethylated fatty acids and alkylphenols on the yeast protoplast cytoplasmic membranes and the extracting ability of the SAC with respect to intracellular proteins of intact yeast cells, were studied. It was shown that the lytic activity of the SAC under study depends on the overall effect of the size of their hydrophobic and hydrophylic fragments rather than on the level of the hydrophylic-lypolytic equilibrium of SAC. The absence of correlation between the lytic activity and the extracting ability of SAC is accounted for by the differences in the mechanisms of membrane degradation under the action of SAC on the protoplasts and intact cells. The data obtained support the previously made assumption that the correlation between the size of SAC mycelles and that of the cell wall pores is the limiting factor of the SAC induced protein extraction from the intact cells.     

Peculiar reversed-phase HPLC behaviour of cationic alkylcobalt(III) complexes holding a tridentate Schiff base ligand

Reversed-phase (RP) chromatographic behaviour of a series of acid-sensitive cationic alkylcobalt(III) chelates with both [N₂O] Schiff base and ethylenediamine has been studied. Their retention times depend on the water content of the mixed eluents in an unusual parabolic manner, which is ascribed to the biphylic nature of the structures in question. Optimal conditions for RP HPLC quantitative analysis of these rather labile organocobalt complexes have been developed. Their decomposition in solutions under ambient conditions has been surveyed using this technique.     

Natural killer cells can enhance the proliferative responses of B lymphocytes

In addition to lytic activity against malignant and virally transformed target cells, recent evidence has suggested that natural killer (NK) cells can modulate immune activities such as the suppression of B cell responses through noncytotoxic means. Using human B cells and highly purified autologous NK cells, we have demonstrated that NK cells can substantially augment the proliferative responses of B cells stimulated with the surface immunoglobulin crosslinking agents anti-IgM or Staphylococcus aureus Cowan strain I (SAC). This "enhancer" activity of NK cells was quite potent and was observed at an NK:B cell ratio as low as 0.05. Peak blastogenic responses of B cells cocultured with NK cells in the presence of B cell activators were observed at 2-3 days, similar to the responses of B cells in the absence of NK cells. Using the inhibitor of DNA synthesis mitomycin C, we determined that B cells and not NK cells were proliferating in cocultures of these lymphocytes stimulated with SAC. Activated B cells neither prevented the lysis of the isotope-labeled NK-sensitive target cell line K562 nor formed conjugates with NK cells, suggesting that cell contact was not a prerequisite for the effect. These studies have further expanded the functional repertoire of NK cells to include enhancer as well as suppressor and lytic activities.     

S-allyl cysteine prevents CCl(4)-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50-200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

S-allyl cysteine prevents CCl4-induced acute liver injury in rats

Aged garlic extract (AGE) possesses multiple biological activities. We evaluated the protective effect of S-allyl cysteine (SAC), one of the organosulfur compounds of AGE, against carbon tetrachloride (CCl₄)-induced acute liver injury in rats. SAC was administrated intraperitoneally (50–200 mg/kg). SAC significantly suppressed the increases of plasma ALT and LDH levels. SAC also attenuated histological liver damage. CCl₄ administration induced lipid peroxidation accompanied by increases in the plasma malondialdehyde and hepatic 4-hydroxy-2-nonenal levels, and SAC dose-dependently attenuated these increases. The hepatic total level of hydroxyoctadecadienoic acid (HODE), a new oxidative stress biomarker, was closely correlated with the amount of liver damage. These results suggest that SAC decreased CCl₄-induced liver injury by attenuation of oxidative stress, and may be a better therapeutic tool for chronic liver disease.     

Protective effect of S-allyl-l-cysteine against endoplasmic reticulum stress-induced neuronal death is mediated by inhibition of calpain

Endoplasmic reticulum (ER) stress, implicated in various neurodegenerative processes, increases the level of intracellular Ca²⁺ and leads to activation of calpain, a Ca²⁺-dependent cysteine protease. We have shown previously that S-allyl-l-cysteine (SAC) in aged garlic extracts significantly protects cultured rat hippocampal neurons (HPNs) against ER stress-induced neurotoxicity. The neuroprotective effect of SAC was compared with those of the related antioxidant compounds, l-cysteine (CYS) and N-acetylcysteine (NAC), on calpain activity in HPNs and also in vitro. SAC, but not CYS or NAC, reversibly restored the survival of HPNs and increased the degradation of α-spectrin, a substrate for calpain, induced by tunicamycin, a typical ER stress inducer. Activities of μ- and m-calpains in vitro were also concentration dependently suppressed by SAC, but not by CYS or NAC. At submaximal concentration, although ALLN (5 pM), which blocks the active site of calpain, and calpastatin (100 pM), an endogenous calpain-inhibitor protein, additively inhibited μ-calpain activity in vitro in combination with SAC, the effect of PD150606 (25 μM), which prevents interaction of Ca²⁺ with the Ca²⁺-binding site of calpain, was unaffected by SAC. In contrast, SAC (1 mM) significantly reversed the effect of PD150606 at a concentration that elicited supramaximal inhibition (100 μM), but did not affect ALLN (1 nM)- and calpastatin (100 nM)-induced inhibition of μ-calpain activity. These results suggest that the protective effects of SAC against ER stress-induced neuronal cell death are not attributable to antioxidant activity, but to suppression of calpain through interaction with its Ca²⁺-binding site.     

