The use of alternative medicine by patients with amyotrophic lateral sclerosis.

The use of complementary and alternative medicine (CAM) is increasing in all industrialised countries, especially in patients with chronic and incurable diseases. However, no data are available on the use of CAM by patients with amyotrophic lateral sclerosis (ALS). The German Association for Neuromuscular Diseases (DGM) mailed out a questionnaire on CAM to 350 ALS patients, 171 of whom completed and returned the survey (response rate 49%). The use of CAM was reported by 92 patients (54%). There were no significant demographic differences between users and nonusers. The patients used 73 different methods or substances; some tried up to 11 different treatments. The most widely used methods were: acupuncture (47%), homeopathy (40%), naturopathy (24%) and esoteric treatments (20%). The lower the patients' expectations from CAM, the better was the subjectively perceived effect. In most cases (60%), alternative treatments were performed by a physician. Patients spent on average 4000 (approximately US$4500) on CAM, generally without reimbursement. CAM is most often used in addition to conventional treatments and may be part of the patients' coping strategy. Open communication between patients and physicians is essential to warn the patients of medically or financially hazardous treatments. Future research should look at the possible palliative effects of CAM on symptom control and quality of life of patients and families.

[Ventilation--and what then? Report of experiences from the viewpoint of the German Society of Muscular Diseases e.V. DGM]. / Beatmung--und was dann? Erfahrungbericht aus Sicht der Deutschen Gesellschaft für Muskelkranke e. V. DGM.

Without doubt respiratory improves life conditions and quality of life of people suffering from neuromuscular disease. Concerning medical-technical possibilities there are only few problems. The respiratory machines, developed during the last years, allow an outpatient treatment with special masks as well as with help of tracheotomy. Yet the medical concept has only small effects if there is something wrong with the psychosocial and psychological conditions. Considering the fact that respiratory prolongs patients life but does not cure, there is a serious problem for patients, their families, the nursing staff and-more and more doctors. For the patients and their families as well as the nursing staff it is necessary to get qualified at time for the outpatient situation already before dismission from hospital. Afterwards they need additional advice and support for solving the daily-life-problems and for saving care-quality in an extensive sense.

Cytoplasmic and mitochondrial forms of yeast adenylate kinase 2 are N-acetylated.

Yeast major adenylate kinase (Aky2p), encoded by a single gene, occurs in two subcellular compartments, mitochondria and cytoplasm. Only 6-8% of the protein which has no cleavable presequence is imported into the organelle (Bandlow et al. (1988) Eur. J. Biochem. 178, 451-457). In the wild type two AKY2-derived signals (a major and a minor one) were detected by a monospecific antibody after two-dimensional gel electrophoresis and Western blotting. The signals reflected identical electrophoretic mobilities and were absent from an AKY2-disrupted strain suggesting that they were due to differently modified forms of Aky2p. Two similar signals were found in a mutant defective in protein N-acetylation, however, the pI values of both spots were shifted towards alkaline pH by one charge. This indicated that both forms of Aky2p were N-acetylated in the wild type and that their charge difference was not caused by incomplete N-acetylation. This observation furthermore suggested that, in the wild type, two different modifications exist one of which is N-acetylation. The second modification remains unidentified. We analysed the influence of protein N-acetylation on mitochondrial import. Both versions of Aky2p occurred in the cytoplasm and in mitochondria. Their proportion was unchanged in the N-acetylation mutant showing that neither modification affected the efficiency of import of adenylate kinase into mitochondria. It is discussed that N-acetylation occurs during or immediately after translation in the cytoplasm so that import of adenylate kinase may ensue co-translationally.

Evaluation of the metal ion requirement of the human deoxyhypusine hydroxylase from HeLa cells using a novel enzyme assay.

Hypusine synthesis in the eukaryotic initiation factor 5A is a unique two-step posttranslational modification. After deoxyhypusine is generated by the deoxyhypusine synthase, the deoxyhypusine hydroxylase (EC 1.14.99.29) catalyzes the formation of mature hypusine. A rapid assay for monitoring the deoxyhypusine hydroxylase activity was established, employing the oxidative cleavage of the hypusyl residue and subsequent extraction of the generated aldehydes. As metal ion chelators have been reported to inhibit the deoxyhypusine hydroxylase, the mechanism of this inhibition and the effect of transition metal ions on enzyme activity were investigated. A ferric ion appears to be essential for enzymatic activity, the inhibition of which is entirely attributed to the metal ion binding capacity of the chelators.

