The causative genes of two types of hereditary doparesponsive dystonia (DRD) due to dopamine (DA) deficiency in the nigrostriatum DA neurons have been elucidated. Autosomal dominant DRD (AD-DRD) was originally described by Segawa as hereditary progressive dystonia with marked diurnal fluctuation (HPD). We cloned the human GTP cyclohydrolase I (GCH1) gene, and mapped the gene to chromosome 14q22.1–q22.2 within the HPD/DRD locus, which had been identified by linkage analysis. GCH1 is the rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor for tyrosine hydroxylase (TH), which is the first and rate-limiting enzyme of DA synthesis. We proved that the GCH1 gene is the causative gene for HPD/DRD based on the identification of mutations of the gene in the patients and decreases in the enzyme activity expressed in mononuclear blood cells to 2–20% of the normal value. About 60 different mutations (missense, nonsense, and frameshift mutations) in the coding region or in the exon-intron junctions of the GCH1 gene have been reported in patients with AD-DRD all over the world. Recent findings indicate that the decreased GCH1 activity in AD-DRD may be caused by the negative interaction of the mutated subunit with the wild-type one, i. e., a dominant negative effect, and/or by decreases in the levels of GCH1 mRNA and protein caused by inactivation of one allele of the GCH1 gene. Autosomal recessive DRD (ARDRD) with Segawa's syndrome was discovered in Germany. The AR-DRD locus was mapped to chromosome 11p15.5 in the chromosomal site of the TH gene. In the AR-DRD with Segawa's syndrome, a point mutation in TH (Gln381Lys) resulted in a pronounced decrease in TH activity to about 15% of that of the wild type. Several missense mutations in the TH gene have been found in AR-DRD in Europe. The phenotype of AR-DRD with the Leu205Pro mutation in the TH gene, which produces a severe decrease in TH activity to 1.5% of that of the wild type, was severe, not dystonia/Segawa's syndrome, but early-onset parkinsonism. However, a marked improvement of all clinical symptoms with a low dose of L-dopa was reported in ARDRD/ parkinsonism patients. These findings on DRD indicate that the nigrostriatal DA neurons may be most susceptible to the decreases in GCH1 activity, BH4 level, TH activity, and DA level, and that DRD is the DA deficiency without neuronal death in contrast to juvenile parkinsonism or Parkinson's disease with DA cell death.

June 1, 2005

In Vitro Transcription of a TATA-Less Promoter: Negative Regulation by the Not1 Protein

Ursula Oberholzer, Martine A. Collart

Page range: 1365-1370

Abstract

Genetic experiments in the yeast Saccharomyces cerevisiae have identified the five Not proteins as global repressors of transcription which preferentially repress core promoters which do not contain a canonical TATA sequence. Recently, the Ccr4 and Caf1 proteins, required for non-fermentative gene expression, were found to be associated with the five Not proteins in 1.2 and 2 MDa Ccr4-Not complexes. These Ccr4-Not complexes, as many other global regulators of transcription, appear to regulate transcription both positively and negatively in vivo . To further characterize the activity of Not1p, the only essential known protein of the Ccr4-Not complex, and determine whether it can act directly as a transcriptional repressor, we established an in vitro transcription system in which the HIS3 TATA-less promoter can be efficiently transcribed. We demonstrate that transcription from the HIS3 TATA-less promoter can be specifically increased in vitro by preparing nuclear extracts from a conditional mutant of the NOT1 gene and analyzing transcription after shifting the nuclear extracts to the restrictive temperature. This result is the first demonstration that one of the Not proteins directly represses transcription. Moreover, it now defines an experimental system in which TATA-less transcription initiation and repression by the Ccr4-Not complex can be studied further.

