Evidence for a plasma-membrane-bound nitrate reductase involved in nitrate uptake of Chlorella sorokiniana

Anti-nitrate-reductase (NR) immunoglobulin-G (IgG) fragments inhibited nitrate uptake into Chlorella cells but had no affect on nitrite uptake. Intact anti-NR serum and preimmune IgG fragments had no affect on nitrate uptake. Membrane-associated NR was detected in plasma-membrane (PM) fractions isolated by aqueous two-phase partitioning. The PM-associated NR was not removed by sonicating PM vesicles in 500 mM NaCl and 1 mM ethylenediaminetetraacetic acid and represented up to 0.8% of the total Chlorella NR activity. The PM NR was solubilized by Triton X-100 and inactivated by Chlorella NR antiserum. Plasma-membrane NR was present in ammonium-grown Chlorella cells that completely lacked soluble NR activity. The subunit sizes of the PM and soluble NRs were 60 and 95 kDa, respectively, as determined by sodium-dodecyl-sulfate electrophoresis and western blotting.Abbreviations EDTA ethylenediaminetetraacetic acid - FAD flavine-adenine dinucleotide - IgG immunoglobulin G - NR nitrate reductase - PM plasma membrane - TX-100 Triton X-100     

Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane-Bound Polysomes

Abstract: Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[³⁵S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α¹, α², β¹, and α²) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to α-tubulin. Only trace amounts of α- and β-tubulin and actin were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and α- and β-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α- and β-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to α-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.     

Nuclear matrix bound terminal deoxynucleotidyl transferase in rat thymus nuclei. I. A possible site for TdT mediated function

Approximately 80% of the terminal deoxynucleotidyl transferase (TdT) in thymus glands from 3–4 week old rats was found to be localized in the nucleus and the remaining 20% in the cytosol. Following endogenous nuclease digestion of the thymus nuclei, 70–85% of the nuclear TdT could be removed by low salt and high salt extractions, whereas 15–30% of the enzyme remained tightly bound to the residual nuclear matrix. Low salt and high salt extracts of the nuclei contained a mixture of 58, 56, 45 and 44 kDa species of TdT whereas only 58 kDa species of the enzyme was found to be associated with the matrix. In addition to TdT, 20–25% of the nuclear DNA polymerase was also tightly bound to the isolated nuclear matrix. These observations lead us to propose that besides being the site of DNA replicationvia-matrix bound replicational complexes [Van der Velden H.M.W. & Wanka F., Molecular Biology Reports 12 (1987): 69], nuclear matrix may also be the site of TdT mediated function and that matrix bound TdT and free TdT could be the functional and nonfunctional forms of the enzyme, respectively, in the thymus gland.Abbreviations dNTP deoxyribonucleoside triphosphate - DTT dithiothreitol - Ig immunoglobulin - PMSF phenylmethylsulfonylfluoride - rNTP ribonucleoside triphosphate - SDS sodium dodecyl sulphate - TCR T cell receptor - TdT terminal deoxynucleotidyl transferase - VDJ variable, diversity and joining segments of Ig or TCR genes     

Soluble Leptin Receptor and Soluble Receptor‐Bound Fraction of Leptin in the Metabolic Syndrome

Objective: In obesity, plasma leptin is high and soluble leptin receptor (sOb‐R) levels are low, resulting in a low fraction of bound leptin. The aim of this study was to investigate the influence of insulin resistance (IR) and the metabolic syndrome (MS) on sOb‐R concentration and the bound‐free ratio of leptin. Research Methods and Procedures: sOb‐R, leptin levels, and homeostasis model assessment (HOMA) index for IR were determined in 76 middle‐aged obese or overweight men. Results: Concentration of sOb‐R and soluble receptor‐bound fraction of leptin were lowest in the highest tertile of HOMA‐IR. sOb‐R and the bound‐free ratio of leptin correlated with HOMA‐IR, leptin concentration, and waist‐to‐hip ratio independently of age, BMI, and fat mass. Leptin and waist‐to‐hip ratio were the sole independent determinants of sOb‐R concentration, and BMI, HOMA‐IR, and visceral adipose tissue were independent determinants of the bound fractin of leptin. sOb‐R concentration and the bound fraction of leptin decreased with increasing numbers of components of the MS, resulting in lower sOb‐R concentration and a lower fraction of bound leptin in men with the MS. Discussion: IR and abdominal obesity are associated with low sOb‐R concentration and low bound‐free ratio of leptin independent of fat mass. Low sOb‐R concentration and low bound‐free ratio of leptin segregate with components of the MS. We suggest that low sOb‐R levels and a low fraction of specifically bound leptin are markers of leptin resistance, which is independently associated with IR and abdominal obesity and may constitute an additional component of the MS.     

带扩散的Barbour血吸虫病模型的定性分析(英文)

这篇文章主要考虑由常微分方程组和偏微分方程组构成的Barbour血吸虫病模型.偏微分系统是反映空间和时间分布的反应扩散系统.对模型的定性性质进行了分析.利用比较原理得出解的一致有上界性.同时利用能量方法证明出椭圆系统在扩散系数的一定范围内没有非常数的正稳态解.     

