Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe

Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature. In a mutant dis1 background (dis1-288), loss of function of Msc1, a fission yeast homolog of the KDM5 family of proteins, suppresses the growth defect and promotes normal mitosis. Genetic analysis implicates a histone deacetylase (HDAC)–linked pathway in suppression because HDAC mutants clr6-1, clr3∆, and sir2∆, though not hos2∆, also promote normal mitosis in the dis1-288 mutant. Suppression of the dis phenotype through loss of msc1 function requires the spindle checkpoint protein Mad2 and is limited by the presence of the heterochromatin-associated HP1 protein homolog Swi6. We speculate that alterations in histone acetylation promote a centromeric chromatin environment that compensates for compromised dis1 function by allowing for successful kinetochore-microtubule interactions that can satisfy the SAC. In cells arrested in mitosis by mutation of dis1, loss of function of epigenetic determinants such as Msc1 or specific HDACs can promote cell survival. Because the KDM5 family of proteins has been implicated in human cancers, an appreciation of the potential role of this family of proteins in chromosome segregation is warranted.     

Molecular origin of the pH dependence of tyrosine D oxidation kinetics and radical stability in photosystem II

A role for redox-active tyrosines has been demonstrated in many important biological processes, including water oxidation carried out by photosystem II (PSII) of oxygenic photosynthesis. The rates of tyrosine oxidation and reduction and the Tyr/Tyr reduction potential are undoubtedly controlled by the immediate environment of the tyrosine, with the coupling of electron and proton transfer, a critical component of the kinetic and redox behavior. It has been demonstrated by Faller et al. that the rate of oxidation of tyrosine D (Tyr_D) at room temperature and the extent of Tyr_D oxidation at cryogenic temperatures, following flash excitation, dramatically increase as a function of pH with a pK_a of ≈ 7.6 [Faller et al. 2001 Proc. Natl. Acad. Sci. USA 98, 14368-14373; Faller et al. 2001 Biochemistry 41, 12914-12920]. In this work, we investigated, using FTIR difference spectroscopy, the mechanistic reasons behind this large pH dependence. These studies were carried out on Mn-depleted PSII core complexes isolated from Synechocystis sp. PCC 6803, WT unlabeled and labeled with ¹³C₆-, or ¹³C₁(4)-labeled tyrosine, as well as on the D2-Gln164Glu mutant. The main conclusions of this work are that the pH-induced changes involve the reduced Tyr_D state and not the oxidized Tyr_D state and that Tyr_D does not exist in the tyrosinate form between pH 6 and 10. We can also exclude a change in the protonation state of D2-His189 as being responsible for the large pH dependence of Tyr_D oxidation. Indeed, our data are consistent with D2-His189 being neutral both in the Tyr_D and Tyr_D states in the whole pH6-10 range. We show that the interactions between reduced Tyr_D and D2-His189 are modulated by the pH. At pH greater than 7.5, the ν(CO) mode frequency of Tyr_D indicates that Tyr_D is involved in a strong hydrogen bond, as a hydrogen bond donor only, in a fraction of the PSII centers. At pH below 7.5, the hydrogen-bonding interaction formed by Tyr_D is weaker and Tyr_D could be also involved as a hydrogen bond acceptor, according to calculations performed by Takahashi and Noguchi [J. Phys. Chem. B 2007 111, 13833-13844]. The involvement of Tyr_D in this strong hydrogen-bonding interaction correlates with the ability to oxidize Tyr_D at cryogenic temperatures and rapidly at room temperature. A strong hydrogen-bonding interaction is also observed at pH 6 in the D2-Gln164Glu mutant, showing that the residue at position D2-164 regulates the properties of Tyr_D. The IR data point to the role of a protonatable group(s) (with a pK_a of ≈ 7) other than D2-His189 and Tyr_D, in modifying the characteristics of the Tyr_D hydrogen-bonding interactions, and hence its oxidation properties. It remains to be determined whether the strong hydrogen-bonding interaction involves D2-His189 and if Tyr_D oxidation involves the same proton transfer route at low and at high pH.     

