Biomedical subjects

H W Katinger

Publications and source records attributed to H W Katinger.

7 recordsLinked to original sources

PubMed1990

High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures.

A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 10(7) cells per ml were obtained. The described system is scaleable.

Animals↗

PubMed1990

Detection of the trans activity of the plum pox virus NIa-like protease in infected plants.

The NIa-like protein of plum pox virus is a protease with high sequence specificity that is autocatalytically released from the viral polyprotein. In order to determine whether the protease is active in trans we constructed a fusion protein consisting of the C-terminal region of the plum pox virus polyprotein and the staphylococcal Protein A. The authentic protease recognition sequence Asn-Val-Val-Val-His-Gln-Ala occurs in the centre of this protein fusion. This protein was cleaved specifically by extracts of plum pox virus-infected plants due to the strong activity of the viral protease making it a useful tool for diagnostic purposes.

Amino Acid Sequence↗

PubMed1989

Expression of the plum pox virus coat protein region in Escherichia coli.

A cDNA complementary to the 3' end of plum pox virus (PPV) RNA was sequenced. The sequence was investigated for the presumable coat protein cistron by computer-aided translation. A fragment containing the stop codon of the polyprotein gene and a putative virus-specific protease cleavage site was subcloned into an E. coli expression vector. It is shown by immunological analysis that the coat protein cistron is located within the subcloned region.

Amino Acid Sequence↗

PubMed1989

The complete nucleotide sequence of plum pox virus RNA.

The complete nucleotide sequence of the RNA of an aphid non-transmissible plum pox virus (PPV-NAT) isolate has been determined from five overlapping cDNA clones. cDNA prepared by primer extension was used to determine the 5' terminus. The assembled RNA is 9741 nucleotides in length, excluding a 3' terminal poly(A) sequence. One large open reading frame starts at nucleotide positions 36 to 38 and is terminated with an UAG codon at positions 9522 to 9524. The putative start codon is located at positions 147 to 149. The encoded polyprotein has a predicted Mr of 353.8K. Comparison of cistrons from tobacco vein mottling virus and tobacco etch virus with those predicted for PPV-NAT indicated a similar genome organization. A highly conserved sequence of 12 nucleotides was found in the 5' non-coding region of these three potyviruses. The potential polyadenylation signal from yeast (UAUGU) was found in the 3' non-coding region of PPV-NAT and several other members of the potyvirus group.

Amino Acid Sequence↗

PubMed1988

Early screening for anti-plum pox virus monoclonal antibodies with different epitope specificities by means of gold-labelled immunosorbent electron microscopy.

The technique of gold-labelled immunosorbent electron microscopy for the initial screening of monoclonal antibodies 10 days after cell fusion from 96-well culture plates is described. The technique is used to identify clones that secrete antibodies binding on the surface of the virion or to viral subunits, and compared to ELISA and Western blotting. High sensitivity was demonstrated.

Antibodies, Monoclonal↗

PubMed1979

Experience with testing for mycoplasma with five different methods.

Five methods for mycoplasma-content detection in cell-cultures are established in this laboratory. Results with about 20 different cell lines continuously grown in this laboratory in some determinations indicate that four methods give good correspondence. A microbiological method gave contrary results in many cases. It is possible that new infections will grow on mycoplasma broth but the older infections are adapted to cell-culture and give negative results on artificial medium. New low-level infections cannot be detected by the other methods which do not include any efficient enrichment step. The most convenient method in our opinion is a DNA-staining method according to Chen because this is a very quick, inexpensive and easily performable process. In practice it seems necessary to check cell cultures in two ways, firstly with a quick method and additionally with the microbiological test to ensure the detection of new low-level infections.

Animals↗

PubMedDate not supplied

A PCR membrane spot assay for the detection of plum pox virus RNA in bark of infected trees.

A procedure for sensitive detection of plum pox virus RNA in infected bark of trees is described. The method is based on the extraction of bark material with buffer containing proteinase K followed by partial purification of RNA using QUIAGEN anion exchange resin. The RNA is then reverse transcribed, the single stranded cDNA is amplified by the polymerase chain reaction using biotinylated deoxynucleotides as label. The amplified cDNA can subsequently be detected by spotting the reaction mixture onto a nitrocellulose membrane. After fixation and washing the incorporated label is detected enzymatically using streptavidin-alkaline phosphatase. It was shown that this non-radioactive detection system is more sensitive than ELISA and a DNA/RNA hybridization test using 32P-labelled probes. It is also possible to detect plum pox virus infection with this assay in trees in the non-vegetative period.

Base Sequence↗