Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography

The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using¹²⁵I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP -Fetoprotein - BSA bovine serum albumine - FCS Fetal calf serum - HRP horseradish peroxidase - OPD o-phenylenediamine dihydrochloride - I.P. isoelectric point - IEF isoelectric focussing - PBS Phosphate buffered saline     

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.     

The HYP2 gene of Saccharomyces cerevisiae is essential for aerobic growth: characterization of different isoforms of the hypusine-containing protein Hyp2p and analysis of gene disruption mutants

In Saccharomyces cerevisiae, hypusine-containing proteins are encoded by two closely related genes, HYP1 and HYP2, which are regulated reciprocally by oxygen and heme. We have purified the aerobically expressed hypusine-containing proteins from yeast. The three proteins detected (two isoforms, which differ in their pI values, and a degradation product thereof, lacking the N-terminal 10 amino acid residues) are all encoded by HYP2. The N-terminus of both isoforms is formed by acetylation of a serine residue after cleavage of the first methionine. Cells mutant for hyp2 are unable to grow aerobically. However, under anaerobic conditions these mutants display no obvious phenotype, presumably because the strictly anaerobically expressed HYPI gene product (Hyp1p) is present. This implies that Hyp1p and Hyp2p fulfill very similar functions. In fact, Hyp1p can substitute for Hyp2p under aerobic conditions, when expressed under the control of the GAL1 promoter in hyp2 mutant cells.Abbreviations HYP1 and HYP2 S. cerevisiae genes encoding hypusine-containing protein Hyplp and Hyp2p, respectively     

A population model for the alkali fly at Mono Lake: depth distribution and changing habitat availability

The densities of alkali fly larvae and pupae were measured in relation to depth and substrate type at six locations around Mono Lake. Samples representing a mixture of different bottom features were taken to a depth of 10 m (33 ft) using SCUBA. This is at or near the depth limit of fly larvae and pupae. The biomass of larvae and pupae on hard substrate were maximum and approximately equal at depths of 0.5 m and 1 m, substantially lower at intermediate depths of 3 m and 5 m, and over an order of magnitude further reduced at 10 m. Densities of flies on hard or rocky substrates (mainly calcareous tufa deposits), were significantly greater than those found on soft substrates such as mud or sand, at all but the greatest depth surveyed.Bathymetric maps of the areas of hard and soft substrate occurring at different lake depths were used to estimate the fly population size over the whole lake, based on the density distribution of larvae and pupae with depth on different substrates. The mapped areas of soft and hard substrates were also calculated for different lake levels, and applying the same procedure, a population model comparing the abundance of flies at different lake levels was developed. This habitat-based population model predicts that the abundance of the alkali fly is maximized at 6380 ft (1945 m) lake surface elevation. Most of the tufa substrate submerged at this lake level will become exposed and unavailable as habitat as the lake declines to 6370 ft (1942 m). In late 1991, the lake level was just over 6374 ft (1943 + m).     

FISCHERELLIN,A NEW ALLELOCHEMICAL FROM THE FRESHWATER CYANOBACTERIUM FISCHERELLA MUSCICOLA1

The benthic cyanobacterium Fischerella muscicola (Thur.) Gom. UTEX 1829 produces a secondary metabolite, fischerellin, that strongly inhibits other cyanobacteria and to a lesser extent members of the Chlorophyceae. Eubacteria are not affected. The major active compound is lipophilic and exhibits a molecular ion at m/z 408. It is heat- and acid-stable but decomposes in 1 M sodium hydroxide (80° C. 1 h). Fischerellin inhibits the photosynthetic but not the respiratory electron transport of cyanobacteria and chlorophytes. Its site of action is located in PS II. Two other species of Fischerella also produce fischerellin, indicating that the synthesis of such allelochemicals might be characteristic of the genus.     

