The functions of Sco proteins from genome-based analysis

Sco proteins are widespread proteins found in eukaryotic as well as in many prokaryotic organisms. The 3D structure of representatives from human, yeast, and Bacillus subtilis has been determined, showing a thioredoxin-like fold. Sco proteins have been implicated mainly as copper transporters involved in the assembly of the CuA cofactor in cytochrome c oxidase. Some mutations have been identified in humans that lead to defective cytochrome c oxidase formation and thus to fatal illnesses. However, it appears that the physiological function of Sco proteins goes beyond assembly of the CuA cofactor. Extensive analysis of completely sequenced prokaryotic genomes reveals that 18% of them contain either Sco proteins but not CuA-containing proteins or vice versa. In addition, in several cases, multiple Sco-encoding genes occur even if only a single potential Sco target is encoded in the genome. Genomic context analysis indeed points to a more general role for Sco proteins in copper transport, also to copper enzymes lacking a CuA cofactor. To obtain further insight into the possible role of Sco in the assembly of other cofactors, a search for Cox11 proteins, which are important for CuB biosynthesis, was also performed. A general framework for the action of Sco proteins is proposed, based on the hypothesis that they can couple metal transport and thiol/disulfide-based oxidoreductase activity, as well as select between either of these two cellular functions. This model reconciles the variety of experimental observations made on these proteins over the years, and can constitute a basis for further studies.     

Inhibition of TGF-beta2 with AP 12009 in recurrent malignant gliomas: from preclinical to phase I/II studies

Transforming growth factor-beta2 (TGF-beta2) is known to suppress the immune response to cancer cells and plays a pivotal role in tumor progression by regulating key mechanisms including proliferation, metastasis, and angiogenesis. For targeted protein suppression the TGF-beta2-specific antisense oligodeoxynucleotide AP 12009 was developed. In vitro experiments have been performed to prove specificity and efficacy of the TGF-beta2 inhibitor AP 12009 employing patient-derived malignant glioma cells as well as peripheral blood mononuclear cells (PBMCs) from patients. Clinically, the antisense compound AP 12009 was assessed in three Phase I/II-studies for the treatment of patients with recurrent or refractory malignant (high-grade) glioma WHO grade III or IV. Although the study was not primarily designed as an efficacy evaluation, prolonged survival compared to literature data and response data were observed, which are very rarely seen in this tumor indication. Two patients experienced long-lasting complete tumor remissions. These results implicate targeted TGF-beta2-suppression using AP 12009 as a promising novel approach for malignant gliomas and other highly aggressive, TGF-beta-2-overexpressing tumors.     

Effector and target cells in the assessment of natural cytotoxic activity of rhesus monkeys

In humans, decreased natural killer cell (NK) activity has been associated with stressful life events, whereas acute arousal and disturbance frequently has been reported to result in increased NK activity. This bidirectional immune modulation prompted us to investigate the effects of a social stressor on the lymphocyte cytolytic activity of 31 infant rhesus monkeys. The first of three studies evaluated the effects of an 8 hr maternal separation on the infants' cytolytic response against the K562 target-cell line. A finding of increased lytic activity indicated a need for a longer evaluation—after a 24 hr separation—and an additional assessment of two other target-cell lines, Raji and Daudi. The observation of decreased lytic responses to Raji and Daudi, in association with increased lysis of K562, warranted a third study to delineate which rhesus effector cells were responsible for lysis of the K562 and Raji target cells. By isolating cell subsets, it was possible to observe that the majority of unprimed cytotoxic activity resided in the CD3- population of cells, but that the CD3 + CD8 + population also mediated a significant amount of cytotoxicity against both targets. In conclusion, these findings support earlier studies indicating that maternal separation results in significant immune alterations in infant monkeys. However, the complex nature of changes in cytotoxic responses during prolonged stress revealed that different lymphocyte populations engage in parallel and compensatory alterations. © 1996 Wiley-Liss, Inc.     

New method for the resolution of the enantiomers of 5,6-Dihydroxy-2-Methyl-Aminotetralin by selective derivatization and HPLC analysis: Application to biological fluids

A new chiral derivatization procedure for the HPLC resolution of chiral catecholamines and structurally related compounds is described. The homochiral reagent, (+)-(R)-1-phenylethyl isocyanate (RPEIC), was added to separate and quantitate the enantiomers of rac-5,6-dihydroxy-2-methyl-aminotetralin, the main metabolite of rac-5,6-diisobutyryl-2-methyl-aminotetralin, a potent dopamine agonist, by reversed-phase HLPC analysis. To avoid catecholamine degradation in the basic reaction medium and to obtain the selective and quantitative derivatization of the amino group of the compound, the reversible complex formation between diphenylborinic acid (DPBA) and the catechol group, in alkaline medium, was performed before homochiral isocyanate addition. The RPEIC derivatization was completed in 30 min and then the DPBA complex was dissociated by adding dilute acid. The structure of intermediates and urea derivatives was confirmed by mass spectrometry. The use of an electrochemical detector, operating in redox mode, allowed HPLC quantitation of enantiomers at the nanogram level in plasma and urine. The derivatization procedure is also suitable for other catecholamine-related compounds. © 1996 Wiley-Liss, Inc.     

