Search PubMed⌕ Search

Biomedical subjects

G Schäfer

Publications and source records attributed to G Schäfer.

At least 19 recordsLinked to original sources
PubMed2002

Impact of fish oil enriched total parenteral nutrition on DNA synthesis, cytokine release and receptor expression by lymphocytes in the postoperative period.

A prospective randomized study on sixty patients was conducted to investigate the effects of a fish oil containing total parenteral nutrition (TPN) regimen in the postoperative period on lymphocyte subset distribution, proliferation, cytokine production and interleukin-2 receptor (IL-2R) expression. Patients who underwent large bowel surgery were divided into three groups. Nineteen patients received TPN with fish oil (0.2 g/kg body weight per day) plus soybean oil (1.0 g/kg per day), twenty patients received soybean oil (1.2 g/kg per day), and twenty-one patients who were on a fat-free regimen served as the control group. Natural killer (NK) cells, total, B-, T-, T4-, T8-lymphocytes, proliferation of lymphocytes, in vitro production of IL-2, IFN-gamma, TNF-alpha, and IL-2R expression were measured. Fish oil administration did not affect subset distribution and proliferation of lymphocytes. Production of interleukin-2 (IL-2), interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) was augmented, and IL-2R expression less enhanced compared with the controls. It is concluded that administration of 0.2 g/kg per day fish oil after a moderate surgical stress is not immunosuppressive, but enhances the production of IFN-gamma, TNF-alpha and possibly IL-2.

Aged↗
PubMed2001

Independent replication of the plasmids pRN1 and pRN2 in the archaeon Sulfolobus islandicus.

The 5.4-kb and 6.9-kb plasmids pRN1 and pRN2 from the crenarchaeon Sulfolobus islandicus are name-giving for a small family of archaeal plasmids. Both plasmids have hitherto been supposed to be dependent on each other because they are always found together in their natural host. Here we demonstrate that each of the plasmids can stably propagate and replicate on its own independent of the other plasmid. Moreover, we could show that in vivo the plasmids bear tightly bound proteins.

DNA Replication↗
PubMed2001

Sulfocyanin and subunit II, two copper proteins with novel features, provide new insight into the archaeal SoxM oxidase supercomplex.

The isolation of a fully functional SoxM terminal oxidase supercomplex from the archaeon Sulfolobus acidocaldarius has failed thus far and several of its constituents have only been predicted genetically, such as the small Cu protein sulfocyanin and the subunit II bearing a Cu(A) center. Here we report the recombinant expression of sulfocyanin and prove its transcription in Sulfolobus as well as its presence in the enriched complex. It reveals a redox potential of +300 mV and spectroscopic features that are characteristic of type I copper centers. It is highly thermostable and firmly attached to the complex by one putative transmembrane anchor. Surprisingly, subunit II is completely missing from the isolated complex and behaves as an easily dissociable constituent which is a unique case within the terminal oxidase family. Its loss into the soluble phase upon cell disruption can be considered the reason for the inactivity of the isolated membrane complex.

Archaeal Proteins↗
PubMed2001

NaZn(H2O)2[BP2O8].H2O: a novel open-framework borophosphate and its reversible dehydration to microporous sodium zincoborophosphate N.

Crystals of NaZn(H2O)2[BP2O8].H2O were grown under mild hydrothermal conditions at 170 degrees C. The crystal structure (solved by X-ray single-crystal methods: hexagonal, P6(1)22 (no. 178), a = 946.2(2), c= 1583.5(1) pm, V= 1227.8(4).10(6) pm3, Z = 6) exhibits a chiral octahedral-tetrahedral framework related to the CZP topology and contains helical ribbons of corner-linked borate and phosphate tetrahedra. Investigation of the thermal behavior up to 180 degrees C shows a (reversible) dehydration process; this leads to the microporous compound Na[ZnBP2O8].H2O, which has the CZP topology. The crystal structure of Na[ZnBP2O8].H2O was determined by X-ray powder diffraction by using a combination of simulated annealing, lattice-energy minimization, and Rietveld refinement procedures (hexagonal, P6(1)22 (no. 178), a = 954.04(2), c = 1477.80(3) pm, V= 164.88(5).10(6) pm3, Z = 6). The essential structural difference caused by the dehydration concerns the coordination of Zn2- changing from octahedral to tetrahedral arrangement.