Calculation of IR dichroic values and order parameters from molecular dynamics simulations and their application to structure determination of lipid bilayers

In polarized infrared (IR) absorption experiments, dichroic values are used to study the structure and orientation of lipid molecules. From computer simulations, we obtained angular distributions of IR transition moment (TM) orientations of the stretch vibrations of CH₂ groups of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholin (POPC) lipid bilayers in the gel (L ) and fluid (L ) phases. From these distributions, we calculated dichroic absorption values, as well as order parameters. We established a connection between the dichroic ratio R ^ATR , which is measured in IR-ATR setups, with the dichroic ratio D and the order parameter S _zz . The calculated values compare well with experimental results for the fluid phase. In addition, we computed angular distributions of transition moments with respect to the tail director orientation for the gel and the fluid phases. Only small differences were found between the distributions in the symmetric stretch orientation, the asymmetric stretch orientation, and the C-H bond orientation of CH₂ groups. The distributions of tail directors of POPC showed average tilts of 14.7° in the gel phase and 32.9° in the fluid phase. We developed a theory which makes it possible to calculate average tilt angles of tail directors in the gel phase from dichroic absorption values obtained from IR measurements for a wide range of lipids. Legendre coefficients were calculated from TM distributions. Order parameters, defined as the second Legendre polynomial, were found to closely approximate the TM distribution in lipid bilayers in the fluid phase.Abbreviations MD molecular dynamics - IR infrared - ATR attenuated total reflection - TM transition moment - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-cholin - POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-phatidylglycerol - DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholin - NMR nuclear magnetic resonance     

Constitutive expression of two apple (Malus x domestica Borkh.) homolog genes of LIKE HETEROCHROMATIN PROTEIN1 affects flowering time and whole-plant growth in transgenic Arabidopsis

Fruit trees, such as apple (Malus × domestica Borkh.), are woody perennial plants with a long juvenile phase. The biological analysis for the regulation of flowering time provides insights into the reduction of juvenile phase and the acceleration of breeding in fruit trees. In Arabidopsis, LIKE HETEROCHROMATIN PROTEIN1 (LHP1) is involved in epigenetic silencing of the target genes such as flowering genes. We isolated and characterized twin apple LHP1 homolog genes, MdLHP1a and MdLHP1b. These genes may have been generated as a result of ancient genome duplication. Although the putative MdLHP1 proteins showed lower similarity to any other known plant LHP1 homologs, a chromo domain, a chromo shadow domain, and the nuclear localization signal motifs were highly conserved among them. RT-PCR analysis showed that MdLHP1a and MdLHP1b were expressed constantly in developing shoot apices of apple trees throughout the growing season. Constitutive expression of MdLHP1a or MdLHP1b could compensate for the pleiotropic phenotype of lhp1/tfl2 mutant, suggesting that apple LHP1 homolog genes are involved in the regulation of flowering time and whole-plant growth. Based on these results, LHP1 homolog genes might have rapidly evolved among plant species, but the protein functions were conserved, at least between Arabidopsis and apple. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conserved Hydrophobic Clusters on the Surface of the Caf1A Usher C-Terminal Domain Are Important for F1 Antigen Assembly

The outer membrane usher protein Caf1A of the plague pathogen Yersinia pestis is responsible for the assembly of a major surface antigen, the F1 capsule. The F1 capsule is mainly formed by thin linear polymers of Caf1 (capsular antigen fraction 1) protein subunits. The Caf1A usher promotes polymerization of subunits and secretion of growing polymers to the cell surface. The usher monomer (811 aa, 90.5 kDa) consists of a large transmembrane β-barrel that forms a secretion channel and three soluble domains. The periplasmic N-terminal domain binds chaperone-subunit complexes supplying new subunits for the growing fiber. The middle domain, which is structurally similar to Caf1 and other fimbrial subunits, serves as a plug that regulates the permeability of the usher. Here we describe the identification, characterization, and crystal structure of the Caf1A usher C-terminal domain (Caf1A_C). Caf1A_C is shown to be a periplasmic domain with a seven-stranded β-barrel fold. Analysis of C-terminal truncation mutants of Caf1A demonstrated that the presence of Caf1A_C is crucial for the function of the usher in vivo, but that it is not required for the initial binding of chaperone-subunit complexes to the usher. Two clusters of conserved hydrophobic residues on the surface of Caf1A_C were found to be essential for the efficient assembly of surface polymers. These clusters are conserved between the FGL family and the FGS family of chaperone-usher systems.     

