NdhO,a Subunit of NADPH Dehydrogenase,Destabilizes Medium Size Complex of the Enzyme in Synechocystis sp. Strain PCC 6803

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.     

Cell penetrating peptide TAT can kill cancer cells via membrane disruption after attachment of camptothecin

Attachment of traditional anticancer drugs to cell penetrating peptides is an effective strategy to improve their application in cancer treatment. In this study, we designed and synthesized the conjugates TAT-CPT and TAT-2CPT by attaching camptothecin (CPT) to the N-terminus of the cell penetrating peptide TAT. Interestingly, we found that TAT-CPT and especially TAT-2CPT could kill cancer cells via membrane disruption, which is similar to antimicrobial peptides. This might be because that CPT could perform as a hydrophobic residue to increase the extent of membrane insertion of TAT and the stability of the pores. In addition, TAT-CPT and TAT-2CPT could also kill cancer cells by the released CPT after they entered cells. Taken together, attachment of CPT could turn cell penetrating peptide TAT into an antimicrobial peptide with a dual mechanism of anticancer action, which presents a new strategy to develop anticancer peptides based on cell penetrating peptides.     

Oxygen concentration-dependent induction of a 140-kDa protein in magnetic bacterium Magnetospirillum magnetotacticum MS-1

Abstract The magnetic bacterium Magnetospirillum magnetotacticum prefers a microaerobic habitat and should be able to sense oxygen. Therefore, the bacterium was cultured under atmospheres containing 0–5% O₂ and analyzed for oxygen-dependent changes in the levels of its protein components by sodium dodecyl sulfate-polyccrylamide gel electrophoresis (SDS-PAGE). The analysis revealed a marked anaerobic induction of a 140-kDa protein, which was suppressed when M. magnetotacticum was switched from microaerobic (<1% O₂) to aerobic (>1% O₂) growth conditions. Although its function remains to be determined, the 140-kDa protein may serve as a useful tool to gain insight into the physiology of the organism.     

β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Shear Stress-induced Redistribution of Vascular Endothelial-Protein-tyrosine Phosphatase (VE-PTP) in Endothelial Cells and Its Role in Cell Elongation

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.     

Biodegradation by activated sludge and toxicity of tetracycline into a semi-industrial membrane bioreactor

Much attention has been devoted recently to the fate of pharmaceutically active compounds such as tetracycline antibiotics in soil and water. Tetracycline (TC) biodegradability by activated sludge derived from membrane bioreactor (MBR) treating swine wastewater via CO₂-evolution was evaluated by means of modified Sturm test, which was also used to evaluate its toxicity on carbon degradation. The impact of tetracycline on a semi-industrial MBR process was also examined and confronted to lab-scale experiments. After tetracycline injection in the pilot, no disturbance was detected on the elimination of organic matters and ammonium (nitrification), reaching after injection 88% and 99% respectively; only denitrification was slightly affected. Confirming the ruggedness and the superiority of membrane bioreactors over conventional bioreactors, no toxicity was observed at the considered level of TC in the pilot (20 mg TOC L⁻¹), while at lab-scale sodium benzoate biodegradation was completely inhibited from 10 mg TOC L⁻¹ TC. The origin of the activated sludge showed a significant impact on the performances, since the ultimate biodegradation was in the range −50% to −53% for TC concentrations in the range 10–20 mg TOC L⁻¹ with conventional bioreactor sludge and increased to 18% for 40 mg TOC L⁻¹ of TC with activated sludge derived from the MBR pilot. This confirmed the higher resistance of activated sludge arising from membrane bioreactor.     

Large-ligand adsorption to membranes III. Cooperativity and general ligand shapes

In previous reports (Stankowski, S. (1983) Biochim. Biophys. Acta 735, 341–351 and 352–360) the ordinary Scatchard-type analysis has been shown to yield erroneous results when applied to the binding of large molecules to membranes or cells. Formulae have been given to treat the limiting cases of very thin and of very bulky ligands. These results are now extended to include ligands of any shape and cooperative interactions. As an example, data on the cooperative binding of polymyxin to charged lipid bilayers are reevaluated. Adsorption with concomitant incorporation of the large molecule into the membrane is also considered.     

Membrane lipid composition and susceptibility to bile salt damage

Erythrocyte membranes with low sphingomyelin: choline-containing phospholipid ratios haemolyse at low concentrations of the bile salt, glycocholate. Erythrocytes with higher sphingomyelin: choline-containing phospholipid ratios require progessively greater concentrations of the bile salt for lysis.Sublytic concentrations of glycocholate remove phospholipid and acetylcholinesterase from the membranes. Membranes with low sphingomyelin: choline-containing phospholipid ratios lose both particulate (microvesicles of distinct composition) and ‘solubilized’ material, the particulate form predominating. The proportion of particulate material falls with increase of the membrane sphingomyelin: choline-containing phospholipid ratio and those membranes of highest sphingomyelin: choline-containing phospholipid ratio lose material predominantly in ‘solubilized’ form.Sheep erythrocytes treated to increase their content of phosphatidylcholine (and thereby reduce their membrane sphingomyelin: choline-containing phospholipid ratio) become more susceptible to lysis by glycocholate.These observations indicate a correlation between membrane lipid composition and the perturbation of membranes with bile salt; they also point to possible features of membranes capable of surviving exposure to the high bile salt concentrations of the biliary tract.     

Errata

Human erythrocytes were exposed to oxidative stress by iodate and periodate. Oxidation causes a time- and concentration-dependent increase in membrane permeability for hydrophilic molecules and ions. The induced leak discriminates nonelectrolytes on the basis of molecular size and exhibits a very low activation energy (E_a = 1–4 kcal · mol^?1). These results are reconcilable with the formation of aqeous pores. The pore size was approximated to be between 0.45 and 0.6 nm. This increase in permeability is reversible upon treatment with dithioerythritol. Blocking of membrane thiol groups with N-ethylmaleimide protects the membranes against leak formation. The oxidation causes dithioerythritol-reversible modification of membrane proteins as indicated by the gel electrophoretic behavior. These modifications can also be suppressed by blocking the membrane thiol groups with N-ethylmaleimide. About half of the membrane methionine is oxidized to acid hydrolysis-stable derivatives. A fast saturating increase in diene conjugation was observed in whole cells but not in isolated membranes, with only minor degradation of fatty acid chains. The oxidation of cell membrane lipids as well as oxidation of cell surface carbohydrates are not involved in leak formation. Taken together with earlier data (Deuticke, B., Poster, B., Lütkemeier, P., and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196–210), these findings indicate that formation of disulfide bonds by different oxidative mechanisms results in leaks with similar properties.