Association of BK viremia with human leukocyte antigen mismatches and acute rejection, but not with type of calcineurin inhibitor.

INTRODUCTION: BK viremia and polyomavirus-associated nephropathy (PVN) represent a significant problem after kidney transplantation. Both are associated with intensified immunosuppression, but other risk factors and the impact of a screening program on outcome are incompletely understood. METHODS: Here, we report on the short- and long-term outcome of a cohort of patients, who were transplanted in 2006/2007 and included in a newly introduced systematic 3-monthly screening for BK viremia at the University Hospital Zurich. In patients testing positive for BK viremia, screening frequency was intensified and immunosuppression reduced. Patients with suspected PVN underwent transplant biopsy. RESULTS: Among 152 included patients, 49 (32%) tested positive for BK viremia, but only 8 developed biopsy-proven PVN. BK viremia had a significant impact on estimated glomerular filtration rate and proteinuria in the first 2 years. Acute rejection episodes and the number of human leukocyte antigen (HLA) mismatches were the strongest independent predictors of BK viremia in a multiple logistic model. In contrast, no particular immunosuppressive agent or regimen was associated with enhanced risk. CONCLUSION: Taken together, systematic BK viremia screening led to detection of a high percentage of viremic patients. With adjustment of immunosuppression, an excellent outcome was achieved. The independent association of HLA mismatches with BK viremia suggests impaired polyomavirus immunosurveillance in highly mismatched allografts.

N-terminal truncation of the variable subunit stabilizes spinach ferredoxin:thioredoxin reductase.

The variable subunit of spinach ferredoxin:thioredoxin reductase (FTR) has an extended N-terminus compared to FTRs from other sources and this was proposed to contribute to the instability of the protein. We constructed two N-terminal truncation mutants of recombinant FTR by removing 16 or 24 residues from the variable subunit. The mutant proteins are readily expressed and show half-saturation values (S(0.5)) for ferredoxin and thioredoxin f comparable to WT. However, truncation increases significantly their stability. Using the stabilized FTR an exposed Cys on its thioredoxin contact surface could be substituted without altering its properties, whereas the replacement of an active site Cys by Ser completely destabilized the protein.

Oxidation-reduction and activation properties of chloroplast fructose 1,6-bisphosphatase with mutated regulatory site.

The concentration of Mg(2+) required for optimal activity of chloroplast fructose 1,6-bisphosphatase (FBPase) decreases when a disulfide, located on a flexible loop containing three conserved cysteines, is reduced by the ferredoxin/thioredoxin system. Mutation of either one of two regulatory cysteines in this loop (Cys155 and Cys174 in spinach FBPase) produces an enzyme with a S(0.5) for Mg(2+) (0.6 mM) identical to that observed for the reduced WT enzyme and significantly lower than the S(0.5) of 12.2 mM of oxidized WT enzyme. E(m) for the regulatory disulfide in WT spinach FBPase is -305 mV at pH 7.0, with an E(m) vs pH dependence of -59 mV/pH unit, from pH 5.5 to 8.5. Aerobic storage of the C174S mutant produces a nonphysiological Cys155/Cys179 disulfide, rendering the enzyme partially dependent on activation by thioredoxin. Circular dichroism spectra and thiol titrations provide supporting evidence for the formation of nonphysiological disulfide bonds. Mutation of Cys179, the third conserved cysteine, produces FBPase that behaves very much like WT enzyme but which is more rapidly activated by thioredoxin f, perhaps because the E(m) of the regulatory disulfide in the mutant has been increased to -290 mV (isopotential with thioredoxin f). Structural changes in the regulatory loop lower S(0.5) for Mg(2+) to 3.2 mM for the oxidized C179S mutant. These results indicate that opening the regulatory disulfide bridge, either through reduction or mutation, produces structural changes that greatly decrease S(0.5) for Mg(2+) and that only two of the conserved cysteines play a physiological role in regulation of FBPase.

Routine experimental system for defining conditions used in photodynamic therapy and fluorescence photodetection of (non-) neoplastic epithelia.