Purification and properties of the formate dehydrogenase and characterization of the<Emphasis Type="Italic"> fdhA</Emphasis> gene of<Emphasis Type="Italic"> Sulfurospirillum multivorans</Emphasis>

The soluble periplasmic subunit of the formate dehydrogenase FdhA of the tetrachloroethene-reducing anaerobe Sulfurospirillum multivorans was purified to apparent homogeneity and the gene (fdhA) was identified and sequenced. The purified enzyme catalyzed the oxidation of formate with oxidized methyl viologen as electron acceptor at a specific activity of 1683 nkat/mg protein. The apparent molecular mass of the native enzyme was determined by gel filtration to be about 100 kDa, which was confirmed by the fdhA nucleotide sequence. fdhA encodes for a pre-protein that differs from the truncated mature protein by an N-terminal 35-amino-acid signal peptide containing a twin arginine motif. The amino acid sequence of FdhA revealed high sequence similarities to the larger subunits of the formate dehydrogenases of Campylobacter jejuni, Wolinella succinogenes, Escherichia coli (FdhN, FdhH, FdhO), and Methanobacterium formicicum. According to the nucleotide sequence, FdhA harbors one Fe₄/S₄ cluster and a selenocysteine residue as well as conserved amino acids thought to be involved in the binding of a molybdopterin guanidine dinucleotide cofactor.Abbreviations Fdh Formate dehydrogenase - PCE Tetrachloroethene     

Bioavailability of anthocyanidin-3-glucosides following consumption of red wine and red grape juice

Pharmacokinetic parameters and the bioavailability of several dietary anthocyanins following consumption of red wine and red grape juice were compared in nine healthy volunteers. They were given a single oral dose of either 400 mL of red wine (279.6 mg total anthocyanins) or 400 mL of red grape juice (283.5 mg total anthocyanins). Within 7 h, the urinary excretion of total anthocyanins was 0.23 and 0.18% of the administered dose following red grape juice and red wine ingestion, respectively. Pharmacokinetic parameters derived from plasma and urine concentrations exhibited higher variability after ingestion of red grape juice. Compared to red grape juice anthocyanins, the relative bioavailability of red wine anthocyanins was calculated to be 65.7, 61.3, 61.9, 291.5, 57.1, and 76.3% for the glucosides of cyanidin, delphinidin, malvidin, peonidin, petunidin, and its sum (referred to as total anthocyanins), respectively. Bioequivalence was established for none of the anthocyanins. On a low level, urinary excretion of anthocyanins was fast, and the excretion rates seem to exhibit monoexponential characteristics over time after ingestion of both red grape juice and red wine. Due to low bioavailability, any significant contribution of anthocyanins to health protecting properties of red wine or red grape juice seems questionable, but the clinical relevance of these findings awaits further investigation.     

Y14 and hUpf3b form an NMD-activating complex

Messenger RNAs with premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). In mammals, PTCs are discriminated from physiological stop codons by a process thought to involve the splicing-dependent deposition of an exon junction complex (EJC), EJC-mediated recruitment of Upf3, and Upf2 binding to the N terminus of Upf3. Here, we identify a conserved domain of hUpf3b that mediates an interaction with the EJC protein Y14. Tethered function analysis shows that the Y14/hUpf3b interaction is essential for NMD, while surprisingly the interaction between hUpf3b and hUpf2 is not. Nonetheless, hUpf2 is necessary for NMD mediated by tethered Y14. RNAi-induced knockdown and Y14 repletion of siRNA-treated cells implicates Y14 in the degradation of beta-globin NS39 mRNA and demonstrates that Y14 is required for NMD induced by tethered hUpf3b. These results uncover a direct role of Y14 in NMD and suggest an unexpected hierarchy in the assembly of NMD complexes.     

The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells,as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics

An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RV Delta UL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.     

Biodegradation of phenols by microalgae

Two green microalgae, Ankistrodesmus braunii and Scenedesmus quadricauda, degraded phenols (each tested at 400 mg ml^–1) selected from olive-oil mill wastewaters, within 5 days, with a removal greater than 70%. Green algae may, therefore, represent an alternative to other biological treatment used for the biodegradation of phenol-containing wastewaters.     

The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: A comparison with radioactivity

A nonradioactive labelling and detection method for plant genomic DNA analysis is compared to the radioactive method. The radioisotopes are replaced by a nucleotide, digoxigenin-11-dUTP, and the signal detection is accomplished by the enzymatic reaction of alkaline phosphatase, conjugated to anti-digoxigenin antibodies, with the chemiluminescent substrate AMPPD (3-(2-spiroadamantane)-4-methoxy-4(3 phosphorytoxy) phenyl-1, 2-dioxetane). The sensitivity of the radioactive and nonradioactive methods are directly compared using identical Southern blots subjected to the radioactive and nonradioactive detection. The advantages of this nonradioactive method are discussed.