[Head impulse test and vibratory test in the diagnosis of vertigo]. / Kopfimpulstest und Vibrationstest in der Schwindeldiagnostik.

One of the most important questions within the field of vertigo-diagnosis is the proof or the exclusion of a vestibular impairment, the spectrum of appropriate diagnostic tools has been expanded by the head impulse test and the vibratory test in the last years. The head impulse test is a method to examine the functionality of single semicircular canals. As clinical "bedside test" it is an already established part of the diagnostic procedures, as an apparative method with registration and quantitative analysis, however, it is available for general in-office use only recently. The vibratory test is a method for provoking non-spontaneous nystagmus. As a basis of a sophisticated vestibular diagnosis the test is less suitable, however, it is an absolutely valuable method to detect peripheral or central vestibular imbalances. In this regard the vibratory test is superior to other methods as for example the head shaking test. In the following article an overview concerning the physiological, methodical, and clinical aspects of the head impulse test and the vibratory test will be given.

Arenaviruses and hantaviruses: from epidemiology and genomics to antivirals.

The arenaviruses and hantaviruses are segmented genome RNA viruses that are hosted by rodents. Due to their association with rodents, they are globally widespread and can infect humans via direct or indirect routes of transmission, causing considerable human morbidity and mortality. Nevertheless, despite their obvious and emerging importance as pathogens, there are currently no effective antiviral drugs (except ribavirin which proved effective against Lassa virus) with which to treat humans infected by any of these viruses. The EU-funded VIZIER project (Comparative Structural Genomics of Viral Enzymes Involved in Replication) was instigated with an ultimate view of contributing to the development of antiviral therapies for RNA viruses, including the arenaviruses and bunyaviruses. This review highlights some of the major features of the arenaviruses and hantaviruses that have been investigated during recent years. After describing their classification and epidemiology, we review progress in understanding the genomics as well as the structure and function of replicative enzymes achieved under the VIZIER program and the development of new disease control strategies.

Rieske iron-sulfur proteins from extremophilic organisms.

Proteins located on the outside of the membranes of organisms thriving under extreme conditions like high or low pH, or high salinity face special challenges maintaining their structural integrity. This review is focused on the Rieske iron-sulfur proteins from these organisms. Rieske proteins are essential subunits of the cytochrome bc-complexes, which are often of crucial importance for the energy metabolism of the cells. On the basis of the available data we propose strategies by which these proteins are able to stabilize their noncovalent bound cofactor and adapt to the function under extreme conditions.

New genes encoding subunits of a cytochrome bc1-analogous complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius.

The soxL gene from Sulfolobus acidocaldarius (DSM 639) encodes a Rieske iron-sulfur protein. In this study we report the identification of two open reading frames in its downstream region. The first one, named soxN, codes for a membrane protein bearing a resemblance to the b-type cytochromes of the cytochrome bc1 and b6f complexes. The protein is predicted to contain at least 10 transmembrane helices and features the two conserved histidine pairs coordinating the heme groups of these cytochromes. The second open reading frame, named odsN, encodes a soluble protein of unknown function. The genomic region displays a complex transcription pattern. Northern blot and RT-PCR analyses revealed the presence of mono- and bi-cistronic transcripts as well as a tri-cistronic transcript of soxL and cbsAB, encoding the mono-heme cytochrome b558/566. Phylogenetic analyses of the genes of the soxLN pair and of other archaeal gene pairs encoding Rieske iron-sulfur proteins and b-type cytochromes revealed an identical branching patterns for both protein families, suggesting an evolutionary link of these genes provided by the functional interaction of the proteins. On the basis of the findings of this study and the previously studied properties of the soxL and cbsA proteins, we propose the occurrence of a novel cytochrome bc1-analogous complex in the membranes of Sulfolobus, consisting of the cytochrome b homolog soxN, the Rieske protein soxL, the high potential cytochrome cbsA, as well as the non-redox-active subunits cbsB and odsN.

Characterization of a non-haem ferritin of the Archaeon Halobacterium salinarum, homologous to Dps (starvation-induced DNA-binding protein).

