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Biomedical subjects

A Mountain

Publications and source records attributed to A Mountain.

36 records · Page 2Linked to original sources
PubMed1993

Expression, purification and characterization of B72.3 Fv fragments.

The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv.

Animals↗
PubMed1992

Engineering antibodies for therapy.

For most MAb-based therapies single doses of MAbs or MAb conjugates will not be curative. Rodent MAbs are highly immonogenic in almost all patients. The HAMA response abrogates efficacy and can cause toxicity in organs of clearance, especially for MAb-cytotoxic agent conjugates. Humanization is the most promising generally applicable approach to overcoming the immunogenicity of rodent MAbs. Chimerization reduces immunogenicity in patients significantly, but not completely. Full humanization of rodent antibodies with retention of most of their antigen binding activity is now a routine procedure. The studies with 4D5 (Kelley et al., 1992), however, illustrate that even when antigen binding activity is retained, humanization may affect the overall conformation of the antibody in ways which influence its interaction with cells (for example when the antigen is internalized or involved in signal transduction) and hence its in vivo properties. As yet there are not sufficient data to judge whether full humanization will (in practical terms) completely overcome the immunogenicity problem in patients, but these data will be available within a year. Antibody fragments are the most promising general approach to manipulating the pharmacokinetics and biodistribution of therapeutic MAbs. Such fragments are clearly superior to whole IgGs for tumour detection and will very likely prove superior for tumour therapy also. MAb targeting of highly potent cytotoxic agents to tumours represents a much-needed approach to improving therapeutic ratios in cancer treatment. Radioisotopes and highly potent low molecular weight drugs are the most promising cell-killing agents for MAb targeting, and conjugation technology suitable for clinical use of some of the best of these agents has now been developed. Very encouraging data have already been obtained in clinical studies of haematopoietic malignancies with MAb-isotope conjugates. Tumour loading data from clinical studies suggest that killing of solid tumours in patients will be achievable in the near future with repeated administration of humanized antibody fragments carrying the superior isotopes or highly potent drugs which are now available.

Animals↗
PubMed1992

Selection of a thermostable variant of chloramphenicol acetyltransferase (Cat-86).

The moderate thermophile Bacillus stearothermophilus was used as a host in which to detect more thermostable variants of the B.pumilus chloramphenicol acetyltransferase (Cat-86) protein. Seventeen mutants were isolated and detected by their ability to grow in the presence of chloramphenicol at a previously restrictive temperature (58 degrees C). The genes encoding these proteins were sequenced; all 17 mutants carried the same C to T transition that conferred an amino acid substitution of alanine by valine at position 203 of the protein sequence. The wild-type and one mutant Cat-86 protein were purified to homogeneity using affinity chromatography, and kinetic and thermal stability studies were undertaken. Both enzymes had similar sp. act. in the region of 215 U/mg, with Km values for chloramphenicol in the range 13.8-15.4 microM and for acetyl CoA in the range 13.6-15.5 microM. The A203V mutant shows greater stability than the wild-type Cat-86 protein at temperatures above 50 degrees C and appears to pass through a transition state between 48 and 50 degrees C.

Bacillus↗
PubMed1990

Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance.

In order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPG1000 (5.0 kb) which carries both a selectable marker (chloramphenicol resistance from Staphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonas putida expressed from a cloned promoter of Bacillus phage SPO2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8.6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPG1000 insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.

Bacillus subtilis↗
PubMed1986

Cloning in Escherichia coli of a Bacillus subtilis arginine repressor gene through its ability to confer structural stability on a fragment carrying genes of arginine biosynthesis.

The structural stability of a previously isolated recombinant plasmid pUL720 was examined. pUL720 contains an insert in pBR322 of 23.8 kbp comprising 4 EcoR1 fragments of sizes 12 kbp and 6 kbp, both of which are homologous to the B. subtilis genome, and 5 kbp and 0.8 kbp (of unknown origin). The 12 kbp fragment, which encodes the arginine biosynthesis genes argA-F-cpa, cannot be cloned in isolation in a high copy vector in E. coli but can be inserted into a low copy vector pGV1106 to generate pUL800. Deletion analysis of pUL720 indicated that the 5 kbp and 0.8 kbp fragments were not necessary to maintain plasmid stability. The 6 kbp fragment, when cloned into the EcoR1 site in pACYC184 to generate pUL2030, permitted the cloning in trans in pBR322 of the 12 kbp fragment or subclones containing the instability region. The minimum inhibitory concentration of kanamycin determined in the B. subtilis argC-neo transcriptional fusion pUL730 and expression of the argF gene product, ornithine carbamoyltransferase (OCTase), in pUL800 were reduced by approximately 3 and 2 fold respectively under conditions of arginine excess and in the presence of pUL2030. B. subtilis partial diploids were constructed by transforming parental and arginine hydroxamate resistant (Ahr) mutants with pUL2100, a plasmid generated by inserting the 6 kbp fragment into the integration vector pJH101. The 6 kbp fragment complemented and restored parental type levels of OCTase in ahrC mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine↗
PubMed1986

Sequence analysis of the Bacillus subtilis argC promoter region.