June 1, 2005

Fast Control of DNA Replication in Response to Hypoxia and to Inhibited Protein Synthesis in CCRF-CEM and HeLa Cells

Gudrun Probst, Hans-Jörg Riedinger, Peter Martin, Michael Engelcke, Hans Probst

Page range: 1371-1382

Abstract

In order to elucidate whether data about the fast regulation of DNA replication in dependence on oxygen supply and on a functioning protein synthesis, previously elaborated with Ehrlich ascites cells, are valid for human cells too, we repeated key experiments with CCRF-CEM and HeLa cells. The most important techniques employed were DNA fibre autoradiography and alkaline sedimentation analyses of growing (pulselabeled) daughter strand DNA. It was found that CCRF-CEM and HeLa cells responded to transient hypoxia and to transient inhibition of protein synthesis in an almost identical fashion. Scheduled replicon initiations were reversibly suppressed and the progress rates of replication forks, which were already active before the respective inhibitory conditions were established, were reversibly slowed down. The inclusion of the fork progress rate in the response differs from Ehrlich ascites cells, which respond only by suppressing initiation. Further circumstances of the fast oxygen dependent response, concerning the behaviour of ribonucleotide reductase and of the dNTP pools, revealed no significant differences among the three cell lines. The striking identity of the response of each of the cell lines to hypoxia and to inhibited protein synthesis prompts the suspicion that converging fast regulatory pathways act on the cellular replication machinery. The phenomena as such seem to be rather widespread among mammalian cells.

June 1, 2005

The Inhibition of NF-B Activation Pathways and the Induction of Apoptosis by Dithiocarbamates in T Cells Are Blocked by the Glutathione Precursor N-Acetyl-L-Cysteine

Paula C. Fernandez, Joel Machado, Volker T. Heussler, Catherine Botteron, Guy H. Palmer, Dirk A.E. Dobbelaere

Page range: 1383-1394

Abstract

Nuclear factor-κB regulates genes that control immune and inflammatory responses and are involved in the pathogenesis of several diseases, including AIDS and cancer. It has been proposed that reactive oxygen intermediates participate in NF-κB activation pathways, and compounds with putative antioxidant activity such as N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) have been used interchangeably to demonstrate this point. We examined their effects, separately and combined, on different stages of the NF-κB activation pathway, in primary and in transformed T cells. We show that NAC, contrary to its reported role as an NF-κB inhibitor, can actually enhance rather than inhibit IκB degradation and, most importantly, show that in all cases NAC exerts a dominant antagonistic effect on PDTC-mediated NF-κB inhibition. This was observed at the level of IκB degradation, NF-κB DNA binding, and HIV-LTR-driven reporter gene expression. NAC also counteracted growth arrest and apoptosis induced by dithiocarbamates. Antagonistic effects were further observed at the level of jun-NH 2 -terminal kinase, p38 and ATF-2 activation. Our findings argue against the widely accepted assumption that NAC inhibits all NF-κB activation pathways and shows that two compounds, previously thought to function through a common inhibitory mechanism, can also have antagonistic effects.

June 1, 2005

Xylose Utilisation: Cloning and Characterisation of the Xylose Reductase from Candida tenuis

Beate Häcker, Andreas Habenicht, Michael Kiess, Ralf Mattes

Page range: 1395-1403

Abstract

Xylose reductases catalyse the initial reaction in the xylose utilisation pathway, the NAD(P)H+H + dependent reduction of xylose to xylitol. In this work, the xylose reductase gene from Candida tenuis CBS 4435 was cloned and successfully expressed in E. coli . From the purified and partially sequenced protein primers were deduced for PCR. The fragment obtained was used for Southern blot analysis and screening of a subgenomic library. The clone containing the open reading frame was sequenced; the gene consisted of 969 nucleotides coding for a 322 amino acids protein with a molecular mass of 36 kDa. Putative regulatory signals were identified with the help of a Saccharomyces cerevisiae regulatory sequence database. In order to express the xylose reductase in E. coli , the gene was placed under positive and negative control. At low temperatures, the xylose reductase was expressed in soluble and active form up to about 10% of the soluble protein; with rising temperatures formation of visible inclusion bodies occurred. In refolding experiments we were able to recover the major portion of xylose reductase activity from the pellet fraction.