An iron-rich protein was isolated from the Archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC 7942. It consists of 20 kDa subunits, forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 103 Fe ions/mol of holoprotein. CD spectra are consistent with an alpha-helical contribution of 58%. The UV/visible spectrum provides no evidence for the presence of haem groups. This protein exhibits features of a non-haem-type bacterial ferritin although it shares only little sequence homology with non-haem bacterial ferritin.

Mutant pso8-1 of Saccharomyces cerevisiae, sensitive to photoactivated psoralens, UV radiation, and chemical mutagens, contains a rad6 missense mutant allele.

A novel mutant isolate of Saccharomyces cerevisiae, sensitive to photoactivated mono- and bi-functional psoralens, to UV at 254 nm (UVC), and to nitrosoguanidine, was found to complement the photoactivated psoralen-sensitivity phenotype conferred by the pso1- pso7 mutations and was therefore named pso8-1. A constructed pso8-1 rad4-4 double mutant was super-sensitive to UVC, thus indicating a synergistic interaction of the two mutant alleles. Molecular cloning via complementation of the pso8 mutant's sensitivity phenotype and genetic studies revealed that pso8 is allelic to RAD6. While a pso8-1 mutant had low mutagen-induced mutability, homoallelic diploids showed nearly wild-type sporulation. Sequence analysis of the mutant allele showed pso8-1 to contain a novel, hitherto undescribed T-->C transition in nucleotide position 191, leading to a substitution by leucine of a highly conserved proline at position 64, Rad6-[P64L], which may have severe consequences for the tertiary structure (and hence binding to Rad18p) of the mutant protein.

A comprehensive phylogenetic analysis of Rieske and Rieske-type iron-sulfur proteins.

The Rieske iron-sulfur center consists of a [2Fe-2S] cluster liganded to a protein via two histidine and two cysteine residues present in conserved sequences called Rieske motifs. Two protein families possessing Rieske centers have been defined. The Rieske proteins occur as subunits in the cytochrome bc1 and cytochrome b6f complexes of prokaryotes and eukaryotes or form components of archaeal electron transport systems. The Rieske-type proteins encompass a group of bacterial oxygenases and ferredoxins. Recent studies have uncovered several new proteins containing Rieske centers, including archaeal Rieske proteins, bacterial oxygenases, bacterial ferredoxins, and, intriguingly, eukaryotic Rieske oxygenases. Since all these proteins contain a Rieske motif, they probably form a superfamily with one common ancestor. Phylogenetic analyses have, however, been generally limited to similar sequences, providing little information about relationships within the whole group of these proteins. The aim of this work is, therefore, to construct a dendrogram including representatives from all Rieske and Rieske-type protein classes in order to gain insight into their evolutionary relationships and to further define the phylogenetic niches occupied by the recently discovered proteins mentioned above.

Crystallization and preliminary crystallographic analysis of Rieske iron-sulfur protein II (soxF) from sulfolobus acidocaldarius.

An archaeal Rieske iron-sulfur protein has been crystallized for the first time. The genetically constructed soluble form of the soxF protein was expressed in E. coli. It contains a correctly inserted [2Fe--2S] cluster. The authentic soxF protein is part of a terminal oxidase complex in the respiratory chain of the hyperthermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639). The enzyme crystallizes in the space group P6(1) or P6(5), with unit-cell parameters a = b = 80.19, c = 75.69 A. A complete data set has been collected to 1.64 A resolution at 100 K.

[Vascular origin of cerebellopontine angle syndrome]. / Vaskuläre Ursachen des rising dbl quote, left (low)Kleinhirnbrückenwinkel-Syndroms"?

