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R Reinhardt

Publications and source records attributed to R Reinhardt.

At least 19 recordsLinked to original sources
PubMed2003

Complete genome sequence of the marine planctomycete Pirellula sp. strain 1.

Pirellula sp. strain 1 ("Rhodopirellula baltica") is a marine representative of the globally distributed and environmentally important bacterial order Planctomycetales. Here we report the complete genome sequence of a member of this independent phylum. With 7.145 megabases, Pirellula sp. strain 1 has the largest circular bacterial genome sequenced so far. The presence of all genes required for heterolactic acid fermentation, key genes for the interconversion of C1 compounds, and 110 sulfatases were unexpected for this aerobic heterotrophic isolate. Although Pirellula sp. strain 1 has a proteinaceous cell wall, remnants of genes for peptidoglycan synthesis were found. Genes for lipid A biosynthesis and homologues to the flagellar L- and P-ring protein indicate a former Gram-negative type of cell wall. Phylogenetic analysis of all relevant markers clearly affiliates the Planctomycetales to the domain Bacteria as a distinct phylum, but a deepest branching is not supported by our analyses.

Adaptation, Physiological↗
PubMed2003

Early biochemical and histological alterations in rat corticoencephalic cell cultures following metabolic damage and treatment with modulators of mitochondrial ATP-sensitive potassium channels.

The present study was aimed at characterizing alterations of the nucleotide content and morphological state of rat corticoencephalic cell cultures subjected to metabolic damage and treatment with modulators of mitochondrial ATP-dependent potassium channels (mitoK(ATP)). In a first series of experiments, in vitro ischemic changes of the contents of purine and pyrimidine nucleoside diphosphates and triphosphates were measured by high performance liquid chromatography (HPLC) and the corresponding histological alterations were determined by celestine blue/acid fuchsin staining. As an ischemic stimulus, incubation with a glucose-free medium saturated with argon was used. Ischemia decreased the levels of adenosine, guanine and uridine triphosphate (ATP, GTP, UTP) and increased the levels of the respective dinucleotides ADP and UDP, whereas the GDP content was not changed. Both 5-hydroxydecanoate (5-HD) and diazoxide failed to alter the contents of nucleoside diphosphates and triphosphates, when applied under normoxic conditions. 5-HD (30 microM) prevented the ischemia-induced changes of nucleotide and nucleoside levels. Diazoxide (300 microM), either alone or in combination with 5-hydroxydecanoate (30 microM) was ineffective. Pyruvate (5 mM) partially reversed the effects of ischemia or ischemia plus 2-deoxyglucose (20mM) in the incubation medium. Diazoxide (300 microM) and 5-HD (30 microM) had no effect in the presence of pyruvate (5mM) and 2-deoxyglucose (20mM). Staining the cells with celestine blue/acid fuchsin in order to classify them as intact, reversibly or profoundly injured, revealed a protective effect of 5-HD. When compared with 5-HD, diazoxide, pyruvate and 2-deoxyglucose had similar but less pronounced effects. In conclusion, these results suggest a protective role of 5-hydroxydecanoate on early corticoencephalic nucleotide and cell viability alterations during ischemia.

ATP-Binding Cassette Transporters↗
PubMed2002

Early biochemical and histological changes during hyperbaric or normobaric reoxygenation after in vitro ischaemia in primary corticoencephalic cell cultures of rats.

In a first series of experiments, the morphological changes of corticoencephalic cells by ischaemia were determined by staining with celestine blue-acid fuchsin in order to classify cells as intact, dark basophilic (supposedly reversibly injured) and preacidophilic or acidophilic (profoundly injured). Hypoxia and glucose-deprivation (in vitro ischaemia) markedly decreased the number of intact cells and correspondingly increased the number of both reversibly and profoundly damaged cells. The morphological characteristics indicated a partial recovery during reoxygenation either in the absence or presence of glucose and irrespective of whether normobaric or hyperbaric oxygen was used. In a second series of experiments, nucleoside triphosphate and diphosphate levels were determined in corticoencephalic cultures by high-performance liquid chromatography. Hypoxia in combination with glucose-deficiency markedly decreased the ATP:ADP, GTP:GDP and UTP:UDP ratios. A still larger fall of these ratios was observed both after normobaric and hyperbaric reoxygenation. In contrast, both normobaric and hyperbaric reoxygenation in the presence of glucose led to an almost complete recovery near the control normoxic values. In conclusion, the histological changes were not adequately reflected by changes in the nucleoside triphosphate:diphosphate ratios and, in addition, hyperbaric oxygen had neither favourable nor unfavourable effects on the early morphological and functional restitution of ischaemically damaged cells under the conditions of the present study.

