Urs Pauli

Detection of bovine viral diarrhea (BVD) virus using the polymerase chain reaction

The approach of reverse transcription (RT) followed by the polymerase chain reaction (PCR) was used to amplify three different fragments of the bovine viral diarrhea virus (BVDV) genome. Two sets of primers framed two different regions within the gene coding for protein p80, the third set of primers was selected to amplify cDNA within the envelope glycoprotein (gp53) region. All three sequences could be detected in the homologous strain (NADL), whereas only some of the fragments could be amplified in heterologous strains of BVDV. RNA extracted from infected cells as well as RNA extracted from viral particles could be detected using RT-PCR. The detection limit was 10(-1)-10(-2) TCID50 in ethidium bromide stained gels and could be further enhanced to 10(-2)-10(-4) TCID50 by hybridization after Southern transfer. The speed and the sensitivity of this method might be of relevance for diagnostic purposes as well as for studies on epidemiology and pathogenesis of infection with BVD virus.

Improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: PK(15)

Close similarities of various physiological parameters makes the pig one of the preferred animal models for the study of human diseases, especially those involving the cardiovascular system. Unfortunately, the use of pig models to study diseases such as viral hemorrhagic fevers and endotoxic shock syndrome have been hampered by the lack of the necessary immunological tools to measure important immunoregulatory cytokines such as tumor necrosis factor (TNF). Here we describe a TNF-bioassay which is based on the porcine kidney cell line PK(15). Compared to the widely used murine fibroblastoid cell line L929, the PK(15) cell line displays a 100-1000-fold higher sensitivity for porcine TNF-alpha, a higher sensitivity for human TNF-alpha, and a slightly lower sensitivity for murine TNF-alpha. Using a PK(15) bioassay we can detect recombinant TNF-alpha as well as cytotoxic activity in the supernatants of lipopolysaccharide (LPS)-activated porcine monocytes at high dilutions. This suggests that the sensitivity of the test should permit the detection of TNF in biological specimens such as pig serum.

Genomic heterogeneity of small ruminant lentiviruses detected by PCR.

In order to detect a large spectrum of small ruminant lentiviruses, primers for PCR were chosen in conserved parts of the LTR and GAG genes of Icelandic Visna virus 1514 and of the POL gene of caprine arthritis-encephalitis virus. This set of primers was tested in six different caprine arthritis-encephalitis virus (CAEV)- and Maedi-Visna virus isolates of Dutch, American and Swiss origin. The LTR primers allowed the detection of the corresponding fragments of all isolates. The GAG primers allowed amplification of the corresponding fragments of all but the Swiss Maedi-Visna virus strain OLV. Using the POL primers, one Maedi-Visna- and two caprine arthritis-encephalitis virus strains were detected after one round of amplification. Sequencing of the GAG and POL amplification products and comparison to Icelandic Visna virus and CAEV strain CO revealed total heterogeneity of 38% for the GAG- and 28% for the POL fragment. The virus strains studied fall into two groups which are more closely related to one another than to Icelandic Visna virus.

Porcine tumor necrosis factor alpha: cloning with the polymerase chain reaction and determination of the nucleotide sequence

We have cloned the gene for the porcine tumor necrosis factor alpha (TNF-alpha) utilizing the polymerase chain reaction (PCR). Total RNA from stimulated monocytes was used to generate TNF-alpha-specific single-stranded cDNA, with reverse transcriptase and a conserved consensus primer, starting at the TNF-alpha translation stop codon. After adding a second conserved consensus primer to the propeptide region, we amplified the cDNA between the two primers. The isolated fragment was cloned and sequenced. Comparison of the nucleotide sequence indicated an 85% sequence similarity to the human TNF-alpha gene. Eighteen amino acids of the deduced mature peptide sequence of porcine TNF-alpha were different from those in the sequence of man. The technique used in this work allows rapid cloning of specific genes from total RNA, and, additionally, screens for full-length transcripts during the amplification procedure.

Induction of nitric oxide synthase in bovine mononuclear phagocytes is differentiation stage-dependent.

Bovine monocytes and monocyte-derived macrophages (MDM) activated by various means were assessed for induction of inducible nitric oxide synthase (iNOS), using the Griess assay, Northern blotting and reverse transcription/polymerase chain reaction (RT-PCR). Interferon-gamma (IFN-gamma) induced little, if any, iNOS expression and NO production in MDM, although these cells responded to IFN-gamma in other regards. In contrasts, MDM produced copious amounts of NO when stimulated with LPS or Salmonella dublin, and this was paralleled by high steady state levels of iNOS mRNA. Heat-killed Listeria monocytogenes induced more iNOS mRNA and nitrite than IFN-gamma, but much less than L. mono-cytogenes and IFN-gamma combined. Monocytes differed from M phi with respect to iNOS induction and nitrite production in several regards: (i) LPS and S. dublin induced only low levels of iNOS mRNA and nitrite in monocytes, although cells responded to these stimuli in various other ways: (ii) IFN-gamma alone induced in monocytes iNOS mRNA generation and NO formation, although to a low and variable degree; (iii) upon maximal stimulation (e.g. by L. monocytogenes and IFN-gamma combined), monocytes produced much less nitrite than MDM, and mRNA levels were lower. Regulation of macrophage iNOS varies considerably between species. We provide the first evidence in any species that the steady state levels of iNOS mRNA and NO generation in monocytes and macrophages activated by various means depend on the stage of mononuclear phagocyte differentiation.