SALINITY AND NUTRIENT LIMITATIONS ON GROWTH OF BENTHIC ALGAE FROM TWO ALKALINE SALT LAKES OF THE WESTERN GREAT BASIN (USA)1

Enrichment cultures of littoral benthic algae from Mono Lake, California, and Abert Lake, Oregon, were grown under conditions of varied salinity and nutrient content. Field-collected inocula were composed mainly of diatoms and filamentous blue-green and green algae. The yield of long-term cultures (30 days) showed tolerance over a broad salinity range (50–150 g·L^?1) for Mono Lake-derived algae. Algae from Abert Lake had a lower range of tolerance (25–100 g·L^?1) Organic content and pigment concentrations of algae from both lakes were also reduced above the tolerated salinity level. Within the range of salinity tolerance for Mono Lake algae, initial growth rates and organic content were reduced by increased salinity. The effects of macro- and micronutrient enrichment on algal growth in Mono Lake water were also tested. Only nitrogen enrichment (either as ammonium or nitrate) stimulated algal growth. Although the benthic algae cultured here had wide optima for salinity tolerance, the rates of growth and storage were limited by increased salinity within the optimum range. Although the lakes compared had similar species composition, the range and limits of tolerance of the algae were related to salinity of the lake of origin.     

High-resolution mapping of the S and Z loci of Phalaris coerulescens.

Self incompatibility (SI) in Phalaris coerulescens is gametophytically determined by two unlinked multi allelic loci (S and Z). Neither the S nor Z genes have yet been cloned. As part of a map-based cloning strategy, high-resolution maps of the S and Z regions were generated from distorted segregating populations using RFLP probes from wheat, barley, oat, and Phalaris. The S locus was delimited to 0.26 cM with two boundary markers (Xwg811 and Xpsr168) and cosegregated with Xbm2 and Xbcd762. Xbcd266 was the closest marker linked to Z (0.9 cM). A high level of colinearity in the S and Z regions was found in both self-incompatible and -compatible species. The S locus was localized to the subcentromere region of chromosome 1 and the Z locus to the long arm end of chromosome 2. Several rice BAC clones orthologous to the S and Z locus regions were identified. This opens the possibility of using the rice genome sequence data to generate more closely linked markers and identify SI candidate genes. These results add further support to the conservation of gene order in the S and Z regions of the grass genomes.     

Molecular basis of intercellular adhesion in the biofilm-forming Staphylococcus epidermidis

The Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA-specific antiserum. PIA was shown to be a linear β-1,6-linked glucosaminoglycan composed of at least 130 2-deoxy-2-amino-D-glucopyrano-syl residues of which 80–85% are N-acetylated, the rest being non-N-acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC-carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co-transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide-polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N-acetylglucosaminyltransferase involved in lipo-chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N-acetylglucosaminyltransferase activity in the formation     

Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway

Benzothiadiazole (BTH) is a novel chemical activator of disease resistance in tobacco, wheat and other important agricultural plants. In this report, it is shown that BTH works by activating SAR in Arabidopsis thaliana. BTH-treated plants were resistant to infection by turnip crinkle virus, Pseudomonas syringae pv ‘tomato’ DC3000 and Peronospora parasitica. Chemical treatment induced accumulation of mRNAs from the SAR-associated genes, PR-1, PR-2 and PR-5. BTH treatment induced both PR-1 mRNA accumulation and resistance against P. parasitica in the ethylene response mutants, etr1 and ein2, and in the methyl jasmonate-insensitive mutant, jar1, suggesting that BTH action is independent of these plant hormones. BTH treatment also induced both PR-1 mRNA accumulation and P. parasitica resistance in transgenic Arabidopsis plants expressing the nahG gene, suggesting that BTH action does not require salicylic acid accumulation. However, because BTH-treatment failed to induce either PR-1 mRNA accumulation or P. parasitica resistance in the non-inducible immunity mutant, nim1, it appears that BTH activates the SAR signal transduction pathway.