Dietrich Simon

Identification of Upregulated Genes in Scrapie-Infected Brain Tissue

ABSTRACT The pathogenesis of scrapie, and of neurodegenerative diseases in general, is still insufficiently understood and is therefore being intensely researched. There is abundant evidence that the activation of glial cells precedes neurodegeneration and may thus play an important role in disease development and progression. The identification of genes with altered expression patterns in the diseased brain may provide insight on the molecular level into the process which ultimately leads to neuronal loss. Differentially expressed genes in scrapie-infected brain tissue were enriched by the suppression subtractive hybridization technique, molecularly cloned, and further characterized. Northern blotting and nucleotide sequencing confirmed the identities of 19 upregulated genes, 11 of which were unknown to be affected by scrapie. A considerable number of these 19 genes, namely those encoding interferon-inducible protein 10 (IP-10), 2′,5′-oligo(A) synthetase, Mx protein, IIGP protein, major histocompatibility complex classes I and II, complement, and β2-microglobulin, were inducible by interferons (IFNs), suggesting that an IFN response is a possible mechanism of gene activation in scrapie. Among the newly found genes, that coding for 2′,5′-oligo(A) synthetase is of special interest because it could contribute to the apoptotic loss of neuronal cells via RNase L activation. In addition, upregulation of the chemokine IP-10 and B-lymphocyte chemoattractant mRNAs was seen at relatively early stages of the disease and was sustained throughout disease development.

Role of Interleukin-1 in Prion Disease-Associated Astrocyte Activation

Prion-induced chronic neurodegeneration has a substantial inflammatory component, and the activation of glia cells may play an important role in disease development and progression. However, the functional contribution of cytokines to the development of the gliosis in vivo was never systematically studied. We report here that the expression of interleukin-1beta (IL-1beta), IL-1beta-converting enzyme, and IL-1 receptor type 1 (IL-1RI) is up-regulated in a murine scrapie model. The scrapie-induced gliosis in IL-1RI(-/-) mice was characterized by an attenuated activation of astrocytes in the asymptomatic stage of the disease and a reduced expression of CXCR3 ligands. Furthermore, the accumulation of the misfolded isoform of the prion protein PrP(Sc) was significantly delayed in the IL-1RI(-/-) mice. These observations indicate that IL-1 is a driver of the scrapie-associated astrocytosis and possibly the accompanying amyloid deposition. In addition, scrapie-infected IL-1RI-deficient (IL-1RI(-/-)) mice showed a delayed disease onset and significantly prolonged survival times suggesting that an anti-inflammatory therapeutical approach to suppress astrocyte activation and/or glial IL-1 expression may help to delay disease onset in established prion infections of the central nervous system.

Highly Infectious Purified Preparations of Disease-Specific Amyloid of Transmissible Spongiform Encephalopathies Are Not Devoid of Nucleic Acids of Viral Size

An efficient purification protocol for infectivity causing a transmissible spongiform encephalopathy (TSE) is described. From fractions purified by this protocol about 3 x 10(8) LD50 but only 3 ng of nucleic acids per gram of brain material can be isolated from all TSE-affected brains (hamster, human, sheep, cattle). By PAGE such fractions from brains of infected and control hamsters contained only one distinct nucleic acid band of 1.5 kg together with some broader smear of nucleic acid material. Although distilled water was used for such purifications, quite often a similar nucleic acid band was isolated from blanks containing no brain material. In all instances this material proved to be DNA. The result challenges the potentially important claim that purified infectious preparations of TSE-specific amyloid are free of nucleic acids of viral size. Nucleic acids isolated by other groups from diseased brain were not detected in preparations isolated by the new protocol. The application of this purification protocol in future studies will be helpful to decide whether TSEs are caused by agents containing nucleic acid or by protein only.

Identification of cDNAs from Japanese pufferfish (Fugu rubripes) and Atlantic salmon (Salmo salar) coding for homologues to tetrapod prion proteins

We identified cDNAs coding for homologues to tetrapod prion proteins (PrPs) in Atlantic salmon (Salmo salar) and Japanese pufferfish (Fugu rubripes), which were termed ‘similar to PrPs’ (stPrPs). Besides significant sequence homologies the fish stPrPs display characteristic structural features in common with tetrapod PrPs. In addition, two stPrPs were shown to be highly expressed in brain tissue. None of the so far identified PrP‐homologues of fish resembles doppel. Hence, the duplication of the PrP gene, which generated doppel, may have occurred not in fish but later in the tetrapod lineage. The identification of fish PrPs provides a basis to address concerns about a possible susceptibility of fish to prion infections.