Adenine Nucleotides↗
PubMed2002

The genome sequence of Schizosaccharomyces pombe.

We have sequenced and annotated the genome of fission yeast (Schizosaccharomyces pombe), which contains the smallest number of protein-coding genes yet recorded for a eukaryote: 4,824. The centromeres are between 35 and 110 kilobases (kb) and contain related repeats including a highly conserved 1.8-kb element. Regions upstream of genes are longer than in budding yeast (Saccharomyces cerevisiae), possibly reflecting more-extended control regions. Some 43% of the genes contain introns, of which there are 4,730. Fifty genes have significant similarity with human disease genes; half of these are cancer related. We identify highly conserved genes important for eukaryotic cell organization including those required for the cytoskeleton, compartmentation, cell-cycle control, proteolysis, protein phosphorylation and RNA splicing. These genes may have originated with the appearance of eukaryotic life. Few similarly conserved genes that are important for multicellular organization were identified, suggesting that the transition from prokaryotes to eukaryotes required more new genes than did the transition from unicellular to multicellular organization.

Base Sequence↗
PubMed2002

CSF evaluation in primary CNS lymphoma patients by PCR of the CDR III IgH genes.

BACKGROUND: Cytologic evaluation of CSF does not consistently detect malignant cells in patients with primary CNS lymphoma (PCNSL). The potentially more sensitive molecular assessment of monoclonality has not been shown in CSF samples. METHODS: The authors studied nested PCR of the complementary determining region III (CDR III) on 76 CSF specimens of patients with PCNSL. Patients with systemic disseminated B-cell non-Hodgkin's lymphoma (n = 17) and 17 patients with no history of lymphoma were compared. PCR products were evaluated by automated fluorescent fragment analysis (ALF). RESULTS: In 68 patients with PCNSL, the authors analyzed the first obtained CSF sample. Nevertheless, 60 patients were taking corticosteroids. In 16 PCNSL samples, amplifiable DNA was not yielded. Taking into account that at least two independent assays have to be performed, CDR III PCR consistently revealed monoclonal products in eight PCNSL and polyclonal results in 52 PCNSL specimens. CDR III PCR detected no monoclonal PCR products in patients without history of lymphoma. In 10 patients with PCNSL, the PCR result and the CSF cytology were discordant. Concerning therapeutic impact, leptomeningeal tumor spread did not predict tumor response in this group of patients with PCNSL. CONCLUSIONS: This study performed CDR III PCR as a routine diagnostic technique applicable even on CSF samples with low cell counts. These data present low incidence of leptomeningeal involvement in this subset of pretreated PCNSL patients. Because the CSF evaluation did not predict outcome in our patients, further analysis in patients with PCNSL should focus on CSF samples that are obtained very early after diagnosis.

Adult↗
PubMed2002

Alterations of purine and pyrimidine nucleotide contents in rat corticoencephalic cell cultures following metabolic damage and treatment with openers and blockers of ATP-sensitive potassium channels.