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.     

Dynamics of the active site architecture in plant-type ferredoxin-NADP reductases catalytic complexes

Kinetic isotope effects in reactions involving hydride transfer and their temperature dependence are powerful tools to explore dynamics of enzyme catalytic sites. In plant-type ferredoxin-NADP⁺ reductases the FAD cofactor exchanges a hydride with the NADP(H) coenzyme. Rates for these processes are considerably faster for the plastidic members (FNR) of the family than for those belonging to the bacterial class (FPR). Hydride transfer (HT) and deuteride transfer (DT) rates for the NADP⁺ coenzyme reduction of four plant-type FNRs (two representatives of the plastidic type FNRs and the other two from the bacterial class), and their temperature dependences are here examined applying a full tunnelling model with coupled environmental fluctuations. Parameters for the two plastidic FNRs confirm a tunnelling reaction with active dynamics contributions, but isotope effects on Arrhenius factors indicate a larger contribution for donor–acceptor distance (DAD) dynamics in the Pisum sativum FNR reaction than in the Anabaena FNR reaction. On the other hand, parameters for bacterial FPRs are consistent with passive environmental reorganisation movements dominating the HT coordinate and no contribution of DAD sampling or gating fluctuations. This indicates that active sites of FPRs are more organised and rigid than those of FNRs. These differences must be due to adaptation of the active sites and catalytic mechanisms to fulfil their particular metabolic roles, establishing a compromise between protein flexibility and functional optimisation. Analysis of site-directed mutants in plastidic enzymes additionally indicates the requirement of a minimal optimal architecture in the catalytic complex to provide a favourable gating contribution.     

Membrane association and activity of 15/16-membered peptide antibiotics: Zervamicin IIB, ampullosporin A and antiamoebin I

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K⁺ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER > AMP >> ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC_16:1PC, diC_18:1PC, C_16:0/C_18:1PC, diC_20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.     

Molecular mechanism of 2-APB-induced Ca influx in external acidification in PC12

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca²⁺ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca²⁺]_i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca²⁺ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca²⁺]_i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca²⁺]_i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca²⁺]_i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca²⁺ and SOC channel blockers such as SK&F96365, La³⁺ and Gd³⁺. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca²⁺]_i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca²⁺]_i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.     

Molecular structure and vibrational and chemical shift assignments of 5-(2-Hydroxyphenyl)-4-(p-tolyl)-2,4-dihydro-1,2,4-triazole-3-thione by DFT and <Emphasis Type="Italic">ab initio</Emphasis> HF calculations

The molecular geometry, vibrational frequencies, gauge including atomic orbital (GIAO) ¹H and ¹³C chemical shift values and several thermodynamic parameters of 5-(2-Hydroxyphenyl)-4-(p-tolyl)-2,4-dihydro-1,2,4-triazole-3-thione in the ground state have been calculated by using the Hartree-Fock (HF) and density functional method (DFT/B3LYP) with 6–31G(d), 6–31 + G(d,p) and LANL2DZ basis sets. The results of the optimized molecular structure are presented and compared with the experimental X-ray diffraction. The computed vibrational frequencies are used to determine the types of molecular motions associated with each of the experimental bands observed. Also, calculated ¹H chemical shift values compared with the experimental ones. The data of the title compound display significant molecular structure and IR, NMR analysis provide the basis for future design of efficient materials having the of 1,2,4-triazole core.     