June 1, 2005

Xylose Utilisation: Cloning and Characterisation of the Xylitol Dehydrogenase from Galactocandida mastotermitis

Andreas Habenicht, Hassan Motejadded, Michael Kiess, Angelika Wegerer, Ralf Mattes

Page range: 1405-1411

Abstract

We cloned and successfully expressed the gene for xylitol dehydrogenase from Galactocandida mastotermitis in Escherichia coli . The amino acid sequence revealed that the enzyme belongs to the superfamily of zinc containing, medium-chain alcohol dehydrogenases. The enzyme catalyses the second step in the xylose utilising pathway converting xylose to xylulosephosphate. Xylulose-phosphate is further degraded by the transaldolase and transketolase reactions of the pentose phosphate pathway. The purified xylitol dehydrogenase from G. mastotermitis was subjected to partial amino acid sequence analysis. The resulting amino acid information was then used to construct oligonucleotide probes for PCR amplification. The PCR product was used to screen a genomic library. The identified xdh gene includes one short intron at its 5′ end. Putative regulatory signals were identified with the help of Saccharomyces cerevisiae regulatory sequence databases. An intronless xdh transcript, cloned by RT-PCR, was actively expressed in pBTac1 at 37°C to approximately 8% of the soluble E. coli protein. Furthermore, the kinetic parameters were determined and conditions were found to stabilise the soluble and active protein.

June 1, 2005

Thermodynamic Properties and DNA Binding of the ParD Protein from the Broad Host-Range Plasmid RK2/RP4 Killing System

Monika Oberer, Helmut Lindner, Otto Glatter, Christoph Kratky, Walter Keller

Page range: 1413-1420

Abstract

ParD is a small, acidic protein from the partitioning system of the plasmid RK2/RP4. The ParD protein exhibits specific DNA binding activity and, as the antidote component of a toxin-antidote plasmid addiction system, ParD forms a tight complex in solution with its toxin antagonist, the ParE protein. Unopposed ParE acts as a toxin that causes growth retardation and killing of plasmid cured cells. ParD negatively autoregulates its expression by binding to an operator sequence in the parDE promoter region. This DNA binding activity is crucial for the regulation of the relative abundance of toxin and antidote which ultimately determines life or death for the bacterial host and its daughter cells. In light scattering studies and gel filtration chromatography we observed the existence of a stable dimer of ParD in solution. The stoichiometry of ParD-DNA complex formation appeared to be 4:1, the molecular mass of the complex was 72.1 kDa. The α-helical content of ParD as determined by CD-spectrometry was 35%. The protein exhibited high thermostability with a T M of 64°C and ΔH of 25 kcal/mol as shown by differential scanning calorimetry. Upon complex formation the T M increased by 10°C. The thermal unfolding of the ParD protein was highly reversible as observed in repeated DSC scans of the same sample. The recovery of the native fold was proven by CDspectroscopy.

June 1, 2005

Dipeptidyl Peptidase III from Rat Liver Cytosol: Purification, Molecular Cloning and Immunohistochemical Localization

Iwao Ohkubo, Yao-Hua Li, Toshinaga Maeda, Yoshio Yamamoto, Takuya Yamane, Pei-Ge Du, Katsuji Nishi

Page range: 1421-1430

Abstract

Dipeptidyl peptidase III (DPP III) was purified to homogeneity from rat liver cytosol. The calculated molecular weight of the purified enzyme was 82845.6 according to TOF-MS and 82000 on non-denaturing PAGE, and 82000 on SDS-PAGE in the absence or presence of Β-mercaptoethanol. These findings suggest that the enzyme exists in a monomeric form in rat liver cytosol. The enzyme rapidly hydrolyzed the substrate Arg-Arg- MCA and moderately hydrolyzed Gly-Arg-MCA in the pH range of 7.5 to 9.5. The K m , k cat and k cat / K m values of DPP III at optimal pH (pH 8.5) were 290μM, 18.0 s −1 and 62.1 s −1 .nm −1 for Arg-Arg-MCA and 125μM, 4.53 s −1 and 36.2 s −1 .nm −1 for Ala-Arg-MCA, respectively. DPP III was potently inhibited by EDTA, 1,10-phenanthroline, DFP, PCMBS and NEM. These findings suggest that DPP III is an exo-type peptidase with characteristics of a metallo- and serine peptidase. For further information on the molecular structure, we screened a rat liver cDNA library using affinity-purified anti-rat DPP III rabbit IgG antibodies, determined the cDNA structure and deduced the amino acid sequence. The cDNA, designated as λRDIII-11, is composed of 2640 bp and encodes 738 amino acids in the coding region. Although the enzyme has a novel zinc-binding motif, HEXXXH, DPP III is thought to belong to family 1 in clan MA in the metalloprotease kingdom. The DPP III antigen was detected in significant amounts in the cytosol of various rat tissues by immunohistochemical examination.