Rat corticoencephalic cell cultures were investigated by high performance liquid chromatography for changes in the levels of adenosine 5'-triphosphate (ATP), guanosine 5'-triphosphate (GTP), uridine 5'-triphosphate (UTP), cytidine 5'-triphosphate (CTP), and the respective nucleoside diphosphates. Hypoxia was induced by gassing the incubation medium for 30 min with 100% argon. Removal of glucose was caused by washing the cultures in glucose-free medium at the beginning of the 30 min incubation period. Whereas hypoxia or glucose-deficiency alone failed to alter the nucleotide levels, the combination of these two manipulations was clearly inhibitory. Diazoxide (300 microM) an opener of ATP-dependent potassium channels (K(ATP)) did not alter the nucleotide contents either in a normoxic and glucose-containing medium, or a hypoxic and glucose-free medium. By contrast, the K(ATP) channel antagonist tolbutamide (300 microM) aggravated the hypoxic decrease of nucleotide levels in a glucose-free medium, although it was ineffective in a normoxic and glucose-containing medium. Hypoxia and glucose-deficiency decreased the ATP/ADP and UTP/UDP ratios, but failed to change the GTP/GDP ratio. Diazoxide and tolbutamide (300 microM each) had no effect on the nucleoside triphosphate/diphosphate ratios either during normoxic or during hypoxic conditions. In conclusion, corticoencephalic cultures are rather resistant to in vitro ischemia. Although they clearly respond to the blockade of plasmalemmal K(ATP) channels (plasmaK(ATP)) by tolbutamide, these channels appear to be maximally open as a consequence of the fall in intracellular nucleotides and, therefore, diazoxide has no further effect.

Animals↗
PubMed2001

Analysis of 41 kb of the DNA sequence from the right arm of chromosome II of Schizosaccharomyces pombe.

We report the complete sequence of cosmid c18A7 (41 046 bp insert), located on the right arm of chromosome II of the Schizosaccharomyces pombe genome. The sequence, which partially overlaps with cosmids SPBC4F6 and SPBC336, contains 16 open reading frames (ORFs) capable of coding for proteins of at least 100 amino acid residues in length (one partial) and one small nucleolar RNA (snoRNA). Four known genes were found: swi10 (encoding a mating-type switching protein also involved in nucleotide excision repair); dim1 (encoding a dimethyladenosine transferase); arf1 (encoding ADP-ribosylation factor 1); and pol3 (cdc6) the partial fragment, encoding the 125 kDa catalytic subunit of the DNA polymerase type B. Six ORFs similar to known proteins were found. They include a transporter of the major facilitator superfamily class, a vacuolar sorting protein, an asparagine synthase, a nuclear protein, a reticulum oxidoreductin and a heat shock protein. Each protein product of the other six ORFs has conserved domains and can be assigned a molecular, but not a biological, function. The sequence has been submitted to the EMBL database under Accession No. AL080287.

Amino Acid Sequence↗
PubMed2001

RAI1 is a novel polyglutamine encoding gene that is deleted in Smith-Magenis syndrome patients.

The human chromosomal band 17p11.2 is a genetically unstable interval. It has been shown to be deleted in patients suffering from Smith-Magenis syndrome. Previous efforts of physical and transcriptional mapping in 17p11.2 and subsequent genomic sequencing of the candidate interval allowed the identification of new genes that might be responsible for the Smith-Magenis syndrome. In this report, one of these genes named RAI1, the human homologue of the mouse Rai1 gene, has been investigated for its contribution to the syndrome. Expression analysis on different human adult and fetal tissues has shown the existence of at least three splice variants. Moreover, the most interesting feature of the gene is the presence of a polymorphic CAG repeat coding for a polyglutamine stretch in the amino terminal domain of the protein.

Abnormalities, Multiple↗
PubMed2001

Analysis of chimerism during the early period after allogeneic peripheral stem cell transplantation.

As there are few reports on early evaluation of chimerism, we assessed fluorescence short tandem repeats (STR) by polymerase chain reaction (PCR) assays to analyse donor and recipient characteristics at early time points after peripheral stem cell transplantation (PBSCT). Peripheral blood of 13 patients was analysed in 1- to 2-day intervals starting from the day of PBSCT. Donor and recipient allelic patterns were determined by a commercially available multiplex STR assay that simultaneously evaluates four or five gene loci. Mixed chimerism appeared in all patients during days 1-9 after transplantation and preceded haematologic engraftment for 3-12 days. Even patients without myeloablative conditioning therapy (n=4) revealed donor allelic patterns within 1-5 days. Nine patients changed during the following days to a complete donor allelic pattern and had an uncomplicated post-transplant disease course. Four patients did not consistently retain complete donor chimerism; two of them relapsed within the next 3 months, one died from septicemia within 7 days, and the fourth, transplanted for aplastic anaemia, is still in complete remission. Overall, STR analysis using a simple and comparatively cheap multiplex system permits the detection of chimerism very early after transplantation and may provide relevant information that correlates with the clinical follow-up.