A common method to induce enhanced short-term endogenous porphyrin synthesis and accumulation in cell is the topical, systemic application of 5-aminolevulinic acid or one of its derivatives. This circumvents the intravenous administration of photosensitizers normally used for photodynamic therapy (PDT) of fluorescence photodetection. However, in the majority of potential medical indications, optimal conditions with respect to the porphyrin precursor or its pharmaceutical formulation have not yet been found. Due to ethical restrictions and animal right directives, the number of available test objects is limited. Hence, definition and use of nonanimal test methods are needed. Tissue and organ cultures are a promising approach in replacing cost intensive animal models in early stages of drug development. In this paper, we present a tissue culture, which can among others be used routinely to answer specific questions emerging in the field of photodynamic therapy and fluorescence photodetection. This technique uses mucosae excised from sheep paranasal sinuses or pig bladder, which is cultured under controlled conditions. It allows quasiquantitative testing of different protoporphyrin IX precursors with respect to dose-response curves and pharmacokinetics, as well as the evaluation of different incubation conditions and/or different drug formulations. Furthermore, this approach, when combined with the use of electron microscopy and fluorescence-based methods, can be used to quantitatively determine the therapeutic outcome following protoporphyrin IX-mediated PDT.

Picrotoxin blockade of invertebrate glutamate-gated chloride channels: subunit dependence and evidence for binding within the pore.

Glutamate-gated chloride channels have been described in nematodes, insects, crustaceans, and mollusks. Subunits from the nematode and insect channels have been cloned and are phylogenetically related to the GABA and glycine ligand-gated chloride channels. Ligand-gated chloride channels are blocked with variable potency by the nonselective blocker picrotoxin. The first two subunits of the glutamate-gated chloride channel family, GluClalpha and GluClbeta, were cloned from the free living nematode Caenorhabditis elegans. In this study, we analyze the blockade of these novel channels by picrotoxin. In vitro synthesized GluClalpha and GluClbeta RNAs were injected individually or coinjected into Xenopus oocytes. The EC50 values for picrotoxin block of homomeric GluClalpha and GluClbeta were 59 microM and 77 nM, respectively. Picrotoxin block of homomeric GluClbeta channels was promoted during activation of membrane current with glutamate. In addition, recovery from picrotoxin block was faster during current activation by glutamate. A chimeric channel between the N-terminal extracellular domain of GluClalpha and the C-terminal membrane-spanning domain of GluClbeta localized the higher affinity picrotoxin binding site to the membrane-spanning domains of GluClbeta. A point mutation within the M2 membrane-spanning domain of GluClbeta reduced picrotoxin sensitivity >10,000-fold. We conclude that picrotoxin blocks GluCl channels by binding to a site accessible when the channel is open.

Role of the [Fe4S4] cluster in mediating disulfide reduction in spinach ferredoxin:thioredoxin reductase.

Thioredoxin reduction in plant chloroplasts is catalyzed by a unique class of disulfide reductases which use a one-electron donor, [Fe2S2]2+,+ ferredoxin, and has an active site involving a disulfide in close proximity to a [Fe4S4]2+ cluster. In this study, spinach ferredoxin:thioredoxin reductase (FTR) reduced with stoichiometric amounts of reduced benzyl viologen or frozen under turnover conditions in the presence of thioredoxin is shown to exhibit a slowly relaxing S = 1/2 resonance (g = 2.11, 2.00, 1.98) identical to that of a modified form of the enzyme in which one of the cysteines of the active-site disulfide is alkylated with N-ethylmaleimide (NEM-FTR). Hence, in accord with the previous proposal [Staples, C.R., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A.-L., Schürmann, P., Knaff, D.B., and Johnson, M.K. (1996) Biochemistry 35, 11425-11434], NEM-FTR is shown to be a stable analogue of a one-electron-reduced enzymatic intermediate. The properties of the Fe-S cluster in NEM-FTR have been further investigated by resonance Raman and electron nuclear double resonance spectroscopies; the results, taken together with the previous UV-visible absorption, variable temperature magnetic circular dichroism, and resonance Raman data, indicate the presence of a novel type of [Fe4S4]3+ cluster that is coordinated by five cysteinates with little unpaired spin density delocalized onto the cluster-associated cysteine of the active-site disulfide. While the ligation site of the fifth cysteine remains undefined, the best candidate is a cluster bridging sulfide. On the basis of the spectroscopic and redox results, mechanistic schemes are proposed for the benzyl viologen-mediated two-electron-reduction of FTR and the catalytic mechanism of FTR. The catalytic mechanism involves novel S-based cluster chemistry to facilitate electron transfer to the active-site disulfide resulting in covalent attachment of the electron-transfer cysteine and generation of the free interchange cysteine that is required for the thiol-disulfide interchange reaction with thioredoxin.