An alternative approach for the synthesis of fluorogenic substrates of endo-beta-(1-->4)-xylanases and some applications

Fluorogenic substrates of endo-beta-(1-->4)-xylanases (EXs), 4-methylumbelliferyl beta-glycosides of xylobiose and xylotriose were synthesized from fully acetylated oligosaccharides using the alpha-trichloroacetimidate procedure. A commercially available syrup containing xylose and xylo-oligosaccharides was used as the starting material. Both fluorogenic glycosides were found to be suitable substrates for EXs, particularly for sensitive detection of the enzymes in electrophoretic gels and their in situ localization on sections of fruiting bodies of some plants, such as tomato, potato and eggplant, all of the family Solanaceae.     

Stimulation and clustering of cytochrome b5 reductase in caveolin-rich lipid microdomains is an early event in oxidative stress-mediated apoptosis of cerebellar granule neurons

The apoptosis of cerebellar granule neurons (CGN) induced by low potassium in the extracellular medium is a model of neuronal apoptosis where an overshot of reactive oxygen species (ROS) triggers the neuronal death. In this work, using dihydroethidium and L-012 as specific dyes for superoxide anion detection we show that this ROS overshot can be accounted by an increased release of superoxide anion to the extracellular medium. The amplitude and time course of the increase of superoxide anion observed early during apoptosis correlated with the increase of the content of soluble cytochrome b(5), a substrate of the NADH-dependent oxidase activity of the cytochrome b(5) reductase associated with lipid rafts in CGN. Western blotting and immunofluorescence microscopy approaches, including fluorescence energy transfer, pointed out an enhanced clustering of cytochrome b(5) reductase within caveolins-rich lipid rafts microdomains. Protein/protein docking analysis suggests that cytochrome b(5) reductase can form complexes with caveolins 1α, 1β and 2, playing electrostatic interactions a major role in this association. In conclusion, our results indicate that overstimulation of cytochrome b(5) reductase associated with lipid rafts can account for the overshot of plasma membrane-focalized superoxide anion production that triggers the entry of CGN in the irreversible phase of apoptosis. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Effects of changes in the shape of the intracellular action potential on the peak-to-peak ratio of single muscle fibre potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet feasible, and consequently, knowledge about certain IAP characteristics of these IAPs is still limited. The ratio between the amplitudes of the second and first phases (the so-called peak-to-peak ratio, PPR) of a single fibre action potential (SFAP) is known to be closely related to the IAP profile. The PPR of experimentally recorded SFAPs has been found to be largely independent of changes in the fibre-to-electrode (radial) distance. The main goal of this paper is to analyze the effect of changes in different aspects of the IAP spike on the relationship between PPR and radial distance. Based on this analysis, we hypothesize about the characteristics of IAPs obtained experimentally. It was found that the sensitivity of the SFAP PPR to changes in radial distance is essentially governed by the duration of the IAP spike. Assuming that, for mammals, the duration of the IAP rising phase lies within the range 0.2-0.4 ms, we tentatively suggest that the duration of the IAP spike should be over approximately 0.75 ms, with the shape of the spike strongly asymmetric. These IAP characteristics broadly coincide with those observed in mammal IAPs.     

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.     

Elucidating the mechanisms of coupled electron transfer and catalytic reactions by protein film voltammetry

Protein film voltammetry, the direct electrochemistry of redox enzymes and proteins, provides precise and comprehensive information on complicated reaction mechanisms. By controlling the driving force for a reaction (using the applied potential) and monitoring the reaction in real time (using the current), it allows thermodynamic and kinetic information to be determined simultaneously. Two challenges are inherent to protein film voltammetry: (i) to adsorb the protein or enzyme in a native and active configuration on the electrode surface, and (ii) to understand and interpret voltammetric results on both a qualitative and quantitative level, allowing mechanistic models to be proposed and rigorous experiments to test these models to be devised. This review focuses on the second of these two challenges. It describes how to use protein film voltammetry to derive mechanistic and biochemically relevant information about redox proteins and enzymes, and how to evaluate and interpret voltammetric results. Selected key studies are described in detail, to illustrate their underlying principles, strategies and physical interpretations.     