Journal Article↗
PubMed2001

First expression and characterization of a recombinant CuA-containing subunit II from an archaeal terminal oxidase complex.

The branched respiratory chain of the archaeon Sulfolobus acidocaldarius contains a supercomplex, SoxM, consisting of a bc1-like subcomplex and a terminal oxidase moiety, including a subunit II analogous polypeptide, SoxH. However, the latter component has never been identified in preparations of SoxM. We demonstrate the presence of an mRNA transcript by Northern analysis. We succeeded in cloning and expressing the respective gene with truncated N-terminus by deleting a 20 AS membrane anchor, which resulted in a water-soluble purple copper protein, which was further characterized. The recombinant subunit II of the SoxM complex contains a correctly inserted binuclear CuA cluster as revealed by UV/vis and EPR spectroscopy. The protein is highly thermostable and displays a redox potential of +237 mV. In recombinant form, the metal interacts with cytochrome c as an artificial electron donor; the physiological electron donor is still unknown, since S. acidocaldarius does not contain any c-type cytochromes. The purple copper center of SoxM shows an interesting pH dependency with a pKa at 6.4, suggesting protonation of the Cu-ligating histidines. Further lowering the pH causes a reversible transition into another cluster form with concomitant liberation of one copper. It may thus provide a model for the study of cluster rearrangements in response to pH.

Amino Acid Sequence↗
PubMed2001

Ferredoxins from the archaeon Acidianus ambivalens: overexpression and characterization of the non-zinc-containing ferredoxin FdB.

Two ferredoxin genes, fdA and fdB, from the extremely thermoacidophilic crenarchaeon Acidianus ambivalens have been sequenced; the sequences share 86% similarity. Whereas the deduced protein sequence of the ferredoxin FdA clearly contains a zinc-binding motif, the corresponding sequence of the FdB is devoid of this motif. Thus far, only the zinc-containing ferredoxin, FdA, from A. ambivalens has been chemically and functionally characterized from its native source. Using RT-PCR and Northern blot analysis, we show that both ferredoxins are expressed by A. ambivalens under either anaerobic or aerobic growth conditions. The zinc-free ferredoxin, FdB, was overexpressed in E. coli and purified to homogeneity. Using EPR spectroscopy, we could demonstrate that FdB contains one [3Fe-4S](1+/0) and one [4Fe-4S](2+/1+) cluster. The reduction potential of the [3Fe-4S](1+/0) cluster was determined as -235+/-10 mV, at pH 6.5, by EPR-monitored redox titration. The high melting temperature of 108+/-2 degrees C of FdB determined by CD spectroscopy reveals that it is not the binding of the Zn2+ that induces the extreme thermostability of these ferredoxins.

Amino Acid Sequence↗
PubMed2000

The crystal structure of the conserved GTPase of SRP54 from the archaeon Acidianus ambivalens and its comparison with related structures suggests a model for the SRP-SRP receptor complex.

BACKGROUND: Protein targeting to the endoplasmic reticulum in eukaryotes and to the cell membrane in prokaryotes is mediated by the signal recognition particle (SRP) and its receptor (SR). Both contain conserved GTPase domains in the signal-peptide-binding proteins (SRP54 and Ffh) and the SR proteins (SRalpha and FtsY). These GTPases are involved in the regulation of protein targeting. Most studies so far have focussed on the SRP machinery of mammals and bacteria, leaving the SRP system of archaea less well understood. RESULTS: We report the crystal structure of the conserved GTPase (NG-Ffh) from the thermophilic archaeon Acidianus ambivalens at 2.0 A resolution and of the Thr112-->Ala mutant, which is inactive in GTP hydrolysis. This is the first structure of an SRP component from an archaeon and allows for a detailed comparison with related structures from Escherichia coli and thermophilic bacteria. In particular, differences in the conserved consensus regions for nucleotide binding and the subdomain interfaces are observed, which provide information about the regulation of the GTPase. These interactions allow us to propose a common signalling mechanism for the SRP-SR system. CONCLUSIONS: The overall structure of SRP-GTPases is well conserved between bacteria and archaea, which indicates strong similarities in the regulation of the SRP-targeting pathway. Surprisingly, structure comparisons identified a homodimeric ATP-binding protein as the closest relative. A heterodimer model for the SRP-SR interaction is presented.