Role of cationic residues in cytolytic activity: modification of lysine residues in the cardiotoxin from Naja nigricollis venom and correlation between cytolytic and antiplatelet activity

Cardiotoxins and postsynaptic neurotoxins from snake venoms have similar primary, secondary, and tertiary structures. Cardiotoxins, however, in contrast to neurotoxins, exhibit general cytotoxicity. Comparison of the distribution of hydrophobic and charged amino acid residues in the three-dimensional structures of lytic cardiotoxins and nonlytic neurotoxins indicates the presence of a cationic site associated with a hydrophobic surface in cardiotoxins, but not in neurotoxins. A cationic site flanked by a hydrophobic site is a common structural feature shared by a wide variety of unrelated cytolysins and is predicted to determine the lytic activity of a large group of cytolysins. To determine the essential nature of the cationic site in cardiotoxin CTX-1 from Naja nigricollis crawshawii venom, we modified the positive charges of nine Lys residues to negative, neutral, or positive charges by succinylation, carbamylation, or guanidination, respectively. Circular dichroism studies indicated that these modifications did not affect the conformation of the cardiotoxin. Binding of the modified cardiotoxins to phospholipids was demonstrated by changes in the intrinsic fluorescence of native and modified CTX-1 after binding to phospholipid vesicles, and by resonance energy transfer with anthracene-phospholipid vesicles. Phospholipid binding was not affected by these modifications, but their binding preference was determined by the electrostatic interactions between the polypeptide and phospholipid. Both positively charged native and guanidinated CTX-1 showed direct lytic activity on human erythrocytes and platelets, whereas the succinylated or carbamylated derivatives did not show lytic activity. The loss of lytic activity cannot be related to conformational changes or phospholipid binding abilities of the modified cardiotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence

The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing were observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.     

Antimicrobial benzodiazepine‐based short cationic peptidomimetics

Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydrophobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiazepine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic ‘back’, and two arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was discovered which has MICs of 12.5 µg/ml against Staphylococcus aureus and 25 µg/ml against Escherichia coli, similar to the well‐known antimicrobial peptide MSI‐78. In contrast to MSI‐78, the above mentioned compound has lower lytic effect against mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics of AMPs for therapeutic and biomedical applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

An enzyme-release assay for the assessment of the lytic activities of complement or antimicrobial peptides on extracellular Toxoplasma gondii

A method is described which allows the evaluation of the membrane lytic activity of either complement or antimicrobial peptides against the extracellular stage of the human protozoan parasite Toxoplasma gondii. The assay is based on lacZ transgenic parasites, determining the activity of released cytoplasmic beta-galactosidase into the culture supernatant upon membrane disintegration. This method was used to evaluate the lytic activities of (i) complement which is a natural defense mechanism in infected hosts against extracellular parasites, and (ii) antimicrobial peptides which have not been evaluated against T. gondii before. The results show that the assay provides a simple and convenient way to assess the membrane lytic activity of such compounds and that T. gondii, like other protozoan parasites, is vulnerable to the membrane-lytic effect of antimicrobial peptides.     

Action of surface-active substances of biological membranes. III. Comparison of hemolytic activity of ionic and nonionic surfactants

The hemolytic action of a number of homologous series of cationic surfactants on human erythrocytes was measured. The hemolytic effects of anionic, nonionic and cationic surface-active agents are compared. The relationship which exists between the key physicochemical properties of surfactants (critical micelle concentration, hydrophile-lipophile balance) and their hemolytic capacities is discussed. The parameters required to compare the actions of various surfactants on different cellular membranes are considered in relation to the study of the correlation between the surfactant lytic effects and the features of the membrane molecular organization.     

