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V Chisholm

Publications and source records attributed to V Chisholm.

13 recordsLinked to original sources
PubMed2001

Generation of a humanized, high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic.

Blocking the cofactor function of human tissue factor may be beneficial in various coagulation-mediated diseases. The murine antibody D3 binds to the membrane proximal substrate interaction region of human tissue factor and blocks tissue factor function even in the presence of bound factor VIIa. The cloned murine D3 antibody was humanized and affinity matured by exchanging amino acids in the complementarity determining regions as well as in the antibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (KD 0.1 nM) as compared to the original murine and chimeric versions. Depending on the particular disease, different pharmacokinetic properties of the antibody may be required and, therefore, several antibody variants-- F(ab), F(ab')2, IgG2, IgG4 and IgG4b-were generated. In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor VIIa-mediated activation of factors IX and X (e.g. D3H44-F(ab')2, IC50(F.X) 47 pM). In addition, D3H44-F(ab')2 completely prevented fibrin deposition in a human ex vivo thrombosis model under venous blood flow conditions (IC50 37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases which involve tissue factor activity, e.g. acute coronary syndrome and venous thrombosis.

Amino Acid Sequence↗
PubMed2000

Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells.

Mammalian cells are often used for the expression of recombinant proteins. The process of screening transfected cells randomly for high producing clones is tedious and time consuming. We evaluated using green fluorescent protein (GFP) for selection of high producing clones by fluorescence-activated cell sorter (FACS) to reduce screening effort. We expressed neurotrophin-3 (NT3), deoxyribonuclease (DNase), or vascular endothelial growth factor (VEGF) with GFP in Chinese hamster ovary cells. The vector expressed the desired secreted protein and the selectable marker, dihydrofolate reductase, in one expression unit and the intracellular GFP in a second expression unit. Transfected cells were grown in selection medium and sorted by FACS. High fluorescence clones were obtained and found to produce high amounts of the desired protein; VEGF productivity correlated well with GFP fluorescence in 48 clones. Further studies demonstrated that productivity correlated very well with RNA of the desired protein. For comparison, we randomly picked and screened 144 VEGF clones, and the highest producing VEGF clone obtained produced 0.7 pg/cell/day. In contrast, the highest producing VEGF clone obtained by FACS sorting produced 4.4 pg/cell/day. FACS sorting therefore selected high producing clones efficiently. Since an assay for the desired protein is not required, high producing clones for a protein of unknown function can be obtained by FACS sorting followed by measuring the RNA level of the desired protein in the highly fluorescent clones.

Animals↗
PubMed1999

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human beta1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or alpha2, 3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of >/=90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Animals↗
PubMed1998

Vascular endothelial growth factor is essential for corpus luteum angiogenesis.

The development and endocrine function of the ovarian corpus luteum (CL) are dependent on the growth of new capillary vessels. Although several molecules have been implicated as mediators of CL angiogenesis, at present there is no direct evidence for the involvement of any. Here we report the unexpected finding that treatment with truncated soluble Flt-1 receptors, which inhibit vascular endothelial growth factor (VEGF) bioactivity, resulted in virtually complete suppression of CL angiogenesis in a rat model of hormonally induced ovulation. This effect was associated with inhibition of CL development and progesterone release. Failure of maturation of the endometrium was also observed. Areas of ischemic necrosis were demonstrated in the corpora lutea (CLs) of treated animals. However, no effect on the preexisting ovarian vasculature was observed. These findings demonstrate that, in spite of the redundancy of potential mediators, VEGF is essential for CL angiogenesis. Furthermore, they have implications for the control of fertility and the treatment of ovarian disorders characterized by hypervascularity and hyperplasia.

Animals↗
PubMed1997

Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders.

Vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis associated with tumors and other pathological conditions, including proliferative diabetic retinopathy and age-related macular degeneration. The murine anti-human VEGF monoclonal antibody (muMAb VEGF) A.4.6.1 has been shown to potently suppress angiogenesis and growth in a variety of human tumor cells lines transplanted in nude mice and also to inhibit neovascularization in a primate model of ischemic retinal disease. In this report, we describe the humanization of muMAb VEGF A.4.6.1. by site-directed mutagenesis of a human framework. Not only the residues involved in the six complementarity-determining regions but also several framework residues were changed from human to murine. Humanized anti-VEGF F(ab) and IgG1 variants bind VEGF with affinity very similar to that of the original murine antibody. Furthermore, recombinant humanized MAb VEGF inhibits VEGF-induced proliferation of endothelial cells in vitro and tumor growth in vivo with potency and efficacy very similar to those of muMAb VEGF A.4.6.1. Therefore, recombinant humanized MAb VEGF is suitable to test the hypothesis that inhibition of VEGF-induced angiogenesis is a valid strategy for the treatment of solid tumors and other disorders in humans.

Adrenal Cortex↗
PubMed1996

High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector.