Molecular characterization of a cDNA encoding functional human deoxyhypusine synthase and chromosomal mapping of the corresponding gene locus.

Deoxyhypusine synthase is essentially required for the post-translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation. From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase. DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa. This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli. Using a panel of somatic rodent-human cell hybrids we localized the deoxyhypusine synthase gene to human chromosome 19.

Isolation and structural characterization of different isoforms of the hypusine-containing protein eIF-5A from HeLa cells.

Posttranslational modification of a specific lysine residue in eukaryotic initiation factor 5A (eIF-5A) is essential for cell viability and proliferation. The product of this modification is hypusine, an amino acid unique to eIF-5A. We have purified and characterized one major and three minor isoforms of human eIF-5A from HeLa cells. The main form, which accounts for approximately 95% of the total eIF-5A, carries hypusine at position 50 and is amino-terminally acetylated as determined by amino acid composition analysis and electrospray ionization mass spectrometry. Analytical gel filtration indicates that this protein variant possesses a native apparent molecular weight that lies between that expected for a monomeric and dimeric form. Nevertheless, several experiments confirm this protein to be monomeric. It is further shown that eIF-5A have well-defined secondary structure. Both the far-UV circular dichroism spectrum as well as secondary structure predictions using different algorithms suggest this protein to have predominantly beta-sheet structure. Two plausible models for the packing of the secondary structure elements are presented. In contrast to the main form, all three minor isoforms of eIF-5A are characterized by acetylation of the epsilon-amino group of lysine at position 47. The minor isoforms are distinguishable by their state of modification of the lysine residue at position 50. Whereas the main form occurs in both the cytoplasmic and the nuclear fraction of HeLa cells, the minor isoforms were not detectable in the preparation of the nuclear fraction. Therefore, acetylation of lysine at position 47 might play a controlling role in the distribution of the minor isoforms to the nucleus.

The polypeptide chain of eukaryotic initiation factor 5A occurs in two distinct conformations in the absence of the hypusine modification.

Eukaryotic initiation factor 5A (eIF-5A) requires posttranslational modification of lysine at position 50 to hypusine for its biological activity. We have expressed an unmodified variant of eIF-5A in Escherichia coli and show that it has structural properties different from those of the native protein in terms of its near- and far-UV circular dichroism spectra and its equilibrium unfolding transition with guanidinium chloride. In contrast to the hypusine-modified protein, which unfolds in a two-state process, the complex unfolding transition of unmodified eIF-5A suggests that this variant occurs in two differently folded conformations, F1 and F2. Both conformations are populated under near-physiological conditions at a ration of 60 to 40, respectively. Equilibrium unfolding consists of parallel events: unfolding of F1 to one or several intermediate states (I), and unfolding of F2 to the unfolded state (U). Although the establishment of each of these individual equilibria is fast, the interconversion is slow at guanidinium chloride concentrations between 0 M and 3 M. Kinetic analysis reveals activation energies of 24.3 kcal mol-1 for the reaction of F1 and F2 and 24.1 kcal mol-1 for the reaction of F2 to F1. Both F1 and F2 possess well-defined secondary and tertiary structure. However, the tertiary structures of the two conformations differ as indicated by their distinct near-UV circular dichroism spectra. These differences may be restricted to the C-terminal part of the protein as 2-dimensional 1H-NMR spectra of unmodified eIF-5A reveal no doubled set of proton resonances for aromatic amino acid and histidine residues, of which almost all are located in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of human deoxyhypusine synthase from HeLa cells.

Post-translational modification of a specific lysine residue in eukaryotic initiation factor 5A is essential for cell viability. The amino acid hypusine, which is the product of this modification, is derived in two subsequent enzyme-catalyzed reactions. We have purified and characterized the enzyme responsible for the first step in hypusine modification, deoxyhypusine synthase, from HeLa cells. The human enzyme is multimeric with a native apparent molecular weight of 150,000 consisting of subunits of 41,000. The amino acid sequences of its peptide fragments share high sequence identity with a hypothetical protein (YHRO68w) on chromosome VIII of Saccharomyces cerevisiae. This protein appears to be the deoxyhypusine synthase of yeast.

Detection of nonfunctional overexpression gene products using two-dimensional gel electrophoresis with a narrowed pH range.

A commonly used technique in the analysis of yeast protein function is the overexpression of cloned genes. In the case that overexpression does not lead to an altered phenotype a stable synthesis of the protein has to be demonstrated. Here an example is shown where overexpression of the yeast HYP2 genes, coding for a hypusine-containing protein, was alternatively analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) alone or by two-dimensional (2-D) gel electrophoresis, using the narrowed pH range of 4.5-5.4. The results of the SDS-PAGE suggested a stable overproduction of HYP gene product, whereas 2-D analysis revealed an accumulation of nonfunctional protein isoforms.

Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.

The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.

The function of the hypusine-containing proteins of yeast and other eukaryotes is well conserved.

The hypusine-containing protein (Hypp) is highly conserved in evolution, from man to archaebacteria, but is not found in eubacteria. Hypp is essential for the viability for yeast cells, where two forms are encoded by the genes HYP1 and HYP2. The hypusine-containing protein Hyp2p, encoded by the HYP2 gene in yeast, is present under both aerobic and anaerobic conditions, whereas Hyp1p synthesis is restricted to anaerobiosis. hyp1 disruption mutants grown under anaerobic conditions reveal no detectable alteration in phenotype relative to wild-type strains. We demonstrate that either Hyp1p or Hyp2p alone is sufficient for normal growth under both metabolic conditions. Moreover, Hypp from various eukaryotic species (slime mold, alfalfa and man) carries the lysine to hypusine modification when expressed in yeast and can substitute functionally for Hyp2p in strains disrupted for HYP2, indicating a highly conserved function of this protein. In contrast, the archaebacterial Hypp expressed in yeast is neither modified by hypusine, nor does it allow growth of cells deficient for yeast Hypp.

Determination and mutational analysis of the phosphorylation site in the hypusine-containing protein Hyp2p.

Electrospray mass spectrometry of the purified isoforms of the hypusine-containing protein of Saccharomyces cerevisiae Hyp2p suggested a phosphorylation of the acidic isoform, which was confirmed by phosphatase treatment. The phosphorylation site was mapped to the N-acetylated serine residue in position no. 1 by mass spectrometric analysis of enzymatic fragments. Mutation of this serine residue gives rise to only the basic isoform, confirming our protein chemical data. As this mutation has no effect on cell viability or growth rate, the unphosphorylated isoform is sufficient to exert the essential in vivo function of Hyp2p.

The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants.

In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYP1 gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.

Detection of the hypusine-containing protein (HP = eIF-5A) in crude yeast extracts by two-dimensional western blots.

The hypusine-containing protein (HP) with its unique modification of a specific lysine residue resulting in the amino acid hypusine is highly conserved among all eukaryotes and is also found in Archaebacteria. Studies of the protein function in translational processes showed a stimulatory effect in the methionyl puromycin assay, but not in in vitro translation of native mRNA. It was therefore also designated as eIF-5A. To further investigate the role of HP in cellular metabolism, we purified the protein from Saccharomyces cerevisiae and raised polyclonal antibodies in chicken. Immunoglobulin preparations from the eggs of the immunized hens were used for Western blot analysis of HP in crude yeast extracts. For those studies, the soluble protein fraction of the yeast was resolved on two-dimensional gels (first dimension: isoelectric focusing using an immobilized pH gradient (IPG), pH 4-7; second dimension: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 12% T) and subsequently blotted onto Fluorotrans membrane. Anodic versus cathodic application of the extracts of the IPG strips was compared.

Isolation and analysis of the human 46-kDa mannose 6-phosphate receptor gene.

From a genomic library in EMBL 3, two overlapping clones for the human 46-kDa mannose 6-phosphate receptor (MPR46) were isolated, which span the entire coding sequence. The human MPR46 gene is distributed over 12 kb and is divided into seven exons (110-1573 bp). All the intron/exon borders agree with the consensus sequences of splice junctions. Exon 1 codes for a 5' untranslated sequence. The ATG initiation codon begins with the second nucleotide in exon 2. A signal sequence of 26 amino acid residues is followed by the extracytoplasmic (luminal) domain, which extends to exon 5. The transmembrane domain of the receptor spans exons 5 and 6 and the cytoplasmic domain is encoded by exons 6 and 7. The latter domain also codes for an extended 3' untranslated sequence. The transcription-initiation site was defined by primer extension. The sequence upstream of the cap site has strong promoter activity and contains structural elements characteristic of promoters found in housekeeping genes. No correlation between the genomic organization and known protein domains of the MPR46 was apparent. Moreover, the sequence of about 150 amino acids within the luminal domain of MPR46, which is homologous to the 15 repeats that constitute the luminal domain of the 300-kDa mannose 6-phosphate receptor (MPR300), does not correlate with intron/exon borders. MPR46 and MPR300 have therefore diverged from a common ancestral gene before introduction of the present intron sequences.