Amino acid sequence of the maize ferredoxin:thioredoxin reductase variable subunit.

The ferredoxin:thioredoxin reductase (FTR) is the essential enzyme of the light dependent regulatory system controlling enzyme activities in oxygenic, photosynthetic cells. This protein is composed of two dissimilar subunits, a catalytic subunit containing a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site, and a variable subunit, whose function is not known yet. Whereas size and primary structure of the catalytic subunit from different organisms seem to be well conserved, they are quite variable for the variable subunit. Here we report the complete amino acid sequence of the variable subunit of maize (Zea mays) FTR established by protein sequencing. The subunit contains 97 residues and has a calculated molecular mass of 10939 Da. A sequence comparison shows 40% identity with the variable subunit from spinach and 38% with the one from Anacystis. The identical residues are grouped in three consensus domains, one near the N-terminus, one in the middle of the subunit and one near the C-terminus. We have obtained some evidence indicating that the N-terminal consensus domain is possibly involved in the interaction with the catalytic subunit.

The function and properties of the iron-sulfur center in spinach ferredoxin: thioredoxin reductase: a new biological role for iron-sulfur clusters.

Thioredoxin reduction in chloroplasts is catalyzed by a unique class of disulfide reductases which use a [2Fe-2S]2+/+ ferredoxin as the electron donor and contain an Fe-S cluster as the sole prosthetic group in addition to the active-site disulfide. The nature, properties, and function of the Fe-S cluster in spinach ferredoxin:thioredoxin reductase (FTR) have been investigated by the combination of UV/visible absorption, variable-temperature magnetic circular dichroism (MCD), EPR, and resonance Raman (RR) spectroscopies. The results indicate the presence of an S = 0 [4Fe-4S]2+ cluster with complete cysteinyl-S coordination that cannot be reduced at potentials down to -650 mV, but can be oxidized by ferricyanide to an S = 1/2 [4Fe-4S]3+ state (g = 2.09, 2.04, 2.02). The midpoint potential for the [4Fe-4S]3+/2+ couple is estimated to be +420 mV (versus NHE). These results argue against a role for the cluster in mediating electron transport from ferredoxin (Em = -420 mV) to the active-site disulfide (Em = -230 mV, n = 2). An alternative role for the cluster in stabilizing the one-electron-reduced intermediate is suggested by parallel spectroscopic studies of a modified form of the enzyme in which one of the cysteines of the active-site dithiol has been alkylated with N-ethylmaleimide (NEM). NEM-modified FTR is paramagnetic as prepared and exhibits a slow relaxing, S = 1/2 EPR signal, g = 2.11, 2.00, 1.98, that is observable without significant broadening up to 150 K. While the relaxation properties are characteristic of a radical species, MCD, RR, and absorption studies indicate at least partial cluster oxidation to the [4Fe-4S]3+ state. Dye-mediated EPR redox titrations indicate a midpoint potential of -210 mV for the one-electron reduction to a diamagnetic state. By analogy with the properties of the ferricyanide-oxidized [4Fe-4S] cluster in Azotobacter vinelandii 7Fe ferredoxin [Hu, Z., Jollie, D., Burgess, B. K., Stephens, P. J., & Münck, E. (1994) Biochemistry 33, 14475-14485], the spectroscopic and redox properties of NEM-modified FTR are interpreted in terms of a [4Fe-4S]2+ cluster covalently attached through a cluster sulfide to a cysteine-based thiyl radical formed on one of the active-site thiols. A mechanistic scheme for FTR is proposed with similarities to that established for the well-characterized NAD(P)H-dependent flavin-containing disulfide oxidoreductases, but involving sequential one-electron redox processes with the role of the [4Fe-4S]2+ cluster being to stabilize the thiyl radical formed by the initial one-electron reduction of the active-site disulfide. The results indicate a new biological role for Fe-S clusters involving both the stabilization of a thiyl radical intermediate and cluster site-specific chemistry involving a bridging sulfide.

An amino acid substitution in the pore region of a glutamate-gated chloride channel enables the coupling of ligand binding to channel gating.