Stabilization of Membrane Bound Enzyme Profiles and Lipid Peroxidation by Withania Somnifera Along with Paclitaxel on Benzo(a)pyrene Induced Experimental Lung Cancer

The present study was aimed to evaluate the therapeutic effects of Withania somnifera along with paclitaxel on lung tumor induced by benzo(a)pyrene in male Swiss albino mice. The levels of ATPase enzymes and lipid peroxidation were evaluated in lung cancer bearing mice, in erythrocyte membrane and tissues. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases (Na⁺/K⁺-ATPases, Mg²⁺-ATPases and Ca²⁺-ATPases) were determined. The alterations of these enzyme activities in membrane and tissues were indicative of the tumor formation caused by benzo(a)pyrene (50 mg/kg body weight, orally) in cancer bearing animals. The activities of these enzymes were reversed to near normal control values in animals treated with Withania somnifera (400 mg/kg b.wt, orally) along with paclitaxel (33 mg/kg b.wt, i.p). Treatment with Withania somnifera along with paclitaxel altered these damage mediated through free radicals, and the treatment displays the protective role of these drugs by inhibiting free radical mediated cellular damages. Over, based on the data providing a correlation Withania somnifera along with paclitaxel provide stabilization of membrane bound enzyme profiles and decreased lipid peroxidation against benzo(a)pyrene induced lung cancer in mice.     

Differential response of iron metabolism to oxidative stress generated by antimycin A and nitrofurantoin

The close interrelationship of oxidative stress and iron is evident by the influence of intracellular reactive oxygen species on iron metabolism. Oxygen radicals can lead to release of iron from iron-sulfur proteins and ferritin, and can damage iron-containing enzymes such as mitochondrial aconitase. Treatment of HepG2 human hepatoma cells with antimycin A has two effects relating to iron depending on the concentrations of antimycin A: increase of the labile iron pool and stimulation of non-transferrin-bound iron uptake. Whereas the first could also be generated with nitrofurantoin, the stimulation of non-transferrin-bound iron uptake was only seen with antimycin A and needed considerably higher concentrations. Pretreatment of the cells with ebselen, which scavenges peroxides, reverted only the effect of nitrofurantoin on the labile iron pool. Depletion with iron chelators before or after treatment with antimycin A diminished the stimulation of non-transferrin-bound iron uptake. We conclude that the generation of oxygen radicals in the mitochondria leads to the liberation of iron from mitochondrial enzymes, which enters the labile iron pool. But high concentrations of antimycin A leading to the stimulation of non-transferrin-bound iron uptake is possibly not related to the inhibition of the respiratory chain.     

Transmembrane proton translocation by cytochrome c oxidase

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O₂-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O₂ reduction and how these reactions might be energetically coupled to proton pumping across the membrane.     

Influenza A virus-induced caspase-3 cleaves the histone deacetylase 6 in infected epithelial cells

Histone deacetylase 6 (HDAC6) is a multi-substrate cytoplasmic enzyme that regulates many important biological processes. Recently, some reports have implicated HDAC6 in viral infection. However, nothing is known about its regulation in virus-infected cells. The data presented here for the first time demonstrate the caspase-3-mediated cleavage of HDAC6 in influenza A virus (IAV)-infected cells. HDAC6 polypeptide contains the caspase-3 cleavage motif DMAD-S at the C-terminus, and is a caspase-3 substrate. The cleavage removes most of the C-terminal ubiquitin-binding zinc finger domain from HDAC6, which could be significant for HDAC6’s role in IAV-induced apoptosis in infected cells.     

The Contribution of a Covalently Bound Cofactor to the Folding and Thermodynamic Stability of an Integral Membrane Protein

The factors controlling the stability, folding, and dynamics of integral membrane proteins are not fully understood. The high stability of the membrane protein bacteriorhodopsin (bR), an archetypal member of the rhodopsin photoreceptor family, has been ascribed to its covalently bound retinal cofactor. We investigate here the role of this cofactor in the thermodynamic stability and folding kinetics of bR. Multiple spectroscopic probes were used to determine the kinetics and energetics of protein folding in mixed lipid/detergent micelles in the presence and absence of retinal. The presence of retinal increases extrapolated values for the overall unfolding free energy from 6.3 ± 0.4 kcal mol^− 1 to 23.4 ± 1.5 kcal mol^− 1 at zero denaturant, suggesting that the cofactor contributes 17.1 kcal mol^− 1 towards the overall stability of bR. In addition, the cooperativity of equilibrium unfolding curves is markedly reduced in the absence of retinal with overall m-values decreasing from 31.0 ± 2.0 kcal mol^− 1 to 10.9 ± 1.0 kcal mol^− 1, indicating that the folded state of the apoprotein is less compact than the equivalent for the holoprotein. This change in the denaturant response means that the difference in the unfolding free energy at a denaturant concentration midway between the two unfolding curves is only ca 3-6 kcal mol^− 1. Kinetic data show that the decrease in stability upon removal of retinal is associated with an increase in the apparent intrinsic rate constant of unfolding, k_u^H2O, from ~1 × 10^− 16 s^− 1 to ~1 × 10^− 4 s^− 1 at 25 °C. This correlates with a decrease in the unfolding activation energy by 16.3 kcal mol^− 1 in the apoprotein, extrapolated to zero SDS. These results suggest that changes in bR stability induced by retinal binding are mediated solely by changes in the activation barrier for unfolding. The results are consistent with a model in which bR is kinetically stabilized via a very slow rate of unfolding arising from protein-retinal interactions that increase the rigidity and compactness of the polypeptide chain.