Superoxide oxidase and reductase activity of cytochrome b559 in photosystem II

This study provides evidence for the superoxide oxidase and the superoxide reductase activity of cytochrome b₅₅₉ (cyt b₅₅₉) in PSII. It is reported that in Tris-treated PSII membranes upon illumination, both the intermediate potential (IP) and the reduced high potential (HP^red) forms of cyt b₅₅₉ exhibit superoxide scavenging activity and interconversion between IP and HP^red form. When Tris-treated PSII membranes were illuminated in the presence of spin trap EMPO, the formation of superoxide anion radical (O₂⁻) was observed, as confirmed by EPR spin-trapping spectroscopy. The observations that the addition of enzymatic (superoxide dismutase) and non-enzymatic (cytochrome c, α-tocopherol and Trolox) O₂⁻ scavengers prevented the light-induced conversion of IP ↔ HP^red cyt b₅₅₉ confirmed that IP and HP^red cyt b₅₅₉ are reduced and oxidized by O₂⁻, respectively. Redox changes in cyt b₅₅₉ by an exogenous source of O₂⁻ reconfirmed the superoxide oxidase and reductase activity of cyt b₅₅₉. Furthermore, the light-induced conversion of IP to HP^red form of cyt b₅₅₉ was completely inhibited at pH > 8 and by chemical modification of the imidazole ring of histidine residues using diethyl pyrocarbonate. We proposed that a change in the environment around the heme iron, induced by the protonation and deprotonation of His²² residue generates a favorable condition for the oxidation and reduction of O₂⁻, respectively.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Effect of selenium-enriched malt on hepatocarcinogenesis, paraneoplastic syndrome and the hormones regulating blood glucose in rats treated by diethylnitrosamine

233 SD rats weighing 100 approximately 120 g were divided randomly into 6 groups. The animals in group I and group II received 0.1 mg/kg selenium in the form of sodium selenite only and served as the negative control and positive control, respectively. Animals in groups III, IV and V were fed with selenium as Se-enriched malt supplemented diets (0.3, 1 and 3 mg/kg), and group VI with selenium by using sodium selenite supplemented diets (3 mg/kg). Animals of groups II approximately VI were induced hepatoma by diethylnitrosamine (100 mg/l) for 16 weeks, then drunk with sterilized water for 2 more weeks. Subsequently, the effects of Se-enriched malt and sodium selenite on hepatoma nodules, relative liver weight, the liver function indices including alanine aminotransferase (ALT), alkaline phosphatase (ALP), albumin (ALB), total bilirubin (TBIL), and the tumor markers, named as gamma-glutamyltranspeptidase (GGT), alpha-fetoprotein (AFP), insulin-like growth factor-II (IGF-II) were recorded. The calcium concentration, glucose content in plasma and values of the hormones regulating blood glucose, such as insulin, glucagons and thyroid hormones (3,5,3'-tetraiodothyronine, T(3); 3,5,3'5'-tetraiodothyronine, T(4)) were observed as well. At the same time, the correlations between the concentration of plasma glucose and related hormones were also analyzed. The results indicated that Se-enriched malt showed a better chemopreventive efficiency in decreasing the number of hepatoma nodules, relative liver weight and the contents of AFP, GGT, IGF-II, ALT, ALP and TBIL in the plasma, and delaying the descent of hormones in the serum, names as insulin, glucagons, T(3) and T(4) than those feeding with sodium selenite. Effect of Se-enriched malt excelled sodium selenite in the aspects of deadening the descent of glucose concentration in the plasma and the rise of calcium concentration in the serum of the rats with hepatoma induced by diethylnitrosamine. The values of glucose and calcium were significantly related to those items fore-named. In conclusion, the function of Se-enriched malt in deadening the lesion and delaying the development of hepatoma of rats induced by diethylnitrosamine was better than that of sodium selenite. Hypoglycemia and hypercalcemia were significantly correlated with the multifactors mentioned above.     

Membrane topology of loop 13-14 of the Na/glucose cotransporter (SGLT1): A SCAM and fluorescent labelling study

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na⁺/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES⁽⁻⁾ and MTSET⁽⁺⁾ had no effect on either cotransport or Na⁺ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K_i^Pz and K_m^αMG for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K_i^Pz values than wtSGLT1 with minimal changes in K_m^αMG, the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.