Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers: Sepharose 4B versus flat sheet and hollow fibre membranes

Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B. The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E. coli derived endotoxin from buffers and bovine serum albumin solutions. The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline. The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120 000 in protein-free solutions and 40 to 33 000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration. The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose. The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.     

Genetic and biochemical properties of an extracellular neutral metalloprotease from Staphylococcus hyicus subsp. hyicus

The gene encoding the extracellular neutral metalloprotease ShpI from Staphylococcus hyicus subsp. hyicus was cloned. DNA sequencing revealed an ORF of 1317 nucleotides encoding a 438 amino acid protein with Mr of 49698. When the cloned gene was expressed in Staphylococcus carnosus, a 42 kDa protease was found in the culture medium. The protease was purified from both S. carnosus (pCAshp1) and S. hyicus subsp. hyicus. The N-terminal amino acid sequences of the two proteases revealed that ShpI is organized as a pre-pro-enzyme with a proposed 26 amino acid signal peptide, a 75 amino acid hydrophilic pro-region, and a 337 amino acid extracellular mature form with a calculated Mr of 38394. The N-termini showed microheterogeneity in both host strains. ShpI had a maximum proteolytic activity at 55°C and pH 7.4–8.5. The protease, which had a low substrate specificity, could be inhibited by metal- and zinc-specific inhibitors, such as EDTA and 1,10-phenanthroline. Insensitivity to phosphoramidon separates ShpI from the thermolysin-like family. The conserved Zn²⁺ binding motif, the only homology to other proteases, and the reactivation of the apoenzyme by Zn²⁺, indicated that Zn²⁺ is the catalytic ion. Ca²⁺ very probably acts as a stabilizer. We also demonstrated the presence of a second extracellular protease in S. hyicus subsp. hyicus.     

Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.     

The most abundant soluble basic protein of the stylar transmitting tract in potato (Solanum tuberosum L.) is an endochitinase

An abundant, pistil-specific basic protein has been purified and characterized from potato (Solanum tuberosum L.). A polymerase chain reaction (PCR) probe was generated for the corresponding gene using oligonucleotides based on internal peptide sequences of the protein, and the PCR probe was further employed to isolate cDNA and genomic clones. The sequence of the gene exhibits up to 70% similarity to previously described endochitinase class 1a protein sequences, and the purified protein possesses chitinase {poly[1, 4-(N-acetyl--D-glucosaminide)] glucanohydrolase, EC 3.2.1.14} activity. The protein, termed SK2, has been located by immunocytochemistry to the intercellular matrix of the stylar transmitting tract. Immunoblot analysis has shown SK2 to be distinct from the wound-induced chitinases of potato. The SK2-class of chitinase is restricted in its distribution within the Solanaceae to the sub-family Solanoidae, which includes cultivated tomato and potato species. It was absent from the Cestroidae species tested (Petunia hybrida, Nicotiana tabacum). A role for SK2 endochitinase in protecting the ovary against pollen-tubemediated pathogen ingress is proposed.Abbreviations FPLC fast protein liquid chromatography - IEF isoelectric focussing - PCR polymerase chain reaction We are indebted to Drs. E. Kombrink, J. Logemann, J. Schmidt and Mr. G. Jach (MPI für Züchtungsforschung, Köln, Germany) for advice on chitinase assays. The technical assistance of Ms. U. Seul and Mrs. B. Piegeler is gratefully acknowledged. Electron microscopy was carried out under the supervision of Drs. Brian Wells and Keith Roberts, (John Innes Centre for Plant Science Research, Norwich, UK). This research was supported by the Deutsche Forschungsgemeinschaft SPP Mechanisms of Hybrid Breeding, and the EC BRIDGE programme.     

Identification and sequence analysis of the soxB gene essential for sulfur oxidation of Paracoccus denitrificans GB17.

The coding region for lithotrophic sulfur oxidation (Sox) in Paracoccus denitrificans GB17 was identified by isolation of a transposon Tn5-mob mutant with a Sox- phenotype (strain TP19). The corresponding wild-type region was cloned previously (G. Mittenhuber, K. Sonomoto, M. Egert, and C. G. Friedrich, J. Bacteriol. 173:7340-7344, 1991). Sequence analysis of a 2.5-kb subclone that complemented strain TP19 revealed that Tn5-mob was inserted into a coding region for a 553-amino-acid polypeptide named SoxB. This polypeptide had an M(r) of 60.573, including a possible signal peptide. The function of the SoxB protein of P. denitrificans GB17 appeared to be identical to that of enzyme B of the thiosulfate-oxidizing enzyme system of Thiobacillus versutus. The amino acid compositions of the two proteins were identical, and the amino acid sequences of three internal peptides of enzyme B as determined by Edman degradation were identical to corresponding sequences of the deduced SoxB protein of P. denitrificans GB17.     

Pulsed-field gel electrophoresis of the genomic restriction fragments of coagulase-negative staphylococci

Abstract The genomes of 47 coagulase-negative staphylococcal strains assigned to different species were analysed by pulsed-field electrophoresis. The strains were clustered on the basis of their similarity in the Sma I restriction patterns into various groups, each group consisting of the type strain and the strains whose Sma I restriction patterns were similar to that of the type strain. The Sma I restriction groups seem to correspond to the following species: Staphylococcus warneri, S. hominis, S. xylosus, S. lugdunensi, S. kloosii, S. haemolyticus, S. lentus, S. cohnii, S. equorum, S. chromogenes, S. saprophyticus, S. simulans, S. carnosus, S. capitis and S. auricularis . The species S. sciuri, S. caseolyticus, S. gallinarum, S. epidermidis and S. schleiferi were represented only by their type strains and showed no similarity in their Sma I restriction patterns neither to each other nor to all the species investigated here. Thus, the classification of coagulase-negative staphylococcal strains into the above species seems to be confirmed also by genome restriction analysis carried out by pulsed-field gel electrophoresis.     

X-inactivation pattern in carriers of X-linked retinitis pigmentosa: A valuable means of prognostic evaluation?

In a large family with X-linked retinitis pigmentosa 2 (XLRP2), we reexamined 7 obligate carrier females and 6 daughters of obligate carriers, whose linkage relationships suggested that they carried the XLRP2 gene. The phenotype varied from totally normal eyes through mild retinal changes to complete loss of vision. The X-inactivation analysis was carried out with the highly informative probe M27 on DNA from blood lymphocytes. This probe detects a locus DXS 255 that is differentially methylated on the active and inactive X chromosomes. In 5 blind heterozygotes (aged 43 to 68 years), we found that the X chromosome carrying the RP2 gene was methylated and active in nearly all their cells. The opposite X inactivation pattern was found in a carrier female (aged 45 years) who gave normal findings on eye examination. Carriers with less skewed X inactivation had a less severe clinical outcome. However, we found little or no correlation between their phenotypes and the methylation status of their X chromosomes. Our results suggest that it may be possible to develop a predictive test that could identify cases with severe outcome and perhaps cases with normal outcome.     

Processing of asparagine-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells

The levels of ß1,2-N-acetylglucosaminyltransferase(GlcNAc-T) I and II activities in cultured cells from Bombyxmori(Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda(Sf-9 and Sf-21) were investigated. Apart from initial experimentswith Man