INTRODUCTION: The seventh and eighth cranial nerves course toward the internal auditory canal within the cerebellopontine angle. Lesions in this region are usually related to malfunctions of these cranial nerves. Although an acoustic schwannoma is one of the main etiologies of cerebellopontine angle pathology, various inflammatory processes and vascular anomalies even though rare must be considered. PATIENTS/METHODS: We describe 5 cases with vascular loops of the basilar or vertebral arteries as a possible cause for hearing loss, vertigo and pulsatile tinnitus. In two cases the vascular lesion was confirmed at surgery, in which a decompression procedure was performed. The work-up for each patient included an auditory test battery and electronystagmography. Imaging studies included MRI and angiography in two cases. RESULTS/CONCLUSIONS: Our experiences show that while the cerebellopontine angle syndrome is mostly caused by benign tumors an abnormal vascular loop has to be considered in any differential diagnosis.

Allelism of Saccharomyces cerevisiae genes PSO6, involved in survival after 3-CPs+UVA induced damage, and ERG3, encoding the enzyme sterol C-5 desaturase.

Sequencing of the yeast gene that complemented the sensitivity to the photoactivated monofunctional 3-carbethoxypsoralen of the pso6-1 mutant strain revealed that the ERG3 locus, encoding sterol C-5 desaturase involved in biosynthesis of ergosterol, is allelic to PSO6. Disruption of the ERG3 gene yielded an erg3Delta mutant viable in ergosterol-containing YEPD media with the same pleiotropic mutant phenotype known for pso6-1 and erg3 mutants, including sensitivity to hydrogen peroxide and paraquat. Thus, the erg3/pso6 yeast mutant seems to be more sensitive than the WT to 3-CPs+UVA because of the oxidative damage contributed by this treatment and not because of an impaired repair of the furocoumarin-thymine monoadducts formed in the DNA. We found a significant increase of petites amongst erg3Delta and pso6-1 yeast mutant strains grown in conditions where respiration was mandatory. Mutant pso6-1, with its lowered content of ergosterol, exhibited enhanced synthesis of chitin that was maldistributed and not confined to the bud scars. Chitin overproduction in pso6/erg3 mutants resulted in hypersensitivity to Calcofluor White.

[Bechterew phenomenon in the human]. / Bechterew-Phänomen beim Menschen.

BACKGROUND: Vestibuloocular and vestibulospinal reactions following the bilateral loss of vestibular function are designated as Bechterew's phenomenon. In vestibular physiology, under experimental conditions, this phenomenon has played an important role for understanding the mechanisms underlying vestibular compensation. However, clinical reports about this phenomenon in humans are extremely rare. PATIENTS AND METHODS: Bechterew's phenomen was observed throughout every stage of its development in a patient with consecutive bilateral loss of vestibular function. The disorder was documented by electronystagmography and posturography. DISCUSSION AND CONCLUSIONS: Bechterew's phenomenon depends on different states of vestibular function developing in a certain succession, whereby the varying relations between failure and compensation are decisive. The phenomen very impressively demonstrates the resilience of the central nervous system in adapting to varying peripheral functional states. From a clinical standpoint, knowledge of these relationships is important for understanding vestibular compensation.

A novel Rieske iron-sulfur protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum: sequencing of the gene, expression in E. coli and characterization of the protein.

The crenarchaeon Pyrobaculum aerophilum is with an optimal growth temperature of 100 degrees C one of the most thermophilic organisms known to possess an aerobic respiratory chain. The analysis of DNA sequences from the Pyrobaculum genome project lead to the identification of an open reading frame potentially coding for a Rieske iron-sulfur protein. The complete gene (named parR) was cloned and sequenced. The deduced amino acid sequence displays unusual amino acid exchanges and a so far unknown sequence insertion. The N-terminus shows similarities to bacterial signal sequences. Several forms of the gene were expressed in E. coli in order to verify the classification as a Rieske protein and to facilitate biophysical studies. Soluble, thermo-stable proteins with correctly inserted iron-sulfur clusters were expressed from two versions of the gene. The delta1-23 truncated holo-protein is redox active. It displays the typical spectroscopic properties of a Rieske protein. The redox potential was determined to be +215 mV at pH 6.5 and is pH dependent above pH 7.5 revealing the influence of two protonation equilibria with pKa values of 8.1 and 9.8. Phylogenetic analysis demonstrates that the parR protein clusters together with the two other available archaeal Rieske sequences from Sulfolobus on a separate branch of the phylogenetic tree apart from the proteins from thermophilic bacteria like Aquifex and Thermus.