We have constructed expression vectors for Chinese hamster ovary (CHO) cells that produce both selectable marker and recombinant cDNA from a single primary transcript via differential splicing. These vectors produce stable CHO cell clones that, when pooled, produce abundant amounts of secreted recombinant proteins compared with the amounts produced by conventional expression approaches that have selectable marker and the cDNA of interest under control of separate transcription units. Our vectors divert most of the transcript to product expression while linking it, at a fixed ratio, to dihydrofolate reductase (DHFR) expression to allow selection of stable transfectants. Pools of clones with increased expression of the product gene can be efficiently generated by selection in methotrexate. The high level of expression from pools allows convenient and rapid production of milligram amounts of recombinant proteins.

Animals↗
PubMed1994

Diabetes: its differential impact on child and family.

We argue that there are two groups of diabetic children who cope differently with the treatment regimen, (a) those who maximize their metabolic control and whose difficulties are associated with better control, and (b) those who maintain control less tightly and whose difficulties are associated with poorer control. This hypothesis was investigated with a sample of 47 diabetic children and their mothers. Analyses confirmed the hypothesis, indicating two distinct groups based on the type of symptom (emotional versus conduct) the child exhibited. Symptom type was associated with mother's knowledge of diabetes, diabetic control, scores received on a standardized measure of psychological disturbance and diabetes-specific concerns. The comparison of these two diabetic groups proved more sensitive to differences in diabetic adjustment than did comparisons made on the basis of psychiatric disturbance.

Adaptation, Psychological↗
PubMed1990

A project in diabetes education for children.

Forty-eight families with children less than 13 years old attending a paediatric diabetic clinic volunteered for a 2-year randomized crossover trial to determine whether an informal education programme (diabetic club) could improve diabetic control. Group A attended the diabetic club for 10 afternoons of informal education in the first year, while Group B continued at the routine clinic (5 visits per year). For the second year Group A returned to the clinic, Group B attended the club. Glycosylated haemoglobin (HbA1) remained stable while attending the club but rose significantly (p less than 0.01) while attending the clinic in both groups (HbA1 at baseline, 1 year, and 2 years: Group A, 9.6 (SD 1.2), 9.6(1.4), 10.7(2.1)%; Group B 8.9(1.3), 10.4(1.4), 10.5(1.4)% (normal reference range 4.7-7.9%)). Other indices of control were unchanged. Diabetic problem-solving scores of parents improved (p less than 0.01) but their knowledge of diabetes did not correlate with their child's HbA1. Dietary intake showed a reduction in percentage of energy taken as fat (40% vs 37.7%, p less than 0.05) during club attendance. The percentage of parents reporting helpful social contact between families increased during their club year (Group A 50 to 78%, Group B 32 to 57%, p less than 0.001). Psychological measurements remained unchanged. An education programme for diabetic children may stabilize diabetic control in the short term but this effect is not sustained. The main benefit was the support provided by increased social contact with families of other diabetic children within the informal framework of the diabetic club.

Child↗
PubMed1986

Functional complementation between mutations in a yeast suppressor tRNA gene reveals potential for evolution of tRNA sequences.

Successive rounds of mutagenesis of a Schizosaccharomyces pombe strain bearing the UGA-reading sup3 tRNASer suppressor have been carried out for two cycles of inactivation and reactivation of the suppressor. The suppressor phenotype at each stage was found to involve different combinations of three mutations, A30, A53, and A67, in the sup3-UGA gene. Single mutations A30 and A53 inactivate the suppressor as does the presence of all three mutations. A67 by itself is phenotypically neutral, but in combination with either A30 or A53 suppressor function is restored. The frequency with which these and other complementation events occur in S. pombe demonstrates a significant potential for nucleotide sequence evolution in tRNA. Differential expression of the S. pombe genes in Saccharomyces cerevisiae suggests that the two yeasts have diverged at the transcriptional and RNA processing level. Processing of the mutant tRNA precursors in S. cerevisiae reveals a hierarchy of structural domains within the tRNA that vary in their importance for RNase P cleavage.

Alleles↗
PubMed1984

Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor.

The nucleotide sequences of a Schizosaccharomyces pombe opal suppressor serine tRNA gene (sup9-e) and of 12 in vivo-generated mutant genes, which have lost the ability to suppress UGA mutations, have been determined. Analysis of the expression of these genes in Saccharomyces cerevisiae in vitro and in vivo systems has revealed defects in tRNA gene transcription and precursor tRNA processing. Single base changes in the D-loop, the intron and the extra arm affect the efficiency of splicing of the tRNA precursors while an anti-codon stem mutation may affect the accuracy of this process. Two mutations which occur in the intervening sequence of the sup9-e gene allow an alternate tRNA base pairing configuration. Transcription of the sup9-e gene and of the adjacent tRNAMet gene (located 7 bp downstream) is essentially abolished in vivo by a G----A19 mutation in the tRNASer gene, suggesting that tRNAMet may be derived solely via processing of the tRNASer-tRNAMet dimeric precursor.

Ascomycota↗