The mechanism of action of antioxidants against lipoprotein peroxidation, evaluation based on kinetic experiments

Peroxidation of blood lipoproteins is regarded as a key event in the development of atherosclerosis. Hence, attenuation of the oxidative modification of lipoproteins by natural and synthetic antioxidants in vivo is considered a possible way of prevention of cardiovascular disorders. The assessment of the susceptibility of lipoproteins to oxidation is commonly based on in vitro oxidation experiments. Monitoring of oxidation provides the kinetic profile characteristic for the given lipoprotein preparation. The kinetic profile of peroxidation is characterized by three major parameters: the lag preceding rapid oxidation, the maximal rate of oxidation (V(max)) and the maximal accumulation of oxidation products (OD(max)). Addition of antioxidants alters this pattern, affecting the kinetic parameters of oxidation. In particular, antioxidants may prolong the lag and/or decrease the V(max) and/or decrease the OD(max). Such specific variation of the set of kinetic parameters may provide important information on the mechanism of the inhibitory action of a given antioxidant (scavenging free radicals, metal-binding or other mechanisms). Numerous natural and synthetic compounds were reported to inhibit oxidation of lipoproteins. Based on the analysis of reported effects and theoretical considerations, we propose a simple protocol that relates the kinetic effects of a given antioxidant to the mechanism of its action.     

Oxygen availability as a possible limiting factor in LDL oxidation

Kinetic studies of copper-induced LDL peroxidation commonly assume that the availability of molecular oxygen in the reaction media is not a limiting factor. The present study reveals that this assumption is valid only at low LDL concentrations. At high LDL concentrations, accumulation of oxidation products, as monitored spectroscopically under conditions of various oxygen concentrations in the medium, comes to a halt when the oxygen concentration in the solution, as measured by an oxygen electrode, decreases to near zero levels. Bubbling of the oxygen into the solution results in resumption of peroxidation. These results are important with respect to the ex vivo assaying of lipoprotein peroxidation because many previous studies have been conducted with LDL concentrations that corresponded to polyunsaturated fatty acid concentrations in access of the concentration of molecular oxygen. The possible pathophysiological significance of the results of this study has yet to be evaluated.     

Is aromaticity essential for trapping the catalytic histidine 447 in human acetylcholinesterase?

Replacement of both the acyl pocket residue Phe295 as well as residue Phe338, adjacent to the catalytic His447 in human acetylcholinesterase (HuAChE), resulted in a 680-fold decline in catalytic activity due to conformational destabilization of the histidine side chain [Barak et al. (2002) Biochemistry 41, 8245]. A possible restriction of this catalytically nonproductive mobility of His447 in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Simulations suggested that of the 12 aliphatic residues substituted at position 295, including hydrophobic and polar amino acids, only methionine was capable of maintaining the catalytically viable conformation of His447. Examination of the reactivities of the actual F295X/F338A HuAChEs showed that indeed the F295M/F338A enzyme was only 2-fold less reactive than the F338A mutant toward acetylthiocholine, while enzymes substituted by the similarly bulky residues leucine and isoleucine were catalytically impaired. Furthermore, only the F295M/F338A enzyme exhibited wild-type-like reactivity toward covalent modifiers of the catalytic Ser203 including the methylphosphonate soman and transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), as well as a facile dealkylation of the F295M/F338A-soman adduct. A different behavior was observed for bulkier ligands which introduce a deformation in the acyl pocket, and therefore their activity seems only marginally affected by the positioning of His447. The findings emphasize the importance of the precise positioning of His447 for catalysis and indicate that, in the absence of aromatic "trapping", restriction of the histidine mobility in F295X/F338A HuAChEs requires a combination of steric interference and a specific polar interaction. The results also underscore the role of the acyl pocket subsite of cholinesterases in maintaining the catalytically viable conformation of the catalytic histidine.     