June 1, 2005

Genomic Expansion Across the Albumin Gene Family on Human Chromosome 4q Is Directional

Hitomi Nishio, Hamdi K. Hamdi, Achilles Dugaiczyk

Page range: 1431-1434

Abstract

The albumin gene family arose in a series of duplication events which gave rise to symmetry in its structure. The four genes are tandemly linked on human chromosome 4q in the order: 5′ ALB-5′ AFP-5′ ALF-5′ DBP-centromere, and their introns display a symmetrical and repetitive pattern that is shared by members of the gene family. These repetitive motifs provide an internal reference, allowing observations of evolutionary changes within a single line (human) of evolutionary descent. The four genes and three intergenic regions between them increase in size as they get closer to the centromere. An invasion by multiple repetitive DNA elements may account, in part, for this expansion.

June 1, 2005

Comparison of the Tamoxifen Regulated Chimeric Cre Recombinases MerCreMer and CreMer

Chrysi Verrou, Yong Zhang, Christa Zürn, Wolfgang W.A. Schamel, Michael Reth

Page range: 1435-1438

Abstract

The site-directed recombinase Cre can be employed to delete or assemble genes in the genome of living cells. We have constructed expression vectors for chimeric Cre recombinases carrying a mutated hormone binding domain either at the C-terminus only (CreMer) or at both the N and C-termini (MerCreMer). The chimeric Cre proteins can be activated by culturing transfected cells with 4-hydroxytamoxifen. In transfected embryonic stem (ES) cells, we compared the extent of recombination of a floxed gene with the expression levels of the chimeric Cre proteins. Our data demonstrate that the bulky MerCreMer protein is not less active than the CreMer protein. Thus, the tighter control and low background activity of the MerCreMer enzyme is not due to a generally low recombinase efficiency.

June 1, 2005

The Human Cathepsin F Gene a Fusion Product between an Ancestral Cathepsin and Cystatin Gene

Thomas Wex, Heike Wex, Dieter Brömme

Page range: 1439-1442

Abstract

Human cathepsin F is a novel papain-like cysteine protease of unknown function. Here, we describe the complete human cathepsin F (CTSF) gene which is composed of 13 exons. In addition to a previous report, two novel upstream located exons whose splice sites interrupted the propeptide of cathepsin F within the ‘cystatin-like’ domain, recently described by Nagler et al . (Biochem. Biophys. Res. Comm. 257 , 313–318, 1999) were identified. A comparison of the genomic structures between this novel part of the cathepsin F gene and those of several cystatin genes revealed striking similarities, supporting the hypothesis that the cathepsin F gene resulted from a gene fusion between an ancestral cystatin and cathepsin gene.

June 1, 2005

Characterization of a New Member of the TNF Family Expressed on Antigen Presenting Cells

Catherine Tribouley, Marco Wallroth, Vivien Chan, Xavier Paliard, Eric Fang, George Lamson, David Pot, Jaime Escobedo, Lewis T. Williams

Page range: 1443-1447

Abstract

The TNF family is involved in the regulation of the immune system, and its members have been implicated in a variety of biological events such as apoptosis, cell proliferation, differentiation and survival. Here we present a new member of the TNF family, tumor necrosis factor super family member 20 (TNFSF20) that we have identified from the expressed sequence tag (EST) database and characterized. The human protein is a 285 amino acid long type II transmembrane protein and is 19% homologous to TNF in its extra-cellular domain. TNFSF20 is expressed at the surface of antigen presenting cells such as cells of the macrophagemonocyte lineage and dendritic cells. After treatment with bacterial lipopolysaccharide (LPS), TNFSF20 expression is downregulated at the surface of the expresssing cells, suggesting that the membrane-bound protein gets cleaved, and that a soluble factor is released in the extra-cellular compartment. The soluble form of the recombinant TNFSF20 induces proliferation of resting peripheral blood monocytes (PBMC) and cell death of activated lymphocytes. TNFSF20 might therefore play a critical role in the regulation of cell-mediated immune responses.

