Dynamics of firefly luciferase inhibition by general anesthetics: Gaussian and anisotropic network analyses

The new crystal structures of the product-bound firefly luciferase combined with the previously determined substrate-free structures allow for a detailed analysis of the dynamics basis for the luciferase enzymatic activities. Using the Gaussian network model and the anisotropic network model, we show here that the superposition of the three slowest anisotropic network model modes, consisting of the bending, rotating, and rocking motions of the C-domain, accounts for large rearrangement of domains from the substrate-free (open) to product-bound (closed) conformation and thus constitutes a critical component of the enzyme's functions. The analysis also offers a unique platform to reexamine the molecular mechanism of the anesthetic inhibition of the firefly luciferase. Through perturbing the protein backbone network by introducing additional nodes to represent anesthetics, we found that the presence of two representative anesthetics, halothane and n-decanol, in different regions of luciferase had distinctively different effects on the protein's global motion. Only at the interface of the C- and N-domains did the anesthetics cause the most profound reduction in the overall flexibility of the C-domain and the concomitant increase in the flexibility of the loop, where the substitution of a conserved lysine residue was found experimentally to lead to >2-3 orders of magnitude reduction in activity. These anesthetic-induced dynamics changes can alter the normal function of the protein, appearing as an epiphenomenon of an "inhibition". The implication of the study is that a leading element for general anesthetic action on proteins is to disrupt the modes of motion essential to protein functions.     

The study on the interaction between phytosterols and phospholipids in model membranes

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.     

Distribution of triterpene acids and their derivatives in organs of cowberry (Vaccinium vitis-idaea L.) plant

Wild berries of the genus Vaccinium have become increasingly popular in human health promotion due to their nutritional and medicinal properties. Some striking divergence of opinion about the content of triterpenoids in these plants still exists, meanwhile, this very large class of natural isoprenoids exhibits a wide range of biological activities and hence is of growing research interest. An investigation of triterpenoidal constituents from the cowberry (Vaccinium vitis-idaea L.) plant led to the isolation of two isomeric acids: oleanolic and ursolic and the occurrence of their derivatives in this plant was demonstrated for the first time. Free triterpene acids as well as small amounts of their bound forms (presumable glycosides and glycoside esters) occur in fruits and the vegetative part of the plant, however, in various amounts and different ratios. The total content of both acids was the highest in organs regarded as traditional herbal resources, namely fruits and leaves (1 and 0.6% of dry mass, respectively), whereas it was markedly lower in stems and rhizomes. However, the rhizomes were in turn the plant organ containing relatively the highest amount of the bound forms of both acids (0.01% of dry mass). Ursolic acid was dominant in the whole plant, but the ratio of oleanolic to ursolic acid was significantly different in individual organs, decreasing from the upper (fruits 1:2.4, leaves 1:2) to the lower (stems 1:3.5, rhizomes 1:5.2) parts of the plant. This pattern of distribution of triterpenoids in the plant may have an important physiological and ecological meaning.     

Comparison of the localization and post-translational modification of Campylobacter coli CjaC and its homolog from Campylobacter jejuni, Cj0734c/HisJ

Campylobacter is an asaccharolytic microorganism which uses amino acids as a source of carbon and energy. CjaC/HisJ is a ligand-binding protein, a component of the ABC transport system. Campylobacter CjaC/HisJ is post-translationally modified by glycosylation. The number of glycosylation motifs present in the CjaC protein is species-specific. C. coli CjaC has two and C. jejuni one motif (E/DXNYS/T) which serves as a glycan acceptor. Although the two C. coli CjaC motifs have identical amino-acid sequences they are not glycosylated with the same efficiency. The efficacy of CjaC glycosylation in Escherichia coli containing the Campylobacter pgl locus is also rather low compared to that observed in the native host. The CjaC localization is host-dependent. Despite being a lipoprotein, CjaC is recovered in E. coli from the periplasmic space whereas in Campylobacter it is anchored to the inner membrane.     