Adult↗
PubMed2000

Physical and transcriptional mapping of the 17p13.3 region that is frequently deleted in human cancer.

Studies of chromosomal losses at 17p13 have suggested the presence of at least two distinct regions for tumor suppressor genes, the TP53 region at 17p13.1 and a more distal region at 17p13.3. Within the latter region, Hypermethylated in Cancer 1 (HIC1) is located, a likely candidate for a tumor suppressor gene that has also been suggested to play a role in the pathogenesis of Miller-Diecker syndrome (MDS). However, single-gene isolation efforts have retrieved additional genes from 17p13.3 that could play a role in tumorigenesis. This indicates that the full potential of this chromosomal region with respect to disease-related genes has not yet been exhausted and that there may exist still unknown genes that contribute to tumorigenesis or to the complex MDS phenotype. To provide a basis for the systematic isolation and evaluation of such genes, we established a physical map over 1.5 Mb of 17p13.3 and assigned 29 transcriptional units within this region.

Abnormalities, Multiple↗
PubMed2000

Sequence and functional comparison in the Beckwith-Wiedemann region: implications for a novel imprinting centre and extended imprinting.

The clustered organization of most imprinted genes in mammals suggests coordinated genetic and epigenetic control mechanisms. Comparisons between human and mouse will help in elucidating these mechanisms by identifying structural and functional similarities. Previously we reported on such a comparison in the central part of the mouse imprinting cluster on distal chromosome 7 with the homologous Beckwith-Wiedemann syndrome (BWS) gene cluster on human chromosome 11p15.5. Here we focus on the adjacent sequences of 0.5 Mb including the KCNQ1/Kcnq1 and CDKN1C/Cdkn1c genes, which are implicated in BWS, and on one of the proposed boundary regions of the imprinting cluster. As in the previously analysed central region, this part of the cluster exhibits a highly conserved arrangement and structure of genes. The most striking similarity is found in the 3' part of the KCNQ1/Kcnq1 genes in large stretches of mostly non-coding sequences. The conserved region includes the recently identified KCNQ1OT1/Kcnq1ot1 antisense transcripts, flanked by a strikingly conserved cluster of LINE/Line elements and a CpG island which we show to carry a maternal germline methylation imprint. This region is likely to be the proposed second imprinting centre (IC2) in the BWS cluster. We also identified several novel genes inside and outside the previously proposed boundaries of the imprinting cluster. One of the genes outside the cluster, Obph1, is imprinted in mouse placenta indicating that at least in extra-embryonic tissues the imprinting cluster extends into a larger domain.

Alternative Splicing↗
PubMed2000

Sequence conservation and variability of imprinting in the Beckwith-Wiedemann syndrome gene cluster in human and mouse.

In human and mouse most imprinted genes are arranged in chromosomal clusters. This linked organization suggests coordinated mechanisms controlling imprinted expression. We have sequenced 250 kb in the centre of the mouse imprinting cluster on distal chromosome 7 and compared it with the orthologous Beckwith-Wiedemann gene cluster on human chromosome 11p15.5. This first comparative imprinting cluster analysis revealed a high structural and functional conservation of the six orthologous genes identified. However, several striking differences were also discovered. First, compared with the mouse the human sequence is approximately 40% longer, mostly due to insertions of two large repetitive clusters. One of these clusters encompasses an additional gene coding for a homologue of the ribosomal protein L26. Second, pronounced blocks of unique direct repeats characteristic of imprinted genes were only found in the human sequence. Third, two of the orthologous gene pairs Tssc4/TSSC4 and Ltrpc5/LTRPC5 showed apparent differences in imprinting between human and mouse, whereas others like Tssc6/TSSC6 were not imprinted in either organism. Together these results suggest a significant functional and structural variability in the centre of the imprinting cluster. Some genes escape imprinting in both organisms whereas others exhibit tissue- and species-specific imprinting. Hence the control of imprinting in the cluster appears to be a highly dynamic process under fast evolutionary adaptation. Intriguingly, whereas imprinted genes within the cluster contain CpG islands the non-imprinted Ltrpc5 and Tssc6/TSSC6 do not. This and additional comparisons with other imprinted and non-imprinted regions suggest that CpG islands are key features of imprinted domains.