S-allyl cysteine attenuated CCl4-induced oxidative stress and pulmonary fibrosis in rats

This study examined effects of S-allyl cysteine (SAC) on carbon tetrachloride (CCl4)-induced interstitial pulmonary fibrosis in Wistar rats. CCl4 (0.5 ml/kg) was intraperitoneally injected into rats twice a week for 8 weeks, and SAC (50, 100, or 200 mg/kg), N-acetyl cysteine (NAC, 200 or 600 mg/kg), or L-cysteine (CYS, 600 mg/kg) were orally administrated to rats everyday for 8 weeks. SAC significantly reduced the increases of transforming growth factor beta, lipid peroxides, AST, and ALT in plasma, induced by CCl4. Although CCl4 is mainly metabolized by hepatic cytochrome P450, CCl4 induced systemic inflammation and some organ fibrosis. SAC dose-dependently and significantly attenuated CCl4-induced systemic inflammation and fibrosis of lung. SAC also inhibited the decrease of thiol levels, the increase of inducible nitric oxide synthase expression, the infiltration of leukocytes, and the generation of reactive oxygen species in lungs. Although NAC and CYS attenuated CCl4-induced pulmonary inflammation and fibrosis, the order of preventive potency was SAC > NAC > CYS according to their applied doses. These results indicate that SAC is more effective than other cysteine compounds in reducing CCl4-induced lung injury, and might be useful in prevention of interstitial pulmonary fibrosis.     

Ultracytochemical study of lytic complex insertion in the glycocalyx of red cells during immune hemolysis mediated by complement

Ruthenium red (RR), a cationic dye and an ultrastructural tracer of cell membrane permeability, was used on sheep red blood cells after lysis produced by a specific antibody and guinea pig complement. In addition to the opacification of the glycocalyx, RR stained structures related to lytic complexes, which appeared as rod-like structures with variable dimensions (generally 45 nm in width, 75 nm in height) inserted in the glycocalyx of red cells. They extended across the external layer of the trilaminar plasma membrane without reaching the internal layer or the cytoplasm. RR staining visualized the internal configuration of the lytic complexes and revealed small channels measuring 10 nm in diameter localized within the complexes. These lytic complexes are thought to correspond to membrane attack complex of complement. To the best of our knowledge, this is the first report of ultrastructural positive staining of lytic complexes in thin sections, allowing visualization of their internal configuration and their insertion in the plasma membrane glycocalyx.     

Ameliorative potential of S-allyl cysteine on oxidative stress in STZ induced diabetic rats

Increased oxidative stress and impaired antioxidant defense mechanism are important factors in the pathogenesis and progression of diabetes mellitus and other oxidant-related diseases. The present study was undertaken to evaluate the possible protective effects of S-allyl cysteine (SAC) against oxidative stress in streptozotocin (STZ) induced diabetic rats. SAC was administered orally for 45 days to control and STZ induced diabetic rats. The effects of SAC on glucose, plasma insulin, thiobarbituric acid reactive substances (TBARS), hydroperoxide, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH/GSSG ratio were studied. The levels of glucose, TBARS, hydroperoxide, and GSSG were increased significantly whereas the levels of plasma insulin, reduced glutathione, GSH/GSSG ratio, superoxide dismutase, catalase and GPx were decreased in STZ induced diabetic rats. Administration of SAC to diabetic rats showed a decrease in plasma glucose, TBARS, hydroperoxide and GSSG. In addition, the levels of plasma insulin, superoxide dismutase, catalase, GPx and reduced glutathione (GSH) were increased in SAC treated diabetic rats. The above findings were supported by histological observations of the liver and kidney. The antioxidant effect of SAC was compared with glyclazide, a well-known antioxidant and antihyperglycemic drug. The present study indicates that the SAC possesses a significant favorable effect on antioxidant defense system in addition to its antidiabetic effect.     

S-Allyl-L-cysteine attenuates cerebral ischemic injury by scavenging peroxynitrite and inhibiting the activity of extracellular signal-regulated kinase

S-Allyl-L-cysteine (SAC) has been shown to reduce ischemic injury due to its antioxidant activity. However, the antioxidant property of SAC has been controversial. The present study investigated the neuroprotective mechanism of SAC in cerebral ischemic insults. SAC decreased the size of infarction after transient or global ischemic insults. While it did not alter the N-methyl-D-aspartate excitotoxicity, SAC significantly scavenged the endogenously or exogenously produced ONOO- and reduced ONOO- cytotoxicity. In contrast, SAC has much lower scavenging activity against H2O2, O2*(-) or NO. Further, SAC inhibited the activity of extracellular signal-regulated kinase (ERK) increased in cultured neurons exposed to oxygen-glucose deprivation or in rat brain tissue after transient middle cerebral artery occlusion. The neuroprotective effect of SAC was mimicked by the ERK inhibitor U0125. The present results indicate that SAC exert its neuroprotective effect by scavenging ONOO- and inhibiting the ERK signaling pathway activated during initial hypoxic/ischemic insults.     

Modulation of melittin-induced lysis by surface charge density of membranes.