Transcription of HIV1 is inhibited by DNA methylation

A possible role of DNA methylation as a factor in HIV latency was studied by methylating a HIV1-LTR-CAT plasmid in vitro and measuring its expression after transfection on Vero cells. Methylation with a eukaryotic DNA methylase resulted in a 70% inhibition of chloramphenicol acetyltransferase expression, in the absence as well as in the presence of the HIV1 trans-activator protein TAT in the cell. A similar degree of transcription inhibition was obtained by methylation of the only Hpa II site at position-143 in the HIV1-LTR with the bacterial Hpa II methylase. In contrast to the effect by eukaryotic methylation, the inhibition by Hpa II methylation could be partially reversed by cotransfection of the TAT gene. The reason may lie in an about 40% demethylation at the Hpa II site which was concomitantly observed.

DNA methylation inhibits transcription by RNA polymerase III of a tRNA gene, but not of a 5S rRNA gene

Methylation of cytosine in the DNA inhibits the transcription by RNA polymerase II in higher eukaryotes, but has no influence on RNA polymerase I transcription. The effect on RNA polymerase III was unknown, so far. Two polymerase III genes: a type 1 5S rRNA gene and a type 2 tRNA gene were methylated in vitro with a purified eukaryotic DNA methyltransferase (EC2.1.1.37) and their transcription was analyzed in Xenopus oocytes. The 5S rRNA gene, an oocyte 5S rRNA gene from X. laevis which is subject to developmental inactivation, was not affected by methylation. Conversely, transcription of the tRNA gene was 80% inhibited by methylation with the eukaryotic methyltransferase. HhaI and HpaII methylation left its transcription unaffected.

l-serine dehydrataste from rat liver. Purification and some properties

1. 1. l-Serine dehydratase l-serine hydrolyase (deaminating), EC 4.2.1.13] from rat liver was purified to electrophoretic homogeneity by a new method. 2. 2. After the last purification step two isoenzymes can be distinguished by DEAE-cellulose chormatography as well as by analytical disc electrophoresis. The same isoenzymes can be demonstrated after electrophoresis of a crude extract from rat liver. The smalles subunit of both isoenzymes has a mol. wt of about 35 000. This is in agremment with previous reports (Inoue, H., Kasper, C. B. and Pitot, H. C. (1971) J. Biol. Chem. 246, 2626–2632). 3. 3. From gel filtration experiments in the presence of the substrate l-serine it is concluded that the active form of l-serine dehydratase consists of two subunits. At low enzyme concentrations, the enzyme dissociates into its subunits; K+ and NH4+ counteract this process.

Methylation of the enhancer region of avian sarcoma virus long terminal repeat suppresses transcription

The effect of methylation of an enhancer on transcription was studied. A 245 bp enhancer‐containing a fragment of the LTR of the avian sarcoma virus was methylated in vitro and ligated back into a vector which lacked the upstream enhancer sequence. The transient expression in QT6 cells indicated that methylation of the enhancer‐containing sequence severely reduced the extent of transcription.

Identification of One of the L-Serine Dehydratase Isoenzymes from Rat Liver as L-Homoserine Dehydratase,

Abstract The higher molecular weight L-serine dehydratase was purified from rat liver and fractionated into two active components by Sephadex G-200 gel chromatography. On the basis of molecular weight, ultracentrifugal pattern and substrates specificity of the major component, it was concluded that this component is identical to L-homoserine dehydratase. Some misinter-pretations which have been derived from the work on L-serine dehydratase in animal tissues are pointed out.

Interactions of l-serine dehydratase from rat liver with its coenzyme and substrates

With l-serine dehydratase (l-serine hydrolase (deaminating) EC 4.2.1.13) the ratio of the activities on l-threonine and on l-serine varies with the pyridoxal 5′-phosphate (PLP) and enzyme concentration if the enzyme is incubated with one of the substrates at a time. Incubating the enzyme with both substrates together, the TS ratio remains constant. To explain this phenomenon, the PLP-enzyme dissociation constants were determined under various conditions. K+ and NH4+ decrease the PLP dissociation constants of the enzyme. Dilution of the enzyme increases the constant, probably due to the fact that the enzyme dissociates into its subunits which have a lower affinity for PLP. Tris and the substrates compete for PLP with the enzyme because of Schiffs base formation. It can be concluded that the TS ratio varies because with the range in the PLP-enzyme dissociation constant, the enzyme is not always saturated with PLP, and the two substrates compete differently for PLP with the enzyme.