The strict molybdate-dependence of glucose-degradation by the thermoacidophile Sulfolobus acidocaldarius reveals the first crenarchaeotic molybdenum containing enzyme--an aldehyde oxidoreductase.

In order to investigate the effects of trace elements on different metabolic pathways, the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius (DSM 639) has been cultivated on various carbon substrates in the presence and absence of molybdate. When grown on glucose (but neither on glutamate nor casein hydrolysate) as sole carbon source, the lack of molybdate results in serious growth inhibition. By analysing cytosolic fractions of glucose adapted cells for molybdenum containing compounds, an aldehyde oxidoreductase was detected that is present in the cytosol to at least 0.4% of the soluble protein. With Cl2Ind (2,6-dichlorophenolindophenol) as artificial electron acceptor, the enzyme exhibits oxidizing activity towards glyceraldehyde, glyceraldehyde-3-phosphate, isobutyraldehyde, formaldehyde, acetaldehyde and propionaldehyde. At its pH-optimum (6.7), close to the intracellular pH of Sulfolobus, the glyceraldehyde-oxidizing activity is predominant. The protein has an apparent molecular mass of 177 kDa and consists of three subunits of 80.5 kDa (alpha), 32 kDa (beta) and 19.5 kDa (gamma). It contains close to one Mo, four Fe, four acid-labile sulphides and four phosphates per protein molecule. Methanol extraction revealed the existence of 1 FAD per molecule and 1 molybdopterin per molecule, which was identified as molybdopterin guanine dinucleotide on the basis of perchloric acid cleavage and thin layer chromatography. EPR-spectra of the aerobically prepared enzyme exhibit the so-called 'desulpho-inhibited'-signal, known from chemically modified forms of molybdenum containing proteins. Anaerobically prepared samples show both, the signals arising from the active molybdenum-cofactor as well as from the two [2Fe-2S]-clusters. According to metal-, cofactor-, and subunit-composition, the enzyme resembles the members of the xanthine oxidase family. Nevertheless, the melting point and long-term thermostability of the protein are outstanding and perfectly in tune with the growth temperature of S. acidocaldarius (80 degrees C). The findings suggest the enzyme to function as a glyceraldehyde oxidoreductase in the course of the nonphosphorylated Entner-Doudoroff pathway and thereby may attribute a new physiological role to this class of enzyme.

The glutamine synthetase from the hyperthermoacidophilic crenarcheon Sulfolobus acidocaldarius: isolation, characterization and sequencing of the gene.

The glutamine synthetase (EC 6.3.1.2) from the hyperthermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was purified to homogeneity, characterized and the glnA gene isolated and sequenced. The amount of enzyme present in the cytosolic fraction from Sulfolobus cells showed a strong variation depending on the carbon and nitrogen sources in the growth medium. The enzyme was found to be a dodecameric protein composed of identical subunits of 52 kDa. It was stable at 78 degrees C in the presence of Mn2+ ions. The catalytic activity was regulated solely by feed-back inhibition through L-alanine and glycine and not by adenylylation. No evidence for the presence of isoenzymes was found. Sequence comparison showed that the Sulfolobus protein is most closely related to the glutamine synthetases of the I-beta type despite its regulatory properties and the finding that the known euryarcheal glutamine synthetase sequences belong to the I-alpha subgroup of these enzymes. Our phylogenetic analysis suggests that the gene duplication leading to the development of the I-alpha and I-beta enzymes preceded the separation of the archea and the bacteria.

[Mechanism of benign, peripheral, paroxysmal positional vertigo (BPPV)]. / Der Mechanismus des benignen, peripheren, paroxysmalen Lagerungsschwindels (BPPV).