A previously characterised promoter region upstream from the Bacillus subtilis argC gene was sequenced. The in vivo position of transcription start point (+1), was determined by mung-bean-nuclease mapping. The nucleotide (nt) sequences in the '-10' (TATAAT) and '-35' (TTGAAT) regions closely resemble consensus promoter sequences recognised by B. subtilis sigma 43 and Escherichia coli sigma 70 RNA polymerases. Between +9 and -64 are three imperfect inverted repeats with high homology to the E. coli arginine biosynthetic gene putative operator sequences (ARG boxes) [Cunin et al., Nucl. Acids Res. II (1985) 5007-5019] and which contain variable intra-repeat distances. Upstream from the '-35' region, extending as far as -71, is a 97% AT-rich sequence. The argC mRNA has a short leader region containing a B. subtilis ribosome-binding site 8 nt upstream from a TTG start codon for an open reading frame (ORF). The deduced amino acid sequence for this ORF contains regions of homology to that for the E. coli argC N-terminal region.

Arginine↗
PubMed1986

Gene sequence encoding early enzymes of arginine synthesis within a cluster in Bacillus subtilis, as revealed by cloning in Escherichia coli.

From a partial Sau3A gene library of Bacillus subtilis chromosomal DNA in the expression plasmid pRK9, four hybrid plasmids were isolated carrying overlapping segments of the argA-argF-cpa cluster. The complementation patterns within Escherichia coli arginine auxotrophs of these hybrids and deletion derivatives provided the gene order argC-argA-argE-argB-argD-cpa-argF.

Arginine↗
PubMed1985

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli.

The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.

Base Sequence↗
PubMed1984

Transcription analysis of a Bacillus subtilis arg gene following cloning in Escherichia coli in an initially unstable hybrid plasmid.

Following shotgun cloning of EcoRI fragments of Bacillus subtilis DNA in pBR322, a hybrid plasmid pUL710 was isolated which complements argC but no other auxotrophs of E. coli K12. Restriction mapping, Southern blotting and other evidence suggest that pUL710 carries an insert of 1.6 kbp, and derives, by deletion of both vector and insert sequences, from a larger but unstable initial hybrid which carried a 12 kbp EcoRI fragment from the B. subtilis chromosome. RecE-dependent integration of pUL710 into the B. subtilis chromosome demonstrated homology between the insert DNA and the argO locus of B. subtilis. pUL710 was found to confer appreciable tetracycline resistance even though the deletion presumed to stabilise the hybrid had inactivated the tet promoter. The results of analysis by Tn5 mutagenesis, transcriptional fusions and run-off in vitro transcription suggest that both the cloned argC gene and the tetracycline gene in pUL710 are expressed from a B. subtilis promoter located very close to the EcoRI cloning site.

Aldehyde Oxidoreductases↗
PubMed1984

Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis.

Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNa originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.

Arginine↗
PubMed1984

Bacillus subtilis 168 mutants resistant to arginine hydroxamate in the presence of ornithine or citrulline.

Mutations in Bacillus subtilis 168 have been isolated that confer resistance to arginine hydroxamate in the presence, but not absence, of ornithine. Seven such Ahor mutants have been studied in detail. In common with certain classes of Ahr mutant (resistant to arginine hydroxamate in the absence of arginine precursors) described previously, these Ahor mutants showed little or no inducibility of enzymes of arginine catabolism. Mutants that showed no inducibility were unable to utilize arginine or ornithine as sole nitrogen source. The only biosynthetic enzyme to show any consistent differences in activity from the parent was ornithine carbamoyltransferase, whose level was slightly elevated in cells grown in the presence of ornithine or citrulline. PBS1 transduction crosses showed that two of the ahor mutations map at the ahrA locus, while a third (unique in its resistance to arginine hydroxamate in the presence of citrulline) mapped at a hitherto undescribed locus closely linked to metC, designated ahrD.

Arginine↗
PubMed1980

Map locations of some mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168.

Mutations conferring resistance to arginine hydroxamate in Bacillus subtilis 168 have been located on the genetic map by PBS1-mediated transduction. The majority of these mutations, belonging to classes 1, 2 and 4 of Harwood and Baumberg (1977) and affecting only expression of arginine catabolic enzymes, map at a locus designated ahr A cotransducible with cysA, purA and sacA. The order of markers in this region appears to be sacA-ahrA-purA-cysA. Certain anomalies were observed in the properties of Pur+ transductants from crosses with an Ahr donor and a purA recipient. A single ahr mutation (class 3), also affecting only arginine catabolism, maps between ctrA and sacA at a locus designated ahrB. Two others (class 6), affecting simultaneously enzymes of both arginine biosynthesis and catabolism, map between lys and aroD at a locus designated ahrC. Preliminary attempts to define the nature of functional products specified by these ahr loci suggest that a protein is encoded at ahrA.

Arginine↗