STRUCTURAL SPECIALIZATION OF THE SITE OF RESPONSE TO VECTORIAL PHOTO-EXCITATION IN THE SOLAR-TRACKING LEAF OF LAVATERA CRETICA

Structural features of the pulvinus of the solar-tracking leaf of Lavatera cretica L. that are involved in its capacity for omnidirectional and fully reversible bending in response to vectorial excitation of the lamina were studied by light- and scanning electron microscopy. Pulvini that had bent in the plane at right angles to the midvein were bisected along that plane and the symmetrical tissues of the expanded and contracted flanks were compared. The pulvinus contains a central vascular core and exhibits a transversely furrowed exterior. These specialized features enable the fully mature tissue of this region of the petiole to bend reversibly. The epidermis, chlorenchyma, peripheral collenchyma, and cortical parenchyma in the pulvinus form concentric, radially symmetric sheaths around the vascular core and exhibit structural features in their cell walls that would allow considerable changes in cell volume and consequently enable the omnidirectional bending of this pulvinus. Thickened wall portions of the pulvinar epidermis and peripheral collenchyma exhibit a highly specialized architecture, consisting of alternating thick and thin strips, that enhances their flexibility, while maintaining mechanical support. Cell walls of the chlorenchyma and the cortical parenchyma are thin and capable of reversible infolding. Those of the cortical parenchyma also exhibit numerous prominent transverse pit fields, indicative of anisotropic orientation of their microfibrillar lattice transverse to the pulvinar axis. This orientation is compatible with elastic deformation of the cortical parenchyma cells along the pulvinar axis. Filament-like cytoplasmic strands were observed along the walls of pulvinar motor cells, predominantly transverse to the pulvinar axis, but their function (if any) in volume changes of these cells is unknown.     

High curvature and jerk analyses of arm ataxia

We investigated high curvature analysis (HCA) and integrated absolute jerk (IAJ) for differentiating healthy and cerebellopathy (CB) patients performing pointing tasks. Seventeen CB patients and seventeen healthy controls were required to move a pointer at their preferred pace between two 50.8 cm laterally spaced targets while standing with their arm extended in front of their body. HCA was used to quantify the frequency of sharp turns in the horizontal-plane (anterior-posterior and medio-lateral) velocity trajectory of the hand-held pointer. IAJ was assessed by integration of absolute jerk (second time derivative of velocity) time histories in the anterior-posterior and medio-lateral directions. HCA scores and IAJ scores were then compared between CB patients and healthy controls; for both analyses, higher scores indicate less smooth movements. We hypothesized that CB patients would have less smooth movement trajectories than healthy controls due to upper extremity ataxia associated with cerebellar disease and degeneration. We found that CB patients had higher HCA scores than healthy controls (P = 0.014). Although CB patients had higher IAJ scores in both anterior-posterior (P = 0.060) and medio-lateral (P = 0.231) directions compared to the healthy controls, the differences were not significant. The difference in sensitivity between the HCA and the IAJ analysis might be explained by primitive neural activation commands, ubiquitous though only evident with some cerebellar dysfunctions, which produce sub-movements which are themselves minimal jerk curves. We conclude that HCA may be a useful tool for quantifying upper extremity ataxia in CB patients performing a repeated pointing task. Received: 17 December 1999 / Accepted in revised form: 7 July 2000     

Does ovarian ecdysone stimulate mosquitoes to synthesize vitellogenin?

Physiological amounts of 20-hydroxyecdysone do not initiate vitellogenin synthesis in unfed, non-vitellogenic mosquitoes. Injecting more than 10,000 times the physiological amount induced synthesis, but considerably less than was induced by a blood meal. A dose of 20-hydroxyecdysone which exceeded the physiological level only several hundred times, did not sustain vitellogenin synthesis, when blood-fed mosquitoes were ovariectomized just prior to injection. Transplanting ovaries from vitellogenic to non-vitellogenic females did not initiate synthesis of vitellogenin in the recipient. In vitro, neither 20-hydroxyecdysone nor the ovaries of vitellogenic females were able to induce synthesis of vitellogenin in non-vitellogenic fat bodies. These experiments suggest that ecdysteroid, released by the ovaries, does not initiate ovarian development in mosquitoes.     