BACKGROUND: Positioning nystagmus of the peripheral benign type (BPPV) has long been considered to be due to cupulolithiasis-i.e. attachment of inorganic material to the cupula-of the posterior vertical semicircular canal. Meanwhile it has generally been recognized that not all characteristics of this type of nystagmus/vertigo can be explained by assuming a gravity-dependent reaction of the posterior canal. Canalolithiasis-i.e. floating material within the canal, heavier than endolymphe-of the posterior semicircular canal is now widely regarded as the cause of BPPV. Again, however, this is a concept far too simple to explain most of the properties of BPPV. METHODS: Clearly defined, carefully performed positioning and positional maneuvers were carried out on 79 patients suffering from typical BPPV in order to reveal those positioning movements and positions necessary to elicit BPPV. RESULTS: Detailed analysis of those positioning maneuvers necessary to elicit BPPV clearly reveals that canalolithiasis cannot be the cause of BBPV. CONCLUSIONS: BPPV is a combination of positioning, and positional-dependent reactions in which the otolithic organs, and particularly the sacculus, seem to be involved.

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius. A novel highly glycosylated, membrane-bound b-type hemoprotein.

In this study we re-examined the inducible cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schäfer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only detectable in cells grown at low oxygen tensions. The composition of the growth medium also exerts significant influence on the cytochrome b558/566 content of S. acidocaldarius membranes. The cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely. It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane. The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine. A second gene (cbsB) was found to be cotranscribed. The latter displays extreme hydrophobicity and is thought to form a functional unit with cytochrome b558/566 in vivo, although it did not copurify with the latter. Sequence comparisons show no similarity to any entry in data banks indicating that this cytochrome is indeed a novel kind of b-type hemoprotein. A cytochrome c analogous function in the pseudoperiplasmic space of S. acidocaldarius is discussed.

Studies of the electron transport chain of the euryarcheon Halobacterium salinarum: indications for a type II NADH dehydrogenase and a complex III analog.

The components involved in the respiratory system of the euryarcheon Halobacterium salinarum were investigated by spectroscopic and polarographic techniques. Previous results about the cytochrome composition could be verified. However, under low oxygen tension, the expression of a d-type cytochrome was detected. Membranes exerted an NADH- and succinatecytochrome-c oxidoreductase as well as an NADH and succinate oxidase activity. These activities could be blocked by the following inhibitors: 7-jodocarboxylic acid, giving evidence for the presence of a type II NADH dehydrogenase, antimycin A, and myxothiazol, indicating the presence of a complex III analog, and the typical succinate dehydrogenase (SDH) and terminal oxidase inhibitors. Complex I inhibitors like rotenone and annonine were inactive, clearly excluding the presence of a coupled NADH dehydrogenase. In addition, no [Fe-S] resonances in the region of the NADH dehydrogenase (NDH) clusters could be observed after NADH addition. One of the terminal oxidases could be shown to act as a cytochrome-c oxidase with a Km value of 37 microM and an activation energy of 23.7 kJ/mol. The relative molecular mass of the endogenous c-type cytochrome could be determined as 14.1 kD. The complex III analog could be enriched after detergent extraction with Triton X-100 and hydroxylapatite (HTP) chromatography. The partially purified complex contained a Rieske iron-sulfur cluster, b- and c-type cytochromes, and was catalytically active in the decylubiquinone-cytochrome-c oxidoreductase assay.

Expression of the Solfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted [2FE-2S] cluster in Escherichia coli.

The Rieske protein II (Schmidt et al., 1996, FEBS Lett. 388, 43-46) from the thermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was expressed in E. coli cells. The full length protein was strictly bound to the E. coli membranes and could only be removed by detergent treatment indicating the presence of a membrane anchor. The iron sulfur cluster was correctly inserted into a fraction of the full length protein and much more effectively into a soluble form created by the deletion of the 45 N-terminal amino acids. The soluble form of the protein displayed the typical spectroscopic properties of a respiratory Rieske protein. The midpoint potential was +375 mV determined by CD redox potentiometry. The presented data demonstrate that the structure of the recombinant protein is very similar or identical to the authentic protein making this a powerful model system for the studies of Rieske proteins by site directed mutagenesis.