Differentiation of rat bone marrow cells into macrophages under the influence of mouse L929 cell supernatant.

Bone marrow cells (BMC) flushed from femora of Lewis rats were cultured in Dulbecco's modification of Eagle's medium supplemented with mouse L929 cell supernatant as a source of colony-stimulating factor (CSF). Differentiation of macrophage progenitor cells into macrophages (M phi) and expression of various markers were kinetically assessed. The proportion of M phi increases from approximately 4% in freshly isolated BMC to 100% after 7-8 days of cell culture. These cells, termed bone marrow cell-derived macrophages (BMDM phi), adhere to and spread on plastic surface; exhibit M phi morphology; stain intensely for nonspecific esterase; are able to phagocytose latex particles, IgG-sensitized erythrocytes, and C3-coated red cells; and express receptors for IgG and C3. A subpopulation of BMDM phi expresses MHC class II antigens as demonstrated by immunofluorescence using MRC OX6 and MRC OX17 monoclonal antibodies which recognize antigens coded in the I-A or I-E subregion of the MHC, respectively. Collectively, our results show that supernatant from mouse L929 cells supports and is continuously required for proliferation and differentiation of rat BMC into typical M phi, and suggest that mouse CSF cross-reacts with the putative receptor on rat M phi.

Specificity of ganglioside binding to rat macrophages.

The binding specificity of rat alveolar macrophages (AM phi) for sheep erythrocytes (E) coated with gangliosides GM1, GM2, GM3, GD1a, GD1b or GT1b was analyzed in a rosette assay by studying the inhibitory effect of gangliosides, various carbohydrates, IgG, C3b-like C3, and fibronectin in this assay. The uptake of gangliosides by E was calculated from radioactivity measurements using 3H-labeled gangliosides. The different gangliosides were taken up by E at 37 degrees C to a similar extent. Uptake of 3H-labeled GM2 correlated linearly to its concn in the incubation medium. Erythrocytes pretreated with the same molar concn of GM2, GD1a, GD1b or GT1b were bound to AM phi to the same degree reaching a maximum of about 90% rosette forming cells. A mean of 17.8% AM phi-bound GM3-coated E. Treatment of E with asialo-GM2 (GA2) or GM1 did not induce significant rosette formation. A dose-dependent inhibition of rosette formation was observed when AM phi were preincubated at 0 degree C with GM2, GM3, GD1a, GD1b or GT1b, but not with GM1 or GA2 Of the tested carbohydrates, sialyl-lactose had a strong inhibitory effect, while lactose was completely ineffective. N-acetyl-neuraminic acid, N-glycolyl-neuraminic acid and N-acetyl-galactosamine were slightly inhibitory. A series of other carbohydrates including highly negatively charged compounds, as well as fibronectin, IgG or C3b-like C3 did not show significant inhibition. Our data indicate the expression of a receptor on rat AM phi recognizing carbohydrates containing sialic acid at or near the non-reducing terminus.

Surface phenotypes of human peripheral blood mononuclear cells from patients with gastrointestinal carcinoma.

Peripheral blood mononuclear cells (PBMC) from 40 patients with gastrointestinal carcinoma (GIC), 13 patients with primary carcinoma in other localizations(non-GIC), and from 57 apparently healthy donors were isolated by Ficoll-Paque gradient centrifugation. The separated cells were stained with several monoclonal antibodies and subjected to analysis on a fluorescence-activated cell sorter. A decreased percentage of PBMC expressing T cell antigens was noted amongst GIC patients, and was mainly due to a reduction of the Leu 2a subset, thus, leading to an increase in the Leu 3a/Leu 2a ratio from 1.4 to 2.1 Non-GIC patients had decreased numbers of both T helper and suppressor cells. Amongst PBMC from GIC and non-GIC patients a statistically increased percentage of cells expressed LeuM 2 (P less than 0.001), LeuM 3 (P less than 0.001), OKM 1 (P less than 0.005), VEP 9 (P less than 0.001), and HLA-DR (P less than 0.001) antigens compared to healthy controls. The percentage of cells bearing these monocyte/macrophage antigens correlated well with the number of cells having monocyte morphology, stained for non-specific esterase, phagocytosed latex particles, and expressed Fc IgG receptor. Our results demonstrate clearly that tumor-bearing patients have an increased relative number of monocytes. The data suggest that cells of the macrophage lineage may be involved in defense mechanisms and changes of the immune system evoked by various tumors.

