Plasma non-transferrin-bound iron uptake by the small intestine leads to intestinal injury and intestinal flora dysbiosis in an iron overload mouse model and Caco-2 cells

Iron overload often occurs during blood transfusion and iron supplementation, resulting in the presence of non-transferrin-bound iron(NTBI) in host plasma and damage to multiple organs, but effects on the intestine have rarely been reported. In this study, an iron overload mouse model with plasma NTBI was established by intraperitoneal injection of iron dextran. We found that plasma NTBI damaged intestinal morphology, caused intestinal oxidative stress injury and reactive oxygen species(ROS) acc...     

Call Characteristic Network Reveal Geographical Patterns of Call Similarity:Applying Network Analysis to Frog’s Call Research

Individual’s phenotypic traits are the results of adaptation to ecological conditions.Therefore,different selection pressures caused by heterogeneous environments may result in phenotypic difference,especially for individuals in different geographical populations.Here,we illustrated for the first time to use social network analysis(SNA)for examining whether geographical proximity predicts the similarity patterns in call characteristics among populations of an anuran species.We recorded calls from 150 male dorsal-striped opposite-fingered treefrogs(Chiromantis doriae)at 11 populations in Hainan Province and one population in Guangdong Province in China's Mainland,and we measured eight acoustic variables for each male.Mantel test didn’t show a correlation between geographical proximity and the similarity in call characteristics among populations.In addition,we failed to find correlations between a population’s eigenvector centrality and the distance to its nearest neighbor,nor between the coefficient of variation of similarity in call characteristics of a population and the average distance to all other populations.Nevertheless,three acoustic clusters were identified by the Girvan-Newman algorithm,and clustering was partially associated with geography.Furthermore,the most central populations were included in the same cluster,but the top betweenness populations were located within different clusters,suggesting that centrality populations are not necessary bridging between clusters.These results demonstrate the potential usefulness of the SNA toolbox and indicate that SNA helps to uncover the patterns that often overlooked in other analytical methods.By using SNA in frog’s call studies,researchers could further uncover the potential relationship in call characteristics between geographical populations,further reveal the effects of ecological factors on call characteristics,and probably enhance our understanding of the adaptive evolution of acoustic signals.     

Prediction of the subcellular location of prokaryotic proteins based on a new representation of the amino acid composition

A new representation of protein sequence is devoted in this paper, in which each protein can be represented by a 20-dimensional (20D) vector of unit length. Inspired by the principle of superposition of state in quantum mechanics, the squares of the 20 components of the vector correspond to the amino acid composition. Using the new representation of the primary sequence and Bayes Discriminant Algorithm, the subcellular location of prokaryotic proteins was predicted. The overall predictive accuracy in the jackknife test can be 3% higher than the result of using amino acid composition directly for the database of sequence identity is less than 90%, but 5% higher when sequence identity is less than 80%. The higher predictive accuracy indicates that the current measure of extracting the information from the primary sequence is efficient. Since the subcellular location restricting a protein's possible function, the present method should also be a useful measure for the systematic analysis of genome data. The program used in this paper is available on request.     

Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product

The methylotrophic yeast Pichia pastoris, which proved successful in producing many heterologous proteins, was used to express an insulin precursor. A transformant with a high copy number of the gene integrated into the chromosome was obtained by the dot-blotting method. In high-density fermentation using a simple culture medium composed mainly of salt and methanol, the expression level reached 1.5 g/L. A simple two-step method was established to purify the expression product from the culture medium with an overall recovery of about 80%. After tryptic transpeptidation, human insulin with full receptor binding capacity and biological activity was obtained. In the presence of zinc, the recombinant human insulin could be crystallized in the rhombohedral form.     

Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice

The serine-threonine kinase Akt, also known as protein kinase B (PKB), is an important effector for phosphatidylinositol 3-kinase signaling initiated by numerous growth factors and hormones. Akt2/PKBbeta, one of three known mammalian isoforms of Akt/PKB, has been demonstrated recently to be required for at least some of the metabolic actions of insulin (Cho, H., Mu, J., Kim, J. K., Thorvaldsen, J. L., Chu, Q., Crenshaw, E. B., Kaestner, K. H., Bartolomei, M. S., Shulman, G. I., and Birnbaum, M. J. (2001) Science 292, 1728-1731). Here we show that mice deficient in another closely related isoform of the kinase, Akt1/PKBalpha, display a conspicuous impairment in organismal growth. Akt1(-/-) mice demonstrated defects in both fetal and postnatal growth, and these persisted into adulthood. However, in striking contrast to Akt2/PKBbeta null mice, Akt1/PKBalpha-deficient mice are normal with regard to glucose tolerance and insulin-stimulated disposal of blood glucose. Thus, the characterization of the Akt1 knockout mice and its comparison to the previously reported Akt2 deficiency phenotype reveals the non-redundant functions of Akt1 and Akt2 genes with respect to organismal growth and insulin-regulated glucose metabolism.     

Residue Gly1326 of the N-type calcium channel alpha 1B subunit controls reversibility of omega-conotoxin GVIA and MVIIA block

We recently reported that amino acid residues contained within a putative EF hand motif in the domain III S5-H5 region of the alpha(1B) subunit affected the relative barium:calcium permeability of N-type calcium channels (Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region partially overlaps with residues previously implicated in block of the channel by omega-conotoxin GVIA, we assessed the effects of mutations in the putative EF hand domain on channel block by omega-conotoxin GVIA and the structurally related omega-conotoxin MVIIA. Both of the toxins irreversibly block the activity of wild type alpha(1B) N-type channels. We find that in addition to previously identified amino acid residues, residues in positions 1326 and 1332 are important determinants of omega-conotoxin GVIA blockade. Substitution of residue Glu(1332) to arginine slows the time course of development of block. Point mutations in position Gly(1326) to either arginine, glutamic acid, or proline dramatically decrease the time constant for development of the block. Additionally, in the G1326P mutant channel activity was almost completely recovered following washout. A qualitatively similar result was obtained with omega-conotoxin MVIIA, suggesting that common molecular determinants underlie block by these two toxins. Taken together the data suggest that residue Gly(1326) may form a barrier, which controls the access of peptide toxins to their blocking site within the outer vestibule of the channel pore and also stabilizes the toxin-channel interaction.     

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.     

Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.     

Full-contact domain labeling: identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein

Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogues. The N-terminal half and the C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Competitive displacement experiments with dibutyryl, dioctanoyl, or dipalmitoyl derivatives of PtdIns(4,5)P(2) suggested that, in addition to the inositol headgroup, a diacylglyceryl moiety was important for binding; these analogues also inhibited the gelsolin-severing activity of F-actin. In addition to the previously identified PtdIns(4,5)P2 binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-terminal half by mapping the photocovalently modified peptide fragments. Moreover, increasing concentrations of Ca(2+) decreased the binding of the photolabile analogues to the C-terminal phosphoinositide binding site on gelsolin. A molecular model of the binding of PtdIns(4,5)P2 within two folded repeats of gelsolin has been calculated using these data.