A bioassay for the detection of tumor necrosis factor from eight different species: evaluation of neutralization rates of a monoclonal antibody against human TNF-α

We have evaluated a recently developed bioassay based on porcine kidney (PK(15)) cells for the detection of tumor necrosis factor-alpha (TNF-alpha) from eight different species. This test could also be used to measure human TNF-beta with similar sensitivity when compared to the widely used L929 bioassay.

Cloning and expression in mammalian cells of porcine tumor necrosis factor alpha: examination of biological properties

We have cloned a full length complementary DNA (cDNA) of the porcine tumor necrosis factor alpha (pTNF-alpha) gene and expressed it in porcine and murine cells. Total RNA obtained from lipopolysaccharide (LPS) stimulated porcine peripheral blood mononuclear cells was reverse transcribed with a specific antisense pTNF-alpha primer to generate a single stranded cDNA which was subsequently amplified by the polymerase chain reaction utilizing an additional pTNF-alpha specific sense primer. The resulting double stranded cDNA was introduced into the pBMGNeo expression vector and transfected by electroporation in porcine (PK(15)) and murine (L929) cell lines. TNF-alpha bioactivity was detected in the supernatant of the transfected cells using a standard L929 bioassay or a PK(15) bioassay. The activity was zinc inducible as expected for a gene controlled by a metallothionein promoter. The bioactivity was not lowered by an anti-mouse TNF-alpha antiserum neutralizing murine, but not human TNF-alpha and a broad immunoreactive band of 17-19 kD was detected using an anti-mouse TNF-alpha serum suitable for immunoblotting. This newly developed tool will allow us to investigate the role of TNF-alpha in pathogenesis of viral infections and gram-negative sepsis.

PORCINE TNF : A REVIEW

One of the classical animal model systems in the study of important cardiovascular diseases, such as septic shock is that of swine. Important mediators of such disease states are different cytokines, among them tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta). This review aims to summarize the current knowledge about the primary structure and the regulation of the porcine TNF genes, as well as methods used to measure their respective proteins.

Regions in the ribosomal minichromosome of Physarum polycephalum are protected from restriction nucleases; protection is insensitive to high salt in the G phase and sensitive in the M phase of the cell cycle

In the central spacer region of the extrachromosomal ribosomal DNA of the slime mold Physarum polycephalum, four small regions of related sequence are completely inaccessible to restriction endonucleases (HinfI and MboI). In addition, some sequences neighboring the inaccessible ones, are partially inaccessible to restriction enzymes and micrococcal nuclease. Taking advantage of the natural synchrony of Physarum plasmodia, we found that this protection is present throughout the cell cycle. Treatment with high salt (2.5 M-NaCl) of nuclei from the G2 phase of the cell cycle left the protection essentially unchanged. When nuclei from the M phase were treated with salt, the protection was abolished. The inaccessible sites are located close to the origins of replication of the rDNA.

Modifications in molecular mechanisms associated with control of cell cycle regulated human histone gene expression during differentiation

Histone proteins are preferentially synthesized during the S-phase of the cell cycle, and the temporal and functional coupling of histone gene expression with DNA replication is mediated at both the transcriptional and posttranscriptional levels. The genes are transcribed throughout the cell cycle, and a 3–5-fold enhancement in the rate of transcription occurs during the first 2 h following initiation of DNA synthesis. Control of histone mRNA stability also accounts for some of the 20–100fold increase in cellular histone mRNA levels during S-phase and for the rapid and selective degradation of the mRNAs at the natural completion of DNA replication or when DNA synthesis is inhibited.Two segments of the proximal promoter, designated Sites I and II, influence the specificity and rate of histone gene transcription. Occupancy of Sites I and II during all periods of the cell cycle by three transacting factors (HiNF-A, HiNF-C., and HiNF-D) suggests that these protein-DNA interactions are responsible for the constitutive transcription of histone genes. Binding of HiNF-D in Site II is selectively lost, whereas occupancy of Site I by HiNF-A and -C persists when histone gene transcription is down regulated when cells terminally differentiate. These results are consistent with a primary role for interactions of HiNF-D with a proximal promoter element in rendering cell growth regulated human histone genes transcribable in proliferating cells.