Archaea↗
PubMed2000

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose.

Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639) has been described as a novel highly glycosylated membrane-bound b-type hemoprotein [Hettmann, T., Schmidt, C. L., Anemüller, S., Zähringer, U., Moll, H., Petersen, A. & Schäfer, G. (1998) J. Biol. Chem. 273, 12032-12040]. The purified cytochrome b558/566 was characterized by MALDI MS as a 64-kDa (glyco)protein expressing 17% glycosylation. Detailed chemical studies showed that it was exclusively O-mannosylated with monosaccharides and N-glycosylated with at least seven hexasaccharide units having the same unique structure. The hexasaccharide was released by cleavage with peptide:N-glycosidase (PNGase) F and found to consist of two residues each of Man and GlcNAc and one residue each of Glc and 6-deoxy-6-sulfoglucose (6-sulfoquinovose). The last sugar has been known as a component of glycolipids of plants and some prokaryotes, but has not been hitherto found in bacterial glycoproteins. Digestion with trypsin/pronase gave a mixture of glycopeptides with the same Asn-linked hexasaccharide chain, from which an N-glycosylated Tyr-Asn dipeptide was purified by gel chromatography and anion-exchange HPLC. Studies of the degradation products using methylation analysis, ESI MS, MALDI MS, and 1H and 13C NMR spectroscopy, including 1H,13C HMQC and NOESY experiments, established the structure of the unique Asn-linked hexasaccharide chain of cytochrome b558/566.

Cytochrome b Group↗
PubMed2000

[Self-reported substance abuse related emergencies: frequency and nature].

OBJECTIVE: The aim of this study was to estimate the frequency and nature of self-reported and drug-related emergencies. METHODS: 47 patients of a ward for opiate detoxification were interviewed about their experiences with drug-related emergencies. Typical categories had to be found like overdoses, seizures, accidents and suicide attempts respectively. RESULTS: 68% had own experience with drug-related emergency. A majority suffered opiate overdose with different extensions as unconsciousness or breath-depression. Alcohol and polydrug use was associated with overdose. Drug-related accidents were only reported by men. Half the number of drug-related emergencies were treated in hospital. Most emergencies occurred alone either in a home environment or outside. CONCLUSION: Harm reduction interventions like observed user rooms should be established. Furthermore other strategies to reduce the number of emergencies as sharing naloxon or resuscitation programs in wards for detoxification could also be an effective method to prevent near fatal or fatal overdoses in dependent subjects.

Accidents, Traffic↗
PubMed2000

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.

Crystallization↗
PubMed2000

The hyper-thermostable Fe-superoxide dismutase from the Archaeon Acidianus ambivalens: characterization, recombinant expression, crystallization and effects of metal exchange.

An iron-containing superoxide dismutase (SOD; EC 1.15.1.1) of the hyperthermophilic archaeon Acidianus ambivalens (Aa-SOD) has been purified and characterized and the gene has been cloned and sequenced. The SOD from the facultatively aerobic member of the crenarchaeota could be expressed in E. coli. Both, the native as well as the heterologously overproduced protein turned out to have extraordinarily high melting temperatures of 128 degrees C and 124.5 degrees C, respectively. To the best of our knowledge, this is the highest directly measured melting temperature of a native protein. Surprisingly, neither the native nor the recombinant superoxide dismutase displays 100% occupation of the metal coordination sites. Obviously it is not the incorporation of a metal ion that confers the extreme thermostability. Expression of the superoxide dismutase in the presence of different metals such as Fe, Co, Ni, Mn and Cu offered the possibility of studying the hitherto unknown cofactor preference of iron-superoxide dismutase. The recombinant enzyme displayed the highest preference for incorporation of cobalt although iron is used as the natural cofactor. Spectroscopic analysis by EPR, atomic absorption and UVNis spectroscopy as well as activity measurements and differential scanning calorimetry of the metal substituted superoxide dismutases were performed. However, the superoxide dismutase of A. ambivalens is active only with iron but may incorporate other metals equally well in the catalytic center without loss of conformational stability or heat tolerance. The co-form of the enzyme could be crystallized.