不同浓度壳聚糖对炭黑曲霉和硫色镰刀菌生长和发育的影响

【背景】壳聚糖是广泛存在于甲壳动物的一种多糖,具有广谱的抗真菌活性,但壳聚糖是否影响炭黑曲霉(Aspergillus carbonarius)和硫色镰刀菌(Fusarium sulphureum)生长和发育尚未见报道。【目的】明确不同浓度壳聚糖对A. carbonarius和F. sulphureum生长和发育的影响。【方法】通过在PDA培养基中添加不同浓度壳聚糖,测定两种真菌的菌落直径、生物量和菌丝干重,观察产孢量、孢子萌发和芽管长度,比较抑菌的差异。【结果】壳聚糖处理可显著改变两种真菌的菌落形态,处理浓度越高菌落皱缩和变形越明显;壳聚糖还可以有效抑制两种真菌的菌落生长、菌丝干重和菌丝生物量,抑制效果呈明显的浓度依赖,对F. sulphureum的抑制效果更好。壳聚糖可抑制两种真菌的产孢量、孢子萌发和芽管伸长,处理浓度越高抑制效果越好,对F. sulphureum的抑制效果更为明显。壳聚糖对A. carbonarius和F. sulphureum的EC₅₀值分别为0.12 mg/mL和0.075 mg/mL。【结论】壳聚糖可有效抑制A. carbonarius和F. sulphureum的生长发育,抑制效果呈浓度依赖,F. sulphureum对壳聚糖更为敏感。     

History of Lipizzan horse maternal lines as revealed by mtDNA analysis

Sequencing of the mtDNA control region (385 or 695 bp) of 212 Lipizzans from eight studs revealed 37 haplotypes. Distribution of haplotypes among studs was biased, including many private haplotypes but only one haplotype was present in all the studs. According to historical data, numerous Lipizzan maternal lines originating from founder mares of different breeds have been established during the breed''s history, so the broad genetic base of the Lipizzan maternal lines was expected. A comparison of Lipizzan sequences with 136 sequences of domestic- and wild-horses from GenBank showed a clustering of Lipizzan haplotypes in the majority of haplotype subgroups present in other domestic horses. We assume that haplotypes identical to haplotypes of early domesticated horses can be found in several Lipizzan maternal lines as well as in other breeds. Therefore, domestic horses could arise either from a single large population or from several populations provided there were strong migrations during the early phase after domestication. A comparison of Lipizzan haplotypes with 56 maternal lines (according to the pedigrees) showed a disagreement of biological parentage with pedigree data for at least 11% of the Lipizzans. A distribution of haplotype-frequencies was unequal (0.2%–26%), mainly due to pedigree errors and haplotype sharing among founder mares.     

Of mice and mammoths: generality and antiquity of the island rule

Aim

We assessed the generality of the island rule in a database comprising 1593 populations of insular mammals (439 species, including 63 species of fossil mammals), and tested whether observed patterns differed among taxonomic and functional groups.

Location

Islands world‐wide.

Methods

We measured museum specimens (fossil mammals) and reviewed the literature to compile a database of insular animal body size (S_i = mean mass of individuals from an insular population divided by that of individuals from an ancestral or mainland population, M). We used linear regressions to investigate the relationship between S_i and M, and ANCOVA to compare trends among taxonomic and functional groups.

Results

S_i was significantly and negatively related to the mass of the ancestral or mainland population across all mammals and within all orders of extant mammals analysed, and across palaeo‐insular (considered separately) mammals as well. Insular body size was significantly smaller for bats and insectivores than for the other orders studied here, but significantly larger for mammals that utilized aquatic prey than for those restricted to terrestrial prey.

Main conclusions

The island rule appears to be a pervasive pattern, exhibited by mammals from a broad range of orders, functional groups and time periods. There remains, however, much scatter about the general trend; this residual variation may be highly informative as it appears consistent with differences among species, islands and environmental characteristics hypothesized to influence body size evolution in general. The more pronounced gigantism and dwarfism of palaeo‐insular mammals, in particular, is consistent with a hypothesis that emphasizes the importance of ecological interactions (time in isolation from mammalian predators and competitors was 0.1 to > 1.0 Myr for palaeo‐insular mammals, but < 0.01 Myr for extant populations of insular mammals). While ecological displacement may be a major force driving diversification in body size in high‐diversity biotas, ecological release in species‐poor biotas often results in the convergence of insular mammals on the size of intermediate but absent species.