Many of the subunits of ligand-gated ion channels respond poorly, if at all, when expressed as homomeric channels in Xenopus oocytes. This lack of a ligand response has been thought to result from poor surface expression, poor assembly, or lack of an agonist binding domain. The Caenorhabditis elegans glutamate-gated chloride channel subunit GluClbeta responds to glutamate as a homomeric channel while the GluClalpha subunit is insensitive. A chimera between GluClalpha and GluClbeta was used to suggest that major determinants for glutamate binding are present on the GluClalpha N terminus. Amino acid substitutions in the presumed pore of GluClalpha conferred direct glutamate gating indicating that GluClalpha is deficient in coupling of ligand binding to channel gating. Heteromeric channels of GluClalpha+beta may differ from the prototypic muscle nicotinic acetylcholine receptor in that they have the potential to bind ligand to all of the subunits forming the channel.

Molecular biology and electrophysiology of glutamate-gated chloride channels of invertebrates.

In this chapter we summarize the available data on a novel class of ligand-gated anion channels that are gated by the neurotransmitter glutamate. Glutamate is classically thought to be a stimulatory neurotransmitter, however, studies in invertebrates have proven that glutamate also functions as an inhibitory ligand. The bulk of studies conducted in vivo have been on insects and crustaceans, where glutamate was first postulated to act on H-receptors resulting in a hyperpolarizing response to glutamate. Recently, glutamate-gated chloride channels have been cloned from several nematodes and Drosophila. The pharmacology and electrophysiological properties of these channels have been studied by expression in Xenopus oocytes. Studies on the cloned channels demonstrate that the invertebrate glutamate-gated chloride channels are the H-receptors and represent important targets for the antiparasitic avermectins.

Amino acid sequence of spinach ferredoxin:thioredoxin reductase catalytic subunit and identification of thiol groups constituting a redox-active disulfide and a [4Fe-4S] cluster.

Ferredoxin:thioredoxin reductase is a [4Fe-4S] protein involved in the light regulation of carbon metabolism in oxygenic photosynthesis. This enzyme catalyses the reduction of thioredoxins with light-generated electrons. Ferredoxin:thioredoxin reductase is composed of two dissimilar subunits, a catalytic subunit, and a variable subunit. The catalytic subunit of spinach ferredoxin:thioredoxin reductase, which contains the redox-active disulfide bridge, was sequenced by conventional protein sequencing techniques and the functional roles of all eight cysteine residues were examined by chemical modifications. The polypeptide chain with a calculated molecular mass of 12,959 Da consists of 113 amino acids and has a calculated isoelectric point of 5.30. Six of the eight cysteine residues are clustered as Cys-Pro-Cys and Cys-His-Cys groups. Cys19 and Cys27 are free cysteines with no catalytic function, Cys54 and Cys84 constitute the redox-active disulfide bridge of the active site, and the remaining four, Cys52, Cys71, Cys73, and Cys82 bind the Fe-S cluster.

The oxidation-reduction properties of spinach thioredoxins f and m and of ferredoxin:thioredoxin reductase.

Oxidation-reduction midpoint potentials have been determined, using cyclic voltammetry, for the active-site disulfide/dithiol couples of spinach thioredoxins f and m and of spinach ferredoxin:thioredoxin reductase (FTR) and for a component likely to be the [4Fe-4S] cluster of FTR. Values for the midpoint potentials (n = 2) of -210 +/- 10 mV were determined for both thioredoxins f and m. Two redox centers were detected in FTR, with midpoint potential values of -230 +/- 10 mV (n = 2) and +340 +/- 30 mV, respectively. Alkylation of the active-site cysteines of FTR by treatment of the enzyme with N-ethylmaleimide (NEM) eliminates the component with the -230 mV midpoint potential, allowing one to assign this value to the active site disulfide/dithiol couple. Inasmuch as the only other electron-carrying center known to be present in FTR is the [4Fe-4S] cluster, it appears likely that the high-potential component can be attributed to this redox moiety. The midpoint potential value of the high-potential feature shifts slightly, to +380 +/- 20 mV, in the NEM-treated enzyme.

Reduction of ferredoxin:thioredoxin reductase by artificial electron donors.

The ferredoxin:thioredoxin reductase is an essential enzyme of the light dependent regulatory system in oxygenic photosynthesis. It is composed of two dissimilar subunits and contains a 4Fe-4S cluster and a redox-active disulfide bridge. Artificial electron donors of redox potentials below -300 mV are capable of reducing the disulfide bridge. Based on our results we speculate that a group of more negative potential than the disulfide bridge is the first acceptor of the electrons in FTR. The chemical reduction of FTR has been used successfully for the detection of the enzyme during its purification.