Phosphorus NMR spectroscopy was used to characterize the importance of electrostatic interactions in the lytic activity of melittin, a cationic peptide. The micellization induced by melittin has been characterized for several lipid mixtures composed of saturated phosphatidylcholine (PC) and a limited amount of charged lipid. For these systems, the thermal polymorphism is similar to the one observed for pure PC: small comicelles are stable in the gel phase and extended bilayers are formed in the liquid crystalline phase. Vesicle surface charge density influences strongly the micellization. Our results show that the presence of negatively charged lipids (phospholipid or unprotonated fatty acid) reduces the proportion of lysed vesicles. Conversely, the presence of positively charged lipids leads to a promotion of the lytic activity of the peptide. The modulation of the lytic effect is proposed to originate from the electrostatic interactions between the peptide and the bilayer surface. Attractive interactions anchor the peptide at the surface and, as a consequence, inhibit its lytic activity. Conversely, repulsive interactions favor the redistribution of melittin into the bilayer, causing enhanced lysis. A quantitative analysis of the interaction between melittin and negatively charged bilayers suggests that electroneutrality is reached at the surface, before micellization. The surface charge density of the lipid layer appears to be a determining factor for the lipid/peptide stoichiometry of the comicelles; a decrease in the lipid/peptide stoichiometry in the presence of negatively charged lipids appears to be a general consequence of the higher affinity of melittin for these membranes.     

Synthesis of neutral and cationic tripyridylporphyrin-D-galactose conjugates and the photoinactivation of HSV-1

Neutral and cationic tripyridylporphyrin-D-galactose conjugates were synthesized and their antiviral activity against herpes simplex virus type 1 (HSV-1) was evaluated. At non-cytotoxic concentrations the studied compounds show significant antiviral activity after photoactivation. The influence of photoactivation on drug treated cells was also analyzed, at different times of infection with HSV-1, for a neutral (1b) and a cationic glycoporphyrin (3b) derivative. The results show that the inhibition of the viral yield is more dependent on photoactivation for compound 1b than for compound 3b. These two compounds also differ in the inhibitory effect during the viral replicative cycle: while compound 3b inhibits the viral yield at all the addition times assayed, compound 1b is more efficient in later times of infection.     

Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/C(Cdc20)), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.     

Re-examination of siRNA specificity questions role of PICH and Tao1 in the spindle checkpoint and identifies Mad2 as a sensitive target for small RNAs

The DNA-dependent adenosine triphosphatase (ATPase) Plk1-interacting checkpoint helicase (PICH) has recently been implicated in spindle checkpoint (SAC) signaling (Baumann et al., Cell 128(1):101–114, 2007). Depletion of PICH by siRNA abolished the SAC and resulted in an apparently selective loss of Mad2 from kinetochores, suggesting a role for PICH in the regulation of the Mad1–Mad2 interaction. An apparent rescue of SAC functionality by overexpression of PICH in PICH-depleted cells initially seemed to confirm a role for PICH in the SAC. However, we have subsequently discovered that all PICH-directed siRNA oligonucleotides that abolish the SAC also reduce Mad2 mRNA and protein expression. This reduction is functionally significant, as PICH siRNA does not abolish SAC activity in a cell line that harbors a bacterial artificial chromosome driving the expression of murine Mad2. Moreover, we identified several siRNA duplexes that effectively deplete PICH but do not significantly affect SAC functionality or Mad2 abundance or localization. Finally, we discovered that the ability of overexpressed PICH to restore SAC activity in PICH-depleted cells depends on sequestration of the mitotic kinase Plk1 rather than ATPase activity of PICH, pointing to an underlying mechanism of “bypass suppression.” In support of this view, depletion or inhibition of Plk1 also rescued SAC activity in cells harboring low levels of Mad2. This observation suggests that a reduction of Plk1 activity partially compensates for reduced Mad2 levels and argues that Plk1 normally reduces the strength of SAC signaling. Collectively, our results question the role of PICH in the SAC and instead identify Mad2 as a sensitive off target for small RNA duplexes. In support of the latter conclusion, our evidence suggests that an off-target effect on Mad2 may also contribute to explain the apparent role of the Tao1 kinase in SAC signaling (Draviam et al., Nat Cell Biol 9(5):556–564, 2007).     

The problem of artificial gravity: The current state and prospects

The review deals with the use of artificial gravity in manned space flights. The need for studying this problem is substantiated, with special emphasis on its implications for future interplanetary flights. The deconditioning of astronauts and a loss of their tolerance to gravitational loads despite the use of various preventive procedures are briefly discussed. The efficiency of artificial gravity generated by a short-arm centrifuge (SAC) is evaluated; the possibility of the use of an SAC in space flights (the effect of the main parameters of G-load on humans, and its tolerability, efficiency, etc.) is considered. Both Russian and foreign data are presented on the use of SAC for simulating microgravity effects under ground-based conditions (immersion and ANOH) and in experiments on board biosatellites. It is emphasized that all the data (both original and the data in the literature) testify to the efficiency of SAC as a preventive and therapeutic facility alleviating the negative effects of simulated microgravity. The problems that have not been resolved to date are also presented.