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.

Cloning and nucleotide sequence of the alpha-galactosidase cDNA from Cyamopsis tetragonoloba (guar).

Polyadenylated mRNA was purified from the aleurone cells of Cyamopsis tetragonoloba (guar) seeds germinated for 18 h and used for the construction of a cDNA library. Clones with the alpha-galactosidase encoding gene were identified using oligo-nucleotide mixed probes based on the NH2 terminal amino acid sequence and on the sequence of an internal peptide. The nucleotide sequence of the cDNA clone showed that the enzyme is synthesized as a precursor with a 47 amino acid NH2 terminal extension. This pre-sequence most likely functions to target the protein outside the aleurone cells into the endosperm. Based upon structural features, it is proposed to divide the precursor into a pre-(signal sequence) part and a glycosylated pro-part comparable with those of the yeast mat A/alpha factor and killer factor. A comparison of the derived amino acid sequence of this alpha-galactosidase from plant origin revealed significant stretches of homology with respect to the amino acid sequences of the enzymes from Saccharomyces cerevisiae and from human origin but only to a minor extent compared with the alpha-galactosidase from Escherichia coli.

The limited host range of an Agrobacterium tumefaciens strain extended by a cytokinin gene from a wide host range T-region.

The host range of Agrobacterium tumefaciens strain LBA649 (pTiAg57) is limited to grapevine and a few other plant species. Its host range was extended through the introduction of the T-region from the wide host range octopine plasmid pTiAch5. In contrast, R prime plasmids harboring the entire wide host range virulence region were unable to achieve this effect. Via site-directed mutagenesis a search was performed to identify the T-DNA genes which were responsible for the observed host range extension. Inactivation of one of the onc-genes (the cyt gene) was found to abolish the capacity of the T-region to extend the host range of LBA649. Therefore, we cloned the cyt gene into a disarmed T-region plant vector and used it in complementation studies with pTiAg57 via the binary vector strategy. We show that the mere presence of the cyt gene from a wide host range Ti plasmid is sufficient to extend the host range of LBA649 to certain plants. We conclude that the limited host range of LBA649 is not caused by a lack of recognition of plants but is mainly due to the absence or inactivity of a cyt gene in the T-region of pTiAg57.

Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli.

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery.

Non-oncogenic plant vectors for use in the agrobacterium binary system.

Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.

Kinetics of mRNA and protein synthesis of genes controlled by the 1,4-beta-endoxylanase A promoter in controlled fermentations of Aspergillus awamori.

In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-beta-endoxylanase A (EXLA)-overproducing strain. Directly after the D-xylose pulse, exIA mRNA was synthesized, and it reached a constant maximal level after 45 to 60 min. This level was maintained as long as D-xylose was present. The kinetics of mRNA synthesis of the genes encoding Thermomyces lanuginosa lipase (lplA) and Escherichia coli beta-glucuronidase (uidA), which were also under the control of the exlA promoter, were similar to those observed for exlA mRNA. The repression of exlA expression was analyzed by pulsing D-glucose to a D-xylose-limited continuous culture. Immediately after the glucose pulse, the exlA mRNA level declined rapidly, with a half-life of approximately 20 to 30 min, and it reached a minimal level after 60 to 90 min. The time span between mRNA synthesis and the secretion of proteins was determined for EXLA and lipase. In both cases, mRNA became visible after approximately 7.5 min. After 1 h, both proteins became detectable in the medium but the rate of secretion of EXLA was faster than that of lipase.

Expression of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha.

The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 microns plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the alpha-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the alpha-galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active alpha-galactosidase enzyme was efficiently secreted (greater than 85%) and after methanol induction, the expression level was 42 mg/l. Amino-terminal sequencing of the purified alpha-galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted alpha-galactosidase was glycosylated and had a sugar content of 9.5%. The specific activity of the alpha-galactosidase produced by H. polymorpha was 38 U mg-1 compared to 100 U mg-1 for the guar alpha-galactosidase. Deglycosylation of the H. polymorpha alpha-galactosidase restored the specific activity completely.