Amino Acid Sequence↗
PubMed2000

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Base Sequence↗
PubMed2000

Criteria for gene identification and features of genome organization: analysis of 6.5 Mb of DNA sequence from human chromosome 21.

To establish criteria for and the limitations of novel gene identification, to identify novel genes of potential relevance to Down Syndrome and to investigate features of genome organization, 6. 550kb. In total, 41 novel gene models were predicted, and for a subset of these, RT-PCR experiments helped to verify and refine the models, and were used to assess expression in early development and in adult brain regions of potential relevance to Down syndrome. Results suggest generally low and/or restricted patterns of expression, and also reveal examples of complex alternative processing, especially in brain, that may have important implications for regulation of protein function. Analysis of complete gene structures of the known genes identified a number of very large introns, a number of very short intergenic distances, and at least one potentially bi-directional promoter. At least 3/4 of known genes and 1/2 of predicted genes are associated with CpG islands. For novel genes, three cases of overlapping genes are predicted. Results of these analyses illustrate some of the complexities inherent in mammalian genome organization and some of the limitations of current sequence analysis technologies. They also doubled the number of potential genes within the region.

Adult↗
PubMed2000

Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia.

The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region.

Acute Disease↗
PubMed2000

Induction of putative tumor-suppressing genes in Rat-1 fibroblasts by oncogenic Raf-1 as evidenced by robot-assisted complex hybridization.

The growth factor receptor-dependent protein kinase Raf-1 is activated by GTP-bound Ras, thereby activating the mitogen-activated protein kinase pathway. To study the role of Raf in transformation we transduced Rat-1 cells with a tetracycline-regulatable retroviral vector encoding the constitutively active oncogenic C-terminal fragment of the human Raf-1 protein. Using subtractive hybridization of mRNAs from induced and noninduced cells and robot-assisted screening by complex hybridization, Raf-induced genes with various different characteristics of induction were investigated. Among the strongly induced genes were those involved in carcinogenesis such as metalloproteinases 3, 10 and 13, cathepsin L, ornithine decarboxylase, and putative tumor-suppressing genes such as monocyte chemoattracting protein 1, interferon-induced protein 10, a recently identified 2'-5' oligoadenylate synthetase-like protein, and plasminogen activator inhibitor type 2. Other components of the plasminogen activator system were not induced. Plasminogen activator inhibitor type 2 is a down-regulator of the proteolytic cascade consisting of various metalloproteinases, some of which are induced by a carboxy-terminal Raf mutant (RafCT). In conclusion, RafCT induces factors which act in a conflicting manner in respect of carcinogenesis, especially within the proteolytic system of the extracellular matrix.

Animals↗
PubMed2000

Tissue gene expression analysis using arrayed normalized cDNA libraries.

We have used oligonucleotide-fingerprinting data on 60,000 cDNA clones from two different mouse embryonic stages to establish a normalized cDNA clone set. The normalized set of 5,376 clones represents different clusters and therefore, in almost all cases, different genes. The inserts of the cDNA clones were amplified by PCR and spotted on glass slides. The resulting arrays were hybridized with mRNA probes prepared from six different adult mouse tissues. Expression profiles were analyzed by hierarchical clustering techniques. We have chosen radioactive detection because it combines robustness with sensitivity and allows the comparison of multiple normalized experiments. Sensitive detection combined with highly effective clustering algorithms allowed the identification of tissue-specific expression profiles and the detection of genes specifically expressed in the tissues investigated. The obtained results are publicly available (http://www.rzpd.de) and can be used by other researchers as a digital expression reference.

Algorithms↗