Stimulation of H+transport activity of vacuolar H+ATPase by activation of H+PPase in Kalanchoë blossfeldiana

In Kalanchoë blossfeldiana cv. Tom Thumb the initial rate of ATP-dependent H+-transport into tonoplast vesicles was stimulated up to three times if the H+-ATPase (EC 3.6.1.3) was energized a few minutes after pre-energization of the H+-PPase (EC 3.6.1.1). H+-PPase-activated ATP-dependent H+-transport was observed in plants of K. blossfeldiana cultivated in short day (SD) or long day (LD) conditions expressing different degrees of crassulacean acid metabolism (CAM). However, based on the higher activity and protein amount of H+-PPase and H+-ATPase present in the vacuolar membrane of SD plants the maximum H+-transport activity in the stimulated mode of the H+-ATPase was significantly higher in tonoplast vesicles of SD plants than of LD plants. Hence, a co-ordinated action of the H+-PPase and H+-ATPase at the tonoplast of Kalanchoë could allow a higher transport capacity at the vacuolar membrane when plants perform high CAM. Immunoprecipitation experiments with an antiserum raised against the A-subunit of the vacuolar H+-ATPase of Mesembryanthemum crystallinum L. showed that in SD and LD plants of K. blossfeldiana the H+-PPase was co-precipitated with the vacuolar H+-ATPase holoenzyme. The co-percipitation of the two transport proteins indicates a close structural localization of the H+-PPase and the A-subunit of the vacuolar H+-ATPase.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.     

Quantitative and time-course analysis of microbial degradation of 1H,1H,2H,2H,8H,8H–perfluorododecanol in activated sludge

A methylene group in the fluorinated carbon backbone of 1H,1H,2H,2H,8H,8H–perfluorododecanol (degradable telomer fluoroalcohol, DTFA) renders the molecule cleavable by microbial degradation into two fluorinated carboxylic acids. Several biodegradation products of DTFA are known, but their rates of conversion and fates in the environment have not been determined. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitatively investigate DTFA biodegradation by the microbial community in activated sludge in polyethylene terephthalate (PET) flasks, which we also determined here showed least adsorption of DTFA. A reduction in DTFA concentration in the medium was accompanied by rapid increases in the concentrations of 2H,2H,8H,8H–perfluorododecanoic acid (2H,2H,8H,8H–PFDoA), 2H,8H,8H-2-perfluorododecenoic acid (2H,8H,8H-2-PFUDoA), and 2H,2H,8H-7-perfluorododecenoic acid and 2H,2H,8H-8-perfluorododecenoic acid (2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA), which were in turn followed by an increase in 6H,6H–perfluorodecanoic acid (6H,6H–PFDeA) concentration, and decreases in 2H,2H,8H,8H–PFDoA, 2H,8H,8H-2-PFUDoA, and 2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA concentrations. Accumulation of perfluorobutanoic acid (PFBA), a presumed end product of DTFA degradation, was also detected. Our quantitative and time-course study of the concentrations of these compounds reveals main routes of DTFA biodegradation, and the presence of new biodegradation pathways.

     

Characterization of H+/OH− currents in phospholipid vesicles

In pure phospholipid vesicles, the conductivity of H⁺/OH^– ions exceeds that for other simple inorganic ions. Protons achieve electrochemical equilibrium across egg phosphatidylcholine vesicles within tens of minutes. When pH gradients are established across vesicles, transmembrane potentials develop. Conversely, the establishment of transmembrane potentials leads to the formation of pH gradients. When the phenomenological permeability of H⁺/OH^– ions in vesicles is estimated, values are obtained that are much greater (six orders of magnitude larger) than those for Na⁺ or K⁺. A wide range in the values for this permeability has been reported; however, much of the discrepancy can be attributed to differences in the vesicle systems and experimental conditions. The H⁺/OH^– current appears to be modulated by changes in membrane dielectric constant. However, the dependence of this current on the pH gradient and on the membrane voltage argues against simple diffusion mechanisms as the source of the H⁺/OH^– current. In addition, in vesicle systems the H⁺/OH^– current shows a surprising invariance to changes in the membrane dipole potential, an observation that argues against the role of simple carriers for H⁺ and OH^– ions.     

Quantitation of γH2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming γH2AX, is an early response to DNA double-strand breaks¹. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)². Overall, DSB induction results in the formation of discrete nuclear γH2AX foci which can be easily detected and quantitated by immunofluorescence microscopy². Given the unique specificity and sensitivity of this marker, analysis of γH2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of γH2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, γH2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer³. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of γH2AX foci ex vivo and in vivo^4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of γH2AX foci in mouse tissues.Download video file.^{(284M, mp4)}     

Determination of Hα,Hβ and Hβ,C′ coupling constants in13C-labeled proteins

Summary A sensitive method to assign H protons stereospecifically as well as to determine rotamer populations about ₁, in two 3D experiments is presented. The SOFT-HCCH-COSY experiment allowed us to measure the³J(H,C) couplings, using constant time evolution of C in t₂ and C_aliphatic-selective decoupling during t₃. The SOFT-HCCH-E.COSY experiment allowed us to measure the³J(H,H) couplings, using constant time evolution of C in t₂, a small flip angle¹H excitation pulse in the second mixing time, and double-band-selective decoupling (aliphatic and carbonyl carbons) during t₃. The method was applied to ribonuclease T₁.     

Structural variability in the genome of phage ?H of Halobacterium halobium

Summary The partially circularly permuted, terminally redundant structure of the DNA of phage H has been confirmed by a cleavage map for the restriction enzymes PstI, ClaI, BglII, HindIII, and, partially, BamHI.Six variants of phage H have been isolated from 71 single plaques. Their genomes differ by several insertions, a deletion, and an inversion of a DNA segment with a minimal length of 11 kb. The inversion occurs with high frequency in variants carrying at the flanks of the invertible DNA in verted repeats of a 1.8 kb DNA element which shares sequence homology with the DNA of H. halobium and may be involved in the extreme variability of its genome.