Anti-IL-8 autoantibody:IL-8 immune complexes suppress spontaneous apoptosis of neutrophils

Our previous studies demonstrated that a significant fraction of interleukin-8 (IL-8) in lung fluids from patients with acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) is associated with anti-IL-8 autoantibodies (anti-IL-8:IL-8 immune complexes). Neutrophils have been implicated in the pathogenesis of ALI/ARDS, and moreover, it is well-established that apoptosis of neutrophils is delayed in patients with ALI/ARDS. The aim of this study was, therefore, to examine the role of anti-IL-8:IL-8 immune complexes in modulating spontaneous apoptosis of normal human neutrophils. Apoptosis was assessed by evaluating morphological changes, measuring enzymatic activity of caspase-3, and determining the extent of DNA degradation. We found that samples containing anti-IL-8:IL-8 immune complexes but not samples from which these complexes were removed inhibited neutrophil apoptosis. Furthermore, the former samples or effectively anti-IL-8:IL-8 complexes induced an increase in the level of antiapoptotic protein, Bcl-X(L). In contrast, levels of proapoptotic proteins Bax and Bak were decreased in the same conditions. Activity of both caspase-3 and caspase-9 was also suppressed by anti-IL-8:IL-8 complex-containing samples. Finally, we established that IgG receptor, FcgammaRIIa, mediates antiapoptotic activity of anti-IL-8:IL-8 complexes and that the key components of the FcgammaRIIa signaling pathway, Src, Syk, PI3 kinase, and ERK, may be involved in regulation of neutrophil apoptosis by the complexes. These studies demonstrate for the first time that anti-IL-8:IL-8 immune complexes have the ability to prolong neutrophil life.     

Expression of vascular endothelial growth factor and its receptors in the porcine corpus luteum during the estrous cycle and early pregnancy

The present study was undertaken to determine the expression of vascular endothelial growth factor (VEGF) and its receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase-1/kinase insert domain-containing receptor (Flk-1/KDR), in the porcine corpus luteum (CL) during the estrous cycle and early pregnancy. Immunohistochemical studies localized proteins of VEGF ligand-receptor system in the cytoplasm of luteal cells and in some blood vessels. Western blot analysis revealed significantly higher levels of VEGF protein during early and mid-luteal phase (vs. late luteal phase; P<0.001 and P<0.01, respectively). Quantification of VEGF mRNA in the CL showed increased mRNA levels during entire luteal phase (vs. Days 16-17; P<0.05). Expression of Flt-1 protein remained high during luteal phase (P<0.001), but the mRNA levels tended to increase from the early to the late luteal phase. Elevated protein expression of Flk-1/KDR was found in the mid-luteal phase (vs. Days 16-17; P<0.05). However, induction of Flk-1/KDR mRNA expression occurred earlier, in early luteal phase. The lowest VEGF, Flt-1 and Flk-1/KDR mRNA and protein levels were observed in regressed CL (P<0.001). During pregnancy, VEGF, Flt-1 and Flk-1/KDR mRNA and protein expression was comparable to the mid-luteal phase. In conclusion, the present study has demonstrated dynamic expression of VEGF and its receptors in the porcine CL during the estrous cycle and early pregnancy. These data suggest that the VEGF ligand-receptor system may play an important role in the development and maintenance of the CL in pigs.     

Urinary excretion rates of 8-oxoGua and 8-oxodG and antioxidant vitamins level as a measure of oxidative status in healthy, full-term newborns

The aim of the present study was to evaluate the oxidative status in healthy full-term children and piglets. Urinary excretion of 8-oxoGua (8-oxoguanine) and 8-oxodG (8-oxo-2'-deoxyguanosine) were determined using HPLC/GS/MS methodology and concentrations of vitamins A, C and E with HPLC technique. The levels of 8-oxoGua in urine samples were about 7-8 times higher in newborn children and piglets when compared with the level of adult subjects, while in the case of 8-oxodG the difference was about 2.5 times. The levels of vitamin C and E in umbilical cord blood of newborn children significantly depend on the concentration of these compounds in their mother's blood. However, the values of vitamin C in human's cord blood were about 2-times higher than in respective mother blood, while the level of vitamin E showed an opposite trend. The results suggest that: (i) healthy, full-term newborns are under potential oxidative stress; (ii) urinary excretion of 8-oxoGua and 8-oxodG may be a good marker of oxidative stress in newborns; and (iii) antioxidant vitamins, especially vitamin C, play an important role in protecting newborns against oxidative stress.