Ribosomal heterogeneity from chromatin diminution in Ascaris lumbricoides.

The genome of Ascaris lumbricoides encodes both germline- and soma-specific proteins homologous to the eukaryotic small ribosomal protein (Rp) S19. The two Ascaris homologs differ by 24 amino acid substitutions and are both components of the small ribosomal subunits. In oocytes, the germline RpS19 homolog (RpS19G) predominates. During chromatin diminution, however, the gene is eliminated from all presomatic cells, and RpS19G is replaced by the product of the somatic gene (RpS19S). Chromatin diminution in A. lumbricoides causes a change in the protein composition of ribosomes during development and represents an alternative means of gene regulation.

Extremely stable transcripts may compensate for the elimination of the gene fert-1 from all Ascaris lumbricoides somatic cells.

The single-copy gene fert-1 becomes eliminated from all somatic cells during the process of chromatin diminution in Ascaris lumbricoides var. suum. By using Northern blot and in situ hybridization techniques, we have analyzed its rather unusual expression pattern. Different splicing and 3' end formation events generate in a developmentally regulated manner various poly(A)+ and poly(A)- fert-1 RNA species. The lack of any significant open reading frame in most of its RNA products indicates that fert-1 may function as structural RNA rather than encoding a protein. Fert-1 transcripts are produced in the precursors of the gametes, but degraded at the time of meiosis and not passed on to the zygote. Embryonic transcription of fert-1 sets in as soon as the female nucleus has completed its meiosis. Our data thus demonstrate that the Ascaris transcription apparatus is active prior to the general onset of zygotic transcription, which we think takes place in the four- to six-cell-stage embryos. Upon elimination of fert-1 gene from the somatic cells, most of its transcripts disappear. Two short fert-1 RNA products, however, are stably maintained throughout development until the second larval stage, which is more than 1 month after the elimination of their coding sequences. Possible functions of fert-1 are discussed.

Chromatin diminution in nematode development.

Chromatin diminution in Parascaris and Ascaris represents the classical case of a developmentally programmed genome rearrangement. The process is very specific with respect to ontogenetic timing and chromosomal localization, and involves chromosomal breakage, new telomere formation and DNA degradation. Recent evidence from Ascaris lumbricoides var. suum suggests that chromatin diminution might have a function in gene regulation.

Eliminated chromatin of Ascaris contains a gene that encodes a putative ribosomal protein.

Chromatin diminution in the nematodes Parascaris equorum and Ascaris lumbricoides leads to the formation of somatic cells that contain less DNA than the germ-line cells. We present molecular evidence for the coding potential of germ-line-specific DNA. We report on a cDNA clone that codes for a putative ribosomal protein (ALEP-1, for A. lumbricoides eliminated protein 1). That the corresponding gene is located in the eliminated portion of the genome indicates a difference in germ-line and somatic ribosomes of A. lumbricoides and P. equorum. Elimination of the ALEP-1 gene from all somatic cells in its fully active state may represent an alternative way to gene regulation.

Type I-like intervening sequences are found in the rDNA of the nematode Ascaris lumbricoides.

The intervening sequences in the large ribosomal RNA gene of Ascaris lumbricoides var. suum show many similarities to the type I insertions, previously found only in some insect species. They include structural features, but also a presumed transcriptional inactivity in vivo: No transcript of the rDNA intervening sequence in A. lumbricoides could be detected in Northern and dot blot hybridizations. However, the primary structure of the Pol I promoter region is well conserved in interrupted and uninterrupted genes. Moreover, genes with an intervening sequence are correctly initiated in a whole-cell in vitro extract from Ascaris oogonia. Hence, the presence of the intervening sequence alone does not seem to account for a transcriptional inhibition in rRNA genes. As with the type I insertions of insect rDNA, some copies of the A. lumbricoides intervening sequence are also present in locations outside the rDNA cluster. About 50% of the extraribosomal copies are found in a repetitive sequence of the genome, and additional copies are inserted in unique sequences. These striking analogies to type I insertions are discussed, and lead to the conclusion that the two phenomena are undoubtedly related. This is the first report proving the presence of a type I-like insertion element outside of the class Insecta.