June 1, 2005

Vascular Endothelial Growth Factor (VEGF) and Its Receptors in Tumor-Bearing Dogs

Patrick Scheidegger, Wolfgang Weiglhofer, Stéphanie Suarez, Barbara Kaser-Hotz, Rudolf Steiner, Kurt Ballmer-Hofer, Rolf Jaussi

Page range: 1449-1454

Abstract

The molecular biology of the angiogenic growth factor, vascular endothelial growth factor (VEGF), has been studied in the dog. All major isoforms of VEGF are present in the dog. The amino acid sequences are identical between human and dog in the loop regions that are responsible for receptor binding. Accordingly, the VEGF receptors of dogs and humans are very similar and permit functional exchange of the growth factor. Here we show that canine VEGF activates human endothelial cells to the same extent as human VEGF. Similarly, the two proteins display identical cell binding properties. The VEGF receptor 1 (Flt-1) shows the same alternative splicing in humans and dogs and is overexpressed in the majority of tumors in both species. VEGF occurs also in canine tumors in similar relative quantities as in human malignancies. Based on the literature and our study we suggest that the molecular biology and the function of the VEGF signaling system are virtually identical in humans and canines and in healthy as well as in disease conditions.

June 1, 2005

Molecular Cloning and Characterization of a cDNA Encoding a Transferrin Homolog from Bombyx mori

Eun Young Yun, Seok Woo Kang, Jae Sam Hwang, Tae Won Goo, Sang Hyun Kim, Byung Rae Jin, O-Yu Kwon, Keun Young Kim

Page range: 1455-1459

Abstract

We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori . The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

June 1, 2005

Mitochondria-Derived and Extra-Mitochondrial Human Type-1 Porin Are Identical as Revealed by Amino Acid Sequencing and Electrophysiological Characterisation

Ulrike Stadtmüller, Jana Eben-Brunnen, Angela Schmid, Dörte Hesse, Simone Klebert, Hartmut D. Kratzin, Jan Hesse, Bodo Zimmermann, Susanne Reymann, Friedrich P. Thinnes, Roland Benz, Hilde Götz, Norbert Hilschmann

Page range: 1461-1466

Abstract

In mammalian cells porin channels are localised in both mitochondrial outer membranes and extra-mitochondrial membranes. We isolated mitochondria-derived porin of a human lymphoblastoid B cell line, determined its amino acid sequence and characterised its channel properties. Interestingly, the amino acid sequence of this porin preparation and, correspondingly, its electrophysiological characteristics in a reconstituted system were identical to those of ‘Porin 31HL’, the human type-1 porin purified from a crude membrane preparation of the same cell line using a different purification protocol. The results raise questions about targeting, insertion and orientation of human type-1 porin in different membranes.

July 27, 2005

Acknowledgement

Page range: 1467-1468

August 4, 2005

Content Index

Page range: 1469-1477

August 4, 2005

Author Index

Page range: 1479-1482

July 27, 2005

Subject Index

Page range: 1483-1495

About this journal

Objective
Biological Chemistry keeps you up-to-date with all new developments in the molecular life sciences. In addition to original research reports, authoritative reviews written by leading researchers in the field keep you informed about the latest advances in the molecular life sciences. Rapid, yet rigorous reviewing ensures fast access to recent research results of exceptional significance in the biological sciences.

Topics

general biochemistry
pathobiochemistry
structural biology
molecular and cellular biology
genetics and epigenetics
molecular medicine
cancer research
virology
immunology
plant molecular biology and biochemistry
novel experimental methodologies

Article formats
Research Articles, Short Communications, Reviews and Minireviews
Reviews are published by invitation only, but suggestions to the Editor-in-Chief are welcome.

The journal is associated with the German Society for Biochemistry and Molecular Biology (GBM).