Amino Acid Sequence↗
PubMed1999

K

Tetrahedral framework structures with topologies that show close relationships to the feldspar group or to gismondine are formed by the zincoborophosphates A[ZnBP(2)O(8)] (see picture). The compounds are readily accessible under mild hydrothermal conditions (T(max)=170 degrees C) and are thermally remarkably stable (for example, 940 degrees C for A=Cs). Metalloborophosphates are a new class of compounds, which are expected to provide access to zeolite-like microporous systems.

Journal Article↗
PubMed1999

The unusual iron sulfur composition of the Acidianus ambivalens succinate dehydrogenase complex.

The succinate dehydrogenase complex of the thermoacidophilic archaeon Acidianus ambivalens was investigated kinetically and by EPR spectroscopy in its most intact form, i.e., membrane bound. Here it is shown that this respiratory complex has an unusual iron-sulfur cluster composition in respect to that of the canonical succinate dehydrogenases known. The spectroscopic studies show that center S3, the succinate responsive [3Fe-4S]1+/0 cluster of succinate dehydrogenases, is not present in membranes prepared from aerobically grown A. ambivalens, nor in partially purified complex fractions. On the other hand, EPR features associated to the remaining centers, clusters S1 ([2Fe-2S]1+/2+) and S2 ([4Fe-4S]2+/1+), could be observed. Similar findings were made in other archaea, namely Acidianus infernus and Sulfolobus solfataricus. Kinetic investigations showed that the A. ambivalens enzyme is reversible, capable of operating as a fumarate reductase - a required activity if this obligate autotroph performs CO2 fixation via a reductive citric acid cycle. Sequencing of the sdh operon confirmed the spectroscopic data. Center S3 ([3Fe-4S]) is indeed replaced by a second [4Fe-4S] center, by incorporation of an additional cysteine, at the cysteine cluster binding motif (CxxYxxCxxxC-->CxxCxxCxxxC). Genomic analysis shows that genes encoding for succinate dehydrogenases similar to the ones here outlined are also present in bacteria, which may indicate a novel family of succinate/fumarate oxidoreductases, spread among the Archaea and Bacteria domains.

Amino Acid Sequence↗
PubMed1999

The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization.

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ >> Mn2+ >> Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophilus (T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization. The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase.

Bacterial Proteins↗
PubMed1999

Refined crystal structure of a superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius at 2.2 A resolution.

The extremely thermostable superoxide dismutase from the hyperthermophilic archaeon Sulfolobus acidocaldarius was crystallized and the three-dimensional structure was determined by X-ray diffraction methods. The enzyme crystallized in the monoclinic spacegroup C2 with the cell dimensions a=168.1 A, b=91.3 A, c=85.7 A, beta=91.4 degrees. The diffraction limit of these crystals was 2.2 A. The crystals were very stable in the X-ray beam and measured diffraction data of a single crystal had a completeness of 99.5 % up to a resolution of 2.2 A. The crystal structure of S. acidocaldarius superoxide dismutase was solved by Patterson search methods using a dimer of Thermus thermophilus superoxide dismutase as a search model. The asymmetric unit accommodates three dimers. Two dimers form a tetramer by using only local symmetries; the third dimer forms a tetramer as well, however, by using the crystallographic 2-fold symmetry. The three-dimensional structure of the S. acidocaldarius dismutase has typical features of tetrameric dismutases. Secondary structure elements as well as residues important for the catalytic activity of the enzyme were found to be highly conserved. The model was refined at a resolution of 2.2 A and yielded a crystallographic R-value of 17.4 % (Rfree=22.3 %). A structural comparison of the two extremely stable tetrameric dismutases from S. acidocaldarius and Aquifex pyrophilus with the less stable enzyme from T. thermophilus and Mycoplasma tuberculosis revealed the structural determinants which are probably responsible for the high intrinsic stability of S. acidocaldarius dismutase. The most obvious factor which may give rise to the extraordinary thermal stability of S. acidocaldarius dismutase (melting temperature of about 125 degreesC) is the increase in intersubunit ion pairs and hydrogen bonds and, more importantly, the significant reduction of solvent-accessible hydrophobic surfaces, as well as an increase in the percentage of buried hydrophobic residues.

Amino Acid Sequence↗
PubMed1999

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.

Amino Acid Sequence↗